Scientific Anual Report 2011 - NKI / AvL

THE 

NETHERLANDS 

CANCER 

INSTITUTE 

SCIENTIFIC 

ANNUAL 

REPORT 2011

SCIENTIFIC ANNUAL REPORT 2011

4 

Patron HM Queen Beatrix

SCIENTIFIC ANNUAL REPORT 2011 

THE NETHERLANDS CANCER INSTITUTE 

CANCER RESEARCH LABORATORY AND CANCER HOSPITAL 

www.nki.nl 

5

6 COPYRIGHT 

Scientifi c Annual Report 2011 

Illustrations and unpublished data in these reports may not be 

used without permission of the author. 

Copyright © 

The Netherlands Cancer Institute 

Antoni van Leeuwenhoek Hospital 

Plesmanlaan 121 

1066 CX Amsterdam 

The Netherlands 

Phone: +31 20 512 9111 

ISSN 1387-8611 

COLOPHON 

Coordinators 

Suzanne Corsetto 

Henri van Luenen 

Monique Duyndam 

Photograph HM The Queen Beatrix 

Enquiries/permission: 

Rijksvoorlichtingsdienst, afd Pers en Publiciteit/Foto 

Postbus 20009 

2500 EA Den Haag 

Photo Ruud Taal/Capital Press 

Copyright RVD 

Photograph AJM Berns: Loek Zuijderduin 

Other photographs and illustrations: 

Audiovisual services 

The Netherlands Cancer Institute/ 


Printed by Drukkerij Badoux, Houten

CONTENTS 

Board Members 8 

Research Divisions 9 

Research Facilities 11 

Introduction 12 

Education in Oncology 16 

Division of Biochemistry 18 

Division of Cell Biology I 24 

Division of Cell Biology II 31 

Division of Experimental Therapy 37 

Division of Gene Regulation 48 

Division of Immunology 54 

Division of Molecular Biology 62 

Division of Molecular Carcinogenesis 74 

Division of Molecular Genetics 80 

Division of Psychosocial Research and Epidemiology 90 

Division of Diagnostic Oncology 102 

Division of Medical Oncology 115 

Division of Radiotherapy 127 

Division of Surgical Oncology 149 

Biometrics Department 162 

Clinical Trials 167 

Invited Speakers 182 

Projects 184 

Personnel Index 203 

7

8BOARD MEMBERS 

BOARD MEMBERS 

BOARD OF DIRECTORS 

AJM Berns, chairman and director of research 

S Rodenhuis, director clinical research and development 

WH Van Harten, director organization and management 

BOARD OF GOVERNORS 

W Kok, president (until June 1, 2011) 

T De Swaan, president (from June 1, 2011) 

HCJ Van der Wielen, vice-president (until June 1, 2011) 

EH Swaab, vice-president (from June 1, 2011) 

JP Balkenende 

GH Blijham 

FH Schröder 

MC Smeets 

PC Van der Vliet 

MJ Van Mourik 

SCIENTIFIC ADVISORY COUNCIL 

AJM Berns, chairman 

JJ Neefjes, secretary 

S Rodenhuis 

B Van Steensel 

T Ruers 

J Jonkers 

INTERNATIONAL SCIENTIFIC ADVISORY BOARD 

T De Lange, Leon Hess Professor, The Rockefeller 

University, New York, USA 

RA Flavell, Professor of Immunobiology, Yale 

University School of Medicine, New Haven, USA 

WGJ Hol, Professor of Biochemistry and Biological 

Structure, University of Washington, Seattle, USA 

J Mendelsohn, President MD Anderson Cancer Center, 

University of Texas, Houston, USA 

P Nurse, Professor of Microbiology, President of 

The Rockefeller University, New York, USA 

R Nusse, Professor of Developmental Biology, Stanford 

University, Stanford, USA 

HL Ploegh, Professor of Biology, Whitehead Institute 

for Biomedical Research, Cambridge, USA 

S. Powell, Chairman, Department of Radiation 

Oncology, Memorial Sloan-Kettering Cancer Center, 

New York, USA 

K Vousden, Director, Beatson Institute for Cancer 

Research, Glasgow, UK 

RA Weinberg, Professor of Biology, Massachusetts 

Institute of Technology, Whitehead Institute for 

Biomedical Research, Cambridge, USA 

President of Board of Governors W Kok (until June 1, 2011) 

President of Board of Governors T de Swaan (from June 1, 2011) 

NATIONAL SCIENTIFIC ADVISORY BOARD 

DD Breimer, Professor of Pharmacology, 

Leiden University 

JL Bos, Professor of Physiological Chemistry, 

University of Utrecht 

EGE De Vries, Professor of Medical Oncology, 

University of Groningen 

JHF Falkenburg, Professor of Experimental Hematology, 

Leiden University 

CG Figdor, Professor of Experimental Immunology, 

Radboud University Nijmegen 

JHJ Hoeijmakers, Professor of Molecular Genetics, 

Erasmus University Rotterdam 

P Lambin, Professor of Radiation Oncology, 

Maastricht University 

RH Medema, Professor of Experimental Oncology, 

Utrecht University 

CJH Van de Velde, Professor of Surgical Oncology, 

Leiden University

RESEARCH DIVISIONS 

Biochemistry 

Titia Sixma (head) 

Thijn Brummelkamp 

Sach Mukherjee 

Anastassis Perrakis 

Caroline Kapper (offi ce manager) 

Cell Biology I 

Arnoud Sonnenberg (head) 

John Collard 

Metello Innocenti 

Kees Jalink 

Wouter Moolenaar 

Ed Roos 

Patty Lagerweij (offi ce manager) 

Cell Biology II 

Jacques Neefjes (head) 

Rob Michalides 

Huib Ovaa 

Peter Peters 

Marieke van der Velde (offi ce manager) 

Experimental Therapy 

Fiona Stewart (head) 

Jan Schellens 

Marjanka Schmidt 

Marcel Verheij 

Jelle Wesseling 

Wilbert Zwart 

Thea Eggenhuizen (offi ce manager) 

Gene Regulation 

Reuven Agami (head) 

Jan-Hermen Dannenberg 

Fred van Leeuwen 

Bas van Steensel 

Suzanne Corsetto (offi ce manager) 

Immunology 

Jannie Borst (head) 

Christian Blank 

John Haanen 

Heinz Jacobs 

Ton Schumacher 

Florry Vyth-Dreese 

Renske de Jong (offi ce manager) 

Molecular Biology 

Hein Te Riele (head) 

Piet Borst (honorary staff member) 

Jos Jonkers 

Sabine Linn 

Alfred Schinkel 

Lodewyk Wessels 

Tom de Knegt (offi ce manager) 

Linda Römer (secretary) 

Molecular Carcinogenesis 

René Bernards (head) 

Roderick Beijersbergen 

Rob Wolthuis 

Patty Lagerweij (offi ce manager) 

Molecular Genetics 

Maarten van Lohuizen (head) 

Anton Berns 

Jacqueline Jacobs 

Anna-Pavlina Haramis 

John Hilkens 

Paul Krimpenfort 

Daniel Peeper 

Margriet Snoek 

Erica Delwel (offi ce manager) 

Marie Anne van Halem (secretary) 

Psychosocial Research and Epidemiology 

Flora van Leeuwen (head) 

Neil Aaronson 

Eveline Bleiker 

Wim van Harten 

Michael Hauptmann 

Matti Rookus 

Sanne Schagen 

Marjanka Schmidt 

Yvonne Driessen-Ruwaard 

(offi ce manager) 

Diagnostic Oncology 

Marcel Stokkel (head) 

Priscilla Axwijk 

Philippe Baars 

Olga Balague Ponz 

Peter Besnard 

Annegien Broeks 

Annemarie Bruining 

Daphne de Jong 

Kenneth Gilhuijs 

Birthe Heeres 

Stijn Heijmink 

Kees Hoefnagel 

Frans Hogervorst 

Jeroen de Jong 

Irma Kluijt 

Petra de Koekkoek – Doll 

Wim Koops 

Tiny Korse 

Robert Kröger 

Charlotte Lange 

Claudette Loo 

Saar Muller 

Jettie Muris 

Petra Nederlof 

Willem Nooijen 

Renee van Pel 

Philip Pevenage 

BOARD MEMBERS 

Warner Prevoo 

Efraim Rosenberg 

Marielle Ruijs 

Joyce Sanders 

Michiel Sinaasappel 

Ferida Sivro 

Jelle Teertstra 

Renato Valdes Olmos 

Hester van Boven 

Fijs van Leeuwen 

Philip Pevenage 

Olaf van Tellingen 

Loes van Velthuysen 

Lizet van der Kolk 

Senno Verhoef 

Erik Vegt 

Wouter Vogel 

Gonneke Winter-Warnars 

Jelle Wesseling 

Ingrid Westerveld - de Zwart 

Bart van de Wiel 

Amir Yazdi 

Christine Arkes (secretary) 

Carla van Tiggelen (secretary) 

Jose Overwater (secretary core facility) 

Medical Oncology 

John Haanen (head) 

Joke Baars 

Paul Baas 

Jos Beijnen 

André Bergman 

Christian Blank 

Jan Paul de Boer 

Willem Boogerd 

Henk Boot 

Dieta Brandsma 

Wieneke Buikhuisen 

Sjaak Burgers 

Annemieke Cats 

Leo Gualtherie van Weezel 

Michel van den Heuvel 

Alwin Huitema 

Martijn Kerst 

Monique van Leerdam 

Sabine Linn 

Anne Lukas 

Serena Marchetti 

Sjoerd Rodenhuis 

Jan Schellens 

Gabe Sonke 

Jacqueline Stouthard 

Neeltje Steeghs 

Babs Taal 

Margot Tesselaar 

Marchien van der Weide 

Mariëlle de Kwant (secretary) 

Karin van Leuveren (secretary) 

9

10 

RESEARCH DIVISIONS 

Radiotherapy 

Marcel Verheij (head) 

Berthe Aleman 

Harry Bartelink 

José Belderbos 

Monique Bloemers 

Gerben Borst 

Roel de Boer 

Luc Dewit 

Paula Elkhuizen 

Rick Haas 

Olga Hamming-Vrieze 

Wilma Heemsbergen 

Edwin Jansen 

Joost Knegjens 

Han Krewinkel 

Joos Lebesque 

Ben Mijnheer 

Luc Moonen 

Arash Navran 

Heike Peulen 

Floris Pos 

Coen Rasch 

Babs Reichgelt 

Peter Remeijer 

Nicola Russell 

Govert Salverda 

Christoph Schneider 

Jan-Jakob Sonke 

Joep Stroom 

Judi van Diessen 

Marcel van Herk 

Baukelien van Triest 

Corine van Vliet-Vroegindeweij 

Marnix Witte 

Thelma Witteveen 

Frits Wittkämper 

Patricia Haye-Fewer 

(section coordinator) 

Surgical oncology 

Theo Ruers (head) 

Marc Van Beurden 

Michiel Van den Brekel 

Arend Aalbers 

Axel Bex 

Bas Van Rhijn 

Biljana Zupan-Kajcovski 

Brigitte Drost 

Christoph Hahn 

Daphne Hompes 

Dirk Buitelaar 

Emiel Rutgers (head) 

Frits van Coevorden 

Germaine Relyveld 

Henk Van de Poel 

Hester Oldenburg 

Houke Klomp 

Ingeborg Vergouwe 

Inka Nieuweboer-Krobotova 

J Joris Hage 

Johanna van Sandick 

Jos van der Hage 

Julia ten Cate 

Karin Ariese 

Katina Efthymiou 

Koen Peeters 

Lenie Hulshoff 

Leonie Woerdeman 

Marianne Crijns 

Marianne Piek-den Hartog 

Marie-Jeanne Baas-Vrancken Peeters 

Marieke van der Berg 

Martine van Huizum 

Michael Srámek 

Michel Wouters 

Omgo Nieweg 

Peter Schutte 

Richard Meijer 

Sandra Huissoon 

Simon Horenblas 

Vic Verwaal 

Wietze Van der Veen 

Wim Meinhardt 

Rachel Tjoa-Bakker (management 

assistant) 

Biometrics department 

Otilia Dalesio 

Financial Administration 

Frieda Boekweit 

General Facilities, ICT and Personnel 

Department 

Eric de Wilde 

General Research Coordination 

Jacques Neefjes, deputy scientifi c 

director 

Henri van Luenen, director of 

operations

RESEARCH FACILITIES 

Animal Pathology & Histology 

Sumiati Baatje 

Lex de Vrije 

Ellen Riem 

Ji-Ying Song 

Jelrik van der Meer 

Martin van der Valk 

Joost van Ooij 

Cryogenic storage 

Minze Dijkstra 

Erwin Kambey 

Digital microscopy 

Lenny Brocks 

Lauran Oomen 

Electron microscopy 

Hans Janssen 

Nico Ong 

Flowcytometry 

Anita Pfauth 

Frank van Diepen 

Glassware 

Esther Holman 

Francis Makatipu-Kambey 

Anita de Bois-Bakker 

Laboratory animals 

Marco Breuer 

Jan Kleinsma 

Filip Mulkens 

Roel Sneeper 

Tatjana Westphal 

and our animal caretakers 

Library 

Suzanne Bakker 

Irene Benne 

Mieke de Mots 

Meena Kanhai 

Truud Kroeskamp 

Microarray & deep sequencing 

Shan Baban 

Wim Brugman 

Ron Kerkhoven 

Roel Kluin 

Marja Nieuwland 

Iris de Rink 

Bernd van der Veen 

Arno Velds 

Molecular pathology & biobank 

Linde Braaf 

Annegien Broeks 

Renate de Groot 

Ingrid Hofl and 

Donne Majoor 

Jose Overwater 

Peptide synthesis 

Dris el Atmioui 

Henk Hilkmann 

Protein expression & purifi cation 

Patrick Celie 

John de Widt 

Magda Stadnik 

Radionuclide Center 

Theo Lamers 

Desiree Verwoerd 

Robotics & screening 

Roderick Beijersbergen 

Bram Gerritsen 

Pasi Halonen 

Cor Lieftink 

Ben Morris 

Secretarial support 

Suzanne Corsetto 

Renske de Jong 

Tom de Knegt 

Yvonne Driessen-Ruwaart 

Caroline Kapper 

Patty Lagerweij 

Marieke van der Velde 

Marie Anne van Halem 

Sequencing 

Roelof Pruntel 

Abderrahim Ajouaou 

Statistics 

Michael Hauptmann 

Martha Lopez-Yurda 

Wilma Heemsbergen 

Technology transfer 

Monique Duyndam 

Hylke Galama 

Rik Grosveld 

Frank Hoorn 

Koen Verhoef 

Transgenesis & cryopreservation 

Paul Krimpenfort 

Fina van de Ahé 

Rahmen Bin Ali 

11 

RESEARCH FACILITIES

12 

INTRODUCTION 

INTRODUCTION 

I am pleased to present our Scientifi c Annual Report 2011. 

It provides an overview of the scientifi c activities at The 

Netherlands Cancer Institute - Antoni van Leeuwenhoek 

Hospital (NKI-AVL). Additional information can be found 

at www.nki.nl. A more broadly accessible overview of our 

activities with illustrations can be found in our brochure 

(available on our website). 

The NKI-AVL is a Comprehensive Cancer Centre, 

combining hospital and research laboratories under one 

roof in a single independent organization. The hospital 

comprises 180 beds, an outpatient clinic and a large 

radiotherapy department. Facilities for clinical research 

include a large patient database, clinical data management, 

extensive diagnostic facilities, a pharmacy with a 

production unit for experimental drugs, and active research 

groups in medical, surgical and diagnostic oncology, 

radiotherapy, pharmacy, epidemiology and psychosocial 

oncology. The laboratory covers all major areas of cancer 

research, with special emphasis on cell-based screens, 

mouse tumor models, cell biology, structural biology, and 

immunology. Translational research is an integral activity 

of many research groups and is fostered by collaborations 

between clinical and basic scientists. 

Clinical activities soared in 2011. In order to accommodate 

the growing number of patients the Board of Governors 

has authorized a 70% expansion of the clinical capacity 

(by 2020). Two other major developments are worth 

mentioning. The national children oncology centre (NKOC) 

will be built near the academic children hospital in Utrecht 

rather than near the NKI-AVL, this to secure the direct 

access to other medical disciplines required for children 

treated for cancer. The NKI-AVL entered in discussions 

with the University Medical Centre Utrecht (UMCU) to 

establish a joint cancer hospital directly adjacent to the 

UMCU and the NKOC. Discussions are ongoing with 

much enthusiasm, as such joint entity would benefi t both 

organizations, further increasing the critical mass as 

well as bringing together complementary expertise in the 

area of radiology and radiotherapy, an area of increasing 

importance for minimal invasive and non-invasive 

interventions in cancer. In 2011 we started the building 

activities of a satellite radiotherapy centre in Hoofddorp, 

we concluded the renovation of the old research building 

and initiated the construction of a new animal facility. 

We fi nally decided not to participate together with the 

Erasmus University, the University Medical Centre Leiden, 

and the Delft Technical University in the establishment 

of a proton therapy centre in Delft, as we believe that with 

the arrival of smaller proton therapy units in the near 

future it is better to opt for a more compact and economic 

unit on our premises. We made limited progress with the 

establishment of a service facility together with UMC- 

Utrecht, Hubrecht Institute, and Erasmus MC to provide 

GMP deep sequencing services for tumors of individual 

patients due to diffi culties to attract funding for this 

initiative. However, it remains high on our wish list as 

it constitutes an integral part of our plans to advance 

Director of Research Ton Berns 

personalized medicine. Furthermore, we have succeeded 

in fi lling most of the vacancies, even those of our intensive 

care unit. Since the shortage of medical experts and skilled 

nursing staff is expected to further increase in the years 

to come we have to make the NKI-AVL stand out as “the 

place to be” for employees. This is an important task for the 

institute and the high ranking of the NKI-AVL on the list of 

best employers in the health care sector does help. 

We concluded 2011 again with a profi t for the hospital. As 

a result no harsh measures with regard to personnel and 

services will have to be taken even though we are facing a 

gradual reduction in reimbursement for our services. Also 

our research expenditure remained within budget in 2011. 

However, this could only be achieved by not fi lling the 

positions of staff members that left the institute. 

Due to the economic situation the research budget of the 

Ministry of Health, Welfare and Sport will be cut yearly 

up to a total percentage of 6% by the end of 2014. While 

it is evident that the government has to reduce costs, 

it is disappointing that in the current political climate 

investment in research has a relatively low priority and 

appears fully focused on short-term valorisation of scientifi c 

research. Many other countries clearly choose to increase 

the support for bottom-up research initiatives as they 

realize that high quality research is critical for securing 

the future prosperity of a country. Fortunately, the Dutch 

Cancer Society has been more receptive to our arguments 

and we have negotiated a new 5-year agreement for core 

funding which will result in a gradual increase of their 

contribution to the research budget. 

Together with the Dutch Cancer Society we have also 

established a foundation that specifi cally will raise funds 

for the NKI. The funds raised by this foundation will 

be critical to maintain our core funding and thereby 

the infrastructure of the institute at an internationally 

competitive level. 

In addition to the core funding the NKI acquired support 

through external grants, donations, and research 

agreements. The contribution of these sources to the total 

budget has steadily increased over the years and represents 

around 65% of the total budget in 2011. For 2011 and 

following years these include several EU-funded projects 

such as Adaptive and innovative Radiation Treatment 

FOR improving Cancer patients’ treatment outcome 

(ARTFORCE), and a program aimed to introduce and

Table 1 

Core research funding NKI-AVL by the Dutch Cancer Society and the Ministry of Health, Welfare and Sport 

in the period 2003-2011 in million Euro’s. 

quality control advanced diagnostic methods for cancer 

patients, thus permitting further stratifi cation of patients 

on the basis of a detailed molecular characterization of their 

tumors and enrollment into defi ned clinical trials within 

Europe (EUROCANPLATFORM). 

HIGHLIGHTS 

The institute continues to have a strong scientifi c output; 

2011 was again a productive year. It is always diffi cult to 

estimate the impact of research when the results are still 

fresh. The research highlights summarised here serve 

primarily as a sampling of work currently on-going in 

the Institute. A more complete and detailed account of 

specifi c projects can be found in the reports of individual 

group leaders in this annual report and on the website 

(www.nki.nl). 

The Bernards group found a molecular explanation for the 

limited therapeutic benefi t of the selective BRAF inhibitor 

vemurafenib in BRAF V600E mutant colon tumors. Using 

a RNA interference-based genetic screen they found that 

inhibition of the epidermal growth factor receptor (EGFR) 

shows strong synergy with BRAF V600E inhibition in 

colon cancer. Inhibition of EGFR by the drugs cetuximab 

or gefi tinib is also strongly synergistic with BRAF V600E 

inhibition in mouse xenografts of colon cancer, suggesting 

that patients with a BRAF mutant colon cancer might 

benefi t from combination therapy consisting of BRAF- and 

EGFR inhibitors. The synergy of these two cancer drugs 

is explained by the observation that BRAF V600E inhibition 

causes a rapid feedback activation of EGFR, which supports 

continued proliferation in the presence of BRAF V600E 

inhibition. 

Petra Paul and colleagues in the group of Neefjes published 

the results of an extensive siRNA screen that resulted in 

new biological insights into the biochemical pathways 

controlling MHC class II antigen presentation, a pathway 

important in anti-tumor and pathogen immune responses 

as well as auto-immunity. 

Koen van de Wetering in the group of Piet Borst developed 

a simple method to fi nd endogenous substrates of drug 

transporters, some of which are involved in drug resistance 

of cancer cells. From cells that contain a large amount of 

transporters in the cell membrane, he can produce vesicles 

in which the orientation of the transporter now permits 

import of compounds into the vesicle. When the vesicles 

are incubated in body fl uids (plasma, urine, bile) the 

natural substrates of the transporter present in the fl uid 

will be transported into the vesicles. The nature of these 

substrates is then determined by a sophisticated form 

of Mass Spectrometry. With this new “Transportomics” 

method a number of new endogenous substrates of ABCtransporters, 

such as MRP2, has been identifi ed. 

The Te Riele group has further refi ned its gene modifi cation 

technology by single-stranded oligonucleotides. 

By using so-called MAMA primers for quantitative PCR, 

they are now able to clonally purify cells carrying a single 

base pair modifi cation. As a proof of principle they have 

generated a G to C substitution in the MYCN gene in 

mouse embryonic stem cells. 

13 

INTRODUCTION 

Year 2003 2004 2005 2006 2007 2008 2009 2010 2011 

Cancer Society 9.1 8.9 8.6 8.3 8.8 9.3 10.0 10.8 11.7 

Ministry Health 10.1 9.2 9.3 9.4 9.5 9.6 10.2 10.3 10.5 

Total* 19.2 18.1 17.9 17.7 18.3 18.9 20.2 21.1 22.2 

Silvia Ariotti in the groups of Schumacher and Haanen 

used intravital imaging to visualize the behavior of 

memory T cells. She demonstrated that after infection or 

vaccination, antigen-specifi c T cells stay behind within the 

skin and continuously patrol the epidermis. This fi nding 

suggests that vaccination against pathogens such as HPV 

will benefi t from the induction of a local memory T cell 

pool at sites of potential viral entry. 

Nomograms for predicting the need for extra radiation dose 

in breast cancer have been developed and made publicly 

available through the web. These nomograms predict both 

the local recurrence as well as the side effects of radiation 

for early breast cancer and are based upon a major breast 

cancer trial of the EORTC in which 5600 patients were 

recruited. 

Kate Sutherland in my own group, using a mouse 

model of small cell lung cancer, showed that this 

tumor predominantly originates from the relatively few 

neuroendocrine cells present in lung but that, although at 

a much lower frequency, rare progenitors with an alveolar 

marker profi le might also serve as its cell of origin. Paul 

Krimpenfort provided conclusive evidence that the DCC 

gene, which was earlier claimed to be irrelevant for tumor 

progression, is actually important in metastatic spread. 

The data reconcile a long dispute on the role of DCC in 

cancer by illustrating that the effect of DCC loss only 

becomes apparent when p53 is also functionally inactivated.

14 

INTRODUCTION 

In the Verheij group, Albert Van Hell showed that liposomal 

co-formulation of the short-chain lipid N-octanoylglucosylceramide 

(GC) and doxorubicin markedly improved 

the drug uptake and response of a chemoresistant breast 

tumor model (Wcre;Cdh1 F/F ;Trp53 F/F ) to doxorubicin 

treatment. In contrast, drug accumulation within normal 

heart tissue was not elevated. In vitro model membrane 

studies and in silico molecular dynamics simulation 

experiments revealed that the energetic barrier for the 

hydrophilic part of the doxorubicin to translocate to the 

opposite side of the membrane was reduced by two-fold due 

to the presence of short-chain lipids. These data suggest 

that the therapeutic window of cancer therapeutics can be 

increased by concomitant targeting of the cellular plasma 

membrane. 

The Epidemiology group (Matti Rookus, Flora van 

Leeuwen) started a new prospective cohort study among 

nurses in the Netherlands (the Nightingale Study). The 

major interest is to investigate the infl uence of shift work 

on the risk of breast cancer; reduced nocturnal levels of 

melatonin during shift work may increase endogenous 

estrogens levels. The recruitment was very successful, with 

over 60,000 women already participating. 

The Epidemiology group also reported on the risk of 

ovarian malignancies in our nationwide OMEGA cohort 

of 251152 women who started subfertility treatment in the 

12 collaborating IVF-clinics in the Netherlands between 

1980 and 1994 (collaboration with CW Burger, Erasmus 

MC Rotterdam). The risk for invasive ovarian cancer in 

IVF-treated women was not signifi cantly increased, but for 

borderline tumors of the ovary a 4-fold increased risk was 

found compared to sub-fertile women not treated with IVF. 

As part of the Breast Cancer Association Consortium, the 

group of Marjanka Schmidt has identifi ed and validated 27 

SNPs associated with breast cancer susceptibility. This year 

they showed that two SNPs, P53 R72P and MDM2 SNP309, 

which play an important role in the p53 response pathway, 

were associated with worse outcome of breast cancer 

within subgroups of tumors displaying a ‘‘more aggressive 

phenotype’’ gene expression profi le. 

QUALITY OF RESEARCH 

The quality of research can be monitored in several ways. 

Our scientifi c productivity as assessed by objective 

bibliometric parameters (citations and impact of scientifi c 

articles published by the NKI staff) has shown a steady 

increase over time (Table 2). It is satisfying to note that the 

Institute retains its internationally prominent position in 

cancer research. 

While a high citation score is gratifying, it is only one 

measure of scientifi c productivity. The quality of our 

work should also be gauged by the invitations of our staff 

to present at international scientifi c meetings, awards 

obtained by our staff and signifi cant grants that were 

obtained. We score high on all these accounts. 

Table 2 

Short-term citations and impact of scientifi c articles published by the NKI research staff 1996 - 2009/2011 

Publication year # publications Citations* Citations/ Impact** 

cited or with publication 

impact factor@ 

1996 260 4056 15.6 1520 

1997 274 4174 14.1 1811 

1998 276 3138 14.2 1392 

1999 280 3474 12.4 1766 

2000 301 4314 16.0 1699 

2001 339 4944 16.2 1554 

2002 407 7436 21.3 2324 

2003 357 5084 15.4 1963 

2004 347 5254 16.2 2018 

2005 399 6261 17.3 2442 

2006 432 6302 16.2 2584 

2007 429 5515 13.8 2590 

2008 445 5671 13.9 2553 

2009 472 7405 15.7 3122 

2010 475@ 2945 

2011 407@ 2825 

* Citations in the two years after publication, excluding self-citations. Starting 1989 citation analysis has been carried out 

online. This allows detection (and elimination) of all self-citations. Before 1989 this pruning was limited to fi rst 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. 

# From 2008 the online publication data is used as criteria for the year of publication.

The quality of research of the groups in each division is 

evaluated approximately every 5 years by an external site 

visit team. In 2011 we have organized a site visit for the 

Animal Facility. On May 11, the site visit team formed by 

Catheryn O’Brien (WEHI Melbourne), Martin Hrabe de 

Angelis (Hemholtz Zentrum, München) and Gerald de 

Haan (UMCG Groningen), evaluated our facility. They were 

very appreciative for the way we support the research under 

the diffi cult conditions imposed by an out-dated building 

and the bureaucratic constrains imposed by Dutch law and 

they gave valuable advice how we could further improve 

the support to investigators. We will implement their 

suggestions where possible. 

HONORS AND APPOINTMENTS 

The NKI-AVL cannot award university degrees. However, 

many of our staff members hold special part-time chairs 

at Dutch Universities. This allows them to award PhD 

degrees to graduate students receiving their training at The 

Netherlands Cancer Institute. In 2011, 31 staff members 

had professorships at one of the Dutch Universities. Huib 

Ovaa received the ‘Golden Medal’ of the Royal Dutch 

Chemical Society KNCV) for his achievements. Sander 

van Kasteren working with Huib Ovaa received the “The 

Biochemical Society Early Career Research Award” of the 

UK Biochemical Society. Ilana Berlin and Carolyn de Graaf 

received an EMBO long-term fellowship. Bas van Steensel 

acquired the prestigious ERC grant and I received the 

Josephine Nefkens award for Cancer Research. 

There were substantial changes in our clinical staff mostly 

due by retirements. Fortunately, we have been able to 

recruit a number of excellent successors. We also recruited 

new group leaders for the research. Thijn Brummelkamp 

was appointed as tenured staff member in the Division of 

Biochemistry, Sach Mukherjee as tenured staff member 

of the bioinformatics group, Wilbert Swart became junior 

group leader in the Division of Molecular Pathology. We 

were fortunate to recruit René Medema from the University 

of Utrecht to become director of Research, starting January 

2012. At the end of 2011 I retired from that position but will 

continue as senior group leader in the Division of Molecular 

Genetics. Several other internal appointments are worth 

mentioning, Jos Jonkers became head of the Division of 

Molecular Pathology, and Daniel Peeper will head the new 

division of Molecular Oncology following the reshuffl ing of 

groups upon relocating of research groups to the renovated 

research building. 

Staff of the NKI-AVL fulfi lled numerous functions in 

national and international organisations, on boards of 

scientifi c journals, as members of study sections, and 

as organisers or co-organisers of scientifi c meetings, 

workshops and congresses. 

OUTLOOK AND ACKNOWLEDGEMENTS 

I am confi dent that the NKI-AVL will continue to fl ourish, 

even in the face of all the uncertainties that are associated 

with the increasingly troublesome economic crisis. We have 

been very successful in securing external grants but these 

grants do hardly contribute to the overhead costs thereby 

putting increasing strain on the core budget. Although the 

contribution of the Dutch Cancer Society will increase in 

the coming years, this will be offset by a lower contribution 

of the government. Therefore, it is imperative that we 

raise additional funds through other routes. The newly 

established NKI-AVL research fund offers perspectives in 

this regard. 

More than ever breakthroughs will come from the 

close collaboration of basic and clinical investigators. A 

continuous fl ow of ideas in both directions is necessary to 

understand at the molecular level why new well-conceived 

treatments often do not work or quickly result in resistance. 

This knowledge should translate into new and better 

treatment regimes. Exciting times are ahead of us. My time 

as director of the NKI is now over. The 12 years I did spend 

in this position have been very gratifying for me personally, 

thanks to the wonderful colleagues that make the NKI 

what it is. I am very thankful for their support, enthusiasm 

and camaraderie. They have made the time fl y. With René 

Medema the NKI has a new energetic director with new 

enthusiasm and ideas. I am convinced that with him the 

NKI will have a great future ahead. 

I want to end by thanking all NKI-AVLers and those who 

continue to support us: The Dutch Cancer Society, the 

most reliable and signifi cant sponsor of our research; the 

Ministry of Health, Welfare and Sport, which provides a 

substantial core grant to the Institute and has fi nancially 

supported us during the renovations of our research 

buildings, and, last but not least, the many individuals who 

provide us with fi nancial, moral, and practical support. 

Only with their help we can continue to develop new ideas 

that can improve the perspectives of cancer patients. I hope 

you are inspired by this report. 

Ton Berns 

Director of Research 

15 

INTRODUCTION

16 

EDUCATION IN ONCOLOGY 


The Netherlands Cancer Institute offers a variety of 

opportunities for practical and theoretical training to 

(trainee) technicians, University Master students, PhD 

students and post-doctoral fellows. Research and clinical 

staff and their group members are involved in theoretical 

and practical training. Many staff members have joint 

appointments as professors at Dutch universities and an 

even larger number contribute to the regular curriculum 

at various universities. The research divisions attract 

students from universities throughout the country. The 

NKI has a formal affi liation with the Science faculty of the 

University of Amsterdam (UvA) and is committed to make 

a contribution to Master student teaching. The institute 

participates in the Oncology Graduate School Amsterdam, 

together with the medical faculties of the UvA and the Free 

University (VU), referred to as Academic Medical Center 

(AMC) and VU medical center (VUmc), respectively. 

All educational activities are supervised by the Teaching 

Committee, which consists of Jannie Borst (chair and dean 

Master students), Hein te Riele (general affairs), Roderick 

Beijersbergen (Master course), Fred van Leeuwen, John 

Hilkens (HLO students and publicity), Titia Sixma (dean 

PhD students) and Fons Balm (clinical teaching). Peter 

Peters functions as dean for the post-docs. 

MASTER STUDENTS 

The program in Experimental Oncology attracts Master 

students of all national universities, see http://www.nki. 

nl/Research/Career/Masters+students. Students generally 

have a background in (Medical) Biology, Health Sciences, 

Chemistry, Pharmacology, Medicine, or Psychology. The 

program offers combined practical and theoretical training 

in various aspects of experimental oncology. Practical 

training includes participation in ongoing research projects 

for a minimum of 4 months. In 2011, 45 Dutch university 

Master students and 3 students from abroad completed 

a placement of 5-9 months at the biomedical research 

divisions. The students came primarily from the Free 

University Amsterdam (VU) (24) and the University of 

Amsterdam (UvA) (13), but also from Utrecht University (4), 

Eindhoven (1), Leiden (1), Maastricht (1), and Groningen (1). 

The institute also provides practical training opportunities 

to trainee technicians, who stay for similar periods of 

time as the university students and like these, often make 

signifi cant contributions to research progress of the PhD 

students and post-docs who supervise them. There is an 

increasing demand from Universities for placing Bachelor 

students for the three month internship that concludes 

their program and we have accommodated 3 of them this 

year. The core element of theoretical training is the course 

in Experimental Oncology, given twice yearly (Table 1). 

This course is compulsory for Master students who do 

an internship at the NKI, but in addition attracts many 

students from throughout the country. We routinely host 

about 40 students per course. It includes lectures and 

tutorials given by our highest level clinical and research 

professionals and is rated very highly in University 

evaluations. 

Table 1 - Course in Experimental Oncology 

Epidemiology F van Leeuwen, M 

Schaapveld 

Surgery E Rutgers 

Radiodiagnostics E Vegt 

Pathology D de Jong 

Molecular diagnostics* R Kerkhoven, S Linn 

Conventional pharmacotherapy S Rodenhuis 

Radiotherapy** M Verheij 

DNA damage response and 

apoptosis* C Vens, J Borst 

Tumor development* R Beijersbergen 

Genomic instability* H te Riele 

Signal transduction* W Moolenaar 

Cell cycle R Bernards 

Oncogene-tumor suppressor 

gene interactions D Peeper 

Cell division R Wolthuis 

Tumor microenvironment K de Visser 

Mouse models of cancer* J Jonkers 

Immunology and 

immunotherapy* T Schumacher, J Haanen 

Analysis of protein structure T Sixma 

Rational drug development A Huitema 

Drug delivery* A Schinkel 

Epigenitica J Dannenberg 

* including tutorial ** including tour 

PHD STUDENTS 

The PhD students at the NKI-AVL participate in the 

Oncology graduate school Amsterdam (OOA) together with 

the oncology departments of the VuMC and the AMC. The 

number of registered students has been rising rapidly in the 

past 2 years. In 2011, the institute had ~ 184 PhD students 

registered with the OOA. Of these, 24 students defended a 

PhD thesis at a Dutch university. 

Students participate in research of their group and in 

interdepartmental work discussions. In addition, the 

students follow the OOA training program, which consists 

of courses (Table 2) and an annual retreat. Apart from 

courses on different topics in cancer research, the OOA 

offers a course on scientifi c English. Students with no 

prior background in cancer research can participate in the 

Experimental Oncology course. 

Part of the training of the NKI students are discussions with 

experts in the fi eld of oncology. The Friday morning seminar 

speakers take their lunch with a delegation of the students. 

Several times per year each graduate student gets the 

opportunity to exchange views with experts in the fi eld. 

The PhD student retreat focuses entirely on the research of 

the graduate students themselves. At this retreat, fi rst year 

students present their work in the form of a poster and older 

students give presentations. Moreover students are in charge 

of chairing sessions and discussions as well as peer review, 

giving a prize for the best poster and best presentation. In 

this manner, the retreat provides training in presentation 

and interaction skills, but it also provides an overview of 

the research in the OOA at an early stage of the student’s 

career. This provides a good opportunity for translational

interaction and bottom-up research, allowing the graduate 

students themselves to contribute signifi cantly to interaction 

between different research groups. 

Senior graduate students can participate in a joint retreat 

with other cancer institutes in Europe. In 2011, this event 

was hosted by CrUK in Glasgow, with participants from 

among others British CrUK institutes, the Italian IFOM 

and the Spanish CNIO. This retreat gives students the 

opportunity to compare notes among excellent cancer 

institutes throughout Europe. 

The progress of the research is monitored annually by a 

supervisory committee. This committee has independent 

members within and outside the division. The committee 

discusses progress with the supervisor and student separately 

and participates in a joint discussion of the research. After 

two years of PhD research the student, supervisor and 

committee evaluate the state of the project in a midterm 

review. At this more elaborate meeting the likelihood of 

achieving a PhD within a reasonable time frame is discussed. 

This meeting can be used to redefi ne goals if necessary. 

The students are represented in the OIO-council that meets 

with the Dean of graduate student affairs on a regular 

basis, as well as upon special request. They also mediate 

communication between the graduate students and research 

manager or board of directors. 

Table 2 - OOA graduate student courses 

March and English Writing and Presenting in Biomedicine 

September Total of 24 students 

12-14 Annual Graduate Student Retreat, Texel 

Oktober Titia Sixma 

128 participants of NKI-VU-AMC 

11-18 Basic Medical Statistics 

November Michael Hauptmann, Wilma Heemsbergen, 

Marta Lopez Yurda 

47 participants 

21 November- Apoptosis 

1 December Jannie Borst, Marcel Verheij, Jan Paul 

Medema, Eric Eldering 

23 participants 

POSTDOCS 

Know your talents! Refl ect on your work and yourself! 

Dare to try new things! These were the most heard pieces 

of advice at this year’s PCDI Postdoc Retreat. Over 100 

postdocs and fi nal-year PhD students gathered at Kapellerput 

in Heeze from February 16-18 to refl ect on their current 

position, explore possibilities for their next career step, 

discuss developments in the scientifi c world, broaden their 

horizons, fi nd out how varied careers in the different areas 

of the Life Sciences can be and how important it is to think 

ahead and choose a career path that suits them best. To give 

postdocs a head start on career development, PCDI offered 

them three days packed with keynote lectures by successful 

(ex)academics on their personal careers, workshops to 

discover and enhance their talents, a forum discussion and 

networking sessions with PhDs who pursued different career 

paths within and outside the Life Sciences. 

In the fi rst session, we learned about two contrasting career 

paths. Prof. Ritsert Jansen, a renowned academic, gave a 

17 


whole list of practical tips on developing one’s talent for 

science and Dr. Roel Breuls conveyed what he had learned 

from his research experiences and used that in his current 

career as a coach and advisor to scientists. 

The next two speakers stressed the importance of 

maintaining a good work/private life balance. Prof. 

Susanne Pedersen talked about her unusual career path 

to becoming a PI and Dr. Katarina Radosevic spoke about 

her challenging climb on the career ladder at the biotech 

company Crucell. Prof. Peter Peters in his talks stressed 

the importance of knowing what your transferrable skills 

are, as they are important assets in any career path one 

chooses. Many researchers were surprised to learn about 

the many transferable skills listed in his presentation. 

The last two keynote speakers shared their insights on the 

challenging journey leading to the foundation of their own 

companies. Dr. Christianne Rijcken was right in the middle 

of the process of founding her own company, whereas Dr. 

Chrétien Herben had already left his company and is using 

his experience in science and his expertise in starting a 

company to shift his career towards knowledge valorisation. 

Being aware of your valuable transferable skills, hidden 

talents, and core qualities is very important and therefore 

was the central theme of the fi rst session of workshops. 

Since it is also vital to know how to ‘present yourself’, the 

focus of the second parallel workshops addressed the style 

of presenting and the many (online) instruments available 

to land your dreamjob. In the last workshop session 

participants improved or learned a (new) transferable skill 

of choice: non-verbal behaviour, grant writing, negotiation, 

project management or networking. 

The fi nal day of the retreat was aimed at broadening the 

horizons of young researchers and discussing with them, 

beyond their own research project, about the future role of 

scientists. Prior to our forum discussion on the ‘Scientist of 

the Future’, two forum panel members, Prof. Frank Miedema 

and Prof. Bas Haring, explained their views on the future 

scientist. Frank Miedema mainly stressed that politics plays 

a large role in today’s science, and that the PR aspects of 

a scientist’s job is becoming increasingly important. Bas 

Haring used the art of storytelling, with stories serving as 

metaphores for important characteristics he thought the 

scientist of the future should have: scientists should be able to 

see the bigger picture of their research, and scientists should 

not only work to produce as many papers as possible, but also 

regularly take time to refl ect on their career and research. 

To give postdocs the opportunity to make informed choices 

about their next career step, the retreat was fi nalized by PCDI 

with a networking session which allowed the participants to 

talk to 30 representatives (all PhDs in the Life Sciences) from 

a diverse range of potential professions within and outside 

academia. Four alternative career paths were represented: 

(i) non-research in academia, (ii) small/medium and (iii) 

large biotech companies, and (iv) government, non-profi ts 

and education. One of the most heard comments in the 

networking session, echoing similar statements by some of 

the keynote speakers, was that many of the representatives 

had followed their heart when making career decisions. For 

more information please visit www.pcdi.nl 

Under the leadership of Peter Peters the retreats have been 

attended by more then 1200 postdocs over the last 12 years. 

This one was the last one under his responsibility.

18 

BIOCHEMISTRY 

Division head, group leader Titia Sixma 

Titia Sixma PhD Group leader 

Marcello Clerici PhD Post-doc 

Alex Fish PhD Post-doc 

Rick Hibbert PhD Post-doc 

Prakash Rucktooa PhD Post-doc 

Mariano Stornaiuolo PhD Post-doc 

Alex Faesen MSc PhD student 

Mark Vargas MSc PhD student 

Francesca Mattiroli MSc PhD student 

Judith Smit MSc PhD student 

Flora Groothuizen MSc PhD student 

Danny Sahtoe MSc PhD student 

Michael Uckelman PhD student 

Pim van Dijk Technical staff 

Herrie Winterwerp Technical staff 

Figure 1: 

Crystal structure of the C-terminal 

domain of Usp7 reveals how the fi ve Ubl 

domains are arranged in a 2+1+2 manner. 

Our biochemical data show that the 

C-terminal end of this domain is 

critical for activation of the active site, 

stimulating ubiquitin binding and 

rearranging the catalytic triad 

(Faesen et al., Mol Cell, 2011) 

Publications 

Akdemir A, Rucktooa P, Jongejan A, 

Elk R, Bertrand S, Sixma TK, Bertrand D, 

Smit AB, Leurs R, de Graaf C, de Esch IJ. 

Acetylcholine binding protein (AChBP) as 

template for hierarchical in silico screening 

procedures to identify structurally novel 

ligands for the nicotinic receptors. Bioorg 

Med Chem 2011;19:6107-19 

Edink E, Rucktooa P, Retra K, Akdemir 

A, Nahar T, Zuiderveld O, van Elk R, 

Janssen E, van Nierop P, van Muijlwijk- 

DIVISION OF BIOCHEMISTRY 

STRUCTURAL BIOLOGY 

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

Understanding the mechanisms of underlying processes will help to determine 

where the errors occur and how they can be treated. We study protein structures by 

X-ray crystallography to provide three-dimensional structures and we complement 

this work by biochemical and biophysical analysis of protein function. This leads 

to insights in molecular mechanisms that we validate in cells. In addition our 

structures provide targets for drug design studies. In our group we focus primarily 

on proteins involved in ubiquitin conjugation, particularly in stress response and 

DNA repair pathways. In addition we study DNA mismatch repair pathways and 

nicotinic receptor signaling. 

DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring 

genomic stability. Defects in the mismatch repair cascade in humans predispose 

to hereditary non-polyposis colorectal cancer and are associated with a variety 

of sporadic cancers. DNA mismatch repair is initiated by the protein MutS (in 

Escherichia coli) or its MSH homologs. MutS recognizes and binds to mismatches or 

unpaired bases that have escaped the proofreading capacity of the DNA replication 

machinery or are present in the genome during recombinatorial events between 

non-fully complementary DNA strands. 

We make use of variants of MutS that specifi cally maintain the dimer or tetramer 

state of these proteins in order to be able to provide quantitative data on the 

mismatch recognition cycle. In 

collaboration with the groups of 

Terence Strick (Paris), Peter 

Friedhoff (Giessen) and Joyce 

Lebbink (Rotterdam) this 

allows a precise analysis 

of the steps involved in 

mismatch recognition and 

the initiation of repair. Also 

these mutants allowed novel 

crystallographic analysis of the 

proteins with and without DNA, leading to new insight into the state of the protein 

prior to DNA binding. We use crystallography and SAXs to defi ne the states that are 

observed and we use it to create novel tools to study the initiation of DNA mismatch 

repair. 

Ubiquitin and SUMO conjugation Ubiquitin conjugation is critical for signaling 

in almost all cellular processes, including DNA repair, apoptosis, cell cycle, 

chromatin regulation and endocytosis. Since these processes are of so important 

for cellular stability, deregulation of ubiquitin-dependent processes often leads 

to cancer. Structure analysis of the proteins involved in ubiquitin and SUMO 

conjugation could help to development of novel drugs inhibiting critical pathways in 

ubiquitin conjugation. 

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

one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade 

of enzymes. Correct ubiquitination requires the complex spatial arrangement of 

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

manner. We are particularly interested in the factors that determine the rate, the 

specifi city and the selectivity of the E2/E3 dependent reaction. In particular we 

are analyzing the ubiquitination of H2A in nucleosomes, both by the polycomb 

repressive complex and by the DNA damage response proteins, RNF8 and RNF168. 

Unravelling these processes in vitro has resulted in some unexpected insights that 

we are currently validating in cells. 

We have a strong interest in deubiquitinating enzymes (DUBs). There are fi ve

classes of DUBs, but we focus mainly on the ubiquitin specifi c subclass. Here 

we expressed a set 12 different DUBs in order to characterize them catalytically. 

In collaboration with Huib Ovaa we are making use of novel tools to study these 

enzymes which allowed the analysis of all diubiquitin pairs for this type of analysis. 

A detailed comparison of kinetics and chain specifi city revealed very large intrinsic 

differences between different DUBs. Effects of regulators can be either at the level of 

kcat or on Km. 

Interestingly the role of the ubiquitin-like Ubl domains in a number of DUB 

enzymes is highly variable. Although these domains have a similar fold to ubiquitin, 

they do not resemble ubiquitin closely in their sequence. The Ubl domain that is 

internal to the Usp4 catalytic domain, however, does compete for ubiquitin binding, 

partially inhibiting the activity of the catalytic domain. Interestingly, this inhibition 

can be relieved by addition of an inactive DUB, such as USP39. 

In contrast the C-terminal domain of Usp7/HAUSP is important due to the activating 

properties on catalytic activity. This HAUSP Ubl domain (HUBL) is a 56 kD domain 

that consists of fi ve consecutive Ubl domains. We solved its crystal structure and 

analyzed how it can activate the catalytic domain, by interaction with a switching 

loop that increases affi nity for ubiquitin and remodels the catalytic triad to its active 

state. 

We could show that USP7 exists in a dynamic equilibrium that can be locked in 

its active state by interaction with an allosteric activator, GMPS. This binds to 

HUBL123, and promotes the interaction between the catalytic domain and HUBL45, 

thus activating the catalysis. 

Nicotinic Acetylcholine receptor homolog AChBP The acetylcholine binding 

protein (AChBP) is a homolog of the extracellular domain of the nicotinic 

acetylcholine receptor, and a representative of the cys-loop receptor family. It 

remains the best model for atomic resolution structures of ligand binding to this 

pharmaceutically important family of ion channels. 

In collaboration with the group of Iwan de Esch (VU) novel interactors for AChBP 

we identifi ed novel scaffolds for interaction with this protein, with unexpected 

binding properties. Currently we are studying variants of these compounds in order 

to understand the mode of interaction. 

Since the nicotinic acetylcholine receptor is responsible for addiction to smoking it 

has been targeted for compounds that can aid in smoking cessation. We have solved 

crystal structures of AChBP in complex with several compounds that have clinical 

effi cacy in smoking cessation, and compare them to nicotine. These structures 

reveal the mode of binding and explain where further development of such 

compounds is possible. Mutant analysis was performed to determine the specifi city 

for different receptor subtypes. Our data show that important determinants are not 

in the fi rst binding shell of the ligands. In this manner structural studies of a series 

of reference compounds including toxins and anti-smoking compounds provide 

insight in the details of subunit specifi city of the nicotinic receptor homologs. 

Figure 2: Crystal structure of 

Aplysia AChBP showing that a 

fragment can be grown into 

the socalled ‘lobeline pocket’ 

(Edink et al, JACS, 2011). 

This interaction requires 

rearrangement of the side 

chain of Tyrosine 91. 

These studies of fragment 

growing were supported 

by structural analysis, 

thermodynamic and binding 

data. 

Publications (continued) 

19 

BIOCHEMISTRY 

Koezen J, Smit AB, Sixma TK, Leurs R, de 

Esch IJ. Fragment growing induces 

conformational changes in acetylcholinebinding 

protein: 

a structural and thermodynamic analysis. 

J Am Chem Soc. 2011;133:5363-71 

Faesen AC, Dirac AM, Shanmugham A, 

Ovaa H, Perrakis A, Sixma TK. Mechanism of 

USP7/HAUSP Activation by Its C-Terminal 

Ubiquitin-like Domain and Allosteric 

Regulation by GMP-Synthetase. Mol Cell 

2011;44:147-59 

Hibbert RG, Huang A, Boelens R, Sixma 

TK. E3 ligase Rad18 promotes 

monoubiquitination rather than ubiquitin 

chain formation by E2 enzyme Rad6. 

Proc Natl Acad Sci U S A 2011;108:5590-5 

Huang A, Hibbert RG, de Jong RN, 

Das D, Sixma TK, Boelens R. Symmetry and 

asymmetry of the RING-RING dimer of 

Rad18. J Mol Biol 2011;410:424-35 

Inagaki A, Sleddens-Linkels E, van 

Cappellen WA, Hibbert RG, Sixma TK, 

Hoeijmakers JH, Grootegoed JA, Baarends 

WM. Human RAD18 interacts with 

ubiquitylated chromatin components and 

facilitates RAD9 recruitment to DNA double 

strand breaks. PLoS One 2011;6:e23155 

Luna-Vargas MP, Christodoulou E, Alfi eri 

A, van Dijk WJ, Stadnik M, Hibbert RG, 

Sahtoe DD, Clerici M, Marco VD, Littler D, 

Celie PH, Sixma TK, Perrakis A. Enabling 

high-throughput ligation-independent cloning 

and protein expression for the family of 

ubiquitin specifi c proteases. J Struct Biol 

2011;175:113-9 

Luna-Vargas MP, Faesen AC, van Dijk WJ, 

Rape M, Fish A, Sixma TK. Ubiquitin-specifi c 

protease 4 is inhibited by its ubiquitin-like 

domain. EMBO Rep 2011;12:365-72 

Monti MC, Cohen SX, Fish A, 

Winterwerp HH, Barendregt A, Friedhoff P, 

Perrakis A, Heck AJ, Sixma TK, van den 

Heuvel RH, Lebbink JH. Native mass 

spectrometry provides direct evidence for DNA 

mismatch-induced regulation of asymmetric 

nucleotide binding in mismatch repair protein 

MutS. Nucleic Acids Res 2011;39:8052-64 

Winkler I, Marx AD, Lariviere D, Heinze 

RJ, Cristovao M, Reumer A, Curth U, Sixma 

TK, Friedhoff P. Chemical trapping of the 

dynamic MutS-MutL complex formed in 

DNA mismatch repair in Escherichia coli. 

J Biol Chem 2011;286:17326-37 

Faesen AC, Luna-Vargas MP, Geurink 

PP, Clerici M, Merkx R, van Dijk WJ, 

Hameed DS, El Oualid F, Ovaa H, Sixma TK. 

The Differential Modulation of USP Activity 

by Internal Regulatory Domains, Interactors 

and Eight Ubiquitin Chain Types. Chem Biol. 

2011;18:1550-61

20 

BIOCHEMISTRY 

Group leader Anastassis Perrakis 

Anastassis Perrakis PhD Group leader 

Christophe Caillat PhD Post-doc 

Eleonora von Castelmur PhD Post-doc 

Krista Joosten PhD Post-doc 

Robbie Joosten PhD Post-doc 

Leonie van Zeijl PhD Post-doc 

Jens Haussmann MSc PhD Student 

Tatjana Heidebrecht Technical staff 

Publications 

Hausmann J, Kamtekar S, 

Christodoulou E, Day JE, Wu T, Fulkerson 

Z, et al. Structural basis of substrate 

discrimination and integrin binding by 

autotaxin. Nat Struct Mol Biol. 2011;18:198- 

204 

Moolenaar WH, Perrakis A. Insights into 

autotaxin: how to produce and present a 

lipid mediator. Nat Rev Mol Cell Biol. 

2011;12:674-9 

Albers HM, Hendrickx LJ, van Tol RJ, 

Hausmann J, Perrakis A, Ovaa H. 

Structure-based design of novel boronic 

acid-based inhibitors of autotaxin. J Med 

Chem. 2011;54:4619-26 

Heidebrecht T, Christodoulou E, 

Chalmers MJ, Jan S, Ter Riet B, Grover RK, 

et al. The structural basis for recognition of 

base J containing DNA by a novel DNA 

binding domain in JBP1. Nucleic Acids Res. 

2011 

Pefani DE, Dimaki M, Spella M, 

Karantzelis N, Mitsiki E, Kyrousi C, et al. 

Idas, a novel phylogenetically conserved 

geminin-related protein, binds to geminin 

and is required for cell cycle progression. J 

Biol Chem. 2011;286:23234-46 

Joosten RP, Joosten K, Cohen SX, Vriend 

G, Perrakis A. Automatic rebuilding and 

optimization of crystallographic structures in 

the PDB. Bioinformatics. 2011 

Read RJ, Adams PD, Arendall WB, 3rd, 

Brunger AT, Emsley P, Joosten RP, et al. A 

new generation of crystallographic validation 

tools for the protein data bank. Structure. 

2011;19:1395-412 

STRUCTURAL BIOLOGY 

This year much work has been invested on proteins involved in mitotic progression 

and DNA replication licensing. Last year’s breakthroughs on our long-term in-house 

collaborations on Autotaxin (with Wouter Moolenaar and Huib Ovaa, division of 

Cell Biology) and on JBP1 (with Piet Borst, division of Molecular Biology) have also 

made good progress. Our adventures in X-ray crystallography model building and 

refi nement took a planned turn towards structural bionformatics and our fi rst 

results in re-building the structures of the Protein Data Bank are now available. 

Structural studies of proteins involved in mitotic progression The Spindle 

Assembly Checkpoint (SAC) is a protein network that ensures that the cell does not 

proceed with separating the sister chromatids in mitosis before all chromosomes 

have been aligned and attached to the spindle machinery. 

Our focus has been on understanding the role of the regulatory domain of Mps1 

kinase, one of the SAC kinases. The MPS (MonoPolar Spindle) family of kinases is 

a dual specifi city protein kinase in vitro, capable of autophosphorylation on serine, 

threonine and tyrosine residues. Mps1 allows resolution of merotelic attachments 

and ensures that single kinetochores achieve the strength of checkpoint signalling 

suffi cient to prevent premature anaphase onset and chromosomal instability. We 

have determined the structure of the N-terminal domain of Mps1, which adopts the 

TPR fold, also found in the Bub1 and BubR1 family of kinases. 

In collaboration with the group of Geert Kops (UMC Utrecht), we have shown with 

site directed mutagenesis and domain deletion and swapping studies, that the TPR 

domain of Mps1 is important for localization of Mps1 to the kinetochores. Using 

both cell based assays and in vitro assays using purifi ed proteins, we establish for 

the fi rst time a physical interaction between the spindle assembly checkpoint and 

kinetochores. 

Geminin and its homologues in DNA replication licensing A new twist to 

our research on the role of Geminin in replication licensing was provided by the 

discovery of two homologue of Geminin: Idas and Lygeas (in Greek mythology, 'Ιδας 

and Λυγʹεας are the cousins of the two Gemini). Lygeas (who unfortunately made 

its fi rst appearance in scientifi c literature under the more mundane name GemC1) 

directly mediates replication initiation through TopBP1- and Cdk2-dependent 

recruitment of Cdc45 onto replication origins. We want to investigate the hypothesis 

if the assigned functions of Geminin in proliferation and development are possibly 

through the combined action of Geminin with its two homologue proteins. 

Structural studies of Autotaxin The role of the signaling phospholipid 

lysophosphatidic acid (LPA) and Autotaxin (ATX), the protein that produces LPA 

from lysophosphatidylcholine (LPC), was established over the last three decades, 

largely owing to the efforts of the group of Wouter Moolenaar at the NKI. LPA and 

ATX have been shown by numerous studies to be involved in cancer metastasis and 

other pathogenic situations, such as infl ammation and neuropathic pain. ATX is 

the protein ecto-nucleotide phosphodiesterase 2 (ENPP2), a 100 kDa glycoprotein, 

capable of both lysoPLD and nucleotide pyrophosphatase activities. We determined 

the structure of ATX last year. 

Autotaxin hydrolyzes different species of LPC (with different alkyl chain lengths) 

and some other lipids, as well as nucleotide substrates. We have shown that both 

nucleotides and lipids partially share the same binding pocket, but the alkyl-chain 

of lipid substrates form additional hydrophobic contacts with ATX. This model 

implies that the LPA product likely has higher affi nity for ATX than any nucleotide 

substrate, a hypothesis consistent with the previous fi nding that LPA acts as an 

inhibitor of ATX activity against a variety of substrates, but not of LPC hydrolysis. 

Our fi ndings also suggest that further analysis of structural determinants of 

substrate discrimination could lead to the identifi cation of molecules that inhibit the 

hydrolysis of specifi c substrates, e.g. long-chain rather than short-chain LPC species. 

The group of Huib Ovaa has developed a series of small-molecule boronic acid 

inhibitors of ATX-mediated LPC hydrolysis, that lower plasma LPA levels in mice 

following intravenous administration. We also determined a structure of ATX in

complex with one of these inhibitors, HA155, which enables us to correlate the activity 

of this inhibitor with its binding mode. We show that the boron atom on one end of 

the inhibitor forms a reversible covalent bond with the nucleophile hydroxyl group 

of the active site threonine nucleophile. The other end of HA155, a hydrophobic 

fl uoro-benzene, is pointing directly into the deep hydrophobic pocket, oriented 

perpendicular to the protein surface. Based on that structure our collaborators were 

able to engineer a new generation of ATX inhibitors with different binding properties. 

Our new focus is to understand the interaction of Autotaxin with cell-surface 

integrins and heparin in the cell surface. Our data on the interaction of ATX with 

platelet integrins and more recent data on heparin and heparan sulfate affi nity 

(see the report of W. Moolenaar group) suggest a mechanism for the delivery of LPA 

close to the cell surface, and we wish to study the molecular mechanism and the 

importance of this mechanism in ATX and LPA mediated signaling. 

fi gure 3 

Structural studies of JBP1 The JBP1 protein, discovered by Piet Borst and 

colleagues, binds to DNA that contains base J (β-D-glucosyl-hydroxymethyluracil). 

JBP1 has been shown to be essential for survival in many major protozoa 

human pathogens such as T. brucei (sleeping sickness), T. cruzi (Chagas’ 

disease) and Leishmania species (various types of Leishmaniasis). A partial 

mammalian homologue of JBP1, harbouring the thumidine hydroxylase region 

of JBP1, exists in the TET proteins that are involved in myeloid leukemia and 

necessary for the synthesis and maintenance of the important epigenetic marker 

hyroxymethylcytosine. 

We showed that JBP1 recognizes J-containing DNA through a 160-residue domain, 

DB-JBP1, with ten thousand-fold preference over normal DNA. The crystal structure 

of DB-JBP1 revealed a novel helix-turn-helix variant fold, a “helical bouquet” with 

a “ribbon” helix responsible for DNA binding, as shown in fi gure 4. Mutation of a 

single residue (Asp525) in that helix abrogates specifi city towards J-DNA, rendering 

mutated JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in 

Leishmania. Based on mutational analysis and H/D-exchange mass-spectrometry 

data, a model of JBP1 bound to J-DNA was constructed, and validated by smallangle 

X-ray scattering. Our results open possibilities for utilizing JBP1 as a drug 

target and a tool for detecting the important mammalian epigenetic marker 

hydroxymethylcytosine. 

More recently, transient kinetic data suggested that the binding of JBP1 to DNA NA 

is followed by a conformation change, that we wish to study in more detail, as s we 

believe it represents a movement of the hydroxylase domain towards the DNA, A, 

getting it into position to hydroxylate thymidine right after the recognition of f 

pre-existing J-base in the vicinity. 

Methods for X-ray crystallography We are focusing on bringing upto-date 

the crystallographic models in the Protein Data Bank (PDB), that 

were deposited there over several decades, and were created using the 

methods and software available at the time. The state of the art software of 

nowadays is used in a fully automated procedure, PDB_REDO, that we have 

designed to improve the accuracy and correct errors of existing models. The fi rst 

results of the new PDB_REDO, incorporating a decade of knowledge and algorithms gorithms 

that were used to build the ARP/wARP software, show marked improvement t of the fi gure 4 

quality of both old but also also new models in the PDB. 


21 

BIOCHEMISTRY 

Perrakis A, Musacchio A, Cusack S, 

Petosa C. Investigating a macromolecular 

complex: the toolkit of methods. J Struct 

Biol. 2011;175:106-12 

Perrakis A, Daenke S, Stuart DI, 

Sussman JL. From SPINE to SPINE-2 

complexes and beyond. J Struct Biol. 

2011;175:105 

Busso D, Peleg Y, Heidebrecht T, 

Romier C, Jacobovitch Y, Dantes A, et al. 

Expression of protein complexes using 

multiple Escherichia coli protein coexpression 

systems: A benchmarking study. J 

Struct Biol. 2011 

Luna-Vargas MP, Christodoulou E, 

Alfi eri A, van Dijk WJ, Stadnik M, Hibbert 

RG, et al. Enabling high-throughput 

ligation-independent cloning and protein 

expression for the family of ubiquitin specifi c 

proteases. J Struct Biol. 2011;175:113-9 

Kryshtafovych A, Moult J, Bartual SG, 

Bazan JF, Berman H, Casteel DE, et al. 

Target highlights in CASP9: Experimental 

target structures for the critical assessment of 

techniques for protein structure prediction. 

Proteins. 2011;79 Suppl 10:6-20 

Monti MC, Cohen SX, Fish A, 

Winterwerp HH, Barendregt A, Friedhoff P, 

et al. Native mass spectrometry provides 

direct evidence for DNA mismatch-induced 

regulation of asymmetric nucleotide binding 

in mismatch repair protein MutS. Nucleic 

Acids Res. 2011;39:8052-64 


Ovaa H, Perrakis A, Sixma TK. Mechanism 

of USP7/HAUSP activation by its 

C-terminal ubiquitin-like domain and 

allosteric regulation by GMP-synthetase. 

Mol Cell. 2011;44:147-59

22 

BIOCHEMISTRY 


Linn SC, Wesseling J. Molecular tests for 

breast-cancer diagnosis? Lancet Onol 

2009;10:314-5 

Group leader Thijn Brummelkamp 

Thijn Brummelkamp PhD Group leader 

Gregor Obernosterer PhD Post-doc 

Vincent Blomen MSc PhD student 

Jacqueline Staring MSc PhD student 

Lucas Jae MSc PhD student 

Bianca Gapp MSc PhD student 

Publications 

Carette JE, Guimaraes CP, Wuethrich I, 

Blomen VA, Varadarajan M, Sun C, Bell G, 

Yuan B, Muellner MK, Nijman SM, Ploegh HL, 

Brummelkamp TR. Global gene disruption in 

human cells to assign genes to phenotypes by deep 

sequencing. Nat Biotechnol 2011;29:542-546 

Carette JE, Raaben M, Wong AC, Herbert 

AS, Obernosterer G, Mulherkar N, Kuehne AI, 

Kranzusch PJ, Griffi n AM, Ruthel G, Dal Cin P, 

Dye JM, Whelan SP, Chandran K, 

Brummelkamp TR. Ebola virus entry requires 

the cholesterol transporter Niemann-Pick C1. 

Nature 2011;477:340-343 

Papatheodorou P, Carette JE, Bell GW, 

Schwan C, Guttenberg G, Brummelkamp TR, 

Aktories K. Lipolysis-stimulated lipoprotein 

receptor (LSR) is the host receptor for the binary 

toxin Clostridium diffi cile transferase (CDT). 

Proc Natl Acad Sci U S A 2011;108:16422-16427 

Reiling JH, Clish CB, Carette JE, 

Varadarajan M, Brummelkamp TR, Sabatini 

DM. A haploid genetic screen identifi es the 

major facilitator domain containing 2A 

(MFSD2A) transporter as a key mediator in the 

response to tunicamycin. Proc Natl Acad Sci U 

S A 2011;108:11756-11765 

Schlegelmilch K, Mohseni M, Kirak O, 

Pruszak J, Rodriguez JR, Zhou D, Kreger BT, 

Vasioukhin V, Avruch J, Brummelkamp TR, 

Camargo FD. Yap1 acts downstream of 

alpha-catenin to control epidermal 

proliferation. Cell 2011;144:782-795 

Figure 5: Outline of Haploid Genetic Screen. 

Mutagenized cells are treated with Clostridium 

diffi cile Toxin and resistant cells are selected. 

Gene-trap insertion sites are recovered from the 

resistant population and sequenced in parallel. 

Insertion sites are aligned to human genome 

and insertions sites close to each other are 

designated a high proximity index. 18 gene 

trap insertions cluster in Lipolysis-stimulated 

lipoprotein receptor (LSR). Mutations shown 

as red triangles (for details see Papatheodorou 

et al., PNAS 2011). 

EXPERIMENTAL BIOMEDICAL GENETICS 

The research in our laboratory focuses on the following topics: the development 

and application of a novel genetic model system to identify genes that play a role in 

human disease and the role of the Hippo signalling pathway in mammals. 

Haploid genetic screens Even though human cells are widely used as experimental 

tool, the human genome in somatic cells is largely refractory to effi cient mutagenesis 

due to its diploid nature. To address this we have developed an entirely novel genetic 

model system based on insertional mutagenesis in haploid or near-haploid human 

cells. We have shown that this platform enables the generation of null alleles for most 

human genes and can be used to pinpoint genes that are involved in phenotypes 

of interest. Combined with parallel sequencing approaches, this method generates 

high-density genetic overviews of genes required for nearly any selectable cell trait. 

Importantly, we can carry out and analyze a genetic screen in which we interrogate 

millions of mutant alleles in a period of weeks in a cost-effective manner. In the last 

years, our lab has completed more than 50 different systematic haploid screens in which we 

identifi ed gene products required for the induction of apoptosis, numerous critical host 

factors used by viruses and bacteria, chemical-genetic interactions that provide insight 

in compound modes of action and novel components of signalling pathways. 

The entry route of Ebola Virus Infections by the Ebola and Marburg fi loviruses cause 

a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are 

available. Virus entry is mediated by the viral glycoprotein, which attaches viral particles 

to the cell surface, delivers them to vesicles called endosomes, and catalyzes fusion 

between viral and endosomal membranes. It was known that additional host factors in 

the endosomal compartment were required for viral entry. However, despite considerable 

efforts, these host factors could not be identifi ed using conventional methods. Using 

a haploid genetic screen we identifi ed 15 proteins affecting traffi cking and lysosome 

function as critical host factors required for entry. Unexpectedly this screen pointed out 

rare individuals whose cells cannot be infected by Ebola virus: patients with Niemann- 

Pick C1 disease. We showed that viral attachment and the earliest steps of entry occurred 

normally in these cells but viral escape from lysosomes required the NPC1 protein, 

in an independent manner from its function as cellular cholesterol transporter. The 

unanticipated role for the hereditary disease gene NPC1 in viral entry, infection and 

pathogenesis may lead to the development of anti-fi lovirus therapeutics. 

Host factors hijacked by Clostridium difficile Clostridium diffi cile infections 

are the most frequent cause of infectious diahrrea in all hospitals worldwide. 

Hypervirulent strains produce a toxin called C. diffi cile transferase or binary toxin. 

This toxin can bind to and intoxicate human cells but the cellular receptor that it 

recognizes was unknown. Using a haploid insertional mutagenesis screen we have 

identifi ed the Lipolysis-stimulated lipoprotein receptor (LSR) as a critical host cell 

factor for intoxication. LSR is highly expressed in the intestine and is used by the 

toxin to bind, enter and intoxicate human cells. 

The Hippo signalling pathway How tissues stop growing upon reaching their 

correct size remains a mystery in biology. The barriers that tissues encounter when 

they reach their fi nal size may bear relevance to neoplastic cells during the early 

stages of tumorigenesis. Drosophila genetics increased our understanding of the 

biology of organ size control. The Hippo pathway has emerged as a key pathway 

that controls tissue expansion through the regulation of cell proliferation and death. 

Interestingly, all components of the Hippo pathway are conserved in mammals 

and some have been implicated in cancer. We use genetic mouse models and 

biochemical experiments to address how this signalling pathway regulates tissue 

size in mammals and how it contributes to tumorigenesis.

STATISTICAL SYSTEMS BIOLOGY 

The Mukherjee group focuses on statistical approaches in molecular and systems 

biology. Our efforts encompass both specifi c biological questions, addressed in 

collaboration with experimental groups, and methodological research motivated by 

such questions. 

Systems biology approaches offer the promise of quantitative models that shed light 

on aspects of basic cancer biology. Equally, there is much potential in model-based 

approaches that exploit high-throughput data to explore the heterogeneity of cancer 

and assist in targeting complex therapies to patients. However, while the promise 

of systems approaches is clear, the challenges posed by noisy and incomplete data, 

inherent biological variability and complex underlying processes and dynamics 

remain substantial. Our efforts are aimed at developing and exploiting statistical 

methods that can help to surmount some of these challenges. Ongoing projects 

include: 

Data-driven characterization of signaling networks How is the genomic 

heterogeneity of cancer manifested at the level of signaling network topologies? 

Are cancers “re-wired” due to genomic aberrations? And if so, how? Are signaling 

network topologies predictive of response to therapeutic agents that target nodes 

in the networks? Addressing these questions requires proteomic assays that can 

interrogate multiple signaling proteins through time, under a range of conditions 

and perturbations, and computational approaches by which to model the data. 

We are working on both theoretical and applied aspects of these questions, in 

collaboration with the Beijersbergen, Bernards, Wessels and Jonkers laboratories at 

the NKI, and the laboratories of Paul Spellman and Joe Gray (OHSU Knight Cancer 

Institute, USA) and Gordon Mills (MD Anderson Cancer 

Center, USA). Furthermore, we are exploring whether 

tumor subtypes show evidence of shared signaling 

topology and whether network analyses can help to 

uncover novel subtypes. 

High-dimensional genome analysis Proteins bind 

to DNA in a manner that depends on chromatin 

organization. At the same time, DNA binding data for 

pairs of proteins can shed light on interplay between those 

proteins. In collaboration with the van Steensel laboratory, 

we are combining both views, by developing models that 

simultaneously describe dependence of binding scores 

across the genome and between proteins. The aim of this 

work is twofold. First, to understand whether a highdimensional 

view, with models that describe binding 

for more than one hundred proteins at once, can shed 

light on genome organisation. Second, to develop an 

understanding of protein-protein interplay that is specifi c 

to genomic region. 

Stochastic transitions in induction processes Several 

approaches to regenerative and personalized medicine 

involve inducing alternative cell fates from embryos, 

tissues, biopsies or blood samples. These processes 

involve global changes in transcriptional state that remain 

incompletely understood. When transitions between cell 

states occur stochastically, at any particular time the population will be a mixture 

of cells in different states. Then, it is challenging to relate data from genome-wide 

assays, such as microarrays, that are averages over the heterogeneous population, 

to underlying cell states and transitions. At the same time, the prospect to carry out 

genome-wide assays with single or small numbers of cells remains limited. We are 

developing stochastic models by which to shed light on transition processes of this 

kind. In collaboration with the Jaenisch laboratory (Whitehead Institute, USA) we 

are exploring these ideas in the context of cellular reprogramming. 


23 

BIOCHEMISTRY 



2009;10:314-5 

Group leader Sach Mukherjee 

Sach Mukherjee DPhil Group leader 

Nicolas Städler PhD Post-doc 

Steven Hill MMath MSc Post-doc 

Chris Oates BA MSc PhD student 

Anas Rana MSci MSc PhD student 

Figure 6: A stochastic model to describe 

state-transition dynamics and state-specifi c 

gene expression signatures during 

reprogramming.

24 

CELL BIOLOGY I 

Publications (continued) DIVISION OF CELL BIOLOGY I 

Division head, group leader Arnoud Sonnenberg 

Arnoud Sonnenberg PhD Group leader 

Michael Ports PhD Post-doc 

Ruben Postel PhD Post-doc 

Pablo Secades PhD Post-doc 

Severine Laval PhD Post-doc 

Evelyne Frijns MSc PhD student 

Dirk-Jan de Groot MSc PhD student 

Mirjam Ketema MSc PhD student 

Coert Margadant MSc PhD student 

Norman Sachs MSc PhD student 

Maaike Kreft Technical staff 

Ingrid Kuikman Technical staff 

RECEPTORS FOR MATRIX ADHESION 

The main objective of our group is to study the mechanisms involved in cell 

adhesion. We are specifi cally interested in understanding the signifi cance of and 

characterizing the interactions that take place between cells and the extracellular 

matrix component laminin in both the epidermis and the glomerulus of the kidney. 

A second line of research involves the outer nuclear membrane protein nesprin-3, 

which binds to the cytoskeletal linker protein plectin, thereby establishing a link 

between the intermediate fi lament system and the nucleus. The physiological roles 

of these proteins and their interaction are studied in model organisms, including 

mice and zebrafi sh. 

Regulation of α5β1 trafficking Cell adhesion to the extracellular matrix is 

mediated by heterodimeric transmembrane proteins called integrins. Integrin 

function depends on conformational rearrangements that increase affi nity for ligand 

(“activation”), and by intracellular traffi cking. We investigate how activation and 

traffi cking of the fi bronectin receptor, integrin α5β1, are regulated by two NPxY 

motifs in the β1-cytoplasmic tail. We therefore generated disrupting mutations in 

the fi rst NPxY (β1 Y783A ), the second NPxY (β1 Y795A ), or both (β1 Y783/795A ), and stably 

expressed these mutants in β1-defi cient cells. Wild-type α5β1 induces epithelial-tomesenchymal 

transition (EMT)-like events characterized by disruption of cell-cell 

contacts, cell scattering, increased motility, organization of a fi bronectin network, 

and a contractile, fi broblast-like morphology with multiple protrusions. This 

phenotype is recapitulated by expression of β1 Y795A , but not by β1 Y783A or β1 Y783/795A , 

indicating that the fi rst but not the second NPxY motif is absolutely required for 

α5β1 function. However, cell surface levels and total-protein levels of β1 Y795A and 

β1 Y783/795A are low (50% and 70% of wild-type, respectively), and they accumulate 

in late endosomes/lysosomes, where they colocalise with internalised fi bronectin, 

suggesting that the second NPxY motif regulates integrin traffi cking. Indeed, 

whereas internalisation rates for all mutants are similar to that of wild-type α5β1, 

recycling to the plasma membrane of β1 Y795A and β1 Y783/795A , but not β1 Y783A , is 

considerably impaired. Instead, a large part of the internal pool of these mutants is 

degraded. Together, these data show that the fi rst NPxY motif regulates activation 

of α5β1, whereas the second NPxY regulates recycling of ligand-bound α5β1 to the 

plasma membrane. 

Regulation of hemidesmosome disassembly by growth factors receptors 

During wound healing, hemidesmome (HD) disassembly enables keratinocyte 

migration and proliferation. Hemidesmosome dynamics can be altered downstream 

of EGFR activation following the phosphorylation of integrin β4 residues, S1356 

and S1364, which reduces the interaction with plectin. This event, however, 

is insuffi cient to drive complete HD disassembly. We have used a FRET-based 

assay to demonstrate that the connecting segment (CS) and carboxy-terminal tail 

(C-tail) of the β4 cytoplasmic domain come into close proximity and facilitate the 

formation of a tertiary binding platform for plectin. Additionally, analysis of β4 

mutants containing either phospho-mimicking or non-phosphorylatable residues at 

threonine 1736 (T1736) in the C-tail show that this residue regulates the interaction 

with the plectin-plakin domain, since binding of β4 to the plectin-plakin domain 

was prevented when T1736 was replaced by aspartic acid but not by alanine. 

Furthermore, the aspartic acid mutation of β4 T1736 impaired hemidesmosome 

formation in PA-JEB/β4 keratinocytes. Phosphorylation of integrin β4 at T1736 

was shown to be regulated by PKD1, which requires translocation to the plasma 

membrane and subsequent activation. In conclusion, we identifi ed PKD1 as a 

novel player in the regulation of HD disassembly, a dynamically regulated process 

involving phosphorylation of β4 on S1356 and S1364, and T1736.

Blood pressure influences end-stage renal disease of Cd151-knockout mice 

Podocytes of the kidney adhere tightly to the underlying glomerular basement 

membrane (GBM) in order to maintain a functional fi ltration barrier. The clinical 

importance of podocyte binding to the GBM via an integrin-laminin-actin axis has 

been illustrated in models with altered function of α3β1 integrin, integrin-linked 

kinase, laminin-521, and α-actinin 4. We expanded on the podocyte-GBM binding 

model by showing that the main podocyte adhesion receptor, integrin α3β1, interacts 

with the tetraspanin CD151 in situ in mice. Deletion of Cd151 in mouse glomerular 

epithelial cells led to reduced adhesive strength to laminin by redistributing α3β1 

at the cell-matrix interface. Moreover, in vivo podocyte-specifi c deletion of Cd151 

led to glomerular nephropathy. Although global Cd151-null B6 mice were not 

susceptible to renal disease, increasing blood and transcapillary fi ltration pressure 

induced nephropathy in these mice. Importantly, blocking angiotensin-converting 

enzyme in renal disease–susceptible global Cd151-null FVB mice prolonged their 

median life span. Together these results establish CD151 as a crucial modifi er of 

integrin-mediated adhesion of podocytes to the GBM and show that blood pressure 

is an important factor in the initiation and progression of Cd151 knockout–induced 

nephropathy. 

Nesprin-3 in zebrafish and mice The nuclear envelope is thought to be connected 

to the cytoskeleton by members of the nesprin protein family. Whereas nesprin-1, 

nesprin-2 and nesprin-4 anchor the nucleus to actin fi laments and microtubules, 

nesprin-3 mediates a connection of the nuclear envelope with the intermediate 

fi lament system. Studies using transfected cells have shown that this indirect 

interaction is established by the binding of nesprin-3 to the cytoskeletal linker 

protein plectin. To study the function of nesprin-3 in vivo, we screened a library of 

ENU-mutagenized zebrafi sh for nesprin-3 mutations. A single nesprin-3 null mutant 

was isolated which carried a premature stop codon in exon 4. Loss of nesprin-3 

results in a reduced association of the keratin fi lament system with the nucleus in 

basal keratinocytes but does not lead to an obvious defect in the organization of the 

epidermis. Moreover, the nesprin-3-defi cient zebrafi sh develop normally and are 

viable and fertile. Additionally, we have generated nesprin-3 knockout mice and, 

similarly to the nesprin-3-defi cient zebrafi sh, these mice do not display an overtly 

abnormal phenotype. Immunofl uorescent analysis of nesprin-3 distribution in wildtype 

mice, revealed a strong co-localization of nesprin-3 with plectin and vimentin 

at the nuclear perimeter in Sertoli cells of the testis. In the nesprin-3 knockout 

mice there is no such nuclear localization of plectin and vimentin. Current studies 

are aimed at establishing a role of nesprin-3 in the force transmission between the 

extracellular matrix and the nucleus. 

Figure 1: Regulation of integrin-mediated adhesion 

Publications 

25 


Postel R, Ketema M, Kuikman I, 

De Pereda JM, Sonnenberg A. Nesprin-3 

augments peripheral nuclear localization 

of intermediate fi laments in zebrafi sh. 

Identifi cation of amino acids essential for 

nesprin-3 interaction with plectin. J. Cell 

Sci 2011;124:755-764 

Ortega E., Buey RM, Sonnenberg A, 

De Pereda JM. The structure of the plakin 

domain of plectin reveals a non-canonical 

SH3 domain interacting with its fourth 

spectrin repeat. J Biol Chem 

2011;286:12429-12438 

Raymond K, Cagnet S, Kreft M, 

Janssen H, Sonnenberg A, Glukova MA. 

Control of the mammary myoepithelial cell 

contraction/relaxation cycle by α3β1 

integrin signaling. EMBO J 2011;30:1896- 

1906 

Kittrell FS, Carletti MZ, Kerbawy S, 

Heestand J, Xian W, Zhang M, Lamarca 

H.L, Sonnenberg A, Rosen JM, Medina 

D, Behbod F. Prospective isolation and 

characterization of committed and 

multipotent progenitors from an 

immortalized cell line, COMMA-D. 

Breast Cancer Res 2011;13:R41 

Chapman HA, Li X, Alexander JP, 

Brumwell A, Lorizio W, Tan K, 

Sonnenberg A, Wei Y, Vu TH. Integrin 

α6β4 identifi es an adult distal lung 

epithelial population with regenerative 

potential in mice. J Clin Invest 

2011;121:2855-2862 

Margadant C, Monsuur HN, Norman 

JC, Sonnenberg A. Mechanisms of 

integrin activation and traffi cking. 

Curr. Opin. Cell Biol 2011;23:607-614 

Zhang F, Michaelson JE, Moshiach S, 

Sachs N, Zhao W, Sun Y, Sonnenberg A, 

Lahti J.M, Huang H, Zhang XA. 

Tetraspanin CD151 maintains vascular 

stability by balancing the forces of cell 

adhesion and cytoskeletal tension. Blood 

2011;118: 4274-4284 

Ketema M, Sonnenberg A. Nesprin-3: 

a versatile connector between the nucleus 

and the cytoskeleton. Biochem Soc Trans 

2011;39:1719-1724 

Sachs N, Claessen N, Aten J, Kreft M, 

Teske GJD, Koemann A, Zuurbier CJ, 

Janssen H, Sonnenberg A. Blood pressure 

infl uences end-stage renal disease of 

Cd151 knockout mice. J Clin Invest 

2012;122:348-358

26 





2009;10:314-5 

Group leader Wouter Moolenaar 

Wouter Moolenaar PhD Group leader 

Maaike Alderliesten PhD Post-doc 

Elisabetta Argenzio PhD Post-doc 

Maikel Jongsma PhD Post-doc 

Elisa Matas-Rico PhD Post-doc 

Dalila Elouarrat MSc PhD student 

Anna Houben Msc PhD student 

Michiel van Veen PhD student 

LIPID GROWTH FACTOR SIGNALING 

The major focus of our research is on bioactive lysophospholipids, most notably the 

lipid growth factor lysophosphatidic acid (LPA), its signaling properties, biosynthesis 

and role in health and disease. LPA signals through six known G protein-coupled 

receptors, termed LPA 1-6, showing both overlapping and distinct signaling 

properties. LPA signaling infl uences proliferation, migration and other functions 

of numerous cell types, both normal and malignant. LPA is produced extracellulary 

by a secreted lysophospholipase D (lysoPLD), named autotaxin (ATX), originally 

identifi ed as an autocrine motility factor for melanoma cells. The ATX-LPA 

signaling axis is vital for embryonic development and, when hyperactive, promotes 

tumor progression and metasis in mice. As such, ATX qualifi es as an attractive 

target for therapy. Our longstanding collaboration with the structural biology group 

of Anastassis Perrakis (Division of Biochemistry) has led to the elucidation of 

the crystal structure of ATX, which has yielded new insights into ATX structureactivity 

relationships. Furthermore, in collaboration with chemist Huib Ovaa and 

collaborators, potent inhibitors of ATX have been generated that are capable of 

lowering plasma LPA levels in vivo. Our current research focuses on ATX structurefunction 

relationships as well as the characterization of novel LPA receptors and 

downstream signaling events. These studies should lead to novel ways of interfering 

with ATX-LPA receptor signalling and with undue LPA production in the tumorstroma 

microenvironment. 

Autotaxin structure-function analysis Autotaxin (ATX) produces LPA from 

extracellular lysophosphatidylcholine. While the LPA-generating activity of ATX has 

been well characterized, the molecular basis of substrate recognition and catalysis by 

ATX, and how it interacts with target cells has been elusive. A four-way collaboration 

with the groups of Anastassis Perrakis (Division of Biochemistry), Andrew Morris 

(University of Kentucky, USA) and Huib Ovaa (Division of Cell Biology) has led to 

the elucidation of the crystal structure of ATX, alone and in complex with a smallmolecule 

inhibitor. We have identifi ed a hydrophobic lipid-binding pocket and a 

nearby open tunnel that may have a key role in substrate entrance or/and product 

release. We have mapped key residues required for catalysis and selection between 

nucleotide and phospholipid substrates. ATX was found to interact with cell-surface 

integrins via its N-terminal somatomedin-B-like somatomed domains, using 

an atypical mechanism. These rresults 

defi ne determinants 

of substrate discrimination by ATX and its family 

members, suggest hhow 

ATX promotes 

localized lo LPA signaling, and 

eenable 

new approaches 

tto 

target ATX by small- 

mmolecule 

inhibitors. 

Figure 2: The autotaxin-LPA 

receptor signaling axis. 

(A) Autotaxin (ATX) converts 

extracellular lysophosphatidylcholine 

(LPC) into bioactive LPA, which in 

turn acts on specifi c G protein-coupled 

receptors to activate multiple signaling 

cascades. 

(B) Structures of LPA and LPC.

ATX isoforms 

ATX exists in distinct alternatively spliced isoforms. The longest isoform, termed 

ATXα, differs from the canonical form (ATXβ) by having a polybasic insert of 

unknown function in the catalytic domain. We fi nd that secreted ATXα undergoes 

proteolytic cleavage at the insert by the endoprotease furin. However, cleaved ATXα 

remains structurally and functionally intact due to strong interactions within 

the catalytic domain. Furthermore, using ELISA and surface plasmon resonance 

assays, we fi nd that ATXα binds to heparin with much higher affi nity than does 

ATXβ. Heparin enhanced the lysophospholipase D activity of ATXα up to twofold. 

These results suggest that ATXα, owing to its basic insert, may preferentially 

bind to heparan sulfate proteoglycans at the cell surface to promote localized LPA 

production and signaling, a process that might be fi ne-tuned by furin and furinlike 

proteases. Our fi ndings raise the possibility that ATXα and ATXβ may act on 

distinct LPA receptors, depending on their membrane localization, a scenario that is 

currently under investigation. 

LPA receptor signaling CLIC4 as a new player 

LPA receptors strongly couple to the G(13)-RhoA pathway to regulate the actin 

cytoskeleton. We discovered that LPA-induced RhoA activation is accompanied 

by rapid recruitment of “Chloride Intracellular Channel” protein 4 (CLIC4) to the 

plasma membrane. CLIC4 is a soluble protein structurally related to omega-type 

glutathione-S-transferases (GSTs) and implicated in various biological processes, 

ranging from chloride channel formation to vascular tubulogenesis. However, 

its function(s) and regulation remain elusive. Intriguingly, CLIC4 translocation 

depends on conserved residues, including a reactive cysteine, the equivalents of 

which are critical for the enzymatic function of GSTs. This suggests that membranetargeted 

CLIC4 may catalyse a yet unknown chemical reaction. Through yeast twohybrid 

screening and biochemical studies, we have identifi ed and validated novel 

binding partners of CLIC4 that may shed new light on its possible function(s) as a 

signaling intermediate. 

Figure 3: Agonist-induced increase in intracellular cAMP levels in B16 melanoma cells. cAMP was 

monitored in real-time using a FRET-based biosensor (CFP-Epac-YFP). Left panel shows the separate 

CFP and YFP signals. Right panel shows increases in [cAMP], measured as increases in the ratio 

CFP/YFP (loss of FRET due to Epac unfolding). αMSH denotes α-melanocyte-stimulating hormone. 

Opposing action of the LPA 5 receptor 

LPA is a chemoattractant for many cell types. LPA-induced cell migration is 

primarily mediated by the classical LPA 1 and LPA 2 receptors. Unexpectedly, 

however, LPA completely inhibits the transwell migration of B16 melanoma cells, 

with alkyl-LPA being 10-fold more potent than acyl-LPA. The anti-migratory 

response to LPA is highly polarized and dependent on protein kinase A (PKA) 

activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 

depletion from the plasma membrane. We fi nd that LPA-induced chemorepulsion 

is mediated specifi cally by the alkyl-LPA-preferring LPA 5 receptor, which raises 

intracellular cAMP via a noncanonical pathway. Our results defi ne LPA 5 as an 

anti-migratory receptor and they point to a mechanism of chemorepulsion likely 

to be relevant for tumor cells that overexpress LPA 5, acting to override positive 

chemotactic signals. 

Publications 

27 


Moolenaar WH, Hla T. SnapShot: 

Bioactive Lysophospolipids. Cell 2011 

(in press) 

Moolenaar WH, Perrakis A. Insights 

into autotaxin: how to produce and present 

a lipid mediator. Nat Rev Mol Cell Biol. 

2011;12:674-79 


Christodoulou E, Day J, Wu T, Fulkerson 

Z, Albers HM, van Meeteren LA, Houben 

AJ, van Zeijl L, Jansen S, Andries M, Hall 

T, Kasiem M, Harlos K, Vander Kooi CW, 

Smyth SS, Ovaa H, Bollen M, Morris AJ, 

Moolenaar WH, Perrakis A. Structural 

basis for substrate discrimination and 

integrin binding by autotaxin. Nat Struct 

Mol Biol. 2011;18:198-204 

Jongsma M, Matas-Rico E, 

Rzadkowski A, Jalink K, Moolenaar WH. 

LPA is a chemorepellent for B16 

melanoma cells: action through the 

cAMP-elevating LPA5 receptor. Plos One 

2011;e29260 

Houben AJ, Moolenaar WH. 

Autotaxin and LPA receptor signaling 

in cancer. Cancerz Metastasis Rev. 

2011;30:557-65 

Houben AJ, Van Meeteren LA, 

Van Wijk XM, Van Zeijl L, Van de 

Westerlo EM, Hausmann J, Fish A, 

Van Kuppevelt TH, Perrakis A, 

Moolenaar WH. The long isoform of 

autotaxin: intradomain cleavage by furin 

and high-affi nity binding to heparin. 

J Biol Chem 2011 (in press) 

Dusaulcy R, Rancoule C, Grès S, 

Wanecq E, Colom A, Guigné C, van 

Meeteren LA, Moolenaar WH, Valet P, 

Saulnier-Blache JS. Adipose-specifi c 

disruption of autotaxin enhances 

nutritional fattening and reduces plasma 

lysophosphatidic acid. J Lipid Res. 

2011;52:1247-55 

Stortelers C, Moolenaar WH. 

The global gene expression program 

of LPA-stimulated fi broblasts. In: 

Lysophospholipid Receptors: Signaling and 

Biochemistry. Eds.: Chun J, Hla T, 

Moolenaar WH, Spiegel S. Publisher: 

John Wiley (in press)

28 





2009;10:314-5 

Group leader Kees Jalink 

Kees Jalink PhD Group leader 

Bram van den Broek PhD Staff physicist 

Linda Henneman PhD Post-doc 

Marcel Raspe PhD Post-doc 

Daan Visser MSc PhD student 

Kasia Kedziora MSc PhD student 

Daniele Leyton Puig PhD student 

Jeffrey Klarenbeek MSc Technical staff 

BIOPHYSICS OF CELL SIGNALING 

Employing advanced imaging and other biophysical techniques, we study cell 

signaling events with high spatial and temporal resolution. Electrophysiological 

(e.g. patch clamping) and advanced imaging (e.g. Fluorescence Resonance Energy 

Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross 

Correlation Spectroscopy (FCCS) and photorelease of caged compounds) are used in 

research projects in our group as well as in a number of collaborations within and 

outside our institute. We also contribute to the development of hardware, software 

and FRET sensors for various intracellular messengers. 

The cation channel TRPM7 in the control of invadosomes and invasive 

migration In an ongoing collaboration with the group of Dr F.N. van Leeuwen 

(Nijmegen) we continued our research into the role of the atypical ‘channel-kinase’ 

TRPM7 as novel component of invadosomes and as determinant of cell adhesion 

and migration. TRPM7 is a membrane ion channel fused to a protein kinase 

domain which functions as a mechanosensor and regulator of local Ca 2+ entry. 

Strikingly, forced expres sion of TRPM7 in neuro blastoma cells is suffi cient to induce 

invadosome formation. Phospholipase C signaling triggers TRPM7-mediated Ca 2+ 

infl ux and enhances invado some formation. Thus, TRPM7 may function as a master 

regulator of invadosomes under the control of GPCR signals. 

We found that TRPM7 confers a highly invasive phenotype on otherwise noninvasive 

neuroblastoma cells, both in vitro (time-lapse imaging, Transwell 

assays) and in vivo (tail-vein injection in nude mice). Conversely, RNAi-mediated 

knockdown of TRPM7 strongly suppresses migration in MDA-MB-231 breast 

carcinoma cells, a model for invasive breast cancer. Moreover, mRNA expression 

profi ling of 246 human breast carcinoma specimens reveals that high expression 

of TRPM7 at the time of diagnosis predicts a poor prognosis and is correlated with 

distant metastasis. This result has now been confi rmed in large published databases 

and, by qPCR, in an independent set of tumor biopsies in Nijmegen. These fi ndings 

provide strong support for a role of TRPM7 in tumor cell dissemi nation. 

Mass spectrometry analysis of TRPM7 immuno-complexes identifi ed some 40 

proteins implicated in cytoskeletal regulation, cell adhesion and -migration. The 

large majority of these proteins localizes to invadosomes. Proteins associated with 

Ca 2+ /PLC signaling are amply represented in the set, suggesting an important 

role for these intracellular messengers. Consistent with this notion we fi nd that 

TRPM7 medi ates local Ca 2+ infl ux to specifi cally activate PLCδ 1 in invadosomes, 

leading to PIP 2 hydrolysis and sustaining the TRPM7 open-channel state. Our 

current investigations address, by combining biophysical readout techniques with 

mutational analysis of the TRPM7 C terminus, the details of TRPM7 sensitivity to 

Ca 2+ and phosphoinositides, and the exact role of PLCδ 1 which appears to mediate 

a Ca 2+ -infl ux dependent feedback loop in the activation of TRPM7. We have also 

started analyzing localization and role of novel invadosome components identifi ed 

in the mass spec screen. Strikingly, a high percentage of the protein components 

identifi ed in the mass spec screen also correlate signifi cantly with poor outcome. 

T-Epac-VV, a superior FRET sensor for the second messenger cAMP We also 

continued further development of FRET sensors in the lab. Among most noticeable 

developments are further improvements in our library of FRET construction vectors. 

Such vectors contain a FRET donor, polylinker and FRET acceptor and are ideal 

‘one-step’ construction blocks to build FRET sensors by simply inserting the protein 

of your choice in the polylinker. A large choice of donors and acceptors are available. 

Using our new additions to this library, we created mTurquoise-Epac-Venus-Venus, a 

cAMP sensor that appears superior to all other sensors in every way. Signal-to-noise 

ratio is enormous and should defi nitely be high enough to allow microscopy screens.

Super resolution imaging This year we obtained funding to purchase a Leica 

SR-GSD super resolution microscope, along with two PhD students to operate and 

improve the setup. In light microscopy, it has been known for over a century that the 

laws of physics limit the achievable resolution to ~ 250 nm. At this resolution, many 

details of the cell architecture as out of reach. Super-resolution light microscopy 

employs a set of ‘tricks’ to circumvent this limitation to achieve resolution down to 

~ 10 nm (see fi gure 4). In this project, we optimize preparation techniques for SR 

microscopy and employ the equipment in running research lines, both within our 

group as well as in collaborations with other groups at the NKI. 

Figure 4: Super-resolution imaging. Multi-color labeled specimen, when imaged by wide-fi eld 

microscopy, yield blurry, defraction-limited resolution. SR microscopy starts by depleting the fl uorescent 

ground state with a high-intensity laser. When most fl uorophores are in the dark state, the remaining 

fl uorophores can be localized with very high precision. This process is repeated until all molecule 

coordinates are recorded, yielding the dataset for reconstruction of the image. Following terminal 

bleaching of all labels, the preparation is either (A) stripped of probes and relabeled for the same 

proteins, yielding much better S/N, or (B) labeled for a (partially) different set of proteins, enabling to 

reconstruct location of all relevant proteins within the complex. 

Publications 

29 


Kuil J, Steunenberg P, Chin PT, 

Oldenburg J, Jalink K, Velders AH, 

Van Leeuwen FW. Peptide-functionalized 

luminescent iridium complexes for lifetime 

imaging of CXCR4 expression. 

Chembiochem 2011;12:1897-903 

Klarenbeek JB, Goedhart J, Hink MA, 

Gadella TW, Jalink K. A mTurquoise-based 

cAMP sensor for both FLIM and 

ratiometric read-out has improved 

dynamic range. PLoS ONE 2011;6:e19170 

Jongsma M, Matas Rico E, 

Rzadkowksi A, Jalink K, Moolenaar WH. 

LPA is a chemorepellent for B16 

melanoma cells: action through the 

cAMP-elevating LPA5 receptor. PLoS ONE 

(accepted)

30 





2009;10:314-5 

Group leader Metello Innocenti 

Metello Innocenti PhD Group leader 

Zhen Liu PhD Post-doc 

Tadamoto Isogai MSc PhD student 

Rob van der Kammen MSc Technical staff 

Publication 

Galovic M, Xu D, Areces LB, van der 

Kammen R, Innocenti M. Interplay 

between N-WASP and CK2 optimizes 

clathrin-mediated endocytosis of EGFR. J 

Cell Sci. 2011;124:2001-12 

ACTIN DYNAMICS IN CANCER CELLS 

The polymerization of actin monomers into fi laments produces mechanical force to 

sculpture protrusions and invaginations on membranes. Cellular actin cannot selfassemble 

into fi laments without proteins referred to as actin nucleators, which are 

essential for actin-based processes to occur in vivo. Not surprisingly, actin dynamics 

are key to migration of normal and cancer cells: actin-based extensions of the plasma 

membrane appear at the onset of migration, mark the presumptive leading edge 

and support persistent cell motility. Two types of actin-based protrusions have been 

observed in mammalian cells: lamellipodia/ruffl es and fi lopodia. 

Lamellipodia and ruffl es are veil-shaped or upward-curled protrusions of the 

plasma membrane, respectively, consisting of a dense meshwork of branched actin 

fi laments nucleated by the Arp2/3 complex. The Arp2/3 complex generates this actin 

array upon being switched on by the WAVE family of nucleation-promoting factors 

(NPFs). Lamellipodia and ruffl es emerge from the leading edge of crawling cells and 

are essential for cells adopting mesenchymal-type movement to migrate. 

Filopodia are fi nger-like and highly dynamic extensions of the cell surface. The 

growth of tightly bundled linear actin fi laments produces a pushing force towards 

the plasma membrane resulting in the protrusion of a fi lopodium. The Formin 

mDia2 has been shown to control fi lopodium initiation. The role of fi lopodia in cell 

migration remains unclear. 

Regulation of WAVE activity Although WAVE plays an essential role not only in 

cell migration but also in a host of actin-based processes, a mechanistic explanation 

for its high versatility is still lacking. We have identifi ed a new WAVE-binding 

protein that confers functional specifi city on WAVE and dictates its contribution to 

macropinocytosis. 

Function and regulation of mDia2 In order to fully understand the biological 

function(s) and the regulation of mDia2, we have isolated the mDia2 interactome. 

This information is helping us dissect the mechanics controlling mDia2 activity. 

Moreover, it suggests new roles for mDia2. 

Mechanisms of formation and roles of filopodia in cell migration The 

mechanisms whereby fi lopodia are generated and the function of fi lopodia in cell 

migration remain mysterious. To tackle these issues, we have generated a genetically 

modifi ed cell line that allows us to induce fi lopodium formation in vitro. Loss-offunction 

genetic and proteomic screens will be performed to inventory fi lopodiumregulatory 

proteins and to reveal the fi lopodium protein signature, respectively. 

Role of the Arp2/3 complex in vivo and in cell culture The Arp2/3 complex has a 

pivotal role in the migration of mesenchymal 

cells, which requires lamellipodia and 

ruffl es. Whether or not it also contributes 

to the ameboid/round-mode of movement 

or to any other process is unknown. Most 

importantly, the role of the Arp2/3 complex in 

physio-pathology has not been addressed yet. 

Conditional Arp2/3-complex knockout mice 

allow us to investigate the roles of the Arp2/3 

complex both at the organismal and cellular 

levels. 

Figure 5: Club-like fi lopodia induced by mDia2ΔC. 

Serum-starved HeLa cells overexpressing Flag-mDia2ΔC 

stained with rhodamine-phalloidin (green) and 

anti-Flag antibodies (red) to detect F-actin and mDia2, 

respectively. Note that mDia2 localizes at the tip 

of fi lopodia.

DIVISION OF CELL BIOLOGY II 

CHEMICAL BIOLOGY IN ANTIGEN PRESENTATION, 

INFECTION-INDUCED TUMORS AND ANTI-CANCER DRUGS 

Our aim is to understand the molecular basis of a series of biological processes 

related to cancer and to defi ne lead structures for manipulation. Our approach is 

gene inactivation by siRNA technology and multi-parallel detection of the resulting 

phenotypes. As silencing mimics the effects of chemical inactivation, identical 

screens are performed with dedicated chemical libraries and the results of the 

two activities are integrated to defi ne a minimal set of potential target-novel lead 

structures. These are subsequently confi rmed under in vitro conditions with 

isolated proteins. In collaboration with the Ovaa (at the Division of Cell Biology) and 

the Overkleeft (at LIC, Leiden University) groups, lead structures will be further 

optimized for further testing in vitro and in vivo. By concentrating on novel enzyme 

families, we hope to provide prove-of-principle for manipulation of new enzyme 

classes in cancer. 

Classically, we worked on MHC class I and MHC class II molecules and the activities 

in these fi elds will fi rst be summarized before the activities in recently developed 

areas will be discussed. 

Antigen presentation by MHC class I molecules MHC class I molecules are 

required for antigen presentation in the form of peptides from viral and tumor 

antigens to the immune system. This system is essential in tumor vaccination 

strategies. We have studied the details of peptide loading onto MHC class I 

molecules by expanding the size of anchor residues. We showed that such peptides 

can bind to MHC class I but not in a stable fashion. We are making crystals of 

such MHC class I-peptide combinations and perform further protein chemistry 

experiments to decipher the details of peptide binding both with purifi ed 

components as well as in microsomes. These experiments may reveal the details of 

peptide loading onto MHC class I molecules and yield tools allowing manipulating 

these. 

Cross-presentation by dendritic cells is required for priming of CTL and proper 

immune responses to infection and cancer. We have scanned libraries of inhibitors 

for de-ubiquitin enzymes for compounds manipulating cross-presentation. We 

have identifi ed activators and – using ubiquitin probes – are defi ning the target(s). 

These will be the basis of new cell biology aiming to understand the basis of crosspresentation 

and deliver tools for further manipulation of these responses. 

Antigen presentation by MHC class II molecules MHC class II molecules are 

lookalikes of MHC class I molecules and also present peptides from antigens. 

However, MHC class II molecules are expressed on immune cells and activate the 

immune response and especially antibody production. In the past we had defi ned 

various aspects of this process. To defi ne new molecules in this pathway and to 

generate new biology, we performed a fl ow-based siRNA screen for factors affecting 

expression and peptide loading of MHC class II. To subcluster the resulting list of 

hits, we performed qPCR-based and microscopy-based analyses of the hits. These 

revealed new proteins involved in transcriptional control as well as other molecules 

involved in the cell biology of MHC class II molecules. We then designed new 

technologies to place the resulting clusters in pathways that explain the selective 

tissue specifi c expression of MHC class II. We then tested whether we could explain 

in molecular terms a long standing issue: how is the intracellular distribution of 

MHC class II molecules controlled in dendritic cells (DC) when these are activated 

by ‘danger signals’ such as LPS. We integrated the results from the fl ow based, 

microscopy based and qPCR screen with expression array data to defi ne a small 

selection of 12 hits that could explain this phenotype. These hits were tested by 

silencing in immature DC, in effect making immature DC with a mature phenotype 

with respect to MHC class II distribution, yielding 8 concurring hits. These 

included many unknown and we selected the novel GTPase Arl14/ARF7 for further 

31 

CELL BIOLOGY II 

Division head, group leader Jacques Neefjes 

Jacques Neefjes PhD Group leader 

Ilana Berlin PhD Post-doc 

Gosia Garstka PhD Post-doc 

Victoria Menendez PhD Post-doc 

Charlotte Sadaka PhD Post-doc 

Tiziana Scanu PhD Post-doc 

Robbert Spaapen PhD Post-doc 

Jeroen Bakker MSc PhD student 

Marlieke Jongsma MSC PhD student 

Baoxu Pang MSc PhD student 

Petra Paul MSc PhD student 

Izhar Salomon MSc PhD student 

Sjoerd van Deventer MSc PhD student 

Rik van der Kant MSc PhD student 

Amy Wu MSc PhD student 

Ruud Wijdeven MSc PhD student 

Lennert Janssen BSc Technical staff 

Publications 

Kessler JH, Khan S, Seifert U, Le Gall S, 

Chow KM, Paschen A, Bres-Vloemans SA, 

de Ru A, van Montfoort N, Franken KL, 

Benckhuijsen WE, Brooks JM, van Hall T, 

Ray K, Mulder A, Doxiadis II, van Swieten 

PF, Overkleeft HS, Prat A, Tomkinson B, 

Neefjes J, Kloetzel PM, Rodgers DW, 

Hersh LB, Drijfhout JW, van Veelen PA, 

Ossendorp F, Melief CJ. Antigen processing 

by nardilysin and thimet oligopeptidase 

generates cytotoxic T cell epitopes. Nat 

Immunol 2011;12:45-53 

Kok M, Zwart W, Holm C, Fles R, 

Hauptmann M, Van ‘t Veer LJ, Wessels LF, 

Neefjes J, Stal O, Linn SC, Landberg G, 

Michalides R. PKA-induced phosphorylation 

of ERalpha at serine 305 and high PAK1 

levels is associated with sensitivity to 

tamoxifen in ER-positive breast cancer. 

Breast Cancer Res Treat 2011;125:1-12 

Neefjes J, Jongsma ML, Paul P, Bakke O. 

Towards a systems understanding of MHC 

class I and MHC class II antigen 

presentation. Nat Rev Immunol 

2011;11:823-36

32 



Paul P, van den Hoorn T, Jongsma ML, 

Bakker MJ, Hengeveld R, Janssen L, 

Cresswell P, Egan DA, van Ham M, ten 

Brinke A, Ovaa H, Beijersbergen RL, Kuijl 

C, Neefjes J. A Genome-wide 

Multidimensional RNAi Screen Reveals 

Pathways Controlling MHC Class II Antigen 

Presentation. Cell 2011;145:268-83 

Van den Hoorn T, Paul P, Jongsma ML, 

Neefjes J. Routes to manipulate MHC class 

II antigen presentation. Curr Opin Immunol 

2011;23:88-95 

Verweij FJ, van Eijndhoven MA, 

Hopmans ES, Vendrig T, Wurdinger T, 

Cahir-McFarland E, Kieff E, Geerts D, van 

der Kant R, Neefjes J, Middeldorp JM, 

Pegtel DM. LMP1 association with CD63 in 

endosomes and secretion via exosomes limits 

constitutive NF-kappaB activation. EMBO J 

2011;30:2115-29 

Spaapen RM, Neefjes J. Immuno-waste 

exposure and further management. 

Nat Immunol 2012;13:109-11 

Van den Hoorn T, Paul P, Janssen L, 

Janssen H and Neefjes J. Dynamics within 

tetraspanin pairs and MHC class II 

expression. J. Cell Science (in press) 

Figure 1: Towards understanding 

proteasome dynamics. Monitoring yeast 

cells during the entry and exit from 

starvation allows us to follow in- and 

export of the proteasome (green) from the 

nucleus (blue), as well as the formation 

and clearance of foci (a potential storage 

mechanism). Furthermore, the RITE 

technique enabled assessment of the 

degradation and synthesis of the 

proteasome. This assay was used in a 

genome wide screen to identify the genes 

involved in the regulation of proteasome 

dynamics. 

study. Using again the results from the genome-wide screen with domain search 

programs, in vitro protein reconstitution experiments, yeast two hybrid and protein 

pull-down combined with mass spec, we defi ned a novel pathway controlling MHC 

class II transport. ARF7 is interacting with a novel effector ARF7EP that binds to the 

actin attaching protein MYO1e to prevent transport. The regulation of this pathway 

is of interest as it controls MHC class II processes such as the activation of T and B 

cells. 

The results of the screen are currently applied to defi ne new GAPs controlling this 

pathway, new DUBs and their targets defi ning multivesicular body formation and 

fusion and new proteins controlling transport of MHC class II molecules. The data 

set generated above is essential in this endeavor to generate new biology. 

Manipulating proteasomes as a new anti-cancer target Proteasomes are huge 

self-compartimentalized proteases that are responsible for the majority of protein 

destruction in cells. For this reason, proteasomes are intrinsically coupled to the 

cell cycle, differentiation and transformation and inhibitors for these have entered 

the clinic as new entities in the treatment of multiple myeloma. How large protein 

multimers like the proteasome are destroyed is unclear. We have established in yeast 

a system allowing the conversion of proteasomes with a green tag for those with 

a red tag. The ratio green over red fl uorescence denotes the ratio old versus new 

proteasomes and thus indicates proteasome complex turn-over. We have crossed the 

yeast with a yeast knock-out library and isolated mutants with effects on proteasome 

transport, aggregation and turn-over by confocal microscopy. These mutants include 

many factors with mammalian homologues and these will be defi ned to determine 

their role in mammalian proteasome turn-over. In addition, we are screening for 

compounds also allowing the inhibition of hits defi ned by our screen. Proteasome 

turn-over activators will slow down cell growth and may be interesting leads for 

further development. 

Chemical biology of anti-cancer compounds Topo-isomerase inhibitors such as 

doxorubicin or etoposide are compounds extensively used in the treatment of cancer 

patients. Doxorubicin and Etoposide inhibit the exact same reaction to initiate DNA 

double strand breaks. Yet, Doxorubicin is considerably more effective as Etoposide 

coinciding with more side effects, most notably cardiotoxicity. In this chemical 

biology approach we used a third drug, aclarubicin, which is like doxorubicin an 

anthracyclin but does not generate DNA double stranded breaks. A comparison of 

the three drugs then allows statements on 

the role of double stranded break formation 

(shared by doxorubicin and etoposide) as well 

as statements on the unique anthracyclin 

structure (shared between the two 

anthracyclin compounds). We showed that 

histones were transiently leaving chromatin 

of non-mitotic cells only when cells were 

exposed to doxorubicin. This is due to the 

anthracyclin structure rather than double 

strand break formation as result of topo-II 

inhibition. Using FAIRE-Seq, we showed 

that histones are released from gene-rich areas and active transcriptional start site, 

thus refl ecting ‘open chromatin structures’. As also H2Ax is released, doxorubicin – 

but not etoposide – strongly attenuates DNA repair of double strand breaks induced 

by the compounds. We show that the result of these manipulations is massive 

alterations of the transcriptome in tissue culture cells as well as in vivo. Especially 

the heart of mice is affected by doxorubicin, which may correlate to the altered 

transcriptome. By performing a ChIP with g-H2AX, we show that DNA repair is not 

equally occurring over the genome and that doxorubicin delays DNA repair mainly 

in open chromatin structures that may thereby more dramatically effected during 

treatment. 

Using novel technologies on an ‘old’ anti-cancer compound allow the identifi cation 

of novel cell biological mechanisms occurring in response to doxorubicin.

LABORATORY FOR DRUG INNOVATION AND MOLECULAR 

IMMUNE TECHNOLOGY: CHEMICAL BIOLOGY OF 

UBIQUITINATION, PROTEOLYSIS AND ANTIGEN 

PRESENTATION 

The ubiquitin-proteasome system and antigen presentation The proteasome 

is a multi-catalytic proteolytic machine that is abundant and responsible for the 

turnover of many critical regulatory proteins including tumor suppressor proteins 

and cell cycle regulators. The destructive force of the proteasome as an important 

determinant of protein half-life is regulated by ubiquitination. Substrates are 

tagged with chains of ubiquitin (Ub) molecules that signal for destruction by the 

proteasome. Ubiquitin is a 76 amino acid protein that can be conjugated onto 

substrates to guide protein destruction. The majority of proteins are targeted 

for proteasomal proteolysis by Ub polymers. Despite a wealth of literature on 

ubiquitination of proteasome substrates, little is known about the degradation 

process at a more detailed molecular level; ubiquitination status and protein stability 

currently cannot be predicted. It is clear however, that a ubiquitin code exists and 

that protein turnover by the proteasome is a tightly regulated and complex process 

that includes not only the complexity of the proteasome but also Ub polymer 

formation and remodeling and Ub recycling. Because of this complexity, tools that 

allow detailed studies of the effects of ubiquitination status on protein turnover are 

urgently needed. 

Our lab aims to develop tools to profi le cellular enzymatic activities associated with 

post-translational modifi cation of proteins with ubiquitin and we study proteasome 

activity, antigen production and antigen presentation by designing tools that 

interfere with individual components of these systems. We search for inhibitors of 

enzymatic activities and ligands of receptors both by high throughput screening 

of small molecule compound collections using in vitro biochemical screens and 

cell-based assays and by rational chemical design followed by chemical optimization 

and all biochemistry further required. Ligands and inhibitors form the basis for the 

development of research probes that we use to achieve our goals. 

Research is centered on one central theme: chemistry and biology of ubiquitinmediated 

proteolysis and MHC class I antigen presentation, and divided into three 

main topics: 

(1) ubiquitin chain chemistry, biochemistry and proteomics 

(2) proteasome activity 

(3) MHC class I antigen presentation 

To study these, we are developing and synthesizing unique reagents to detect or 

interfere with the ubiquitin system, proteolysis and with MHC class I mediated 

antigen presentation. 

Isopeptide-linked ubiquitin and ubiquitin-like assay reagents Deubiquitinating 

enzymes (DUBs) are proteases that remove ubiquitin (Ub) from proteins. Because 

the deregulation of DUBs is linked to the occurrence of a variety of diseases, they 

are of interest as potential drug targets. Consequently, good assay reagents are 

required to report enzymatic activity and inhibition. In line with this, we have 

recently developed a chemical ligation approach to a previously reported fl uorescence 

polarization (FP) assay for DUB activity. These assay reagents are based on a 

fl uorophore labeled lysine or peptide (fi gure 2b) linked to Ub or Ubl via an isopeptide 

bond (fi gure 2a), and have two characteristics that make them very powerful for 

(high-throughput) investigations of DUB catalytic action. First, it is the only reported 

assay reagent that incorporates an isopeptide linkage. Secondly, its physiological 

relevance (and potentially its affi nity for a deconjugating enzyme) can be enhanced 

by functionalizing the assay reagent with substrate-derived elements around the 

isopeptide linkage. 

We further functionalized our FP reagents by introducing a peptide sequence 

derived from a known ubiquitinated substrate (fi gure 2b). These context-specifi c 

reagents resemble the native environment that a Ub(l) protease encounters better 

compared to a single lysine residue. 

Group leader Huib Ovaa 

Huib Ovaa PhD Group leader 

33 


Boris Rodenko PhD Senior post-doc 

Sander van Kasteren DPhil Post-doc 

Alessia Amore PhD Post-doc 

Celia Berkers PhD Post-doc 

Farid El Oualid PhD Post-doc 

Paul Geurink PhD Post-doc 

Remco Merkx PhD Post-doc 

Anass Znabet PhD Post-doc 

Harald Albers MSc PhD student 

Reggy Ekkebus MSc PhD student 

Rieuwert Hoppes MSc PhD student 

Annemieke de Jong MSc PhD student 

Yves Leestemaker MSc PhD student 

Dharjath Hameed MSc PhD student 

Henk Hilkmann Ing Technical staff 

Dris El Atmioui Ing Research assistant 

Gabrielle van Tilburg BSc Research assistant 

Kim Wals Ing Research assistant 

Publications 

Albers HM, Hendrickx LJ, van Tol RJ, 

Hausmann J, Perrakis A, Ovaa H. 

Structure-based design of novel boronic 

acid-based inhibitors of autotaxin. J Med 

Chem 2011;54:4619-26 

Berkers CR. Probing proteasome activity 

and function Cancer diagnostics and 

mechanism of antigen processing. Leiden 

University, 2010 

de Jong A, Schuurman KG, Rodenko B, 

Ovaa H, Berkers CR. Fluorescence-based 

proteasome activity profi ling. Methods Mol 

Biol 2012;803:183-204 

El Oualid F, Hameed DS, El Atmiou D, 

Hilkman H, Ovaa H. Synthesis of Atypical 

Diubiquitin Chains. Methods Mol Biol 

2011;832 


Ovaa H, Perrakis A, Sixma TK. Mechanism 

of USP7/HAUSP activation by its 

C-terminal ubiquitin-like domain and 

allosteric regulation by GMP-synthetase. 

Mol Cell 2011;44:147-59 

Frederiks F, Stulemeijer IJ, Ovaa H, van 

Leeuwen F. A modifi ed epigenetics toolbox 

to study histone modifi cations on the 

nucleosome core. Chembiochem 

2011;12:308-13

34 




Christodoulou E, Day JE, Wu T, Fulkerson 

Z, Albers HM, van Meeteren LA, Houben 

AJ, van Zeijl L, Jansen S, Andries M, Hall 

T, Pegg LE, Benson TE, Kasiem M, Harlos 

K, Kooi CW, Smyth SS, Ovaa H, Bollen M, 

Morris AJ, Moolenaar WH, Perrakis A. 

Structural basis of substrate discrimination 

and integrin binding by autotaxin. Nat 

Struct Mol Biol 2011;18:198-204 

Nijholt DA, de Graaf TR, van Haastert 

ES, Oliveira AO, Berkers CR, Zwart R, 

Ovaa H, Baas F, Hoozemans JJ, Scheper 

W. Endoplasmic reticulum stress activates 

autophagy but not the proteasome in 

neuronal cells: implications for Alzheimer’s 

disease. Cell Death Differ 2011;18:1071-81 



Cresswell P, Egan DA, van Ham M, 

Ten Brinke A, Ovaa H, Beijersbergen RL, 

et al. Marizomib, a proteasome inhibitor 

for all seasons: preclinical profi le and a 

framework for clinical trials. Curr Cancer 

Drug Targets 2011;11:254-84 



Cresswell P, Egan DA, van Ham M, ten 


C, Neefjes J. A Genome-wide 

Multidimensional RNAi Screen Reveals 

Pathways Controlling MHC Class II 

Antigen Presentation. Cell 2011;145:268-83 

Van Kasteren SI, Berlin I, Colbert JD, 

Keane D, Ovaa H, Watts C. A 

Multifunctional Protease Inhibitor 

To Regulate Endolysosomal Function. 

ACS Chem Biol 2011;6:1198-204 

Licchesi JDF , Mieszczanek J, 

Mevissen TE, Rutherford TJ, Akutsu M, et 

al. An ankyrin-repeat ubiquitin-binding 

domain determines TRABID’s specifi city 

for atypical ubiquitin chains. Nat Struct 

Mol Biol 2011;19:62-71 

Faesen AC, Luna-Vargas MPA, Geurink 

PP, Clerici M, Merkx et al. The differential 

modulation of USP activity by internal 

regulatory domains, interactors and 

seven Ub-chain types. Chem & Biol 

2011;18:1550-61 

Geurink PP, El Oualid F, Jonker A, 

Hameed DS, Ovaa H. A General 

Chemical Ligation Approach Towards 

Isopeptide-linked Ubiquitin and 

Ubiquitin-like Assay Reagents. 

ChemBioChem 2012;13:293-7 

Chemical reporters of UPS activity Pharmacological interference with UPSmediated 

protein degradation holds much promise. However, the only example 

of pharmacological modulation of the UPS approved for use in the clinic so far is 

the proteasome inhibitor bortezomib and tools to study proteasome action are in 

demand. A chemical approach using irreversible covalent inhibitors equipped with 

reporter groups offers several advantages over traditional approaches, including their 

applicability to any cell line or tissue. We recently developed such probes which have 

been shown to provide information that correlates directly with the functional state 

of enzyme active sites: active forms only and not latent or (auto)inhibited activities 

are reported. Using fl uorescent proteasome activity reporters we have analyzed 

proteasome activity in mouse tissues, we were able to monitor pharmacological 

inhibition in vivo and we were able to visualize active proteasomes in (primary) 

human cells both by confocal microscopy and fl ow cytometry. 

A broad-spectrum endosomal protease inhibitor To address the problem of 

functional redundancy between the proteases in the endo-lysosomal pathway (where 

inhibition of an enzyme can be compensated for by another enzyme), we developed 

a single inhibitor that inhibits all endo-lysosomal proteases (fi gure 3). This inhibitor 

could dramatically reduce the degradation of exogenous proteins taken up in this 

manner. Using the inhibitor, EGFR-signaling could be extensively prolonged and 

unstable antigens could be rescued from over-degradation leading to improved 

antigen presentation. 

Figure 2A: Graphic representation of an FP assay. 

When a fl uorophore that is covalently attached to a small molecule, such as a small peptide, is excited 

by polarized light, it will emit largely depolarized light. When it is bound to a high molecular weight 

molecule, such as Ub or a Ubl, the emitted light is much less depolarized. By following the change in 

fl uorescence polarization the activity can be monitored. P: polarization. 

(B) FP assay with full length USP7 and ubiquitinated PTEN[5-21] derived peptide FP substrate 

(100 nM). 

Figure 3: A broad-spectrum endosomal protease inhibitor

NANOBIOLOGY 

A major objective of modern structural biology is to appreciate cellular organization 

by elucidating the spatial arrangement of macromolecular complexes within a cell. 

The clue of the treatment of various diseases is inside nanomachines within the cell. 

Investigating these machines becomes more attractive when there is a possibility 

to realize these experiments without isolation. Obtaining high-resolution images at 

2-3nm from cryo-electron microscopy tomograms (cryo-ET) from native cells will 

give immense information. We have therefore made a main focus in our research 

line on visualization of nanomachines in their native cellular environment and 

strive to integrate this in cancer research. 

The cellular nanocosm is made up of numerous types of macromolecular complexes 

or biological nanomachines. These form functional modules that are organized into 

complex subcellular networks. Information on the structure of these nanomachines 

has mainly been obtained by analyzing isolated structures, using imaging 

techniques such as X-ray crystallography, NMR, or single particle cryo-electron 

microscopy (cryo-EM SPA). 

Figure 4: Example of a protein 

complex analyzed by Musa Sani 

using single particle cryo-electron 

microscopy 

Yet there is a strong need to 

image biological complexes 

in a native state and within 

a cellular environment, 

in order to gain a better 

understanding of their 

functions. Emerging 

methods and instruments 

in EM are now making this 

goal reachable. Cryo-ET bypasses the need for conventional fi xatives, dehydration 

and stains, so that a close-to-native environment is retained. As this technique is 

approaching macromolecular resolution, it is possible to create maps of individual 

macromolecular complexes. Atomic structures made by X-ray and NMR can 

be ‘docked’ or fi tted into the lower resolution particle density maps to create a 

macromolecular atlas of the cell under normal and pathological conditions. The 

majority of cells, however, are too thick to be imaged in an intact state and therefore 

methods such as ‘high pressure freezing’ and ‘cryo-sectioning of unperturbed 

vitreous fully hydrated samples’ have been introduced in our laboratory for cryo-ET. 

This year we continued on improving methods for visualizing nanomachines in a 

close-to-physiological, cellular context. EM is in a renaissance and we initiated with 

a Dutch team of colleagues large infrastructure grants and created the Netherlands 

Center for Nanoscopy (NeCEN) where two FEI Titan Krios cryo-EMs (each more 

then 7 million Euro) in a new building are now commissioned for its use early 2012. 

The opening was October 12 this year. For more information see www.necen.nl. 

Cryo-ET of vitreous cryo-sections is one of the most suitable methods for exploring 

the 3D organization of biological samples that are too large to be imaged in an 

intact state. Producing good quality vitreous cryo-sections, however, is challenging. 

We focused on the major obstacles to success: contamination in and around the 

microtome, and attachment of the ribbon of sections to an electron microscopic 

grid support fi lm. We then explored vitreous cryo-section-induced compression at 

the macromolecular level using electron cryo-tomography and focused on the 80S 

ribosomes. We provided perspectives on the continued advancements in cryogenic 

sample preparation for vitreous cryo-sectioning, image collection and post-image 

processing. 

Mycobacteria Chronic infl ammation can contribute to tumorigenesis and 

metastatic progression. The latter, including macrophages and lymphocytes, are 

important elements of the tumor microenvironment. Mycobacterium tuberculosis 

Group leader Peter Peters 

Peter Peters PhD Group leader 

35 


Nicole van der Wel PhD Associate staff scientist 

Sue Godsave PhD Post-doc / EU project manager 

Pekka Kujala PhD Post-doc 

Alicia Lammerts van Buren PhD Post-doc 

Massimiliano Maletta PhD Post-doc 

Musa Sani PhD Post-doc 

Matthijn Vos PhD Post-doc 

Abdallah Abdallah PhD Post-doc 

Pavel Afanasyev MSc PhD student 

Karin de Punder MSc Technical staff 

Hans Jansen BSc Technical staff 

Maaike van Zon BSc Technical staff 

Nico Ong Research assistant 

Publications 

Abdallah AM, Bestebroer J, Savage ND, 

de Punder K, van Zon M, Wilson L, Korbee 

CJ, van der Sar AM, Ottenhoff TH, van der 

Wel NN, Bitter W, and Peters PJ. 

Mycobacterial Secretion Systems ESX-1 and 

ESX-5 Play Distinct Roles in Host Cell Death 

and Infl ammasome Activation. Journal of 

immunology (Baltimore, Md.: 1950). 

2011;187:4744-53 

Bos E, SantAnna C, Gnaegi H, Pinto RF, 

Ravelli RB, Koster AJ, de Souza W, and 

Peters PJ. A new approach to improve the 

quality of ultrathin cryo-sections; its use for 

immunogold EM and correlative electron 

cryo-tomography. Journal of structural 

biology. 2011;175 62-72 

Daleke MH, Cascioferro A, de Punder K, 

Ummels R, Abdallah AM, van der Wel N, 

Peters PJ, Luirink J, Manganelli R, Bitter W. 

Conserved Pro-Glu (PE) and Pro-Pro-Glu 

(PPE) protein domains target LipY lipases of 

pathogenic mycobacteria to the cell surface 

via the ESX-5 pathway. J Biol Chem. 

2011;286:19024-34 

De Lau W, Barker N, Low TY, Koo BK, Li 

VS, Teunissen H, Kujala P, Haegebarth A, 

Peters PJ, van de Wetering M, Stange DE, 

van Es JE, Guardavaccaro D, Schasfoort RB, 

Mohri Y, Nishimori K, Mohammed S, Heck 

AJ, Clevers H. Lgr5 homologues associate 

with Wnt receptors and mediate R-spondin 

signalling. Nature. 2011;476:293-7

36 



Kujala P, Raymond CR, Romeijn M, 

Godsave SF, van Kasteren SI, Wille H, 

Prusiner SB, Mabbott NA and Peters, PJ. 

Prion uptake in the gut: identifi cation of 

the fi rst uptake and replication sites. PLOS 

Pathog 2011;7:12 

Pierson J, Vos M, McIntosh JR, and 

Peters PJ. Perspectives on electron 

cryo-tomography of vitreous cryo-sections. 

Journal of electron microscopy (Tokyo). 

2011;60 Suppl 1;60 

Pierson J, Ziese U, Sani M, and Peters 

PJ. Exploring vitreous cryo-section-induced 

compression at the macromolecular level 

using electron cryo-tomography; 80S yeast 

ribosomes appear unaffected. Journal of 

structural biology. 2011;173:345-9 

Saurí A, Oreshkova N, Soprova Z, 

Jong WS, Sani M, Peters PJ, Luirink J, van 

Ulsen P. Autotransporter β-domains have 

a specifi c function in protein secretion 

beyond outer-membrane targeting. J Mol 


Figure 5: Model of T7SS-Induced 

Translocation, Necrosis, and IL-1β 

Secretion 

Non-pathogenic (A) and pathogenic 

mycobacteria (B) are phagocytosed. 

Non-pathogenic mycobacteria remain 

phagolysosomal and membrane bound. 

In time pathogenic mycobacteria 

translocate to the cytosol through 

ESX-1-secreted effector proteins. 

Phagosomal translocation results in 

cytosolic release of active cathepsin B 

and ESX-5 effector proteins. 

is a devastating pathogen, but controlled use of its cell wall activates macrophages 

in ways that can be harnessed for therapy. For example, M. bovis Bacille Calmette- 

Guérin (BCG) is one of the most widely used antitumor adjuvant therapies in 

humans. Injection of BCG into the bladder mediates regression of transitional cell 

carcinomas by stimulating a vigorous local immune response, bathing tumors in 

cytokines and activated immune cells. 

The discovery of cytosolic mycobacteria by our group (van der Wel, Cell 2008) 

challenged the paradigm that these pathogens exclusively localize within 

the phagosome of host cells. As yet the biological relevance of mycobacterial 

translocation to the cytosol remained unclear. In our current study we used 

electron microscopy techniques to establish a clear link between translocation and 

mycobacterial virulence. Pathogenic, patient-derived mycobacteria strains were 

found to translocate to the cytosol, while non-pathogenic phenotypes did not. We 

were then able to link cytosolic translocation with pathogenicity by introducing the 

ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal 

M. bovis BCG. Furthermore, we show that translocation is dependent on the 

11-amino acid C-terminus of the early-secreted antigen ESAT-6, a 6kDa secreted 

ESX-1 protein. Together these data demonstrated that the ability to translocate from 

the phagolysosome to the cytosol determines mycobacterial virulence. 

ESX-5 is also a type VII secretion systems (T7SS) of pathogenic mycobacteria that 

play a role in pathogenesis. We showed that a functional ESX-5 T7SS is required for 

inducing caspase-independent cell death in macrophages. Host cell death induction 

was dependent on mycobacterial internalization and translocation to the cytosol, 

but independent of TNF-α and TLR-2 signaling. Addition of cathepsin B inhibitors 

abrogated both M. tuberculosis wild-type and their ESX-5 mutants induced death. 

Our study established a role for an ESX-5 T7SS in cathepsin B-dependent cell death 

by pathogenic mycobacteria. 

Prions After oral exposure, prions are thought to enter Peyer’s patches via M cells 

and accumulate fi rst upon follicular dendritic cells (FDCs) before spreading to the 

nervous system. How prions are initially acquired from the gut lumen is not known. 

Using high-resolution cryo-immunogold electron microscopy, we reported this 

year the traffi cking of the prion protein (PrP) toward Peyer’s patches of wild-type 

and PrP-defi cient mice. PrP was transiently detectable at 1 day post feeding (dpf) 

within large multivesicular LAMP1-positive endosomes of enterocytes in the follicleassociated 

epithelium (FAE) and subsequently detected on vesicles in late endosomal 

compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP 

labelling was observed on the plasma membranes of FDCs in germinal centres of 

Peyer’s patches from wild-type mice only, identifying FDCs as the fi rst sites of PrP 

conversion and replication. PrP was detected on vesicles displaying FAE enterocytederived 

A33 protein, implying transport towards FDCs in association with FAEderived 

vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons 

within neighboring submucosal and myenteric plexi. Together, these data identifi ed 

a novel M cell-independent mechanism for prion transport to initiate conversion and 

replication upon FDCs and subsequent infection of enteric nerves. 

We continued our successful collaboration with the laboratory of Hans Clevers 

(Hubrecht Institute, Utrecht).

DIVISION OF EXPERIMENTAL THERAPY 

RADIATION-INDUCED VASCULAR DAMAGE 

Vascular damage and fi brosis can lead to serious late complications in cancer 

patients after radiotherapy. Vascular damage manifests as atherosclerosis in large 

vessels and telangiectasia (dilated, thin-walled blood vessels prone to bleeding) 

and perfusion defects in capillaries. Microvascular damage is also associated 

with infl ammation and fi brosis. In our studies we focus on the mechanisms of 

development of late, radiation-induced tissue damage, with the ultimate goal of 

designing appropriate intervention strategies to inhibit or prevent this. 

Radiation-induced microvascular damage and fibrosis development 

(collaboration with Rob Coppes, UMCG, Groningen) The mechanisms whereby 

telangiectasia develop are largely unclear. The vascular phenotype suggests that 

aberrant repair responses, including TGF-β signalling, are involved. In addition to 

vascular injury, irradiation causes excessive connective tissue formation (fi brosis). 

Fibrosis develops mainly through the actions of TGF-β and its downstream targets. 

We used mouse kidneys (a microvascular rich organ) as a model to study the 

molecular mechanisms and cellular alterations leading to radiation-induced 

telangiectasia and fi brosis. These studies identifi ed the TGF-β co-receptor endoglin 

as being critically involved in both processes. Mice expressing reduced levels of 

endoglin (Eng +/- mice) developed less radiation-induced vascular damage and less 

fi brosis. Eng +/- mice showed lowered TGF-β levels and decreased expression of 

downstream target genes related to fi brosis (Serpine1, Ctgf, and Col3a1). However, 

this was not suffi cient to explain the vascular phenotype in Eng +/- mice as vascular 

repair genes (Vegfa, Id1) were unaffected. 

Further studies showed that irradiation triggered leukocyte infi ltration, which was 

reduced in Eng +/- mice. As leukoctyes are a rich source of cytokines, chemokines 

and growth factors, we investigated whether these cells might infl uence vascular 

repair after irradiation. Immunohistochemical studies showed that the infi ltrate in 

the irradiated kidneys mainly consisted of macrophages. Comparison of the mRNA 

expression profi le in irradiated in wild type and Eng +/- kidneys showed that Eng +/- 

mice expressed reduced levels of the pro-infl ammatory/angiogenic/fi brotic cytokines 

interleukin 6 (Il6) and interleukin 1 beta (Il1β). Staining of irradiated kidneys 

confi rmed that these molecules are indeed produced by macrophages. Moreover, 

when we studied the ability of isolated leukocyte precursors from the bone marrow 

of wild type and Eng +/- mice to respond to LPS stimulation, we detected impaired 

upregulation of Il6 and Il1β in cells derived from Eng +/- mice. These results suggest 

that pro-infl ammatory cytokine production by macrophages contributes to the 

vascular and fi brotic phenotype after irradiation. Future studies will investigate how 

endoglin regulates the immune response in other irradiated tissues and whether 

modulating macrophage infi ltration and cytokine production leads to a reduction in 

the development of normal tissue damage after irradiation. 

Radiation induced cardiac damage (collaboration with Sylvia Heeneman, 

Cardiovascular Research Institute, Maastricht and Mat Daemen, Department of 

Pathology, AMC, Amsterdam) Radiation has been identifi ed as an independent 

risk factor for cardiovascular damage in long-term survivors of cancer treated with 

radiotherapy. The goal of this study is to investigate the functional and structural 

alterations of radiation-induced heart diseases, with the aim of identifying suitable 

intervention strategies. 

Single doses of 2, 8 or 16 Gy were delivered to the hearts of wild type C57BL/6J 

mice and damage was evaluated at 20, 40 and 60 weeks, relative to age-matched 

controls. Single photon emission computed tomography (SPECT/CT) and ultrasound 

were used to measure cardiac geometry and function, which was related to histo- 

37 

EXPERIMENTAL THERAPY 

Division head, group leader Fiona Stewart 

Fiona Stewart PhD Group leader 

Paul Baas MD PhD Academic staff 

Nicola Russell MD PhD Academic staff 

Fijs van Leeuwen PhD Academic staff 

Saske Hoving PhD Post-doc 

Marion Scharpfenecker PhD Post-doc 

Ingar Seemann MSc PhD student 

Karin de Cortie Technical staff 

Ben Floot Technical staff 

Johannes te Poele Technical staff 

Nils Visser Technical staff

38 


Publications 

Begg AC, Stewart FA, Vens C. 

Strategies to improve radiotherapy with 

targeted drugs. Nat Rev Cancer 

2011;11:239-53 

Hoving S, Heeneman S, Gijbels MJJ, 

te Poele JAM, Pol JFC, Gabriels K, Russell 

NS, Daemen MJ, Stewart FA. Antiinfl 

ammatory and anti-thrombotic 

intervention strategies using atorvastatin, 

clopidogrel and knock-down of CD40L do 

not modify radiation-induced 

atherosclerosis in ApoE null mice. 

Radiother Oncol 2011;101:100-8 

Scharpfenecker M, Floot B, Korlaar R, 

Russell NS, Stewart FA. ALK1 

heterozygosity delays development of late 

normal tissue damage in the irradiated 

mouse kidney. Radiother Oncol 

2011;99:349-55 

Seemann I, Gabriels K, Visser NL, 

Hoving S, te Poele JA, Pol JF, Gijbels MJ, 

Janssen BJ, van Leeuwen FW, Daemen 

MJ, Heeneman S, Stewart FA. Irradiation 

induced modest changes in murine cardiac 

function despite progressive structural 

damage to the myocardium and 

microvasculature. Radiother Oncol 2011 

Stewart FA, Hoving S, Russell NS. 

Vascular damage as an underlying 

mechanism of cardiac and cerebral toxicity 

in irradiated cancer patients. Radiat Res. 

2010; 174:865-9 

Rosenfelder N, Stewart F, Brada M. 

Vascular effects of radiation in the central 

nervous system. In: Shrieve DC, Loeffl er 

JS (eds). Human Radiation Injury. 

Philadelphia: Lippincott Williams & 

Wilkins, 2011 

Westenberg AH, van der Sangen MJC, 

Bijker N, Stenfert Kroese MC, Stewart 

FA, Rodenhuis CC, Hurkmans CW. 

Hypofractionering bij primair operable 

mammacarcinoom. Ned Tijd Oncol 

2011;8:297-301 

morphology and microvascular damage. Functional cardiac imaging demonstrated 

decreases in end diastolic and systolic volumes (EDV, ESV), while the ejection 

fraction (EF) was increased at 20 and 40 weeks after 2-16 Gy. Cardiac blood volume 

was decreased after irradiation. Histological examination revealed infl ammatory 

changes at 20 and 40 weeks after 8-16 Gy. Microvascular density in the left ventricle 

was decreased at 40 and 60 weeks after 8 and 16 Gy, with functional damage to 

remaining microvasculature manifest as decreased alkaline phosphatase, increased 

von Willebrand factor, albumin leakage from vessels and amyloidosis. A dose of 16 

Gy lead to sudden death between 30 and 40 weeks in 38% of mice. These results 

show that irradiation with 2 and 8 Gy induced modest changes in murine cardiac 

function within 20 weeks but this did not deteriorate further, despite progressive 

structural and microvascular damage. This indicates that heart function can 

compensate for signifi cant structural damage, although higher doses, eventually 

lead to sudden death. 

Cardiac damage in C57BL/6J mice is also being compared with atherosclerosis prone 

ApoE -/- mice, and Eng +/- mice (defi cient in TGF-β signaling and microvascular 

repair). Functional damage after irradiation was very similar in all three strains. 

However, decreases in microvascular density occurred earlier in Eng +/- and ApoE -/- 

mice and there was more severe loss of alkaline phosphatase expression (indicative 

of microvascular damage) than in wild type mice. In addition, hearts from ApoE -/- 

mice developed endocardial foam cell accumulation (initial stages of atherosclerosis) 

and fatty lesions in mid-sized coronary arteries at 20 weeks after irradiation. This 

would be expected to lead to more severe cardiotoxicity at later times. 

In ongoing experiments we are attempting to stimulate vascular recovery after 

cardiac irradiation, either by injection of bone-marrow derived endothelial precursor 

cells during the initial stages of vascular damage, or by feeding the irradiated mice 

thalidomide, which acts as an infl ammatory and immune modulator. 

Angiogenic ti ssue response in patients with malignant mesothelioma after 

treatment with cisplatin, pemetrexed and Axitinib (collaboration with Arjan 

Griffi oen, VUMC, Amsterdam ) Malignant pleural mesothelioma (MPM) have 

a high microvessel density and express increased levels of vascular endothelial 

growth factor receptors VEGFR1-3, and other angiogenic factors like platelet-derived 

growth factor (PDGF). Recent studies reported a negative correlation between the 

microvessel density and VEGF levels in MPM biopsies, and survival. Axitinib is a 

potent kinase inhibitor of VEGFR1-3 and PDGFR-b. A prospective, randomized, 

phase I/II trial has been initiated in the NKI (coordinator P Baas) to determine 

safety and effi cacy of the addition of Axitinib to standard chemotherapy for patients 

with previously untreated MPM. A major limitation in evaluation of ‘targeted 

agents’, is the lack of non-invasive methods to determine effi cacy in an early stage. 

We therefore initiated a feasibility study to investigate the effects of Axitinib on 

tumor vascularisation. Thoracoscopic biopsies (obtained before treatment and 

after three treatment courses) were collected and angiogenic parameters, e.g. 

microvessel density, proliferating endothelial cells and expression and activation of 

VEGFR2, were assessed. Moreover, plasma samples were taken during the treatment 

course and VEGF protein levels were determined by ELISA. Preliminary results 

demonstrate a reduction in vessel density and endothelial cell proliferation in the 

Axitinib-treated tumors. Moreover, VEGFR2 protein expression and activity and 

VEGF protein levels correlated with treatment response. We are now including more 

patients in this study and results will be correlated with clinical chemistry data and 

CT or X-ray scans.

STRATEGIES TO ENHANCE CHEMOSENSITIVITY AND 

RADIOSENSITIVITY 

Improving drug delivery by sphingolipids Anti-cancer therapy is often 

suboptimal due to inability to deliver suffi cient levels of cytostatics into tumor cells. 

The cellular plasma membrane is an effective barrier for exogenous compounds 

to accumulate in the tumor cell. We identifi ed a well-defi ned class of sphingolipid 

analogs that effectively address this barrier, catalyzing drug-membrane traversal 

preferential for tumor cell membranes. 

A liposomal co-formulation of the short-chain lipid N-octanoyl-glucosylceramide 

(GC) and doxorubicin was applied in a genetically engineered mouse (GEM) 

breast tumor model (Wcre; Cdh1 F/F ;Trp53 F/F ). These tumors, which are resistant 

to a variety of conventional and biological cytostatic therapies, responded to 

doxorubicin treatment when combined with the membrane modulation strategy. Coadministration 

of GC generated a sustained anti-tumor response and signifi cantly 

improved overall survival (30 days versus 11 days for control; p < 0.005). Using 

a nuclear isolation procedure, we showed that the presence of GC enhanced the 

intracellular tumor accumulation in vivo by a factor 1.9 (p < 0.05). In contrast, 

the accumulation within normal heart tissue was not elevated. The data are in 

agreement with in vitro experiments using cultured cardiac myocytes. When 

compared to either clinically available formulations of doxorubicin (free or PEGylated 

liposomal doxorubicin) a favorable effi cacy, pharmacokinetic and toxicity profi le was 

obtained by co-formulation of doxorubicin with GC. 

In order to elucidate the mechanism of action of the sphingolipid analogues, in 

vitro and model membrane studies and in silico molecular dynamics simulation 

experiments were designed. We demonstrated that short-chain 

sphingolipids act at the level of the plasma membrane, independently 

of lipid microdomain (raft) formation or membrane proteins. In 

artifi cial lipid membranes of well-defi ned compositions the shortchain 

sphingolipids enhanced doxorubicin-membrane traversal 

similar to cell membranes; N-octanoyl-glucosylceramide elevated the 

translocation rate of doxorubicin by a factor 1.93 (p < 0.05). Molecular 

dynamics simulations, in collaboration with Rijks Universiteit 

Groningen, revealed that the energetic barrier for the hydrophilic part 

of the doxorubicin to translocate to the opposite side of the membrane 

reduced by two-fold (p < 0.05) due to the presence of short-chain lipids. 

This phenomenon of facilitated traversal is explained by a transient 

hydrophilic gateway (hemifusion) through which amphiphilic drugs 

can traverse (see fi gure 1). 

Taken together, short-chain sphingolipids catalyze the transbilayer 

movement of amphiphilic cytostatic drugs by a transient drug-membrane gateway, 

and the short-chain lipid GC improves the therapeutic ratio in multi-drug 

resistant GEM breast tumors by enhancing the intracellular tumor accumulation 

of doxorubicin while limiting normal tissue exposure. Our data suggest that the 

therapeutic window of classical or newly developed cancer therapeutics can be 

widened by concomitant targeting of the cellular plasma membrane. Currently, 

extensive toxicology studies are carried out to prepare the translation of this concept 

in a clinical setting. 

Targeting NAD + biosynthesis to enhance radiation-induced cell death 

APO866 is a highly specifi c non-competitive inhibitor of nicotinamide 

phosphoribosyltransferase (NAMPT), a key enzyme in the regulation of NAD + 

biosynthesis. Inhibition of NAMPT reduces cellular NAD + levels. Among NAD + 

consuming enzymes are Poly(adenosine-diphosphate[ADP]-ribose)polymerases 

(PARPs) and Sirtuins. NAD + is involved in numerous biochemical processes, 

including regulation of DNA repair, replication, transcription and apoptosis. 

APO866 is thought to preferentially target tumor cells because of their high NAD + 

turnover rates and increased dependence on NAD + compared to normal cells. 

We investigated the cytotoxic effect of APO866 alone and in combination with 

radiation on tumor cells in vitro and in vivo. APO866, in nanomolar concentrations, 

induces a time- and dose-dependent depletion of cellular NAD + levels in a 

39 


Group leader Marcel Verheij 

Marcel Verheij MD PhD Group leader 

Baukelien van Triest MD PhD Academic staff 

Conchita Vens PhD Senior scientist 

Albert van Hell PhD Post-doc 

Caroline Verhagen MD PhD student 

Manon Verwijs Technical staff 

Shuraila Zerp MSc Technical staff 

Gerben Koning PhD Erasmus MC, Rotterdam 

Lília Cordeiro Pedrosa PhD student 

Erasmus MC, Rotterdam 

Fi Figure 1: Short-chain Sh t h i sphingolipids hi li id 

facilitate traversal of the widely used 

anti-cancer agent doxorubicin over cell 

membranes. Over a time period of 120 

nanoseconds, in the presence of the 

short-chain lipid (left) a transient 

hydrophilic gateway is formed by a process 

resembling hemifusion, through which the 

hydrophilic part of the doxorubicin 

translocates. In absence of the short-chain 

lipids (right), doxorubicin is not able to 

traverse the membrane over a similar 

period of time.

40 


Publications 

Begg AC, Stewart FA, Vens C. 

Strategies to improve radiotherapy with 

targeted drugs. Nat Rev Cancer 

2011;11:239-53 

Van Lummel M, Van Blitterswijk WJ, 

Vink SR, Veldman RJ, van der Valk MA, 

Schipper D, Dicheva BM, Eggermont 

AM, ten Hagen TL, Verheij M, Koning 

GA. Enriching lipid nanovesicles with 

short-chain glucosylceramide improves 

doxorubicin delivery and effi cacy in solid 

tumors. FASEB J 2011;25:280-9 

panel of human tumor cell lines (U937, J16, LNCaP, PC3 and A431). In vitro, 

APO866-mediated reduced NAD + levels resulted in enhanced cytotoxicity and, 

in combination with ionizing radiation, decreased clonogenic survival. This was 

not caused by radiation-induced enhancement of dsDNA breaks, as determined by 

γ-H2AX nuclear foci formation, nor by induction of apoptosis. In vivo experiments 

with PC3 xenografts, treated with the APO866/radiation combination, showed that 

the combination was more effective than APO866 or radiation alone. No signifi cant 

toxicity was observed after any of these treatments and tumors from animals treated 

with APO866 showed signifi cant reduced NAD + levels. Taken together, APO866 

treatment depletes NAD + and enhances tumor cell kill in combination with radiation 

in vitro and in vivo. 

Mechanisms and modulation of radiosensitivity Interfering with DNA repair 

or exploiting DNA repair defects arising spontaneously in tumors are promising 

approaches for tumor-targeted radiosensitization. PARP inhibitors are examples of 

potential radiosensitizers. Poly(ADP-ribosyl)ation processes are induced by DNA 

damage, such as caused by radiation, and are catalyzed by the PARP enzymes. 

Poly(ADP-ribosyl)ation facilitates base excision and single strand break repair (BER/ 

SSBR). As a consequence, inhibition of PARP1 and PARP2 results in chemo- and 

radiosensitization. Single agent activity has been previously demonstrated to result 

from cellular defects in homologous recombination (HR) driven repair of DNA 

double strand breaks (such as caused by BRCA mutations). We tested the potential 

of such small molecule inhibitors to sensitize to radiation and alkylating agents in 

several cell lines with genetic defects in other DNA repair processes. We found that 

PARP inhibition induced radio and chemosensitization depending on the DNA 

repair status and the function of both BER and HR. Considerable radiosensitization 

was evident at PARP inhibitor concentrations that are signifi cantly lower than those 

showing single agent activity. 

These studies support the use of radiotherapy with low dose PARP inhibitors and 

emphasize the need for development of DNA repair biomarkers for evaluation of 

agent activity and for patient selection. We have therefore initiated studies to test 

the potential of tumor and blood sample based biomarkers for DNA repair activity 

and damage response. γH2AX and RAD51 foci after ex vivo radiation are related 

to double strand break repair and a BER assay is currently being developed and 

assessed. 

Identifying DNA repair defects in tumors for chemo- and radiosensitization 

Combining the crosslinker cisplatin with radiation is the standard therapy option 

for patients with advanced squamous cell carcinoma of the head and neck (HNSCC). 

DNA crosslink repair is dependent on the activity of the Fanconi (FANC) pathway. 

Hence integrity of this repair pathway is essential to cell survival upon exposure 

to crosslinking agents such as mitomycin C (MMC). Fanconi Anemia patients, 

with defi cits in the FANC pathway, show a large increase in incidence of HNSCC, 

suggesting involvement of the FANC DNA repair pathway in the development of 

sporadic HNSCC. We therefore set out to search for somatic DNA repair defects in 

sporadic HNSCC. 

Functional integrity of the FANC pathway was fi rst evaluated in a panel of 30 

primary HNSCC tumor cell lines by testing for MMC hypersensitivity, MMC 

induced G2 cell cycle blocks and defects in the ubiquitination of the FANCD2 

protein. Functional defects in crosslink repair were found in a signifi cant proportion 

of the HNSCC cell lines. DNA sequencing analysis confi rmed that many of those 

defective cell lines carried deleterious mutations in one or more of 10 different 

FANC genes. Current efforts focus on the de-convolution and validation of the 

functional signifi cance of these mutations. 

We then attempted to confi rm the DNA repair defects observed in cell lines by 

analyzing a large set of HNSCC tumor biopsies. For this purpose a capture array 

was designed which was enriched for exons of over 300 genes involved in DNA 

repair and known to play a role in HNSCC. Two patient cohorts with defi ned tumor 

characteristics and treatment parameters have been selected and largely sequenced. 

Sequencing of additional patient material to complement the study is ongoing and 

we are approaching the completion of the mutation analyses of these cohorts.

PHARMACODYNAMICS OF ANTI CANCER DRUGS 

Multi-parameter flow cytometry method for the determination of DNA and 

phosphorylated extracellular-signal-regulated kinase (pERK) in circulating 

tumor cells (CTCs) Density gradient centrifugation of peripheral blood, followed 

by magnetic anti-epithelial cell adhesion molecule Micro Beads were used for 

the selective isolation of CTCs. Multiparameter fl ow cytometry was used for 

identifi cation and enumeration of CTCs and determination of their pERK and 

DNA content. The assay specifi city was 0.26 ± 0.29 false positive CTCs per 8 ml 

peripheral blood (n = 18 healthy volunteers in triplicate). Recovery was 75% over a 

linear range of 2 – 10000 spiked tumor cells. Within and between assay variation 

was within 12.6%. The lower limit of quantifi cation was 2 tumor cells per 8 ml 

peripheral blood. Formaldehyde-methanol fi xed samples could be stably stored for 

4 months at -80°C. The applicability of the method was demonstrated by successful 

enumeration of CTCs, and the determination of DNA, and pERK in patients with 

metastatic cancer. We showed a signifi cant upregulation of pERK in CTCs after ex 

vivo stimulation by epidermal growth factor indicating the presence of a functional 

signal transduction pathway in CTCs. 

Dihydropyrimidine dehydrogenase (DPD) and Thymidylate synthase (TS) 

enzyme activity radio assay in human peripheral blood mononuclear cells 

(PBMCs) The DPD activity assay was developed using 3 H labeled thymine as 

substrate combined with high performance liquid chromatography and on line 

radioisotope detection. HPLC is used to separate the 3 H-thymine from the enzyme 

reaction product 3 H-dihydrothymine. The decrease in the peak area of the substrate 

was used for the quantifi cation of DPD activity in PBMCs. The TS activity assay is 

based on transfer of a methyl-group from the cofactor methylenetetrahydrofolate 

onto the substrate 3 H-deoxyuridine monophosphate under release of tritium water, 

which is measured with scintillation counting after removal of the unreacted 

substrate with activated charcoal. Both assays were extensively optimized for 

sensitivity, and subsequently validated for specifi city, recovery, linearity, accuracy, 

precision, sensitivity and stability. This resulted in the fi rst assay capable of 

detecting TS activity in resting (G 0 phase of the cell cycle) PBMCs from healthy 

volunteers. 

Detection and enumeration of mature circulating endothelial cells (CEC) and 

endothelial progenitor cells (EPC) Markers expressed by CECs and EPCs are in 

development to apply as a biomarker of angiogenesis in the clinic. The endothelial 

subpopulations are defi ned as: CD31+/CD34+/CD146+/CD133- for CECs and CD31+/ 

CD34+/CD146+/CD133+ for EPCs. Magnetic CD146-conjugated beads are used 

to isolate the CEC and EPC fraction from human peripheral blood. The isolated 

fraction is stained with anti-CD45 to distinguish from hematopoietic blood cells and 

endothelial markers CD31, CD34 and progenitor marker CD133. CECs and EPCs are 

being detected and enumerated by fl ow cytometry. 

Phase 1 dose escalation study of MEK inhibitor RO4987655, administered 

orally as monotherapy in patients with advanced tumors The RAS/RAF/ 

MEK/ERK signaling pathway is dysregulated in many human cancers. RO4987655 

is a potent, highly selective ATP non-competitive MEK inhibitor. The Maximum 

Tolerable Dose (MTD) has been established at 8.5 mg bidaily doses (BID). Target 

inhibition by pERK suppression has been measured at this dose. Preliminary results 

in patients show that RO4987655 is moderately well tolerated. Skin rash is frequently 

reported as adverse effect. Transient eye toxicity (serous retinal detachment) also 

occurs. A substantial number of patients show stable disease. 

Phase 1 dose escalation study of Wee1 inhibitor MK1775 in both monotherapy 

and in combination with gemcitabine, cisplatin or carboplatin in adult 

patients with advanced solid tumors MK-1775 (MK) is an inhibitor of the Wee1 

kinase regulating the G2 checkpoint leading to chemo-sensitization in p53 defi cient 

tumor cells. Downregulation of pCDC2 upon treatment with MK and chemotherapy 

are used as biomarker. Target engagement is observed at MTD doses. Preliminary 

41 


Group leader Jan Schellens 

Jan Schellens MD PhD Group leader 

Jos Beijnen PhD Academic staff 

Serena Marchetti MD PhD Academic staff 

Irma Meijerman PhD External staff 

Lot Devriese MD MSc PhD student 

Suzanne Leijen MD PhD student 

Geert Frederix MSc PhD student 

Bart Jacobs MSc PhD student 

Didier Meulendijks MSc PhD student 

Dick Pluim Technical staff 

Artur Burylo Technical staff 

Structural collaborations: 

Group Laura Van ’t Veer PhD 

Daphne de Jong MD PhD, Department 

of Pathology 

Publications 

Deenen MJ, Cats A, Beijnen JH, 

Schellens JH. Part 1: background, 

methodology, and clinical adoption of 

pharmacogenetics. Oncologist. 2011;16: 

811-9 


Schellens JH. Part 2: pharmacogenetic 

variability in drug transport and phase I 

anticancer drug metabolism. Oncologist 

2011;16:820-34 


Schellens JH. Part 3: Pharmacogenetic 

variability in phase II anticancer drug 

metabolism. Oncologist. 2011;16:992-1005 



variability in anticancer pharmacodynamic 

drug effects. Oncologist 2011;16:1006-20 

Deenen MJ, Klümpen HJ, Richel DJ, 

Sparidans RW, Weterman MJ, Beijnen JH, 

Schellens JH, Wilmink JW. Phase I and 

pharmacokinetic study of capecitabine and 

the oral mTOR inhibitor everolimus in 

patients with advanced solid malignancies. 

Invest New Drugs 2011 

Deenen MJ, Terpstra WE, Cats A, Boot 

H, Schellens JH. Standard-dose tegafur 

combined with uracil is not safe treatment 

after severe toxicity from 5-fl uorouracil or 

capecitabine. Ann Intern Med. 

2010;153:767-8

42 



Deenen MJ, Tol J, Burylo AM, 

Doodeman VD, de Boer A, Vincent A, 

Guchelaar HJ, Smits PH, Beijnen JH, 

Punt CJ, Schellens JH, Cats A. 

Relationship between single nucleotide 

polymorphisms and haplotypes in DPYD 

and toxicity and effi cacy of capecitabine in 

advanced colorectal cancer. Clin Cancer 

Res. 2011;17:3455-68 

Devriese LA, Bosma AJ, Van de 

Heuvel MM, Heemsbergen W, Voest EE, 

Schellens JH. Circulating tumor cell 

detection in advanced non-small cell 

lung cancer patients by multi-marker 

QPCR analysis. Lung Cancer 2011 

De Vries NA, Buckle T, Zhao J, 

Beijnen JH, Schellens JH, Van Tellingen 

O. Restricted brain penetration of the 

tyrosine kinase inhibitor erlotinib due to 

the drug transporters P-gp and BCRP. 

Invest New Drugs 2010 

Devriese LA, Voest EE, Beijnen JH, 

Schellens JH. Circulating tumor cells as 

pharmacodynamic biomarker in early 

clinical oncological trials. Cancer Treat 

Rev. 2011;37:579-89 

Joerger M, deJong D, Burylo A, 

Burgers JA, Baas P, Huitema AD, Beijnen 

JH, Schellens JH. Tubuline, BRCA1, 

ERCC1, Abraxas, RAP80 mRNA 

expression, p53/p21 immunohistochemistry 

and clinical outcome in patients with 

advanced non small-cell lung cancer 

receiving fi rst-line platinum-gemcitabine 

chemotherapy. Lung Cancer 2011;74:310-7 

Molloy TJ, Devriese LA, Helgason 

HH, Bosma AJ, Hauptmann M, Voest EE, 

Schellens JH, Van’t Veer LJ. A 

multimarker QPCR-based platform for the 

detection of circulating tumour cells in 

patients with early-stage breast cancer. Br 

J Cancer. 2011 7;104:1913-9 

Leijen S, Soetekouw PM, Jeffry Evans 

TR, Nicolson M, Schellens JH, Learoyd 

M, Grinsted L, Zazulina V, Pwint T, 

Middleton M. A phase I, open-label, 

randomized crossover study to assess the 

effect of dosing of the MEK 1/2 inhibitor 

Selumetinib (AZD6244; ARRY-142866) 

in the presence and absence of food in 

patients with advanced solid tumors. 

Cancer Chemother Pharmacol. 

2011;68:1619-28 

results show that MK-1775 with chemotherapy is well tolerated and a substantial 

number of patients show stable disease or partial remission as best response. 

A Phase II, Pharmacological study with Wee1 inhibitor MK-1775 in 

combination with Carboplatin in patients with p53 mutated epithelial 

ovarian cancer that show relapse or progression after standard first line 

treatment with Carboplatin – Paclitaxel Combination Therapy MK-1775 is a 

chemosensitizer, especially for p53 defi cient tumor cells. Epithelial ovarian cancers 

often harbor p53 abnormalities. First line therapy of ovarian cancer consists of 

carboplatin or platinum containing therapy. Re-exposing patients with early relapse 

to carboplatin in combination with Wee1 inhibitor MK-1775 is an ultimate proof 

of principal study. Ca-125 marker responses have already been observed in a few 

patients. 

A Phase I/II and pharmacological study NAMI-A, a novel Ruthenium Anti- 

Cancer Agent and Gemcitabine Combination second-line therapy in Non- 

Small Cell Lung cancer patients NAMI-A, a ruthenium derivative, was well 

tolerated in a Phase I trial. As Gemcitabine is more effective in the treatment of 

NSCLC when administered in combination with a heavy metal compound, and as 

NAMI-A was reported to be effective against solid tumor metastases in various 

mouse models, the toxic and therapeutic effects of this combination are being 

studied. 450 mg/m 2 NAMI- A in combination with 1000 mg/m 2 of Gemcitabine 

given in a 3 weekly schedule, is the MTD dose and more favorable when given in a 

three weekly than in a four weekly schedule. Bone marrow toxicity often prevented 

administration of the 3 rd dose in a four weekly schedule. Pharmacokinetics were as 

observed in a previous study with NAMI-A. 

A phase I open label, dose-escalation study of the phosphoinositide 3-kinase 

inhibitor (GSK2126458) in subjects with solid tumor or lymphoma Patients 

are currently being pre-screened for PIK3CA amplifi cations/mutations and KRAS 

mutations before inclusion in this study. To reach higher daily doses without an 

increase in toxicities, the study also incorporated bi-daily dosing instead of once 

daily. Toxicities include diarrhoea, nausea, increased insulin and rash. The study 

has expanded the dose escalation part with several other arms, which include 

pharmacodynamic and exploratory cohorts. Tumor PIK3CA mutational analysis was 

implemented for patient enrichment in this trial. 

A phase I open-label, multicenter study to assess the safety, tolerability, 

pharmacokinetics and preliminary anti-tumor activity of ascending doses of 

AZD5363 under adaptable dosing schedules in patients with advanced solid 

malignancies This study started in December 2010 and so far 10 patients have been 

included. Main toxicities observed were diarrhoea and skin rash. Pharmacokinetics 

appeared to be linear over the tested dose-range. 

An open-label, Phase I/IIa, dose escalating study of 2B3-101 in patients with 

solid tumors and brain metastases or malignant glioma A glutathione (GSH) 

pegylated liposomal doxorubicin hydrochloride formulation (2B3-101) improves the 

brain penetration of doxorubicin and therefore improves overall survival of patients 

with metastases in the brain). This fi rst clinical study is currently recruiting the fi rst 

cohort of 3 patients from 35 planned. The aim is to examine the safety profi le of the 

drug, determine MTD and recommended dose for the phase II, pharmacokinetic 

profi le and preliminary tumor activity. 

Phase I Multiple Ascending Dose (MAD) study of RO5458640, a humanized 

monoclonal antibody against the TNF-Like Weak Inducer of Apoptosis 

(TWEAK) ligand, in patients with advanced solid tumours RO5458640 is a 

potentially fi rst-in-class anticancer drug targeting the Fn14 receptor ligand TWEAK. 

Preliminary results from the fi rst three dose levels show that RO5458640 is being 

well tolerated by patients. The fi rst data on pharmacokinetics are also favorable, as 

exposure increases linearly with increasing dose.

MOLECULAR AND GENETIC BREAST CANCER EPIDEMIOLOGY 

This research line spans two divisions, the Division of Experimental Therapy and 

the Division of Psychosocial Research and Epidemiology; group members are 

divided over these divisions depending on their focus of work. 

Genetic determinants of breast cancer risk and breast cancer outcome A 

large part of genetic breast cancer susceptibility and prognosis is as yet unexplained. 

We are continuously validating newly identifi ed single nucleotide polymorphisms 

(SNPs) within the Breast Cancer Association Consortium (BCAC; >50,000 

breast cancers; PI D Easton, Cambridge). This year we identifi ed and validated 14 

more variants, totaling 27 breast cancer susceptibility SNPs, some of these being 

specifi cally associated with risk for certain breast cancer subtypes defi ned by 

immunohistochemical markers (ER, PR, HER2, CK5/6, EGFR). It is becoming clear 

that non-genetic risk factors are also associated with specifi c breast cancer subtypes. 

The BCAC established that reproductive factors and BMI are most clearly related 

to hormone receptor positive tumors and suggest that distinctive associations for 

core basal phenotype may underlie much of this heterogeneity. Future studies in 

BCAC will investigate this in more detail, e.g. the MINDACT lifestyle study. In 

this study, we started to collect risk factor questionnaires and screening data for 

the Dutch MINDACT patients (collaboration Laura van ‘t Veer and Emiel Rutgers). 

The identifi cation of SNPs for prognosis prediction has shown to be a challenge as 

effects may be small and/or may only be important in combination with somatic 

events in the same pathway. As a proof of principal, we showed that two SNPs, P53 

R72P and MDM2 SNP309, playing an important role in the p53 response pathway, 

were associated with worse outcome of breast cancer within subgroups of tumors 

displaying a ‘‘more aggressive phenotype’’ gene expression profi le. 

In collaboration with the Epidemiology Groups at the PSOE Division and clinical 

groups, we are focusing on prognosis and long-term outcome of BRCA1/2 

carriers among young (

44 



Mook S, Van ‘t Veer LJ, Rutgers EJ, 

Ravdin PM, van de Velde AO, van 

Leeuwen FE, Visser O, Schmidt MK. 

Independent prognostic value of screen 

detection in invasive breast cancer. J Natl 

Cancer Inst. 2011;103:585-97 

Lin WY, et al. A role for XRCC2 gene 

polymorphisms in breast cancer risk and 

survival. J Med Genet. 2011;48:477-84 

Yang XR, et al. Associations of breast 

cancer risk factors with tumor subtypes: a 

pooled analysis from the Breast Cancer 

Association Consortium studies. J Natl 

Cancer Inst. 2011;103:250-63 

Stevens KN, et al. Evaluation of 

variation in the phosphoinositide-3-kinase 

catalytic subunit alpha oncogene and 

breast cancer risk. Hum Mutat. 2011 

Figueroa JD, et al. Associations of 

common variants at 1p11.2 and 14q24.1 

(RAD51L1) with breast cancer risk and 

heterogeneity by tumor subtype: fi ndings 

from the Breast Cancer Association 

Consortium. Hum Mol Genet. 

2011;20:4693-06 

Stevens KN, et al. Common breast 

cancer susceptibility loci are associated 

with triple-negative breast cancer. Cancer 


Milne RL, et al. Confi rmation of 5p12 

as a susceptibility locus for progesteronereceptor-positive, 

lower grade breast cancer. 

Cancer Epidemiol Biomarkers Prev. 

2011;20:2222-31 

Haiman CA, et al. A common variant 

at the TERT-CLPTM1L locus is associated 

with estrogen receptor-negative breast 

cancer. Nat Genet. 2011;43:1210-4 

declare that they do not agree with the use of their tissue on the basis of information 

which in most hospitals is available in a general information leafl et. At present, the 

extent of information about research with residual tissue in the hospital leafl ets 

differs widely in Dutch hospitals. Our earlier research, in limited cancer patient 

groups, showed that patients are unaware of residual tissue storage for their own 

clinical benefi t and possible use in research, and are unsatisfi ed with the current 

information procedure (i.e. opt-out), though only a minority wished to refuse 

research. Many patients wish to complement the current opt-out procedure with 

short verbal information by a member of hospital staff. We have recently started 

a new project in which we will evaluate three consent procedures in larger groups 

of cancer and non-cancer patients. In a randomized trial, three different consent 

procedures will be offered to 1800 patients in four different hospitals in order to 

determine patients’ preference for and feasibility of these procedures.

MOLECULAR MARKERS TO PREDICT CHEMOSENSITIVITY IN 

BREAST CANCER 

Development of clinically useful and feasible tests using molecular 

markers to predict the response of primary breast cancers to specific 

chemotherapeutic agents or combinations Preoperative chemotherapy for locally 

advanced breast cancer may lead to excellent responses in some tumors and to no or 

partial response at all in others. Therefore there is a strong need for tests that can 

predict chemotherapy response. In the CTMM BreastCare project we aim to develop 

such tests based on molecular markers. 

Our group has previously employed array Comparative Genomic Hybridization 

(aCGH) to assess the genomic patterns of BRCA1-mutated breast cancers. It is 

reasonable to assume that this pattern indicates ‘BRCAness’ and thus serves as a 

marker for homologous recombination defi ciency. We have shown that this so-called 

BRCA1-likeaCGH profi le is also present in about half of all triple negative sporadic 

breast cancers and is predictive for benefi t from intensive chemotherapy with 

DNA cross-linking agents. As aCGH is a rather complex method, we translated the 

BRCA1aCGH profi le to a Multiplex Ligation-dependent Probe Amplifi cation (MLPA) 

assay, to identify both BRCA1-mutated breast cancers and sporadic cases with a 

BRCA1-likeaCGH profi le. The assay performed equally well in identifying BRCA1 

mutated as BRCA1-like patients. Since the MLPA assay y can identify y BRCA1-mutated 

and BRCA1-like sporadic breast cancer patients, this method could be both applied 

in clinical genetic testing, but also as a predictor of treatment benefi t. BRCA1-like 

tumors are highly sensitive e to chemotherapy with DNA damaging agents, and also 

to PARP-inhibitors. The MLPA assay is rapid and robust, can easily be multiplexed, 

and works well with DNA derived derived from paraffi n-embedded tissues. Therefore, Therefore, 

we now have an optimal tool ool for studying BRCA1-like status in clinical samples 

(see fi gure 2). 

So far, we employed the 

MLPA BRCA1-like assay 

in two projects. First, 

we studied BRCA1-like 

tumors and conventional 

dose chemotherapy 

sensitivity in more detail. 

We compared clinical 

factors and survival rates 

in a uniform cohort of 

triple negative breast tumors ors treated with alkylating agents. BRCA1-like tumors 

occurred in younger patients nts and were more often node negative, negative which are features 

shared with tumors in BRCA1-mutation carriers. We did not observe a difference 

in survival between BRCA1-like and non-BRCA1-like triple negative breast cancers 

after treatment with conventional dose chemotherapy with alkylating agents. 

These results confi rm our previous fi ndings that BRCA1-like tumors have similar 

sensitivity to anthracycline-based adjuvant chemotherapy as other triple-negative 

tumors. In the future, it will be important to establish whether BRCA1-like tumors 

also share the exquisite sensitivity of BRCA-mutated tumors to PARP-inhibitors. 

Second, we are prospectively validating the BRCA1-like status and chemosensitivity 

in an ongoing clinical trial. Patients with triple negative breast cancer having a 

BRCA1-like MLPA profi le are randomized between high dose chemotherapy with 

alkylating agents and conventional dose chemotherapy. 18 Patients have now been 

included in the trial. 

Finally, ongoing work is the collection of tumor biopsies of all patients neoadjuvantly 

treated at the Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital. 

Every year we obtain biopsies from around 130 patients, which can be triple negative, 

ER+ and HER2+. For these last two groups we are also interested in the concept 

of BRCAness. This work is now ongoing. Besides our BRCAness projects, we are 

planning to perform other techniques, such as sequencing, to fi nd other predictive 

markers for chemotherapy sensitivity. 

45 


Group leader Jelle Wesseling 

Jelle Wesseling MD PhD Group leader 

Jos Jonkers PhD Academic staff 

Petra Nederlof PhD Academic staff 

Sjoerd Rodenhuis MD PhD Academic staff 

Lodewyk Wessels PhD Academic staff 

Esther Lips PhD Post-doc 

Petra ter Brugge PhD Post-doc 

Jorma de Ronde PhD student 

Petra Somer-Kristel Technical staff 

Lennart Mulder Technical staff 

Eline van der Burg Technical staff 

Figure 2. Translation of the aCGH classifi er 

into a MLPA assay. 

The most important genomic regions of the 

original aCGH based classifi er (3q22-27, 

5q12-14, 6p23-22, 12p13, 12q21-23, 13q31-34) 

(A) were mapped to a set of 34 MLPA probes 

(B).

46 


Publications 

Lips EH, Mulder L, Hannemann J, 

et al. Indicators of homologous 

recombination defi ciency in breast cancer 

and association with response to 

neoadjuvant chemotherapy. Ann Oncol. 

2011;22:870-6 

Lips EH, Laddach N, Savola SP, et al. 

Quantitative copy number analysis by 

Multiplex Ligation-dependent Probe 

Amplifi cation (MLPA) of BRCA1associated 

breast cancer regions identifi es 

BRCAness. Breast Cancer Res. 

2011;13:R107 

Lips EH, Mulder L, De Ronde JJ, et al. 

Neoadjuvant chemotherapy in ER+ 

HER2- breast cancer: response prediction 

based on immunohistochemical and 

molecular characteristics. Breast Cancer 

Res Treat. 2011 

Vollebergh MA, Lips EH, Nederlof 

PM, et al. An aCGH classifi er derived 

from BRCA1-mutated breast cancer 

and benefi t of high-dose platinum-based 

chemotherapy in HER2-negative 

breast cancer patients. Ann Oncol. 

2011;22:1561-70 

Figure 3: Expression of BRCA1 in a TNBC 

xenograft model showing BRCA1 promoter 

hypermethylation (T127). A, Fusion point 

of the BRCA1- STAT3 fusion transcript 

found in a cisplatin resistant T127 tumor 

(T127 cis1) B, Fusion point of the BRCA1- 

ACACA fusion transcript found in a 

nimustine resistant T127 tumor (T127 

nim2). C, Expression of BRCA1 protein is 

found in T127 cis1, but not in untreated 

control tumors (ctrl). 

Response and resistance in BRCA1 deficient TNBC xenograft models to 

alkylating agents and PARP inhibition Triple negative breast cancer (negative 

for ER, PR, and HER2; TNBC) tends to rapidly develop resistance to conventional 

therapy and targeted therapy is not available. In the development of new therapies, 

the availability of in vivo models is essential. Xenograft models, in which human 

tumor tissue is engrafted directly into recipient mice, may be very useful for this 

purpose (Marangoni et al, Clin Can Res 2007;13:3989). 

We have set up a panel of xenografts for human TNBC by implanting fresh breast 

cancer fragments in immunocompromized mice. After outgrowth, tumors are 

propagated by serially transplantation and are characterized based on histology, 

IHC, gene expression profi le, aCGH, Brca1 promoter methylation, mRNA and 

protein. 

We have identifi ed several TNBC xenografts defi cient for BRCA1, through mutations 

in the gene leading to a truncated, non-functional protein, or through epigenetic 

mechanisms, leading to downregulation of BRCA1 expression. Since BRCA1 plays 

an important role in the repair of double strand breaks, BRCA1 defi cient tumors are 

thought to be especially sensitive to alkylating agents that induce DNA crosslinks 

and subsequent DNA breaks (Silver et al, J Clin Oncol 2010;28:1145, Byrski et al, J Clin 

Oncol 2010;28:375). 

We have tested the response and development of resistance in BRCA1 defi cient 

TNBC xenografts. For this, we used 2 models that lack BRCA1 expression due to 

BRCA1 promoter hypermethylation and also 1 model that contains a BRCA1 gene 

mutation (c.2210delC) and consequently expresses a truncated, non-functional 

protein. We tested the alkylating agents cisplatin, nimustine and melphalan and 

the PARP inhibitor olaparib. We started therapeutic intervention studies on the 

xenografts when tumors reached a size of 200mm 3 . Treatment was discontinued 

when tumors reached a size of 50% or less of starting size. In case of relapse 

treatment was started again after relapse to starting size. 

BRCA1 defi cient models responded very well to treatment with alkylating agents, 

with complete disappearance of palpable lesion in most cases after 21 days of 

treatment. However, some tumors did relapse and had to be treated again. After 

relapse, most tumors remain sensitive to cisplatin, but resistance to cisplatin did 

occur. Tumors that relapsed after melphalan, nimustine or olaparib treatment 

quickly developed resistance. 

We started investigating resistance mechanisms in these BRCA1 defi cient 

xenograft models. We found that development of resistance in these models is often 

accompanied by expression of BRCA1, whereas untreated tumors do not express any 

BRCA1. In the models characterized by lack of BRCA1 expression due to promoter 

hypermethylation, resistant tumors that express BRCA1 often show demethylation 

of the BRCA1 promoter. In 2 resistant tumors, hypermethylation of the promoter 

was still present, but fusion transcripts were found with the BRCA1 mRNA preceded 

by one or several exons of an unrelated gene (fi gure 3A, B). This fusion transcript 

results in a full length BRCA1 protein as seen on western blot (fi gure 3C). 

In the BRCA1 mutated model, resistance in some of the tumors is also accompanied 

by expression of BRCA1, while untreated tumors do not express BRCA1. In these 

tumors, sequencing showed the presence of additional small deletions (5-23 bp) 

around the site of the original 1bp deletion. These deletions restored the BRCA1 

reading frame, allowing expression of a nearly full length protein. This mechanism 

is similar to that found in BRCA mutated tumors that develop therapy resistance in 

patients (Norquist et.al. JCO 2011;29:3008). 

In conclusion, TNBC xenograft models are very suitable to study treatment response 

and resistance. The models show that BRCA1 defi cient tumors are sensitive to 

alkylating agents and PARP inhibition. Re-expression of BRCA1 due to promoter 

demethylation or genetic rearrangements is found as a mechanism of resistance to 

cisplatin.

TOWARDS A MOLECULAR UNDERSTANDING OF BREAST 

CANCER AND INDIVIDUALIZED ENDOCRINE TREATMENT 

Luminal breast tumors represent 75% of all breast cancer cases, and most breast 

cancer-related deaths are still observed in this hormone receptor positive subgroup. 

Since different subgroups of patients respond to different modalities of endocrine 

treatment, an individualized treatment selection would have a signifi cant clinical 

benefi t. 

Our lab is focused on identifying predictive criteria for ‘tailored endocrine 

treatment’ in breast cancer, and pursues a swift transfer of our fi ndings into clinical 

practice. We aim to not only elucidate a molecular mechanism that underlies 

endocrine resistance, but also to provide a rationale for endocrine treatment 

selection in an individualized manner. Our research design involves studies on cell 

lines as well as primary tumor tissue, enabling us to achieve mechanistic insights 

into endocrine responsiveness in a physiologically and clinically relevant context. 

Ubiquitin ligase siRNA screen for Faslodex-resistance This project is being 

performed in collaboration with the research groups of Roderick Beijersbergen and 

Jacques Neefjes. Faslodex is a synthetic Estrogen Receptor (ERα) ligand that is used 

for the treatment of metastatic breast cancer and induces ERα degradation through 

a yet unknown mechanism. We have performed an siRNA-based knocked down 

screen and identifi ed 15 hits were as essential for Faslodex-induced ERα degradation. 

These hits will be deconvoluted, verifi ed in cell biological assays, and tested as 

potential biomarkers. 

PKA phosphorylation redirects ERα to promoters of a unique gene 

set associated to tamoxifen resistance Protein Kinase A-induced ERα 

phosphorylation at serine residue 305 can induce tamoxifen resistance and cell 

growth in breast cancer. How this phospho-modifi cation causes tamoxifen resistance 

is unclear. We identifi ed the molecular complex that is required for PKA-induced 

ERα phosphorylation, and found AKAP13 as an essential protein to bring the ERα 

and PKA in the same molecular complex for the receptor to be phosphorylated. In 

addition, we found that a Serine 

305 phospho-modifi cation of the 

ERα (ERαS305-P) redirects the 

receptor to new transcriptional 

start sites affecting the 

transcriptome (fi gure 4). 

This translates into a 26gene 

expression classifi er that 

identifi es breast cancer patients 

with a poor disease outcome 

after tamoxifen treatment, 

of which we could identify the 

underlying molecular pathway 

involved. These genes are 

specifi cally upregulated in 

ERαS305-P cells exposed to 

tamoxifen to induce cell growth. 

ERα–DNA binding landscape in primary human breast tumor samples Most 

research on breast cancer involves artifi cial cell line models. In collaboration with 

the research groups of Jason Carroll (Cancer Research UK, Cambridge) and Sabine 

Linn, we have developed protocols to assess the genome-wide chromatin interactions 

of the Estrogen Receptor from core needle biopsy breast cancer samples (fi gure 5). 

We are currently applying these protocols on breast cancer samples from the AFTER 

study (Anastrozole +/- Fulvestrant or Tamoxifen Exposure - Response in Molecular 

Profi le; NCT00738777). The chromatin binding patterns of the Estrogen Receptor 

will be coupled to gene expression data and immunohistochemistry, in order to 

understand this novel layer of Estrogen Receptor regulation in breast cancer and to 

correlate this pattern with treatment success. 

Publications 

47 


Junior group leader Wilbert Zwart 

Wilbert Zwart PhD Junior group leader 

Xanthippi Alexi PhD Post-doc 

Renée de Leeuw MSc PhD student 

Marjolein Droog MSc PhD student 

Cristiane Bentin Toaldo BSc Technical staff 

Zwart W, Theodorou V, Kok M, Canisius 

S, Linn S, Carroll JS. Estrogen Receptorcofactor-chromatin 

specifi city in the transcriptional 

regulation of breast cancer EMBO J 2011 

Krijgsman O, Roepman P, Zwart W, 

Carroll JS, Tian S, de Snoo FA, Bender RA, 

Bernards R, Glas AM. A diagnostic gene 

profi le for molecular subtyping of breast 

cancer associated with treatment response. 

Breast Cancer Res Treat. 2011 

Zwart W, Theodorou V, Carroll JS. 

Estrogen receptor-positive breast cancer: 

a multidisciplinary challenge. Wiley 

Interdiscip Rev Syst Biol Med. 2011;3:216-30 

Figure 4: (left) Reprogramming of ERα binding 

patterns by PKA. A) Snapshot of ERα/ 

chromatin interactions as analyzed by ChIP-seq. 

B) Venn diagram of ERα binding events after 

Estradiol stimulation (green) versus Serine 305 

phosphorylated ERα after activation of PKA 

(blue). C) the geneset with proximal Serine 305 

phosphorylated ERα’s can be used as a classifi er 

to identify breast cancer patients with a poor 

outcome after tamoxifen treatment. 

Figure 5: (below) ERα ChIP-seq from core 

needle biopsies of breast tumor. 

A) Examples of ERα binding events in a 

cell line and two biopsy samples. 

B) endocrine treatment changes binding.

48 

GENE REGULATION 

Division head, group leader Reuven Agami 

Reuven Agami PhD Group leader 

Rani Elkon PhD Post-doc 

Nicolas Léveillé PhD Post-doc 

Gijs van Haaften PhD Post-doc 

Mathias Jenal PhD Post-doc 

Shih-Cheng Chen PhD Post-doc 

Fabrizio Loayza PhD Post-doc 

Hayley Moore PhD Post-doc 

Jarno Drost MSc PhD student 

Marieke van Kouwenhove MSc PhD student 

Arnold Bos MSc PhD student 

Carlos Melo MSc PhD student 

Mariette Schrier PhD Technical staff 

Joachim Oude Vrielink Technical staff 

DIVISION OF GENE REGULATION 

MICRORNAS AND RNA BINDING PROTEINS IN CANCER 

Our main research objective is to understand the cancerous process in humans and 

identify essential novel cancerous genes. The knowledge we gather will allow us to 

design novel therapeutic approaches. 

Most human tumors harbor multiple genetic alterations that activate oncogenes, 

inhibit tumor suppressors and induce genomic instability. As each tumor contains 

many genetic alterations, the study of the contribution of each alteration to the 

cancerous phenotype was obscured. In the past, we developed and successfully 

used an RNA interference (RNAi) approach to inactivate genes in mammalian cells. 

Recently, we used this RNAi system to characterize the tumor suppressor activity of 

the bromodomain containing protein 7 (BRD7) (Drost J et al., 2010; Mantovani F et 

al., 2010). 

We also initiated studies to identify cancerous microRNAs (miRNAs), an emerging 

gene family encoding for endogenous small RNAs. We developed novel and unique 

genetic tools to screen for cancer-causing and cancer-preventing miRNAs, and 

discovered and characterized the roles of the miR-372, miR-221, miR-135 and miR- 

192/194 families in tumor growth and metastasis. We continue working along this 

line of research. 

Interestingly, we noticed that the regions surrounding many functional miRNA 

targets could be highly conserved throughout evolution. We hypothesized that these 

regions recruit RNA-binding proteins (RBPs) that regulate miRNA function. Using 

functional genetic screens we identifi ed RBPs that control the activity of miRNAs. 

We propose that the genetic interaction between miRNAs and RBPs determines 

developmental processes, cellular proliferation, and cancer progression. 

Functional genetic approaches identify novel tumor suppressor genes 

Oncogene-induced senescence is a p53-dependent defense mechanism against 

uncontrolled proliferation. Consequently, many human tumors harbor p53 

mutations and others show a dysfunctional p53 pathway, frequently by unknown 

mechanisms. Using functional genetic screens with RNAi libraries we identifi ed 

BRD7 (bromodomain-containing 7) as a protein whose inhibition allows full 

neoplastic transformation in the presence of wild-type p53 (fi gure 1A). In human 

breast tumors harboring wild-type, but not mutant, p53, the BRD7 gene locus was 

frequently deleted and low BRD7 expression was found in a subgroup of tumors. 

Functionally, BRD7 is required for effi cient p53-mediated transcription of a subset 

of target genes. BRD7 interacts with p53 and p300 and is recruited to target gene 

promoters, affecting histone acetylation, p53 acetylation and promoter activity. 

Thus, BRD7 suppresses tumorigenicity by serving as a p53 cofactor required for the 

effi cient induction of p53-dependent oncogene-induced senescence. 

Selective inhibition of miRNA accessibility by RBM38 is required for p53 

activity MicroRNAs (miRNAs) interact with 3’-untranslated regions (3’UTRs) of 

messenger RNAs (mRNAs) to restrict expression of most protein coding genes 

during normal development and cancer. RNA-binding proteins (RBPs) can 

control the biogenesis, stability, and activity of miRNAs. We identifi ed RBM38, a 

ubiquitously expressed RNA binding protein) in a genetic screen for RBPs whose 

expression control miRNA access to target mRNAs. RBM38 is induced by p53 and 

its activity to modulate miRNA-mediated repression is required for proper function 

of p53. In large cohorts of human breast cancer, reduced RBM38 expression by 

promoter hypermethylation strongly correlated with wild-type p53 status. Thus, 

our results indicate a novel layer of gene regulation by p53, which is required for its 

tumor suppressive function.

3’UTR-mediated gene silencing of the Mixed Lineage Leukemia (MLL) gene 

Translocations involving the Mixed Lineage Leukemia (MLL) gene generate in-frame 

fusions of MLL with more than 50 different partner genes (PGs). Common to all 

MLL translocations is the exchange not only of coding regions, but also of MLL and 

PG 3´-untranslated regions (3ÚTRs). As a result, the MLL-PG fusion is normally 

highly expressed and considered the main driver of leukemia development, whereas 

the function of the PG-MLL fusions in leukemic disease is unclear. As 3ÚTRs 

have been recognized as determinant regions for regulation of gene expression, 

we hypothesized that loss of the MLL 3ÚTR could have a role in generating high 

MLL-PG levels and leukemia development. Indeed, our fi ndings indicate that MLL 

translocations relieve oncogenic MLL-PG fusions from the repressive MLL-3ÚTR, 

contributing to higher activity of these genes and leukaemia development (Gomez- 

Benito et al., 2011). 

Figure 1: A. BRD7 interacts with p53 and acetylated histones, and is required for effi cient p53 

transcriptional activity. B. Interplay between RNA-Binding proteins (RBP) and miRNA activity on 

target mRNA. C. A schematic representation of MLL translocations with a partner gene (PG) at the 

mRNA level. Domains of MLL are marked. 

Publications 

49 


Caporali A , Meloni M, Vollenkle C, 

Bonci D, Sala-Newby GB, Addis R, 

Spinetti G, Losa S, Masson R, Baker AH, 

et al. Deregulation of microRNA-503 

contributes to diabetes mellitus-induced 

impairment of endothelial function and 

reparative angiogenesis after limb 

ischemia. Circulation 2011;123:282-291 

Denby L, R amdas V, McBride MW, 

Wang J, Robinson H, McClure J, 

Crawford W, Lu R, Hillyard DZ, Khanin R, 

et al. miR-21 and miR-214 are consistently 

modulated during renal injury in rodent 

models. The American journal of 

pathology 2011;179:661-672 

Gomez-Beni to M, Loayza-Puch F, 

Oude Vrielink JA, Odero MD, and Agami 

R. Correction: 3’UTR-Mediated Gene 

Silencing of the Mixed Lineage Leukemia 

(MLL) Gene. PLoS One 2011;6 

van Kouwen hove M, Kedde M and 

Agami R. MicroRNA regulation by 

RNA-binding proteins and its implications 

for cancer. Nat Rev Cancer 2011;11:644- 

656 

Leveille N , Elkon R, Davalos V, 

Manoharan V, Hollingworth D, Oude 

Vrielink J, le Sage C, Melo CA, Horlings 

HM, Wesseling J, et al. Selective 

inhibition of microRNA accessibility by 

RBM38 is required for p53 activity. Nat 

Commun 2011;2:513 

Lerner M, Lundgren J, Akhoondi S, 

Jahn A, Ng HF, Moqadam FA, Oude 

Vrielink JA, Agami R, Den Boer ML, 

Grander D et al. MiRNA-27a controls 

FBW7/hCDC4-dependent cyclin E 

degradation and cell cycle progression. Cell 

Cycle 2011;10:2172-2183

50 


Group leader Bas van Steensel 

Bas van Steensel PhD Group leader 

Mario Amendola PhD Post-doc 

Carolyn de Graaf PhD Post-doc 

Guillaume Filion PhD Post-doc 

Jop Kind PhD Senior Post-doc 

Alexey Pindyurin PhD Post-doc 

Joris van Arensbergen PhD Post-doc 

Ulrich Braunschweig PhD Post-doc 

Dominika Bijosˇ MSc PhD student 

Wouter Meuleman MSc PhD student 

Daniel Peric-Hupkes MSc PhD student 

Joke van Bemmel MSc PhD student 

Ludo Pagie PhD Bioinformatician 

Wendy Talhout BAS Technical staff 

Arantxa Rosado BSc Technical staff 

CHROMATIN GENOMICS 

Chromatin is probably the most complex molecular ensemble in the cell. It 

consists of genomic DNA together with all directly or indirectly associated protein 

and RNA molecules. This includes histones, DNA-binding factors (DBFs), the 

basal transcription machinery and its nascent transcripts, replication and repair 

machineries that copy and maintain DNA, and many other molecules that interact 

with any of these components. All of these components work in concert, and 

cannot be fully understood unless they are studied in their complete context. In 

addition, the spatial organization of interphase chromosomes is thought to be of key 

importance for genome expression and maintenance. Yet, this three-dimensional 

chromosome organization and its impact on gene regulation and other functions are 

still poorly understood. 

In order to gain insight into these fundamental processes, we take a broad 

integrative genomics approach, using both fruit fl y and mammalian cells as model 

systems. We conduct our studies in the living cell, in the context of the entire 

genome. We develop and apply new genomics techniques to reveal the interplay 

among many chromatin proteins, and to visualize the architecture of chromosomes 

inside the nucleus. 

Chromatin Protein Discovery Project In 2008 we started the Chromatin 

Protein Discovery Project, which aims to identify and functionally characterize 

hitherto unknown chromatin proteins in Drosophila. We selected more than 100 

candidate proteins by computational predictions that take into account protein 

domain structure, interactions with known chromatin proteins, and likelihood of 

nuclear localization. For each of these we generated full-genome, high-resolution 

DamID maps, which reveal whether each protein interacts with specifi c parts of the 

genome. This yielded informative binding maps of 42 novel proteins. By extensive 

computational analysis of these binding maps we predicted the functions and 

molecular interactions for each protein. Several of these predictions are further 

evaluated by wet-lab experiments. Thus, this project broadens our view of chromatin 

by identifying and annotating dozens of novel components. 

The network of interactions in chromatin Our previous work has led to the 

insight that chromatin in Drosophila consists of fi ve principal types, which are 

defi ned by distinct combinations of proteins and histone marks. These chromatin 

types are distributed along the genome in domains that can be >100kb long. We 

identifi ed a repressive chromatin type that covers about half of the genome and lacks 

classic heterochromatin markers. Furthermore, transcriptionally active euchromatin 

consists of two types that differ in molecular organization and regulate distinct 

classes of genes (Filion, van Bemmel, Braunschweig, et al. Cell 2010;143:212-224). 

What are the mechanisms that drive the formation of these different chromatin 

types at distinct locations along the genome? And how does each chromatin type 

contribute to the regulation of genes? In order to elucidate these mechanisms, we 

are developing several new strategies. First, we use computational methods such as 

Bayesian Network Inference to construct models of the interaction network, based 

on genome-wide DamID maps of more than 100 chromatin proteins. Second, we are 

developing a new parallel reporter gene assay which enables us to mechanistically 

dissect the impact of specifi c chromatin environments on gene expression for 

thousands of locations in the genome. Third, we are studying the effects on genome 

rearrangements such as chromosome inversions on the domain organization of 

chromatin. Fourth, we have begun to explore the role of long non-coding RNAs in 

specifi c chromatin types. Together, these strategies will provide insight into the 

fundamental principles of the molecular organization of chromatin. 

Spatial organization of Polycomb chromatin domains The local composition 

of chromatin may be tightly linked to the spatial folding of chromosomes. In 

order to study this, we closely collaborated with Dr. Bas Tolhuis and in the lab of 

Prof. Dr. Maarten van Lohuizen (Division of Molecular Genetics). We adapted the

Chromosome Conformation Capture on Chip (4C) assay to systematically map 

chromosomal interactions in Drosophila melanogaster larval brain tissue. We 

focused on genomic loci bound by the repressive Polycomb Group (PcG) proteins. 

Our results demonstrate that PcG target genes interact extensively and highly 

specifi cally with each other in nuclear space. Notably, interactions are mostly limited 

to genes on the same chromosome arm, and by studying a chromosome with a large 

pericentric inversion we demonstrate that a topological rather than a sequencebased 

mechanism is responsible for this constraint. These results reveal that many 

interactions among PcG target genes exist and that these interactions are guided by 

overall chromosome architecture. 

Mechanisms and dynamics of genome – nuclear lamina interactions The 

nuclear lamina (NL) is a fi brous protein sheet at the nucleoplasmic surface of the 

nuclear envelope. By DamID mapping, we previously found that the genome of 

mammalian cells is associated with the NL through ~ 1,300 Lamina-Associated 

Domains (LADs). These LADs tend to be large (median size 0.5Mb) and harbor 

thousands of genes that are transcriptionally repressed (Guelen et al 2008, Nature 

453:948-951). We hypothesize that the NL provides an anchoring scaffold that helps 

to organize the genome in nuclear space. 

In collaboration with Dr. Lodewyk Wessels (Division of Molecular Biology) and Prof. 

Dr. Marcel Reinders (Delft University of Technology) we have analyzed the sequence 

properties and evolutionary conservation of LADs. We found that the positions of 

LADs along the genome are extremely conserved between mouse and human, in 

particular those LADs that are cell-type invariant. This high degree of conservation 

suggests that LADs are essential for genome function. Sequence analysis suggests 

that simple AT-richness may be a major determinant of LADs. We are currently 

testing this hypothesis using in vivo and in vitro assays. 

Finally, we have developed a novel method to visualize and track LADs in living 

cells. By expressing a Dam-Lamin fusion protein in human cells, genomic DNA that 

contacts the NL becomes adenine-methylated. Co-expression of a methyl-adenine 

binding protein fused to GFP then results into fl uorescent tagging of these genomic 

regions. Because adenine-methylation is stable, this strategy enables us to follow 

the fate of these regions over time. So far, this has revealed that during most of 

interphase, LADs can move locally, but they remain close to the nuclear periphery. 

We are currently tracking the fate of LADs throughout the cell cycle. This new 

technology allows us to directly visualize the dynamics of genome organization in 

living cells. 

Publications 

51 


van Steensel B. Chromatin: 

constructing the big picture. EMBO J. 

2011;30:1885-95 

Tolhuis B, Blom M, Kerkhoven RM, 

Pagie L, Teunissen H, Nieuwland M, 

Simonis M, de Laat W, van Lohuizen M, 

van Steensel B. Interactions among 

Polycomb domains are guided by 

chromosome architecture. PLoS Genet. 

2011;7:e1001343 

Peric-Hupkes D, van Steensel B. Role 

of the nuclear lamina in genome 

organization and gene expression. Cold 

Spring Harb Symp Quant Biol. 

2010;75:517-24 

Figure 2. Single-cell visualization of DNA 

contacting the nuclear lamina. By 

expression of a Dam-Lamin fusion protein, 

all DNA that interacts with the lamina is 

stably marked by adenine-methylation. 

This is then visualized by co-expression of 

a methyl-A-binding protein fused to GFP. 

Left panel: GFP signal. Right panel, same 

with the position of the lamina indicated 

by the dotted line (based on staining of 

Lamin B1, not shown).

52 


Group leader Fred van Leeuwen 

Fred van Leeuwen PhD Group leader 

Iris Stulemeijer PhD Post-doc 

Kitty Verzijlbergen MSc PhD student 

Marit Terweij MSc PhD student 

Hanneke Vlaming MSc PhD student 

Tibor van Welsem Technical staff 

Publications 

Verzijlbergen KF, van Welsem T, Sie D, 

Lenstra TL, Turner DJ, Holstege FCP, 

Kerkhoven RM, van Leeuwen F. A barcode 

screen for epigenetic regulators reveals a role for 

the NuB4/HAT-B histone acetyltransferase 

complex in histone turnover. PLoS Genetics 


Vlaming H, van Leeuwen F. Cross-talk 

between aging and the epigenome. Epigenomics 

(in press) 

van Leeuwen F, Frederiks F, Terweij M, 

De Vos D, Bakker BM. News About Old 

Histones - A Role for Histone Age in Controlling 

the Epigenome. Cell Cycle (in press) 

De Vos D, Frederiks F, Terweij M, van 

Welsem T, Verzijlbergen KF, Iachina E, de 

Graaf EL, Maarten Altelaar AF, Oudgenoeg G, 

Heck AJ, Krijgsveld J, Bakker BM, van Leeuwen 

F. Progressive methylation of ageing histones by 

Dot1 functions as a timer. EMBO Reports. 

2011;12:956-62 

Radman-Livaja M, Verzijlbergen KF, 

Weiner A, van Welsem T, Friedman N, 

Rando OJ, van Leeuwen F. Patterns and 

mechanisms of ancestral histone protein 

inheritance in budding yeast. PLoS Biology. 


Stulemeijer IJ, Pike BL, Faber AW, 

Verzijlbergen KF, van Welsem T, Frederiks F, 

Lenstra TL, Holstege FC, Gasser SM, van 

Leeuwen F. Dot1 binding induces chromatin 

rearrangements by histone methylationdependent 

and -independent mechanisms. 

Epigenetics & Chromatin. 2011;4:2 

Frederiks F, Stulemeijer IJ, Ovaa H, van 

Leeuwen F. A modifi ed epigenetics toolbox to 

study histone modifi cations on the nucleosome 

core. ChemBioChem. 2011;12:308-13 

EPIGENETIC REGULATION OF GENE EXPRESSION 

In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational 

modifi cations of the histone proteins can affect chromatin structure 

and function and thereby regulate gene expression and DNA damage response. 

Changes in chromatin modifi cation can also result in heritable changes in gene 

expression and these epigenetic changes can lead to cancer. The mechanisms by 

which epigenetic imprints are established or prevented are still poorly understood. 

Many chromatin modifi ers are conserved from yeast to humans. Our group uses 

the budding yeast Saccharomyces cerevisiae as a powerful model system to identify 

new epigenetic regulators and to unravel the molecular mechanisms by which 

chromatin-modifying enzymes affect chromatin structure and function. 

Function and regulation of histone H3 lysine 79 (H3K79) methylation by Dot1 

We previously discovered a novel histone methyltransferase Dot1, which can add one, 

two, or three methyl groups to lysine 79 of histone H3. Dot1 infl uences heterochromatin 

structure and the DNA damage response, and has been implicated in 

oncogenic transformation in mammals. A major goal of our research is to understand 

the function and regulation of H3-Lys79 methylation. We determined that Dot1 is a 

non-processive enzyme in vivo. This uncommon mechanism affects the function of 

the methylated lysine and determines how methylation can be regulated. In yeast, in 

agreement with the methylation mechanism, the degree of methylation of H3-Lys79 

increases progressively on histones and depends on the growth rate of cells, suggesting 

that this methyl mark constitutes a timer mechanism. Since human and yeast 

Dot1 are structurally very similar, we expect that similar rules apply to human cells. 

Chromatin dynamics When a cell divides, parental histones (containing the 

epigenetic marks) and newly synthesized (unmodifi ed) histones are assembled 

onto the daughter DNA strands in a manner that reproduces the chromatin and 

transcription states that existed prior to chromosome duplication. The mode of 

histone inheritance is still unclear, however, and recent studies have shown that 

chromatin can in fact be dynamic. We developed a novel assay in yeast to determine 

protein turnover and inheritance in vivo. This universally applicable assay, called 

Recombination-Induced Tag Exchange (RITE) involves differential labeling of 

existing and newly synthesized proteins by a genetic epitope-tag switch (see 

Figure). Using RITE we found that histones throughout the genome are subject 

to extensive replication-independent exchange, suggesting that histones and their 

post-translational marks are not permanent residents in chromatin. In addition, 

we have mapped the inheritance of ancestral histones in replicating cells. They 

were found to be retained non-randomly with a preference for the 5’ ends of long 

poorly transcribed genes. In agreement with the biochemical analysis of bulk 

histones, the genomic location of old histones roughly correlates with the location 

of trimethylated H3K79. Together, these fi ndings identify the inheritance of aging 

histones in replicating cells as a potential mechanism to modulate the epigenetic 

landscape. We have recently developed high-throughput methods based on barcode 

screens to identify proteins responsible 

for histone deposition and eviction. 

We expect that our studies will provide 

novel mechanistic insights into the role 

of histones in maintaining and erasing 

epigenetic patterns and gene expression 

programs. 

Figure 3: Recombination-Induced Tag Exchange 

(RITE): a genetic assay to determine protein 

stability, inheritance, and turnover in vivo. 

The epitope tag on histone H3 can be switched 

at the genomic level from an old to a new tag in 

arrested yeast cells. Upon re-entry into the cell 

cycle old histone proteins disappear and new 

histone proteins accumulate.

THE ROLE OF CLASS I HDACS IN TRANSCRIPTION, 

DEVELOPMENT AND TUMORIGENESIS 

Cancer is a genetic disease in which epigenetic aberrations contribute to initiation 

and progression of human malignancies. In contrast to genetic mutations, 

epigenetic changes are reversible and therefore an attractive target for anti-cancer 

therapy. Histone deacetylases (HDACs), enzymes involved in removing acetyl 

groups from lysine residues, are important drug targets in cancer therapy. Small 

molecule inhibitors of HDACs (HDACi) have entered the clinic in the treatment of 

hematological malignancies and are being tested in clinical trials involving other 

malignancies. However, little is known about the role of individual HDACs in 

normal hematopoiesis and tumorigenesis. 

Our group focuses on dissecting the function of HDACs in development and 

tumorigenesis by using mice and cells carrying Hdac1 and Hdac2 conditional 

knockout alleles. By deleting these genes in normal cells as well as tumor cells we 

wish to discover anti-tumor relevant targets of HDACs and study their specifi c and 

redundant functions in transcription regulation, development and tumorigenesis. 

Hdac1 and Hdac2 collectively maintain hematopoietic stem cells We 

previously showed that bone-marrow specifi c ablation of both Hdac1 and Hdac2 

(DKO) resulted in lethal bone marrow cytopenia associated with anemia, and 

thrombocytopenia, while loss of Hdac1 or Hdac2 did not affect hematopoiesis, 

indicating that Hdac1 and Hdac2 have redundant functions in hematopoiesis 

(Wilting et al., 2010). Analysis of hematopoiesis in DKO mice revealed a dramatic 

reduction of nearly all hematopoietic lineages and absence hematopoietic stem cells 

(HSCs) and their immediate progenitors. Subsequently, competitive bone marrow 

transplantation assays showed that Hdac1 and Hdac2 collectively maintain HSC and 

early hematopoietic progenitors in a cell- autonomous manner. 

Hdac1 and Hdac2 dosage dependent tumor suppression While HDACs are 

mostly viewed as tumor promoting enzymes, our preliminary results suggest a 

previously unanticipated gene dosage dependent tumor suppressive role for Hdac1 

and Hdac2. Mice carrying an allelic series of Hdac1 and Hdac2 in the thymus 

showed progressive reduction of global Hdac-activity in Hdac1 +/Δ ; Hdac2 Δ/Δ , 

Hdac1 Δ/Δ ; Hdac2 +/+ and Hdac1 Δ/Δ ; Hdac2 +/Δ thymocytes, which correlated with 

perturbed thymocyte development and lymphomagenesis (fi gure 4A,B). Mice 

harbouring these thymocytes developed with up to 100% incidence and a mean 

latency of 10 weeks, aggressive, monoclonal lymphoblastic lymphomas, which 

presented in all cases chromosome 15 trisomy associated with elevated Myc oncogene 

expression (fi gure 4B, C). Moreover, analysis of pre-leukemic thymocytes and 

tumors showed a critical role for an Hdac1/2 activity threshold to prevent aberrant 

transcription of genes collaborating with the Myc oncoprotein in lymphomagenesis, 

providing mechanistic insight into the tumor suppressive function of Hdac1 and 

Hdac2. 

Paradoxically, complete loss of Hdac1 and Hdac2 in thymocytes resulted in a defi ned 

differentiation block during early thymocyte development, suggesting that Hdac1 

and Hdac2 are obligate haplo-insuffi cient in tumor suppression. These fi ndings 

have important implications for the treatment of cancer patients with HDACi, since 

they suggest that full inhibition of Hdac1/2-activity is critical to prevent de novo 

tumorigenesis. Future work will address the mechanisms underlying the tumor 

suppressive function of Hdac1 and Hdac2 and will provide us with new drug targets 

that, when inhibited or activated, can prevent transformation of normal cells and 

drive malignant cells into apoptosis or a cell cycle arrest. 

Figure 4: A. Progressive reduction of global histone deacetylase activity in thymocytes carrying 

compound Hdac1 and Hdac2 null alleles. B. Tumorigenesis in mice harboring compound Hdac1/Hdac2 

defi cient thymocytes correlated with the level of global HDAC activity. C. Thymocte specifi c deletion of 

Hdac1/Hdac2 resulted in T-cell lymphomas as indicated by CD4/CD8 fl ow cytometry analysis. 

53 


Junior group leader Jan-Hermen Dannenberg 

Jan-Hermen Dannenberg PhD Junior group 

leader 

Richard Heideman MSc Post-doc 

Roel Wilting MSc PhD student 

Eva Yanover BSc Technical staff 

Publication 

Richard Heideman. Gene control and 

remodeling during hematopoiesis, 2011. 

Thesis, University of Amsterdam, 

Amsterdam

54 

IMMUNOLOGY 

Division head, group leader Jannie Borst 

Jannie Borst PhD Group leader 

Yanling Xiao MD PhD Senior post-doc 

Jonathan Coquet PhD Post-doc 

Ulf Geumann PhD Post-doc 

Bert van de Kooij MSc PhD student 

Rogier Rooswinkel MSc PhD student 

Elise Veraar MSc PhD student 

Nikolina Babala MSc Technical staff 

Madelon Paauwe MSc Technical staff 

Evert de Vries BSc Technical staff 

DIVISION OF IMMUNOLOGY 

LYMPHOCYTE ACTIVATION AND SURVIVAL 

Our interest is to determine how cells decide between living and dying. We focus 

on the mechanism of action of TNF receptor family members, since these govern 

such decisions. Lymphocytes are our main cell type of interest, since throughout 

their existence, they mostly live “on the edge” between life and death. Our work is 

inspired by the desire to improve cancer immunotherapy. The second aim of our 

work is to contribute to the design of novel therapies aimed at killing cancer cells by 

activating apoptotic pathways. 

TNF receptor family members and control of the immune response From our 

work, TNF receptor family member CD27 and its ligand CD70 have emerged as 

interesting targets to improve anti-tumor immunity. This costimulatory receptor/ 

ligand pair promotes the generation and maintenance of effector CD8 T cells, the 

formation of memory CD8 T cells and their secondary expansion. 

To determine by which molecular mechanisms CD27 directs the T cell response, we 

have identifi ed CD27 target genes and followed them up by functional experiments 

in vitro and in vivo. In this way, we have found that CD27 promotes the survival 

of effector CD8 T cells at tissue sites via autocrine IL-2 signaling and promotes 

clonal expansion of CD8 T cells at the priming site via Bcl-xL and the Pim-1 serine/ 

threonine kinase. In addition, CD27 directs the expression of specifi c chemokines in 

primed CD8 T cells, which according to our recent fi ndings promote generation of 

the effector T cell pool by their impact during priming, possibly by the attraction of 

T cells to dendritic cells. 

In CD4 T cells, CD27 target genes are diagnostic for a T helper-1 effector function. 

CD27 signaling also prevents the formation of T helper-17 cells, a pro-infl ammatory 

CD4 T cell subset that causes auto-immune diseases. CD27 signaling does so 

by silencing IL-17 gene expression by histone modifi cation. We have identifi ed 

a signaling pathway that is instrumental in this and are performing chromatin 

immunoprecipitation experiments, followed by deep sequencing to identify the 

range of target genes that are affected by CD27 signaling. Furthermore, we have 

found that the CD27-CD70 pathway is involved CD4 T cell-dependent memory 

programming of CD8 T cells, which proceeds via a novel multimembrane spanning 

molecule, whose mechanism of action is currently being explored. 

With the aid of our recently generated CD70-defi cient mice, we discovered that 

CD27-CD70 interactions in the thymus contribute to the generation of regulatory 

CD4 T cells (Treg). It has long been known that CD70 is expressed constitutively 

in the thymic medulla of mice and humans, but the functional relevance was 

previously unclear. Since Treg suppress anti-tumor immune responses, the impact 

of CD27 on this subset is particularly relevant. CD27 does not impact on FoxP3 

expression, nor on suppressive function of Treg, as read out in vitro. It does promote 

Treg expansion in chronic infection though, indicating that we have to be aware of 

potential stimulation of Treg when using CD27 agonist reagents, which are currently 

under consideration for clinical application in cancer immunotherapy. 

Constitutive expression of CD70 is a hallmark of an activated immune system in 

both mice and humans. From our studies in CD70 transgenic mice, we know that 

this drives immune activation, with dramatically detrimental effects. We have 

discovered that CD70 expression at the cell surface is carefully controlled, apparently 

to avoid immune activation. In dendritic cells, the MHC Class II chaperone 

Invariant chain brings CD70 to late endosomal MHC Class II compartments, from 

which it can be mobilized to the cell surface, together with MHC Class II. We have 

now developed technology to study this process in real time, in primary dendritic 

cells transduced with fl uorescently-tagged molecules. 

By analysis of CD70 transgenic mice, we have discovered a novel link between 

CD27-CD70 and bone homeostasis. CD27 hallmarks a newly defi ned osteoclast 

progenitor and that sustained engagement of CD27 on these cells by CD70 on 

activated immune cell inhibits osteoclast development, leading to an increased

trabecular bone mass and perturbation of the bone marrow niche. This negative 

feedback by the CD27-CD70 pathway may serve to counteract the osteolytic effects 

of chronically activated immune cells. 

Conclusion Our fi ndings indicate that targeting the CD27-CD70 costimulatory axis is 

of interest not only in the context of anti-tumor immunity, but also in auto-immune 

or infl ammatory disease. Follow up in the human system should further validate 

this. In collaboration with researchers from former Organon, we study reagents that 

target the human molecules with the aim to apply them clinically. 

Apoptosis signaling and cancer therapy Pro-apoptotic agents are of great interest 

for cancer therapy, provided that they can act in a tumor-specifi c fashion. The TRAIL 

death receptors conform to this condition; they are not toxic on normal tissue and 

induce apoptotic cell death in many different tumor types. Although the exact 

mechanisms underlying this tumor-specifi city are not known, agonist reagents that 

target the two TRAIL receptors in human have moved rapidly through preclinical 

testing and are now in Phase I and II clinical trials. Interestingly, TRAIL receptor 

agonists act synergistically with conventional and novel therapeutics in many cases, 

by a variety of mechanisms. 

We have identifi ed a ubiquitin ligase that determines the steady-state cell surface 

expression of TRAIL receptor-1, but not TRAIL receptor-2. This ligase targets TRAIL 

receptor-1 for degradation, by promoting its endocytosis. We have identifi ed the 

mechanistic details of this process, including the defi nition of a lysine residue in the 

receptor tail that is essential for interaction with and ubiquitination by the ligase. We 

have identifi ed another molecule that impacts on intracellular transport of TRAIL 

receptor-1 in the biosynthetic route. Both mechanisms attenuate apoptosis sensitivity 

of the tumor cells and are therefore of clinical relevance. 

Another important target for tumor therapy are the inhibitory Bcl-2 family members 

that block apoptosis at the level of the mitochondrial membrane. In response to 

apoptotic stimuli, the mitochondrial membrane is permeabilized, resulting in the 

release of mediators that activate the capases, which execute cell death. BH3 domainonly 

proteins bring about this membrane permeabilization, by activating the effector 

molecules Bax and Bak. The inhibitory Bcl-2 family members block this, by physical 

interaction with their pro-apoptotic relatives. This mechanism is intensely studied, 

since novel targeted reagents are based upon it. BH3 domain mimetic drugs act 

tumor-specifi cally according to the principle of “oncogene” addiction. 

We have (re-)defi ned the target specifi city of the BH3 mimetic ABT-737 for all six 

inhibitory Bcl-2 family members, as individually expressed in p53 profi cient and 

defi cient T-ALL. Three Bcl-2 proteins are completely untargeted by this agent, 

raising the question how their resistance can be overruled. By inducible expression 

of BH3-only proteins, we have defi ned which of these can induce apoptosis in cells 

that express the untargeted proteins. This identifi ed the BH3 proteins that should be 

induced or additionally targeted by novel mimetics to overcome treatment resistance. 

We have found that ubiquitin- and proteasome dependent turnover of inhibitory 

proteins is also an important parameter in their activity and are studying the 

mechanisms involved. To better delineate the selectivity of Bcl-2 family interactions, 

we have used and validated a technique to fi x interactions as they occur in the 

mitochondrial membrane. The comprehensive interaction profi le that we have 

obtained extends current knowledge and allows for rational intervention to overrule 

apoptosis resistance caused by inhibitory Bcl-2 family proteins. 

Publications 

55 

IMMUNOLOGY 

Salek-Ardakani S, Flynn R, Arens R, 

Yagita H, Smith GL, Borst J, 

Schoenberger SP, Croft M. The TNFR 

family members OX40 and CD27 link 

viral virulence to protective T cell vaccines 

in mice. J Clin Invest. 2011;121:296-307 

Maas C, de Vries E, Tait SW, Borst J. 

Bid can mediate a pro-apoptotic response 

to etoposide and ionizing radiation 

without cleavage in its unstructured loop 

and in the absence of p53. Oncogene. 

2011;30:3636-47

56 

IMMUNOLOGY 


Group leader Ton Schumacher 

Ton Schumacher PhD Group leader 

Gavin Bendle PhD Post-doc 

Andreas Hollenstein PhD Post-doc 

Shalin Naik PhD Post-doc 

Leila Perie PhD Post-doc 

Jan Rohr MD Post-doc 

Remko Schotte PhD Post-doc 

Marit van Buuren MSc PhD student 

Carmen Gerlach MSc PhD student 

Sander Kelderman MSc PhD student 

Carsten Linnemann MSc PhD student 

Riccardo Mezzadra MSc PhD student 

Laura Bies Technical staff 

Daisy Philips Technical staff 

Nienke van Rooij Technical staff 

Mireille Toebes Technical staff 

Jos Urbanus Technical staff 

Publications 

Hombrink P, Hadrup SR, Bakker A, 

Kester MG, Falkenburg JH, von dem 

Borne PA, Schumacher TN, Heemskerk 

MH. High-throughput identifi cation of 

potential minor histocompatibility antigens 

by MHC tetramer-based screening: 

feasibility and limitations. PLoS One. 


Blank CU, Hooijkaas AI, Haanen JB, 

Schumacher TN. Combination of targeted 

therapy and immunotherapy in melanoma. 

Cancer Immunol Immunother. 

2011;60:1359-71 

Linnemann C, Schumacher TN, Bendle 

GM. T-cell receptor gene therapy: critical 

parameters for clinical success. J Invest 

Dermatol. 2011;131:1806-16 

Unger WW, Velthuis J, Abreu JR, Laban 

S, Quinten E, Kester MG, Reker-Hadrup S, 

Bakker AH, Duinkerken G, Mulder A, 

Franken KL, Hilbrands R, Keymeulen B, 

Peakman, M, Ossendorp F, Drijfhout JW, 

Schumacher TN, Roep BO. Discovery of 

low-affi nity preproinsulin epitopes and 

detection of autoreactive CD8 T-cells using 

combinatorial MHC multimers. J 

Autoimmun. 2011 

DISSECTING AND MANIPULATING ANTIGEN-SPECIFIC T CELL 

IMMUNITY 

The aim of our research is straightforward 1). To design novel technologies that 

can be used to examine and modify antigen-specifi c T cell immunity 2). To use 

these tools to unravel and manipulate the molecular processes underlying immune 

recognition by T lymphocytes. Within these projects, a main focus is on the design 

and testing of novel concepts for adoptive immunotherapy. 

Dissecting antigen-specific T cell immunity There is now substantial evidence 

that therapeutic manipulation of immune reactivity can result in clinically 

meaningful effects on human cancer. For example, T cell responses induced by 

either anti-CTLA4 treatment or infusion of ex vivo expanded tumor-infi ltrating 

T cells (TIL therapy) has shown substantial activity in metastatic melanoma. 

Importantly, at present we do not know which cytotoxic T cell reactivities mediate 

cancer regression. Furthermore, as the number of potential melanoma-associated 

antigens to which these responses can be directed is very high, classical strategies to 

map cytotoxic T cell reactivity do not suffi ce. Knowledge of such reactivities would 

be of obvious use, both to monitor current therapies and to design more targeted 

strategies that selectively aim to induce immune reactivity against these antigens. 

In the past years we have developed a broadly applicable “MHC-based toolkit” for the 

parallel detection of dozens of different T cell populations within a single sample. 

Having established this technological framework, we have used it to determine 

the effects of two forms of immunotherapy on the cytotoxic T cell repertoire in 

melanoma patients. In a fi rst project, we have assessed the composition of TIL 

products used for melanoma therapy and have assessed whether TIL therapy 

induces a detectable alteration in the melanoma-reactive T cell repertoire in 

patients. These analyses have demonstrated that virtually all TIL products contain 

(unique combinations of) T cell populations specifi c for a panel of some 150 shared 

melanoma-associated epitopes. Strikingly however, the combined magnitude of 

these T cell responses is very low. In subsequent work we have likewise addressed 

whether the melanoma-specifi c T cell repertoire is enhanced in patients that are 

treated with anti-CTLA4 antibody. As is the case for TIL therapy, anti-CTLA4 

therapy leads to an increase in the number of T cell responses against the shared 

melanoma-associated antigens. However, also in this case, the combined magnitude 

of these T cell responses is very low. These data provide indirect evidence for 

the hypothesis that recognition of patient-specifi c neo-antigens that arise as a 

consequence of mutations may be a signifi cant component of treatment-induced 

melanoma-specifi c T cell reactivity. Analysis of this intriguing possibility will be an 

important focus area in the coming years. 

Dissection of T cell immunity through genetic tagging and intravital 

imaging The ability to visualize antigen-specifi c T cell responses and to determine 

the differentiation pathways of different subsets of T cells is essential for our 

understanding of pathogen- and vaccine-induced immunity. While MHC tetramer 

technology makes it possible to follow the development of immunity at the T cell 

population level, it doesn’t allow the analysis of cell fate and cellular differentiation 

pathways. 

To allow such lineage tracking, we have developed technologies with which 

individual T cells can be tagged with genetic barcodes. This tagging technology 

relies on the use of oncoretroviral and lentiviral libraries containing thousands of 

different DNA ‘barcodes’. Infection of progenitor cells of interest by these libraries 

of viral vectors and subsequent analysis of the barcodes present within the cell 

populations that arise from them can then be utilized to reveal lineage relationships. 

In the past year, we have used this barcode labeling strategy, together with second 

generation sequencing, to measure the numerical output of individual antigenspecifi 

c T cells, and to address which (types of) progeny are generated by individual 

multipotent progenitors (MPP) in vivo. With respect to the fi rst question: the data 

obtained demonstrate that antigen-specifi c T cell responses are dominated by the 

output of only a very small number of naïve antigen-specifi c T cells, and that this 

strong bias in clonal output is established early during infection. With respect to the

second question: the data obtained demonstrate that individual MPP, cells which 

previously have been defi ned as multipotent on the basis of in vitro assays, only 

produce a restricted set of cell types in vivo. Furthermore, the type of output that is 

generated by individual MPP falls into a number of discrete classes, consistent with 

the existence of intrinsic or environmentally imposed progenitor subclasses. 

The vast majority of studies that analyze antigen-specifi c T cell responses – 

including those described above- primarily assess the development of antigenspecifi 

c T cell responses within the blood compartment. To also allow the analysis 

of T cell reactivity at the sites of action, the Haanen group and our group have 

developed an intravital imaging system in which virus-specifi c T cell responses can 

be followed in skin. Recent work on the imaging of memory T cells that remain in 

skin following clearance of infection indicates that the antigen-specifi c T cells that 

stay put take on a dendritic morphology and show a remarkable crawling behavior 

in between skin keratinocytes, for many months after the infection has resolved. 

This continuous migration allows these cells to contact large numbers of skin cells 

through time, and we have demonstrated that these migrating memory T cells can 

identify rare antigen-expressing epithelial cells in vivo. These data demonstrate 

the existence of a specialized antigen-specifi c T cell population of “skin patrolling 

memory T cells”. 

Adoptive T cell therapy (collaboration with Haanen lab) The cornerstone of our 

translational work is the development and evaluation of adoptive T cell therapies 

for human cancer. The MHC-based monitoring strategies described above will 

be utilized to evaluate T cell reactivity in the clinical trial for TIL therapy that has 

recently been started by the Haanen group. Furthermore, the T cell reactivities 

that are visualized in these analyses form a very useful starting point for the 

further development of our second approach for adoptive T cell therapy; the 

genetic engineering of T cell reactivity. Specifi cally, in the past years our group 

has developed the retroviral introduction of antigen-specifi c T cell receptors into 

peripheral T cells as a means to induce tumor-specifi c T cell immunity. We have now 

developed the pharmaceutical process that will be utilized to generate Mart-I reactive 

T cells for a fi rst NKI TCR gene therapy trial, and this trial will commence in 2012. 

In parallel, we are preparing for a substantially larger clinical program in which we 

aim to test the clinical value of a series of different T cell receptors that target various 

tumor-associated antigens. Towards this goal, we have isolated antigen-specifi c T cell 

populations that target a series of different cancer/testis antigens. Furthermore, we 

have recovered the TCR genes encoded, using a deep sequencing based “TCR gene 

capture” approach. In subsequent work, we will evaluate the large panel of TCRs 

that we have obtained with respect to safety and effi cacy, in order to select a series of 

TCR genes that will be used for future TCR gene therapy trials. In addition, we are 

currently evaluating alternative, transposon-based, gene transfer strategies to create 

the TCR-modifi ed T cells that can be used in such clinical studies. 


57 

IMMUNOLOGY 

Jorritsma A, Schotte R, Coccoris M, de 

Witte MA, Schumacher TN. Prospects and 

limitations of T cell receptor gene therapy. 

Curr Gene Ther. 2011;11:276-8 

Brackenridge S, Evans EJ, Toebes M, 

Goonetilleke N, Liu MK, di Gleria K, 

Schumacher TN, Davis SJ, McMichael AJ, 

Gillespie GM. An early HIV mutation 

within an HLA-B*57-restricted T cell 

epitope abrogates binding to the killer 

inhibitory receptor 3DL1. J Virol. 

2011;85:5415-22 

Gerlach C, van Heijst JW, Schumacher 

TN. The descent of memory T cells. Ann NY 

Acad Sci. 2011;1217:139-53 

Oosterhuis K, Ohlschläger P, van den 

Berg JH, Toebes M, Gomez R, 

Schumacher TN, Haanen JB. Preclinical 

development of highly effective and safe 

DNA vaccines directed against HPV 16 E6 

and E7. Int J Cancer. 2011;129:397-406 

Oosterhuis K, van den Berg JH, 

Schumacher TN, Haanen JB. DNA 

vaccines and intradermal vaccination by 

DNA tattooing. Curr Top Microbiol 

Immunol.2012;351:221-50 

Schulte I, Hitziger T, Giugliano S, 

Timm J, Gold H, Heinemann FM, 

Khudyakov Y, Strasser M, König C, 

Castermans E, Mok JY, van Esch WJ, 

Bertoletti A, Schumacher TN, Roggendorf 

M. Characterization of CD8+ T-cell 

response in acute and resolved hepatitis A 

virus infection. J Hepatol. 2011;54:201-8 

Figure 1: Visualization of TCR gene 

rearrangements in melanoma-specifi c T 

cells by TCR gene capture. Circles represent 

the TCR alpha and beta loci, all 

long-range rearrangements detected are 

depicted. In the example shown both TCR 

alpha alleles and one TCR beta allele have 

undergone recombination.

58 

IMMUNOLOGY 

Group leader John Haanen 

John Haanen MD PhD Group leader 

Florry Vyth-Dreese PhD Senior staff scientist 

Bianca Heemskerk PhD Post-doc 

Pia Kvistborg PhD Post-doc 

Koen Oosterhuis MSc PhD student 

Silvia Ariotti MSc PhD student 

Trees de Jong Technical staff 

Willeke van de Kasteele Technical staff 

Martin van der Maas Technical staff 

Samira Michels Technical staff 

Publications 

Haanen JB. Issues around melanoma. 

Ned Tijdschr Geneeskd. 2011;155:A3836 




Cancer Immunol Immunother. 

2011;60:1359-71 

Lesterhuis WJ, Haanen JB, Punt CJ. 

Cancer immunotherapy-revisited. Nat Rev 

Drug Discov. 2011;10:591-600 

Chapman PB, Hauschild A, Robert C, 

Haanen JB, Ascierto P, Larkin J, Dummer R, 

Garbe C, Testori A, Maio M, Hogg D, 

Lorigan P, Lebbe C, Jouary T, Schadendorf 

D, Ribas A, O’Day SJ, Sosman JA, Kirkwood 

JM, Eggermont AM, Dreno B, Nolop K, Li J, 

Nelson B, Hou J, Lee RJ, Flaherty KT, 

McArthur GA; BRIM-3 Study Group. 

Improved survival with vemurafenib in 

melanoma with BRAF V600E mutation. N 

Engl J Med. 2011;364:2507-16 

Verdegaal EM, Visser M, Ramwadhdoebé 

TH, van der Minne CE, van Steijn JA, 

Kapiteijn E, Haanen JB, van der Burg SH, 

Nortier JW, Osanto S. Successful treatment 

of metastatic melanoma by adoptive transfer 

of blood-derived polyclonal tumor-specifi c 

CD4+ and CD8+ T cells in combination with 

low-dose interferon-alpha. Cancer Immunol 

Immunother. 2011;60:953-63 

IMMUNOTHERAPY AND IMMUNOMONITORING 

The main objective of this line of research is the development of novel T cell 

immunity based strategies that can be translated to clinical application. The focus 

is on treatment of patients with solid tumors, especially melanoma and renal cell 

carcinoma. A second line of research focuses on immunomonitoring. This work is 

performed under supervision of Dr F Vyth-Dreese and Dr P Kvistborg. Its primary 

aim is to evaluate specifi c and cytokine-based immunotherapies, using advanced 

technologies for characterization of immune responses in peripheral blood and at 

the tumor site. These studies are conducted together with the Schumacher lab at the 

NKI-AVL and with national and international collaborators. 

DNA vaccination for the treatment of cancer Phase I study in melanoma patients 

Induction of immunity following DNA vaccination is generally considered a slow 

process. We have shown that DNA delivery to the skin results in a highly transient 

pulse of antigen expression. Based on this information, we developed a novel, rapid 

and potent intradermal DNA vaccination method. By short-interval intradermal 

DNA delivery, robust T cell responses, of a magnitude suffi cient to reject established 

subcutaneous tumors, are generated within 14 days. These results were confi rmed 

in a non-human primate model (in collaboration with BPRC, Rijswijk). We showed 

that DNA tattooing results in 10-100-fold increase in vaccine-specifi c T cells, 

compared to intramuscular vaccination. 

Together with Dr. B Nuijen (Pharmacy) and Profs. W Hennink and G Storm 

(University of Utrecht) we have developed an ex vivo human skin model to optimize 

DNA tattoo vaccination for human skin and to create a platform for testing novel 

DNA formulations for transfection and targeting of human skin cells. Results from 

these studies have been crucial for clinical application of this strategy. A clinical 

grade DNA vaccine has been produced in the GMP DNA plasmid production unit 

(Amsterdam-Biotherapeutics Unit) at the NKI-AVL/Slotervaart Hospital pharmacy 

department. From 2009 till 2011, eight stage IV melanoma patients have been 

enrolled in a fi rst phase I clinical trial with DNA tattoo vaccination. The study 

was amended in 2010 and recently opened in the Leiden University Medical Center 

as well. 

DNA vaccination for the treatment of high risk human papilloma virus (HPV) associated 

cancers Human papilloma virus infection (serotypes 16 and 18) is strongly 

associated with the development of squamous cell cancer of the cervix, but also 

penis, vulva, anus en oropharynx. Because the persistence of oncogenic highrisk 

HPV proteins E6 and E7 is required for carcinogenesis, these viral antigens 

are exquisite targets for immunotherapeutic interventions. Indeed, therapeutic 

vaccinations targeting these viral antigens have shown some promise in woman 

suffering from cervical cancer. 

In the upcoming years (in collaboration with Prof Gemma Kenter, Dr Marc van 

Beurden en Prof Smon Horenblas (all section Surgical Oncology), we will perform 

a phase I/II study in patients with HPV 16-positive squamous cell cancer of the 

penis and cervix, using a novel and potent intradermal DNA vaccination strategy. 

In preclinical studies, we have developed highly immunogenic and safe HPV 16 E6 

and E7 containing DNA vaccines for which we have started GMP production in 

2010 for a fi rst clinical trial. The E7 DNA vaccine has been fi nished and will be 

tested in a phase I/II clinical trial. 

Adoptive immunotherapy program TIL therapy Adoptive therapy with Tumorinfi 

ltrating Lymphocytes (TIL) is based on results from the Surgery Branch, NIH, 

Bethesda, MD, USA and results from the Sheba Medical Center, TeL Aviv, Israel 

showing a 50% objective response rate in heavily pretreated stage IV melanoma 

patients. This treatment combines the ex vivo culture of melanoma-reactive T cells 

from resected metastases with non-myeloablative chemotherapy and high dose bolus 

IL-2. Our goals are to show that this treatment can be given safely at the NKI-AVL, to 

demonstrate in an randomized controlled phase II trial that this treatment improves 

progression-free survival compared to standard chemotherapy and to perform a 

comprehensive analysis of the T cells specifi cities of the melanoma-reactive TIL

prior and after adoptive transfer. We have started a pilot study (n=5) and enrolled 

4 patients in 2011. 

T-cell receptor gene therapy In close collaboration with the Schumacher lab, 

we have selected a highly avid T cell receptor (TCR) specifi c for melanocyte 

differentiation antigen MART-1 26-35. This TCR, called 1D3, has been cloned into a 

retroviral vector (MP-71) and has been produced by a German GMP manufacturer. 

This TCR has been equipped with extra safe guards to prevent mispairing with 

endogenous TCR chains after transduction of peripheral T cells to prevent potential 

side effects. The process of clinical grade culturing and transduction of peripheral 

T cells with the 1D3-MP-71 retrovirus has been validated step-by-step in our GMP 

facility in order to start a phase I clinical study in melanoma patients in 2012. 

Immunomonitoring of DNA tattoo vaccination trial Recently, we analysed biopsy 

samples from the fi rst three cohorts of melanoma patients (n=8) treated in the DNA 

tattoo vaccination trial. In general, higher yields were recovered from biopsies at 

week 6 than at week 2 and in 2 cases more MART-1 modifi ed-specifi c compared 

to MART-1 wt specifi c T cells were detected. In 1/2 cases MART-1 specifi c CD8 T 

cells were grown out from a non-vaccinated site, suggesting a systemic effect of 

vaccination. 

Immunohistological analysis indicated that after vaccination at week 2 and week 

6 enhanced numbers of activated CD8 T lymphocytes were present near the basal 

membrane, whereas pre and wk 6 non-vaccination sites showed a normal skin 

pattern. Confocal microscopic analysis of these biopsies confi rmed that (part of) 

the T cells were activated (GrB+, Ki67+, CD69+) and CXCR3 positive. In 7/8 cases 

vaccination induced accumulation of activated, CD80 positive DC in subepidermal 

areas. 

One patient of cohort 3 showed slidely enhanced PBMC IFNγ responses to MART-1 

peptides, as well as a small systemic MART-1 specifi c CD8 T cell response at week 6 

and this was the only patient with stable disease. 

These results show that DNA tattoo vaccination can induce local CD8 T cell 

responses. 

Immune infiltrates in renal cell carcinoma (RCC) treated with anti-angiogenic 

agents Patients with metastatic RCC are currently treated with targeted therapy 

consisting of tyrosine kinase and mTOR inhibitors, and anti-VEGF mAb. These 

therapies are based on inhibition of angiogenesis, as well as direct tumor-targeting 

and may potentiate anti-tumor immune responses. Tumor specimens obtained from 

RCC patients treated with Sunitinib, Bevacizumab or IFNα show increased immune 

cell infi ltration and apoptosis of tumor and vasculature compared with untreated 

patients. These analyses will be extended to additional patients. 

Detection of mHag-specific T lymphocytes in human tissues We have 

successfully applied in situ tetramer staining for the detection of minor 

Histocompatibility antigen (mHag)-specifi c T cells in a human ex vivo in situ skin 

explant model of Graft versus Host reactivity (in collaboration with groups at the 

Leiden University Medical Center and Utrecht University). Recently, we have 

developed, validated and published a method, using so-called MHC-dextramers, to 

directly enumerate specifi c T lymphocytes in cryopreserved tissues. In addition, we 

have implemented this technique to detected mHag-directed T lymphocytes in skin 

lesions of patients with Graft versus Host Disease. 

Human ex vivo in situ skin model for vitiligo and melanoma In collaboration 

with the AMC and LUMC, a human in situ skin model has been developed to study 

immune factors involved in the development of vitiligo and potential therapy of 

melanoma. Using melanocyte specifi c T cell clones or bulk T cells obtained from 

vitiligo lesions co-cultured with normal skin tissues, the induction of vitiligo could 

be mimicked ex vivo. Recently, we published a study on comparison of tumor 

specifi c T cells in peripheral blood of melanoma patients compared to tumor 

infi ltration profi les. 


59 

IMMUNOLOGY 

Oosterhuis K, Ohlschläger P, van den 

Berg JH, Toebes M, Gomez R, Schumacher 

TN, Haanen JB. Preclinical development of 

highly effective and safe DNA vaccines 

directed against HPV 16 E6 and E7. Int J 

Cancer. 2011;129:397-406 

Kim Y-H, Vyth-Dreese FA, Schrama E, 

Pavel S, Bajema I, Goulmy E and Spierings 

E. Exogenous Addition of Minor H Antigen 

HA-1+ Dendritic Cells to Skin Tissues Ex 

Vivo Causes Infi ltration and Activation of 

HA-1-Specifi c Cytotoxic T Cells. Biol Blood 

Marrow Transplant 2011;17:69-77 

Tjin EPM, Konijnenberg D, Krebbers G, 

Mallo H, Drijfhout JW, Franken K, 

van der Horst C, Bos JD, Nieweg OE, 

Kroon BBR, Haanen JBAG, Melief CJ, 

Vyth-Dreese FA, Luiten RM. T cell immune 

function in tumor, skin and peripheral blood 

of advanced stage melanoma patients: 

implications for immunotherapy. Clin Canc 

Res 2011;17:5736-47 

Kim Y-H, Faaij CMJM, van Halteren 

AGS, Schrama E, Dellemijn TAM, Schøler J, 

Egeler RM, Pavel S, Vyth-Dreese FA, van 

Tol MJD, Goulmy E and E. Spierings E. In 

situ detection of HY-specifi c T cells in acute 

graft-versus-host disease-affected male skin 

after gender mismatched stem-cell 

transplantation. Biol Blood Marrow 

Transplant (in press) 

Figure 2: Production of Tumor Infl itrating 

Lymphocytes (TIL) in clean room.

60 

IMMUNOLOGY 


Group leader Heinz Jacobs 

Heinz Jacobs PhD Group leader 

Peter Krijger MSc PhD student 

Marc Hogenbirk MSc PhD student 

Niek Wit MSc PhD student 

Paul van den Berk Technical staff 

Publications 

Krijger PHL*, Hendel A*, Diamant N, Goren 

Z, Langerak P, Kim J, Reißner T, Lee K Y, 

Geacintov NE, Carell T, Myung K, Tateishi S, 

D’Andrea A, Livneh Z*, and Jacobs H*. PCNA 

ubiquitination is important, but not essential for 

translesion DNA synthesis in mammalian cells. 

PLoS Genet. 2011; 7:e1002262 (*shared 

contribution) 

Krijger PHL, van den Berk PCM, Wit N, 

Langerak P, Jansen JG, Reynaud C-A, de Wind 

N, and Jacobs H. PCNA ubiquitinationindependent 

activation of polymerase η during 

somatic hypermutation and DNA damage 

tolerance. DNA Repair 2011;10:1051-9 

Heideman M*, Lutz J*, Roth E, van den 

Berk P, Müller W, Raman C, Wabl M, Jäck 

H-M*, and Jacobs H*. Pro-B cells sense 

productive immunoglobulin heavy chain 

rearrangement irrespective of polypeptide 

production. PNAS 2011;108:10644-9 (*shared 

contribution) 

Wit N, Krijger PHL, van den Berk PCM, and 

Jacobs H. Lysine residue 185 of Rad1 is a 

topological but not a functional counterpart of 

lysine residue 164 of PCNA. PLoS One 2011; 

6:e16669 

Krijger PHL, Lee K-Y, Wit N, van den Berk 

PCM, Wu X, Roest HP, Maas A, Ding H, 

Hoeijmakers JHJ, Myung K and Jacobs H. 

HLTF and SHPRH are not essential for PCNA 

polyubiquitination, survival and somatic 

hypermutation: Existence of an alternative E3 

ligase. DNA Repair 2011;10:438-44 

Krijger PHL, Wit N, van den Berk PCM, 

Jacobs H. The Fanconi Anemia Core Complex 

is dispensable during Somatic Hypermutation 

and Class Switch Recombination. PLoS One 

2010; 5:e15236 

Heideman MR. Gene control and remodeling 

during hematopoiesis. Thesis, University of 

Amsterdam, September 13th, 2011 

Krijger P. Regulation of Translesion Synthesis 

Polymerases during Somatic Hypermutation 

and DNA Damage Tolerance. Thesis University 

of Amsterdam, June 10th, 2011 

DNA DAMAGE TOLERANCE, PROGRAMMED MUTAGENESIS 

AND LYMPHOMAGENESIS 

B cells have the unique capacity to modify their immunoglobulin genes by 

programmed mutagenesis. Using B cells as a model system, we focus on the 

following aspects in this process: 1. DNA damage tolerance; 2. Decision-making 

between DNA repair and mutagenesis; 3. Targeting specifi city of Activation-Induced 

Deaminase (AID); 4. The role of IgH mRNA in establishing allelic exclusion, 

The role of PCNA ubiquitination in activation of translesion DNA synthesis 

(TLS) DNA damage can block replication and lead to mutations, genomic instability 

and cancer. When the removal of DNA damage and restoration of the original 

sequence prior to replication is impossible, cell utilize DNA damage tolerance 

mechanisms, which help replication to bypass the lesions. A major universal 

tolerance mechanism is TLS, in which specialized low-fi delity DNA polymerases 

elongate the DNA across the lesion. This is a double-edged sword, because the 

price of completion of replication is the risk of increased point mutations opposite 

the lesion. A key element in TLS regulation is the attachment of ubiquitin to the 

PCNA protein, a DNA sliding clamp that tethers the DNA polymerases to DNA, 

which functions to recruit the TLS DNA polymerase to the damaged site in DNA. 

While in yeast this modifi cation of PCNA is crucial for TLS, there is a debate about 

its importance in mammals. Using several independent systems, we recently 

demonstrated the existence of PCNA ubiquitination-dependent and -independent 

TLS activation pathways in mammals. 

Lysine residue 185 of Rad1 is a topological but not a functional counterpart 

of lysine residue 164 of PCNA Monoubiquitylation of the homotrimeric DNA 

sliding clamp PCNA at lysine residue 164 (PCNA K164 ) is a highly conserved, DNA 

damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This 

ubiquitylation event recruits TLS polymerases capable of replicating across damaged 

DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in 

yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of 

Rad9, Rad1, and Hus1, in a DNA damage-inducible manner. Based on the highly 

similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1 K185 ) was identifi ed 

as the only topological equivalent of PCNA K164 . To investigate a potential role of 

posttranslational modifi cations of Rad1 K185 in DNA damage tolerance, we generated 

mice with a conditional deletable Rad1 K185R allele. Our data indicate that Rad1 K185 is 

not a functional counterpart of PCNA K164 . Currently, we use conditional inactivation 

of Rad1, Rad9 and Hus1 to defi ne novel post-translational modifi cations and their 

functions in the mammalian 9-1-1 DNA sliding clamp. 

Pro-B cells sense productive immunoglobulin heavy chain rearrangement 

irrespective of polypeptide production B-lymphocyte development is dictated 

by the protein products of functionally rearranged Ig heavy (H) and light (L) chain 

genes. Ig rearrangement begins in pro-B cells at the IgH locus. If pro-B cells 

generate a productive allele, they assemble a pre-B cell receptor complex, which 

signals their differentiation into pre-B cells and their clonal expansion. Pre-B cell 

receptor signals are also thought to contribute to allelic exclusion by preventing 

further IgH rearrangements. We have shown in two independent mouse models 

that the accumulation of a stabilized μH mRNA that does not encode μH chain 

protein specifi cally impairs pro-B cell differentiation and reduces the frequency of 

rearranged IgH genes in a dose dependent manner. Because non-coding IgH mRNA 

is usually rapidly degraded by the nonsense-mediated mRNA decay machinery, 

we propose that the difference in mRNA stability allows pro-B cells to distinguish 

between productive and nonproductive Ig gene rearrangements and that μH mRNA 

may thus contribute to effi cient H chain allelic exclusion.

COMBINING TARGETED THERAPY AND IMMUNOTHERAPY IN 

MELANOMA AND RENAL CELL CARCINOMA 

We aim to identify mechanisms of tumor immune escape and to develop therapeutic 

protocols to overrule such escape, in combination with targeted therapy. Tumor 

immune escape mechanisms include action of inhibitory molecules on tumor cells 

and their ligands on antigen presenting cells and action of immune regulatory 

cells in the tumor environment. We examine whether blockade of inhibitory 

mechanisms, in possible synergy with small molecule-based targeted therapies, 

improves anticancer immunotherapy. 

Development of an inducible spontaneous murine melanoma model It is 

crucial to test new therapeutic approaches in appropriate in vivo models that 

simulate the human cancer reality. Transplantable tumor models often do not mimic 

the complex interaction between the tumor cell and the tumor microenvironment 

and therefore may have little predictive value for the treatment of cancer patients. 

Spontaneous murine melanoma models are more physiological, but due to the 

late onset of tumor formation less practical for long-term immunotherapeutic 

experiments. By crossing mice that inducibly express in melanocytes the 

BRAFV600E mutation and loss of PTEN, we could induce melanoma with high 

penetrance, short time to onset and lymph node metastasis. We could inhibit tumor 

growth by targeting BRAF or MEK in vivo by oral application of small molecule 

inhibitors. 

Role of co-inhibitory molecules during tumor immune escape Appropriate 

T cell activation depends on TCR ligation and a positive secondary signal. Recent 

work revealed that this secondary signal is not an on-off phenomenon but a signal 

of modulated intensity, which is orchestrated by several co-stimulatory and coinhibitory 

molecules. We and others have shown that one of the ligands (PD-L1) of 

one such a co-inhibitory molecule (PD-1) is highly expressed on tumor cells and 

leads to impaired immune responses. We found an increased PD-L1 expression on 

metastases compared to primary melanoma in human, but no infl uence on overall 

survival, raising the question in which situations PD-L1 inhibits tumor-specifi c T 

cells. In experiments using murine TCR transgenic T cells, we found that low tumor 

antigen expression combined with PD-L1 expression predisposes for the strongest 

inhibition of tumor-specifi c T cells. 

Homeostatically proliferating T cells for the treatment of cancer One approach 

in immunotherapy is the adoptive transfer of tumor-reactive T cells. For effective 

tumor growth control, tumor-reactive T cells should suffi ciently expand and survive, 

without exhaustion. Transfer of naïve peripheral T cells into lymphopenic recipient 

mice results in a slow cytokine-driven proliferation of these T cells. During this 

homeostatic proliferation (HP), T cells acquire effector functions (IFN-production, 

lytic activity), while keeping characteristics of naïve T cells. This results in better 

CD62L-mediated lymph node homing, less anergy induction and better tumor 

growth control compared to naïve or effector T cells. As induction of lymphopenia 

by chemo- or chemoradiotherapy is accompanied by serious adverse events, we aim 

at induction of HP in vitro. We recently achieved in vitro expansion of T cells with 

superior tumor growth control capabilities in vivo (in mice) and characterize them at 

the moment in comparison to naïve, primed and memory T cells. 

Generation of Tumor-infiltrating lymphocytes (TIL) from Renal Cell 

Carcinoma (RCC) after tyrosine kinase inhibitor pretreatment TIL therapy is 

a promising immunotherapeutic approach in melanoma that induces long-lasting 

clinical responses and is currently tested at the NKI-AVL. Culture of TIL from RCC 

has been described before, but failed to induce clinical responses. Reason for this is 

that former protocols lacked preconditioning of the patients to induce lymphopenia 

and combination with small molecules and antibody-mediated blocking of inhibitory 

mechanisms. We aim to expand TIL from Sunitinib-pretreated RCC patients, 

adding blocking antibody to generate higher TIL numbers, while using more gentle 

expansion protocols to prevent exhaustion and negative selection. 

Group leader Christian Blank 

61 

IMMUNOLOGY 

Christian Blank MD PhD Group leader 

Andrew Kaiser PhD Post-doc 

Anna Hooijkaas MSc PhD student 

Aurelie Guislain Technical staff 

Jules Gadiot Technical staff 

Publications 

Vogel WV, Guislain A, Kvistborg P, 

Schumacher TNM, Haanen JBAG, Blank 

CU. Ipilimumab-induced sarcoidosis in a 

patient with metastatic melanoma 

undergoing complete remission. J Clin 

Oncology (in press) 

Hooijkaas AI, Gadiot J, van Boven H, 

Blank CU. Expression of the embryological 

morphogen Nodal in stage III/IV 

melanoma. Melanoma Res 2011;21:491- 

501 



therapy and immunotherapy in 

melanoma. Cancer Immunol Immunother 

2011;60:1359-71 

Krönig H, Falchner KJ, Odendahl M, 

Brackertz B, Conrad H, Muck D, Hein R, 

Blank C, Peschel C, Haller B, Schulz S, 

Bernhard H. PD-1 expression on 

MelanA-reactive T cells increases during 

progression to metastatic disease. Int J 

Cancer 2011 

Gadiot J, Hooijkaas AI, Kaiser AD, van 

Tinteren H, van Boven H, Blank C. 

Overall survival and PD-L1 expression in 

metastasized malignant melanoma. 

Cancer 2011;117:2192-201

62 

MOLECULAR BIOLOGY 

Division head, group leader Hein te Riele 

Hein te Riele PhD Group leader 

Rob Dekker Post-doc 

Henri van de Vrugt Post-doc 

Camiel Wielders PhD Post-doc 

Kamila Wojciechowicz-Grzadka Post-doc 

Sietske Bakker MSc PhD student 

Erika Cantelli PhD student 

Tim Harmsen PhD student 

Tanja van Harn PhD student 

Hellen Houlleberghs PhD student 

Eva Wielders Msc PhD student 

Marleen Dekker Technical staff 

Elly Delzenne-Goette Technical staff 

Sandra de Vries MSc Technical staff 

Anja van der Wal Technical staff 

Publications 

Aarts M and Te Riele H. Progress and 

prospects: oligonucleotide-directed gene 

modifi cation in mouse embryonic stem cells: 

a route to therapeutic application. Gene Ther 

2011;18:213-9 

Wielders EAL, Dekker RJ, Holt I, Morris 

GE and Te Riele H. Characterization of 

MSH2 variants by endogenous gene 

modifi cation in mouse embryonic stem cells. 

Human Mutation 2011;32:389-96 

Johannesma PC, Van der Klift HM, Van 

Grieken NCT, Troost D, Te Riele H, Jacobs 

MAJM, Postma TJ, Heideman DAM, Tops 

CMJ, Wijnen JT, Menko FH. Childhood brain 

tumours due to germ line bi-allelic mismatch 

repair gene mutations. Clin Genet 

2011;80:243-55 

Holt I, Lam LT, Tomé S, Wansink DG, 

Te Riele H, Gourdon G, Morris GE. The 

mouse mismatch repair protein, MSH3, is a 

nucleoplasmic protein that aggregates into 

denser nuclear bodies under conditions of 

stress. J Cell Biochem 2011;112:1612-21 

Dekker M, De Vries S, Aarts M, Dekker 

R, Brouwers C, Wiebenga O, De Wind N, 

Cantelli E, Tonelli R, and Te Riele H. 

Transient suppression of MLH1 allows 

effective single-nucleotide substitution by 

single-stranded DNA oligonucleotides. 

Mutation Res 2011;715:52-60 

Van de Vrugt HJ, Koomen M, Bakker S, 

Berns MA, Cheng NC, van der Valk MA, de 

Vries Y, Rooimans MA, Oostra AB, Hoatlin 

ME, Te Riele H, Joenje H, Arwert F. Evidence 

for complete epistasis of null mutations in 

DIVISION OF MOLECULAR BIOLOGY 

Genetic instability and deregulated cell cycle control are hallmarks of human cancer. 

Our research involves both aspects focusing on (1) the role of the DNA mismatch 

repair and Fanconi anemia genome maintenance pathways in mutation avoidance 

and (2) the role of cell cycle checkpoints in tumor suppression. The principle tools 

include gene modifi cation in murine embryonic stem cells (ESC) and analyses of the 

phenotypic consequences in ESC, mutant mice and cell lines derived thereof. 

DNA MISMATCH REPAIR 

Lynch syndrome/HNPCC (hereditary non-polyposis colorectal cancer) is caused by 

inherited defects in DNA mismatch repair (MMR). The primary function of MMR 

is correction of DNA replication errors, which are recognized by MSH2/MSH6 or 

MSH2/MSH3 protein complexes. Subsequent recruitment of another heterodimeric 

protein complex, MLH1/PMS2, activates exonucleolytic activity to remove the errorcontaining 

DNA strand allowing resynthesis of a new error-free strand. 

Mismatches can also arise by replication of damaged bases such as O 6 -methylguanine. 

This lesion elicits futile cycles of repair, leading to double-strand break formation 

and cell death. Thus, DNA MMR acts anti-mutagenic and mediates the toxicity of 

methylating agents. 

Oligonucleotide-directed gene modification MMR can also recognize 

mismatches that arise artifi cially in cells, e.g., during oligonucleotide-directed 

gene modifi cation. ‘Oligo targeting’ makes use of single-stranded oligodeoxyribonucleotides 

(ssODN) of ~ 35 nucleotides that are identical to the 

chromosomal target sequence except for the nucleotide(s) that comprise(s) the 

desired modifi cation. However, in mouse ESCs, oligo targeting appeared only 

effective in the absence of Msh2 (Dekker et al., NAR 2003;31:e27). Apparently, 

recognition by MMR of the incomplete base paring between the ssODN and its 

chromosomal complement leads to abortion of the gene modifi cation reaction. 

As readout for oligo targeting we have generated ESCs carrying single-copy 

chromosomally-located G418 resistance (neo) or EGFP reporter genes that were 

disabled by a mutation in the start codon. ssODN-mediated restoration of the start 

codon generates G418-resistant or green-fl uorescent cells. Using the EGFP reporter 

we found that anti-sense ssODN immediately yielded green-fl uorescent cells, 

whereas with sense ssODN, green fl uorescent cells only appeared after 48h (Aarts 

and Te Riele, NAR 2010;38:6956). This observation supports a model in which the 

ssODN physically integrates into the host genome during DNA replication. 

ESCs can be rendered permissive for oligo targeting by shRNA-mediated transient 

suppression of either MSH2 (Aarts et al., NAR 2006;34:e147) or MLH1 (Dekker 

et al., Mutation Res 2011;715:52-60). Whereas MSH2 knockdown allows effective 

substitution of 3-4 adjacent nucleotides, single nucleotide substitution can be 

achieved upon knockdown of MLH1. To identify cells carrying a single base-pair 

alteration, we developed a real-time PCR protocol that makes use of a so-called 

mismatch-amplifi cation-mutation-assay (MAMA) primer. The latter was specifi cally 

designed to detect the single nucleotide modifi cation amidst a vast excess of nonmodifi 

ed alleles (Dekker et al., Mutation Res 2011;715:52-60). By this approach we 

have substituted a single base pair in the mouse Mycn gene (fi gure 1). 

Chemical modifi cation by phosphorothioate (PTO) linkages, often used to protect 

ssODN from exonucleolytic degradation and to improve the targeting effi ciency, 

appeared detrimental to cells and impeded the outgrowth of targeted cells. 

Unmodifi ed ssODNs were less harmful and ultimately yielded higher targeting 

frequencies than PTO-ssODNs (Aarts and Te Riele, NAR 2010; 38:6956). 

Unclassified variants of MMR genes Single codon variants of MMR genes are 

widespread in the human population and in (suspected) Lynch syndrome patients 

but their phenotypic consequences are often diffi cult to predict. We use oligo

targeting to recreate suspected variants of MMR genes in ESCs in order to assess 

their MMR capacity. We have thus far analyzed fi ve of such ‘Variants of uncertain 

signifi cance’ (VUS) of MSH2 and found one fully and another partially abrogating 

MMR activity (Wielders et al., Human Mutation 2011;32:389-96). Introduced into 

the germ line of mice, we found this variant to induce tumour formation to the 

same extend as a full Msh2 knockout, identifying it as a deleterious mutation. 

Three variants showed normal MMR capacity, which is remarkable in view of the 

evolutionary conservation of the affected amino acids. Also three MSH6 variants 

carrying codon substitutions at conserved positions showed wild-type activity. 

FANCONI ANEMIA 

Another example of cancer predisposition by inherited defects in DNA repair is 

Fanconi anemia (FA), a recessive disorder characterized by malformations, progressive 

anemia and high incidence of AML and HNSCC. FA is caused by bi-allelic defects in 

either one of 15 genes, FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P. The products 

of these genes are essential for the repair of DNA interstrand crosslinks (ICL). 

To assess the signifi cance of the FA genome maintenance pathway in suppression 

of cancer, we have generated Fancf- and Fancm-defi cient mice. FANCF and FANCM 

are part of the FA core complex that is essential for mono-ubiquitination of FANCD2 

and FANCI. Both, Fancf and Fancm defi ciency caused hypogonadism in mice and 

hypersensitivity to cross-linking agents in mouse embryonic fi broblasts (MEFs), 

thus phenocopying other FA mouse models. Fancf-defi cient female mice were 

highly prone to development of granulosa cell tumors (Bakker et al., J Pathology 

2012;226:28-39). Also Fancm defi ciency caused decreased overall and tumor-free 

survival (Bakker et al., Hum Mol Genet 2009;18:3484), however, no specifi c tumor 

type was seen. At the cellular level, FANCM was found to suppress spontaneous 

(but not induced) sister chromatid exchanges, a role that seems independent of its 

function in the FA core complex. The Fancf and Fancm knockout alleles have been 

crossed into cancer prone Apc +/- and Eμ-Pim transgenic mice to study whether 

defects in the FA pathway accelerate tumorigenesis. 

CELL CYCLE CHECKPOINTS 

Loss of G 1/S control is a frequent if not mandatory event in tumor development. G 1/S 

control relies on the pocket proteins pRB, p107 and p130 that collectively regulate the 

activity of E2F transcription factors. We use MEFs devoid of pocket proteins (TKO 

MEFs) to study residual cell cycle control mechanisms and to identify events that 

promote oncogenic transformation. 

Growth-factor independence TKO MEFs still rely on mitogens for survival 

and proliferation. In the absence of mitogens, many TKO MEFs die whereas the 

surviving fraction arrests in the G 2 phase of the cell cycle. G 2 arrest was effectuated 

through inhibitory interactions of the cyclin-dependent-kinase inhibitors p21 CIP1 

and p27 KIP1 with Cyclins A and B1 (Foijer et al., Cancer Cell 2005;8:455). G 2 arrested 

cells showed high levels of DNA double strand breaks, which were only partially 

repaired when TKO cells were re-stimulated to enter the cell cycle. Moreover, mitotic 

chromosomes showed a ‘railroad’ appearance indicative of defects in centromeric 

sister-chromatid cohesion (Van Harn et al., Genes Dev 2010;24:1377). Furthermore, 

we found aneuploidy in cell cultures derived from TKO cells that had been 

transiently mitogen deprived. Current experiments indicate that mitogen-starved 

TKO cells sustain severe replication stress that contributes to G 2 arrest. 

Anchorage independence Although TKO MEFs are immortal and refractory to 

RAS V12 -induced senescence, they were not transformed by RAS V12 (Vormer et al., 

MCB 2008;28:7263). Apparently, a cell cycle mechanism still operates to restrict 

proliferation of TKO cells in the absence of anchorage. To identify this mechanism, 

we are using both gain-of-function and loss-of-function screening. We found 

that ectopic expression of the immortalizing oncogene TBX2 allowed anchorage- 

63 



murine Fanconi anemia genes Fanca and 

Fancg. DNA Repair 2011;10:1252-61 

Bakker ST, Van de Vrugt HJ, Visser JA, 

Delzenne-Goette A, Van der Wal A, Berns 

MAD, Van de Ven M, Oostra AB, De Vries S, 

Kramer P, Arwert F, Van der Valk M, 

De Winter JP, and Te Riele H. Fancf-defi cient 

mice are prone to develop ovarian tumors. 

J Pathology 2012;226:28-39 

Bakker ST. Fancm, the mouse that 

roared. PhD thesis VU University Amsterdam, 

December 2011 

Figure 1: Oligonucleotide-directed single 

basepair substitution in murine Mycn. 

(A) The Mycn sequence around the G.C 

basepair (*, indicated in bold); the antisense 

ssODN designed to substitute G.C for C.G 

(modifying base G indicated in bold, italics); 

position of the Mycn Forward primer and the 

sequence of the mutation-specific Mycn 

MAMA primer containing a non-matching 

nucleotide at the penultimate position (A in 

bold, italics). After transient suppression of 

MLH1 and introduction of the ssODN, cells 

were seeded onto 96-wells plates at 5000 cells 

per well. Wells containing modifi ed cells were 

identifi ed by real-time PCR using the 

mutation-specifi c Mycn MAMA primer. 

In subsequent rounds, enrichment for 

mutant cells was obtained by reseeding 

positive wells at 1000, 100 and 1 cell per well. 

(B) Real time PCR showing amplification of 

wild-type alleles (blue lines, left) using a 

wild-type sequence-specific primer, and 

mutant alleles using the Mycn MAMA 

primer. The red lines (most right) indicate 

background amplification; the green lines 

indicate the presence of modified sequences. 

Grey lines indicate background amplification 

or a low fraction of modified alleles.

64 


Group leader Piet Borst 

Piet Borst MD PhD Group leader 

Henri van Luenen PhD Academic staff 

Sven Rottenberg DVM PhD Dipl ECVP 

Senior post-doc 

Koen van de Wetering DVM PhD Senior 

post-doc 

Charlotte Guyader PhD Post-doc 

Robert Jansen PhD Post-doc 

Pankaj Tripathi PhD Post-doc 

Nikola Banishki MSc PhD student 

Guotai Xu MSc PhD student 

Serge Zander DVM MSc PhD student 

Marcel de Haas Technical staff 

Liesbeth van Deemter Technical staff 

Sabrina Jan Technical staff 

Ariena Kersbergen Technical staff 

Sunny Sapthu Technical staff 

Wendy Sol Technical staff 

Figure 2: Transport of unknown MRP2substrates 

present in urine in vesicular 

transport experiments. MRP2-containing (A) 

or control (B) inside-out membrane vesicles 

were incubated in the presence of 4 mM ATP 

for 10 min at 37 ˚C in a diluted urine sample 

of an Mrp2-/- mouse. The vesicle content was 

subsequently analysed by an LC/MS based 

targeted-metabolomics screen, specifi cally 

detecting glucuronosyl conjugates. Every colour 

represents the chromatogram of a specifi c mass 

transition in which 176 Da (the glucuronic 

acid moiety) was lost after fragmentation. 

In subsequent experiments we identifi ed the 

glucuronides transported by MRP2 into the 

vesicles as phytoestrogen-glucuronides 

independent growth of RAS V12 -expressing TKO MEFs. For loss-of-function 

screening, we have developed a new technique that allows enzymatic production 

of shRNA libraries from cDNAs extracted from arrested cells. Screening of these 

libraries uncovered several novel suppressors of anchorage-independent growth. 

DNA BASE J 

Base J (β-glucosyl-hydroxymethyluracil), which we discovered in African 

trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993;75:1129-1136), is a base 

present in kinetoplastid fl agellates and in Euglena. It replaces 1% of thymine in 

nuclear DNA and is predominantly located in repetitive sequences, such as telomeric 

repeats. We have characterized a J-binding protein (JBP1) that binds to J-containing 

duplex DNA (Cross et al. EMBO J 1999;18:6573-6581). We have shown that JBP1 is a 

thymidine hydroxylase that catalyses the fi rst step of J biosynthesis, the conversion 

of specifi c T-residues in DNA into hydroxymethyluracil. JBP1 belongs to the family 

of Fe 2+ - and 2-oxoglutarate-requiring dioxygenases, as does a second putative 

hydroxylase, JBP2. In the kinetoplastid Leishmania J is located for > 98% in telomeric 

repeats. A JBP1 KO is lethal. In contrast, JBP2 is dispensable in Leishmania, but JBP2 

KO strains are hypersensitive to bromodeoxyuridine (BrdU). The J level goes down to 

30% of WT in the Leishmania JBP KO strains and during growth in BrdU, Leishmania 

JBP2 KO strains lose even more J (down to 13% of WT). How J loss leads to cell death 

was long unclear. Using immuno-precipitation of J-DNA and deep sequencing, 

we have found the 1% of non-telomeric J in Leishmania at specifi c chromosomeinternal 

positions, partly at transcriptional stops (collaboration with NKI-AVL deep 

sequencing unit and Peter Myler, Seattle). We have shown that loss of this internal J 

leads to massive read-through of RNA Polymerase II transcriptional stops, providing 

a plausible explanation for J-less death. With Jonas Korlach (Pacifi c Biosciences, 

USA) we are using SMRT sequencing to locate the exact positions of J in DNA. With 

Anastassis Perrakis (NKI-AVL) we are trying to determine the structure of JBP1-J- 

DNA complexes by crystallography. The structure of the DNA-binding domain of JBP1 

was solved already. Interestingly, this domain has a unique structure not seen before 

in DNA-binding proteins and the specifi c binding of JBP1 to J-DNA was shown to be 

dependent on a single aspartate residue interacting with the glucose-moiety of base J. 

DRUG TRANSPORTERS 

We are interested in mechanisms of drug resistance in cancer cells and focus on 

resistance caused by increased ATP-dependent transport of drug out of the cell, 

mediated by ATP-binding cassette (ABC) transporters. We have isolated genes for 

these transporters and are characterizing their substrate specifi city and sensitivity 

to inhibitors in transfected cells. To study the physiological function of these 

transporters, we have inactivated genes for several drug transporters by targeted 

gene disruption in mice. We are mainly studying the Multidrug Resistance Protein 

(ABCC) family members MRP2, 3, 4, 5 and 6. 

We have initiated a systematic search for compounds conjugated to glucuronide 

or sulphate that are transported by MRPs by comparing the derivatives in plasma/ 

urine of WT and KO mice using Mass Spectrometry (MS). We have identifi ed 

several glucuronidated and sulphated phyto-estrogens, derived from food, as novel 

MRP2 and MRP3 substrates by this approach. We have also recently identifi ed novel 

substrates of MRP4 and MRP5. We are refi ning the LC/MS analysis to allow the 

identifi cation of all compounds altered in plasma/urine of KO mice. This approach

should also be helpful in fi nding substrates of other MRPs and BCRP (ABCG2). 

We have recently developed a new method to study the substrate spectrum of ABC 

transporters: we incubate extracts of mouse urine with membrane vesicles prepared 

from cells overproducing an ABC transporter and determine the compounds 

transported into the vesicles by LC/MS-based metabolomics. An example of the 

power of this approach is shown in Figure V.2 in which we analyzed the substrates 

taken up by MRP2-containing vesicles from urine. 

DRUG RESISTANCE IN “SPONTANEOUS” MOUSE TUMORS 

In collaboration with Jos Jonkers (NKI-AVL), we are studying resistance mechanisms 

in “spontaneous” mammary tumors arising in mice, conditionally defective in p53 

and Brca1. When treated with the maximum tolerable dose of doxorubicin, docetaxel 

or topotecan, the breast tumors initially respond but eventually always develop 

resistance. Resistance is often associated with upregulation of the Mdr1a and Mdr1b 

genes (Abcb1), which encode drug-transporting P-glycoproteins (P-gps) and we have 

shown with specifi c inhibitors that remarkably low levels of Abcb1 upregulation 

(5-fold the levels in sensitive tumors) suffi ce to make the tumor multidrug resistant. 

We are also using this mouse model to test new anticancer drugs and drug 

combinations. Impressive tumor regression has been obtained with a new inhibitor 

of Poly-ADP-ribose polymerase 1 (PARP1), olaparib, but resistance to this compound 

also arises by Abcb1 upregulation. We have crossed disrupted alleles for the Abcb1 

genes into our mouse model. The tumors without P-gp are hypersensitive to several 

drugs and we are using mice with such tumors to uncover other forms of resistance, 

- notably to doxorubicin, docetaxel and olaparib -, not mediated by P-gp. Loss of 

53BP1, resulting in partial restoration of DNA repair by homologous recombination 

is one such resistance mechanism (fi gure 3). 

In contrast to the results obtained with MDR drugs, we have been unable to obtain 

cisplatin resistance in this tumor model. The tumors respond to each new treatment 

with cisplatin, but are never fully eradicated. Tumor-initiating cells (“stem cells”) are 

not enriched in the “remnants” from which the tumors regrow after chemotherapy. 

We think that the resistance of “remnants” is not due to specifi c biochemical defense 

mechanisms of the putative tumor stem cells, but to the ability of a sub-fraction of 

the cells to go into “hibernation”. We have isolated Brca1 -/- ;p53 -/- tumor cell lines in 

low O 2 that resemble the original tumor, such as B11 cells. Cells surviving cisplatin 

appear to stall in G1/G0. We are studying how these cells escape cisplatin-induced 

death. This system is also used to screen for genes that can increase resistance to 

cisplatin or olaparib. 

Brummelkamp and co-workers have isolated a human cell line that is nearly 

completely haploid. This cell line is very suitable for identifying genes that 

affect resistance in a recessive fashion, i.e. drug import transporters. With Thijn 

Brummelkamp (NKI-AVL) we have started to screen this cell line for recessive genes 

affecting resistance to a wide series of anti-cancer drugs in clinical use. 

Publications 

65 


Beekman CA, Buckle T, van Leeuwen 

AC, Valdes Olmos RA, Verheij M, 

Rottenberg S et al. Questioning the value 

of (99m)Tc-HYNIC-annexin V based 

response monitoring after docetaxel 

treatment in a mouse model for hereditary 

breast cancer. Appl Radiat Isot 

2011;69:656-62 

Drost R, Bouwman P, Rottenberg S, 

Boon U, Schut E, Klarenbeek S, Klijn C, 

Van der Heijden I, van der Gulden H, 

Wientjens E, Pieterse M, Catteau A, 

Green P, Solomon E, Morris J, Jonkers J. 

BRCA1 RING function is essential for 

tumor suppression but dispensable for 

therapy resistance. Cancer Cell 2011 (in 

press) 

Fulop K, Jiang Q, Wetering KV, 

Pomozi V, Szabo PT, Aranyi T et al. 

ABCC6 does not transport vitamin 

K3-glutathione conjugate from the liver: 

Relevance to pathomechanisms of 

pseudoxanthoma elasticum. Biochem 

Biophys Res Commun 2011 

Heidebrecht T, Christodoulou E, 

Chalmers M, Jan S, ter Riet B, Grover R 

et al. The structural basis for recognition 

of J-base containing DNA by a novel 

DNA-binding domain in JBP1. NAR 

2011;39:5715-28 

Krumpochova P, Sapthu S, Brouwers 

JF, De Haas M, de Vos R, Borst P, Van de 

Wetering K. Transportomics: screening for 

substrates of ABC transporters in body 

fl uids using vesicular transport assays. 

FASEB J 2011 (in press) 

Robertson AB, Dahl JA, Vagbo CB, 

Tripathi P, Krokan HE, Klungland A. A 

novel method for the effi cient and selective 

identifi cation of 5-hydroxymethylcytosine 

in genomic DNA. Nucleic Acids Res 


Rottenberg S, Borst P. Drug resistance 

in the mouse cancer clinic. Current Cancer 

Ther Rev 2011 (in press) 

Figure 3: The effect of 53BP1 loss on 

olaparib sensitivity (a) Western blot 

showing 53BP1 levels in Brca1-/- ;p53-/- mouse mammary tumor cells (B11) that 

express a non-targeting scrambled hairpin 

(SCR) or a hairpin against 53bp1. 

(b) Clonogenic assay using olaparib. 

The IC50 is indicated between brackets. 

(c) Overall survival of mice carrying 

53BP1-positive (shSCR) or –negative 

(sh53BP1) tumors treated with 50mg 

olaparib per kg i.p. daily for 28 days or left 

untreated.

66 



Group leader Jos Jonkers 

Jos Jonkers PhD Group leader 

Karin de Visser PhD Associate staff member 

Peter Bouwman PhD Post-doc 

Seth Coffelt PhD Post-doc 

Gilles Doumont PhD Post-doc 

Linda Henneman PhD Post-doc 

Ewa Michalak PhD Post-doc 

Petra ter Brugge PhD Post-doc 

Marieke van de Ven PhD Post-doc 

Martine van Miltenburg PhD Post-doc 

Metamia Ciampricotti MSc PhD student 

Chris Doornebal MSc PhD student 

Rinske Drost MSc PhD student 

Janneke Jaspers MSc PhD student 

Sjors Kas MSc PhD student 

Kelly Kersten MSc PhD student 

Sjoerd Klarenbeek MSc PhD student 

Christiaan Klijn MSc PhD student 

Hanneke van der Gulden Technical staff 

Ingrid van der Heijden Technical staff 

Ellen Wientjens Technical staff 

Ute Boon Research assistant 

Tanya Braumuller Research assistant 

Tissee Hau Research assistant 

Eva Kregel Research assistant 

Mark Pieterse Research assistant 

Eline van der Burg Research assistant 

Publications 

Michalak EM, Jonkers J. Studying therapy 

response and resistance in mouse models for 

BRCA1-defi cient breast cancer. J Mammary 

Gland Biol Neoplasia 2011;16:41-50 

Bouwman P, Drost R, Klijn C, Pieterse 

M, van der Gulden H, Song JY, Szuhai K, 

Jonkers J. Loss of p53 partially rescues 

embryonic development of Palb2 knockout 

mice but does not foster haploinsuffi ciency of 

PALB2 in tumour suppression. J Pathol 

2011;224:10-21 

Ciampricotti M, Vrijland K, Hau CS, 

Pemovska T, Doornebal CW, Speksnijder 

EN, Wartha K, Jonkers J, de Visser KE. 

Development of metastatic HER2+ breast 

cancer is independent of the adaptive immune 

system. J Pathol 2011;224:56-66 

Derksen PW, Braumuller TM, van der 

MOUSE MODELS OF BREAST CANCER 

The focus of our group is on the genetic dissection of human breast cancer through 

the use of genetically engineered mouse models (GEMMs). For this, we have 

developed models for p53-induced breast cancer, BRCA1- and BRCA2- associated 

hereditary breast cancer, and E-cadherin-mutated lobular breast cancer. We are 

using these models to (1) investigate genotype-phenotype relations in mammary 

tumorigenesis; (2) study therapy response and resistance of primary tumors and 

metastases; (3) study the role of infl ammation in breast cancer development and 

therapy response; (4) identify genetic changes underlying breast tumorigenesis. 

Functional assays in BRCA-deficient ES cells To identify factors that mediate the 

growth arrest induced by BRCA1 loss we have performed cellular survival screens 

in ES cells containing selectable conditional knockout alleles of Brca1 and Brca2. 

We found that inactivation of 53BP1 rescues the proliferation and homologousrecombination 

defects of BRCA1-defi cient cells and reverts their hypersensitivity to 

DNA-damaging agents. Notably, loss of 53BP1 expression is also found in subsets of 

sporadic triple-negative and BRCA-associated breast cancers. We are currently using 

our selectable conditional Brca1 knockout ES cells to perform survival screens with 

focused libraries of shRNA vectors targeting DDR factors, chromatin regulators and 

deubiquitinating enzymes. 

We have also used our selectable conditional ES cells to perform functional 

complementation assays for testing human BRCA1 variants of unknown clinical 

signifi cance (VUS). For this, we have engineered our ES cells to enable rapid knockin 

of human BRCA1 cDNAs by recombinase mediated cassette exchange (RMCE). 

Introduction of wild-type hBRCA1 – but not pathogenic hBRCA1 mutants – rescues 

the growth defect of ES cells lacking endogenous BRCA1. We have so far tested 100 

defi ned hBRCA1 VUSs in our functional complementation assay. 

Mouse models for BRCA-associated breast cancer We have previously generated 

GEMMs for BRCA1- and BRCA2-associated hereditary breast cancer. The BRCA1defi 

cient mouse mammary tumors share histopathological and molecular features 

with BRCA1-mutated breast cancers in women: they are highly proliferative, poorly 

differentiated adenocarcinomas that lack expression of hormone receptors (ER, PR) 

and ERBB2. Interestingly, we have found that mammary tumor formation in our 

BRCA1 models is still estrogen-dependent, suggesting that SERMs or aromatase 

inhibitors may be effective agents for chemoprevention of breast cancer in BRCA1mutation 

carriers. 

The central role of BRCA1 and BRCA2 in the DNA damage response (DDR) implies 

that BRCA-defi cient tumors are especially sensitive to DNA-damaging agents. 

In collaboration with Sven Rottenberg and Piet Borst we have used our BRCA1/2 

models to test the anti-tumoral effi cacy of the PARP inhibitor AZD2281 (olaparib), 

which displays selective toxicity to BRCA-defi cient cells. While administration 

of olaparib to mice with BRCA1- or BRCA2-defi cient mammary tumors caused 

tumor shrinkage without signs of toxicity, long-term treatment induced acquired 

drug resistance caused by upregulation of the P-glycoprotein (Pgp) drug effl ux 

transporter. To study Pgp-independent mechanisms of olaparib resistance, we 

have crossed the BRCA1 mammary tumor model onto a Pgp-defi cient background 

and transplanted the resulting BRCA1- and Pgp-defi cient mammary tumors into 

wildtype recipients. Repeated treatment of these mice with olaparib resulted in 

induction of drug resistance without loss of target (PARP) inhibition. In 25% of the 

cases, olaparib resistance was caused by somatic mutations in 53bp1, highlighting 

the role of this DDR factor in therapy resistance. 

To study the effects of specifi c BRCA1 mutations on tumorigenesis and therapy 

response, we have generated mouse mutants mimicking the human BRCA1- 

185delAG, BRCA1-5382insC and BRCA1-C61G founder mutations and crossed these 

3 mouse strains into our BRCA1 mammary tumor model. All 3 mutants fail to 

suppress mammary tumor formation, but show different activities in the DNA 

damage response following treatment of tumors with platinum drugs or PARP 

inhibitors. Whereas BRCA1-null and BRCA1-5382insC tumors never develop 

resistance to cisplatin, the BRCA1-185delA and BRCA1-C61G tumors readily become

esistant while retaining the Brca1 founder mutation, suggesting that BRCA1 RING 

function is required for tumor suppression but dispensable for therapy resistance. 

Mouse models for E-cadherin-mutated invasive lobular breast cancer Loss 

of E-cadherin is associated with invasive lobular carcinoma (ILC), which accounts 

for 10-15% of all breast cancers. To study the causal role of E-cadherin in breast 

oncogenesis, we have generated mouse models for invasive lobular breast carcinoma 

(ILC) based on epithelium-specifi c inactivation of E-cadherin and p53. Compared to 

p53 –/– mammary carcinomas, Ecad –/– ;p53 –/– mammary tumors show a signifi cantly 

reduced latency, a morphological switch from ductal to pleomorphic lobular 

carcinoma, and a phenotypic change from non-invasive to highly invasive and 

metastatic tumors. 

Human ILC is characterized by a high frequency of PI3K pathway mutations. 

To study the role of PI3K activation in lobular breast cancer formation, we have 

generated mice with mammary-specifi c loss of E-cadherin and PTEN. Ecad –/– 

;Pten –/– mammary tumors resemble human classical ILC and develop signifi cantly 

faster than p53 –/– mammary tumors, demonstrating synergism between and 

E-cadherin mutation and PTEN loss. To study the role of PI3K pathway activation 

in maintenance of established ILCs, we are using the Ecad –/– ; p53 –/– and Ecad –/– ; 

Pten –/– mammary tumor models for intervention studies with mTOR inhibitors and 

other PI3K pathway inhibitors. 

The inflammatory tumor-microenvironment and its impact on breast 

cancer development and therapy Immune cells are one of the most abundant 

cell types recruited to the microenvironment of many tumors. Their role during 

tumorigenesis is, however, controversial, as both tumor-protective and tumorpromoting 

properties have been reported. The overall goal of the research group 

of Karin de Visser is to study the role of the adaptive and innate immune system 

in spontaneous breast cancer progression, metastasis formation and therapy 

response. In addition to the MMTV-NeuT mouse model for HER-positive breast 

cancer, the above-mentioned Ecad –/– ; p53 –/– mammary tumor model for human 

ILC is employed. Like human breast cancers, mammary carcinomas arising in this 

mouse model are characterized by abundant presence of immune cells, antibody 

depositions and increased levels of pro-infl ammatory mediators. By genetic 

elimination and pharmacological inhibition of specifi c subsets of the innate and 

adaptive immune system, we have identifi ed a critical role for adaptive immune cells 

in spontaneous metastasis formation. We are currently investigating the underlying 

mechanisms. 

We are also using spontaneous mammary tumor models to study the ability of the 

immune system to modulate chemotherapy response and resistance. To test the role 

of the adaptive immune system in chemotherapy response, we have bred the MMTV- 

NeuT and E-cadherin mammary tumor models onto a T and B cell defi cient Rag2defi 

cient background. Interestingly, absence of the adaptive immune system affected 

neither mammary tumor latency nor responses of established tumors to cisplatin, 

oxaliplatin or doxorubicin. These results stand in contrast to the previously reported 

dependency of the antitumoral effi cacy of oxaliplatin and doxorubicin on anti-tumor 

adaptive immune responses (Tesneire et al., Curr Opin Immunol 2008;20:504-11). 

Identification and validation of novel cancer genes We have employed various 

genomics approaches (aCGH, next-gen sequencing and insertional mutagenesis 

screens) to identify novel cancer genes in our mouse mammary tumor models. We 

have developed several computational algorithms (KC-SMART and comparative KC- 

SMART) to identify networks of co-occurring and mutually exclusive mutations. To 

facilitate functional in vivo validation of candidate cancer genes, we have developed 

together with the Berns group a rapid procedure for RMCE-mediated introduction 

of additional mutations in embryonic stem cells (ESCs) derived from established 

GEMMs of human cancer. The resulting GEMM-ESCs can be used to generate 

experimental mouse cohorts via blastocyst injections. We are currently using this 

procedure to introduce a number of different candidate cancer genes in our BRCA1 

and E-cadherin mammary tumor models. 

67 



Burg E, Hornsveld M, Mesman E, Wesseling 

J, Krimpenfort P, Jonkers J. Mammary-specifi c 

inactivation of E-cadherin and p53 impairs 

functional gland development and leads to 

pleomorphic invasive lobular carcinoma in 

mice. Dis Model Mech 2011;4:347-358 

Schackmann RC, van Amersfoort M, 

Haarhuis JH, Vlug EJ, Halim VA, Roodhart 

JM, Vermaat JS, Voest EE, van der Groep P, 

van Diest PJ, Jonkers J, Derksen PW. 

Cytosolic p120-catenin regulates growth of 

metastatic lobular carcinoma through 

Rock1-mediated anoikis resistance. J Clin 

Invest 2011;121:3176-3188 

Huijbers IJ, Krimpenfort P, Berns A, 

Jonkers J. Rapid validation of cancer genes in 

chimeras derived from established genetically 

engineered mouse models. Bioessays 

2011;33:701-710 

De Jong J, de Ridder J, van der Weyden L, 

Sun N, van Uitert M, Berns A, van Lohuizen 

M, Jonkers J, Adams DJ, Wessels LF. 

Oncogene discovery by direct association of 

insertion features with gene expression. 

Nucleic Acids Res 2011;39:e105 

Koudijs MJ, Klijn C, van der Weyden L, 

Kool J, ten Hoeve J, Sie D, Prasetyanti PR, 

Schut E, Kas S, Whipp T, Cuppen E, Wessels 

L, Adams DJ, Jonkers J. High-throughput 

semi-quantitative analysis of insertional 

mutations in heterogeneous tumors. Genome 

Res 2011;21:2181-2189 

Shimada S, Mimata A, Sekine M, 

Mogushi K, Akiyama Y, Fukamachi H, 

Jonkers J, Tanaka H, Eishi Y, Yuasa Y. 

Synergistic tumor suppressor activity of 

E-cadherin and p53 in a conditional mouse 

model for metastatic diffuse-type gastric 

cancer. Gut 2011 

Drost R, Bouwman P, Rottenberg S, Boon 

U, Schut E, Klarenbeek S, Klijn C, van der 

Heijden I, van der Gulden H, Wientjens E, 

Pieterse M, Catteau A, Green P, Solomon E, 

Morris JR, Jonkers J. BRCA1 RING function 

is essential for tumor suppression but 

dispensable for therapy resistance. Cancer Cell 

2011;20:797-809 

Vollebergh MA, Jonkers J, Linn SC. 

Genomic instability in breast and ovarian 

cancers: translation into clinical predictive 

biomarkers. Cell Mol Life Sci 2012;69:223-245 

Krimpenfort K, Song JY, Proost N, 

Zevenhoven J, Jonkers J, Berns A. Deleted in 

Colorectal Carcinoma (DCC) suppresses 

metastasis formation in p53 defi cient 

mammary tumors. Nature (in press) 

Ciampricotti M, Hau CS, Doornebal CW, 

Jonkers J, de Visser KE. Chemotherapy 

response of spontaneous mammary tumors is 

independent of the adaptive immune system. 

Nat Med (in press)

68 



Group leader Sabine Linn 

Sabine Linn MD PhD Group leader 

Wilbert Zwart PhD Senior post-doc 

Eelke Gort PhD Post-doc 

Karin Beelen MD PhD student 

Rutger Koornstra MD PhD student 

Philip Schouten MSc PhD student 

Marieke Vollebergh MD PhD student 

Mark Opdam Technical staff 

Tesa Severson MSc Technical staff 

Figure 4: Overview of histological patient 

characteristics and the aCGH classifi cation 

per patient (n=249). Characteristics 

studied: Estrogen-receptor (ER) (cut-off 10% 

positive tumor cells); Progesterone-receptor 

(PgR) (cut-off 10% positive tumor cells); 

HER2 status: the study comprised only 

HER2 negative patients; Bloom-Richardson 

Grade I+II (negative) vs. Grade III 

(positive); non-BRCA1-likeCGH (negative) 

vs. BRCA1-like CGH (positive); non-BRCA2likeCGH 

(negative) vs. BRCA2-like CGH 

(positive). 

Legends consist of light grey: negative cases; 

dark grey: positive cases for abovementioned 

characteristic; medium grey: cases unknown 

for above-mentioned characteristic. 

MOLECULAR DISSECTION OF CANCER BY DIFFERENTIAL 

DRUG SENSITIVITY 

In the clinic, we mainly use anticancer drugs based on outcomes of clinical trials that 

have been carried out in the general breast cancer population, whereas little is known 

about the molecular mechanisms underlying differential drug sensitivity. The same 

holds true for other cancer types, including non-small cell lung cancer (NSCLC), 

stomach cancer, ovarian cancer, and colorectal cancer. The focus of our research line 

is to unravel these molecular mechanisms in order to develop tests that may guide 

treatment decisions in the clinic and ultimately improve survival. For this purpose 

we use several genome-wide approaches and molecular techniques, in order to dissect 

the mechanisms that divide clinically well-defi ned cohorts of breast, colorectal, 

stomach, ovarian and NSCLC patients into resistant and sensitive to a particular 

drug. In addition, we have a close collaboration with the groups of Jos Jonkers and 

Piet Borst, who use conditional mouse models for breast cancer, and derived clonal 

cell lines, to study differential chemosensitivity in a controlled fashion. 

A second research line focuses on the impact of prognostic molecular classifi ers on 

adjuvant systemic treatment advice in breast cancer. 

Five year follow-up data of the 70-gene prognosis signature in daily clinical 

practice In collaboration with the Divisions of Diagnostic Oncology, Medical 

Oncology, and Surgical Oncology and Agendia BV, we have demonstrated that the 

5-year distant disease-free survival (DDFS) probabilities confi rmed the additional 

prognostic value of the 70-gene signature to clinico-pathologic factors used in 

AdjuvantOnline (AOL) risk estimations. If in a comparable cohort diagnosed 

today the 70-gene signature would be added to standard guidelines used to select 

patients for adjuvant systemic therapy, a reduction of ~ 30% in the use of adjuvant 

chemotherapy would be seen. Omission of adjuvant systemic therapy as judged 

appropriate by doctors and patients and supported by a low risk 70-gene signature 

test appeared indeed safe. In the group that did not receive any adjuvant systemic 

treatment (chemotherapy nor endocrine therapy) the 70-gene signature low risk – 

AOL low risk group (n=88) had a DDFS of 95.0% (95% CI 90.3-99.9). The 70-gene 

signature low risk – AOL high-risk group (n=70) had a DDFS of 100%. 

Development of a predictive test for tamoxifen resistance in breast cancer 

We have collected suitable primary breast cancer material of ~ 750 postmenopausal 

patients who participated in a randomized trial of adjuvant tamoxifen versus no 

endocrine therapy that had been conducted in the Netherlands from 1982-1994 (IKA 

trial). Tissue microarrays have been constructed of these 750 primary breast cancers. 

For ~ 600 patients we have also isolated tumor DNA to study the role of mutation, 

amplifi cation and methylation of selected genes in tamoxifen resistance. 

Outcome after tamoxifen in breast cancer can be infl uenced by polymorphisms 

in tamoxifen metabolizing enzymes. CYP2C19 is not only involved in 

4-hydroxytamoxifen formation but also in estrogen catabolism. A relatively 

common variant, CYP2C19*2, encodes a non-functional enzyme. We investigated 

the association between common CYP2C19 variants and benefi t from adjuvant 

tamoxifen in patients who had participated in the IKA trial. Patients with a 

CYP2C19*2 allele derived more benefi t from tamoxifen (HR 0.25; p= 0.0003), 

than patients without a CYP2C19*2 allele (HR 0.64; p=0.10) (p for interaction 

0.04) (fi gure 4). In patients who did not receive tamoxifen, CYP2C19*2 was an 

adverse prognostic factor. CYP2C19 is primarily involved in endogenous steroid 

metabolism, and CYP2C19*2 has been associated with higher life-long E1 levels, 

higher breast cancer incidence, and may thereby affect tumorigenesis and prognosis. 

We hypothesize that the presence of a CYP2C19*2 allele may skew tumorigenesis 

towards a molecular subtype that is more often estrogen-dependent. 

Molecular mechanisms underlying sensitivity to high dose alkylating agents 

(collaboration with Petra Nederlof, Division of Diagnostic Oncology, and Sjoerd 

Rodenhuis, Division of Medical Oncology). The inability of breast cancer cells 

defi cient in homologous recombination (HR), such as BRCA1/-2 -mutated cells, to 

repair DNA double strand breaks (DSBs) appears to offer a target for DSB-inducing

therapies, such as platinum agents, intensifi ed alkylating therapy, and poly(ADP) 

ribose polymerase (PARP) inhibitors. Our group previously employed array 

Comparative Genomic Hybridization (aCGH) to assess the genomic profi les of 

BRCA1- and BRCA2-mutated breast cancers. We called these aCGH profi les BRCAlike 

CGH profi les. We demonstrated that high-risk, stage III, HER2-negative breast 

cancer patients with BRCA-like CGH tumors had fi ve times less risk to die from breast 

cancer when they were treated with adjuvant high-dose (HD) carboplatin-thiotepacyclophosphamide 

(CTC) with autologous stem cell rescue, instead of standard 

fl uorouracil-epirubicin-cyclophosphamide (FEC) chemotherapy. One third of the 

stage III, HER2-negative breast cancer patients had a BRCA-like CGH tumor. Half of 

the BRCA-like CGH tumors were hormone receptor positive (fi gure 5). 

In collaboration with the University of Heidelberg, Germany, we are now analyzing 

a second series of 87 high-risk breast cancer patients, who had all been treated 

with HD alkylating chemotherapy with autologous stem cell rescue in the period 

1992-2000. In case of interesting data, we shall add a matched control group of ~ 90 

patients to further validate our earlier fi ndings. 

Translation of the BRCA-like CGH classifier to other cancer types HR defi ciency 

is not limited to breast cancer. Although less obvious than breast and ovarian cancer 

risk, other cancers also occur at increased rates in hereditary breast and ovarian 

cancer families with germline BRCA1 and -2 mutations. These other cancers include 

gastric (BRCA2-associated, RR:2.59), pancreatic (BRCA2-associated, RR:3.51), and 

colon cancer (BRCA1-associated, RR:2.03) (Thompson and Easton;The Breast Cancer 

Linkage Consortium). In collaboration with the NKI Familial Cancer Clinic possible 

BRCA-associated tumors will be collected to investigate a causal effect as well as 

generate tumor type specifi c array CGH profi les. These profi les will subsequently be 

tested in defi ned cohorts of patients who had been treated with either DSB-inducing 

therapy or other therapy and outcome correlated with BRCA-like CGH status. 

Netherlands Breast Cancer Project (NBCP) In collaboration with the Dutch 

Cancer Registry, NABON and BOOG we have initiated a project to fi nd answers for 

clinical and translational research questions that will never be answered anymore 

through prospective clinical trials. For this, we make use of the Dutch Cancer 

Registry, where data of over 150,000 breast cancer patients is stored with clinical 

follow-up. The ultimate aim is to combine clinical data with molecular data of tumor 

material that has been traced back through PALGA, the Dutch nationwide surgical 

pathology registry. 

Identification of druggable targets in lobular breast cancer and triple 

negative breast cancer As participant of the FP7 RATHER consortium, together 

with the Bernards group (Division of Molecular Carcinogenesis), we have initiated 

a project to assess the presence of druggable targets in 150 lobular and 150 triple 

negative breast cancers. We are using high-throughput next generation sequencing, 

together with exon capture technology, to sequence genomic regions of interest 

in breast cancer in all 300 tumor samples. The fi nal goal is to deliver diagnostic 

tests along with the right targeted therapy – chemotherapy combination to improve 

treatment options and outcome for these diffi cult to treat breast cancer subtypes. 

Publications 

69 


De Ruiter MB, Reneman L, Boogerd W, 

Veltman DJ, Caan M, Douaud G, Lavini C, 

Linn SC, Boven E, van Dam FS, Schagen SB. 

Late effects of high-dose adjuvant 

chemotherapy on white and gray matter in 

breast cancer survivors: Converging results 

from multimodal magnetic resonance 

imaging. Hum Brain Mapp. 2011 

Vollebergh MA, Jonkers J, Linn SC. 

Genomic instability in breast and ovarian 

cancers: translation into clinical predictive 

biomarkers. Cell Mol Life Sci. 2011 

Van Schaik RH, Kok M, Sweep FC, van 

Vliet M, van Fessem M, Meijer-van Gelder 

ME, Seynaeve C, Lindemans J, Wesseling J, 

Van ‘t Veer LJ, Span PN, van Laarhoven H, 

Sleijfer S, Foekens JA, Linn SC, Berns EM. 

The CYP2C19*2 genotype predicts tamoxifen 

treatment outcome in advanced breast cancer 

patients. Pharmacogenomics. 2011;12:1137- 

1146 

Schilder CM, Seynaeve C, Linn SC, 

Boogerd W, Beex LV, Gundy CM, Nortier JW, 

van de Velde CJ, van Dam FS, Schagen SB. 

Self-reported cognitive functioning in 

postmenopausal breast cancer patients before 

and during endocrine treatment: fi ndings 

from the neuropsychological TEAM 

side-study. Psychooncology. 2011 

Zwart W, Theodorou V, Kok M, Canisius 

S, Linn S, Carroll JS. Oestrogen receptor-cofactor-chromatin 

specifi city in the 

transcriptional regulation of breast cancer. 

EMBO J. 2011 

Esserman LJ, Shieh Y, Rutgers EJ, Knauer 

M, Retel VP, Mook S, Glas AM, Moore DH, 

Linn S, van Leeuwen FE, Van ‘t Veer LJ. 

Impact of mammographic screening on the 

detection of good and poor prognosis breast 

cancers. Breast Cancer Res Treat. 

2011;130:725-734 

Figure 5: Kaplan Meier survival analysis 

of postmenopausal breast cancer patients 

who had received tamoxifen treatment 

or no adjuvant systemic therapy divided in 

patients without a CYP2C19*2 allele (A) 

and with a CYP2C19*2 allele (B)

70 



Group leader Alfred Schinkel 

Alfred Schinkel PhD Group leader 

Dilek Iusuf MSc PhD student 

Seng Chuan Tang MSc PhD student 

Selvi Durmus MSc PhD student 

Jeroen Hendrikx MSc PhD student 

Ning Xu MSc PhD student 

Anita van Esch Technical staff 

Els Wagenaar Technical staff 

Figure 6: Putative structure of a prototypic 

ABC drug transporter 

Publications 

Van de Steeg E, van Esch A, Wagenaar E, 

van der Kruijssen CMM, van Tellingen O, 

Kenworthy KE, Schinkel AH. High impact of 

Oatp1a/1b transporters on in vivo disposition 

of the hydrophobic anticancer drug paclitaxel. 

Clin Cancer Res. 2011;17:294-301 

Ter Heine R, Van Waterschoot RA, Keizer 

RJ, Beijnen JH, Schinkel AH, Huitema AD. 

An integrated pharmacokinetic model for the 

infl uence of CYP3A4 expression on the in vivo 

disposition of lopinavir and its modulation by 

ritonavir. J Pharm Sci. 2011;100:2508-15 

Kayouka M, Houzé P, Baud FJ, Cisternino 

S, Debray M, Risède P, Schinkel AH, Warnet 

JM. Does modulation of organic cation 

transporters improve pralidoxime activity in 

an animal model of organophosphate 

poisoning? Crit Care Med. 2011;39:803-11 

Poller B, Iusuf D, Sparidans RW, 

Wagenaar E, Beijnen JH, Schinkel AH. 

GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG 

RESISTANCE AND PHARMACOKINETICS 

Our research focuses on genes and proteins that cause drug resistance or drug 

susceptibility in tumors, or infl uence the pharmacological and toxicological behavior 

of anticancer and many other drugs and toxins, including carcinogens. Insight into 

these systems may: i) improve chemotherapy and more generally pharmacotherapy 

approaches for cancer and other diseases; ii) increase insights into factors 

determining susceptibility to carcinogens, and; iii) allow elucidation of physiological 

functions. To study the physiological, pharmacological and toxicological roles of 

the proteins involved, and their interactions, we generate and analyze knockout or 

transgenic mice lacking or overexpressing the relevant genes. 

Impact of drug transporters We have a long-standing interest in plasma 

membrane proteins of the ATP binding cassette (ABC) multidrug transporter 

family, including P-glycoprotein (P-gp, ABCB1/MDR1), MRP2 (ABCC2) and BCRP 

(ABCG2) (Figure 1). These proteins actively export a wide range of anticancer, anti- 

HIV/AIDS, and many other drugs from cells. This ATP-dependent drug extrusion 

can cause multidrug resistance (MDR) in tumor cells. ABCB1, ABCC2 and ABCG2 

all localize to the apical membrane of polarized epithelial cells, resulting in apically 

directed export of drug substrates, and there is considerable overlap in substrate 

specifi city between these transporters. Previous experiments in Abcb1 and Abcg2 

knockout mice indicated that these transporters can protect an organism against 

exogenous toxins and drugs by limiting penetration of substrates into brain, testis, 

and fetus, by restricting uptake of orally administered substrates, and by mediating 

excretion of substrates via liver and intestine. To extend these analyses we have 

generated Abcc2 knockout mice, and crossed these with existing Abcb1, Abcg2 and 

Abcc3 knockout mice in order to elucidate the separate and combined contributions 

of these transporters to pharmacological, toxicological and physiological functions. 

Abcc3 is expressed in the basolateral membrane of hepatocytes and enterocytes, 

so transporting in the opposite direction of Abcb1, Abcg2 and Abcc2. Below we 

describe some representative studies carried out with these mouse strains. 

Impact of Abcc2, Abcc3 and Abcg2 on the oral pharmacokinetics of 

methotrexate ABCC2, ABCC3 and ABCG2 together infl uence the pharmacokinetics 

of the anti-cancer and anti-rheumatic drug methotrexate (MTX) and its toxic 

metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v. MTX administration. We 

now used Abcc2;Abcc3;Abcg2 -/- and corresponding single and double knockout mice 

to investigate the relative impact of these transporters on MTX and 7OH-MTX 

pharmacokinetics after oral MTX administration. The plasma areas under the curve 

(AUC plasma) in Abcg2 -/- and Abcc2;Abcg2 -/- mice were 1.7- and 3.0-fold higher than in 

wild-type mice, respectively, suggesting additive effects of Abcc2 and Abcg2 on oral 

MTX pharmacokinetics. However, the AUC plasma in Abcc2;Abcc3;Abcg2 -/- mice was 

not different from that in wild-type mice, indicating that Abcc3 protein is necessary 

for increased MTX plasma concentrations in the absence of Abcc2 and/or Abcg2. 

Furthermore, MTX liver levels were increased in Abcg2-defi cient strains and MTX 

kidney levels were 2.2-fold increased compared to wild-type in Abcc2;Abcg2 -/- mice. 

Absence of Abcc2 and/or Abcg2 also led to signifi cantly increased liver and kidney 

levels of 7OH-MTX. Our results suggest that inhibition of ABCG2 and/or ABCC2, 

or genetic polymorphisms or mutations reducing expression or activity of these 

proteins may increase the oral availability of MTX. Such conditions may also present 

risk factors for increased MTX-related toxicity in patients treated with oral MTX. 

P-glycoprotein (ABCB1) restricts the brain penetration of the primary active 

tamoxifen metabolites endoxifen and 4-hydroxytamoxifen Tamoxifen is the 

most widely used drug for patients with early-stage breast cancer and estrogenreceptor 

positive tumors. We investigated the impact of ABCB1 on tamoxifen and 

its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen and 

endoxifen.We used in vitro transport assays and Abcb1a/1b -/- mice to investigate 

the impact of ABCB1 on the oral availability and brain penetration of tamoxifen 

and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral

administration of tamoxifen was not changed in the absence of Abcb1. However, 

brain accumulation of tamoxifen, 4-hydroxytamoxifen and N-desmethyltamoxifen 

were modestly, but signifi cantly (1.5-2 fold) increased. Endoxifen, however, displayed 

a 9-fold higher brain penetration. Endoxifen was transported by ABCB1 in vitro. 

Upon direct oral administration of endoxifen, systemic exposure was slightly 

decreased in Abcb1a/1b -/- mice, but brain accumulation of endoxifen was dramatically 

increased (up to 23-fold). Shortly after high-dose intravenous administration (5 or 

20 mg/kg), endoxifen brain accumulation was only 2-fold increased in Abcb1a/1b -/- 

mice, suggesting a partial saturation of Abcb1 at the blood-brain barrier. Endoxifen, 

the clinically most relevant metabolite of tamoxifen, is thus an ABCB1 substrate in 

vitro and in vivo, and Abcb1 can limits its brain penetration. ABCB1 might thus be 

relevant for tamoxifen/endoxifen resistance of ABCB1-positive breast cancer and of 

tumors positioned behind a functional blood-brain barrier. 

Brain accumulation of sunitinib is restricted by ABCB1 and ABCG2 and can be 

enhanced by oral elacridar and sunitinib coadministration Sunitinib is an orally 

active, multi-targeted tyrosine kinase inhibitor used in the treatment of metastatic 

renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We 

investigated the in vivo roles of ABCB1 and ABCG2 in plasma pharmacokinetics 

and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib 

kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. 

We used in vitro transport assays and Abcb1a/1b -/- , Abcg2 -/- and Abcb1a/1b/Abcg2 - 

/- mice to study the roles of Abcb1 and Abcg2 in sunitinib disposition. In vitro, 

sunitinib was a good substrate of murine ABCG2 and a moderate substrate of 

human ABCB1 and ABCG2. In vivo, the systemic exposure of sunitinib after 

oral dosing (10 mg/kg) was unchanged when Abcb1 and/or Abcg2 were absent. 

Brain accumulation of sunitinib was markedly (23-fold) increased in Abcb1a/b/ 

Abcg2 -/- mice, but only slightly (2.3-fold) in Abcb1a/b -/- mice, and not in Abcg2 - 

/- mice. Importantly, a clinically realistic coadministration of oral elacridar and 

oral sunitinib to wild-type mice resulted in dramatically increased sunitinib brain 

accumulation, equaling levels in Abcb1a/1b/Abcg2 -/- mice. This indicates complete 

inhibition of the blood-brain barrier transporters. Brain accumulation of sunitinib is 

effectively restricted by both Abcb1 and Abcg2 activity. Complete inhibition of both 

transporters, leading to dramatically increased brain accumulation of sunitinib, is 

feasible and safe with a clinically realistic oral elacridar/sunitinib coadministration. 

High impact of Oatp1a/1b transporters on in vivo disposition of the 

hydrophobic anticancer drug paclitaxel Organic anion transporting polypeptides 

(OATP, gene name: SLCO) facilitate sodium-independent uptake transport of a 

wide variety of organic endo- and exogenous compounds, such as bile salts, steroid 

and thyroid hormones and their conjugates, and numerous drugs and toxins. 

Members of the OATP1A/1B family have a broad substrate specifi city and are highly 

expressed in the sinusoidal membrane of hepatocytes, or in the apical membrane 

of enterocytes where they might affect liver or intestinal uptake, respectively, of 

drugs, xenobiotics, and endogenous substances. Expression in a number of tumors 

has also been described. These transporters might thus play an important role 

in drug disposition and drug uptake into tumors. The hydrophobic anticancer 

drug paclitaxel (PTX) was recently identifi ed as a substrate for OATP1B3 in vitro. 

Using Slco1a/1b -/- mice which lack all Oatp1a/1b transporters, we investigated the 

role of Oatp1a/1b transporters in the pharmacokinetics of PTX in vivo, as well as 

their impact at different dose levels of PTX and methotrexate (MTX). In spite of 

its hydrophobicity, PTX systemic exposure (at 10 mg/kg) was >2-fold increased in 

Slco1a/1b -/- mice compared with wild-type, whereas PTX liver uptake was ~ 2-fold 

reduced. Oatp1a/1b transporters played a pronounced role in determining plasma 

levels and tissue distribution of MTX and PTX over a broad dose range. These 

transporters thus affect even highly hydrophobic drugs. Variation in OATP1A/1B 

transporter activity, due to genetic variation, inhibition, and/or tumor expression 

might therefore affect toxicity and therapeutic effi cacy of these anticancer drugs. 

71 



Differential impact of P-glycoprotein (ABCB1) 

and breast cancer resistance protein (ABCG2) 

on axitinib brain accumulation and oral 

plasma pharmacokinetics. Drug Metab 

Dispos. 2011;39:729-35 

Poller B, Wagenaar E, Tang SC, Schinkel 

AH. Double-transduced MDCKII cells to study 

human P-glycoprotein (ABCB1) and Breast 

Cancer Resistance Protein (ABCG2) interplay 

in drug transport across the blood-brain 

barrier. Mol Pharm. 2011;8:571-82 

Iusuf D, Teunissen SF, Wagenaar E, 

Rosing H, Beijnen JH, Schinkel AH. 

P-glycoprotein (ABCB1) transports the 

primary active tamoxifen metabolites 

endoxifen and 4-hydroxytamoxifen, and 

restricts their brain penetration. J Pharmacol 

Exp Ther. 2011;337:710-17 

Teunissen SF, Rosing H, Seoane MD, 

Brunsveld L, Schellens JH, Schinkel AH, 

Beijnen JH. Investigational study of tamoxifen 

phase I metabolites using chromatographic 

and spectroscopic analytical techniques. J 

Pharm Biomed Anal. 2011;879:1677-85 

Koolen SL, van Waterschoot RA, van 

Tellingen O, Schinkel AH, Beijnen JH, 

Schellens JH, Huitema AD. From Mouse to 

Man: Predictions of Human Pharmacokinetics 

of Orally Administered Docetaxel From 

Preclinical Studies. J Clin Pharmacol. 2011 (in 

press) 

Van Waterschoot RAB, Schinkel AH. A 

critical analysis of the interplay between 

cytochrome P450 3A and P-glycoprotein: recent 

insights from knockout and transgenic mice. 

Pharmacol Rev. 2011;63:390-410 

Vlaming ML, van Esch A, van de Steeg E, 

Pala Z, Wagenaar E, van Tellingen O, 

Schinkel AH. Impact of abcc2 [multidrug 

resistance-associated protein (MRP) 2], abcc3 

(MRP3), and abcg2 (breast cancer resistance 

protein) on the oral pharmacokinetics of 

methotrexate and its main metabolite 

7-hydroxymethotrexate. Drug Metab Dispos. 

2011;39:1338-44 

Hendrikx JJ, Hillebrand MJ, Thijssen B, 

Rosing H, Schinkel AH, Schellens JH, 

Beijnen JH. A sensitive combined assay for the 

quantifi cation of paclitaxel, docetaxel and 

ritonavir in human plasma using liquid 

chromatography coupled with tandem mass 

spectrometry. J Chromatogr B Analyt Technol 

Biomed Life Sci. 2011;879:2984-90 

Tang SC, Lagas JS, Lankheet NA, Poller B, 

Hillebrand MJ, Rosing H, Beijnen JH, 

Schinkel AH. Brain accumulation of sunitinib 

is restricted by P-glycoprotein (ABCB1) and 

breast cancer resistance protein (ABCG2) 

and can be enhanced by oral elacridar and 

sunitinib coadministration. Int J Cancer. 

2012;130:223-33.

72 



Group leader Lodewyk Wessels 

Lodewyk Wessels PhD Group leader 

Nicos Angelopoulos PhD Post-doc 

Sander Canisius PhD Post-doc 

Theo Knijnenburg PhD Post-doc 

Magali Michaut PhD Post-doc 

Guillem Rigaill PhD Post-doc 

Andreas Schlicker PhD Post-doc 

Hayssam Soueidan PhD Post-doc 

Ewald van Dyk MSc PhD student 

Johann de Jong MSc PhD student 

Christiaan Klijn MSc PhD Student 

Wouter Meuleman MSc PhD student 

Jorma de Ronde MSc PhD student 

Christine Staiger MSc PhD student 

Sidney Cadot Bioinformatician 

Bram Gerritsen Bioinformatician 

Jelle ten Hoeve Bioinformatician 

BIOINFORMATICS AND STATISTICS 

The Bioinformatics and Statistics group provides leadership on the collection and 

analysis of data for the research programs of the institute, by conducting research 

in computational biology and by performing state of the art analyses of a wide array 

of data types. Research topics include stratifying tumors into groups with distinct 

and homogeneous outcome and therapy response; the characterization of genes and 

pathways involved in tumorigenesis and understanding molecular regulatory and 

signaling mechanisms. A number of exemplary projects are presented below in 

more detail. 

Estimating DNA copy number ratio from capture sequencing data Target 

enrichment, also referred to as DNA capture, provides an effective way to focus 

sequencing efforts on a genomic region of interest. This approach is commonly 

employed to interrogate exons, which are likely to harbor variants that have a causal 

role in disease. Capture data is typically used to detect single nucleotide variants and 

small insertions or deletions. However, it can also be used to detect copy number 

alterations (CNAs), which is particularly useful in the context of cancer, where such 

changes occur frequently. We have developed a statistical modeling approach that is 

specifi cally suited for detecting CNAs in capture data. 

In copy number analysis it is common practice to determine ratios between test 

and control samples, but this approach disregards the total coverage and is prone to 

outliers. Rather than modeling the ratio, we instead modeled the coverage of the test 

sample as a linear function of the control sample. Another benefi t of this approach is 

that it is able to deal with regions that are completely deleted, which are problematic 

for methods that use log ratios. To demonstrate the utility of our approach, we 

used capture data to determine copy number for a set of ~ 600 genes in a panel of 

nine breast cancer cell lines. We found high concordance between our results and 

those generated using the Affymetrix SNP6.0 SNP platform. When we compared 

our results to other methods, including ExomeCNV, we found that our approach 

produced better overall correlation with SNP data and was less prone to outliers. 

Pathway construction from the literature and protein-protein interaction 

networks Many key cellular processes, like cell proliferation or differentiation, are 

responses to changes in environment. Signals from the cell surface are transmitted 

downstream by sequential protein interactions, represented as an interaction 

network. These networks contain signaling pathways, representing a consensus, 

expert description of the function of a subset of the interactions. The interactions 

describing these pathways are typically documented in the literature. Reverse 

engineering signaling pathways from experimental data is very hard, as there 

are many possible interactions and typically insuffi cient data to fully support this 

process. In addition, a large number of published studies need to be surveyed to 

fi nd support for interactions detected in the data. For that reason, reliable (semi-) 

automated pathway reconstruction from literature evidence is indispensable. 

We propose a novel approach to characterize, compare and build signaling pathways 

by combining the protein interactome with literature evidence. Our approach 

is based on Latent Semantic Indexing, a method combining text mining and 

dimensionality reduction to identify key concepts in published abstracts. Latent 

Semantic Indexing produces a factor space, which is a vector space of manageable 

dimensionality, in which we can represent both individual articles and signaling 

pathways. We used this factor space to identify pathways by measuring textual 

similarities between articles describing individual interactions. More specifi cally, 

we showed that 1) we are able to distinguish, with high precision, expert-curated 

signaling pathways from randomly connected components; 2) in this space, articles 

can be clustered by pathways, and that 3) complete pathways can be recovered 

from the interactome by expanding a small set of articles describing a subset of 

the interactions constituting a pathway. Our approach is a very useful tool for 

experimentalists, since it allows reliable checking and expansion of existing pathway 

hypotheses and also adds value to experimental results by providing a knowledgebased 

context.

Analysis of genomic signals at multiple scales In the genome, information 

is encoded on a wide range of spatial scales as functional genomic regions come 

in many sizes. As a consequence, measurements derived from the genome will 

exhibit structure at different spatial scales. This should be taken into account when 

analyzing such data. We have introduced a novel approach based on scale space 

theory to analyze genomic signals at different spatial scales. The types of genomic 

signals to which our method is applicable include DNA sequence based data, such 

as CG content and intra-species sequence conservation, as well as (epi-)genomic 

measurements, such as microarray and ChIP-seq data. 

The multi-scale representation of a genomic signal offers a novel way to 

summarize and visualize the information content across genomic scales. Using 

this representation, we can outline interesting properties of an individual genomic 

signal and detect and compare differences between genomic signals. We have 

demonstrated how this approach can uncover connections between functional 

genomic annotations and sequence-associated (binding) patterns at different length 

scales. Additionally, gene-specifi c multi-scale signatures were used in a machinelearning 

framework to predict gene expression and cluster membership. Here, we 

show substantially improved prediction accuracy when compared to using single 

scales. 

Figure 7: The expression of 

two genes, MNAT1 and 

AURKA, was found to 

show an interaction effect 

with respect to patient 

survival. Patients with 

high expression for both 

genes have a poorer 

metastasis-free survival 

than patients with either 

none, or only one of the 

two genes highly expressed. 

The fi gure shows 

Kaplan-Meier curves for a 

cohort of 312 breast cancer 

patients stratifi ed 

according to the joint 

expression of the two genes. 

Copy number co-occurrence analysis identifies high-level genetic interactions 

driving breast tumor development In the course of tumor development, a 

cell incrementally acquires a set of mutations that eventually drive it towards a 

malignant phenotype. Far from being independent, each mutation is selected for 

in the context of and for compatibility with the mutations already present. We have 

developed a computational framework to detect co-occurring events in aCGH data. 

We employed this approach to perform a genome-wide analysis of breast tumours 

to identify high-level interactions between tumorigenic copy number alterations. 

In our cohort, many such interactions were found. Since we have gene expression 

data available for the same samples, we further refi ned the search by integrating 

the gene expression and copy number data. More specifi cally, we investigated, for 

the set of signifi cantly co-occurring regions, whether the gene expression patterns 

of the genes located in the co-occurring regions also show co-occurrence patterns. 

For these genes we subsequently tested whether the co-occurrence was signifi cantly 

associated with survival. This revealed that the co-occurrence of MNAT1 and 

AURKA is strongly associated with survival. More specifi cally, presence of the joint 

activation of these genes dramatically decreases the fi ve year survival of a patient 

(see fi gure 7). This gene pair jointly induces chromosomal instability in a cell while 

inhibiting the normal cellular response to such a state. Our analysis demonstrates 

the importance of evaluating dependences between tumorigenic mutations rather 

than considering these mutations in isolation. 

Publications 

73 


Knijnenburg TA, Roda O, Wan Y, Nolan 

GP, Aitchison JD, Shmulevich I. A regression 

model approach to enable cell morphology 

correction in high-throughput fl ow 

cytometry. Mol Syst Biol. 2011;7:531 

Koudijs MJ, Klijn C, van der Weyden L, 

Kool J, ten Hoeve J, Sie D, Prasetyanti PR, 

Schut E, Kas S, Whipp T, Cuppen E, 

Wessels LFA, Adams DJ and Jonkers J, 

High-throughput semi-quantitative analysis 

of insertional mutations in heterogeneous 

tumors. Genome Res. 2011 

de Jong J, de Ridder J, van der Weyden 

L, Sun N, van Uitert M, Berns A, van 

Lohuizen M, Jonkers J, Adams DJ and 

Wessels LFA, Computational identifi cation 

of insertional mutagenesis targets for cancer 

gene discovery. Nucleic Acids Res. 2011 

Farazi TA, Horlings HM, ten Hoeve J, 

Mihailovic A, Halfwerk H, Morozov P, 

Brown M, Hafner M, Reyal F, van 

Kouwenhove M, Kreike B, Sie D, Hovestadt 

V, Wessels LFA, van de Vijver MJ, and 

Tuschl T. MicroRNA sequence and 

expression analysis in breast tumors by deep 

sequencing. Cancer Res. 2011;71:4443-53 

Kallioniemi O, Wessels LFA, Valencia A. 

On the organization of bioinformatics core 

services in biology-based research institutes. 

Bioinformatics. 2011;27:1345 

Mittempherger L, de Ronde J, 

Nieuwland M, Kerkhoven RM, Simon I, 

Rutgers EJTh, Wessels LFA and Van ‘t Veer 

LJ. Gene expression profi les from formalin 

fi xed paraffi n embedded breast cancer tissue 

are largely comparable to fresh frozen 

matched tissue. PLoS One. 2011;6:e17163 

Borst P, Wessels LFA. Do predictive 

signatures really predict response to cancer 

chemotherapy? Cell Cycle. 2010;9:4836-40 

Vollebergh MA, Lips EH, Nederlof PM, 

Wessels LFA, Schmidt MK, van Beers EH, 

Cornelissen S, Holtkamp M, Froklage FE, 

de Vries EG, Schrama JG, Wesseling J, van 

de Vijver MJ, van Tinteren H, de Bruin M, 

Hauptmann M, Rodenhuis S, Linn SC. An 

aCGH classifi er derived from BRCA1mutated 

breast cancer and benefi t of 

high-dose platinum-based chemotherapy in 

HER2-negative breast cancer patients. Ann 

Oncol. 2010 

Varela I, Klijn C, Stephens PJ, Mudie LJ, 

Stebbings L, Galappaththige D, van der 

Gulden H, Schut E, Klarenbeek S, 

Campbell PJ, Wessels LFA, Stratton MR, 

Jonkers J, Futreal PA, Adams DJ. Somatic 

structural rearrangements in genetically 

engineered mouse mammary tumors. 

Genome Biol. 2010;11:R100

74 

MOLECULAR CARCINOGENESIS 

Publications (continued) DIVISION OF MOLECULAR 

CARCINOGENESIS 

Division head, group leader René Bernards 

René Bernards PhD Group leader 

Katrien Berns PhD Academic staff 

Annette Dirac PhD Senior post-doc 

Valentina Gambino PHD Post-doc 

Michiel van der Heijden MD PhD Senior 

post-doc 

Michael Hölzel MD PhD Senior post-doc 

Sidong Huang PhD Senior post-doc 

Prasanth Kumar PhD Post-doc 

Ian Majewski PhD Post-doc 

Lorenza Mittemperger PhD Post-doc 

Rianne Oosterkamp MD Clinical fellow 

Zheng Xue PhD Post-doc 

Ernst-Jan Geutjes MSc PhD student 

Floris Groenendijk MD PhD student 

Guus Heynen MSc PhD student 

Anirudh Prahallad MSc PhD student 

Chong Sun MSc PhD student 

Annemiek Bes-Gennissen Technical staff 

Astrid Bosma Technical staff 

Marielle Hijmans MSc Technical staff 

Wipawadee Grernrum Technical staff 

Publications 

Ashw orth A, Bernards R. Using 

functional genetics to understand breast 

cancer biology. Cold Spring Harbor 

perspectives in biology. 2010;2:a003327 

Bei jersbergen RL, Bernards R. Functional 

subtyping of breast cancer. Cancer Discovery. 

2011;1:205-206 

Bernards R, Filipowicz W, Livingston 

DM, Mihich E. Twenty-second annual 

Pezcoller Symposium: RNA biology and 

cancer. Cancer Res. 2010;70:10034-7 

Bre nnan D, O’Connor D, Laursen H, 

McGee S, McCarthy S, Zagozdzon R, 

Rexhepaj E, Culhane A, Martin F, Duffy M, 

Landberg G, Ryden L, Hewitt S, Kuhar M, 

Bernards R, RC M, Crown J, Jirstrom K, 

Gallagher W. The cocaine- and 

amphetamine-regulated transcript mediates 

ligand-independent activation of ERα, and 

is an independent prognostic factor in 

node-negative breast cancer. Oncogene. 2011 

(in press) 

FUNCTIONAL GENOMICS 

My group interrogates the genome to identify biomarkers and mechanisms 

of response to cancer drugs. We use both functional genetic screens and next 

generation sequencing technologies to achieve these goals. 

Identification of mechanisms and biomarkers of drug response Both intrinsic 

and acquired unresponsiveness to therapy is a recurring problem in the treatment of 

cancer. It is therefore important to identify the molecular pathways that contribute 

to unresponsiveness to cancer therapeutics. We use loss-of-function genetic screens 

with large sets of shRNA vectors to identify genes that contribute to drug resistance, 

in particular to the new classes of targeted therapeutics. In the past year, we have 

focused on the identifi cation of genes whose suppression contributes to resistance 

to receptor tyrosine kinase (RTK) inhibitory drugs and their downstream signalling 

pathways. Using a large-scale RNAi screen, we identifi ed MED12, a component 

of the transcriptional MEDIATOR complex, as a determinant of response to 

ALK and EGFR inhibitors. MED12 is in part cytoplasmic where it negatively 

regulates TGFβR2 signalling through physical interaction. MED12 suppression 

therefore results in TGFβ activation, which is both necessary and suffi cient for 

drug resistance. TGFβ signalling causes ERK activation and consequently MED12 

suppression also confers resistance to MEK and BRAF inhibitors in other cancers. 

MED12 loss induces an EMT-like phenotype, which is associated with poor 

outcome in primary colon cancer and resistance to MEK inhibitors in a large and 

heterogeneous panel of cancer cell lines. Inhibition of TGFβR signalling restores 

drug responsiveness in MED12 KD cells, suggesting a strategy to treat drug-resistant 

tumours that have undergone a TGFβ-induced EMT. In addition to MED12, we 

also identifi ed two components of the SWI/SNF chromatin-remodeling complex 

(ARID1A and SMARCE1) as markers of response to several targeted cancer 

therapeutics, including ALK and EGFR inhibitors in lung cancer. 

Inhibit ion of the BRAF V600E oncoprotein by the small molecule drug PLX4032 

(vemurafenib) is highly effective in the treatment of melanoma. However, colon 

cancer patients harbouring the same BRAF V600E oncogenic lesion have poor 

prognosis and show only a very limited response to this drug. To investigate the 

cause of the limited therapeutic effect of vemurafenib in BRAF V600E mutant colon 

tumours, we performed a RNA interference-based genetic screen to search for 

kinases whose knockdown synergizes with BRAF V600E inhibition. We found that 

blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with 

BRAF V600E inhibition. In multiple BRAF V600E mutant colon cancers inhibition 

of EGFR by the antibody drug cetuximab or the small molecule drugs gefi tinib or 

erlotinib is strongly synergistic with BRAF V600E inhibition, both in vitro and in 

vivo. Mechanistically, we fi nd that inhibition of BRAF V600E prevents activation of 

the CDC25C phosphatase by MEK-ERK kinases, a phosphatase that de-activates 

EGFR. Thus, BRAF V600E inhibition causes a rapid feedback activation of EGFR, 

which supports continued proliferation in the presence of BRAF V600E inhibition. 

Melanoma cells express low levels of EGFR and are therefore not subject to this 

feedback activation. Consistent with this, we fi nd that ectopic expression of EGFR 

in melanoma cells is suffi cient to cause resistance to PLX4032. Our data suggest 

that BRAF V600E mutant colon cancers (some 8-10% of all colon cancers) for whom 

there are currently no targeted treatment options available, might benefi t from 

combination therapy consisting of BRAF- and EGFR inhibitors (fi gure1). 

Retinoids play key roles in development, differentiation and homeostasis. These 

diverse effects are primarily exerted through regulation of specifi c target genes 

mediated by RAR/RXR nuclear receptor complex. Corepressors and coactivators play 

important roles in creating the proper chromatin environment for transcriptional 

control by RAR/RXR. We have continued to work on mechanisms of resistance to 

retinoic acid (RA) induced differentiation. In earlier studies we had identifi ed ZNF423

Figure 1: BRAF inhibition causes feedback 

activation of EGFR in colon cancer. 

A). In BRAF-mutant colon cancer cells, 

persistent signalling through the BRAF- 

MEK-ERK pathway leads to activation of the 

CDC25C phosphatase, which inactivates the 

EGFR through dephosphorylation, thereby 

limiting EGFR signalling. 

B). In the presence of the BRAF inhibitor 

vemurafenib BRAF-mutant colon cancer 

cells have lost the feedback inhibition of 

EGFR through MEK-ERK-CDC25C 

signalling, thereby hyperactivating EGFR 

signalling through the PI3K-AKT pathway. 

Consequently, colon cancer cells continue to 

proliferate in the presence of BRAF inhibitor. 

Melanoma cells in general do not express 

EGFR and are therefore not susceptible to 

this feedback activation, explaining their 

favourable response to vemurafenib 

monotherapy. 

and the tumour suppressor gene NF1 is critical components of the response to retinoic 

acid in neuroblastoma cells. In the past year, we have focused on the role of a wellestablished 

transcriptional repressor, CtBP2, in the retinoic acid response. We found, 

through a large-scale RNAi genetic screen, that down-regulation of Ctbp2 expression 

in F9 embryocarcinoma cells and mouse embryonic stem cells confers resistance to 

RA-induced differentiation. Mechanistically, we found that CTBP2 associates with 

RAR/RXR at RA target gene promoters and is essential for their transactivation in 

response to RA. This was unexpected, as it suggested that CTBP2 acts as a co-activator 

of RA signalling rather than a co-repressor. We found that CTBP2 is indispensable to 

create a chromatin environment conducive for RAR/RXR-mediated transcription by 

recruiting the p300 histone acetyltransferase. Our data reveal an unexpected function 

of CTBP2 as a coactivator for RAR/RXR in RA signalling. 

We have also started to use insertional mutagenesis screens to identify gain-offunction 

genetic alterations that contribute to resistance to retinoic acid. Using 

a retroviral tagging approach, we have identifi ed Mastermind-like 3 (MAML3) 

as a gene whose over-expression confers resistance to retinoic acid-induced 

differentiation in neuroblastoma cells. We are currently studying the molecular 

mechanism of action of MAML3 in the RA signalling pathway. 

High throughput sequencing In collaboration with Agilent Technologies (Santa 

Clara, CA USA) we developed and validated a “kinome capture” platform that allows 

us to rapidly sequence 586 kinase and kinase-related genes through the enrichment 

of their coding sequences by hybridization selection. We have begun to use this 

platform to identify kinase mutations in lobular breast cancer as part of the RATHER 

project funded by the EU. In collaboration with Agendia, MDAnderson Hospital and 

Astra Zeneca, we have also started sequencing kinomes of colon cancers with the 

aim to identify subgroups of colon cancers that are dependent on specifi c signalling 

pathways. Such studies may help guide the use of tarwgeted agents in these patients. 

In collaboration with the family cancer clinic of our hospital, we have sequenced 

whole exomes of members of a large Dutch family having hereditary diffuse 

gastric cancer (HDGC). This family does not have mutations in one of the known 

predisposition genes for HDGC. Using this approach we have identifi ed in this 

family a loss of function mutation in this family that is intimately linked to the risk 

of HDGC development. 

In collaboration with the Spanish lung cancer physician Dr. Rafael Rosell, we are 

sequencing the primary tumour and several metastases derived from patients with 

EML4-ALK translocated non-small cell lung cancers that developed resistance to 

crizotinib. We aim to reveal mechanism of drug resistance through the analysis of 

kinome alterations in the metastases from these tumours. 

75 



Eich horn PJA, Rodón L, Gonzàlez-Juncà 

A, Dirac A, Gili M, Martínez-Sáez E, Aura 

C, Barba I, Prat A, Cuartas I, Jimenez J, 

García-Dorado D, Bernards R, Baselga J, 

Seoane J. USP15 stabilizes the TGFβ 

receptor I and is responsible for the 

hyperactivation of the TGFβ signal in 

glioblastoma. Nat Med. 2011 (in press) 

Epping MT, Meijer LA, Krijgsman O, 

Bos JL, Pandolfi PP, Bernards R. TSPYL5 

suppresses p53 levels and function by physical 

interaction with USP7. Nat Cell Biol. 

2011;13:102-108 

Geutjes EJ, Kum ar-Bajpe P, Bernards R. 

Targeting the epigenome for treatment of 

cancer. Oncogene. 2011 (in press) 

Geutjes EJ, Tia n S, Roepman P, 

Bernards R. Deoxycytidine kinase is 

overexpressed in poor outcome breast cancer 

and determines responsiveness to nucleoside 

analogs. Breast cancer Res Treat. 2011 

Geutjes EJ. The cancer epigenome: 

towards epigenetic therapy of cancer. (2011) 

Thesis, University of Utrecht 

Krijgsman O, Roepman P, Zwart W, 

Carroll JS, Tian S, de Snoo FA, Bender RA, 

Bernards R, Glas AM. A diagnostic gene 

profi le for molecular subtyping of breast 

cancer associated with treatment response. 

Breast Cancer Res Treat. 2011 

Majewski IJ, Be rnards R. Taming the 

dragon: genomic biomarkers to individualize 

the treatment of cancer. Nat Med. 

2011;17:304-12 

Nijwening JH, G eutjes EJ, Bernards R, 

Beijersbergen RL. The Histone Demethylase 

Jarid1b (Kdm5b) Is a Novel Component of 

the Rb Pathway and Associates with 

E2f-Target Genes in MEFs during 

Senescence. PloS one. 2011;6:e25235

76 



Group leader Roderick Beijersbergen 

Roderick Beijersbergen PhD Group leader 

Pasi Halonen PhD Post-doc 

Kathy Jastrzebski PhD Post-doc 

Jordi Vidal-Rodriguez PhD Post-doc 

Johan Kuiken MSc PhD student 

Klaas de Lint MSc PhD student 

Jeroen Nijwening MSc PhD student 

Bram Gerritsen MSc Bioinformatician 

Cor Lieftink MSc Bioinformatician 

Ben Morris Technical staff 

Wouter Nijkamp Technical staff 

THE RNAI STRATEGY IN TARGET DISCOVERY 

Despite the recent advances in the treatment of cancer, major challenges remain. 

These include the identifi cation of more tumour specifi c targets, the understanding 

of therapy resistance and the ability to predict therapy response. Together these 

goals will allow for a more personalized treatment of cancer that will likely improve 

overall response and survival. Over the last years we have developed and applied 

RNAi technology platforms that allow for the identifi cation of novel tumour specifi c 

drug targets in cancer, the identifi cation of essential components in disease-related 

pathways and the generation of computational models for the prediction of treatment 

response in breast cancer. 

Synthetic lethal interactions For the effective treatment of cancer, there is an 

urgent need for drugs that specifi cally target tumour cells without affecting normal 

cells. With the use of RNA interference, we explore synthetic lethal phenotypes 

in mammalian cells. Synthetic lethal phenotypes are defi ned as a combination 

of two mutations, which by themselves are non-lethal, but together result in a 

lethal phenotype. These interactions can lead to the identifi cation of novel cancer 

drug targets that are only cytotoxic in the context of a tumour specifi c alteration 

and represent “genotype-specifi c” drug targets. We have generated a panel of 

isogenic cell lines derived from primary human BJ fi broblasts that contain single 

or multiple defi ned genetic alterations that together are required for tumourigenic 

transformation of these primary cells. These genetic alterations include, amongst 

others, loss-of-p53, activation of RAS or activation of PI3K. These cell lines have 

been used in high throughput single well assays in combination with large siRNA 

collections targeting over 8000 genes to identify siRNAs that result in enhanced 

lethality only in the background of these tumour specifi c genetic alterations. 

We have identifi ed a number of genes that show a lethal phenotype only in the 

context of RAS mutation. We have analysed the RAS dependent interactions in a 

large panel of lung cancer cell lines in the absence or presence of a mutant RAS 

allele to exclude context dependent effects. For a small number of these genes we 

will generate model systems to study their effects using inducible shRNA constructs 

in vivo in mouse models. At the same time we are unravelling the underlying 

biological pathways for the synthetic lethal phenotype to increase the chance of 

fi nding a druggable target. 

Generation of computational models for predicting response to therapy in 

breast cancer Rationally designed targeted therapies can be applied in personalized 

medicine upon characterization of specifi c dependencies in tumours. A successful 

example of such therapy is the use of an antibody targeting the epidermal growth 

factor receptor-2 (HER2 of ERBB2). Although the clinical benefi t is considerable 

in patients with HER2-positive breast cancer, the overall response rate is modest 

( ~ 30%) as a single therapy or 40-60% when combined with chemotherapy. Although 

multiple resistance mechanisms have been identifi ed, no regimen has been 

developed that overcomes resistance in patients. It has become clear that alterations 

in receptor tyrosine kinases and their downstream signalling pathways can account 

for de novo resistance. Two major cellular pathways downstream of these receptors 

are the phosphatidylinositol 3-kinase (PI3K) and mitogen activated protein kinase 

(MAPK) pathways controlling metabolism, proliferation, survival and motility. 

Both pathways are frequently hyperactivated in cancer and are required for the 

maintenance of the transformed phenotype. As a result a major effort is underway to 

develop inhibitors to components of these pathways. 

PI3K and MAPK signalling pathways are strongly implicated in breast cancer. At 

present, many drugs are in clinical development targeting specifi c components of 

these pathways, although their successes are limited. Complicated interactions with 

other signalling pathways and unexpected feedback loops can limit the effectiveness 

of treatment and, in some instances, even accelerate the tumourigenic process. The 

understanding and modelling of these complex pathways using a systems approach 

can yield predictive models for therapy responses to pathway-targeted therapeutics 

in breast cancer. We are developing in silico models of therapy response in breast

cancer by exploiting normal and tumour cell lines as in vitro model systems for 

the quantifi cation of functional activation of pathway components and associated 

cellular phenotypes. 

Protein phosphorylation is a fundamental mechanism of intracellular signalling. 

Protein kinases, including receptor tyrosine kinases, initiate and relay 

phosphorylation signals along signalling pathways in cells. The quantifi cation of the 

dynamics of kinase activities and the resulting phosphorylations can yield valuable 

information about the activity of these proteins and their pathways and the resulting 

biological phenotypes. We use RNAi for perturbation experiments in combination 

with a phosphor-proteomic analysis platform for quantifi cation of the activation 

status of each individual pathway components (fi gure 2). The data obtained from 

these experiments is used to create and validate a dynamic and quantitative 

computational model of pathway behaviour and phenotypical outcome in relation 

to drug response. With this approach we anticipate to maximize the success rate of 

available targeted therapies against MAPK and PI3K pathway components 

Figure 2: Breast cancer phospho-proteomics Analysis of protein and phosphoprotein expression in 

a panel of breast cancer cell lines in the presence of a specifi c kinase inhibitor measured on a reverse 

phosphoprotein array platform (RPPA). The heatmap indicates the relative differences in the 

phosphoprotein levels after 20 minutes stimulation with EGF in the presence of a kinase inhibitor. 

77 


Publications 

Nijwening JH, Geutjes EJ, Bernards 

R, Beijersbergen RL. The histone 

demethylase Jarid1b (Kdm5b) is a novel 

component of the Rb pathway and 

associates with E2f-target genes in MEFs 

during senescence.PLoS One. 


Paul P, van den Hoorn T, Jongsma 

ML, Bakker MJ, Hengeveld R, Janssen L, 

Cresswell P, Egan DA, van Ham M, Ten 


C, Neefjes J. A. Genome-wide 

multidimensional RNAi screen reveals 

pathways controlling MHC class II antigen 

presentation. Cell. 2011;145:268-83 

Nijwening JH, Kuiken HJ, 

Beijersbergen RL. Screening for 

modulators of cisplatin sensitivity: 

unbiased screens reveal common themes. 

Cell Cycle. 2011;10:380-6 

Heeg S, Hirt N, Queisser A, Schmieg 

H, Thaler M, Kunert H, Quante M, 

Goessel G, von Werder A, Harder J, 

Beijersbergen RL, Blum HE, Nakagawa 

H, Opitz OG. EGFR overexpression 

induces activation of telomerase via PI3K/ 

AKT-mediated phosphorylation and 

transcriptional regulation through 

Hif1-alpha in a cellular model of 

oral-esophageal carcinogenesis. Cancer 

Sci. 2011;102:351-60 

Beijersbergen RL, Bernards R. 

Functional subtyping of breast cancer. 

Cancer Discovery. 2011;1:205-206

78 


Group leader Rob Wolthuis 

Rob Wolthuis PhD Group leader 

Cornelia Rumpf PhD Post-doc 

Michiel Boekhout MSc PhD student 

Linda Clijsters MSc PhD student 

Erik Voets MSc PhD student 

Janneke Ogink Technical staff 

Bas ter Riet Technical staff 

Cinzia de Benedictis Technical staff 

Figure 3: Measuring APC/C activity by 

time-lapse fl uorescence microscopy of 

Cyclin B1 destruction A plasmid encoding 

for fl uorescent cyclin B1 was injected into 

G2-phase cells, a few hours before they 

entered mitosis. The top panel shows a cell 

undergoing mitotic division, followed over 

time by differential interference contrast 

(DIC). The bottom panel shows the 

localisation and quantitative levels of cyclin 

B1 in the fl uorescence channel of the same 

cells. The cell undergoing division rapidly 

degrades cyclin B1, as measured by a 

decrease in fl uorescence signal. This assay 

allows the quantitation of ubiquitindependent 

protein destruction in live cells 

undergoing division and was fi rst established 

in the laboratory of Jon Pines, Gurdon 

Institute, Cambridge, UK 

HOW ONE CELL CREATES TWO: 

MECHANISMS OF CELL DIVISION 

Creating new cells Cell division forms the deadly essence of cancer and should 

therefore be a key target of directed therapy. My group aims to understand how 

cell division works at a molecular level in human cells. We study the major 

transitions in the cell cycle that rely on integrated signalling reactions and passage 

through molecular checkpoints. Such intertwined pathways are clearly sensitive to 

therapeutic intervention, a principle often used in existing cancer therapy, but they 

also contain redundancies and feed-back loops that make drug responses hard to 

predict. Passage through cell division can perhaps best be seen as a sequence of key 

‘cellular decisions’. Such decisions refl ect the balance between positive and negative 

signals and are prone to adaptations and cell-type differences. It is our goal to extract 

the core molecular principles of each of these major hurdles that cells take in the 

division process until they successfully split into two. 

Responses to cell division blockage Equally important in our work is the other 

side of the coin: to understand how cells react when they fail to pass such essential 

cell division hurdles, for instance in response to therapeutic drugs or to naturally 

occurring cellular insults. We therefore also analyse how checkpoints and divisionarrests 

impinge on the fi tness of cells. To this end, we pay much attention to the 

identifi cation and development of molecular probes revealing crucial steps in a 

cell’s decision-taking process. Supported by a recently awarded HFSP grant, we are 

developing new markers of cell division events. We expect that our work will lead 

to a better understanding of specifi c prognostic factors that determine a cancer 

cell’s proliferation capacity. Furthermore, we intend to design more directed ways 

to intervene with cancer-relevant steps in the decision-taking process and start 

predicting which pathways will determine the eventual response to therapy. 

Approaches and goals For these purposes, we use a combination of biochemistry, 

molecular genetics and automated fl uorescence microscopy of normal and cancer 

cells. We are focussing on the decisive pathways in three major cell cycle transitions: 

i) the entry into mitosis from G2-phase; ii) passage through mitosis and the 

formation of two daughter cells (fi gure 3), and iii) re-initiation of DNA replication 

after mitosis (fi gure 4). In 2011, we set-up a new research line to better understand 

adaptation mechanisms invoked when cells are forced to stall during cell division 

attempts. Interestingly, cells that delay in mitosis, e.g. in response to the cancer 

drugs paclitaxel, cannot initiate gene expression to generate a stress response 

because transcription is actively silenced in this cell cycle phase. We found that these 

cells instead employ regulation of de novo protein synthesis to survive a stressful 

delay in the mitotic state. We discovered that this involves a specifi c change in the 

translation initiation machinery resulting in a unique mitotic protein expression 

program, involving cell cycle genes, chaperones and regulators of apoptosis. For this 

project, which will be continued and further completed in 2012, we collaborate with 

the group of Antonis Koromilas (McGill University, Montreal), who studies how 

protein synthesis is regulated under stress conditions. 

Bridging the gap between genomics and cell biology In 2011, my group invested 

considerably in the development of equipment and detection tools for automated 

fl uorescence microscopy at the NKI. These are needed to measure, at suffi cient 

resolution, the dynamic behaviour of key regulatory proteins. Furthermore, we are

continually developing single-cell assays that reveal the specifi c enzymatic activities 

of such regulators. This requires the availability of fast and high-resolution image 

capturing equipment, combined with computer automation to follow fl uorescent 

molecules in large numbers of dividing cells and under normal culture conditions. 

Furthermore, automation of fl uorescence laser beam-based technologies must 

be in place to change protein function at our demand. In the beginning of this 

year, we collaborated with the Biophysics group of Kees Jalink and the NKI Digital 

Microscopy Facility (DMF) to implement an ‘NWO-Groot’ Equipment Grant for a 

Leica SP5-based high sensitivity confocal microscope platform. On this system, socalled 

Matrix Screening becomes possible, a set-up developed by the Jan Ellenberg 

group in Heidelberg for systematic analyses of cell division. The introduction of 

this new-generation confocal platform creates unprecedented opportunities for 

higher-throughput advanced fl uorescence cell analyses at the NKI. In addition, 

thanks to support by the Nefkens Foundation and the NKI, three Applied Precision 

DeltaVision deconvolution cell imaging systems have been tailor-designed by our 

group, to address emerging questions in cell division research posed at the NKI. 

These will be implemented in a new microscopy lab, shared with the group of Rene 

Medema, at the Division of Cell Biology to which our group will move in 2012. 

From G2 to Mitosis Cells pause in G2 phase when the conditions are unfavourable 

for safe cell division, such as after DNA damage, or when cells are too small. The 

decision to enter mitosis is controlled by signalling pathways that direct activation of 

the principal mitotic kinase, cyclin B1-CDK1. We study the mechanisms that trigger 

cyclin B1-CDK1 activation in late G2 and the parameters of an ‘activation threshold’ 

for mitosis. Also phosphatases participate in triggering the G2-M transition. 

A recent project led to the identifi cation of the human Greatwall kinase, MASTL. 

MASTL reduces the mitotic entry threshold and drive mitotic entry of G2 cells by 

inhibiting PP2A activity. By determining the mechanisms that direct mitotic entry, 

we aim to understand how normal and cancer cells may differ in their decisions to 

enter mitosis or stay in G2 phase. 

Passing Mitosis and Initiating S-phase Once cells are in mitosis, they need to 

ensure that all the duplicated chromosomes are attached to the mitotic spindle. 

This is safeguarded by the mitotic spindle checkpoint, which inhibits another key 

enzyme of our interest, a large ubiquitin ligase called the Anaphase-Promoting 

Complex (APC/C) and its activator CDC20. Mitotic entry fi rst requires accumulation 

of regulatory proteins, such as cyclin B1, but these proteins must be destroyed again 

at distinct, consecutive time points for correct cell division (fi gure 3). We found 

that degradation of cyclin A and Nek2A, followed by loss of cyclin B1, GEMININ 

and SECURIN, coordinates mitosis and prepares cells for the next S phase (fi gure 

4). To study the role of GEMININ in more detail, we developed markers of DNA 

replication licensing, which revealed that the spindle checkpoint controls the timing 

of licensing by ensuring simultaneous, APC/C-CDC20 dependent destruction of 

GEMININ and cyclin B1. Altogether, our recent work revealed new roles for APC/Ccooperating 

E2 ubiquitin conjugating enzymes, mechanisms by which APC/C 

substrates are targeted for destruction, and roles of the spindle checkpoint in 

connecting mitosis to the following S phase. An important future research goal will 

be to resolve the factors that determine the fate of cells that are arrested in mitosis 

as a result of prolonged spindle checkpoint activity. It is anticipated that answers to 

these questions could create exciting opportunities to improve anti-cancer therapies. 

79 


Figure 4: Regulation of DNA-replication 

licensing by spindle checkpoint-dependent 

control of GEMININ levels Live cells stably 

expressing both geminin-Cherry and 

Cdt1-Venus were imaged by DIC and 

fl uorescence microscopy. Geminin is 

degraded when the spindle checkpoint is 

switched off. Cdt1-Venus localization acts as 

a marker for DNA licensing. The different 

phases of mitosis as observed by DIC 

microscopy are indicated. (Adapted from 

Clijsters et al, submitted)

80 

MOLECULAR GENETICS 

Division head, group leader 

Maarten van Lohuizen 

Maarten van Lohuizen PhD Group leader 

Elisabetta Citterio PhD Senior post-doc 

Karim Nacerdine PhD Post-doc 

Inka Pawlitzky PhD Post-doc 

Gaetano Gargiulo PhD Post-doc 

Anke Sparmann PhD Post-doc 

Bas Tolhuis PhD Post-doc 

Nienke de Vries PhD Post-doc 

Bart Westerman PhD Post-doc 

Cesare Lancini PhD Post-doc 

Waseem Akthar PhD Post-doc 

Michela Serresi PhD Post-doc 

Joep Vissers MSc PhD student 

Martijn Koppens PhD student 

Santiago Gisler PhD student 

Marleen Blom Technical staff 

Paulien Cornelissen Technical staff 

Danielle Hulsman Technical staff 

Ellen Tanger Technical staff 

Els Verhoeven Technical staff 

Huub van Vugt Technical staff 

Figure 1: Mammary fat pad 

transplantation assay. Ductal outgrowth is 

severely impaired upon transplantation of 

Bmi1-/- (KO) when compared to 

Bmi1+/- (Het). This suggests a severe 

mammary epithelial precursor (stem) cell 

defect in Bmi1 defi cient mice. 

DIVISION OF MOLECULAR GENETICS 

ROLE OF POLYCOMB-GROUP GENES IN TRANSCRIPTIONAL 

REPRESSION, STEM CELL FATE AND TUMORIGENESIS 

Our lab has a long-standing interest in epigenetic gene regulation dictated by 

chromatin modifi cations. We study the mechanism of transcriptional repression 

by Polycomb-group (Pc-G) protein complexes, and the effects of deregulation 

of Pc-G genes on development, Cell cycle control, cancer formation and stem 

cell maintenance. In addition, we are performing large-scale genetic screens in 

primary cells and in cancer-predisposed mice to identify cancer-relevant networks 

of oncogenes and tumor-suppressor genes. Model organisms comprise Mouse and 

Drosophila. 

Functional characterization of Pc-G protein complexes Repressive Pc-G proteins 

and counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are 

involved in maintenance of proper gene expression patterns during development at 

the level of chromatin structure. Pc-G protein complexes control large sets of genes 

including Hox gene clusters and the INK4a/ARF tumor suppressor locus. At least 

two biochemical distinct evolutionary highly conserved Pc-G protein complexes 

can be distinguished. The fi rst (PRC2) contains EzH1/EzH2 (SET domain proteins 

acting as Histone H3 methylases), Su(z)12, Eed and histone deacetylases. The 

second large complex (PRC1) encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2 

and Ring1b/Ring1a together with other more loosely associated proteins is required 

throughout development. To study Pc-G, function we focus on representative 

members of PRC1 and PRC2 in gain- and loss-of-function studies in mice and 

Drosophila. In genetic and biochemical experiments we identifi ed the Bmi1/ 

Ring1b heterodimer as an E3 ubiquitin ligase for monoubiquitination of histone 

H2A (collaboration with G Buchwald and Titia Sixma, division of Biochemistry). 

Conditional Ring1b loss-of-function experiments indicate an essential role for 

maintenance of Pc-G repression in development and stem cell maintenance. An 

outstanding question is how the activity of PcG enzymes is regulated; we recently 

obtained evidence that phosphorylation of Bmi1 is required for E3 ligase activity 

of PRC1 and by mutating the essential phosphorylation sites demonstrated that 

these are important for Bmi1’s oncogenic capacity. In addition we are studying the 

function of the deubequitinating enzyme Usp3 that binds to mono-ubiquinated H2A 

and can remove this mark. A major unresolved question is where and how Pc-G 

complexes bind to chromatin. We have performed genome-wide surveys of where 

PRC1 and PRC2 complexes bind to the Drosophila and mammalian genomes using 

DAMid profi ling on tilling arrays (collaboration with Bas van Steensel, division of 

Gene Regulation). This highlighted binding of both PRC1 and PRC2 to distinct 

domains of 10-140 Kb containing ± 400 target genes in Drosophila and identifi ed 

± 800 mammalian targets. These comprise conserved developmental regulators 

that control differentiation. In recent in vivo 4C (chromatin conformation capture 

on Chip) experiments we demonstrated that these domains interact in vivo in 3D 

nuclear space in Drosophila neural tissues. 

Connections between Pc-G gene repression, control of stem cell fate and 

cancer formation We originally identifi ed Bmi1 as an oncogene that cooperates 

with cMyc in the induction of B and T-cell lymphomas in mice, underscoring the 

connection between deregulation of Pc-G repression and cancer. In contrast, Bmi1 

knockout mice show severe progressive proliferation defects and increased apoptosis 

of lymphoid and myeloid cells, resulting in severe lymphopenia. In addition, also 

Bmi1-defi cient primary embryo fi broblasts (MEFs), neural precursors and many 

other primary cell types show proliferation defects. We demonstrated that these 

defects are in part due to increased levels of the tumor suppressors p16INK4a 

and p19ARF, that are critical regulators of the RB and the p53 tumor suppressor 

pathways. As such, the INK4a/ARF locus acts as an important tumor-prevention

mechanism in normal cells and stem/precursor cells. Using the mammary fat pad 

transplantation model, we revealed the essential role of Bmi1 in mammary epithelial 

stem cells and precursors and ductal tree development (fi gure 1). Genetic studies 

showed that the proliferative defects but not the observed premature differentiation 

upon loss of Bmi1 in mammary epithelial precursors is in part mediated via INK4a/ 

ARF. Importantly, these studies revealed a dual role for Bmi1/Pc-G: controlling 

both proliferation and differentiation. A key characteristic of cancer cells is their 

unlimited self-renewal. In this respect, cancer cells resemble stem cells, and 

accumulating evidence suggests that many forms of cancer may indeed contain cells 

carrying stem cell markers. In studying the proliferation defects in Bmi1 defi cient 

mice we discovered that Bmi1 is required for proliferation and self-renewal of neural 

stem cells. Importantly, loss of the INK4a/ARF locus rescues the proliferation 

& renewal defects, indicating it also is a critical Pc-G target in neural stem cells. 

Using a transplantable Glioma model we demonstrated a critical role for Bmi1 in 

brain tumor maintenance (fi gure 2). Interestingly, Bmi1 acts in this tumor setting 

in an Ink4a/ARF-independent manner on cell adhesion and migration. These 

results, together with the recently established role of Bmi1 in hemapoietic stem cells 

and leukaemic stem cells, suggest a common conserved role for Bmi1-containing 

Polycomb complexes in maintenance and expansion of stem cells or committed 

progenitors and in the pathogenesis of tumors originating from the neoplastic 

transformation of these cells. The possible broader relevance of these fi ndings for 

human cancer is further underscored by the amplifi cation of BMI1 in Mantle cell 

lymphomas and a subset of brain cancers and the overexpression of BMI1 in various 

tumor types including non-small cell lung cancer, breast cancer, prostate cancer 

and liver cancer. Conditional transgenic- and knockout models are currently used 

to investigate the role of Pc-G genes in various tissue stem/progenitors and in solid 

cancers that develop in these tissues. 

In vivo genetic screens to identify new groups of collaborating oncogenes 

or tumor suppressors In close collaboration with Anton Berns (this Division), Jos 

Jonkers (Division of Molecular Biology) and D Adams and A Bradley/The Sanger 

Centre, Hinxton, UK, we have developed high-throughput insertional mutagenesis 

techniques. The power of this approach as a cancer gene discovery platform is 

highlighted by the fi rst completed screens in hemapoietic tumors induced in wild 

type, p53 or p19Arf defi cient mice. We recently extended these screens to p15Ink4b, 

p21 and p27 defi cient mice and to Pten-defi cient mice prone to MMTV-induced 

mammary tumorigenesis. These screens yielded over 10.000 insertion sites 

implicating over 300 loci in tumorigenesis and uncovered new pathway-specifi c 

oncogenes and candidate tumor-suppressors. Cross species comparative analysis 

with a large array-CGH dataset of human cancer cell lines revealed both new and 

novel candidate oncogenes and tumor-suppressor genes. In addition, this screen 

allowed the identifi cation of both mutual exclusive as well as cooperative interactions 

between these genes. The role and mechanism of action of several of these new 

putative oncogenes or tumor suppressors, is under investigation hemapoietic- and 

mammary fat pad cell-transplantation systems. 

Figure 2: Severely reduced 

Glioma formation of 

Bmi1-/- transformed 

astrocytes. Survival curves 

indicate that astrocytes 

oncogenically transformed 

by loss of INK4a/ARF and 

activation of EGF-receptor 

signaling rapidly form 

agressive gliomas whereas tumor formation is delayed upon transplantation of Bmi1-defi cient 

transformed astrocytes orthotopically transplanted in the forebrain of recipient mice. 

Publications 

81 


Westerman BA, Braat AK, Taub N, 

Potman M, Vissers JHA, Blom M, Verhoeven 

E, Stoop H, Gilis A, Velds A, Nijkamp W, 

Beijersbergen R, Huber LA, Loojenga LHJ, 

van Lohuizen M. A genome-wide RNAi screen 

in mouse embryonic stem cells identifi es Mp1 

as a key mediator of differentiation. J. Ex Med 

2011 (in press) 

Martin N, Benhamed M, Nacerddine K, 

Demarque MD, van Lohuizen M, Dejean A, 

Bischof O. Physical and functional interaction 

between PML and TBX2 in the establishment 

of cellular senescence. EMBO J. 2011:10;1038 

de Jong J, de Ridder J, van der Weyden L, 

Sun N, van Uitert M, Berns A, van Lohuizen 

M, Jonkers J, Adams DJ, Wessels LF. 

Computational identifi cation of insertional 

mutagenesis targets for cancer gene discovery. 

Nucleic Acids Res. 2011;39:e105 

Smith LL, Yeung J, Zeisig BB, Popov N, 

Huijbers I, Barnes J, Wilson AJ, Taskesen E, 

Delwel R, Gil J, Van Lohuizen M, So CW. 

Functional crosstalk between Bmi1 and MLL/ 

Hoxa9 axis in establishment of normal 

hematopoietic and leukemic stem cells. Cell 

Stem Cell. 2011;8:649-62 

van der Velden YU, Wang L, Zevenhoven 

J, van Rooijen E, van Lohuizen M, Giles RH, 

Clevers H, Haramis AP. The serine-threonine 

kinase LKB1 is essential for survival under 

energetic stress in zebrafi sh. Proc Natl Acad 

Sci U S A. 2011;108:4358-63 

Ginjala V, Nacerddine K, Kulkarni A, Oza 

J, Hill SJ, Yao M, Citterio E, van Lohuizen M, 

Ganesan S. BMI1 is recruited to DNA breaks 

and contributes to DNA damage induced H2A 

ubiquitination and repair. Mol Cell Biol. 

2011;31:1972-82 

Tolhuis B, Blom M, Kerkhoven RM, Pagie 

L, Teunissen H, Nieuwland M, Simonis M, 

de Laat W, van Lohuizen M, van Steensel B. 

Interactions among Polycomb Domains Are 

Guided by Chromosome Architecture. PLoS 

Genet. 2011;7:e1001343

82 


Group leader Anton Berns 

Anton Berns PhD Group leader 

Paul Krimpenfort PhD Academic staff 

Margriet Snoek PhD Academic staff 

Hilda de Vries PhD Post-doc 

Jitendra Badhai PhD Post-doc 

Ivo Huijbers PhD Post-doc 

Min-chul Kwon PhD Post-doc 

Kate Sutherland PhD Post-doc 

Lorenzo Bombardelli PhD Post-doc 

Andrej Alendar MSc PhD student 

Colin Pritchard MSc Research assistant 

Rahmen Bin Ali Technical staff 

Jan Paul Lambooij Technical staff 

Natalie Proost Technical staff 

Maaike Hanegraaf Technical staff 

Fina van de Ahé Technical staff 

John Zevenhoven Technical staff 

Figure 3: Schematic representation 

of the cells of origin that can give rise to 

the different tumors in lung. 

Solid lines indicate transotions we have 

shown to occur. 

MOUSE MODELS FOR CANCER 

The mouse is used as a model organism for establishing the role of oncogenes and 

tumor suppressor genes in tumor development. By utilizing Cre/Lox mediated 

switching and taking advantage of somatic gene transfer methods, expression of 

multiple oncogenes and tumor suppressor genes are regulated in a tissue-specifi c 

and spatial-temporal fashion permitting accurate modeling of tumorigenesis 

as it is observed in man. It provides the opportunity to carefully defi ne relevant 

genotype-phenotype correlations and permit us to identify new genes involved in 

tumor progression using a variety of techniques, such as array CGH, expression 

profi ling and insertional mutagenesis. Finally, these models constitute an excellent 

experimental system to test new intervention strategies. 

Functional analysis of oncogenes and tumor suppressor genes Our emphasis 

is on lung cancer and mesotheliomas. To achieve (sporadic) activation of oncogenes 

and inactivation of tumor suppressor genes we use Adeno-Cre or Lentivirusmediated 

somatic gene transfer to sporadically switch the conditional oncogenes and 

tumor suppressor gene alleles in the desired tissues. Subsequently, tumor initiation 

and progression is monitored longitudinally. 

Lung tumors We focus on small cell lung cancer (SCLC) and non-small cell 

lung cancer (NSCLC). When Rb and p53 are inactivated specifi cally in lung, SCLC 

develops in nearly 100% of the mice. The marker profi le of these tumors closely 

resembles that of human SCLC. Even similar genomic aberrations are found such 

as the amplifi cation of the L-Myc gene. These tumors are often heterogeneous 

consisting of different cell types that, when cultured in vitro, either grow as 

spheres in suspension or attached to substrate. Cells growing in suspension 

carry neuroendocrine markers whereas the adherent cells show progenitor-like 

features. Interestingly, these phenotypically very diverse cell lines share distinct 

genetic aberrations indicating that they were derived from a common progenitor. 

Subcutaneous grafting of each of the cell types independently gave rise to localized 

tumors that retained the features of the inoculated cells. However, grafting 

mixtures resulted in local growth as well as metastasis of the neuroendocrine 

cells to liver indicating that the non-neuroendocrine cells in the graft endowed 

the neuroendocrine cells with metastatic potential. We have shown that this 

requires activation of the MAPK pathway in neuroendocrine cells through factors 

released from the non-neuroendocrine cells. One of the critical genes induced in 

neuroendocrine cells is PEA3, a gene previously found to be involved in invasion 

and metastasis. PEA3 appears required but is not suffi cient to convey full metastatic 

potential to the neuroendocrine cells. 

The presence of subclones in the tumor with greatly different marker profi les 

raised the question of the cell of origin of SCLC. This is likely also a key factor for 

understanding the behavior of the tumor with respect to malignant progression and 

response to therapy. To gain insight into the cell 

of origin of SCLC and NSCLC we have developed 

a series of Adeno-Cre viruses carrying cell-type 

specifi c promoters driving Cre expression. These 

vectors enable us to switch oncogenes and tumor 

suppressor genes in distinct lung cell types in 

vivo. In this way we can direct Cre expression 

specifi cally to Clara cells, Alveolar type II 

cells, neuroendocrine cells and basal epithelial 

cells. It appeared that Cre driven from a 

neuroendocrine-specifi c promoter in conditional 

Rb/p53 mutant mice gives rise to SCLC with high 

effi ciency, whereas Cre driven from the alveolarspecifi 

c promoter SPC shows a much lower 

incidence and delayed onset of SCLC. Expression 

from a Clara cell-specifi c promoter caused 

mostly hyperplasia in the bronchial epithelial 

lining and resulted only rarely in SCLC. When

similar experiments are performed in conditional mutant KrasG12D mice NSCLC 

is most effi ciently induced upon expression of Cre in alveolar type II cells. Clara cell 

specifi c expression resulted in slower tumor development and the tumors showed 

pronounced papillary features indicating that the cell of origin is an important factor 

for the phenotypic features of the tumor that arises. When tumors were induced 

in compound KrasG12D;p53 mice adenocarcinomas developed after infection with 

all the different viruses, indicating that a wider range of cell types can give rise to 

adenocarcinomas when p53 is concomitantly inactivated (fi gure 3). 

Mesotheliomas Previously we have shown that inactivation of Nf2 and Ink4a/Arf 

or Nf2/p53/Ink4a by intrathoracic Adeno-Cre injection in compound conditional 

knockout mice gives rise to mostly sarcomatoid mesotheliomas and that Ink4a 

plays an important role in the aggressiveness of the tumor. To make this tumor 

model more amenable for in vitro and in vivo screens, we have isolated un-switched, 

phenotypically different cells from the mesothelial lining of these mice. We can 

propagate those cells in vitro and this not only permits effi cient cell type specifi c in 

vitro switching of conditional alleles but also allows controlled introduction of other 

genes or shRNA constructs. Subsequent injection of these cells in recipient mice 

gives rise to mesotheliomas. This permits us to test responses to specifi c inhibitors 

both in vitro and in vivo. We have explored the sensitivity of these different cell types 

to a panel of different drugs. The cell of origin appears a factor infl uencing in the 

drug response of these cells in vitro. 

Parallel to these experiments we are testing protocols to establish cultures from 

mesothelioma specimen of human mesotheliomas. So far it has been diffi cult to 

achieve growth after grafting primary tumor cells in immunodefi cient mice. 

Role of DCC in tumor progression Since its discovery in the early 1990s the DCC 

gene, located on chromosome 18q21, is suggested as tumor suppressor gene as its 

loss is implicated in the majority of advanced colorectal and many other cancers. 

DCC belongs to the family of Netrin-1 receptors, which play a major role in the 

development of the nervous system by guiding axonal outgrowth. The Netrin-1 

receptors were also shown to function as dependence receptors as they deliver 

survival signals in the presence and induce apoptosis in the absence of their ligand. 

However, the role of DCC as a tumor suppressor remains controversial because of 

the rarity of DCC specifi c mutations, and the presence of other tumor suppressor 

genes in the same chromosomal region. We have shown that in a mouse model for 

mammary carcinoma based on somatic inactivation of p53 additional loss of Dcc 

promotes metastasis formation without affecting the phenotype of the primary 

tumor. Furthermore, we demonstrate that in cell cultures derived from p53-defi cient 

mouse mammary tumors Dcc expression controls Netrin-1 dependent cell survival 

providing a mechanistic basis for the enhanced metastatic capacity of tumor cells 

lacking Dcc. In line with these in vitro observations, ‘in vivo’ tumor cell survival 

is enhanced by Dcc loss. Together, our data support DCC ‘s function as a contextdependent 

tumor suppressor that limits survival of disseminated tumor cells. 

ES cell lines from compound mutant mice We have invested in the development 

of methods to accelerate the generation of complex compound mutant mice. 

The concept is to re-derive embryonic stem (ES) cells from available compound 

mutant strains and use these ES cells to introduce additional genetic changes 

by gene targeting or the exchange of expression cassettes. This approach has 

become within reach by the description of defi ned media (2i and 3i) that prevent 

the differentiation of ES cells. Using this approach we have successfully derived 

a series of ES cell lines from the strains we routinely use. Injection of these ES 

cells in pre-implantation embryos results in the majority of cases in chimeras 

with extensive ES cell contribution. We have shown that these chimeras can be 

directly used for tumorigenesis studies and that the latency period is very similar 

whereas the histopathology is identical to that of the original model from which the 

ES cells were derived (fi gure 4). We have equipped these ES cells with exchange 

cassettes permitting introduction of additional genes or shRNAs and begun to 

apply this strategy to interrogate tumor progression mechanisms in the lung and 

mesothelioma models. 

Publications 

83 


Calbo J, van Montfort E, Proost N, 

van Drunen E, Beverloo HB, Meuwissen 

R, and Berns A. A functional role for 

tumor cell heterogeneity in a mouse model 

of small cell lung cancer Cancer Cell. 

2011;19:244-256 

de Jong J, de Ridder J, van der Weyden 

L, Sun N, van Uitert M, Berns A, van 

Lohuizen M, Jonkers J, Adams DJ, and 

Wessels LFA. Computational 

identifi cation of insertional mutagenesis 

targets for cancer gene discovery Nucleic 

Acids Res. 2011;39:e105 

Huijbers IJ, Krimpenfort P, Berns A, 

and Jonkers J. Rapid validation of cancer 

genes in chimeras derived from established 

genetically engineered mouse models 

Bioessays. 2011;33:701-710 

Nawijn MC, Alendar A, and Berns A. 

For better or for worse: the role of Pim 

oncogenes in tumorigenesis Nat Rev 


Sutherland KD, Proost N, Brouns I, 

Adriaensen D, Song J-Y, and Berns A. 

Cell of origin of small cell lung cancer: 

inactivation of Trp 53 and rb1 in distinct 

cell types of adult mouse lung Cancer Cell. 

2011;19:754-764 

Figure 4: Incidence of tumor development in 

small cell lung cancer and mesothelioma in 

the original strains and in the chimeric mice 

produced with ES cells from the strains. The 

similarity in latency illustrates that chimeric 

mice can be directly used in tumor induction 

experiments. This reduces the time to generate 

the models and substantially reduces the number 

of mice needed to conduct experiments.

84 



Group leader Daniel Peeper 

Daniel Peeper PhD Group leader 

Renee van Amerongen PhD Post-doc 

Gireesh Anirudhan PhD Post-doc 

Tristan Gallenne PhD Post-doc 

Kylie Greig PhD Post-doc 

Kristel Kemper PhD Post-doc 

Christelle Lenain MD PhD Post-doc 

Katrin Meissl PhD Post-doc 

Judith Mueller PhD Post-doc 

Patricia Abrao Possik PhD Post-doc 

Marjon Smit PhD Post-doc 

Celia Vogel PhD Post-doc 

Sedef Iskit MSc PhD student 

Joanna Kaplon MSc PhD student 

Sirith Douma MSc Technical staff 

Nils Visser MSc Technical staff 

Publications 

Peeper DS. Ageing: Old cells under 

attack. Nature 2011;479:186-7 

Peeper DS. Oncogene-induced 

senescence and melanoma: where do we 

stand? Pigment Cell Melanoma Res. 

2011;24:1107-11 

Peeper DS. C/EBPβ: lost beyond 

translation. EMBO J. 2011;30:3663-4 

Geiger TR, Song JY, Rosado A, Peeper 

DS. Functional characterization of 

human cancer-derived TRKB mutations. 

PLoS One. 2011;6 

Hömig-Hölzel C, van Doorn R, Vogel 

C, Germann M, Cecchini MG, Verdegaal 

E, Peeper DS. Antagonistic TSC22D1 

variants control BRAF(E600)-induced 

senescence.EMBO J. 2011;30:1753-65 

Smit MA, Peeper DS. Zeb1 is required 

for TrkB-induced epithelial-mesenchymal 

transition, anoikis resistance and 

metastasis. Oncogene. 2011;30:3735-44 

Vizioli MG, Possik PA, Tarantino E, 

Meissl K, Borrello MG, Miranda C, 

Anania MC, Pagliardini S, Seregni E, 

Pierotti MA, Pilotti S, Peeper DS, Greco 

A. Evidence of oncogene-induced 

senescence in thyroid carcinogenesis. 

Endocr Relat Cancer. 2011;18:743-757 

DISSECTING CANCER CELL SIGNALING NETWORKS & 

IDENTIFYING THERAPEUTIC CANCER TARGETS 

Are there any intrinsic cellular mechanisms that protect us against cancer? How 

can we dissect tumor-suppressing genetic networks? How can we effectively identify 

novel cancer genes? Can we use our laboratory results to make a difference in the 

clinic? For example, how can we identify novel and specifi c drug targets? And can 

we fi nd ways to enhance the activity of currently used drugs? In a nutshell, these 

are the fundamental as well as clinically relevant questions that we are taking up 

in the Peeper laboratory, which has two main focuses. First, we are dedicated to 

dissecting signaling mechanisms that protect mammalian cells against oncogenic 

transformation, aiming to increase our understanding of the molecular principles of 

cancer. Second, we are determined to identify novel and specifi c therapeutic targets 

for cancer. 

Both aims are based on the premises that non-malignant cells require multiple (epi-) 

genetic alterations to transform into metastatic tumor cells, and that tumor cells, in 

turn, become dependent on activated oncogenes but also several normal genes for 

their survival and proliferation. To achieve these goals, we make use of advanced 

techniques, including screens with 100.000-vector RNAi libraries and nextgeneration 

sequencing, but we also use classical biochemical and genetic approaches. 

While we are studying a diverse array of tumor types, there is a particular focus 

on melanoma (a highly aggressive skin cancer) and metastatic breast cancer. 

At the cellular level, we have a special interest in several cancer-relevant events: 

(i) override of Oncogene-Induced cellular Senescence (‘OIS’, a potent tumor 

suppressor mechanism limiting the proliferative capacity of incipient cancer cells); 

(ii) suppression of ‘anoikis’ (apoptosis resulting from lack of adhesion, suppression 

of which may contribute to metastasis); (iii) (non-)oncogene addiction and drug 

enhancer and resistance screens (both in the context of ‘Synthetic Lethality’). Our 

results over the past decade demonstrate that these approaches, together, lead to the 

identifi cation of signaling networks of gene products, deregulation of which allows 

for tumorigenesis and metastasis. Our more recent results indicate that among these, 

several factors exist that can be exploited for therapeutic intervention of cancer. 

ONGOING 

I. Breast cancer metastasis: mechanism and drug target identification 

Metastasis commonly underlies the malignancy of cancers, representing the 

principal cause of cancer-treatment failure and patient death. Tumor spread and 

seeding are prevented by several physiologic barriers, including ‘anoikis’: apoptosis 

resulting from lack of adhesion. Indeed, acquiring resistance to anoikis has been 

proposed to represent a general prerequisite for survival of metastases during 

systemic circulation. Based on this cancer hallmark, and with the help of a genomewide 

functional screen, we previously identifi ed TrkB, a neurotrophic tyrosine 

kinase receptor, as a potent suppressor of anoikis and inducer of highly metastatic 

tumors. We have identifi ed several downstream signaling targets of this receptor, 

including the transcription factors Twist, Zeb1 and Snail. Their RNAi-mediated 

silencing strongly suppressed the metastatic potential of tumor cells. 

Taking advantage of this experimental system, we have discovered a novel and 

critical mediator of breast cancer metastasis, the Fra-1 transcription factor. Fra-1 

depletion from human breast cancer cells reduced their metastatic potential by >3 

orders of magnitude. Further, by analyzing the expression of a set of Fra-1 target 

genes we can make accurate predictions on the clinical outcome of breast cancer. 

Using a synthetic lethal drug screen we identifi ed inhibitors of the adenosine 

receptor Adora2B, which are preferentially cytotoxic to metastasizing, Fra-1expressing, 

cells, relative to Fra-1-depleted, non-metastasizing cells. Exploiting the 

prognostic Fra-1 signature we have found several additional factors that may be 

amenable to targeted inhibition. Eventually, this should lead to combined diagnosis/ 

treatment options for the patient. 

II. Oncogene-Induced cellular Senescence (OIS): mechanism and drug 

target identification We are identifying novel OIS pathways whose deregulation 

contributes to cancer in general and melanoma in particular. Also in this setting,

we are combining function-based, genome-wide screens with 

classical molecular biological approaches. For example, we 

study the genetic basis for malignant progression of benign 

moles (nevi) to malignant melanoma. We, in a longstanding 

collaboration with prof. Wolter Mooi (VUmc), were the fi rst to 

discover that moles display many of the classical hallmarks of 

OIS in vivo. In subsequent investigations, using a combination 

of genetic profi ling and laser capture microdissection of 

compound clinical nevus-melanoma specimens, we have found 

that activation of the PI(3)Kinase pathway represents a frequent 

event by which nevi can escape from senescence, thereby 

progressing to melanoma. Targeted inhibition of PI(3)Kinase 

restores senescence features in melanoma cells, raising the 

possibility that these results can be applied clinically. 

Further, gene-expression microarray expression analysis of 

senescent cells and their ‘escapers’ revealed that senescent 

cells adopt an infl ammatory phenotype. Depletion of several 

of these factors, particularly interleukins 6 and 8, abrogated 

the senescence response. The dozens of cytokines and other 

factors that are secreted by senescent cells serve to allow for 

communication between them and their microenvironment. 

Hence, we termed this the Senescence-Messaging Secretome, or SMS. 

We have also recently discovered a functional connection between cellular 

metabolism and OIS. Although it is conceivable that the cellular metabolism is 

deregulated during, and perhaps contributes to, OIS, little is known about such 

communication. By comprehensive metabolic fl ux profi ling and functional analysis 

we unmasked the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) as an 

enzyme critically contributing to OIS. OIS was accompanied by the simultaneous 

downregulation of the inhibitory PDH kinase PDK1 and induction of the PDHactivating 

phosphatase PDP2, leading to the concerted activation of PDH. This 

caused a major shift in metabolite fl ux from glycolysis to the tricarboxylic acid (TCA) 

cycle, increasing pyruvate oxidation. Conversely, bypass of OIS, a rate-limiting 

step towards oncogenic transformation, coincided with PDH inhibition. Enforced 

normalization of PDK1 or PDP2 levels inhibited PDH and abrogated OIS, causing 

continued cell proliferation. These results establish a functional link between OIS 

and a key mitochondrial signaling axis balancing glycolysis and respiration. 

III. Screening for novel therapeutic targets in melanoma We have set up several 

screens to identify novel therapeutic targets for melanoma. Cancer cells commonly 

depend on activated oncogenes, as well as certain non-oncogenes, for their 

proliferation and/or survival, a phenomenon known as (non-)oncogene addiction. 

In that context, exploiting the phenomenon of synthetic lethality (SL) as a mean of 

therapeutic intervention is gaining increasing interest. For example, set out to screen 

for synthetic lethal partners of BRAF E600 . 61 genes whose knockdown resulted in 

selective toxicity to BRAF E600 -expressing cells, which are currently being validated. 

The BRAF kinase inhibitor PLX4032 has generated tremendous excitement recently, 

following dramatic effects in clinical trials of patients with metastatic melanoma 

carrying the BRAFE600 mutation. Despite such early promise, however, patients 

commonly relapse with drug-resistant tumours. There is, therefore, an urgent need 

to fi nd drugs that could be used in combination with PLX4032 to reduce the risk 

of resistance. Moreover, drugs that enhance the clinical effi cacy of PLX4032 could 

enable lower doses of PLX4032 to be used, reducing the risk of side effects such as 

skin lesions that have already been reported for this drug. We are performing largescale 

shRNA screens to fi nd genes and pathways that sensitize melanoma cells to 

PLX4032. 

Along these lines, we are setting up several additional screens, both in vitro and in 

vivo, to fi nd genes that are essential for tumor progression. For this purpose, we 

make use of large-scale RNAi libraries and massive parallel sequencing. Together, 

these approaches should lead to the identifi cation of novel therapeutic targets in 

melanoma and possibly other cancer types. 

85 


Figure 5: Comparison of the glycolytic 

fl ux in cycling and OIS cells. The profi les 

show that OIS is accompanied by a major 

shift in metabolite fl ux from glycolysis to 

the tricarboxylic acid (TCA) cycle, 

increasing pyruvate oxidation. Additional 

data point to a crucial role for the 

mitochondrial gatekeeper pyruvate 

dehydrogenase (PDH) in OIS. 

Figure 6: Images of the lungs (upper panel) 

and haematoxylin-eosin stained sections of 

the lungs (lower panel, scale bar: 100 μm; 

T: Tumor) of mice that were injected 

intravenously with 1.106 human breast 

cancer MDA-MB-231 cells expressing 

independent Fra-1 shRNAs as indicated, 

photographed at 3 months after inoculation. 

The images illustrate the critical requirement 

for Fra-1 for the metastatic activity of breast 

cancer cells.

86 


Group leader John Hilkens 

John Hilkens PhD Group leader 

Gerjon Ikink MSc PhD student 

Mandy Boer Technical staff 

Publication 

Vendel-Zwaagstra A and Hilkens J. Gene 

Discovery by MMTV Mediated Insertional 

Mutagenesis. In: Insertional mutagenesis 

strategies in cancer genetics. Dupuy AJ and 

Largaespada DA, editors. Springer 2011, 

p38-76 

Figure 7: Irs4 but not Irs1 collaborates with 

ErbB2 in anchorage independent growth. 

NMuMG mouse mammary epithelial cells 

were infected with retroviral constructs 

containing mouse Irs4 or Irs1 cDNA or double 

infected with one of the Irs constructs and 

ErbB2 (Neu). The cells were plated in soft 

agar and the number of colonies was counted 

after 13 days to test for anchorage independent 

growth. The fi gure shows the number of 

colonies obtained with two independently 

infected cell cultures with the constructs or 

combination of constructs indicated. Irs4 but 

not Irs1 synergizes with ErbB2. 

GENES INVOLVED IN BREAST CANCER PROGRESSION 

AND METASTASIS 

Transformation of a normal cell into a malignant cancer cell is the result of accumulation 

of multiple genetic changes affecting oncogenes and tumor suppressor genes. 

Insertional mutagenesis, whole genome sequencing, and other technologies have 

identifi ed a large number of genes involved in oncogenesis. However, only a limited 

number of key players in oncogenesis have been extensively studied, while this is 

essential for the development of more effective novel therapeutic strategies. Our aim is 

to identify genes involved in breast cancer by using insertional mutagenesis in mouse 

models and to characterize these genes and the oncogenic pathways they control. 

Identification of mammary cancer genes in mouse models for breast cancer by 

mouse mammary tumor virus induced insertional mutagenesis screens We have 

identifi ed a large number of MMTV common insertion sites (CISs) in ~ 400 mammary 

tumors from wild-type mice, mice conditionally defi cient for Trp53 (p53 - ) in the 

mammary gland and MMTV-NeuNT transgenic (ErbB2 + ) mice using a splinkerette 

PCR combined with the new parallel sequencing platforms (in collaboration with 

the Jonkers lab). Many of the CISs affect genes not previously implicated in cancer 

or breast cancer in particular. These screens revealed that Wnt and Fgf genes are 

activated in almost all wild-type and p53 - tumors but rarely in ErbB2 + tumors. Genes 

belonging to the R-spondin gene family (Rspo2 and Rspo3) were also frequently 

activated wild-type and p53 - tumors but rarely in tumors from ErbB2 + mice. In 

contrast, other genes were found to be signifi cantly more frequently tagged in tumors 

from MMTV infected ErbB2 + mice than in wild-type mice. 

Genes collaborating with ErbB2 We have identifi ed Eras as frequent MMTV target 

gene in mammary tumors from ErbB2 + mice. Eras was signifi cantly more frequently 

tagged and overexpressed in tumors from MMTV infected ErbB2 + mice than in 

tumors from MMTV infected wild-type mice. Ectopic expression of Eras or Irs4, 

another frequent MMTV target, (but not Irs1) in immortalized normal mouse and 

human mammary epithelial cells (NMuMG and MCF-10A) strongly increased cell 

proliferation and anchorage independent growth and rendered the cells tumorigenic 

in nude mice, validating Eras as an oncogene. Eras as well as Irs4 accelerated ErbB2 

induced tumorigenesis when co-expressed in NMuMG cells. Both genes strongly 

activated the PI3-kinase pathway. ERBB2 preferentially forms a heterodimeric complex 

with ERBB3 in which ERBB2 mainly activates the MAPK pathway, while ERBB3 

predominantly activates the PI3K pathway. Indeed, we found ErbB3 highly expressed 

in all tumors that arise in non-MMTV-infected ErbB2 transgenic mice but only in 57% 

of the MMTV infected ErbB2 transgenic mice. Moreover, Eras expression is negatively 

correlated with ErbB3 expression in MMTV induced tumors (p=0.0075). This strongly 

suggests that activation of the PI3K-pathway via ERAS can act as an alternative for 

activation of this pathway through ERBB3. Although Eras is highly homologous to 

H-Ras and K-Ras, the latter Ras oncogenes did not synergize with ErbB2. In contrast 

to Eras, Irs4 was also frequently tagged in tumors in which ErbB3 was activated 

suggesting that Irs4 has an additional function in mouse mammary tumorigenesis. 

Eras mRNA was also expressed to a variable degree in 20% of human breast cancers 

suggesting a possible involvement of this normally silent gene in human breast 

carcinogenesis. Although Irs4 mutations and overexpression was found in T-cell 

lymphomas we did not fi nd expression in a series of 50 human breast cancers. 

R-spondin genes Rspo2 and Rspo3 are frequently tagged by MMTV in mammary 

tumors from wild-type mice. Although both genes strongly synergize with Wnt in 

in vitro reporter assays, tagging of both gene families was not correlated. We have 

generated transgenic mice conditional expressing Rspo3 in the mammary gland. 

These developed mammary tumors with an average latency of ~ 310 days, confi rming 

the oncogenic capacity of Rspo3. Wnt1 transgenic mice developed tumors with a 

latency of 155 day. Crossing Rspo3 and Wnt1 transgenic mice did not signifi cantly 

increase the latency of the Wnt1 induced tumors suggesting that in these transgenics 

the Wnt1 pathway is already maximally activated or Rspo3 has an independent function 

unrelated to that of Wnt genes.

THE CELLULAR RESPONSE TO TELOMERE DYSFUNCTION 

Telomeres are specialized nucleoprotein complexes that protect natural chromosome 

ends from being seen as DNA breaks. Telomeres progressively shorten every cell 

division, eventually causing telomere dysfunction. This triggers a DNA-damagelike 

response that causes cells to die or stop proliferating indefi nitely (senesce). 

This response limits the replicative life span of cells and contributes to organismal 

aging. In addition, it represents an important tumor suppressor mechanism as it 

prevents unlimited outgrowth of potentially cancerous cells. However, dysfunctional 

telomeres are also subject to DNA-repair activities, which in DNA-damage 

checkpoint-defi cient cells can lead to genomic instability and facilitate development 

of cancer. 

Our main aim is to increase our understanding of how mammalian cells precisely 

perceive and respond to loss of telomere function. 

Identification of factors critical for DNA-damage response signaling and 

repair activities at uncapped telomeres If cells with uncapped telomeres 

fail to senesce or die and continue proliferating, DNA-repair activities acting on 

chromosome ends cause chromosome fusions, anaphase bridges and nonreciprocal 

translocations. Such cells with instable genomes are at high risk of developing 

into cancer. Little is known about the precise structure of an uncapped telomere, 

how it is recognized, what precise processing events occur and how these events 

are controlled. To identify novel factors that contribute to telomere-driven genomic 

instability we developed an RNAi loss-of-function genetic screen that we call TIGIRscreen 

for telomere-induced genomic instability regulators. The TIGIR-screen relies 

on telomere uncapping via well-controlled reversible inactivation of the telomeric 

protein TRF2, using a temperature-sensitive mutant TRF2 allele (TRF2ts) in a 

p53-defi cient background. The basis of our approach is that in this system telomere 

fusions and genomic instability eventually become so severe that cells die. However, 

interference with the effi ciency of telomere fusion, such as upon DNA-ligaseIVdefi 

ciency, reads out as enhanced survival. We use this system both for candidatedriven 

approaches and unbiased screens to identify factors critical for DNA-damage 

response signaling and repair activities at uncapped telomeres. 

As part of a hypothesis-driven approach we investigated the involvement of 

ubiquitylation in the telomere-damage response. Ubiquitylation plays an important 

role in the cellular response to DNA lesions but was not known to act at deprotected 

telomeres. Using TRF2 inactivation we could demonstrate that the ubiquitinligase 

RNF8 signifi cantly contributes to telomere-driven genomic instability by 

controlling DNA-damage response and -repair activities at uncapped telomeres 

through ubiquitylation of telomeric chromatin. We found that uncapped telomeres 

accumulate ubiquitylated histone H2A and H2AX in a manner dependent on RNF8. 

The ability of RNF8 to ubiquitylate telomeric chromatin corresponds to its capacity 

to facilitate accumulation of both 53BP1 and phospho-ATM at uncapped telomeres 

and to promote non-homologous end-joining of deprotected chromosome ends. 

Furthermore, depletion of RNF8, as well as of the ubiquitin-ligase RNF168, reduces 

telomere-induced genome instability. This suggests that RNF8 and RNF168 might 

enhance cancer development by contributing to telomere-induced genomic instability. 

In pilot TIGIR-screens, using limited numbers of shRNAs and cells, we retrieved 

several factors with known activity in the telomere-damage response that together 

illustrate that the TIGIR-screen allows identifi cation of factors acting in any phase 

of the telomere-damage response, from upstream in the sensing of uncapped 

telomeres (NBS1, RAD50, ATM), to signal transduction and recruitment of repair 

factors (RNF8, RNF168, 53BP1), until the fi nal joining of uncapped telomeres (DNAligaseIV). 

Importantly, these pilots also identifi ed several TIGIRs with unknown 

roles at telomeres, including a histone methyltransferase and ubiquitin-modifying 

enzyme without a clear established role at DNA breaks. Their mechanism of 

action in the telomere-damage response is now under investigation, as well as 

their potential roles in the response to DNA double-strand breaks and neoplastic 

transformation. In future work we will continue validating and mechanistically 

investigating hits retrieved from pilot TIGIR-screens. Furthermore we will perform 

these screens on a large-scale genome-wide level. 

87 


Junior group leader Jacqueline Jacobs 

Jacqueline Jacobs PhD Junior group leader 

Marieke Peuscher MSc PhD student 

Jaco van der Torre MSc PhD student 

Publication 

Peuscher MH, Jacobs JJL. DNA-damage 

response and repair activities at uncapped 

telomeres depend on RNF8. Nature Cell 


Figure 8: 

a) Telomere deprotection following 3 hrs of 

inactivation of temperature-sensitive TRF2 

(TRF2ts) at 39 degrees induces foci (TIFs) 

of ubiquitylated H2A at telomeres in an 

RNF8-dependent manner. 

b) shRNA-mediated depletion of RNF8 

compromises accumulation of p-ATM and 

53BP1 at uncapped telomeres after 3 hrs of 

TRF2ts inactivation at 39 degrees. 

c) RNF8-depletion impairs NHEJ-mediated 

fusion of uncapped telomeres upon TRF2ts 

inactivation. 

d) Prolonged inactivation of TRF2ts results in 

severe fusion of chromosome ends and cells are 

unable to divide or die (see control). However, 

shRNA-mediated inhibition of Nbs1, RNF8, 

RNF168 or DNA Ligase IV allows cells to 

survive prolonged telomere uncapping by 

TRF2ts inactivation.

88 


Junior group leader Anna-Pavlina Haramis 

Anna-Pavlina Haramis PhD Junior group leader 

Yme van der Velden MSc PhD student 

Liqin (Bruce) Wang Research assistant 

Publications 

Van der Velden YU, Wang L, Zeven- 

hoven J, van Rooijen E, van Lohuizen M, 

Giles RH, Clevers H, Haramis APG. 

The serine-threonine kinase LKB1 is essential 

for survival under energetic stress in 

zebrafi sh. Proc Natl Acad Sci USA. 

2011;108:4358-63 

Van der Velden YU and Haramis APG. 

Insights from model organisms on the 

functions of the tumor suppressor protein 

LKB1: Zebrafi sh chips in. Aging. 

2011;3:363-67 

Figure 9: The intestinal architecture is not 

maintained in lkb1 mutants. Hematoxylin/ 

eosin stained sections of wt and lkb1 intestines 

at 7 days of development. Loss of epithelial 

folding in lkb1 intestines (arrows). 

THE DEVELOPMENTAL ROLES OF ONCOGENES AND TUMOR 

SUPPRESSORS IN ZEBRAFISH 

Our research focuses on the regulation of tissue homeostasis by extrinsic factors 

(energy supply) and intrinsic mechanisms (epigenetic regulation of differentiation) 

using zebrafi sh as our central experimental model. We are following two main lines 

of research: 

Energy homeostasis in development and disease Here, we aim to understand how 

regulation of energy-metabolism impacts on tissue differentiation and homeostasis. 

The molecular pathways that link energy-metabolism with growth-control during 

development are often deregulated in diseases such as obesity, diabetes and cancer. 

Indeed, control of metabolism is a key element of cancer progression and tumor 

growth is energy-dependent. Several tumor suppressors control metabolic pathways 

and metabolic enzymes have emerged as important regulators of tumor growth. 

A tumor suppressor gene that is important for metabolic control is LKB1. LKB1 is 

a serine-threonine kinase that activates several downstream kinases, including the 

AMP-activated kinase (AMPK), a critical energy-checkpoint at the cell and organism 

level. LKB1 is mutated in a cancer-predisposition syndrome, Peutz-Jeghers Syndrome 

(PJS), and in over 30% of lung adenocarcinomas in humans. Since mouse models 

of lkb1 defi ciency are embryonic lethal, the role of LKB1 and control of energymetabolism 

during development has not been explored. Zebrafi sh (Danio rerio) is an 

ideal system to dissect molecular mechanisms and pathways, largely owing to the 

rapid development and availability of readily accessible transparent embryos. The 

major metabolic pathways are very well conserved between humans and zebrafi sh. 

To study control of metabolism during development, we generated and characterized 

lkb1-defi cient zebrafi sh. This work revealed that LKB1 controls whole-body energy 

homeostasis. Zebrafi sh lkb1 mutants, are embryonic-viable, exhibit deregulated 

metabolism, and fast metabolic rate, a characteristic of tumors cells. That led to 

premature exhaustion of energy reserves and profoundly decreased cellular ATP 

levels. The lkb1 mutants became energy-depleted much sooner than food-deprived 

wild type animals and displayed untimely starvation. 

We are currently investigating the impact of deregulated metabolism on tissue 

homeostasis. For instance, in the lkb1 model of premature starvation, metabolic 

organs such as the liver and intestine exhibit severe defects (fi gure 9) whereas other 

tissues appear less affected. We are studying the molecular mechanisms underlying 

this preferential organ response. We established that a major metabolic adaptation 

process occurs in zebrafi sh when the yolk is consumed and that LKB1 has a central 

role in the regulation of this. We aim to identify the LKB1-controlled molecular 

drivers of this ”switch”. 

Epigenetic regulation of tissue homeostasis Polycomb group (PcG) protein 

complexes, which function in the epigenetic regulation of gene expression, control 

numerous developmental processes. Epigenetic silencing mediated by PcG has 

been shown to have crucial roles in differentiation, proliferation, stem-cell fate 

maintenance and cancer. The PcG member Ring1b is an E3 ubiquitin ligase that 

ubiquitinates histone H2A. This mark correlates with the repression of gene 

expression of PcG target genes. Loss of Ring1b in mouse embryonic stem cells 

causes deregulation of key developmental signaling pathways. However, since 

Ring1b-defi cient mice do not survive beyond early development, the impact of these 

aberrations in gene expression in the context of a developing vertebrate organism 

has been poorly understood. 

We recently generated a stable mutant in zebrafi sh Ring1b by using Zinc Finger 

Endonuclease technology. We have established that Ring1b protein levels and 

ubiquitination of H2A are not detected in the mutants. Interestingly, Ring1bdefi 

cient zebrafi sh survive early development and show surprisingly specifi c defects 

in tissue differentiation. These include most prominently, complete absence of headcartilage 

elements and jaw structures, the lack of pectoral fi ns as well as osteoblast 

stem-cell differentiation defects. The zebrafi sh ring1b mutant provides an excellent 

system to dissect the molecular mechanisms linking PRC1-mediated activity and 

specifi c organ development in vertebrates.

INVESTIGATING THE ROLE OF INFLAMMATION IN PROSTATE 

CANCER DEVELOPMENT 

Immune cells are recruited to the microenvironment of many tumor types. Their 

role in cancer development is controversial since both cancer promoting properties 

and protective activity against tumorigenesis has been reported. However, there 

is emerging evidence that infl ammation is an important factor in the etiology 

of prostate cancer. This evidence stems from epidemiological, histopathological 

and molecular studies. Moreover, there is evidence that the infl ammatory 

microenvironment plays a crucial role in the development of castration resistant 

prostate cancer (CRPC). CRPC reprensents late stage disease with disabling 

symptoms and a high mortality. The aim of our research is to address the role of the 

adoptive and innate immune system in prostate cancer development, development of 

CRPC and drug and radiotherapy sensitivity. 

Role of lymphocytes and mast cells in prostate carcinogenesis Both 

overexpression of myc and loss of PTEN are frequent genetic lesions in human 

prostate cancer. Two mouse models are employed for our studies; one model with 

a prostate specifi c expression of human myc and a model with a prostate specifi c 

loss of PTEN. The myc mouse model develops preneoplastic lesions at the age of 

two monts and invasive prostate cancer after nine months. The PTEN mice develop 

invasive lesions much faster and the tumors also metastasize to the abdominal 

lymph nodes and to the lungs. Immune cells are found in low numbers in the 

prostates of myc mice, including lymphocytes, macrophages and mast cells. The 

role of the immune cells in prostate cancer development is addressed by genetic 

elimination and pharmacological elimination or inhibition of specifi c subsets of cells 

and soluble mediators of the immune system. Therefore, myc mice and PTEN mice 

are crossed to B- and T-cell defi cient RAG2 knock out mice and to mast cell defi cient 

W-kit sash mice. The recruitment of macrophages to the microenvironment can 

be inhibited by pharmacological elimination of the macrophages attractant CCL-2. 

Differences in latency of the development of prostate cancer between wild-type and 

immune defi cient mice are strong evidence for a pivotal role of specifi c subsets of 

the immune system in tumorigenesis. Mechanistic studies concentrate on temporal 

and special analysis of immune cell populations and expression of pro-infl ammatory 

soluble mediators in the tumor microenvironment. 

Role of the inflammatory tumor-microenvironment in castration resistant 

prostate cancer development Bulky prostate tumors of PTEN mice expressing 

Luciferase are harvested and small fragments are ortotopically transplanted into the 

prostates of mice with a genetically or pharmacological impaired immune system. 

After outgrowth of the tumors, mice are castrated which results in regression of 

the tumors. However, tumors will regrow and become castration resistant. Tumor 

regression and subsequent growth can be assessed by bioluminescence techniques. 

Differences in the latency of development of castration resistance between wildtype 

and immune defi cient mice are indicative for a role of specifi c subsets of the 

immune system in the development of CRPC. 

89 


Group leader André Bergman 

André Bergman MD PhD Group leader 

Hester van Zeeburg PhD Post-doc 

Johan van Burgsteden MSc Technical staff 

Jane van Heeteren MSc Technical staff

90 

EPIDEMIOLOGY 

Division head, group leader Flora van Leeuwen 

Group leader Matti Rookus 

Flora van Leeuwen PhD Group leader 

Matti Rookus PhD Group leader 

Marjanka Schmidt PhD Junior group leader 

Berthe Aleman PhD Academic staff 

Annegien Broeks PhD Academic staff 

Frans Hogervorst PhD Academic staff 

Nicola Russell PhD Academic staff 

Emiel Rutgers MD PhD Academic staff 

Michael Schaapveld PhD Academic staff 

Laura van ’t Veer PhD Academic staff 

Senno Verhoef PhD Academic staff 

Sandra van den Belt-Dusebout PhD Post-doc 

Annemieke Opstal-van Winden PhD Post-doc 

Anouk Pijpe PhD Post-doc 

Susanne Rebers PhD Post-doc 

Martijn Verheus PhD Post-doc 

Eric Vermeulen PhD Post-doc 

Naomi Boekel MSc PhD student 

Richard Brohet MSc PhD student 

Anja van Eggermond MSc PhD student 

Lieske Schrijver MSc PhD student 

Mandy Spaan MSc PhD student 

Rianne van Nimwegen MSc PhD student 

Sandra van den Broek MSc PhD student 

Janneke Verloop MSc PhD student 

Cherita Sombroek MSc Junior scientifi c 

researcher 

Thea Mooij MSc Statistical analyst 

Marie-José Blom MSc Data manager 

Juliet Boessenkool-Pape MSc Data manager 

Ellen Engelhardt MSc Data manager 

Cora Knol MSc Data manager 

Judith de Lange MSc Data manager 

Alexander Brandenburg MSc Research assistant 

Miranda Gerritsma Research assistant 

Harmke Groot MSc Research assistant 

Esther Janssen Research assistant 

Karen Kooijman Research assistant 

Kiki Jeanson MSc Research assistant 

Kim Kersten MSc Research assistant 

Marianne Kuenen Research assistant 

Merian van Wouwe Research assistant 

Gabey Ouwens Research assistant 

Saskia Pelders MSc Research assistant 

DIVISION OF PSYCHOSOCIAL 

RESEARCH AND EPIDEMIOLOGY 

EPIDEMIOLOGY 

The cancer epidemiology group is currently concentrating on two principal research 

lines: (1) the etiology of hormone-related cancers, with a focus on gene-environment 

interactions; (2) the long-term health consequences of cancer treatment, particularly 

in terms of the risk of developing second malignancies or cardiovascular disease. 

Risk factors for hormone-related cancer In our nationwide cohort study among 

families with a BRCA1/2 mutation and tested in a clinical-genetic center (Hebon 

study), we are studying whether 1) hormonal/life-style factors modify cancer risk 

in BRCA1/2 families, 2) common genetic alterations are associated with the risk 

of breast cancer among BRCA1/2 carriers. The cohort now includes 6978 family 

members (BRCA1/2 carriers, non-carriers and non-tested women and men). 

A new procedure to recruit BRCA1/2 family members in the Hebon study was 

piloted among breast cancer survivors in the MINDACT trial. A website was created 

to host an online questionnaire, to inform high risk family members on medical 

aspects, and to support the Hebon working group that conducts the study (www. 

hebon.nl, in Dutch). In December 2011 900 BRCA1/2 carriers will be invited for a 

second pilot study to test the improved procedure in the intended study group. 

We estimated breast and ovarian cancer risks for BRCA1/2 carriers. In The 

Netherlands the average cumulative breast cancer risk by age 70 years was estimated 

to be 45% (95%CI = 36-52%) for BRCA1 and 27% (95%CI = 14-38%) for BRCA2 

mutation carriers. The corresponding cumulative risks for ovarian cancer were 31% 

(95%CI 17-43%) for BRCA1 and 6% (95%CI 2-11%) for BRCA2 mutation carriers. In 

BRCA1 families breast cancer risk increased with more recent date of birth (P

CI: 0.30-1.15) and 0.42 (95% CI: 0.19-0.94). No differences were seen by ER status 

of the fi rst breast cancer, but power of this stratifi ed analysis was still low. Therefore, 

the effects of tamoxifen and tumor characteristics cannot yet be disentangled. 

We continued the harmonization of the epidemiologic risk factor data for the 

Consortium of Investigators of Modifi ers of BRCA1/2 (CIMBA, 9645 BRCA1/2 carriers). 

Within CIMBA results of the Illumina iSelect iCOGS chip (200k SNPs, selected on 

associations with hormone-related cancers) became available for analyses on more than 

20,000 BRCA1/2 carriers ( ~ 10% from Hebon). SNP*risk factor interactions, found for 

sporadic breast cancer, will be investigated among the BRCA1/2 carriers. 

In 2011, we reported on the risk of ovarian malignancies in our nationwide OMEGA 

cohort of 25,152 women who started subfertility treatment in the 12 collaborating 

IVF-clinics in the Netherlands between 1980 and 1994 (collaboration with CW 

Burger, Erasmus MC Rotterdam). The cohort includes 19,146 women treated with 

in-vitro fertilization (IVF) and a comparison group of 6,006 subfertile women 

not treated with IVF. After a median follow-up of 14.7 years, 77 women had 

developed ovarian malignancies (42 invasive ovarian tumors and 35 borderline 

ovarian tumors). The risk for invasive ovarian cancer in IVF-treated women was 

not signifi cantly increased, neither when compared with the general population 

(SIR, 1.30; 95% CI, 0.86-1.88), nor when compared with the non-IVF group (HR, 

1.51; 95% CI, 0.65-3.54). The risk of borderline ovarian tumors in the IVF group was 

signifi cantly increased compared with the general population (SIR, 1,76: 95% CI 

1.16-2.56) and also compared with to the non-IVF group (HR, 4.23; 95% CI, 1.25- 

14.33), adjusted for age, parity and subfertility cause. The risk of ovarian malignancy 

did not increase with a larger number of IVF cycles, possibly due to lack of power. 

Because borderline ovarian tumors are rare, the cumulative incidence for IVFtreated 

women to develop a borderline ovarian tumor before the age of 55 was low, 

i.e. 0.35% compared with 0.1% in the general population. 

Recently, the original cohort has been linked with the Netherlands Cancer Registry 

to update cancer risk up till July 2009. After a median follow-up of 

16.7 years, 553 women had developed breast cancer (405 in the IVF 

group and 148 in the non-IVF group). In preliminary analyses the 

risk of breast cancer in IVF-treated women was neither increased 

compared with the general population nor compared with the non- 

IVF comparison group (SIR, 0.98 (95% CI, 0.88-1.01); HR, 0.99 

(95% CI, 0.71-1.38)). The risk of breast cancer did not increase with 

more IVF cycles. 

To increase power for the assessment of the risk of hormone-related 

cancers after fertility treatment, we continued the expansion of 

the OMEGA cohort (OMEGA II project, www.omega-onderzoek. 

nl) with 13,500 women who started IVF treatment between 1995 

and 2000 and 5,400 subfertile women not treated with IVF. We 

collected detailed data regarding IVF treatment from the IVF-clinics. 

Data collection regarding lifestyle and reproductive factors and 

health outcomes through internet-based and paper questionnaires 

is ongoing. Approximately 5,800 women have been invited for 

OMEGA II, with a response rate of 50%. Of the participants, 79% 

also sent toenail clippings for future extraction of DNA. We recently 

obtained authorization for linkage with the Dutch Municipal 

Base Administration to obtain current addresses of all remaining eligible cohort 

members to be able to invite them to participate. 

In 2011, we started recruitment for a new prospective cohort study (the Nightingale 

Study) among nurses in the Netherlands in collaboration with the Institute for 

Risk Assessment Sciences (IRAS, Utrecht University) and the BIG-register (of 

the Ministry of Health, Welfare and Sport). Our main interest is to investigate 

the association between shift work and risk of breast cancer. One of the main 

hypotheses is that reduced nocturnal levels of melatonin during shift work increase 

the levels of endogenous oestrogen. It is already known from prospective studies 

that higher endogenous oestrogen levels increase the risk of breast cancer. Following 

a pilot study (N=900 (ex-) nurses, response rate 16%, Spring 2011) we improved 

the procedure. In September 2011 the actual recruitment started by invitation of 

193,029 (ex-) nurses through the BIG-registry. They were asked to complete a web- 

Selected publications 

91 

EPIDEMIOLOGY 

Antoniou A, Beesley J, McGuffog, and 

120 other autors, Rookus M, Easton D. 

Common Breast Cancer Susceptibility Alleles 

and the Risk of Breast Cancer for BRCA1 

and BRCA2 Mutation Carriers: Implications 

for Risk Prediction. Cancer Research 

2010;70:9742-9754 

De Haas EC, Zwart N, Meijer C, 

Suurmeijer AJ, Van der Meer J, Guchelaar 

HJ, Hoekstra HJ, Van Leeuwen FE, Sleijfer 

DTh, Boezen HM, Gietema JA. Association 

of Plasminogen-Activator Inhibitor 1 (PAI-1) 

4G/5G Gene Polymorphism with Survival 

and Chemotherapy-related Vascular Toxicity 

in Non-seminomatous Testicular Cancer. 

Cancer 2010;116:5628-36 

Geenen MM, Bakker PJM, Kremer LCM, 

Kastelein JJP, van Leeuwen FE. Increased 

prevalence of risk factors for cardiovascular 

disease in long-term survivors of acute 

lymphoblastic leukemia and Wilms tumor 

treated with radiotherapy. Pediatr Blood 

Cancer 2010;55:690-697

92 

EPIDEMIOLOGY 


Enciso-Mora V, Broderick P, Ma Y, 

Houlston RS, et al. A genome-wide 

association study of Hodgkin’s lymphoma 

identifi es new susceptibility loci at 2p16.1 

(REL), 8q24.21 and 10p14 (GATA3). Nat 

Genet 2010;42:1126-1130 

Van Dijk IWEM, Oldenburger F, 

Cardous-Ubbink MC, Geenen MM, 

Heinen RC, De Kraker J, Van Leeuwen FE, 

Van der Pal HJH, Caron HN, Koning CCE, 

Kremer LCM. Evaluation of late adverse 

events in long-term Wilms’ tumor survivors. 

Int. J. Radiation Oncology Biol Phys 

2010;78:370-8 

Esserman LJ, Shieh Y, Rutgers EJT, Retèl 

V, Mook S, Glas AM, Moore DH, Linn S, 

Van Leeuwen FE, Van ’t Veer LJ. Impact of 

mammographic screening on the detection of 

good and poor prognosis breast cancer. Breast 

Cancer Res Treat 2011;130:725-34 

Ma YP, Van Leeuwen FE, Cooke R, 

Broeks A, Eciso V, Olver B, Lloyd A, 

Broderick P, Russell N, Janus C, Answordth 

A, Houlston S. FGFR2 genotype and risk of 

radiation-associated breast cancer in 

Hodgkin lymphoma. Blood 2011 (in press) 

Manders P, Pijpe A, Hooning MJ, Kluijt 

I, Vasen HF, Hoogerbrugge N, van Asperen 

CJ, Meijers-Heijboer H, Ausems MG, van 

Os TA, Gomez-Garcia EB, Brohet RM, van 

Leeuwen FE, Rookus MA. Body weight and 

risk of breast cancer in BRCA1/2 mutation 

carriers. Breast Cancer Res Treat 

2011;126:193-202 

Mook S, van ‘t Veer LJ, Rutgers EJ, 

Ravdin PM, van de Velde AO, van Leeuwen 

FE, Visser O, Schmidt MK. Independent 

prognostic value of screen detection in 

invasive breast cancer. J Natl Cancer Inst 

2011;103:585-597 

Oeffi nger KC, van Leeuwen FE, 

Hodgson DC. Methods to assess adverse 

health-related outcomes in cancer survivors. 

Cancer Epidemiol Biomarkers Prev. 

2011;20:2022-34 

Pijpe A, Mulder RL, Manders P, van 

Leeuwen FE, Rookus MA. Validation study 

suggested no differential misclassifi cation of 

self-reported mammography history in 

BRCA1/2 mutation carriers. J Clin 

Epidemiol 2011;64:1434-43 

Schouten L, Steevens J, Huysentruyt C, 

Coffeng C, Keulemans Y, van Leeuwen FE, 

Driessen A, Den Brandt P. Total Cancer 

Incidence and Overall Mortality Are Not 

Increased Among Patients With Barrett’s 

Esophagus. Clinical Gastroenterology and 

Hepatology 2011;9:754-761 

based informed consent form and questionnaire and to donate toenail clippings 

for DNA analyses (e.g. clock genes; optional). Recently, we exceeded the number 

of responders we aimed for (N= 50,000 women (26%)). The questionnaire is still 

online (www.nightingale-studie.nl, in Dutch). 

We closely collaborate with Marjanka Schmidt, Division of Molecular Pathology with 

regard to studies on breast cancer outcome conducted in the ‘BOSOM’ and Hebon 

cohorts, and in the international Breast Cancer Association Consortium (BCAC). 

We also conduct collaborative study on patient consent procedures for the use of 

human materials in clinical research. These studies are reported in the section of 

the Division of Molecular Pathology. 

Late effects of cancer treatment Now that curative treatment is available for a 

substantial group of cancer patients, it is increasingly important to evaluate how the 

occurrence of late complications of treatment affects their long-term survival. We 

aim to evaluate the risk of second cancers and cardiovascular disease (CVD) after 

radio- and chemotherapy (CT) for Hodgkin’s lymphoma (n=3,400), testicular cancer 

(n=3,750) and breast cancer (n ~ 130,000) over a period of up to 40 years after primary 

treatment. 

In 2011, a program grant was awarded to Flora van Leeuwen to make a 

comprehensive assessment of the late effects of treatment for Hodgkin’s lymphoma 

(HL) (Queen Wilhelmina research award). To study the long-term cardiovascular 

disease (CVD) risk following HL treatment, the LICHT study (Late effects: 

Insights into Cardiovascular disease risk after HL Therapy) was set up. This study 

comprises a set of case-control studies, investigating risk factors for valvular disease, 

myocardial infarction, angina pectoris and congestive heart failure in HL survivors. 

The aim of this study is to quantify separate and joint effects of radiation dose 

and volume to the heart, anthracycline dose, other chemotherapy, life style and 

reproductive factors and established CVD risk factors. To estimate the radiation 

dose to the heart and the irradiated heart volume, individual radiation dosimetry 

will be performed in collaboration with Prof. Sarah Darby and Dr. David Cutter, 

Oxford University (UK). For the fi rst two case-control studies, on valvular disease 

and myocardial infarction, we already included 142 and 158 cases, respectively, from 

a large cohort of HL survivors (N=2700) treated between 1965 and 1996. Cases 

were matched on age, sex and calendar year of diagnosis with at least 2 controls who 

also had HL, but did not develop CVD. Preliminary results of the risk of myocardial 

infarction after HL show an increased odds ratio (OR) of 2.77 for patients treated 

with 40 Gy or more at the mediastinum, compared to patients who did not receive 

irradiation on the mediastinum. 

To study the effects of RT, CT, reproductive and genetic factors on the risk of 

breast cancer after HL, we are performing a nationwide case-control study nested 

in a cohort of 1,011 female patients who had been diagnosed with HL before age 

41 (collaboration with Divisions of Experimental Therapy, Annegien Broeks and 

Radiotherapy, Nicola Russell, Berthe Aleman). So far we have identifi ed 170 case 

patients, who were individually matched to 510 controls. All women received a 

questionnaire on lifestyle factors and hormone use and were asked to provide a 

blood sample for genetic analyses. Preliminary analyses were focused on the effects 

of chemotherapy and reproductive factors among patients who received chest 

irradiation, leaving 143 breast cancer cases and 368 controls eligible for analysis. 

Patients treated with RT in combination with CT had a statistically signifi cantly 

lower risk of breast cancer than patients treated with RT alone (OR 0.57, 95% 

confi dence interval (CI) 0.37-0.88). Compared with women treated with RT only, 

the risk of breast cancer appeared to decrease with higher procarbazine dose: 

OR=0.43 (95% CI 0.22-0.85) among patients treated with 4.2-8.4 g/m 2 procarbazine 

and OR=0.30 (95% CI 0.10-0.89) among women given more than 8.4 g/m 2 

procarbazine. A premature menopause (before age 31) after HL treatment was also 

associated with a reduced risk of breast cancer (OR 0.35, 95% CI 0.13-1.46), implying 

that ovarian hormones are crucial to stimulate breast cancer development once 

radiation has produced an intiating event. Hormone replacement therapy and oral 

contraceptive use (versus no use) and use for 7 years or more (versus use for 

Treatment Effects and screening Recommendations) consortium developed an 

informative website about treatment-related adverse effects after HL treatment 

in collaboration with the Dutch Lymphoma Patient Federation. This website was 

launched on March 26, 2011 and evaluated in 2011. The evaluation study was 

conducted in 103 > 5-year HL survivors still attending the outpatient clinic of 

the NKI-ALV (response rate 67%). Results showed that our informative website 

increased knowledge about treatment-related adverse effects in 75% of the 

participants, while levels of distress about these effects were not materially effected. 

The user-friendliness and user-satisfaction of the BETTER website were very good. 

The results of this study are relevant for optimizing the website, but also provide 

more insight into how HL survivors deal with new information on treatmentrelated 

adverse effects. In 2011 the BETTER consortium not only completed the 

development of several national screening guidelines for treatment-related adverse 

effects after HL, but also completed the identifi cation of HL survivors treated in 

the UMCN who are at risk of late effects and eligible to be recalled for medical 

surveillance, based on the screening guidelines. 

Testicular cancer patients remain at increased risk to develop contralateral testicular 

cancer. It is still unclear whether testicular cancer treatment, especially platinumbased 

chemotherapy, affects the risk of developing a contralateral testicular cancer. 

In our expanded Dutch Testicular cancer cohort, currently comprising 3,749 

testicular cancer patients treated in the Netherlands during 1965-1995, with updated 

follow-up, we studied the infl uence of testicular cancer treatment on contralateral 

testicular cancer risk and assessed the impact of a contralateral testicular cancer on 

patient prognosis. After a median follow-up of 18.5 years 87 contralateral testicular 

germ cell tumors were diagnosed, of which 77 were diagnosed 6 months or more 

after the primary testicular germ cell tumor (metachronous tumors). The risk to 

develop a metachronous contralateral testicular tumor was 18 times higher than in 

the general male population. Risk remained elevated up to 20 years after diagnosis, 

at which time the cumulative incidence of contralateral testicular tumors reached 

2.2%. Platinum-based chemotherapy was associated with a signifi cant 2.6 times 

lower contralateral testicular tumor risk among non-seminoma patients. 

Patients with a metachronous contralateral testicular tumor had a 2.2 times higher 

risk to develop a subsequent non-testicular cancer and, consequently, a 1.7-times 

higher risk of death than patients who did not develop a metachronous contralateral 

testicular tumor. 

In a previous cohort study we showed increased risks of cardiac morbidity and 

mortality among breast cancer patients treated between 1970 and 1986. To evaluate 

the long-term cardiovascular morbidity and mortality in survivors of breast cancer 

treated with more contemporary regimens we are conducting two new large cohort 

studies. 

The fi rst cohort is hospital-based and consists of approximately 15,000 patients 

treated between 1970 and 2004 in the Netherlands Cancer Institute or the Erasmus 

MC, Daniel den Hoed Cancer Center. For this cohort detailed treatment information 

and cardiovascular risk factors are being collected. 

The second is a population-based cohort of patients with invasive breast cancer 

(n=97,747) and ductal carcinoma in situ of the breast (DCIS; n=13,545) diagnosed 

between 1989 and 2004 in the Netherlands. First analyses on patients diagnosed 

with DCIS of the breast showed that compared to the general population, DCIS 

patients have a slightly lower risk of death, with a standardized mortality ratio of 

0.93 (95%CI 0.89-0.97) for all causes and 0.77 (95%CI 0.71-0.84) for death from 

cardiovascular diseases. Possible explanations for these lower mortality rates are 

the adoption of a healthier lifestyle after BC diagnosis and/or confl icting risk 

profi les between breast cancer and CVD. We hypothesized that patients treated 

with radiotherapy (RT) for left-sided DCIS would have an increased risk for 

cardiovascular disease than patients treated for right-sided DCIS. However, patients 

treated with RT for left-sided DCIS did not have an increased risk for cardiovascular 

hospital admission (hazard ratio (HR)=0.70, 95%CI 0.49-0.98), or cardiovascular 

intervention (HR=0.70, 95%CI 0.46-1.07) compared to patients treated for rightsided 

DCIS. So, with the current follow-up duration, patients treated with RT for 

left-sided DCIS do not have an increased CVD-risk. 


93 

EPIDEMIOLOGY 

Van den Berg M, Overbeek A, Van der 

Pal H, Versluys B, Bresters D, Van Leeuwen 

FE, Lambalk C, Kaspers G, Van Dulmenden 

Broeder E. Using web-based and 

paper-based questionnaires for collecting 

data on fertility issues among female 

childhood cancer survivors: differences in 

response characteristics. Journal of Medical 

Internet Research 2011;13:e76 

Van der Pal, HJ, van Dalen EC, van 

Delden E, van Dijk IW, Kok WE, Geskus RB, 

Sieswerda E, Oldenburger F, Koning CC, 

van Leeuwen FE, Caron HN, Kremer LC. 

High risk of symptomatic cardiac invents in 

childhood cancer survivors. J Clin Oncol 


Van Leeuwen FE, Klip H, Mooij TM, Van 

de Swaluw AMG, Lambalk CB, Kortman M, 

Laven JSE, Jansen CEM, Helmerhorst FM, 

Cohlen BJ, Willemsen WNP, Smeenk JMJ, 

Simons AHM, Van der Veen F, Evers JLH, 

Van Dop PA, Macklon NS, Burger CW. Risk 

of borderline and invasive ovarian tumours 

after ovarian stimulation for in vitro 

fertilization in a large Dutch cohort. Human 

Reproduction 2011;26:3456-65 

Yang XR, Chang-Claude J, and 150 other 

autors, van Leeuwen FE, Garcia-Closas M. 

Associations of breast cancer risk factors with 

tumor subtypes: a pooled analysis from the 

Breast Cancer Association Consortium 

studies. Natl Cancer Inst. 2011;103:250-63 

Maduro JH, den Dekker HA, Pras E, de 

Vries EG, van der Zee AG, Klokman WJ, 

Reyners AK, van Leeuwen FE, Langendijk 

JA, de Bock GH, Gietema JA. 

Cardiovascular Morbidity after 

Radiotherapy or Chemoradiation in Patients 

with Cervical Cancer. Int J Radiat Oncol 

Biol Phys 2010;78:1337-44 

fi gure 3: Cumulative incidence of 

contralateral testicular cancer for patients 

with a non-seminoma testicular germ cel 

tumor (TGCT) by attained age according to 

age at diagnosis (

94 

EPIDEMIOLOGY 

Group leader Michael Hauptmann 

Michael Hauptmann PhD Group leader 

Marta Lopez PhD Academic staff 

Wilma Heemsbergen PhD Academic staff 

José Meulepas MSc PhD student 

Publications 

Hoebers F, Heemsbergen W, Moor S, 

Lopez M, Klop M, Tesselaar M, Rasch C. 

Reirradiation for head-and-neck cancer: 

delicate balance between effectiveness and 

toxicity. Int J Radiat Oncol Biol Phys 

2011;81:e111-8 

Devriese LA, Bosma AJ, van de Heuvel 

MM, Heemsbergen W, Voest EE, Schellens 

JH. Circulating tumor cell detection in 

advanced non-small cell lung cancer patients 

by multi-marker QPCR analysis. Lung 

Cancer 2012;75:242-247 

Kok M, Zwart W, Holm C, Fles R, 

Hauptmann M, Van ‘t Veer LJ, Wessels LF, 

Neefjes J, Stal O, Linn SC, Landberg G, 

Michalides R. PKA-induced phosphorylation 

of ER alpha at serine 305 and high PAK1 

levels is associated with sensitivity to 

tamoxifen in ER-positive breast cancer. 


Knegjens JL, Pameijer FA, Hauptmann 

M, Balm AJ, Hoebers FJ, De Bois JA, 

Kaanders JH, Verhoef LC, Wijers OB, 

Wiggenraad RG, Buter J, Rasch CR. Tumor 

volume as outcome predictor in 

chemoradiation for advanced head and neck 

cancer. Head Neck 2011;33:375-382 

Van Monsjou HS, Lopez-Yurda M, 

Hauptmann M, Van den Brekel MW, Balm 

AJ, Wreesmann VB. Oral and oropharyngeal 

squamous cell carcinoma in young patients: 


experience. Head Neck 

BIOSTATISTICS 

The group provides state-of-the-art statistical expertise to researchers and doctors 

from a wide range of departments in the Institute and hospital, including the 

design and analysis of clinical trials, prognostic and predictive biomarkers, observer 

agreement, sample size calculations, risk prediction, gene expression, epidemiologic 

studies, and animal and in vitro experiments. Our contribution ranges from ad-hoc 

statistical advice to long-term collaborations. 

Cancer and radiation exposure from diagnostic imaging Radiation doses from 

diagnostic procedures are usually much lower than therapeutic doses. However, 

computed tomography (CT) delivers substantially higher radiation doses than most 

other diagnostic imaging techniques, and its use has increased dramatically during 

the past 10-15 years. Therefore, radiation protection is a concern, particularly among 

children, as they are sensitive to radiation-induced cancer and have a long lifespan 

to express late effects. Within a European consortium (EPI-CT), we are conducting 

a retrospective record-linkage cohort study including at least 40,000 children 

who received one or more electronically archived CT scan in participating Dutch 

hospitals. We will obtain information on all CT scans, including body part scanned, 

indication and radiologist report, as well as technical parameters and the images 

for organ dose estimation. Radiology records will be linked with the Netherlands 

Cancer Registry in order to determine cancer incidence and with other registries to 

collect information on potential confounders (Perinatal Registry of the Netherlands 

for congenital and other inborn disorders, European Bone Marrow Transplant 

registry for use of leukemogenic drugs, Statistics Netherlands for socio-economic 

status). The collected data will be used to evaluate the association between radiation 

exposure from pediatric CT scans and risk of childhood and young adult leukemia, 

to describe nationwide patterns of CT scan use in Dutch children and to contribute 

data to the European pooled study to investigate risk for rarer forms of cancer. The 

statistical analysis of the combined European consortium data, which is expected 

to include about one million children, will also be conducted by our group. The 

study provides the fi rst empirical quantitative assessment of CT-related radiation 

doses and corresponding risk. Besides providing safety information for this popular 

and invaluable imaging procedure, the study will uniquely contribute to etiologic 

knowledge on childhood/young adult leukemia as well as low-dose radiation risks 

and associated mechanisms.

HEALTH-RELATED QUALITY OF LIFE ASSESSMENT AND 

BEHAVIORAL INTERVENTIONS IN CLINICAL ONCOLOGY 

This research line has two primary foci: (1) development of methods and 

applications of health-related quality of life (HRQL) assessment in clinical research 

and clinical practice; and (2) development and testing of behavioral and psychosocial 

interventions to reduce symptom burden and improve the HRQL of patients with 

cancer. 

The health-related quality of life of patients with low-grade glioma This 

observational study investigated the generic and condition-specifi c health-related 

quality of life (HRQL) of low-grade glioma patients (LGG). 195 LGG patients, 

diagnosed on average, 5.6 years earlier, were compared with 100 hematological 

(non-Hodgkin lymphoma and chronic lymphatic leukemia cancer patients (NHL/ 

CLL) and 205 general population controls, comparable with the LGG patients at the 

group level on age, sex and education (healthy controls). Generic HRQL was assessed 

with the SF-36 Health Survey; condition-specifi c HRQL with the Medical Outcomes 

Study Cognitive Function Questionnaire and the EORTC Brain Cancer Module. 

Objective neurocognitive functioning was assessed with a standardized battery 

of neuropsychological tests. No statistically signifi cant differences were observed 

between the LGG and NHL/CLL patients in SF-36 scores. The LGG patients scored 

signifi cantly lower than the healthy controls on 6 of the 8 scales and on the mental 

health component score of the SF-36. Approximately one-quarter of the LGG patients 

reported serious neurocognitive symptoms. Female gender, epilepsy burden, and 

number of objectively assessed neurocognitive defi cits were associated signifi cantly 

with both generic and condition-specifi c HRQL. Clinical variables, including time 

since diagnosis, tumor lateralization, extent of surgery, and radiotherapy did not 

show a consistent relationship with HRQL. The fi ndings from this study indicate 

that LGG patients experience signifi cant problems across a broad range of HRQL 

domains, many of which are not condition-specifi c. However, the neurocognitive 

defi cits and epilepsy that are relatively prevalent among LGG patients are associated 

with negative HRQL outcomes, and thus contribute additionally to the vulnerability 

of this population of cancer patients. 

Cognitive behavioral therapy and physical exercise for climacteric symptoms 

in breast cancer patients experiencing treatment-induced menopause This 

multicenter, randomized trial evaluated the effectiveness of cognitive behavioral 

therapy (CBT), physical exercise (PE) or the combination of CBT/PE in alleviating 

climacteric symptoms in breast cancer patients experiencing treatment-induced 

menopause. Four hundred twenty-two breast cancer patients were randomly 

assigned to a CBT (N = 109), a PE (N = 104), a CBT/PE (N = 106) or a waiting list 

control group (N = 103). Self-report questionnaires were completed at baseline, 12 

weeks (T1) and 6 months (T2) post-study entry. To compare the intervention groups 

with the control group over time, multilevel procedures were used to model the 

series of repeated measurements. Compared to the control group, intervention 

groups (intention-to-treat) showed overall decrease in levels of menopausal 

symptoms (FACT-ES; p

96 

PSYCHOSOCIAL RESEARCH 


Gehring K, Aaronson NK, Taphoorn MJB, 

Sitskoorn MM. Cognitive interventions for 

patients with brain tumors: An update. 

Expert Rev Anticancer Ther 2010;10:1779-95 

Hilarius DL, Kloeg PH, van der Wall E, 

Komen M, Gundy CM, Aaronson NK. 

Cancer-related fatigue: Clinical practice 

versus practice guidelines. Support Care 

Cancer 2011;19:531-538 

Hilarius DL, Kloeg PH, van der Wall E, 

van den Heuvel JJG, Gundy CM, Aaronson 

NK. Chemotherapy-induced nausea and 

vomiting in daily clinical practice: A 

community hospital-based study. Support 

Care Cancer. 2012;20:108-17 

Knols RH, de Bruin ED, Uebelhart D, 

Aufdemkampe G, Schanz U, Stenner-Liewen 

F, Hitz F, Taverna C, Aaronson NK. Effects of 

an outpatient physical exercise program on 

hematopeoetic stem-cell transplantation 

recipients: a randomized clinical trial. Bone 

Marrow Transplant 2011;46:1245-1255 

Snyder CF, Blackford AL, Aaronson NK, 

Detmar SB, Carducci MA, Brundage MD, 

Wu AW. Can patient-reported outcome 

measures identify cancer patients’ most 

bothersome issues? J Clin Oncol 2011;29: 

1216-20 

van der Poll-Franse LV, Mols F, Gundy 

CM, Creutzberg CL, Nout RA, Verdonck-de 

Leeuw IM, Taphoorn MJ, Aaronson NK. 

Normative data for the EORTC QLQ-C30 

and EORTC-sexuality items in the general 

Dutch population. Eur J Cancer 2011;47: 

667-75 

Vermeulen E, Schmidt MK, Cornel M, 

Knoppers BM, van Leeuwen FE, 

Aaronson NK. Connective tissue: Cancer 

patients’attitudes towards medical resarch 

using excised (tumour) tissue. Biosocieties 

2011;6:466-486 

treated in phase III clinical trials; and (2) to pilot test questionnaires for assessing 

the HRQL of mid- to long-term cancer survivors (> 5 years disease free). This study 

will generate practical and ethically acceptable procedures for recruiting patients 

from EORTC clinical trials for long-term HRQL survivorship studies and thereby 

lay the groundwork for a series of future, substantive studies. Approximately 140 

patients will be recruited from each of two EORTC GU Group phase III clinical 

trials – one in testicular and one in prostate cancer. The two samples are drawn from 

three broad geographic/cultural regions: (1) Northern Europe; (2) Southern Europe; 

and (3) the United Kingdom. HRQL is assessed at 3 levels: (1) generic (the SF-36 

Health Survey); (2) cancer-specifi c (the EORTC QLQ-C30 plus condition-specifi c 

modules; and (3) cancer survivor-specifi c (the Impact of Cancer questionnaire). To 

date, 105 prostate cancer survivors (Netherlands: 45; Belgium: 36; France: 24) and 

94 testicular cancer survivors (Netherlands: 36; Italy: 35; Norway: 23) have completed 

the questionnaires. The questionnaires of the prostate cancer survivors from Italy 

and the testicular cancer survivors form the UK are expected in late 2011 and early 

2012. 

Phy sical exercise during chemotherapy to improve physical fitness and 

reduce fatigue (PACES) This multicenter RCT is evaluating the effectiveness of 

two physical exercise interventions in maintaining or enhancing physical fitness 

and in minimizing fatigue in patients undergoing adjuvant chemotherapy for breast 

or colon cancer: (1) a low intensity, home-based, self-management program; and (2) 

a moderate intensity, structured, supervised program. In total, 230 patients will be 

randomized to one of the two intervention groups or to a usual care control group. 

All participants undergo performance tests and complete self-report questionnaires 

at baseline, at the completion of chemotherapy, and at 6 month follow-up. To date, 

170 patients have been randomized into the study, of whom 85 have completed the 

fi rst follow-up (T1) and 20 the 6-month follow-up (T2). Patient recruitment will 

continue until July 2012. 

Behavioral and psychosocial effects of rapid genetic counseling and 

testing (RGCT) in newly diagnosed breast cancer patients This multicenter, 

randomized trial, carried out in collaboration with the University Medical Center 

Utrecht (Dr. Margreet Ausems), is investigating the uptake of RGCT when offered 

routinely to newly diagnosed breast cancer patients who, prior to receiving primary 

treatment, are identifi ed as having at least a 10% risk of carrying a mutation in the 

BRCA1 or BRCA2 gene, and the impact of RGCT on a range of outcomes. Women 

(N = 271, response 77%) recruited from 12 hospitals in the Amsterdam and Utrecht 

regions of the Netherlands have been randomized to either the RGCT group or 

a usual care group (2:1 ratio). The study endpoints include: (1) uptake of RGCT; 

(2) choice of clinical management strategy, including direct bilateral mastectomy 

or delayed preventive contralateral mastectomy; (3) cancer risk perception and 

cancer-related distress; (4) knowledge of genetic aspects of breast cancer; (5) 

decisional satisfaction; (6) HRQL; and (6) satisfaction with RGCT. In 2009, fi nal 

ethical approval was obtained and patient accrual was initiated in all hospitals. 

Questionnaires have been administered at study entry, and at 6 month (response 

92%) and 12 month follow-up (response 88%). A subset of women has been 

interviewed to obtain supplementary, qualitative data. Recruitment was completed 

in January 1 st 2011 and all follow-up data will be available at the beginning of 2012. 

Thus far, 22 carriers have been identifi ed (10% of patients who had DNA testing).

COGNITIVE FUNCTION IN CANCER PATIENTS 

Signifi cant proportions of cancer patients report cognitive changes following therapy 

that interfere with their daily life activities and that can persist into the survivorship 

period. The projects constituting this research line center around the investigation 

of the prevalence, nature and cause of cognitive problems associated with systemic 

therapies, and at the development of strategies to diminish these symptoms. 

Late effects of chemotherapy on cognitive and brain functioning in the 

elderly Studies have shown that chemotherapy-exposed breast cancer patients have 

focal reductions in gray matter volume compared to control subjects, months up 

till several years post-treatment. Whether chemotherapy is associated with brain 

structure in the long run is as yet largely unknown. 

In a collaborative study with the Erasmus MC (MB Breteler) we compared total brain 

volume, total gray and white matter volume, and regional gray matter volume of 

187 women who had been treated with chemotherapy for breast cancer, more than 

20 years before, with that of an age-matched population-based sample of women 

without a history of cancer (n=374). 

We found that breast cancer survivors previously treated with CMF chemotherapy 

had smaller total gray matter volume and total brain volume than reference subjects. 

No group differences were observed in total white matter volume or regional gray 

matter volume. The difference in gray matter volume in chemotherapy-exposed 

survivors was comparable to the effect of almost four years of age on gray matter 

volume loss. Our results suggest that chemotherapy may induce long-lasting effects 

on brain structure. 

Molecular and cellular mechanisms of cognitive impairment following chemotherapy 

for cancer Since chemotherapy is generally given as a cocktail of multiple 

cytotoxic agents, it is diffi cult to state which agents are responsible for the observed 

cognitive impairment. In this study with the Vrije Universiteit (AB Smit) we 

examined the effect of several cytotoxic agents on cognition in a mice model. 

Male C57Bl6 mice were treated with one of the following agents: cyclophosphamide 

(CYC, 150 mg/kg ip), docetaxel (DOC, 33 mg/kg iv), doxorubicin (DOX, 10 mg/kg iv), 

5-fl uorouracil (5-FU, 75 mg/kg ip), methotrexate (MTX, 250 mg/kg iv), or topotecan 

(TOP, 25 mg/kg ip). A control group received saline ip. Behavioral testing started 

two weeks after treatment and included the following tasks: general behavior in an 

automated home cage, open fi eld, novel location recognition (NLR), Barnes maze, 

fear conditioning, and simple choice reaction time task (SCRTT). 

CYC and DOX treatment decreased activity during the dark phase, as measured in 

an automated home cage. CYC, DOC, DOX, and MTX treated animals were not able 

to learn the NLR task. All cytotoxic agents affected learning behavior in the Barnes 

maze. Performance in the SCRTT was decreased in animals treated with CYC, 

DOC, 5-FU, and TOP. 

CYC, DOC, or DOX treatment decreased general activity in the phenotyper. 

However, this did not result in less activity in the other behavioral tasks. All 

cytotoxic agents affected spatial memory as measured in the NLR and Barnes maze. 

5-FU treated animals showed a slower response time in the SCRTT. And also in the 

SCRTT; impairment in response control was seen in animals treated with CYC, 

DOC, or TOP. The results show that, in mice, single treatment with several cytotoxic 

agents causes impairment in various cognitive domains. 

Priming cognitive problems following chemotherapy: The influence of stigma 

consciousness Informing patients about the relation between chemotherapy and 

cognitive problems may contribute to the occurrence of cognitive problems in cancer 

patients. We examined the conditions under which ‘stereotype priming’-effects 

occur and mediating processes. 

In a collaborative study with the Vrije Universiteit (E Das) the infl uence of informing 

patients about the association between cognitive problems and chemotherapy was 

investigated among 126 female breast cancer patients. Half of the patients received 

the introduction that ‘some patients treated with chemotherapy experience cognitive 

problems’; the other half received a neutral introduction. Self-reported cognitive 

97 


Group leader Sanne Schagen 

Sanne Schagen PhD Group leader 

Willem Boogerd MD PhD Academic staff 

Jose Belderbos MD PhD Academic staff 

Olaf van Tellingen PhD Academic staff 

Sabine Linn MD PhD Academic staff 

Hester Oldenburg MD PhD Academic staff 

Rita Roodbergen MSc Academic staff 

Michiel de Ruiter PhD Post-doc 

Riejanne Seigers PhD Post-doc 

Vincent Koppelmans MSc PhD student 

Sanne Menning MSc PhD student 

Myrle Kemperman MSc PhD student 

Heleen Feenstra MSc PhD student 

Chad Gundy MSc Senior statistical analyst 

Jacobien Kieffer MSc Statistical analyst 

Marianne Kuenen Research assistant 

Kim Kersten Research assistant 

Monica Kobus Research assistant 

Key publications 

De Ruiter MB, Reneman L, Boogerd 

W, Veltman DJ, Caan M, Douaud G, 

Lavini C,Linn SC, Boven E, van Dam 

FSAM, Schagen SB. Late effects of 

high-dose adjuvant chemotherapy on white 

and gray matter in breast cancer survivors: 

Converging results from multimodal 

Magnetic Resonance Imaging. Hum Brain 

Mapp. 2011 

Koppelmans V, Breteler MM, Boogerd 

W, Seynaeve C, Gundy C, Schagen SB. 

Neuropsychological performance in breast 

cancer survivors more than 20 years after 

adjuvant chemotherapy. JCO (in press) 

Koppelmans V, de Ruiter MB, van der 

Lijn F, Boogerd W, Seynaeve C, van der 

Lugt A, Vrooman H, Niessen WJ, Breteler 

MB, Schagen SB Global but not focal 

brain gray matter volume reductions in 

long-term breast cancer survivors exposed 

to adjuvant chemotherapy. BCRT 

(in press) 

Koppelmans V, Schagen SB, Poels 

MM, Boogerd W, Seynaeve C, Lugt AV, 

Breteler MM. Incidental fi ndings on brain 

Magnetic Resonance Imaging in long-term 

survivors of breast cancer treated with 

adjuvant chemotherapy. Eur J Cancer. 

2011;47:2531-6

98 



Schilder C, Seynaeve C, Linn SC, 

Boogerd W, Beex LVAM, Gundy CM, 

Nortier JWR, van de Velde CJH, van Dam 

FSAM and Schagen SB. Self-reported 

cognitive functioning in postmenopausal 

breast cancer patients before and during 

endocrine treatment: fi ndings from the 

neuropsychological TEAM side-study. 

Psycho-oncology 2011 

Wefel JS, Vardy J, Ahles T, Schagen 

SB. International Cognition and Cancer 

Task Force recommendations to harmonise 

studies of cognitive function in patients 

with cancer. Lancet Oncol. 2011;12:703-8 

Schagen SB, Das E, Vermeulen I. 

Information about chemotherapyassociated 

cognitive problems contributes 

to cognitive problems in cancer patients. 

Psychooncology. 2011 

Handboek kanker bij ouderen. Kanker 

en kankerbehandeling: cognitief 

functioneren SB Schagen, HAAM Maas. 

De Tijdstroom uitgeverij (in press) 

complaints were measured with the Cognitive Failure Questionnaire. Cognitive 

performance was assessed with the Groningen Fifteen Words Test. Stereotype 

activation was measured by asking patients to complete word fragments, e.g., BR… 

can be completed as BRAIN or BROWN. Stigma consciousness was measured by the 

Stigma Consciousness Questionnaire. 

Priming differentially affected cognitive complaints and test scores depending on 

the level of consciousness of cancer patient stigma. Priming increased cognitive 

complaints for patients high in stigma consciousness and decreased cognitive 

complaints for patients low in stigma consciousness (β = .199, t = 2.40, p = .02). 

In contrast, priming increased cognitive test scores for patients high in stigma 

consciousness, and decreased test scores for patients low in stigma consciousness 

(β = .184, t = 2.05, p

PSYCHOSOCIAL ISSUES IN CANCER GENETICS 

This research line is being conducted in close collaboration with the NKI-AVL 

family cancer clinic. It comprises a number of studies which are focused on two 

psychosocial themes in genetic counseling for cancer: 1) the uptake and long-term 

psychosocial impact of risk-reducing behavior; and 2) early detection of psychosocial 

problems and the development of psycho-educational interventions. 

Screening for psychosocial problems at the family cancer clinic The aim of this 

4 year KWF-study is to develop and evaluate a screening questionnaire as an aid in 

identifying individuals experiencing signifi cant psychosocial problems associated 

with cancer genetic counseling. In 2009, this multidimensional questionnaire was 

developed according to EORTC guidelines for questionnaire module development: 

1) generation of relevant issues, 2) operationalization of these issues into a set 

of items, and 3) questionnaire pre-testing. Twenty-six issues were identifi ed and 

operationalized into items covering six problem-domains: family, children, genetics, 

cancer, practical issues, and emotions. In 2010 and 2011, this so called “Signalquestionnaire” 

has been evaluated for its reliability, validity, sensitivity, specifi city 

and positive predictive value for detecting psychosocial problems and psychosocial 

support needs. For this validation study, 127 counselees completed both the Signalquestionnaire 

and a clinical interview with a psychosocial worker (‘gold standard’). 

This new questionnaire proved to be suffi ciently valid. In 2011, a randomized 

clinical trial has started to evaluate the implementation of this newly developed 

cancer-genetics ‘Signal-questionnaire’, as an aid in 1) facilitating communication on 

psychosocial issues during the genetic counselling session, 2) increasing counselor’s 

awareness of psychosocial problems of the counselee, and 3) improving the 

management of these psychosocial problems during and after the process of genetic 

counselling. This trial is performed in collaboration with the UMCU (M Ausems). 

Preventive total gastrectomy The aim of this cross-sectional, multi-center study 

is to investigate the experiences with, and consequences of gastroscopy screening 

and prophylactic total gastrectomy in CDH1 mutation carriers. Mutations in the 

CDH1 gene are associated with a 70% lifetime risk for diffuse gastric cancer and 

an additional 40% risk for lobular breast cancer in women. The following research 

questions are being addressed: (1) What is the impact of prophylactic gastrectomy 

on quality of life and future planning? (2) What factors infl uence the decision and 

timing of prophylactic gastrectomy? (3) Which sociodemographic, clinical and 

psychological factors are associated with quality of life after gastrectomy? and (4) 

What can we recommend to improve the health care in individuals from CDH1 

families? Six families were identifi ed with a CDH1 mutation. All individuals with 

a CDH1 gene mutation were invited to complete a self-report questionnaire and 

to participate in a semi-structured interview. A comparison group of individuals 

who underwent a total gastrectomy because of cancer were also invited to complete 

99 


Group leader Eveline Bleiker 

Eveline Bleiker PhD Group leader 

Annemieke Cats MD Academic staff 

Daniela Hahn MSc Academic staff 

Irma Kluijt MD Academic staff 

Senno Verhoef MD Academic staff 

Leonie Woerdeman MD Academic staff 

Jessica Baars PhD Post-doc 

Willem Eijzenga MSc PhD student 

Marijke Wevers MD PhD student 

Femme Harinck MD PhD student 

Chad Gundy MSc Senior statistical analyst 

Tanja Nagtegaal MSc Junior scientifi c researcher 

Grace Sidharta MSc Research assistant 

Sophie van der Velden Genetic counselor 

Anja van Rens Genetic counselor 


Ahmed AKJ, Hahn DEE, Hage JJ, Bleiker 

EMA, Woerdeman LAE. Temporary banking 

of the nipple-areola-complex in 97 skin 

sparing mastectomies. Plastic and 

Reconstructive Surgery 2011;127:531-539 

Douma KFL, Bleiker EMA, Vasen HFA, 

Gundy CM, Gerritsma MA, Aaronson NK. 

Psychological distress and quality of life of 

partners of individuals with familial 

adenomatous polyposis (FAP). Psycho- 

Oncology 2011;20:146-154 

Douma KFL, Bleiker EMA, Vasen HFA, 

Gundy CM, Gerritsma MA, Aaronson NK. 

Quality of life and consequences for daily life 

of familial adenomatous polyposis (FAP) 

family members. Colorectal Disease. 

2011;13:669-677 

Harinck F, Nagtegaal T, Kluijt I, Aalfs C, 

Smets E, Poley J-W, Wagner A, Hooft J van, 

Fockens P, Bruno M, Bleiker E. Feasibility of 

a pancreatic cancer surveillance program 

from a psychological point of view. Genetics 

in Medicine 2011 

Lammens CRM, Bleiker EMA, Verhoef 

S, Ausems MGEM, Majoor-Krakauer D, 

Sijmons R, Hes FJ, Gomez Garcia EB, Os 

TAM van, Spruijt L, Luijt RB van der, 

Ouweland A van den, Ruijs M, Gundy C, 

Nagtegaal T, Aaronson NK. Distress in 

partners of individuals diagnosed with or at 

high risk of developing tumors due to rare 

hereditary cancer syndromes”. Psycho- 

Oncology 2011;20:631-638

100 



Lammens CRM, Aaronson NK, Hes 

FJ, Links TP, Zonnenberg BA, Lenders 

JWM, Majoor-Krakauer D, Os TAM van, 

Gómez García EB, Herder W de, Luijt RB 

van der, Ouweland AMW van den, Hest 

LP van, Verhoef S & Bleiker EMA. 

Compliance with periodic surveillance for 

Von Hippel-Lindau disease. Genetics in 

Medicine 2011;13: 519-27 

Nieuwenhuis MH, Douma KFL, 

Bleiker EMA, Aaronson NK, Clevers H, 

Vasen HFA. Clinical evidence for an 

association between familial adenomatoes 

polyposis (FAP) and type II diabetes. 

International Journal of Cancer (in press) 

Wevers MR, Ausems MGEM, Verhoef 

S, Bleiker EMA, Hahn DEE, Hogervorst 

F, Luijt RB van der, Valdimarsdottir HB, 

Hillegersberg R van, Rutgers EJTh, 

Aaronson NK. Behavioral and 

psychosocial effects of rapid genetic 

counseling and testing in newly diagnosed 

breast cancer patients: Design of a 

multicenter clinical trial. BMC Cancer 

2011:11:6,1-9 

the self-report questionnaire. In total, 25 of the 31 CDH1 mutation carriers returned 

the questionnaires (81%). Of these, 20 individuals had undergone prophylactic total 

gastrectomy on average 2.5 years earlier. The high cancer risk was a major factor in 

both deciding for DNA-testing and prophylactic gastrectomy in all respondents. 

Furthermore preliminary results showed that ‘the level of energy’ was the most 

important factor determining functioning and quality of life after prophylactic 

gastrectomy: 65% of the participants experienced reduced excessive fatigue. About 

40% reported moderate to severe impairment in daily activities. Careful counseling 

and follow-up of CDH1-carriers (operated but also non-operated) is warranted. 

Surveillance of the pancreas in high risk individuals The aim of this study 

is to investigate the psychological burden of participating in a pancreatic cancer 

(PC-) surveillance program. Since 2006, a multi-center prospective study, funded 

by ZONMw, is investigating the effectiveness of PC-surveillance (EUS and MRI) 

in high-risk individuals. High-risk individuals are defi ned as (1) fi rst degree 

relatives (FDR) of patients with familial pancreatic cancer (FPC) and (2) carriers of 

a PC-prone gene mutation. In 2009, a psychosocial study arm was added to this 

multicenter surveillance study. The specifi c research questions of the psychosocial 

study are: 1) What is the perceived burden of participation in a PC-surveillance 

program, 2) what are the motivations to participate in such a program, 3) to what 

extent are those participating in the surveillance program worried about developing 

cancer, and 4) which factors are associated with anxiety experienced after an EUS- 

MRI-based surveillance program. Using a retrospective design, individuals were 

invited to complete a questionnaire four weeks after receiving their surveillance 

results. Sixty-nine individuals (85%) completed the questionnaire (54% female; 

mean age 52 yrs). Surveillance with MRI and EUS was reported as “very to extremely 

uncomfortable” by 25% of the respondents. In total 29% was “often” or “almost 

always” concerned about developing cancer. Perceived advantages of surveillance 

outweighed disadvantages according to 88% of respondents. In 2010, a prospective 

arm has been added to the study design. From the retrospective study we may 

conclude that, from a psychosocial point of view, PC surveillance in high-risk 

individuals is feasible. 

Long-term psychosocial and medical impact of breast cancer genetic 

counselling offered soon after diagnosis The aim of this study is to assess the 

long-term psychosocial and medical impact of genetic counselling offered soon 

after diagnosis of breast cancer. Specifi c research questions are: (1) What are the 

current cognitions and levels of distress of breast cancer patients who were actively 

approached for genetic counselling soon after diagnosis 7-14 years ago; (2) What are 

the perceived changes regarding risk management (surveillance and/or preventive 

surgery) and family relations arising from their disease and genetic counselling?; (3) 

How do patients evaluate their responsibility to communicate results from genetic 

counselling to their fi rst-degree relatives, and to what extent did they succeed in 

conveying test results and screening recommendations to these relatives?; and (4) 

What are the characteristics of those patients who have high levels of distress, or 

who have not adequately communicated the genetic counselling results with their 

relatives? The study, which is funded by Pink Ribbon, is performed in collaboration 

with the department of Medical Genetics of the UMCU (M Ausems) and has 

started in February 2011. Today, we have invited 161 women who were diagnosed 

with breast cancer between January 1997 and January 2004, and who were then 

actively approached for genetic counselling, and who had one or more consults for 

genetic counselling for the BRCA genes. They received a self-report questionnaire. 

The current response rate in this group is 63%, and will increase. Furthermore, 

a control group of 172 women who were diagnosed with breast cancer between 

2002 and 2004 and received radiotherapy, but who did not fulfi l the criteria for 

referral for cancer genetic counselling and DNA testing. This project will result in 

recommendations for improving services aimed at enhancing the quality of life and 

surveillance behavior of patients with breast cancer and their fi rst-degree relatives.

EARLY STAGE TECHNOLOGY ASSESSMENT, OPERATIONS 

RESEARCH AND CANCER REHABILITATION 

Early stage technology assessment From 2003 through 2006, a technology 

assessment study was conducted on the introduction of a 70-gene micro array test 

as a prognostic tool in the treatment of node negative breast cancer (the RASTERstudy) 

as a side study of the European MINDACT-study. As the diffusion of this 

technology was in an early stage and the course of development not easy to predict, 

an evaluation approach has been chosen that takes the technology dynamics into 

account, constructive technology assessment (CTA). In 2011, the CEA modelling 

including real life scenarios, a comparison between cost-effectiveness of two 

genomic breast cancer prognosis tests, involving compliance of prescribers and a 

paper on the trade off between investments in research versus further investments 

in development were submitted. Additionally, in cooperation with the University 

of Twente, we have initiated an early stage technology assessment of TIL-transfer 

technology in advanced melanoma. In 2011 Anna Miquel Cases started to work 

as PhD student on early stage technology assessment in the application of 

diagnostic/prognostic markers in neo-adjuvant breast cancer treatment. A series 

of scanning interviews were conducted so far. This is part of the CTMM program 

BREASTCARE. 

Operations improvement in oncology In cooperation with the University of 

Twente, a PhD student (Peter van Berkel) worked on a mathematical analysis and 

scheduling of care pathways within the oncologic hospital setting, and the effect of 

increased focus on effi cient capacity use. A study is ongoing on the comparison of 

characteristics of colorectal surgery pathways using structured effi ciency measures 

and the national colorectal quality registry (DSCA). 

Together with the University of Twente and the Integraal Kanker Centrum Noord- 

Oost, a PhD student works on a project to evaluate the added value of accrediting 

oncology departments in General Hospitals. 

As part of the Eurocan Platform project, work package 12 is directed towards 

the development of scientometrics and a designation system for excellent 

Comprehensive Cancer Centers. A PhD student started on the latter part of this 

project (Abinaya Rajan); she conducted a survey among stakeholders that served as 

input for Delfi -like discussion meeting. This projects aims to draft a pilot Excellence 

Designation System that is to be piloted in the coming years. 

Rehabilitation, physical activity and cancer Survivorship care and rehabilitation 

are important elements of a cancer centre’s program. In 2009, a multidisciplinary 

rehabilitation program was started for breast cancer survivors receiving adjuvant 

treatment. In addition, a rehabilitation program for head-and-neck cancer patients 

has been approved by health insurers and was rolled out beginning of 2010. Finally, 

a major Alpe d’Huzes KWF project was started early 2011, focusing on patient 

empowerment, return to work, tele-monitoring and implementation of relevant 

fi ndings and programs related to physical exercise and supported by innovative IT. 

This program will run for 5 years. The IT supported patient empowerment part 

is staffed with a PhD student and a Post-doc and is lead by the NKI. Furthermore, 

a PhD student located at the University of Twente (Janne Mewes) is working 

on the cost effectiveness and budget impact of multidisciplinary/multi faceted 

rehabilitation. 

101 


Gr oup leader Wim van Harten 

Wim van Harten MD PhD Group leader 

L Steuten PhD Academic Staff 

Peter van Berkel Msc Research staff 

Wineke van Lent Msc PhD student 

Valesca Retèl MSc PhD student 

Wim Groen PhD Post-doc 

Wilma Kuijpers MSc PhD student 

Janne Mewes PhD student 

Abinaya Rajan PhD student 

Anna Miquel Cases PhD student 

Gerke Kleinsmit Research assistant 


Retèl VP, Van der Molen L, Hilgers FJM, 

Rasch CRN, L’Ortye AAMHJ, Steuten LMG, 

Van Harten WH. A cost-effectiveness 

analysis of a preventive exercise program for 

patients with advanced head and neck cancer 

treated with concomitant chemoradiotherapy. 

BMC Cancer, 2011;11:475 

Retèl VP, Joore MA, Van Harten WH. 

Head-to-head comparison of the 70-gene 

signature versus the 21-gene assay: 

Cost-effectiveness and the effect of 

compliance. Breast Cancer Research and 

Treatment, 2011 

Van Berkel PT, Boucherie RJ, Hans EW, 

Hurink JL, Van Lent WAM, Van Harten WH. 

Accounting for inpatient wards when 

developing master surgical schedules. Anesth. 

Analg. 2011;112:1472-9 

Gulmans J, Vollenbroek-Hutten MMR, 

Van Gemert-Pijnen JEWC, Van Harten WH 

Determinants of use and non-use of a 

web-based communication in cerebral palsy 

care: Evaluating the association between 

professionals’ system use and their a priori 

expectancies and background. BMC Medical 

Informatics and Decision Making 2011;11:43

102 

DIAGNOSTIC ONCOLOGY 

Division head Marcel Stokkel 

DEPARTMENT OF CLINICAL 

CHEMISTRY 

Willem Nooijen PhD Academic staff 

Olaf van Tellingen PhD Academic staff 

Tiny Korse PhD Research associate 

Levi Buil Technical staff 

Marian Buning-Kager Technical staff 

Dorothé Linders Technical staff 

Lin Fan PhD student 

DEPARTMENT OF NUCLEAR MEDICINE 

Cornelis Hoefnagel MD PhD Academic staff 

Fijs van Leeuwen PhD Academic staff 

Saar Muller PhD Academic staff 

Michiel Sinaasappel PhD Academic staff 

Ferida Sivro-Prndelj MD Academic staff 

Marcel Stokkel MD PhD Academic staff 

Renato Valdés Olmos MD PhD Academic 

staff 

Erik Vegt MD PhD Academic staff 

Wouter Vogel MD PhD Academic staff 

Oscar Brouwer MD Clinical research fellow 

Bas Koolen MD Clinical research fellow 

Jacob Kist MD Clinical research fellow 

Hanneke Kouwenberg MD Registrar 

Kathleen Weyts MD Registrar 

Hester Arkies MD Registrar 

Liesbeth Salm MD Registrar 

Saskia Baank Technical staff 

Martine Bakker Technical staff 

Natascha Bruin Technical staff 

Christel Feenstra Technical staff 

Bert Pool Technical staff 

Yvonne Pluister Technical staff 

Chelvi Mylvaganan Technical staff 

Lyandra Rooze Technical staff 

Mariska Sonneborn Technical staff 

Colinda Vroonland Technical staff 

DEPARTMENT OF PATHOLOGY 

Hester van Boven MD PhD Head 

Olga Balague Ponz MD PhD Academic staff 

Annegien Broeks PhD Academic staff 

Frans Hogervorst PhD Academic staff 

Daphne de Jong MD PhD Academic staff 

Jeroen de Jong MD PhD Academic staff 

Jettie Muris MD PhD Academic staff 

Petra Nederlof PhD Academic staff 

Renee van Pel MD Academic staff 

Efraim Rosenberg PhD Academic staff 

Loes van Velthuysen MD PhD Academic staff 

Jelle Wesseling MD PhD Academic staff 

Bart van de Wiel MD Academic staff 

DIVISION OF DIAGNOSTIC ONCOLOGY 

DEPARTMENT OF CLINICAL CHEMISTRY 

Pharmacological studies in mice 

Lin Fan, Levi Buil, Mark de Gooijer, Gayathri Chandrasekaran, Ruud Weijer, Eloy Moreno Roig, 

Lisette Hoogendijk, Diana Hanekamp, Alessia Gasparini and Olaf van Tellingen 

High-grade glioma (HGG) / glioblastoma multiforme (GBM) is a complex 

disease with many aberrant pathways working in concert and decades of testing 

numerous agents has failed to deliver drugs that provide a meaningful benefi t 

for these patients. In order to improve this disappointing track record, we need 

to change the current paradigm of drug testing. There are many agents under 

development (for other cancer types) that may also be useful for targeting some of 

the aberrant pathways in GBM, but testing these one at the time, as has been the 

current practice, does not seem to be the most successful way. We have started on 

a project looking at drug combinations using our experimental murine high grade 

glioma models, which was recently supported by a grant of the Dutch foundation 

Stophersentumoren. Our current efforts focus on (1) inhibition of DNA repair 

pathways to enhance the effi cacy of standard chemoradiation therapy and (2) 

concomitant inhibition of the three most commonly activated signal transduction 

pathway that drive the majority of GBMs (PI3K-mTor-, Ras-Mek-Erk- and Ink4a/b- 

CDK4/6-Rb-E2F1 pathways). When selecting candidate agents their ability to 

penetrate the blood-brain barrier (BBB) is an important factor. 

Standard treatment of newly diagnosed high-grade glioma after surgical resection 

involves radiotherapy (RT) and chemotherapy (CT) with temozolomide. For the fi rst 

part on abrogation of DNA repair we are investigating the standard treatment in 

combination with inhibition of PARP by ABT-888 or of Wee1 kinase by PD0166285 

and MK-1775. Importantly, we have mimicked the therapy of patients as closely as 

possible by implementing μ-Image Guided Radiotherapy (μ-IGRT) using the X-Rad 

225Cx (Precision X-Ray Inc). Through cone beam CT guidance this system offers 

precise delivery of high energy beams (225 KVp) of small fi eld sizes (1 - 5 mm), 

minimizing the exposure of normal tissues and allowing the delivery of RT doses 

that can not be given by conventional whole body RT. Here RT was delivered using 

a fractionated schedule (5 Gy per day x 4) in combination with oral temozolomide 

(100 mg/kg/day x 4) alone or with ABT-888 (10 mg/kg/bid x 4), PD0166285 (0.25 

mg/kg/bid x 4 or MK-1775 (20 mg/kg/bid x 4). Treatment of orthotopically injected 

Ink4a/Arf;P53;K-Ras v12 neurosphere-cultured cells (GBM652457) demonstrated 

that the PARP inhibitor ABT-888 signifi cantly improved the response (assessed by 

bioluminescence monitoring) and survival. However, the same combination was 

not more effi cacious against spontaneous Ink4a/Arf;P53;K-Ras v12 tumors relative 

to RT + CT alone. Addition of the Wee1 kinase inhibitors PD0166285 or MK1775 

did not improve the effi cacy of RT+CT against intracranially injected GBM652457 

cells. We are currently investigating the reasons underlying these results. ABT-888, 

PD0166285 and MK-1775 are all substrates of ABCB1 and ABCG2 and especially 

MK-1775 has a very poor BBB penetration. Experiments with combined inhibition of 

signal-transduction pathways involved in high grade gliomas will begin soon. 

Neuroendocrine tumours 

In collaboration with the Division of Medical Oncology (Babs Taal, Paul Baas), Pathology 

(Loes van Velthuysen) and Netherlands Cancer Registry (Otto Visser) 

Epidemiological data of neuroendocrine tumors (NET) are mostly classifi ed 

according to the primary site of the tumor. As histological grade might be more 

informative as refl ection of the biological behavior, we evaluated the epidemiological 

data of NET stratifi ed by grade. 42,186 patients with NET were identifi ed in the 

Netherlands from 1990-2008. Four groups were defi ned: well-differentiated 

NET, grade 1 and 2 (G1NET and G2NET), and poorly differentiated (grade 3) 

neuroendocrine carcinoma, large cell and small cell (LCNEC and SCNEC,

Figure 1: Age-standardized 

incidence rates according to 

gender and histological 

grade in the Netherlands 

from 1990-2008 for grade 

1 and 2 neuroendocrine 

tumors and large cell 

neuroendocrine carcinoma 

respectively). The annual incidence for G1NET remained stable at 2.1/100,000, 

whereas an increase was observed for G1 stomach (0.009 to 0.011), for both small 

(0.30 to 0.39) and large bowel tumors (0.25-0.30), but a decrease for G1 lung tumors 

(0.49 to 0.39). The incidence for G2NET increased from 0.01 in 1990 to 0.16 in 

2008, and for the LCNEC from

104 


Wen Qi PhD Post-doc 

Christian Siedschlag PhD Post-doc 

Lukas Batteau Technical staff 

Tessa Buckle Technical staff 

Anita Paape Technical staff 

Maddalena Rossi Technical staff 

Chantal Beekman Technical staff 

Wei Chen PhD student 

Lotte Elshof PhD student 

Lotte Lutkenhaus PhD student 

Kenneth Pengel PhD student 

Alexander Schmitz PhD student 

Annemarie Schmitz PhD student 

Ingar Seemann PhD student 

Kirsten Zuurmond PhD student 

A new venipuncture was then required. To avoid possible contamination of 

adipocytes originating from the epidermis, the second subsequent tube was used. 

If the vein could not be found, and possibly only skin tissue was aspirated, one ml 

serum from the well performed venipuncture was added to tube from the ceased 

venipuncture. 

Median S-100B level derived from the troublesome venipunctures was 0.18 (range 

0.10-0.63) μg/l and from the well performed venipunctures 0.10 (range 0.04-0.11) 

μg/l (P < 0.0001). Troublesome venipunctures lead to a high risk of increasing 

S-100B levels. To avoid possible contamination of adipocytes originating from 

the epidermis, we recommend to use the second subsequent tube for S-100B 

determination to obtain a reliable S-100B value. 

Non-small cell lung cancers 

In collaboration with the Division of Medical Oncology and Radiotherapy (Michel van den Heuvel, 

Wilma Uyterlinde, Jose Belderbos) 

In all patients with cytological or histological proven locally advanced NSCLC treated 

with concurrent chemoradiation between 2008 and 2010, we investigated the 

relevance of tumor markers. 

Before, during and after treatment Cyfra 21.1; CEA, NSE, SCC and cytokeratine 

fragments (CK8, CK18 and CK19) were measured. In case of missing samples stored 

pretreatment serum were used for analysis. Planned Target Volumes (PTV) and 

Gross Tumour Volumes (GTV) were derived from radiation patient charts. To assess 

the prognostic values of the tumour marker levels, the association with PTV, GTV 

time to progression and overall surival will be investigated. 

Biobank 

In collaboration with the Division of Experimental Therapy (Marjanka Schmidt, Annegien Broeks) 

The biobank that started in 2010 is now in full operation. Each new patient in 

the NKI-AVL will be informed about participation in the biobank and will be 

asked to donate two tubes of blood. Serum and full blood will be stored for future 

investigations in a broad research area. The approval and logistics are under 

responsibility of the Core Facility - Molecular Pathology and Biobank. 

PCR: SNP’s and enzyme deficiency We are investigating the opportunity to 

distinguish wild type DNA from its SNP in a relative cheap manner. We developed 

a HRM assay on the Roche LightCycler 480 and were able to identify a group of 

patients with an UGT2B7*2 (UDP-glucuronosyltransferase 2B7) variant. UGT2B7*2 

exhibited a severely reduced effect on the metabolism of tamoxifen compared to wild 

type UGT2B7. 

We are also investigating the 1896C>T mutation located in the exon 14 of the DPYD 

gene. It’s possible that this polymorphism variant is, besides the IVS14 G>A mutant, 

involved in severe toxicity after capecitabine intake. 

DEPARTMENT OF NUCLEAR MEDICINE 

Clinical Nuclear Medicine 

P Baas, MJ Baas-Vrancken Peeters, A Bex, J Belderbos, NS van den Berg, JP de Boer, S Burgers, 

T Buckle, M Donker, P Elkhuizen, K Gilhuijs, J van der Hage, M van den Heuvel, S Horenblas, 

H Klomp, CM Korse, FWB van Leeuwen, C Lange, C Loo, W Meinhardt, OE Nieweg, H Oldenburg, 

K Pengel, HG van der Poel, S Rodenhuis, ET Rutgers, EE Schaake, M Verheij, B Taal, J Teertstra, 

M Wouters, T Aukema, O Brouwer, CA Hoefnagel, B Koolen, SH Muller, F Sivro, M Sinaasappel, 

MPM Stokkel, RA Valdés Olmos, E Vegt, L Vermeeren, WV Vogel 

The line of technological innovation in nuclear medicine was continued including 

different fi elds such as radioguided intervention, hybrid tracers for sentinel node 

identifi cation, dedicated breast imaging, and new tracers for PET-CT. Generated 

work at the NKI-AVL was twice nominated for the Marie Curie Award at the 

Annual European Nuclear Medicine Congress held in Birmingham (O Brouwer

and M Donker), and received a Scholar-in-training Award from the San Antonio 

Breast Cancer Symposium (B Koolen). Kees Hoefnagel received The Giörgy Hevesy 

Memorial Medal from the Hungarian Society of Nuclear Medicine. Finally, O 

Brouwer received the Vlietstra award for best abstract and presentation at the Dutch 

Society of Urology meeting. 

Sentinel node with fluorescent and radioactive surgical guidance The 

experience with the hybrid tracer ICG- 99m Tc-nanocolloid was extended to other 

areas. 

A reproducibility study with this novel tracer was completed. After a fi rst injection 

of 99m Tc-nanocolloid both lymphoscintigraphy and SPECT were acquired in order 

to localize sentinel nodes. Subsequently, a second injection of bimodal ICG- 99m Tcnanocolloid 

complex followed by the exact imaging protocol was performed. In 25 

patients with melanoma (head/neck, trunk) and penile carcinoma a similar pattern 

of lymphatic drainage was observed without additional sentinel nodes after the 

second injection. The bimodal tracer enabled intraoperative fl uorescence imaging of 

all radioactive sentinel nodes. A similar correlation was observed when the excided 

sentinel nodes were measured. 

In a separate study concerning head and neck melanoma ICG- 99m Tc-nanocolloid 

led to the identifi cation of 25 sentinel nodes in 10 patients. Fluorescence particularly 

improved surgical guidance in areas with sentinel nodes located close to a high 

radioactive background signal such as the injection site. In the patients where blue 

dye was used merely 23% of the sentinel nodes stained blue whereas all of them were 

fl uorescent. 

Sentinel node detection using SPECT/CT and portable imaging devices The 

clinical study concerning stage I testicular cancer was evaluated on the basis of 10 

patients. SPECT-CT identifi ed sentinel nodes in all patients. In 5 patients with right 

testicle cancer sentinel nodes were found aortocaval or paracaval, in one of them 

together with a sentinel node adjacent to the testicular vessels. In patients with left 

testicular cancer SPECT-CT identifi ed para-aortic sentinel nodes in 5 patients, in 

two of them together with sentinel nodes along the testicular vessels. Twenty-six 

sentinel nodes were laparoscopically removed and only in one patient a sentinel node 

contained metastases. No recurrences developed in the 9 patients with a negative 

sentinel node during a median follow-up of 21 months (range 2-50 months). 

By improving low dose CT SPECT/CT was able to accurately indicate sentinel node 

localization in 20 patients with head/neck malignancies (10 melanoma, 10 oral 

cavity carcinoma). In 15 basins optimized low dose CT led to the identifi cation of 

lymph node clusters corresponding to the radioactive areas; in 12 of these basins 2 or 

more sentinel nodes were found at surgery guided by a portable gamma camera. 

Laparoscopic sentinel node biopsy was evaluated in 121 prostate cancer patients after 

transrectal ultrasound guided tracer injection into the prostate. SPECT-CT was used 

for preoperative sentinel localization and a portable gamma camera at the operation 

room. In 49 patients sentinel node metastases were found. In 37 patients sentinel 

nodes were found outside the area of extended pelvic lymph node dissection. Five of 

these sentinel nodes were tumor bearing but only twice exclusively so. 

In 10 patients undergoing sentinel node biopsy preoperative SPECT/CT data 

were automatically registered with the coordinates of a freehand SPECT based 

positioning system by a marker fi xed to an anatomical reference area of the patient. 

The registration succeeded automatically based on segmented CT data. Both virtual 

reality (VR) and augmented reality (AR) modes were tested. Overall average static 

error for the CT was 0.88mm for VR and 2.86mm for AR. For SPECT the errors 

were 5.07mm and 5,8mm respectively. The use of SPECT/CT visualized into the 

freehand-SPECT probe device may facilitate navigation for sentinel node localization 

at the operation room in the future. 

Lymphatic mapping in mutifocal/multicentric breast cancer The multicentric 

study in patients with multiple invasive tumors in one or more quadrants of the 

breast included 50 patients from 4 hospitals in the Netherlands. After a fi rst 99m Tcnanocolloid 

injection in the largest tumor lymphatic mapping was established using 

lymphoscintigraphy and SPECT-CT. Subsequently, a second injection of the tracer 

PUBLICATIONS 

105 


Adank MA, Jonker MA, Kluijt I, van Mil 

SE, Oldenburg RA, Mooi WJ, Hogervorst FB, 

van den Ouweland AM, Gille JJ, Schmidt MK, 

van der Vaart AW, Meijers-Heijboer H, 

Waisfi sz Q. CHEK2*1100delC homozygosity 

is associated with a high breast cancer risk in 

women. J Med Genet. 2011;48:860-863 

Alderliesten T, Loo CE, Pengel KE, 

Oldenburg HSA, Rutgers EJTh, Gilhuijs KGA, 

Vrancken Peeters MTFD. Radioactive Seed 

Localization of Breast Lesions: an Adequate 

Localization Method without Seed Migration. 

The Breast Journal 2011;17:594-601 

Antoniou AC, Kartsonaki C, Sinilnikova 

OM, Soucy P, et. al. Common alleles at 6q25.1 

and 1p11.2 are associated with breast cancer 

risk for BRCA1 and BRCA2 mutation carriers. 

Hum Mol Genet 2011;20:3304-3321 

Aukema T. Surgical implications of novel 

PET technologies in breast cancer, lung cancer 

and melanoma. Thesis, Universitiy of 

Amsterdam, 2011 

de Bonilla-Damiá A, Roberto Brouwer O, 

Meinhardt W, Valdés-Olmos RA. Lymphatic 

Drainage in Prostate Carcinoma assessed by 

Lymphoscintigraphy and SPECT/CT: Its 

importance for the Sentinel Node Procedure. 

Rev Esp Med Nucl. 2011 (in press) 

Beekman CA, Buckle T, van Leeuwen AC, 

Valdés Olmos RA, Verheij M, Rottenberg S, 

van Leeuwen FW. Questioning the value of 

(99m)Tc-HYNIC-annexin V based response 

monitoring after docetaxel treatment in a 

mouse model for hereditary breast cancer. Appl 

Radiat Isot 2011;69:656-662 

Betsalel OT, Rosenberg EH, Almeida LS, 

Kleefstra T, Schwartz CE, Valayannopoulos V, 

Abdul-Rahman O, Poplawski N, Vilarinho L, 

Wolf P, den Dunnen JT, Jakobs C, Salomons 

GS. Characterization of novel SLC6A8 variants 

with the use of splice-site analysis tools and 

implementation of a newly developed LOVD 

database. Eur J Hum Genet 2011;19:56-63 

Bex A, Valdés Olmos RA. GOSTT in 

kidney cancer. In: International Atomic 

Energy Agency, Guided intraoperative 

scintigraphic tumour targeting (GOSTT), 

IAEA 2011 (in press) 

Bex A, Vermeeren L, Meinhardt W, Prevoo 

W, Horenblas S, Valdés Olmos RA. 

Intraoperative sentinel node identifi cation and 

sampling in clinically node-negative renal cell 

carcinoma: initial experience in 20 patients. 

World J Urol 2011;29:793-799 

Boone J, Bex A, Prevoo W. Percutaneous 

Radiofrequency Ablation of a Small Renal 

Mass Complicated by Appendiceal Perforation. 

Cardiovasc Intervent Radiol 2011 (in press)

106 



Brouwer OR, Valdés Olmos RA, 

Vermeeren L, Hoefnagel CA, Nieweg OE, 

Horenblas S. SPECT/CT and a portable 

gamma-camera for image-guided laparoscopic 

sentinel node biopsy in testicular cancer. J 

Nucl Med. 2011;52:551-554 

Brouwer OR, Horenblas S, Nieweg OE, 

Valdés Olmos RA. GOSTT in testicular 

cancer. In: International Atomic Energy 

Agency, Guided intraoperative scintigraphic 

tumour targeting (GOSTT), IAEA 2011 (in 

press) 

Brouwer OR, Klop MWC, Buckle T, van 

den Brekel MWM, Balm FJM, Nieweg OE, 

Valdés-Olmos RA, van Leeuwen FWB. 

Feasibility of sentinel node biopsy in head and 

neck melanoma using a hybrid radioactive and 

fl uorescent tracer. Ann Surg Oncol 2011 

(accepted) 

Bruin SC, He Y, Mikolajewska-Hanclich I, 

Liefers GJ, Klijn C, Vincent A, Verwaal VJ, de 

Groot KA, Morreau H, van Velthuysen ML, 

Tollenaar RA, van ‘t Veer LJ. Molecular 

alterations associated with liver metastases 

development in colorectal cancer patients. Br J 

Cancer 2011 2011;105:281-287 

Buckle T, van den Berg NS, Kuil J, 

Bunschoten A, Oldenburg J, Borowsky AD, 

Wesseling J, Masada R, Oishi S, Fujii N, van 

Leeuwen FWB. Non-invasive longitudinal 

imaging of tumor progression using an 

111Indium labeled CXCR4 peptide antagonist. 

Am J Nucl Med Mol Im 2011 (accepted) 

Buckle T, Brouwer OR, Valdés Olmos RA, 

van der Poel HG, van Leeuwen FWB. 

Relation between tracer deposits and lymphatic 

drainage in prostate cancer patients? J Nucl 

Med 2011 (minor revisions) 

Bunschoten A, Buckle T, Kuil J, Luker KE, 

Luker GD, Nieweg OE, van Leeuwen FWB. 

Targeted non-covalent self-assembled 

nanoparticles based on human serum 

albumin. Biomaterials, 2011 (accepted) 

Courrech Staal EF, Smit VT, van 

Velthuysen ML, Spitzer-Naaykens JM, 

Wouters MW, Mesker WE, Tollenaar RA, van 

Sandick JW. Reproducibility and validation of 

tumour stroma ratio scoring on oesophageal 

adenocarcinoma biopsies. Eur J Cancer 

2011;47:375-382 

followed by the exact same imaging protocol was performed for the other tumors. 

Additional lymphatic drainage was depicted after the second and/or third injection 

in 30 patients (60%). Thirty additional sentinel nodes were seen in the axilla, 11 

in the internal mammary chain and 2 intramammary. Sentinel nodes contained 

metastases in 17 patients (17%). In 5 patients with a tumor-positive node in the axilla 

that was visualized after the fi rst injection, an additionally axillary involved node was 

found after subsequent injection. In two patients, isolated tumor cells were found in 

sentinel nodes only visualized after the second injection. 

Radioguided tumor excision in breast cancer Both radioguided occult lesion 

localization (ROLL) with 99m Tc-nanocolloid and radioguided seed localization (RSL) 

using 125 I seed were evaluated in a group 154 patients receiving breast conserving 

surgery. In 83 patients 99m Tc-nanocolloid was administered the day before surgery 

at the site of a twistmarker positioned into the tumor previous to neoadjuvant 

chemotherapy. In 71 patients a 125 I seed was placed into the tumor before the start 

of chemotherapy. Due to the half life of 60 days the 125 I seed is still traceable after 

completing chemotherapy avoiding the need for an extra localization procedure 

before surgery. Patient, tumor and resected specimen characteristics were 

comparable for both groups. Irradical resection was seen in only 7% for ROLL and 

8% for RSL. 

Radioguided tumor excision using a freehand SPECT probe was continued, in some 

cases combined with sentinel node lymphadenectomy. The principal advantage of 

the freehand SPECT probe in comparison with the standard probe is the generation 

of 3D images in addition to the acoustic signals for both ROLL and RSL. 

PET-CT in breast cancer 18 F-FDG PET-CT was evaluated as a staging procedure 

for primary stage II and III breast cancer in 154 patients. In 25 patients 42 FDG-avid 

additional lesions were seen. In 20 patients (13%) these lesions could be confi rmed. 

In 16 patients lesions were exclusively seen on PET/CT leading to a change in 

treatment in 13 patients (8%). In 129 patients with a negative PET/CT no metastases 

developed during a follow-up of 9 months. PET/CT was superior to conventional 

imaging techniques (chest radiography, liver ultrasonography, bone scintigraphy) as 

a staging modality. 

In 203 out of 214 patients (95%) with stage II and III breast cancer scheduled to 

receive adjuvant chemotherapy, FDG tumor uptake, varying from moderate to 

intense, was considered suffi cient for response monitoring with PET/CT; this was 

seen for 74% of invasive lobular carcinomas and 97% of invasive ductal carcinomas. 

In a univariable analysis primary tumor SUVmax was signifi cantly higher in 

patients with distant metastasis at staging, non-lobular carcinomas, grade 3 tumors, 

and in tumors with negative hormone receptors, high p53-expression, and a high 

proliferation index as defi ned by Ki-67-expression. Also tumors with a high risk 

according to their gene-expression profi le showed signifi cantly higher SUVmax. 

After multiple linear regression triple negative tumors and grade 3 tumors were 

associated with a higher SUVmax. 

18 F-FDG PET-CT for response monitoring during neoadjuvant chemotherapy was 

evaluated in 94 stage II and III breast cancer patients. For the whole group (near) 

complete response at pathology was signifi cantly associated with a decrease in 

FDG tumor uptake (SUVmax) during treatment (area under ROC curve 0.88). In 

a subgroup analysis a signifi cant association was found for ER positive/HER2 nonamplifi 

ed and triple negative tumors but not for HER2 amplifi ed tumors. 

FDG PET/CT was also evaluated in combination with MRI. In 65 patients with 

response or residual disease after chemotherapy the area under ROC was 0.84 for 

MRI alone, and 0.94 in combination with FDG PET/CT. The accuracy was largest 

for triple-negative tumors at both imaging modalities but mRI and PET/CT were 

complementary in the ER+/HER2- and in the HER2+ subgroups. 

Breast cancer imaging with a small PET ring camera The evaluation of the 

fi rst prototype of a small PET ring camera (MAMMI) for dedicated breast imaging 

was evaluated in 32 patients with stage II-III breast cancer patients (European 

project MAMMI EU-037555). FDG-avid primary tumors were visualized in 31 of 32 

consecutive patients. In one patient no primary tumor was visible on FDG PET-CT

nor on MAMMI (nor on MRI). On the basis of the hanging breast position and a low 

FDG dosage MAMMI was able to visualize tumors at a distance in the range of 21 

to 76 mm from the pectoral muscle. Agreement in FDG tumor uptake (SUVmax) 

between MAMMI and PET/CT was 0.86. However SUVmax assessed by MAMMI 

was 2.5 higher than PET/CT. With the installation of the defi nitive prototype in 

March 2011 evaluation of the results for response to neoadjuvant chemotherapy is 

ongoing as well as a separate protocol for patients receiving radiotherapy. 

PET-CT in lung cancer The role of FDG PET-CT for identifi cation of response 

to neoadjuvant erlotinib was evaluated in 60 patients with NSCLC eligible for 

surgical resection. The patients preoperatively received 150 mg erlotinib once 

daily for 3 weeks. PET/CT evaluation revealed metabolic response with more than 

25% SUVmax decrease in 16 patients (27%) whereas CT using RECIST criteria 

showed response in only 4 (7%). Pathologic examination showed signifi cant tumor 

regression in 14 patients (23%). 

FDG PET-CT was also involved for assessment of response in a study combining 

cetuximab with concurrent chemoradiotherapy in patients with NSCLC. Evaluation 

of early response monitoring is ongoing. 

New PET tracers Fluorine-18-methyl-choline (F18-choline), PET/computed 

tomography (CT) is a relative new tracer that has been introduced this year for 

routinely evaluation of prostate cancer patients with signifi cantly increased or 

rapidly increasing prostate-specifi c antigen (PSA) levels. We observed uptake of this 

PET tracer in mediastinal lymph nodes in a signifi cant number of patients. A total 

of 48 consecutive men (mean age: 65.6 years; range: 50-79 years, standard deviation: 

7.1) with histopathologically proven prostate cancer underwent F18-choline PET/ 

CT imaging for restaging. In 27 patients (56.3%), F18-choline PET/CT showed 

positive lymph nodes in the mediastinum (mean standardized uptake values: 

3.75; range: 1.7-13.8, standard deviation: 2.4). Only one patient had histologically 

proven pulmonary metastasis. No signifi cant relationship was observed between 

mediastinal F18-choline lymph node uptake and serum PSA level (P=0.785), initial 

T stage (P=0.555), N stage (P=0.548), M stage (P=0.426), smoking (P=0.537), chronic 

obstructive pulmonary disease (P=0.115) or the presence of tumour recurrence on 

F18-choline PET/CT. Mediastinal lymph node uptake of F18 choline is frequently 

observed, without any signifi cant relationship with tumour characteristics. 

Therefore, interpretation of positive mediastinal lymph node uptake should be done 

carefully. Currently, a study is performed in which the value of F18-choline PET/CT 

is compared with routine bone scintigraphy, to assess the value of both techniques in 

prostate cancer patients in the evaluation of rising PSA levels. 

In collaboration with the department of clinical pharmacy of the Slotervaart 

Hospital, a new radiopharmaceutical Ga68-DOTATATE was developed for 

neuroendocrine tumor imaging. This tracer binds to somatostatinreceptors and can 

be regarded as a tracer with better features than Octreoscan. Currently, the most 

optimal labelling technique is under study and the goal is to introduce this tracer 

for routine use in clinical practice in 2012. Five patients with a suspicion on tumor 

recurrence have been studied so far demonstrating better results than Octreoscan 

(see fi gure 2). 

Figure 2: In111-Octreotide 

scintigraphy (A) and 

Ga68-DOTATATE PET 

(C), illustrating an 

improved detection 

rate of metastases of a 

neuroendocrine tumor 

by means of this new 

PET tracer 

107 



Cserni G, Amendoeira I, Bianchi S, 

Chmielik E, Degaetano J, Faverly D, 

Figueiredo P, Foschini MP, Grabau D, 

Jacquemier J, Kaya H, Kulka J, Lacerda M, 

Liepniece-Karele I, Penuela JM, Quinn C, 

Regitnig P, Reiner-Concin A, Sapino A, van 

Diest PJ, Varga Z, Vezzosi V, Wesseling J, 

Zolota V, Zozaya E, Wells CA. Distinction of 

isolated tumour cells and micrometastasis in 

lymph nodes of breast cancer patients 

according to the new Tumour Node Metastasis 

(TNM) defi nitions. Eur J Cancer 

2011;47:887-894 

Derksen PW, Braumuller TM, van der 

Burg E, Hornsveld M, Mesman E, Wesseling 

J, Krimpenfort P, Jonkers J. Mammaryspecifi 

c inactivation of E-cadherin and p53 

impairs functional gland development and 

leads to pleomorphic invasive lobular 

carcinoma in mice. Dis Model Mech 

2011;4:347-358 

De Munck L, Schaapveld M, Siesling S, 

Wesseling J, Voogd AC, Tjan-Heijnen VC, 

Otter R, Willemse PH. Implementation of 

trastuzumab in conjunction with adjuvant 

chemotherapy in the treatment of nonmetastatic 

breast cancer in the Netherlands. 


De Vreeze RSA, de Jong D, Koops W, 

Nederlof PM, Ariaens A, Haas RL, van 

Coevorden F. Oncogenesis and classifi cation 

of mixed-type liposarcoma: a radiological, 

histopathological and molecular biological 

analysis. Int J Cancer 2011;128:778-786 

De Vreeze RSA, van Coevorden F, 

Boerrigter L, Nederlof PM, Haas RL, Bras J, 

Rosenwald A, Mentzel T, de Jong D. 

Delineation of chondroid lipoma: an 

immunohistochemical and molecular 

biological analysis. Sarcoma 2011 (in press) 

De Zordo T, Ladurner M, Horninger W, 

Heijmink SW, Jaschke W, Aigner F, 

Frauscher F. New ultrasound technologies for 

the diagnostics of prostate cancer. Radiologe 

2011;51:938-946 

Dickinson L, Ahmed HU, Allen C, 

Barentsz JO, Carey B, Futterer JJ, Heijmink 

SW, Hoskin PJ, Kirkham A, Padhani AR, 

Persad R, Puech P, Punwani S, Sohaib AS, 

Tombal B, Villers A, van der Meulen J, 

Emberton M. Magnetic resonance imaging 

for the detection, localisation, and 

characterisation of prostate cancer: 

recommendations from a European consensus 

meeting. Eur Urol 2011;59:477-494

108 



Didraga MA, van Beers EH, Joosse SA, 

Brandwijk KIM, Oldenburg RA, Wessels LFA, 

Hogervorst FBL, Ligtenberg MJ, 

Hoogerbrugge N, Verhoef S, P Devilee, 

Nederlof PM. A non-BRCA1/2 hereditary 

breast cancer sub-group defi ned by aCGH 

profi ling of genetically related patients. Breast 


Emmering J, Vogel WV, Stokkel MP. 

Intramuscular metastases on FDG PET-CT: a 

review of the literature. Nucl Med Commun 


Gilhuijs KGA, Loo CE. MRI-respons 

monitoring van het mammacarcinoom. 

Oncologie up to date 2011;2:4-5 

Fleskens SA, Bergshoeff VE, Voogd AC, 

van Velthuysen ML, Bot FJ, Speel EJ, Kremer 

B, Takes R, Slootweg P. Interobserver 

variability of laryngeal mucosal premalignant 

lesions: a histopathological evaluation. Mod 

Pathol 2011;24:892-898 

Graafl and NM, Valdés Olmos RA, 

Meindhardt W, Bex A, van der Poel HG, van 

Boven HH, Nieweg OE, Horenblas S. 

Sentinel-nodebiopsie voor stadiering van de lies 

bij peniscarcinoom na eerdere primaire 

tumorbehandeling. Tijdschrift voor Urologie 

2011;6:133-136 

Graafl and NM, Teertstra HJ, Besnard AP, 

Boven HH, Horenblas S. Identifi cation of 

High Risk Pathological Node Positive Penile 

Carcinoma: Value of Preoperative 

Computerized Tomography Imaging. J Urol 

2011;185:881-887 

Graafl and N, Valdés Olmos RA, 

Horenblas S. GOSTT in penile cancer. In: 

International Atomic Energy Agency, Guided 

intraoperative scintigraphic tumour targeting 

(GOSTT), IAEA 2011 (in press) 

Haans JJ, de Zwart IM, Eilers PH, Reiber 

JH, Doornbos J, de Roos A, Masclee AA. 

Gastric volume changes in response to a meal: 

Validation of magnetic resonance imaging 

versus the barostat. J Magn Reson Imaging 


Haiman CA, Chen GK, Vachon CM, 

Canzian F, Dunning A, Millikan RC, Wang X, 

et al. A common variant at the TERT- 

CLPTM1L locus is associated with estrogen 

receptor-negative breast cancer. Nat Genet. 


Harinck F, Nagtegaal T, Kluijt I, Aalfs C, 

Smets E, Poley JW, Wagner A, van Hooft J, 

Fockens P, Bruno M, Bleiker EM. Feasibility 

of a pancreatic cancer surveillance program 

from a psychological point of view. Genet Med 


Thyroid cancer and neuroendocrine tumors Chromogranin A (CgA) assay 

and somatostatin receptor scintigraphy (SRS) are implemented in the standard 

workup of neuroendocrine tumors (NETs). A total of 88 consecutive patients with 

histologically confi rmed well-differentiated NETs were included in a study to assess 

the value of SRS and CgA in staging and follow-up patients with well-differentiated 

NETs. CgA values differed signifi cantly between patients with and without clinical 

symptoms (P=0.028), a positive and negative SRS (P=0.005), the different SRS 

scores (P=0.002), the number of lesions (P=0.001), and the presence or absence 

of liver metastasis (P=0.003). The sensitivity, specifi city, positive predictive value, 

and negative predictive value were 78, 93, 98, and 47% for SRS and 62, 100, 100, 

and 35% for CgA; however, by combining the test, all results improved. All patients 

(n=11) referred for routine follow-up had stable CgA values, whereas in one patient 

only the SRS score increased. In the group of patients with a suspicion on tumor 

progression during follow-up (n=14), CgA values increased in nine patients. In this 

group, the SRS score increased in two patients. Despite the higher sensitivity of 

SRS than of CgA in staging and restaging well-differentiated NETs, both tests are 

required at the initial stage. Disease extent, symptoms, and liver metastasis have 

an impact on both SRS results and CgA values. CgA has an important value in the 

assessment of tumor progression during follow-up, whereas the role of SRS in the 

routine follow-up of well-differentiated NETs is limited. 

A KWF grant was received to create a nationwide multicenter study aiming to 

improve diagnosis and treatment of recurrent differentiated thyroid carcinoma. 

Based on prior work it is hypothesised that in vivo tumor characterization with FDG 

PET/CT and Iodine-124 PET/CT will accurately predict response to treatment with 

high dose treatment, resulting in optimized indications for this treatment. As a fi rst 

step, a supervised phantom study with central QC/QA of the novel Iodine-124 PET/ 

CT modality involving all treatment centers in the Netherlands was initiated. 

Cardiotoxicity Left ventricular dyssynchrony is a prognostic parameter in heart 

failure patients, irrespective of its cause such as chemotherapy. It may predict 

response to cardiac resynchronization therapy and may well predict adverse cardiac 

events. Recently, a geometrical approach for dyssynchrony analysis of myocardial 

perfusion scintigraphy (MPS) was introduced. In this study the feasibility of this 

geometric method to detect dyssynchrony was assessed in a population with a 

normal MPS and in patients with documented ventricular dyssynchrony. All 

patients underwent a two-day stress/rest MPS protocol. For the normal population 

time to peak motion was 42.8 ± 5.1 % RR-cycle, SD of peak motion was 3.5 ± 1.4 % 

RR-cycle and bandwidth was 18.2 ± 6.0 % RR-cycle. No signifi cant gender-related 

differences or differences between rest and post-stress acquisition were found for 

the dyssynchrony parameters. Discrepancies between the normal and abnormal 

population were most profound for the mean wall motion (p-value

myocardial wall R2 =0.10). In general, SPECT indices showed moderate correlations 

with the planar uptake. By applying a volumetric segmentation method we were able 

to segment the heart in all patients. SPECT I-123 MIBG quantifi cation was found 

to be highly reproducible and had a moderate to good correlation with the planar 

indices. This study was performed at and in collaboration with the department of 

nuclear medicine, LUMC (thesis: BJ van der Veen). 

Currently, a pilot-study is performed to assess the value of the aforementioned 

techniques in patients with cardiotoxicity due to Herceptin. Patients with a 

decreased or rapidly decreasing LVEF are included in this study and will undergo 

I-123 MIBG scintigraphy of the heart to assess cardiac innervation. Initial results 

have shown that patients with a normal I-123 MIBG study show recovery of the 

LVEF during follow-up, whereas patients with decreased I-123 MIBG uptake have no 

recovery of the LVEF, which may have impact on prognosis. Based on these results, a 

prospective study will be designed. 

Radiotherapy planning Target defi nition for external beam radiotherapy involves 

visual interpretation of multiple imaging modalities, and thus remains a laborious 

and observer-dependent procedure. A novel strategy of computer-assisted tumor 

delineation was developed, based on automatic contouring of FDG-PET and 

planning-CT. In a pilot study with head-neck cancer patients, this strategy led to a 

signifi cant reduction of the total time needed by radiation oncologists to delineate 

GTV (-35-40%), and it reduced interobserver variation (-25-30%), including a strong 

reduction in large deviations by less-experienced observers. Based on these results, 

larger studies with computer-assisted multimodality tumor delineation will be 

designed. 

Surgical fluorescence guidance towards the sentinel lymph node 

Oscar Brouwer, Tessa Buckle, Nynke van den Berg, Fijs van Leeuwen, Martin Klop, Michiel van der 

brekel, Fons, Balm, Simon Horenblas, Henk van de Poel, Omgo Nieweg, Renato Valdes-Olmos 

One clinical precedent for the use of nano-sized imaging agents is the localization 

of the tumor draining sentinel lymph nodes. In this application, radiocolloids such 

as 99mTc-NanoColl are currently used to plan the surgical procedure and to provide 

acoustic guidance during the intervention. By using the bimodal or rather hybrid 

imaging agent ICG- 99m Tc-NanoColl, which is both radioactive and fl uorescent, we 

have been able to integrate both surgical planning and intraoperative fl uorescence 

guidance towards the sentinel lymph nodes. We have currently applied this pocedure 

in > 150 patients with varying malignancies. Currently the technology is being 

dissiminated to other clinical sites. 

We have also shown the value of hybrid imaging agents in achieving navigation 

towards SLNs. Herein a fl uorescence laparoscope was navigated through the 

preoperative 3D SPECT/CT images. The accuracy of this navigation process is 

then validated in real-time using the fl uorescent emission coming from the area of 

interest. As such the hybrid technology helps correct for a common shortcoming in 

surgical navigation, namely intraoperative movement of the areas of interests. 

On top of using the hybrid imaging agent to help improve the surgical accuracy, the 

location of intraprostatic tracer deposids defi ned ex vivo via fl uorescence imaging 

could also be directly correlated with the lymphatic drainage pattern. This study 

revealed that variations in the injection accuracy also resulted in variations of the 

lymphatic drainage pattern. 

Targeted imaging agents for surgical guidance 

Joeri Kuil, Anton Bunschoten, Mark Rood, Tessa Buckle, Aldrik Velders, Lee Josephson, 

Omgo Nieuweg, Fijs van Leeuwen 

For targeted imaging applications we have developed a number of multimodal 

imaging agents that comprise receptor specifi c peptides for e.g. the CXCR4 or α vβ 3 

integrin over expression. Targeting peptides have been used in their monomeric 

form, as multimeric/dendritic structures and/or as self-assembled imaging agents. 

In the latter concept non-covalent interactions with nano-sized proteins are used 

109 



Hartog H, Horlings HM, van der Vegt B, 

Kreike B, Ajouaou A, van de Vijver MJ, 

Marike Boezen H, de Bock GH, van der 

Graaf WT, Wesseling J. Divergent effects of 

insulin-like growth factor-1 receptor expression 

on prognosis of estrogen receptor positive versus 

triple negative invasive ductal breast 

carcinoma. Breast Cancer Res Treat 

2011;129:725-736 

Heijmink SW, Fütterer JJ, Strum SS, Oyen 

WJ, Frauscher F, Witjes JA, Barentsz JO. 

State-of-the-art uroradiologic imaging in the 

diagnosis of prostate cancer. Acta Oncol 

2011;50 Suppl 1:25-38. Review 

Hoeks CM, Barentsz JO, Hambrock T, 

Yakar D, Somford DM, Heijmink SW, 

Scheenen TW, Vos PC, Huisman H, van Oort 

IM, Witjes JA, Heerschap A, Fütterer JJ. 

Prostate cancer: multiparametric MR imaging 

for detection, localization, and staging. 

Radiology 2011;261:46-66. Review 

Hompes D, Prevoo W, Ruers T. 

Radiofrequency ablation as a treatment tool 

for liver metastases of colorectal origin. Cancer 

Imaging 2011;24:23-30. Review 

Im KM, Kirchhoff T, Wang X, Green T, 

Chow CY, Vijai J, Korn J, Gaudet MM, et al. 

Haplotype structure in Ashkenazi Jewish 

BRCA1 and BRCA2 mutation carriers. Hum 

Genet. 2011;130:685-699 

Jones S, Li M, Parsons DW, Zhang X, 

Wesseling J, Kristel P, Schmidt MK, 

Markowitz S, Yan H, Bigner D, Hruban RH, 

Eshleman JR, Iacobuzio-Donahue CA, 

Goggins M, Maitra A, Malek SN, Powell S, 

Vogelstein B, Kinzler KW, Velculescu VE, 

Papadopoulos N. Somatic mutations in the 

chromatin remodeling gene ARID1A occur in 

several tumor types. Hum Mutat 2011 (in 

press) 

Joosse SA, Brandwijk KI, Mulder L, 

Wesseling J, Hannemann J, Nederlof PM. 

Genomic signature of BRCA1 defi ciency in 

sporadic basal-like breast tumors. Genes 

Chromosomes Cancer 2011;50:71-81 

Kluijt I, Siemerink EJ, Ausems MG, van 

Os TA, de Jong D, Simões-Correia J, van 

Krieken JH, Ligtenberg MJ, Figueiredo J, van 

Riel E, Sijmons RH, Plukker JT, van 

Hillegersberg R, Dekker E, Oliveira C, Cats A, 

Hoogerbrugge N; on behalf of the Dutch 

Working Group on Hereditary Gastric 

Cancer. CDH1-related hereditary diffuse 

gastric cancer syndrome: Clinical variations 

and implications for counseling. Int J Cancer. 

2011 (in press)

110 



Kempers MJ, Kuiper RP, Ockeloen CW, 

Chappuis PO, Hutter P, Rahner N, 

Schackert HK, Steinke V, Holinski-Feder E, 

Morak M, Kloor M, Büttner R, Verwiel ET, 

van Krieken JH, Nagtegaal ID, Goossens M, 

van der Post RS, Niessen RC, Sijmons RH, 

Kluijt I, Hogervorst FB, Leter EM, Gille JJ, 

Aalfs CM, Redeker EJ, Hes FJ, Tops CM, van 

Nesselrooij BP, van Gijn ME, Gómez García 

EB, Eccles DM, Bunyan DJ, Syngal S, Stoffel 

EM, Culver JO, Palomares MR, Graham T, 

Velsher L, Papp J, Oláh E, Chan TL, Leung 

SY, van Kessel AG, Kiemeney LA, 

Hoogerbrugge N, Ligtenberg MJ. Risk of 

colorectal and endometrial cancers in 

EPCAM deletion-positive Lynch syndrome: a 

cohort study. Lancet Oncol. 2011;12:49-55 

Kluijt I, Sijmons RH, Hoogerbrugge N, 

Vasen HF, Cats A. Familial gastric cancer: 

diagnosis, treatment and periodic 

surveillance. Ned Tijdschr Geneeskd. 

2011;155:A2731. Review 

Koolen BB, Vegt E, Rutgers EJ, Vogel WV, 

Stokkel MP, Hoefnagel CA, Fioole-Bruining 

A, Vrancken Peeters MJ, Valdés Olmos RA. 

FDG-avid sclerotic bone metastases in breast 

cancer patients: a PET/CT case series. Ann 

Nucl Med. 2011 (in press) 

Koolen BB, Vrancken Peeters MJ, 

Aukema TS, Vogel WV, Oldenburg HS, van 

der Hage JA, Hoefnagel CA, Stokkel MP, 

Loo CE, Rodenhuis S, Rutgers EJ, Valdés 

Olmos RA. 18F-FDG PET/CT as a staging 

procedure in primary stage II and III breast 

cancer: comparison with conventional 

imaging techniques. Breast Cancer Res Treat. 


Koolen BB, Vrancken Peeters MJ, 

Aukema TS, Vogel WV, Oldenburg HS, van 

der Hage JA, Hoefnagel CA, Stokkel MP, 

Loo CE, Rodenhuis S, Rutgers EJ, Valdés 

Olmos RA. 18F-FDG PET/CT as a staging 

procedure in primary stage II and III breast 

cancer: comparison with conventional 

imaging techniques. Breast Cancer Res Treat. 


Koolen SL, van Waterschoot RA, van 

Tellingen O, Schinkel AH, Beijnen JH, 

Schellens JH, Huitema AD. From Mouse to 

Man: Predictions of Human 

Pharmacokinetics of Orally Administered 

Docetaxel From Preclinical Studies. J Clin 

Pharmacol. 2011 (in press) 

Korse CM, Muller M, Taal BG. 

Discontinuation of proton pump inhibitors 

during assessment of chromogranin A levels in 

patients with neuroendocrine tumours. Br J 


Figure 3: Formation of a 

fl uorescent non-covalent 

complex of HSA/ICG, B) 

chemical structures of ICG, 

IR783-CO2H and Cy5, C) 

covalent labeling of HSA 

with Cy5 and subsequent 

formation of a non-covalent 

quenched complex of 

HSA-Cy5 with the NIR-dyes 

ICG or IR783-CO2H, D) 

formation of targeted 

bionanoparticles based upon 

the interaction of IR783labeled 

peptides with HSA. 

Bunschoten et al. 

Biomaterials 2011 

to optimize the pharmacokinetics. FRET interactions are utilized to monitor the 

assembly/disassembly of the individual components that make up the imaging agent 

(see fi gure 3). All imaging agents are validated in vitro and in the preclinical setting 

can be used for in vivo imaging and receptor specifi c surgical guidance. 

DEPARTMENT OF PATHOLOGY 

Discovery, evaluation and validation of prognostic and predictive factors to tailor 

diagnosis and treatment of breast cancer patients 

Theo Ruers, Danny Evers, Claudette Loo, Sabine Linn, Emiel Rutgers, Mila Donker, Bas Koolen, 

Joyce Sanders, Jettie Muris, Petra Nederlof, Jelle Wesseling 

Breast cancer is a heterogeneous disease requiring an individualized approach for 

optimal diagnosis and treatment of breast cancer patients. Therefore, we collaborate 

with a variety of research groups, mainly within the institute, but also worldwide, 

to ensure that our concerted action ultimately will benefi t breast cancer patient 

survival. As such, we team up on the development of new imaging techniques 

(e.g. collaboration with the Theo Ruers group), improvement of existing biomarker 

assessment (e.g. HER2 testing), as well as development of new biomarker tests to 

determine for example the tamoxifen sensitivity in breast cancer positive for the 

estrogen receptor (see for a detailed description the contribution of Sabine Linn). 

Part of Jelle Wesseling’s research aiming at the discovery of factors determining 

sensitivity and resistance to systemic treatment is carried out at the Department of 

Experimental Therapy/Molecular Pathology and is described there. 

MOLECULAR PATHOLOGY 

EGFR mutation in lung carcinomas and response to Iressa therapy 

Daphne de Jong, Petra Nederlof, Erik Thunissen, Michel van den Heuvel, Marieke Vollebergh, 

Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Wieringa-Ariaens 

Gefi tinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine 

kinase (EGFR-TK), has shown potent anti-tumor effects and improved symptom 

and quality-of-life of a subset of patients with advanced NSCLC and a mutation in 

exon 18, 19, 20 or 21 of the EGFR gene (Tyrosine kinase domain). We have evaluated 

specimens of 300 iressa treated patients of a large series of NSCLC from our 

institute and the VU medical centre (Dr Erik Thunissen). We correlated response 

status with tumour characteristics such as EGFR and KRAS mutation status and 

EGFR gene amplifi cation (CISH).

Molecularly supported differentiation models in liposarcoma with 

consequences for diagnostic and clinical management 

Ronald de Vreeze, Henrique Ruijter, Ivon Tielen, Lucie Boerrigter, Aafke Ariaens, Petra Nederlof, 

Rick Haas, Frits van Coevorden, Daphne de Jong 

Liposarcoma can be distinguished in well-defi ned disease entities, characterized 

by morphology and in part by genetic alterations as well-differentiated (WDLS), dedifferentiated 

(DDLS), myxoid, round cell (MRLS), and pleomorphic (PLS). WLS and 

DDLS are characterized by amplifi cation of the 12q13-15 region including MDM2 

and CDK4 genes. MRLS is characterized by a t(12;16) or t(12;22) translocation. These 

classifying markers have been used to explore several clinical and biological 

aspects of these diseases and their interrelations. In rare instances, mixed-type 

liposarcomas with components of MRLS and WDL are seen. On the basis of MRI 

features, these cases were collected from the fi les of the NKI-AVL. Molecular 

analysis showed that these mixed type liposarcomas should not be regarded as 

collision tumors, but as an extreme variant of the morphological spectrum within 

a single biological entity. These fi ndings support a stem cell model with different 

levels of maturation within specifi c disease entities and expands the morphological 

spectrum of MRLS. This stem cell model with tumor progression from intermediate 

stages of development was further studied in a large series covering the spectrum 

from lipoma to high grade pleiomorphic liposarcoma using immunohistochemistry 

and MLPA. Amplifi cation of 12q and in low-grade cases also expression of the target 

genes CDK4 and MDM2 in a subset of PLS as well as in in WDLSand DDLS further 

supports this model. 

THE NETHERLANDS CANCER INSTITUTE 

FAMILY CANCER CLINIC 

Mohamed Achachah, Abderrahim Ajouaou, Majella Boutmy-de Lange, Amber Gorsira, Daniela 

Hahn, Frans Hogervorst, Mobien Kasiem, Irma Kluijt, Lizet van der Kolk, Petra Nederlof, Rob Plug, 

Roelof Pruntel, Anja van Rens, Efraim Rosenberg, Marielle Ruijs, Esther Scheerman, Ivon Tielen, 

Sophie van der Velden, Senno Verhoef 

Like in previous years, also in this year the number of families who have sought 

genetic advice through our hospital has increased substantially, amounting to 

almost 1000 patients for the year 2011. Most families seen at our family cancer clinic 

present with a possible genetic predisposition for breast and/or ovarian cancer. The 

DNA-diagnostic laboratory has screened more than 3500 families for germ line 

mutations in the BRCA1/2 genes since the start in 1995. These families are obtained 

through our Family Cancer Clinic and the Oncogenetic section of the Department 

of Clinical Genetics of the Academic Medical Center. In November the quality 

assurance and control system of the department of Pathology, including the DNAdiagnostic 

laboratory of the Family Cancer Clinic and Molecular Pathology has been 

evaluated by the CCKL (part of Raad voor Accreditatie). A defi nite report is expected 

in the beginning of 2012. 

This year we had an unusual fi nding. A germline BRCA1 mutation carrier was also 

carrier of a BRCA2 pathogenic mutation but this latter mutation seems not to be 

present in all cells and is likely to be a mosaic mutation. Further research is ongoing. 

We have started Next Generation Sequencing, (massive parallel sequencing), of 

a few large families with no BRCA1/2 mutation. The complete exome has been 

sequenced of several family members and interesting fi ndings are being validated 

and pursued. Using Array CGH for hereditary breast/ovarian cancer we study 

whether the genomic aberrations in breast tumors are similar to the specifi c 

aberrations found in BRCA1 and BRCA2 tumors (in collaboration with Petra 

Nederlof, Division of Experimental Therapy and Diagnostic Oncology). More than 

350 a-CGH analyses have been completed, for high risk families and unclassifi ed 

variant analysis mostly. A BRCA2 classifi er has been validated and implemented in 

the routine diagnostics together with the assessment of methylation of the BRCA1 

promotor. This year we had to change from a BAC aCGH platform to a Nimblegen 

111 



Korse CM, Taal BG, Vincent A, van 

Velthuysen ML, Baas P, Buning-Kager JC, 

Linders TC, Bonfrer JM. Choice of tumour 

markers in patients with neuroendocrine 

tumours is dependent on the histological 

grade. A marker study of Chromogranin A, 

Neuron specifi c enolase, Progastrin-releasing 

peptide and cytokeratin fragments. Eur J 

Cancer 2011 (in press) 

Korse CM, Taal BG, Bonfrer JM, Vincent 

A, van Velthuysen ML, Baas P. An elevated 

progastrin-releasing peptide level in patients 

with well-differentiated neuroendocrine 

tumours indicates a primary tumour in the 

lung and predicts a shorter survival. Ann 

Oncol 2011;22:2625-2630 

Korse CM, Bonfrer JM, Prevoo W, Baas P, 

Taal BG. Increase of angiogenic growth factors 

after hepatic artery embolization in patients 

with neuroendocrine tumours. Tumour Biol 

2011;32:647-652 

Kuil J, Buckle T, Yuan H, Oishi S, Fujii N, 

Josephson L, van Leeuwen FWB. Synthesis 

and in vitro evaluation of a bimodal CXCR4 

antagonistic peptide. Bioconjugate Chem 

2011;18:859-864 

Kuil J, Steunenberg P, Oldenburg J, 

Velders AH, van Leeuwen FWB. Peptidefunctionalized 

luminescent iridium 

complexes for life-time imaging of CXCR4 

expression. Chem BioChem 2011;16:1897- 

1904 

Kuil J, Buckle T, Oldenburg J, Yuan H, 

Josephson L, van Leeuwen FWB. Hybrid 

peptide dendrimers for imaging of CXCR4 

expression. Mol Pharm 2011 (accepted) 

Kuiper R P, Vissers LE, Venkatachalam R, 

Bodmer D, Hoenselaar E, Goossens M, 

Haufe A, Kamping E, Niessen RC, 

Hogervorst FB, Gille JJ, Redeker B, Tops 

CM, van Gijn ME, van den Ouweland AM, 

Rahner N, Steinke V, Kahl P, Holinski-Feder 

E, Morak M, Kloor M, Stemmler S, Betz B, 

Hutter P, Bunyan DJ, Syngal S, Culver JO, 

Graham T, Chan TL, Nagtegaal ID, van 

Krieken JH, Schackert HK, Hoogerbrugge 

N, van Kessel AG, Ligtenberg MJ. 

Recurrence and variability of germline 

EPCAM deletions in Lynch syndrome. Hum 

Mutat 2011;32:407-414 

Lammens CR, Aaronson NK, Hes FJ, 

Links TP, Zonnenberg BA, Lenders JW, 

Majoor-Krakauer D, Van Os TA, Gomez- 

Garcia EB, de Herder W, van der Luijt RB, 

van den Ouweland AM, Van Hest LP, 

Verhoef S, Bleiker EM. Compliance with 

periodic surveillance for Von-Hippel-Lindau 

disease. Genet Med 2011;13:519-527

112 



Lammens CR, Bleiker EM, Verhoef S, 

Ausems MG, Majoor-Krakauer D, Sijmons 

RH, Hes FJ, Gómez-García EB, Van Os TA, 

Spruijt L, van der Luijt RB, van den 

Ouweland AM, Ruijs MW, Gundy C, 

Nagtegaal T, Aaronson NK. Distress in 

partners of individuals diagnosed with or at 

high risk of developing tumors due to rare 

hereditary cancer syndromes. Psychooncology 

2011;20:631-638 

Leveille N, Elkon R, Davalos V, 

Manoharan V, Hollingworth D, Oude 

Vrielink J, le Sage C, Melo CA, Horlings HM, 

Wesseling J, Ule J, Esteller M, Ramos A, 

Agami R. Selective inhibition of microRNA 

accessibility by RBM38 is required for p53 

activity. Nat Commun 2011;2:513 

Lin F, Sherris D, Beijnen JH, Van 

Tellingen O. High-performance liquid 

chromatography analysis of a novel 

small-molecule, anti-cancer drug, Palomid 

529, in human and mouse plasma and in 

mouse tissue homogenates. J Chromatogr B 

Analyt Technol Biomed Life Sci 2011 (in 

press) 

Lips EH, Laddach N, Savola SP, 

Vollebergh MA, Oonk AM, Imholz AL, 

Wessels LF, Wesseling J, Nederlof PM, 

Rodenhuis S. Quantitative copy number 

analysis by Multiplex Ligation-dependent 

Probe Amplifi cation (MLPA) of BRCA1associated 

breast cancer regions identifi es 

BRCAness. Breast Cancer Res 2011;27:R107 

Lips EH, M ulder L, de Ronde JJ, Mandjes 

IA, Vincent A, Vrancken Peeters MT, 

Nederlof PM, Wesseling J, Rodenhuis S. 

Neoadjuvant chemotherapy in ER+ HER2breast 

cancer: response prediction based on 


characteristics. Breast Cancer Res Treat 2011 

(in press) 

Lips EH, Mulder L, Hannemann J, 

Laddach N, Vrancken Peeters MT, van de 

Vijver MJ, Wesseling J, Nederlof PM, 

Rodenhuis S. Indicators of homologous 

recombination defi ciency in breast cancer and 

association with response to neoadjuvant 

chemotherapy. Ann Oncol 2011;22:870-876 

Lodder WL , Teertstra HJ, Tan IB, 

Pameijer FA, Smeele LE, van Velthuysen 

ML, van den Brekel MW. Tumour thickness 

in oral cancer using an intra-oral ultrasound 

probe. Eur Radiol 2011;21:98-106 

Loo CE, Straver ME, Rodenhuis S, Muller 

SH, Wesseling J, Vrancken Peeters MJ, 

Gilhuijs KG. Magnetic resonance imaging 

response monitoring of breast cancer during 

neoadjuvant chemotherapy: relevance of 

breast cancer subtype. J Clin Oncol 

2011;29:660-666 

135k oligo array platform. For this reason previous classifi ers had to be tested and 

validated which was a tremendous amount of work. 

For families with hereditary non polyposis colorectal cancer we have mutation 

scanning tests for mismatch repair genes hMLH1, hMSH2 and hMSH6 and the 

MutY (MYH) gene. Microsatellite instability tests and immunohistochemistry 

for the mismatch repair genes is carried out in collaboration with the pathologist 

Daphne de Jong. Furthermore methylation of the hMLH1 promoter and the presence 

of a specifi c somatic mutation in the BRAF gene (V600E) are being assessed. 

About 50% of the micro satellite instable (MSI-high) tumors with absent staining 

of hMLH1 have a methylated promoter. This result has direct consequences for the 

clinical interpretation of the family history data. Interestingly we have identifi ed an 

individual which has a germline methylation of the MLH1 promotor. This is a rare 

event and has been reported previously. 

The Family Cancer Clinic contributes data to several multi-center national and 

international research projects, e.g. GEO-HEBON (gene environment interactions 

in hereditary breast and ovarian cancer, see Division Psychosocial Research and 

Epidemiology), DNA-profi ling by classic cGH and array cGH of breast cancer 

patients (see Division of Experimental Therapy), the BCAC and CIMBA consortiums 

which focus on the contribution of SNPs to cancer risk (HEBON resource, Matti 

Rookus, dept of Psychosocial Research and Epidemiology), studies into the biological 

signifi cance of so called unclassifi ed variants (DNA changes of which it is uncertain 

whether they be pathogenic mutations or polymorphisms) in collaboration with the 

dept Molecular Biology groups of Jos Jonkers (BRCA1 UVs) and Hein te Riele (MMR 

UVs), in national and international collaborations with other DNAdiagnostic 

and research labs, eg ENIGMA for BRA1/2 UVs (Frans Hogervorst), psychosocial 

studies, in collaboration with the department of Psychosocial Research and 

Epidemiology and clinical and genetic research in families with gastrointestinal 

cancer, including stomach cancer and pancreatic cancer (Annemieke Cats, Division 

of Medical Oncology). 

In collaboration with the Academic Medical Centre and other departments of 

Gastroenenteroly and Clinical Genetics families are included in studies of familial 

stomach cancer and pancreatic cancer, for whom periodic screening programs are 

being developed, tried out and evaluated. 

In 2008 a PhD student started a project on the implementation of genetic 

counseling and where necessary rapid DNA-testing in recently diagnosed breast 

cancer patients: the TIME- study. In this multi-centre study in collaboration with 

the Division of Medical Genetics of the Utrecht Medical Centre, patients from 14 

different hospitals have been enrolled. The analysis of the data from 271 included 

patients from a potential of 351 (ie 77% recruitment) has commenced. 

The Family Cancer Clinic has undergone a quality review visit, with a favorable 

preliminary judgment. A defi nite report is expected in February 2012. 

DEPARTMENT OF RADIOLOGY 

The diagnostic-imaging laboratory at the department of Radiology pursues new 

imaging techniques, image processing, multi-modality registration, pattern 

recognition and molecular imaging to improve the sensitivity and specifi city 

of cancer diagnosis, pre-treatment assessment of tumor extent, image-guided 

therapy and response monitoring. The focus of the department was on prostate 

cancer imaging and follow-up and breast cancer imaging and follow-up using MRI 

and on image guided treatment procedures of liver tumors, lung tumors and renal 

tumors. Finally, there is a close clinical collaboration with the department of nuclear 

medicine and radiotherapy in the development of new imaging tools and therapeutic 

options.


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DIVISION OF MEDICAL ONCOLOGY 

The division of Medical Oncology comprises a growing number of research activities 

with focus on translational research, early drug development and clinical research. 

In this report only research from the largest groups is presented. Much of the 

work is multidisciplinary and involves groups from different divisions or different 

institutions both in the Netherlands and abroad. One of the major common themes 

between the different research groups within this division is personalized medicine. 

We expect that in the near future cancer treatment will be individualized, so that 

patients can be offered the optimal treatment for their specifi c type of cancer. 

CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS 

Jan Schellens, Henk Boot, Annemieke Cats, Bastiaan Nuijen, Hilde Rosing, Serena Marchetti, 

Neeltje Steeghs, Alwin Huitema, Jos Beijnen 

BACKGROUND AND OBJECTIVES 

Over the last year signifi cant investments were made in clinical and translational 

research involving “personalized medicine” by expanding the infrastructure 

and the number of trials for patient selection on the basis of tumor derived 

biomarkers determined prior to entry into the trial. Besides this, the department 

remained involved in research in different phases of anticancer drug development, 

concerning: I) Pharmaceutical formulation, II) Bioanalytical method development 

and implementation in clinical pharmacological studies, III) Early clinical phase I/ 

II studies and IV) supportive care studies in patients with breast cancer, V) Health 

Technology Assessment (HTA) of hormonal therapy in breast cancer. 

Research activities of the division of Clinical Pharmacology of the department 

of Medical Oncology, the department of Pharmacy & Pharmacology and the 

department of Experimental Therapy (group Schellens) are closely integrated. 

In 2011 we continued clinical research fully compliant with ICH-GCP guidelines, 

previously certifi ed by the Dutch Ministry of Health. The department of Pharmacy 

& Pharmacology successfully continued its offi cial governmental GLP (in the fi eld 

of analytical chemistry), GMP (formulation and manufacturing of investigational 

cytotoxic drugs) and GDP (ISO9002 for worldwide distribution of clinical supplies) 

licenses. 

I PHARMACEUTICAL FORMULATION 

Three pharmaceutical formulation projects aiming at the development of oral dosage 

forms of the anti-cancer agents docetaxel, paclitaxel, and capecitabine are ongoing. 

So-called solid dispersion formulations were developed for the poorly water-soluble 

taxanes docetaxel (ModraDoc) and paclitaxel (ModraPac). Both formulations are 

currently used in phase I clinical trials. A third, extended-release tablet formulation 

of capecitabine was developed (ModraCape) and a phase I study was initiated 

this year to evaluate its release characteristics in vivo. Furthermore, several 

projects, including pharmaceutical development and/or clinical manufacturing of 

cyclodextrin and liposomal formulations of various compounds are ongoing. Also, a 

number of clinical studies were facilitated by specifi c re-packaging, manufacturing 

of placebo tablets or capsules, and blinding of verum. In the beginning of 2011, 

the manufacturing facilities of the Department of Pharmacy & Pharmacology 

were audited by the Dutch Health Inspectorate (IGZ) and approved. The existing 

Manufacturer’s license was expanded with cellular products, enabling the start of 

a phase I clinical trial with Tumor Infi ltrating Lymphocytes (TIL). During 2011, 

the fi rst patients were treated with this experimental therapy. Validation studies 

for a second cellular therapy, i.e. T-cell receptor (TCR) gene therapy, were fi nalized. 

Pharmaceutical products of HPV E6 and E7 pDNA vaccines were developed and 

manufactured. A research program in collaboration with Utrecht University (UU) 


Division head John Haanen 

115 

MEDICAL ONCOLOGY 

John Haanen MD PhD Head 

Joke Baars MD PhD Academic staff 

Paul Baas MD PhD Academic staff 

André Bergman MD PhD Academic staff 

Jos Beijnen PhD Academic staff 

Christian Blank MD PhD Academic staff 

Willem Boogerd MD PhD Academic staff 

Henk Boot MD PhD Academic staff 

Wieneke Buikhuisen MD PhD Academic 

staff 

Sjaak Burgers MD PhD Academic staff 

Annemieke Cats MD PhD Academic staff 

Jan Paul de Boer MD PhD Academic staff 

Dieta Brandsma MD PhD Academic staff 

Alwin Huitema PhD Academic staff 

Martijn Kerst MD PhD Academic staff 

Marjolein Klous PhD Academic staff 

Monique van Leerdam MD PhD Academic 

staff 

Sabine Linn MD PhD Academic staff 

Anne Lukas MD PhD Academic staff 

Serena Marchetti MD Academic staff 

Bastiaan Nuijen PhD Academic staff 

Sjoerd Rodenhuis MD PhD Academic staff 

Hilde Rosing PhD Academic staff 

Jan Schellens MD PhD Academic staff 

Gabe Sonke MD Academic staff 

Neeltje Steeghs MD PhD Academic staff 

Jacqueline Stouthard MD PhD Academic 

staff 

Babs Taal MD PhD Academic staff 

Margot Tesselaar MD Academic staff 

Michel van den Heuvel MD PhD Academic 

staff 

Marchien van der Weide MD PhD Academic 

staff 

Roel van Gijn PhD Academic staff 

Marije Appels Temporary staff 

Sandra Bakker Temporary staff 

Ilja Bot Temporary staff 

Rogier Boshuizen Temporary staff 

Foke van Delft Temporary staff 

Michael van Geer Temporary staff 

Annebeth Haringhuizen Temporary staff 

Michiel van der Heijden Temporary staff 

Jan van Meerbeeck MD PhD Temporary 

academic staff 

Nienke Miltenburg Temporary staff 

Josine Quispel Temporary staff 

Robert Rhodius Temporary staff 

Quirine van Rossum-Schornagel Temporary 

staff 

Dirkje Sommeijer Temporary staff

116 


Wim van der Steeg Temporary staff 

Hans van Thienen Temporary staff 

Suzan Vrijaldenhoven Temporary staff 

Annette van Zweeden Temporary staff 

Karin Beelen PhD student 

Ellen Derissen PhD student 

Lot Devriese PhD student 

Maarten Deenen PhD student 

Thomas Dorlo PhD student 

Anne Charlotte Dubbelman PhD student 

Geert Frederix PhD student 

Andrew Goey PhD student 

Iris van der Heijden PhD student 

Helgi Helgason PhD student 

Jeroen Hendrikx PhD student 

Nynke Jager PhD student 

Tine Janssens PhD student 

Anita Kort PhD student 

Heinz Josef Klümpen PhD student 

Stijn Koolen PhD student 

Wiete Kromdijk PhD student 

Nienke Lankheet PhD student 

Suzanne Leijen PhD student 

Jelte Meulenaar PhD student 

Johannes Moes PhD student 

Ruud van de Noll PhD student 

Nalini Radhakishun PhD student 

Emilia Sawicki PhD student 

Philip Schouten PhD student 

Rik Stuurman PhD student 

Bas Teunissen PhD student 

Linda Tulner PhD student 

Jolanda van den Hoven PhD student 

Coen van Hasselt PhD student 

Mariska van Vliet PhD student 

Annemieke van Winden PhD student 

Abadi Gebretensae Technical staff 

Michel Hillebrand Technical staff 

Ciska Koopman-Kroon Technical staff 

Luc Lucas Technical staff 

Lianda Nan-Offeringa Technical staff 

Joke Schol Technical staff 

Bas Thijssen Technical staff 

Matthijs Tibben Technical staff 

Marja Merqui-Roelvink Clinical trial manager 

Sandra Adriaansz Nurse practitioner 

Annelies Boekhout Nurse practitioner 

Karina Bucholtz Nurse practitioner i.o. 

Ria Dubbelman Nurse practitioner 

Figure 1: Mean plasma concentration-time 

profi les of oral paclitaxel formulated as 

capsules ModraPac001, or as the drinking 

solution of paclitaxel, taken together with 

the booster drug ritonavir. Left: linear; 

right: logarithmic concentration scale. 

and University of Twente (UT) aiming at the pharmaceutical development of 

non-viral vectors (e.g. lipoplexes and polyplexes) for improvement of DNA-plasmid 

transfection is ongoing. 

II BIOANALYTICAL METHOD DEVELOPMENT + IMPLEMENTATION IN 

PK STUDIES 

We succeeded to develop a liquid-chromatography tandem mass spectrometry (LC- 

MS/MS) method for the determination of ortataxel in human plasma. Ortataxel is a 

new-generation taxane showing responses in paclitaxel and doxetaxel-resistant nonsmall 

cell lung cancer. The bioanalytical method is able to quantify ortataxel levels 

in plasma down to 50 pg/mL. 

Dried blood spots (DBS) for drug analysis offers advantages for the collection, 

storage and shipping of blood samples. In preclinical studies the number of 

experimental animals can be reduced and in clinical studies (i.e. pediatric studies) it 

offers a low-stress sampling option. A DBS method was developed and validated for 

the determination of sunitinib. 

The importance of the development of highly selective bioanalytical methods 

has been demonstrated in the bioanalysis of tamoxifen. Discrepancies in mean 

metabolite concentrations (endoxifen and 4-hydroxytamoxifen) reported in literature 

were investigated. A published LC-MS/MS method was used to analyse a set of 75 

serum samples from patients treated with tamoxifen. Results revealed a factor 2 to 3 

overestimation of the metabolite levels when compared with outcomes from our own 

highly selective method. 

Assays were validated for the determination of a pro-drug of gemcitabine in human 

plasma and gemcitabine and dFdU in plasma and urine. These assays were used to 

support an oral bioequivalence study of the drug. 

A mass balance study with E7080, a new experimental tyrosine kinase inhibitor, 

was completed this year. The metabolite profi ling revealed products of E7080 

oxidation and demethylation in both urine and faeces. Furthermore, products of 

demethylation and glucuronidation in urine and plasma were found. 

The following sample analysis were performed for clinical trials within and outside 

the Institute concerning paclitaxel, docetaxel, topotecan, capecitabine and its 

metabolites, trabectedin, AS703026 (an inhibitor of MEK 1 and MEK2), gemcitabine 

and dFdU, gemcitabine triphosphate, cyclophosphamide (in combination with 

fl udarabine) and its metabolite 4-hydroxy cyclophosphamide, eribulin, and platinum 

(originating from cisplatin, carboplatin and oxaliplatin). 

III EARLY CLINICAL PHASE I/II STUDIES 

III-1 Novel approaches to improve oral bioavailability 

The ‘boosting’ concept of oral docetaxel plus ritonavir, an effi cacious inhibitor of 

gut wall and hepatic CYP3A4, has been further developed this year. The major step 

forward is that the oral capsule formulation of docetaxel, denoted ModraDoc001, 

turns out to have benefi cial pharmaceutical properties in patients. The maximum 

tolerated dose (MTD) in the ongoing phase I study was determined to be 80 mg 

ModraDoc001 plus 200 mg ritonavir. Expansion at the lower dose of 60 mg 

ModraDoc001 plus 200 mg ritonavir is currently performed. Main toxicities 

are diarrhea and general malaise. The novel oral paclitaxel capsule formulation 

(ModraPac001) showed excellent systemic exposure to paclitaxel and has therefore 

been taken further into the clinic (fi gure 1). The phase I study with daily low-dose 

or metronomic ModraPac001 plus booster drug ritonavir is being executed at the 

starting dose-level of BID 2.5 mg ModraPac001 plus 100 mg ritonavir.

III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs 

Currently, we perform thirty-six phase I/II, pharmacological and proof of concept 

studies, which number has signifi cantly increased compared with last year. We 

recruited more than 250 new patients for these studies this year. Twenty-two of these 

studies are two- or multicenter studies. Representative examples are described. 

a. Studies with the poly-ADP-ribose polymerase (PARP1) inhibitor olaparib (AZD2281) 

The ongoing three-center phase I trial to test the safety, optimal dose and schedule 

of AZD2281 when given daily BID in combination with three-weekly carboplatin and 

paclitaxel, or in combination with weekly paclitaxel has entered its 27 th dose-level. 

We demonstrated promising anticancer activity especially in patients with tumors 

harboring BRCA1/2 mutations. Prolonged myelosuppression is the main toxicity of 

the combination. 

b. Phase I studies with oral fi broblast growth factor receptor (FGFR) inhibitors 

The trials with BGJ398 and AZD4547 respectively are ongoing. The study with 

AZD4547 has reached its MTD, which is hyperphosphatemia, skin and nail 

toxicity and general malaise. The hyperphosphatemia seems to indicate that the 

FGFR mediates phosphate excretion in the kidneys. Trials continue with patient 

prescreening for FGFR1,2 mutation or FGFR3 amplifi cation. 

c. Studies with the monoclonal antibody (MoAb) TWEAK 

We started this fi rst in man trial with the MoAb TWEAK directed against the ligand 

of the Fn14 transmembrane protein that activates the MAPK pathway. Prescreening 

on the basis of Fn14 expression determined by IHC precedes patient inclusion. 

Safety up to 1600 mg weekly or every three weeks is excellent. 

d. Studies with the monoclonal antibody (MoAb) against HER3 

We started this three-center phase I and fi rst in man study whereby HER3 

expression is determined on fresh tumor tissue in patients with epithelial tumors. 

The starting dose-level is 100 mg IV every two weeks. Patients may enter the study if 

at least 10% of tumor cells express HER3. 

e. Other phase I studies 

The fi rst-in-man phase I study with the novel androgen receptor down regulating 

molecule AZD3514, indicated for hormone refractory prostate cancer, shows PSA 

declines at dose-levels of 500 mg daily or higher. Currently BID 2000 mg is tested. 

The fi rst-in-man study with the combination of erlotinib and the PI3K inhibitor 

GDC0941 showed remarkable activity in advanced mesothelioma. Intermittent 

dosing of GDC0941, together with continuous daily dosing of erlotinib, was 

implemented as continuous dosing of both compounds was found unsafe. Main 

toxicity is skin toxicity. The fi rst in man study with the MEK inhibitor BO21189 

developed into a phase Ib study in melanoma, NSCLC and colorectal cancer. 

Encouraging activity was seen in BRAF V600E mutated melanoma. Main toxicity 

is EGFR-inhibitor-like skin toxicity. This is also a daily continuous schedule. 

We continued the phase I study with the Wee1 inhibitor MK1775 in combination 

with or gemcitabine, cisplatin or carboplatin. Main toxicities are general malaise, 

nausea and vomiting, which can be controlled well with intensive antiemetic 

medication. This concept is currently been tested in refractory ovarian cancer with 

p53 pathway mutation. Encouraging partial remission was observed in two out of the 

fi rst fi ve recruited patients. 

III-3 Pharmacokinetic and pharmacodynamic (PK/PD) modelling and simulation 

PK/PD modelling and simulation was used to quantify the relationship between 

decline of the left ventricular ejection fraction (LVEF) in response to trastuzumab 

treatment. Patients (n=240) with prior anthracyclin treatment showed a cumulative 

anthracycline dose-dependent increase in sensitivity to LVEF declines. The recovery 

half-life of the LVEF after cessation of therapy was approximately 50 days. The model 

is currently used to establish optimal management and monitoring strategies for 

patients treated with adjuvant trastuzumab. 

The relationship between pharmacokinetics, hematological toxicity and genotype 

117 


Joke Foekema Nurse practitioner 

Emmy Harms Nurse practitioner 

Marjo Holtkamp Nurse practitioner 

Marianne Keessen Nurse practitioner 

Annemieke Koenen Nurse practitioner i.o. 

Maria Kuiper Nurse practitioner 

Elsbeth van der Laan Nurse practitioner i.o. 

Lisette Saveur Nurse practitioner 

Henk Mallo Nurse practitioner 

Annemarie Nol Nurse practitioner 

Suzanne Onderwater Nurse practitioner 

Margaret Schot Nurse practitioner 

Wilma Uyterlinde Nurse practitioner 

Jana van der Sar Nurse practitioner 

Dick Visser Nurse practitioner i.o. 

Marion Zimmerman Nurse practitioner i.o.

118 


Publications 

Ackerstaff AH, Rasch CR, Balm AJ et al. 

Five-year quality of life results of the 

randomized clinical phase III (RADPLAT) 

trial, comparing concomitant intra-arterial 

versus intravenous chemoradiotherapy in 

locally advanced head and neck cancer. Head 

Neck 2011 

Appels NM, Bolijn MJ, van Eijndhoven 

MA, Stephens TC, Beijnen JH, Schellens JH. 

Characterization of the in vitro activity of 

AZD3409, a novel prenyl transferase inhibitor. 

Cancer Chemother Pharmacol. 2011 67:137-45 

Baas P, Rhodius R. Thymoma update 

2011. Eur J Cancer. 2011;47 Suppl 3:S315-6 

Baas P. Diagnosis: ASTER--another fl ower 

in the diagnostic fi eld of lung cancer? Nat Rev 

Clin Oncol. 2011;8:198-9 

Baas P, Belderbos JS, van den Heuvel M. 

Chemoradiation therapy in nonsmall cell lung 

cancer. Curr Opin Oncol. 2011;23:140-9 

Bachner M, Loriot Y, Gross-Goupil M, 

Zucali PA, Horwich A, Germa-Lluch JR, 

Kollmannsberger C, Stoiber F, Fléchon A, 

Oechsle K, Gillessen S, Oldenburg J, 

Cohn-Cedermark G, Daugaard G, Morelli F, 

Sella A, Harland S, Kerst M, Gampe J, 

Dittrich C, Fizazi K, De Santis M. 2-18fl uorodeoxy-D-glucose 

positron emission 

tomography (FDG-PET) for 

postchemotherapy seminoma residual lesions: 

a retrospective validation of the SEMPET 

trial. Ann Oncol. 2011 

Bergamo A, Gaiddon C, Schellens JH, 

Beijnen JH, Sava G. Approaching tumour 

therapy beyond platinum drugs Status of the 

art and perspectives of ruthenium drug 

candidates. J Inorg Biochem. 2011;106:90-99 

Bergman AM, Kerst JM. Ontwikkelingen 

in de systemische behandeling van castratieresistent 

prostaatkanker. NTVO 2011;8:160-8 

Berry DA, Ueno NT, Johnson MM, Lei X, 

Caputo J, Rodenhuis S, Peters WP, Leonard 

RC, Barlow WE, Tallman MS, Bergh J, Nitz 

UA, Gianni AM, Basser RL, Zander AR, 

Coombes RC, Roché H, Tokuda Y, De Vries 

EG, Hortobaghyi GN, Crown JP, Pedrazzoli 

P, Bregni M, Demirer T. High-dose 

chemotherapy with autologous stem-cell 

support as adjuvant therapy in breast cancer: 

overview of 15 randomized trials. J Clin 

Oncol. 2011;29: 3214-23 

Bex A, Blank C, Meinhardt W, van 

Tinteren H, Horenblas S, Haanen J. A phase 

II study of presurgical sunitinib in patients 

with metastatic clear-cell renal carcinoma and 

the primary tumor in situ. Urology. 

2011;78:832-7 

Bex A, Larkin J, Blank C. Non-clear cell 

renal cell carcinoma: how new biological 

insight may lead to new therapeutic 

modalities. Curr Oncol Rep. 2011;13:240-8 

of different genes in drug-associated pathways was studied in non-small cell lung 

cancer patients treated with gemcitabine and cisplatin. The cytidine deaminase *2 

mutation was related to a decreased clearance and thus an increased drug exposure 

of gemcitabine. This study was extended to investigate the relationship between 

the genotype of a range of genes involved in the pharmacodynamic effects of 

gemcitabine and cisplatin on treatment outcome (tumor response and toxicity). 

III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment 

Pharmacogenetic screening of patients treated with 5-FU/Capecitabine 

DYPD*2 genotyping in routine clinical practice was continued. To date a population 

of more than 2000 patients has been genotyped prior to start of capecitabine 

therapy, amongst which twenty-one heterozygotes have been identifi ed. Doseadaptation 

could be performed in seventeen of these patients. Safety was excellent. 

No major toxicities were reported and no toxic deaths occurred. The cost analysis of 

the genotyping approach testing demonstrated that the adaptive dosing in DPYD*2A 

patients is cost-effective. 

IV SUPPORTIVE CARE STUDIES IN PATIENTS WITH BREAST CANCER 

We continued the multicenter cardiac protection trial with the angiotensine II (ATI) 

receptor antagonist candesartan in patients with HER2 positive breast cancer who 

received adjuvant treatment with trastuzumab. This is a double blind randomized 

trial versus placebo. Recruitment was completed this year. In total 210 patients have 

been registered in 19 centers in The Netherlands. 

V HEALTH TECHNOLOGY ASSESSMENT 

In a systematic review, we identifi ed 20 economic evaluations comprising of 5 

different endocrine therapeutic strategies in early breast cancer. Despite similar 

comparators the outcomes of the included studies varied widely. Detailed 

recommendations for standardization in modelling treatment strategies in early 

breast cancer regarding time horizon, carry over effect and incidence of recurrence, 

were established. 

In addition to the wide variety in modelling methods, also a wide variety in cycle 

lengths was observed. In order to eliminate the dependence on fi xed cycle lengths 

or time-horizons, we developed a Markov model in the programme R which 

makes use of an ordinary differential equation solver algorithm. We demonstrated 

that our model was free of bias whereas the use of fi xed cycle lengths or time 

horizons resulted in signifi cant bias. A cycle length of one year resulted in an 

underestimation of 0.016 absolute life years (LYs) and c 251. Time horizons shorter 

than 30 years demonstrated substantial differences in LYs gained (0.098 for 10 years 

compared 0.280 LY for 30 years). 

VI COLLABORATION WITH THE DUTCH MEDICINES EVALUATION BOARD 

We continued the support of the CBG/EMA by assessment of several Rapporteur 

and Co-rapporteur reports and scientifi c advises. 

We also continued the support of the EMA as chairperson of the Scientifi c Advisory 

Group-Oncology of the EMA. 

We continued the scientifi c advices to the minister of Health and the Dutch 

Health Insurance Authority (NZA) as chairperson of the “Committee for 

Pharmaceutical Aid”. 

CLI NICAL IMMUNOTHERAPY AND TARGETED THERAPY 

John Haanen, Christian Blank, Sandra Adriaansz, Henk Mallo, Elsbeth van der Laan, Loes Pronk, 

Marieke Groot-Obbink, W Boogerd, Dieta Brandsma, André Bergman 


The clinical immuno- and targeted therapy group is primarily involved in 

the treatment of melanoma and renal cell carcinoma patients. Translational 

immunotherapy research focuses on adoptive cellular therapies, such as T-cell 

receptor gene therapy and treatment with tumor-infi ltrating lymphocytes (TIL) for

melanoma and DNA and peptide vaccination studies for HPV-related squamous cell 

cancers. For renal cell cancer our group is leading in the development of investigatorinitiated 

phase II/III trials to improve the treatment with small molecule receptor 

tyrosine kinase inhibitors (RTKI), mTOR kinase inhibitors and combinations of 

cytokines and anti-angiogenesis drugs. 

MELANOMA 

Translational research with DNA vaccination in melanoma patients: 

In January 2009 we started a fi rst in man study with DNA tattoo vaccination. 

In this dose-escalating phase I clinical study advanced-stage HLA-A*0201positive 

melanoma patients are treated with an in-house manufactured DNA 

vaccine, pDERMATT, encoding Tetanus Toxin Fragment C (TTFC) fused to an 

immunodominant epitope of the melanocyte differentiation antigen MART-1. The 

DNA vaccine (5 mg/ml) is being tattoed into the epidermis of patients on days 0, 3 

and 6 and a booster vaccination is administered at days 28, 31 and 34. The starting 

dose is 1 mg of DNA applied to a total skin surface of 2 x 2 cm. Dose escalation is 

performed by increasing the skin area that is being tattooed (8 cm2 , 16 cm2 and 

32 cm2 ). In 2010 the 2nd dose cohort (8 cm2 ) with three patients was fi nished and 

two patients have been treated in dose cohort 3 (16 cm2 ) in 2011. The study is now 

also open for enrolment at the Leiden University Medical Center. So far, only local 

vaccination site toxicity (grade 1 and 2) was observed. In biopsies taken one week 

after the last tattoo (both priming and booster) from the vaccination site showed 

the presence of CD8+ MART-1-specifi c T cells, indicating that a cellular immune 

response had been induced. 

Translational research with tumor-infiltrating lymphocytes in melanoma patients 

We are preparing for a clinical phase II study using tumor-infi ltrating lymphocytes 

for patients with metastatic melanoma. This is based on results from single arm, 

single institution trials performed at the Surgery Branch of the NIH, Bethesda, 

USA, and Sheba Medical Institute, Tel Aviv, Israel, which showed a 50% or more 

objective response rate in heavily pre-treated stage IV melanoma patients. 

As of September 2008 the Division of Immunology, Medical Oncology and 

Pharmacy has worked closely together to prepare a TIL trial at the NKI-AVL (see 

also Clinical Pharmacology Section). After receiving approval from the Central 

Committee on Research involving Human Subjects (CCMO) for a pilot study (n=5), 

we enrolled 4 patients in this study in 2011. Toxicity was as expected and objective 

clinical responses were observed (fi gure 2). 

Anti-CTLA4 treatment 

In 2006 we participated (European top includer) in the randomized double-blind 

placebo-controlled phase III trial comparing the combination of Ipilimumab, a 

fully human anti-CTLA4 monoclonal antibody, and gp100 peptide vaccine, to 

Ipilimumab or gp100 vaccine alone. In June 2010, the results were presented 

at ASCO, showing a statistical signifi cant and meaningful overall survival 

improvement of both Ipilimumab arms compared to gp100 vaccine arm. From 

June 2010 – August 2011, Ipilimumab was available at our institute in a Named 

Patient Program for treatment of patients with metastatic melanoma, who have 

failed fi rst line treatment. Over 100 patients have been enrolled in this program and 

blood samples are being analyzed for changes in cellular immune responses upon 

ipilimumab treatment (see section IV). 

BRAF V600E mutated melanoma 

In 2011 we participated in the vemurafenib Expanded Access Program (EAP) for 

patients with BRAF mutated stage IV melanoma. This was based on the positive 

outcome of a multicenter randomized controlled phase III trial, BRIM3, comparing 

standard dacarbazine with an oral BRAF V600E inhibitor in metastatic melanoma 

patients with tumors harbouring this BRAF mutation. We screened more than 100 

patients in the EAP and more than 50 patients were enrolled. Tumor and blood 

samples are collected for translational research. 

In addition, we participated in phase II trial with MEK1/2 inhibitor MEK162 and a 

phase III trial with BRAF V600E/K inhibitor Dabrafenib. 


119 


Bex A, Haanen J. Tilting the AXIS towards 

therapeutic limits in renal cancer. Lancet. 2011 

Van der Bij S, Schaake E, Koffi jberg H, 

Burgers JA, de Mol BA, Moons KG. Markers 

for the non-invasive diagnosis of 

mesothelioma: a systematic review. Br J 





Cancer Immunol Immunother. 2011;60:1359-71 

Boekhout AH, Vincent AD, Dalesio OB, 

van den Bosch J, Foekema-Töns JH, 

Adriaansz S, Sprangers S, Nuijen B, Beijnen 

JH, Schellens JH. Management of Hot 

Flashes in Patients Who Have Breast Cancer 

With Venlafaxine and Clonidine: A Randomized, 

Double-Blind, Placebo-Controlled Trial. 

J Clin Oncol. 2011;29:3862-68 

Boekhout AH, Beijnen JH, Schellens JH. 

Trastuzumab. Oncologist. 2011;16:800-10 

De Boer JP, Raderer M, van Tinteren H, 

Aleman BM, Boot H, de Jong D. Treatment of 

extranodal marginal zone lymphoma of 

mucosa-associated lymphoid tissue with 

fl udarabine: effect on tumor 

microenvironment. Leuk Lymphoma 2011 

Van der Bol JM, Visser TJ, Loos WJ, de 

Jong FA, Wiemer EA, van Aken MO, Planting 

AS, Schellens JH, Verweij J, Mathijssen RH. 

Effects of methimazole on the elimination of 

irinotecan. Cancer Chemother Pharmacol. 

2011;67:231-36 

Boss DS, Glen H, Beijnen JH, de Jong D, 

Wanders J, Evans TRJ, Schellens JHM. 

Serum β-HCG and CA-125 as tumor markers 

in a patient with osteosarcoma: case report. 

Tumori. 2011;109-14 

Boss DS, Witteveen PO, van der Sar J, 

Lolkema MP, Voest EE, Stockman PK, 

Ataman O, Wilson D, Das S, Schellens JH. 

Clinical evaluation of AZD1152, an i.v. 

inhibitor of Aurora B kinase, in patients with 

solid malignant tumors. Ann Oncol. 

2011;22:431-37 

Figure 2: Partial response six weeks 

after TIL therapy in TIL treated 

patient NKI3

120 



Bromberg JE, Doorduijn JK, Baars JW, 

van Imhoff GW, Enting R, van den Bent MJ. 

Acute painful lumbosacral paresthesia after 

intrathecal rituximab. J Neurol 2011 

Bueno-de-Mesquita JM, Sonke GS, van 

de Vijver MJ, Linn SC. Additional value and 

potential use of the 70-gene prognosis 

signature in node-negative breast cancer in 

daily clinical practice. Ann Oncol. 

2011;22:2021-30 

Caron WP, Clewell H, Dedrick R, 

Ramanathan RK, Davis WL, Yu N, Tonda M, 

Schellens JH, Beijnen JH, Zamboni WC. 

Allometric scaling of pegylated liposomal 

anticancer drugs. J Pharmacokinet 

Pharmacodyn. 2011;38:653-69 

Chapman PB, Hauschild A, Robert C, 

Haanen JB, Ascierto P, Larkin J, Dummer R, 

Garbe C, Testori A, Maio M, Hogg D, 

Lorigan P, Lebbe C, Jouary T, Schadendorf 

D, Ribas A, O’Day SJ, Sosman JA, Kirkwood 

JM, Eggermont AM, Dreno B, Nolop K, Li J, 

Nelson B, Hou J, Lee RJ, Flaherty KT, 

McArthur GA; BRIM-3 Study Group. 

Improved survival with vemurafenib in 

melanoma with BRAF V600E mutation. N 

Engl J Med. 2011;364:2507-16 

Courrech Staal EFW, Bloemendal K, 

Bloemer MC, Aleman BMP, Cats A, van 

Sandick JW. Oesophageal cancer treatment in 

a tertiary referral hospital evaluated by 

indicators for quality of care. Eur J Surg 

Oncol, 2011 



Guchelaar HJ, Smits PH, Beijnen JH, Punt 

CJ, Schellens JH, Cats A. Relationship 

between single nucleotide polymorphisms and 

haplotypes in DPYD and toxicity and effi cacy 

of capecitabine in advanced colorectal cancer. 

Clin Cancer Res, 2011;17:3455-68 

Deenen MJ, Schellens JH, Cats A. SNPs 

and haplotypes in DPYD and outcome of 

capecitabine-response. Clin Cancer Res. 

2011;17:5835-36 


Schellens JH. Part 1: background, 

methodology, and clinical adoption of 

pharmacogenetics. Oncologist. 2011;16:811-9 



variability in drug transport and phase I 

anticancer drug metabolism. Oncologist. 

2011;16:820-34 


Schellens JH. Part 3: Pharmacogenetic 

variability in phase II anticancer drug 

metabolism. Oncologist. 2011;16:992-1005 

RENAL CELL CARCINOMA 

In the renal cell carcinoma program we closely collaborate with Dr Axel Bex from 

the urology-oncology group. 

In 2005 we started participation in a treatment-use program of the small molecule 

sunitinib, a multiple receptor tyrosine kinase inhibitor with high affi nity for 

VEGF-R, PDGF-R, c-KIT and FLT3. Since VEGF and PDGF play prominent roles in 

the pathogenesis of sporadic clear cell renal cell cancer, inhibition of kinase activity 

of their receptors appeared to be a rational therapy in this patient population. In 

2011, still 2 out of 53 patients that were treated in this study were on sunitinib, 

indicating that in a minority of patients sunitinib can induce long-term disease 

stabilization. Serum samples from amongst others these 53 patients were used in a 

collaborative study to perform a genome wide association study, analyzing single 

nucleotide polymorphisms that are correlated with toxicity of sunitinib treatment. 

An investigator-initiated study to defi ne the optimal time point for tumor 

nephrectomy in primary metastatic renal cell carcinoma patients is ongoing. A total 

of 20 patients have been treated in this small proof of principle trial and 

translational research analysing the effect of sunitinib on the expansion of TIL in 

vitro and changes in the angiogenesis pathways (in collaboration with Prof. A. 

Griffi oen, VUMC) is ongoing. 

The idea of defi ning the optimal timing for nephrectomy in primary metastatic RCC 

was adopted by the EORTC GU group and has been approved as a randomized 

controlled multicenter phase III trial (NCT01099423) that has started enrolment in 

2010. Patients are being randomized to receive either 3 cycles of sunitinib treatment 

prior to tumor nephrectomy or immediate nephrectomy. Post surgery both groups 

will (re)start sunitinib treatment until disease progression. Study endpoint: 

Progression-free and overall survival. 

BREAST CANCER THERAPY AND RESPONSE PREDICTION 

Sabine Linn, Sjoerd Rodenhuis, Gabe Sonke, Karin Beelen, Jos Beijnen, Jorma De Ronde, Marjo Holtkamp, 

Alwin Huitema, Rutger Koornstra, Esther Lips, Ingrid Mandjes, Lennart Mulder, Jettie Muris, 

Petra Nederlof, Margaret Schot, Philip Schouten, Marieke Vollebergh, Jelle Wesseling, Lodewyk Wessels 


The objective of this program is to develop and improve potentially curative therapy 

for patients with locoregional or oligometastatic breast cancer. For all studies, close 

collaborations are maintained with many other clinical departments and research 

divisions. In 2011, about 150 patients were entered in sixteen medical studies in 

breast cancer, either focusing on the treatment of early breast cancer or on advanced 

disease. 

PREOPERATIVE CHEMOTHERAPY 

The preoperative chemotherapy program continues to accrue over 100 patients per 

year and now includes data and tissues of more than 500 patients. The program is 

the core of a multidisciplinary research effort to optimize response prediction and to 

improve response monitoring. It consists of separate studies for ER+/HER2- breast 

cancer (single institution), Triple Negative tumors (neo-TN randomized multicenter 

trial) and HER2+ tumors (TRAIN multicenter phase II study). The latter trial closed 

in 2011 after accruing 111 patients. 

The effort to identify mRNA gene expression patterns associated with chemotherapy 

resistance has been continued, and lists of candidate genes have been established 

separately for each breast cancer subtype. These can now be tested prospectively, in 

order to enrich the sets for clinically predictive genes. For triple-negative tumors, an 

aCGH signature predictive for a response to bifunctional alkylating agents has been 

defi ned previously. A novel MLPA assay was shown to be of similar effi cacy and is 

now employed to select patients for this treatment modality. As the assay is quick 

and cheap, this has led to a considerable simplifi cation of the trial logistics. The 

study has recruited 40 patients in the previous year and additional centers are being 

added to accelerate inclusion.

ADJUVANT SYSTEMIC THERAPY, PROGNOSIS AND PREDICTION 

The RASTER study (ISRCTN71917916) was a prospective 

community-based observational study to investigate 

the introduction of the prognostic 70-gene signature 

(Mammaprint®) in daily clinical practice. Recently, the 

5-year follow-up data confi rmed the additional prognostic 

value of the 70-gene signature to clinico-pathologic factors 

used in AdjuvantOnline risk estimations (fi gure 3). If in a 

comparable cohort diagnosed today the 70-gene signature 

would be added to standard guidelines used to select 

patients for adjuvant systemic therapy, a reduction of ~ 30% 

in the use of adjuvant chemotherapy would be seen. 

The primary objective of the Matador study is to identify 

predictive gene expression profi les for a disease-free 

survival benefi t of either a docetaxel-containing regimen 

(docetaxel-doxorubicin-cyclophosphamide (TAC)) or an 

accelerated doxorubicin-cyclophosphamide (ACdd) regimen 

(ISRCTN61893718). As such, frozen tissue of the primary 

tumor is mandatory to be eligible. The study, open since 

2004, is running in over 30 centers in the Netherlands 

and ~ 560 patients of the planned 600 patients have been 

included. 

The preoperative window trial to study responsiveness of 

hormone-receptor positive breast cancers to tamoxifen, 

anastrozole or fulvestrant in postmenopausal patients has 

been extended to other centers (NCT00738777). Blood 

and tumor tissue is collected pre and post treatment 

for translational research, including gene expression profi ling. Changes in 

Ki67 expression are used as a read-out for hormonal therapy responsiveness. In 

collaboration with Leiden University Medical Center and the TEAM study group, the 

TEAM II trial has been initiated to investigate the optimal duration of neoadjuvant 

exemestane therapy (currently ~ 66 patients included) and to study the benefi t of 

three years adjuvant oral ibandronate in addition to standard adjuvant systemic 

therapy in postmenopausal patients with hormone-receptor positive early breast 

cancer (currently ~ 640 patients included) (ISRCTN17633610). 

THORACIC ONCOLOGY 

Paul Baas, Sjaak Burgers, Wieneke Buikhuisen, Michel van den Heuvel, Josine Quispel, Jose 

Belderbos, Houke Klomp, Petra Nederlof, Michel Wouters, Jan Schellens 


The department Clinical Research focus on combination studies of chemotherapy, 

radio and radiotherapy, malignant mesothelioma and immunomodulating studies. 

NON-SMALL CELL LUNG CANCER (NSCLC) 

Of the two immunological studies in non-small lung cancer, one maintenance study 

with Selectokine has been fi nalized and is awaiting analysis. The phase II randomised 

study of Stimuvax after chemoradiotherapy or stage IV non-small cell lung cancer 

is ongoing. As fi rst line therapy, the group has joined the international study of 

carboplatin, paclitaxel, bevacizumab plus or minus nitroglycerine patches. As second 

line therapy much attention has been devoted to maintenance studies and testing of 

targeted agents for specifi c subgroups of patients. The subgroups involve patients with 

K-RAS mutated tumors or squamous cell carcinoma. Together with the department of 

Radiation Oncology, concurrent chemoradiotherapy treatments are optimized. Daily 

cisplatin is compared to weekly cisplatin or combined with cetuximab. A database is 

constructed to evaluate the response and toxicity of combined treatment modality. 

In conjunction with the Breast cancer and Head and Neck group a study of olaparib 

combined with radiotherapy in a phase I study has started. Other initiatives in 

non-small cell lung cancer are in conjunction with the European Thoracic Oncology 


121 


Figure Fi 3: Kaplan-Meier K l Mi plots ltf 

for DDFS 

(A) in patient groups defi ned by combined 

MP and AOL risk estimations and 

adjuvant systemic therapy received (B). 

Deenen MJ, Cats A, Beijnen JH, Schellens 

JH. Part 4: pharmacogenetic variability in 

anticancer pharmacodynamic drug effects. 

Oncologist. 2011;16:1006-20 

Deenen MJ, Klümpen HJ, Richel DJ, 

Sparidans RW, Weterman MJ, Beijnen JH, 

Schellens JH, Wilmink JW. Phase I and 

pharmacokinetic study of capecitabine and the 

oral mTOR inhibitor everolimus in patients 

with advanced solid malignancies. Invest New 

Drugs. 2011 (in press) 

Devriese LA, Bosma AJ, van de Heuvel 




by multi-marker QPCR analysis. Lung 

Cancer. 2011 

Devriese LA, Voest EE, Beijnen JH, 

Schellens JH. Circulating tumor cells as 

pharmacodynamic biomarker in early clinical 

oncological trials. Cancer Treat Rev. 

2011;37:579-89 

Dikken JL, van Sandick JW, Swellengrebel 

HAM, Lind PA, Putter H Jansen EPM, Boot 

H, van Grieken NCT, van de Velde CJH, 

Verheij M, Cats A. Neo-adjuvant chemotherapy 

followed by surgery and chemotherapy 

or by surgery and chemoradiotherapy for 

patients with resectable gastric cancer 

(CRITICS). BMC Cancer, 2011:11;329

122 



Dikken JL, van de Velde CJH, Coit DG, 

Shah MA, Verheij M, Cats A. Treatment of 

resectable gastric cancer. Ther Adv 

Gastroenterol, 2011 (in press) 

Dikken JL, Verheij M, Cats A, Jansen 

EPM, Hartgrink HH, van de Velde CJH. 

Extended lymph node dissection for gastric 

cancer from a European perspective. Gastric 


Dikken JL, van de Velde CJH, Verheij M, 

Cats A. Resectabel maagcarcinoom. 

Deel 1. Introductie en chirurgische behandeling. 

Ned Tijdsch Oncol, 2011 (in press) 

Donker M, Hage JJ, Woerdeman LA, 

Rutgers EJ, Sonke GS, Vrancken Peeters MJ. 

Surgical complications of skin sparing mastectomy 

and immediate prosthetic reconstruction 

after neoadjuvant chemotherapy for invasive 

breast cancer. Eur J Surg Oncol. 2011 

Dubbelman AC, Rosing H, Thijssen B, 

Lucas L, Copalu W, Wanders J, Schellens JH, 

Beijnen JH. Validation of high-performance 

liquid chromatography-tandem mass 

spectrometry assays for the quantifi cation of 

eribulin (E7389) in various biological 

matrices. J Chromatogr B Analyt Technol 


Dubbelman AC, Rosing H, Jansen RS, 

Mergui-Roelvink M, Huitema AD, Koetz B, 

Lymboura M, Reyderman L, Lopez-Anaya A, 

Schellens JH, Beijnen JH. Mass Balance 

Study of 14C-eribulin in Patients with 

Advanced Solid Tumours. Drug Metab 

Dispos. 2011 (in press) 

Dubbelman AC, Rosing H, Schellens JH, 

Beijnen JH. Bioanalytical aspects of clinical 

mass balance studies in oncology. Bioanalysis. 

2011;3:2637-55 

Early Breast Cancer Trialists’ 

Colleborative Group (EBCTCG), Davies C, 

Godwin J, Gray R, Clarke M, Cutter D, Darby 

S, McGale P, Pan HC, Taylor C, Wang YC, 

Dowsett M, Ingle J, Peto R. Relevance of 

breast cancer hormone receptors and other 

factors to the effi cacy of adjuvant tamoxifen; 

patient-level meta-analysis of randomized 

trials. Lancet. 2011;378:771-84 


M, Retel VP, Mook S, Glas AM, Moore DH, 

Linn S, van Leeuwen FE, Van ‘t Veer LJ. 



cancers. Breast Cancer Res Treat. 

2011;130:725-734 

Platform for maintenance therapy or with the so-called Lungscape study, in which 

retrospective analysis of resected tumor material is analysed for the presence of 

activating mutations. Also studies with denosumab or the combination of erlotinib 

with bevacizumab in patients with advanced NSCLC are initiated. 

SMALL CELL LUNG CANCER 

For patient with limited stage disease we continue to participate in the phase III 

European Convert study which investigates the effects of concurrent twice daily 

radiotherapy and chemotherapy versus sequential chemotherapy and radiotherapy. 

For extensive stage disease the study with sunitinib as a window of opportunity has 

been fi nalised and turned out to be negative. Currently no other studies are ongoing. 

PLEURAL DISEASES 

The group continues the development of the pleurabank study and the development 

of innovative diagnostic and treatment algorithms. Over 500 patients have given 

consent to biomarker research applying both frozen serum and pleural fl uid 

samples. A national study is initiated comparing indwelling catheters versus 

standard thorax drainage with pleurodesis in patients with malignant effusions. 

MALIGNANT PLEURAL MESOTHELIOMA (MPM) 

The results of the randomised phase III switch maintenance study of Thalidomide 

after standard chemotherapy has been presented at ASCO with a negative result. No 

subgroups where identifi ed for which predictive or prognostic factors in serial serum 

samples could be found so far. As second and third line therapy, a randomized phase 

III study of Vorinostat versus placebo has been evaluated with a negative outcome. 

No subgroups could be identifi ed showing any favourable profi le. The randomized 

phase IB-II study of chemotherapy plus or minus axitinib with translational research 

is fully ongoing. The phase I part has successfully been fi nalized and in 2012 the 

study will be completed. 

In collaboration with the laboratories of Jacques Neefjes and Anton Berns, new 

drugs for treating malignant mesothelioma will be tested in vitro and in animal 

models. 

GASTROENTEROLOGY 

Henk Boot, Annemieke Cats, Monique van Leerdam, Babs Taal, Margot Tesselaar, 

Maarten Deenen, Luc Dewit, Edwin Jansen, Irma Kluijt, Tiny Korse, Corrie Marijnen, 

Didier Meulendijks, Johanna van Sandick, Lisette Saveur, Jan Schellens, Baukelien van Triest, 

Anouk Trip, Steven Vanhoutvin, Marcel Verheij 


The division of Gastroenterology and Hepatology is involved in different phases of 

clinical research, with emphasis on innovative multimodality treatments for gastric, 

anal and rectal cancer, NET and hereditary tumors. 

GASTRIC CANCER 

The international, randomized, controlled, CRITICS-study continues to accrue 

patients with primary resectable gastric cancer and preoperative chemotherapy and 

investigates the role of postoperative chemotherapy versus chemoradiotherapy. The 

study has now included more than half of the 788 required patients, of which most 

have been entered in the NKI-AVL. A quality control program has shown to improve 

lymph node yield in participating institutes and will be further prolonged. 

In patients with primary irresectable gastric cancer (M0) the NARCIS-study 

investigates a possible role for neoadjuvant chemoradiotherapy with carboplatin and 

paclitaxel. This study has recently completed accrual and we will analyze the study 

for feasibility, (pathological) response and R0 resection rate in collaboration with 2 

other Dutch institutes to determine whether this treatment may add to the currently 

available treatment armentarium.

For patients with metastatic gastric cancer we fi nalized a phase I/II study of 

combination chemotherapy with docetaxel, oxaliplatin and capecitabine and 

subsequently initiated a multicenter study with this regimen in combination with 

bevacizumab and trastuzumab for Her2-positive tumors. The fi rst 3 patients have 

started treatment in our institute. 

In a nationwide study, we investigated reasons to opt for (or against) DNA-testing, 

experience with surveillance and prophylactic gastrectomy, decisional confl ict, 

and the effect of gastrectomy on cancer worries in 31 patients with hereditary 

diffuse-type gastric cancer with a CDH1-mutation. We also investigated the impact 

of a prophylactic total gastrectomy on daily life and compared this with patients 

undergoing a gastrectomy for gastric cancer. We are planning to study this further 

prospectively. 

Figure 4: Study design CRITICS study. 

LYNCH SYNDROME 

In patients with dominantly inherited colorectal cancer due to a mismatch repair 

gene mutation, the role of chromoendoscopy on polyp detection and the role of video 

capsule endoscopy on the incidence and prevalence of small bowel neoplasia in 

asymptomatic Lynch syndrome carriers is being investigated in a multicenter study. 

Sofar, 29 and 30 patients have been included in the NKI-AVL. The total number of 

patients included in The Netherlands is 170 and 140, respectively. 

RECTAL CANCER 

In inoperable rectal cancer patients, we test the feasibility of EBRT and increasing 

doses of HDRBT. Currently, 19 patients are included in the study. 

NEUROENDOCRINE TUMORS (NET) 

Several topics have been investigated, which have found their way to the thesis 

of Tiny Korse. The incidence of NET in the Netherlands is increasing, especially 

in gastric and small bowel, but it was found that histological subtyping is more 

important for prognostifi cation than location of the tumor. Survival has improved, 

most impressively in metastatic disease from 30 to 44% at 5-years. A high serum 

level of a novel tumor marker, progastrin-releasing factor was an unfavorable 

prognostic marker and was predominantly found in lung NET. In a large cohort 

(824 NET patients and 282 healthy controls), a combination of 4 tumor markers was 

more informative for prognosis: in grade 1 and 2 Chromogranin A plus Cytokeratin 

fragments, and in grade 3 proGRP and Cytokeratin fragments. 

ANAL CANCER 

A phase I, dose-fi nding study assessed the feasibility and effi cacy of simultaneous 

integrated boost – intensity modulated radiation therapy (SIB-IMRT) with 

concomitant capecitabine and mitomycin-C in locally advanced anal cancer. Patients 

with the GSTP1 313A>G variant allele experienced more frequently more severe 

toxicity, but showed a signifi cantly higher complete response rate of 100% compared 

to 50% in wild type patients. 


123 


Felip E, Gridelli C, Baas P, Rosell R, 

Stahel R; Panel Members. Metastatic 

non-small-cell lung cancer: consensus on 

pathology and molecular tests, fi rst-line, 

second-line, and third-line therapy: 1st ESMO 

Consensus Conference in Lung Cancer; 

Lugano 2010. Ann Oncol. 2011;22:1507-19 

Gast MC, Zapatka M, van Tinteren H, 

Bontenbal M, Span PN, Tjan-Heijnen VC, 

Knol JC, Jimenez CR, Schellens JH, Beijnen 

JH. Postoperative serum proteomic profi les 

may predict recurrence-free survival in 

high-risk primary breast cancer. J Cancer Res 

Clin Oncol. 2011;137:1773-83 

Giesen E, Mager A, van Tinteren H, 

Rodenhuis S, Kerst JM. An alternative 

treatment regimen of advanced seminoma 

with carboplatin, etoposide, and bleomycin 

instead of cisplatin-based therapy. Urol Oncol. 


Goey AK, Sparidans RW, Meijerman I, 

Rosing H, Schellens JH, Beijnen JH. A 

sensitive LC-MS/MS method for the 

quantitative analysis of the Echinacea 

purpurea constituent undeca-2-ene-8,10diynoic 

acid isobutylamide in human plasma. 

J Chromatogr B Analyt Technol Biomed Life 


Graafl and NM, Moonen LM, van Boven 

HH, van Werkhoven E, Kerst JM, Horenblas 

S. Inguinal recurrence following therapeutic 

lymphadenectomy for node positive penile 

carcinoma: outcome and implications for 

management. J Urol. 2011;185:888-93 

Grünwald V, Desar IM, Haanen J, Fiedler 

W, Mouritzen U, Olsen MW, van Herpen 

CM. A phase I study of recombinant human 

interleukin-21 (rIL-21) in combination with 

sunitinib in patients with metastatic renal 

cell carcinoma (RCC). Acta Oncol. 

2011;50:121-6 

Grünwald V, Karakiewicz PI, Bavbek SE, 

Miller K, Machiels JP, Lee SH, Larkin J, Bono 

P, Rha SY, Castellano D, Blank CU, Knox JJ, 

Hawkins R, Anak O, Rosamilia M, Booth J, 

Pirotta N, Bodrogi I; on behalf of the REACT 

Study Group. An international expandedaccess 

programme of everolimus: Addressing 

safety and effi cacy in patients with metastatic 

renal cell carcinoma who progress after initial 

vascular endothelial growth factor receptortyrosine 

kinase inhibitor therapy. Eur J 

Cancer. 2011 

Haanen JB. Issues around melanoma. 

Ned Tijdschr Geneeskd. 2011;155:A3836 

Van Hasselt JGC, Boekhout AH, 

Beijnen JH, Schellens JHM, Huitema ADR. 

Population pharmaco-kinetic-pharmacodynamic 

analysis of trastuzumab-associated 

cardiotoxicity. Clin Pharmacol Ther. 

2011;90:126-32

124 



Hendrikx JJ, Hillebrand MJ, Thijssen B, 

Rosing H, Schinkel AH, Schellens JH, 

Beijnen JH. A sensitive combined assay for the 

quantifi cation of paclitaxel, docetaxel and 

ritonavir in human plasma using liquid 

chromatography coupled with tandem mass 

spectrometry. J Chromatogr B Analyt Technol 


Jänne PA, Boss DS, Camidge DR, Britten 

CD, Engelman JA, Garon EB, Guo F, Wong S, 

Liang J, Letrent S, Millham R, Taylor I, 

Eckhardt SG, Schellens JH. Phase I 

Dose-Escalation Study of the Pan-HER 

Inhibitor, PF299804, in Patients with 

Advanced Malignant Solid Tumors. Clin 


Jansen RS, Rosing H, Schellens JH, 

Beijnen JH. Mass spectrometry in the 

quantitative analysis of therapeutic 

intracellular nucleotide analogs. Mass 

Spectrom Rev. 2011;30:321-43 

Jansen RS, Rosing H, Schellens JH, 

Beijnen JH. Deoxyuridine analog nucleotides 

in deoxycytidine analog treatment: secondary 

active metabolites? Fundam Clin Pharmacol. 

2011;25:172-85 

Janssens T, Brouwers EE, de Vos JP, 

Schellens JH, Beijnen JH. Determination of 

platinum originating from Carboplatin in 

human urine and canine excretion products 

by inductively coupled plasma mass 

spectrometry. J Anal Toxicol. 2011;35:153-61 

Janssens T, Brouwers EE, de Vos JP, 

Schellens JH, Beijnen JH. Determination of 

platinum originating from carboplatin in 

canine sebum and cerumen by inductively 

coupled plasma mass spectrometry. J Pharm 

Biomed Anal. 2011;54:395-400 

Joerger M, Burgers SA, Baas P, Smit EF, 

Haitjema TJ, Bard MP, Doodeman VD, Smits 

PH, Vincent A, Huitema AD, Beijnen JH, 

Schellens JH. Germline polymorphisms in 

patients with advanced nonsmall cell lung 

cancer receiving fi rst-line platinumgemcitabine 

chemotherapy: A prospective 

clinical study. Cancer. 2011 

Joerger M, deJong D, Burylo A, Burgers 

JA, Baas P, Huitema AD, Beijnen JH, 

Schellens JH. Tubuline, BRCA1, ERCC1, 

Abraxas, RAP80 mRNA expression, p53/p21 

immunohistochemistry and clinical outcome 

in patients with advanced non small-cell lung 

cancer receiving fi rst-line platinumgemcitabine 

chemotherapy. Lung Cancer. 

2011;74:310-7 


Joerger M, Burgers JA, Baas P, Doodeman VD, 

Smits PH, Jansen RS, Vainchtein LD, Rosing H, 

Huitema AD, Beijnen JH, Schellens JH. Gene 

polymorphisms, pharmacokinetics, and 

hematological toxicity in advanced non-small-cell 

lung cancer patients receiving cisplatin/ 

gemcitabine. Cancer Chemother Pharmacol. 2011 

Joerger M, Ferreri AJ, Krähenbühl S, Schellens 

JH, Cerny T, Zucca E, Huitema AD. Dosing 

algorithm to target a predefi ned AUC in patients 

with primary central nervous system lymphoma 

receiving high-dose methotrexate. Br J Clin 

Pharmacol. 2011;10:1365-2125 

Kappers I, Klomp HM, Koolen MG, 

Uitterhoeve LJ, Kloek JJ, Belderbos JS, Burgers JA, 

Koning CC. Concurrent high-dose radiotherapy 

with low-dose chemotherapy in patients with 

non-small cell lung cancer of the superior sulcus. 

Radiother Oncol. 2011 

Karakullukcu B, De Boer JP, Van Veen R, 

Wegman J, Tan B. Surgical debulking combined 

with photodynamic therapy to manage residual 

extramedullary plasma-cytoma of the 

nasopharynx. Photodiagnosis Photodyn Ther 

2011;8:264-266 

Keizer RJ, Funahashi Y, Semba T, Wanders J, 

Beijnen JH, Schellens JH, Huitema AD. 

Evaluation of a2-integrin expression as a biomarker 

for tumor growth inhibition for the investigational 

integrin inhibitor E7820 in preclinical and clinical 

studies. AAPS J. 2011;13:230-39 

Keizer RJ, Schellens JH, Beijnen JH, Huitema 

AD. Pharmacodynamic biomarkers in model-based 

drug development in oncology. Curr Clin 


Keizer RJ, Zandvliet AS, Beijnen JH, Schellens 

JHM, Huitema ADR. Two-stage model-based 

design of cancer phase I dose escalation trials: 

evaluation using the phase I program of barasertib 

(AZD1152). Invest New Drugs. 2011(in press) 

Keizer RJ, van Benten M, Beijnen JH, 

Schellens JH, Huitema AD. Piraña and PCluster: 

a modeling environment and cluster infrastructure 

for NONMEM. Comput Methods Programs 

Biomed. 2011;101:72-79 

Kluijt I, Siemerink EJ, Ausems MG, van Os TA, 

de Jong D, Simões-Correia J, van Krieken JH, 

Ligtenberg MJ, Figueiredo J, van Riel E, Sijmons 

RH, Plukker JT, van Hillegersberg R, Dekker E, 

Oliveira C, Cats A, Hoogerbrugge N; on behalf of 

the Dutch Working Group on Hereditary Gastric 

Cancer. CDH1-related hereditary diffuse gastric 

cancer syndrome: Clinical variations and 

implications for counseling. Int J Cancer, 2011 (in 

press) 

Kluijt I, Sijmons RH, Hoogerbrugge N, Vasen 

HF, Cats A. Familiaire maagkanker: richtlijnen 

voor diagnostiek en controles. Ned Tijdschr 

Geneeskd. 2011;155:A2731. Review. 

Klümpen HJ, Samer CF, Mathijssen RH, 

Schellens JH, Gurney H. Moving towards dose 

individualization of tyrosine kinase inhibitors. 

Cancer Treat Rev. 2011;37:251-60 

Koning CC, Aarts MJ, Struikmans H, 

Poortmans PM, Lybeert ML, Jobsen JJ, Coebergh 

JW, Janssen-Heijnen ML, Visser O, Louwman WJ, 

Burgers JA. Mapping Use of Radiotherapy for 

Patients with Non-small Cell Lung Cancer in the 

Netherlands between 1997 and 2008. Clin Oncol 

(R Coll Radiol). 2011 

Koolen BB, Vrancken Peeters MJ, Aukema TS, 

Vogel WV, Oldenburg HS, Van der Hage JA, 

Hoefnagel CA, Stokkel MP, Loo CE, Rodenhuis S, 

Rutgers EJ, Valdés Olmos RA. 18F-FDG PET/CT 

as a staging procedure in primary stage II and III 

breast cancer: comparison with conventional 

imaging techniques. Breast Cancer Res Treat. 2011 

Koolen SL, Witteveen PO, Jansen RS, 

Langenberg MH, Kronemeijer RH, Nol A, 

Garcia-Ribas I, Callies S, Benhadji KA, Slapak CA, 

Beijnen JH, Voest EE, Schellens JH. Phase I Study 

of Oral Gemcitabine Prodrug (LY2334737) Alone 

and in Combination with Erlotinib in Patients with 

Advanced Solid Tumors. Clin Cancer Res. 

2011;17:6071-82 

Koolen SL, van Waterschoot RA, van Tellingen 

O, Schinkel AH, Beijnen JH, Schellens JH, 

Huitema AD. From Mouse to Man: Predictions of 

Human Pharmacokinetics of Orally Administered 

Docetaxel From Preclinical Studies. J Clin 

Pharmacol. 2011 (in press) 

Koppelmans V, Schagen SB, Poels MM, 

Boogerd W, Seynave C, Lugt AV, Breteler MM. 

Incidental fi ndings on brain magnetic resonance 

imaging in long-term survivors of breast cancer 

treated with adjuvant chemotherapy. Eur J Cancer 

2011;47:2531-2536 

Korse CM, Taal BG, Bonfrer JM, Vincent A, van 

Velthuysen ML, Baas P. An elevated progastrinreleasing 

peptide level in patients with welldifferentiated 

neuroendocrine tumours indicates a 

primary tumour in the lung and predicts a shorter 

survival. Ann Oncol. 2011 

Korse CM, Bonfrer JM, Prevoo W, Baas P, Taal 

BG. Increase of angiogenic growth factors after 

hepatic artery embolization in patients with 

neuroendocrine tumours. Tumour Biol. 

2011;32:647-52 

Korse CM, Taal BG, Vincent A, van Veldhuysen 

M-LF, Baas P, Buning-Kager JCGM, Linders TC, 

Bonfrer JMG. Choice of tumor markers in patients 

with neuroendocrine tumors is dependent on the 

histological grade: a marker study of Chromogranin 

A, neuron specifi c enolase, progastrin-releasing 

peptide and cytokeratin fragments. Eur J Cancer 


Korse CM, Muller M, Taal BG. Discontinuation 

of protonpump inhibitors during assessment of 

chromogranin A levels in patients with neuroendocrine 

tumors. Br J Cancer 2011;105:1173-5


Korse CM. Clinical relevance of tumor markers 

and epidemiology of neuroendocrine tumors. Thesis 

Kunst P, Burgers S, Onderwater S, vd Heuvel 

M. Stenting of airways: beware of the 

complications. Ann Thorac Surg. 2011;92:774 

Langenberg MH, Witteveen PO, Roodhart J, 

Lolkema MP, Verheul HM, Mergui-Roelvink M, 

Brendel E, Krätzschmar J, Loembé B, Nol-Boekel 

A, Christensen O, Schellens JH, Voest EE. Phase I 

evaluation of telatinib, a VEGF receptor tyrosine 

kinase inhibitor, in combination with bevacizumab 

in subjects with advanced solid tumors. Ann Oncol. 

2011;22:2508-15 

Lankheet NA, Blank CU, Mallo H, Adriaansz 

S, Rosing H, Schellens JH, Huitema AD, Beijnen 

JH. Determination of sunitinib and its active 

metabolite N-desethylsunitinib in sweat of a 

patient. J Anal Toxicol. 2011;35:558-65 

Leijen S, Soetekouw PM, Jeffry Evans TR, 

Nicolson M, Schellens JH, Learoyd M, Grinsted 

L, Zazulina V, Pwint T, Middleton M. A phase I, 

open-label, randomized crossover study to assess the 

effect of dosing of the MEK 1/2 inhibitor 

Selumetinib (AZD6244; ARRY-142866) in the 

presence and absence of food in patients with 

advanced solid tumors. Cancer Chemother 


Lesterhuis WJ, Haanen JB, Punt CJ. Cancer 

immunotherapy--revisited. Nat Rev Drug Discov. 

2011;10:591-600 

Lips EH, Mulder L, De Ronde, JJ, Mandjes 

IAM, Vincent A, Vrancken Peeters, MTFD, 

Nederlof PM, Wesseling J, Rodenhuis S. 

Neoadjuvant chemotherapy in ER+ HER2− breast 

cancer: response prediction based on 


characteristics. Breast Cancer Res Treat. 2011 

Lips EH, Laddach N, Savola SP, Vollebergh 

MA, Oonk AM, Imholz AL, Wessels LF, Wesseling 

J, Nederlof PM, Rodenhuis S. Quantitative copy 

number analysis by Multiplex Ligation-dependent 

Probe Amplifi cation (MLPA) of BRCA1-associated 

breast cancer regions identifi es BRCAness. Breast 

Cancer Res. 2011 

Loo C, Straver ME, Rodenhuis S, Muller S, 

Wesseling J, Vrancken Peeters MJ, Gilhuijs K. 

MRI response monitoring of breast cancer during 

neoadjuvant chemotherapy: Relevance of breast 

cancer subtype. J Clin Oncol. 2011;20:660-6 

Von Meyenfeldt EM, Prevoo W, Peyrot D, Lai A 

Fat N, Burgers SJ, Wouters MW, Klomp HM. 

Local progression after radiofrequency ablation for 

pulmonary metastases. Cancer. 2011;117:3781-7 

Moes JJ, Koolen SL, Huitema AD, Schellens 

JH, Beijnen JH, Nuijen B. Pharmaceutical 

development and preliminary clinical testing of an 

oral solid dispersion formulation of docetaxel 

(ModraDoc001). Int J Pharm. 2011;420:244-50 

Molloy TJ, Devriese LA, Helgason HH, Bosma 

AJ, Hauptmann M, Voest EE, Schellens JH, van ‘t 

Veer LJ. A multimarker QPCR-based platform for 

the detection of circulating tumour cells in patients 

with early-stage breast cancer. Br J Cancer. 

2011;104:1913-9 

Mulder CJJ, Peeters M, Cats A, Dahele A, 

Terhaar sive Droste J. Digestive oncologist in the 

gastroenterology training curriculum. World J 

Gastroenterol, 2011;17:1109-15 

Nieto M, Borregaard J, Ersbøll J, Ten Bosch 

GJ, van Zwieten-Boot B, Abadie E, Schellens JH, 

Pignatti F. The European medicines agency review 

of pazopanib for the treatment of advanced renal 

cell carcinoma: summary of the scientifi c 

assessment of the committee for medicinal products 

for human use. Clin Cancer Res. 2011;17:6608-14 

O’Brien ME, Konopa K, Lorigan P, Bosquee L, 

Marshall E, Bustin F, Margerit S, Fink C, Stigt JA, 

Dingemans AM, Hasan B, Van Meerbeeck J, Baas 

P. Randomised phase II study of amrubicin as 

single agent or in combination with cisplatin versus 

cisplatin etoposide as fi rst-line treatment in 

patients with extensive stage small cell lung cancer 

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Oosterhuis K, Ohlschläger P, van den Berg JH, 

Toebes M, Gomez R, Schumacher TN, Haanen 

JB. Preclinical development of highly effective and 

safe DNA vaccines directed against HPV 16 E6 and 

E7. Int J Cancer. 2011;129:397-406 

Powles T, Kayani I, Blank C, Chowdhury S, 

Horenblas S, Peters J, Shamash J, Sarwar N, 

Boletti K, Sadev A, O’Brien T, Berney D, Beltran 

L, Haanen J, Bex A. The safety and effi cacy of 

sunitinib before planned nephrectomy in metastatic 

clear cell renal cancer. Ann Oncol. 2011;22:1041-7 

Powles T, Blank C, Chowdhury S, Horenblas S, 

Peters J, Shamash J, Sarwar N, Boleti E, Sahdev 

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metastatic clear cell renal cancer. Eur Urol. 

2011;60:448-54 

Rice D, Rusch V, Pass H, Asamura H, Nakano 

T, Edwards J, Giroux DJ, Hasegawa S, Kernstine 

KH, Waller D, Rami-Porta R; International 

Association for the Study of Lung Cancer International 

Staging Committee and the International 

Mesothelioma Interest Group. Recommendations 

for uniform defi nitions of surgical techniques for 

malignant pleural mesothelioma: a consensus 

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of lung cancer international staging committee 

and the international mesothelioma interest group. 

J Thorac Oncol. 2011;6:1304-12 

Rodenhuis S. Centraliseren van kankerzorg: 

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125 


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Late effects of high-dose adjuvant 

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Schagen SB, Boogerd W, Eisel UIM, 

Buwalda B. Re: Neurocognitive functioning in 

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Van Schaik RH, Kok M, Sweep FC, van 







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Schilder CM, Seynaeve C, Linn SC, 

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Self-reported cognitive functioning in 

postmenopausal breast cancer patients before 

and during endocrine treatment: fi ndings from 

the neuropsychological TEAM side-study. 

Psychooncology. 2011 

Soto E, Keizer RJ, Trocóniz IF, Huitema 

AD, Beijnen JH, Schellens JH, Wanders J, 

Cendrós JM, Obach R, Peraire C, Friberg LE, 

Karlsson MO. Predictive ability of a 

semi-mechanistic model for neutropenia in 

the development of novel anti-cancer agents: 

two case studies. Invest New Drugs. 

2011;29:984-95 

Sparidans RW, Martens I, Valkenburg-van 

Iersel LBJ, den Hartigh J, Schellens JHM, 

Beijnen JH. Liquid chromatography-tandem 

mass spectrometric assay for the PARP-1 

inhibitor olaparib in combination with the 

nitrogen mustard melphalan in human 

plasma. J Chromatogr B. 2011;1851-56 

Stahel R, Thatcher N, Früh M, Le 

Péchoux C, Postmus PE, Sorensen JB, Felip 

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Steeghs N, Mathijssen RH, Wessels JA, 

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Gelderblom H, Guchelaar HJ. Infl uence of 

pharmacogenetic variability on the 

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Steeghs N, Gelderblom H, Wessels J, 

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Stokkel MP, Rietbergen DD, Korse CM, 

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Taal BG, de Herder WW. Nieuwe 

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Taal W, Dubbink HJ, Zonnenberg CB, 

Zonnenberg BA, Postma TJ, Gijtenbeek JM, 

Boogerd W, Groenendijk FH, Kros JM, 

Kouwenhoven MC, van Marion R, 

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Sillevis Smitt PA, Dinjens WN, van den Bent 

MJ. First-line temozolomide chemotherapy in 

progressive low-grade astrocytomas after 

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2011;13:235-241 

Teunissen SF, Rosing H, Seoane MD, 

Brunsveld L, Schellens JH, Schinkel AH, 

Beijnen JH. Investigational study of tamoxifen 

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Teunissen SF, Jager NG, Rosing H, 

Schinkel AH, Schellens JH, Beijnen JH. 

Development and validation of a quantitative 

assay for the determination of tamoxifen 

and its fi ve main phase I metabolites in 

human serum using liquid chromatography 

coupled with tandem mass spectrometry. 

J Chromatogr B Analyt Technol Biomed Life 



Tjin EP, Konijnenberg D, Krebbers G, Mallo H, 

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2011;17:5736-47 

Tran L, Baars J, Damen C, Beijnen J, Huitema 

A. Three spectroscopic techniques evaluated as a 

tool to study the effects of iodination of monoclonal 

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Tran L, Baars JW, De Boer JP, Hoefnagel CA, 

Beijnen JH, Huitema AD. The pharmacokinetics of 

131I-rituximab in a patient with CD20 positive 

non-Hodgkin Lymphoma: evaluation of the effect of 

radioiodination on the biological properties of 

rituximab. Hum Antibodies 2011;20:37-40 

Tran L, Huitema AD, van Rijswijk MH, Dinant 

HJ, Baars JW, Beijnen JH, Vogel WV. CD20 

antigen imaging with 124I-rituximab PET/CT in 

patients with rheumatoid arthritis. Hum 

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Tran L, Vogel WV, Sinaasappel M, Muller S, 

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Huitema AD. The pharmacokinetics of 

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Trotta F, Leufkens HG, Schellens JH, Laing R, 

Tafuri G. Evaluation of oncology drugs at the 

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Van der Veldt AA, Vroling L, de Haas RR, 

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Van der Veldt AA, Eechoute K, Gelderblom H, 

Gietema J, Guchelaar HJ, van Erp NP, van den 

Eertwegh AJ, Haanen JB, Mathijssen RH, Wessels 

JA. Genetic polymorphisms associated with a 

prolonged progression-free survival in patients with 

metastatic renal cell cancer treated with sunitinib. 


Verdegaal EM, Visser M, Ramwadhdoebé TH, 

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Haanen JB, van der Burg SH, Nortier JW, Osanto 

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adoptive transfer of blood-derived polyclonal 

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combination with low-dose interferon-alpha. 

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Vogel WV, Guislain A, Kvistborg P, 

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Metastatic Melanoma Undergoing Complete 

Remission. J. Clin. Oncology (in press). 

Vollebergh MA, Jonkers J, Linn SC. Genomic 

instability in breast and ovarian cancers: 

translation into clinical predictive biomarkers. Cell 

Mol Life Sci. 2011 


Wessels LFA, Schmidt MK, van Beers EH, 


Vries EGE, Schrama JG, Wesseling J, van de Vijver 

MJ, van Tinteren H, de Bruin M, Hauptmann M, 

Rodenhuis S, Linn SC. An aCGH classifi er derived 

from BRCA1-mutated breast cancer and benefi t of 

high-dose platinum-based chemotherapy in 

HER2-negative breast cancer patients. Ann of 

Oncol. 2011;22:1561-70 

Weder W, Stahel RA, Baas P, Dafni U, de 

Perrot M, McCaughan BC, Nakano T, Pass HI, 

Robinson BW, Rusch VW, Sugarbaker DJ, van 

Zandwijk N. The MARS feasibility trial: 

conclusions not supported by data. Lancet Oncol. 

2011;12:1093-4 

Zwart W, Theodorou V, Kok M, Canisius S, 

Linn S, Carroll JS. Oestrogen receptor-co-factorchromatin 

specifi city in the transcriptional 

regulation of breast cancer. EMBO J. 2011

DIVISION OF RADIOTHERAPY 

In 2011, the department has further focused its research activities by defi ning 

two long-term ambitions. First, in ten years from now, most patients treated with 

curative intent will be irradiated according to an adaptive strategy that is based on 

geometrical changes, functional characteristics and genetic profi ling. Second, in ten 

years from now, an experimental therapy is available for all top 5 tumor indications, 

combining radiation with novel radiobiological response modifi ers, preferentially 

originating from our own research. 

Adaptive radiotherapy (ART) strategies have become standard of care in an 

increasing number of indications, allowing modifi cation of the treatment plan on 

the basis of information acquired during (prostate, breast, head and neck) and after 

irradiation (lung). Functional imaging like FDG-PET and fMRI is increasingly 

used in our treatment planning protocols and allows a more precise delineation 

of the target volume and a better prediction of response. The Adaptive and 

innovative Radiation Treatment FOR improving Cancer patients’ treatment outcome 

(ARTFORCE) project is an apposite example of this approach, optimizing treatment 

selection for individual patients based on both investigational biomarkers and the 

use of novel image-guided radiation treatment, for head and neck and lung cancer 

patients. 

In 2011 volumetric rotational radiation delivery (VMAT) has been implemented for 

all major indications, improving dose distributions and reducing treatment times 

signifi cantly. In 2011 the department has also become completely “paper-less”, 

making the radiation treatment chain safer and more effi cient. 

In order to test and rapidly implement the latest hard- and software developments, 

the department has acquired an R&D linear accelerator. In addition to stimulating 

innovation, this linac also serves as a training and instruction site. 

In 2011, for the fi rst time, the number of patients treated in our department will 

exceed 5,000; further growth will be accommodated in a satellite location that will 

open its doors in 2013. 

The Radiotherapy department remains an active proponent of introducing proton 

therapy in The Netherlands and aims at realizing a proton facility within the NKI- 

AVL in collaboration with the National Center for Pediatric Oncology (NKOC) and 

the University Medical Center Utrecht. 

Two co-workers of the division successfully defended their theses: Stella Mook 

(prognostic factors in breast cancer; cum laude) and Bas Kreike (gene expression 

profi les in breast cancer). 

IMAGE ACQUISITION AND PROCESSING 

Tanja Alderliesten, Suzanne van Beek, Anja Betgen, Johan de Boer, Roman Bohoslavsky, Hermine 

Dees-Ribbers, Joop Duppen, Alessia Gasparini, Rutger Heddes, Maarten Hulshof, Rianne de Jong, 

Matthijs Kruis, Simon van Kranen, Joos Lebesque, Patricia Lindsay(1), Corrie Marijnen, Angelo 

Mencarelli, Jasper Nijkamp, Hans van Piggelen, Lennert Ploeger, Floris Pos, Coen Rasch, Peter 

Remeijer, Simon Rit, Peter de Ruiter, Rajko Topolnjak, Jeroen van de Kamer, Corine van Vliet- 

Vroegindeweij, Marnix Witte, Lambert Zijp, Jan-Jakob Sonke, Marcel van Herk 

A new many-modality visualization and registration package for radiotherapy 

research In radiotherapy research, structured and intuitive applications are 

indispensable to effi ciently process the large amounts of image data being collected. 

For instance, our SBRT patients have a baseline PET-CT, a dose plan, multiple CBCT 

scans acquired during treatment and one ore more follow up PET-CT examinations. 

In such a case, it may be required to correlate PET response with the estimated 

delivered dose taking patient deformations into account. Rather than creating a 

specifi c application for this purpose, we are developing a new general-purpose 

framework to serve as a basis for these kind of applications, and other imaging 

research in radiotherapy. 

The key to the new framework is the defi nition of a limited set of independent 


Division head Marcel Verheij 

Marcel Verheij MD PhD Head 

127 

RADIOTHERAPY 

Berthe Aleman MD PhD Academic staff 

Harry Bartelink MD PhD Academic staff 

José Belderbos MD PhD Academic staff 

Monique Bloemers MD Academic staff 

Gerben Borst MD PhD Academic staff 

Roel de Boer PhD Academic staff 

Luc Dewit MD PhD Academic staff 

Paula Elkhuizen MD PhD Academic staff 

Rick Haas MD PhD Academic staff 

Olga Hamming-Vrieze MD Academic staff 

Wilma Heemsbergen PhD Academic staff 

Edwin Jansen MD PhD Academic staff 

Joost Knegjens MD Academic staff 

Han Krewinkel MSc Academic staff 

Joos Lebesque MD PhD Academic staff 

Ben Mijnheer PhD Academic staff 

Luc Moonen MD PhD Academic staff 

Arash Navran MD Academic staff 

Heike Peulen MD Academic staff 

Floris Pos MD PhD Academic staff 

Coen Rasch MD PhD Academic staff 

Babs Reichgelt MD Academic staff 

Peter Remeijer PhD Academic staff 

Nicola Russell MD PhD Academic staff 

Govert Salverda MD Academic staff 

Christoph Schneider PhD Academic staff 

Jan-Jakob Sonke PhD Academic staff 

Joep Stroom PhD Academic staff 

Marcel van Herk PhD Academic staff 

Judi van Diessen MD Academic staff 

Baukelien van Triest MD PhD Academic staff 

Corine van Vliet-Vroegindeweij PhD 

Academic staff 

Marnix Witte PhD Academic staff 

Thelma Witteveen MD PhD Academic staff 

Frits Wittkämper PhD Academic staff 

Rosemarie de Haan MD Temporary staff 

Nieke Harinck MD Temporary staff 

Karel Hinnen MD PhD Temporary staff 

Birgit Hollmann MD Temporary staff 

Monique de Jong MD Temporary staff 

Femke van der Leij MD Temporary staff 

Philip Meijnen MD PhD Temporary staff 

Stella Mook MD PhD Temporary staff 

Marlies Nowee MD PhD Temporary staff 

Brenda Tomasoa MD Temporary staff 

Anouk Trip MD Temporary staff 

Ben Vanneste MD Temporary staff 

Brian Vendel MD Temporary staff 

Wouter Vogel MD PhD Temporary staff 

Francine Voncken MD Temporary staff 

Kim de Vries MD Temporary staff

128 

RADIOTHERAPY 

Sanne Wouterse MD Temporary staff 

Tanja Alderliesten PhD Post-doc 

Chun Chen PhD Post-doc 

Alessia Gasparini PhD Post-doc 

Anton Mans PhD Post-doc 

Anke van Mourik PhD Post-doc 

Edoardo Pasca PhD Post-doc 

Raul Pecharroman Gallego PhD Post-doc 

Roel Rozendaal PhD Post-doc 

Alize Scheenstra PhD Post-doc 

Hanno Spreeuw PhD Post-doc 

Rajko Topolnjak PhD Post-doc 

Johan de Boer MSc PhD student 

Wei Chen PhD student 

Hermine Dees-Ribbers PhD student 

Simon van Kranen MSc PhD student 

Matthijs Kruis PhD student 

Jasper Nijkamp MSc PhD student 

Hua Zhang PhD student 

Barry Doodeman Physician assistent 

Marcel Jonker Physician assistent 

Robin Kalisvaart Physician assistent 

Margriet Kwint Physician assistent 

Sandra Vreeswijk Physician assistent 

Anja Betgen MSc Technical staff 

Josien de Bois Technical staff 

Joop Duppen Technical staff 

Joeri Honnef Technical staff 

Rianne de Jong Technical staff 

Hans Krewinkel Technical staff 

Angelo Mencarelli Technical staff 

Tom Minderhoud Technical staff 

Agnieszka Olszewska MSc Technical staff 

Carmen Panneman MSc Technical staff 

Carolien Peters Technical staff 

Kenneth Pengel MSc Technical staff 

Hans van Piggelen Technical staff 

Lennert Ploeger Technical staff 

Maddalena Rossi MSc Technical staff 

Roy Scheepbouwer Technical staff 

René Tielenburg Technical staff 

Lambert Zijp MSc Technical staff 

high level objects such as scans, viewers, and deformable registration tools that are 

controlled and connected by a scripting language. The working of each specialized 

image processing application is then defi ned in a short script. The framework 

itself processes these scripts, creates the necessary connections between data and/ 

or viewers and handles user-input. A novelty in this approach is the use of data 

channels (array of scans). In the above-mentioned example, there will be channels 

named CT, PET, DOSE and CBCT. There is no limit on the number of objects 

created other than available memory. For specialized tasks such as contouring, 

masking or registration, toolbars can be activated by defi ning their corresponding 

objects. The aim of the design is to fi nd an optimum between fl exibility and 

automation, with straightforward extendibility. 

Reducing trial-and-error with probabilistic IMRT planning for prostate cancer 

Probabilistic planning allows treatment optimization and evaluation without using 

explicit safety margins. This added fl exibility generally leads to better compromises 

when PTV and OARs overlap. We are currently developing a practical planning 

system for future clinical use. Because probabilistic planning has more freedom to 

optimize the plan, we observed that less trial and error is needed to obtain a good 

plan. Our aim of this study was therefore to study the potential reduction in trialand-error 

steps during plan optimization for prostate cancer. 

Probabilistic optimization was implemented by extending the clinical cost functions 

of the Pinnacle TPS research version to include the effect of translational and 

rotational geometrical uncertainties. Random errors effects are incorporated by 

blurring the dose distribution. Systematic error effects are then included by rotating 

and shifting the CTV with respect to the blurred dose. The same geometrical 

uncertainties are applied in our in-house developed evaluation tool to analyze the 

resulting plans. 

In clinical practice, treatment plans are fi rst optimized using a standard set of 

objectives and then manually modifi ed until a clinically acceptable plan is achieved. 

The actual number of additional optimizations depends on the planner’s expertise 

and the particular characteristics of the plan. In this study clinical plans were 

replanned twice, fi rst applying a set of probabilistic objectives, and next our standard 

clinical objectives. Comparing each case with the originally clinical plan allows 

estimation of the potential reduction in trial-and error steps needed to obtain an 

acceptable plan. 

SIB treatment plans from 41 prostate cancer patients were re-optimized using 

probabilistic and standard objectives. 

The typical optimization time for probabilistic and classical plans were 10 and 2 

minutes respectively. The original prescribed dose was 78Gy to the prostate and 

seminal vesicles. Probability of delivering at least 95% of the prescribed dose 

(74.1Gy) to 99% of the target (PD95V99, median) and rectum wall volume receiving 

at least 65 Gy (V65, median) were analyzed. After the fi rst classical optimization 

[PD95V99=72%, V65=16%], and keeping the original clinical plans [PD95V99=87%, 

V65=17%] as reference, planning with probabilistic objectives lead to better plans 

than standard planning, obtaining higher target coverage with similar rectum wall 

dosage [PD95V99 = 87%, V65 = 16%]. 

Probability based treatment planning directly includes geometrical uncertainties 

during optimization without using explicit safety margins. Plans obtained after one 

optimization run achieve acceptable dose-volume metrics more often than those 

using standard objectives. This indicates a potential reduction in trial-and-error 

steps i.e. even though probabilistic planning is computationally more demanding, 

the total plan time is likely to reduce 

Estimation and correction of eddy-current induced geometrical distortions 

of prostate DWI scans Historically, radiotherapy treatment plans were based 

solely on CT data. In recent years, Magnetic Resonance (MR) techniques have 

been incorporated in treatment planning. Diffusion Weighted Imaging (DWI) is 

a promising technique to detect dominant lesions inside the prostate even though 

image quality of these scans is often poor. DWI is very susceptible to eddy-current 

induced geometric distortions, causing misalignments between the original signal 

and the diffusion sensitized signals. These misalignments cause artifacts in the

Figure 1 

Apparent Diffusion 

Coeffi cient (ADC) 

map. In this study, 

we investigate these 

distortions and their 

potential consequences 

for delineation for 

radiotherapy planning. 

Six prostate cancer 

patients underwent a 

DWI scan (Philips Achieva 3.0T), with b-values of 0 and 500 (s/mm2) in 6 directions 

using an endorectal coil. To measure the eddy-current induced misalignments 

we registered the prostate in the different diffusion sensitized images with the 

unsensitized data. In these registered sensitized data we compensated for the 

misalignments using b-spline interpolation. Subsequently, we averaged over the 

sensitized images and used the result to calculate the ADC map. Distortions mainly 

occur in the phase encoding direction. In our dataset, the phase was encoded in the 

LR direction. We measured a mean absolute LR displacement of 1.5mm (maximum 

4.2mm). The mean displacement in the AP direction was 0.4mm (maximum 

1.9mm) and 0.3mm in the CC direction (maximum 1.2mm). Next, we compared 

ADC maps from the original and the compensated data (fi gure 1). Quantitative 

evaluation was performed by calculating the average and the standard deviation 

of the ADC in a homogeneous part of the prostate. For all patients the standard 

deviation dropped after compensation. On average this drop was 5.6% (range 1.6% 

- 8.7%), whilst the average ADC signal increased by 1.8% (range 0% to 3.5%). This 

demonstrates that false contrast created by these distortions, was reduced by the 

correction method. In conclusion, Eddy currents can cause signifi cant distortions in 

DWI and thereby induce false contrast and misregistrations. Compensation for this 

effect is benefi cial when incorporating DWI data into radiotherapy planning. 

Inter & intra fraction bladder volume variation for full and empty bladder: 

a comparison The purpose of this study is to compare inter- and intra-fraction 

bladder volume variation during radiotherapy (RT) for bladder cancer with full 

and empty bladder. Most bladder motion is caused by bladder and rectum fi lling 

during treatment. At our department we use two different protocols for bladder 

cancer RT. For patients with multiple tumors, the clinical target volume (CTV) is 

the whole bladder, treated empty. Most solitary bladder cancers, however, are treated 

with partial bladder RT. These patients are treated with a full bladder to move 

healthy tissue away from the treatment fi elds. For whole bladder RT bony anatomy 

registration is used, and both inter & intra-fraction variation affect the precision. For 

partial bladder RT, markers at the borders of the tumor are used for image guidance. 

In that case the precision is mostly affected by intra-fraction variation. 

We studied bladder volumes of 24 patients with bladder cancer. For all patients, 

one planning CT and on average 7 sets (pre & post treatment) of cone beam CTs 

(CBCTs) were acquired (range 4-14 sets). Eight patients were treated with an ‘empty 

bladder’ (EB) protocol and sixteen patients with a ‘full bladder’ (FB) protocol: they 

were instructed to empty their bladder and drink 250 ml water one hour before the 

planning CT and each treatment fraction. The bladder was delineated on all scans 

by one experienced radiographer. Time trends in bladder volume (V) and volume 

increase, fi lling rate (dV/dt) and bladder wall movement were studied. 

The random variations in bladder volume are similar for both protocols, The average 

time between pre and post irradiation CBCT was 8 minutes. (range 6-18 min) The 

average fi lling rate was 2.1 ml per minute (range 0.02-8.9 ml/minute) which did 

not differ between EB and FB. Five of 24 patients (1 EB, 4 FB) had a signifi cant 

bladder volume decrease over the whole course of treatment and two of them had a 

decrease in fi lling rate. The largest intra-fraction bladder wall movement was found 

in the cranial anterior direction: mean displacement there was 3.2 mm with a SD of 

3.4mm. Intra-fraction wall motion up to 17 mm was found. 

Publications 

129 

RADIOTHERAPY 

Alderliesten MC, Klarenbeek JB, van der 

Luit AH, van Lummel M, Jones DR, Zerp S, 

Divecha N, Verheij M, van Blitterswijk WJ. 

Phosphoinositide phoshatase SHIP-1 

regulates apoptosis induced by edelfosine. Fas 

ligation and DNA damage in mouse 

lymphoma cells. Biochem J 2011;440:127-35 

Alderliesten T, Den Hollander S, 

Jonathan Yang TI, Elkhuizen PHM, van 

Mourik AM, Hurkmans C, Remeijer P, Van 

Vliet-Vroegindeweij C. Dosimetric impact of 

post-operative seroma reduction during 

radiotherapy after breast-conserving surgery. 



Rutgers EJ, Gilhuijs KG, Vrancken Peeters 

MJ. Radioactive seed localization of breast 

lesions: an adequate localization method 

without seed migration. Breast J 2011;17:594- 

601 

Baas P, Belderbos JS, van den Heuvel M. 

Chemoradiation therapy in non-small cell 

lung cancer. Current in Oncol 2011;23:140-49 

Beekman CA, Buckle T, van Leeuwen AC, 

Valdés Olmos RA, Verheij M, Rottenberg S, 

van Leeuwen FW. Questioning the value of 

(99m)Tc-HYNIC-annexin V based response 

monitoring after docetaxel treatment in a 

mouse model for hereditary breast cancer. 

Appl Radiat Isot 2011;69:656-62 

Cats A, Verheij M, van Grieken NCT, van 

de Velde CJH. Maagcarcinoom. In: 

Oncologie 2011. Editors: CJH van de Velde, 

WTA van der Graaf, JHJM van Krieken, 

CAM Marijnen, JB Vermorken, Bohn 

Stafl eu van Loghum. 2011:355-59 

Chai X, van Herk M, van de Kamer JB, 

Hulshof MC, Remeijer P, Lotz HT, Bel A. 

Finite element based bladder modelling for 

image-guided radiotherapy of bladder cancer. 

Med Phys 2011;38:142-50 

Courrech Staal EF, Aleman BM, van 

Velthuysen ML, Cats A, Boot H, Jansen EP, 

van Coevorden F, van Sandick JW. 

Chemoradiation for esophageal cancer: 

Institutional experience with three different 

regimens. Am J Clin Oncol 2011;34:343-49 

De Boer JP, Raderer M, van Tinteren H, 

Aleman BM, Boot H, de Jong D. Treatment 

of extranodal marginal zone lymphoma of 


fl udarabine: effect on tumor 

microenvironment. Leuk Lymphoma 2011; 

52:2262-9 

De Ruysscher D, Belderbos J. Recent 

advances in Radiotherapy for lung cancer. In: 

Lung Cancer Therapy Annual 7. Editor: R 

Stahel 2011 (in press)

130 

RADIOTHERAPY 


De Vreeze RS, de Jong D, Koops W, 

Nederlof PM, Ariaens, Haas RL, van 

Coevorden R. Oncogenesis and classifi cation 



analysis. Int J Cancer 2011;128:778-86 

Defraene G, van den Bergh L, Al- 

Mamgani A, Haustermans K, Heemsbergen 

W, van den Heuvel F, Lebesque JV. The 

benefi ts of including clinical factors in rectal 

normal tissue complication probability 

modelling after radiotherapy for prostate 

cancer. Int J Radiat Oncol Biol Phys 2011 (in 

press) 

Devriese LA, Bosma AJ, van den Heuvel 




by Multi-marker QPCR analysis. Lung 


Dikken JL, van Sandick JW, Swellengrebel 

HA, Lind PA, Putter H, Jansen EPM et al. 

Neo-adjuvant chemotherapy followed by 

surgery and chemotherapy for patients with 

resectable gastric cancer (CRITICS). BMC 

Cancer 2011;11:329 

Dikken JL, Verheij M, Cats A, Jansen 

EPM, Hartgrink HH, van de Velde JH. 

Extended lymph node dissection for gastric 

cancer from a European perspective. Gastric 

Cancer 2011:14:396-8 

Dikken JL, van de Velde CJH, Coit DG, 

Shah MA, Verheij M, Cats A. Treatment of 

resectable gastric cancer. Therapeutic 

Advances in Gastroenterology 2011 (in press) 

Dikken JL, van de Velde CJH Verheij M, 

Cats A. Resectabel maagcarcinoom. Deel 1. 

Introductie en chirurgische behandeling. 

NTvO 2011 (in press) 

Dikken JL,van de Velde CJH, Verheij M, 

Cats A. Resectabel maagcarcinoom. Deel 2. 

Neoadjuvante en adjuvante behandeling. 

NTvO 2011 (in press) 

Figure 2 

Intra-fraction volume variation is small compared to inter-fraction variation. 

However, it should be taken into account, especially for cranial-anterior located 

tumors. Bladder fi lling rate was not different in the EB and FB group. Contrary to 

common believe, both empty and full bladder protocols have similar inter-and intrafraction 

uncertainties. 

Dose-effect relations for predicting acute esophagitis after IMRT concurrent 

chemoradiotherapy of Non Small Cell Lung Cancer based on dose-surface 

distributions Predicting acute esophagitis (AE) in NSCLC patients receiving IMRT 

is challenging. Currently, only dosimetric parameters (e.g. V70, V50, V35) derived 

from conformal treatments are applied in the clinic to limit or predict AE. There is, 

however, a lack of consensus on the most predictive parameter. In this study, we aim 

to analyze the IMRT dose distribution received on the esophagus surface, and relate 

these to AE. 

A group of 31 consecutive patients treated in the Antoni van Leeuwenhoek Hospital 

with AE Grade 0 to 3 (CTCv.3.0) was selected for this study. Twenty patients scored 

as Grade 0-2 were categorized as non-toxic (NT), while 11 patients with Grade 3 were 

categorized as toxic (T). All patients underwent daily concurrent chemoradiotherapy 

using IMRT with 66Gy in 24 fractions. The dosimetric parameter used in 

treatment planning to avoid AE was V35≤65%. The delineated esophagus from the 

planning CT and the delivered dose were available for each patient, allowing a 2D 

esophagus surface dose map (ESDM) to be computed (fi gure 2a). To analyze the 

dose distribution on the esophagus surface, two hypotheses were tested. H1: The 

dose delivered to a specifi c location of the esophagus is associated with AE. H2: 

The local dose distribution, independent of the esophagus anatomy, is associated 

with AE. To test H1, ESDMs of all patients were mapped on an identical esophagus 

grid. To test H2, ESDMs of all patients were mapped according to the location of the 

voxel with the highest dose, accounting for translation along and rotation around the 

length of the esophagus (fi gure 2b). After the inter-patient dose mapping, a voxel-byvoxel 

based t-test, followed by a permutation based multiple comparisons test, were 

conducted to test the dose distribution differences between NT and T patients for 

both hypotheses. Finally, the region that received a signifi cantly higher dose for NT 

than T could be highlighted according to the two-sided signifi cance level of p≤0.05. 

In H1, no signifi cant region was found to be associated with AE. This result 

implies that there is no evidence that the esophagus has a non-homogeneous 

radiosensitivity. This fi nding needs to be further analyzed with clinical or biological 

evidence. In H2, a signifi cant region was found to be associated with AE (fi gure 2c). 

This result suggests that AE is associated with local dose distribution, especially the 

length and circumference of the high dose region on the esophagus. The locations 

of the signifi cant regions suggest that especially the dose to the entire esophagus 

circumference is related to AE. 

The dose that is given to a specifi c esophagus location is not associated with AE, 

while the shape of the local dose distribution is associated with AE. The region that 

gives signifi cantly higher dose to T patients suggests that dose-length-circumference 

parameters may be predictive for AE in high dose IMRT and concurrent 

chemotherapy. 

Using longitudinal and circumferential parameters from esophagus surface 

dose distributions to predict acute esophagitis after concurrent chemo- 

IMRT for lung cancer Acute esophagitis (AE) is a common side effect of high dose 

radiation with concurrent chemoradiotherapy in NSCLC patients. Currently, only 

dosimetric constraints (e.g. V50, V35) derived from 3D conformal treatments are 

applied in the clinic to limit and/or predict AE. There is however, a lack of consensus 

on the most predictive parameter. In a previous study on esophagus surface dose 

maps (ESDM), the local dose distribution was found to be associated with AE, 

suggesting that a higher dose to a longer and/or wider range of esophagus surface 

is associated with severe AE. Therefore, in this study, we analyzed longitudinal 

and circumferential parameters, derived from the ESDM, and relate these to the 

incidence and severity of AE. 

A group of 31 consecutive patients with AE Grade 0 ~ 3 (CTCv.3.0) treated between 

2008 and 2009 was selected for this study. Twenty Grade 0 ~ 2 and 11 Grade 3

patients were categorized as non-toxic (NT) and toxic (T), respectively. All patients 

underwent daily concurrent chemoradiotherapy using IMRT in 2.75Gy×24 fractions. 

The esophagus cost function used for treatment planning was V35≤65%. For all 

patients an ESDM of the planned dose was computed. Next, two sets of parameters 

were extracted: 1) the length of the esophagus that received more than DGy dose 

over more than X% of its circumference on the axial slices and 2) the percentage 

of the circumference that received more than DGy dose over more than Lcm of its 

length. Univariate binary logistic regression was applied to select the most predictive 

parameter in either set. 

The lengths and circumferences in each set are highly correlated. The best 

longitudinal parameter derived from the univariate regression was the length 

of the esophagus that received more than 30Gy dose over more than 70% of its 

circumference (p=0.056, OR=1.210). This parameter has a mean of 10.0cm, with 

a range of 1.5 to 19.2cm. The best circumferential parameter was the percentage 

of the circumference that received more than 65Gy dose over more than 3cm of its 

length (p=0.029, OR=17.023). This parameter has a mean of 58%, with a range of 0 

to 100%. Both parameters were superior to the dosimetric parameters V35 (p=0.085, 

OR=1.040) or V50 (p=0.120, OR=1.035), which are often used in the clinic. 

This preliminary study shows that in NSCLC patients treated with concurrent 

chemoradiation, AE toxicity is associated with longitudinal and circumferential 

parameters describing the shape of the dose distribution on the esophagus. 

However, more data are required to compare the additional predictive value of these 

parameters with conventional dosimetric parameters derived from DVHs (fi gure 3). 

Figure 3 

Targeting accuracy of image guided radiation of intracranial tumors in mice 

quantified using gold nanoparticles Technological advances have dramatically 

improved the conformality and accuracy of radiotherapy (RT) of cancer patients. 

Recently, micro image-guided RT (μIGRT) systems have been developed that 

integrate small beam geometries, cone beam CT (CBCT) guidance and robotic 

corrections. These μIGRT systems therefore bridge the technology gap between 

pre-clinical radiation research and clinical radiation therapy. The purpose of 

this work was to quantify radiation delivery accuracy of a μIGRT system (X-RAD 

225Cx, Precision X-Ray Inc.) in vivo by using gold nanoparticles (AuNPs), injected 

intracranially with U87 tumor cells, as contrast agent. 

AuNPs (30-100 μg/mL, 150 nm, in saline solution) embedded in gel were imaged 

using CBCT (40-120 kVp). AuNPs were subsequently stereotactically injected in 

nude mice with (n=3) or without (n=5) U87 tumor cells, 3 mm below the skullcap. 

CBCT scans were taken every 3-5 days with different scanning techniques for up to 

2 weeks. The tumor marked by the AuNPs was targeted by selecting its position on 

the CBCT reconstructed scan. The computer controlled couch consequently moved 

to place the selected position at the isocenter, which was irradiated with a 5 mm 

diameter fi eld or star shots (8 equi-angular spaced beams, 1 mm diameter fi eld). 

γH2Ax and HE staining were performed on the slices around the tumor. 

Adequate contrast is found in vitro for each imaging technique (maximum 

relative contrast C=2.4 at 40 kVp). In vivo, no signal was detected for AuNPs 

alone. An increased contrast of the tumor region (C=0.49 for 40 kVp) was found 

for AuNPs+U87. This signal remains detectable at day 15 after injection. No 

abnormalities in mouse behaviour were observed. The presence of AuNPs increased 


131 

RADIOTHERAPY 

Egelmeer AG, Velazquez ER, de Jong JM, 

Oberije C, de Jong MC, Rasch C, Hoebers F 

et al. Development and validation of a 

nomogram for prediction of survival and local 

control in laryngeal carcinoma patients 

treated with radiotherapy alone: a cohort 

study based on 994 patients. Radiother Oncol 

2011;100:108-15 

Emmering J, Vogel WV, Stokkel MP. 

Intramuscular metastases on FDG PET-CT: a 

review of the literature. Nucl Med Commmun 

2011(in press) 


M, Retel VP, Mook S et al. Impact of 

mammographic screening on the detection of 

good and poor prognosis breast cancers. Breast 


Farazi TA, Horlings HM, Ten Hoeve JJ, 

Mihailovic A, Kreike B, van de Vijver MJ et 

al. MicroRNA sequence and expression 

analysis in breast tumors by deep sequencing. 

Cancer Research 2011;71:4443-53 

Fitton I, Cornelissen SA, Duppen JC, 

Steenbakkers RJ, Peeters ST, Hoebers FJ, 

Kaanders JH, Nowak PJ, Rasch CR, van Herk 

M. Semi-automatic delineation using 

weighted CT-MRI registered images for 

radiotherapy of nasopharyngeal cancer. Med 

Phys 2011;38:4662-6 

Fuller CD, Nijkamp J, Duppen JC, Rasch 

CR, Thomas JR Jr, Wang SJ, Okunieff P, 

Jones WE 3rd , Baseman D, Patel S, 

Demandante CG, Harris AM, Smith BD, 

Katz AW, McGann C, Harper JL, Chang DT, 

Smalley S, Marshall DT, Goodman KA, 

Papanikolaou N, Kachnic LA. Prospective 

randomized double-blind pilot study of 

site-specifi c consensus atlas implementation 

for rectal cancer target volume delineation in 

the cooperative group setting. Int J Radiat 

Oncol Biol Phys 2011;79:481-9 

Graafl and NM, Moonen LMF, van Boven 

HH, van Werkhoven E, Kerst M, Horenblas 




management. Journal of Urology 

2011;185:888-93 

Guckenberger M, Kestin LL, Hope AJ, 

Belderbos J, Werner-Wasik M, Yan D, Sonke 

J-J, Bissonnette JP, Wilbert J, Xiao Y, Grills 

IS. Stereotactic body radiotherapy for early 

stage non-small cell lung cancer is safe and 

effective irrespective of pre-treatment 

pulmonary function. J Thor Oncol 2011 (in 

press) 

Haas RLM, de Klerk G. An illustrated case 

of doxorubicin-induced radiation recall 

dermatitis and a review of the literature. Neth 

J Med 2011;69:72-5

132 

RADIOTHERAPY 


Haimovitz-Friedman A, Yang TJ, Thin 

TH, Verheij M. Imaging radiotherapyinduced 

apoptosis. Radiat Res 2011 (in press). 

Haverkort MA, van de Kamer JB, Pieters 

BR, van Tienhoven G, Assendelft E, Lensing 

AL, van Herk M, de Reijke TM, Stoker J, 

Koning CC. Position verifi cation for the 

prostate: effect on rectal wall dose. Int J 

Radiat Oncol Biol Phys 2011;80:462-8 

Hilbers FS, Boekel NB, van den Broek AJ, 

van Hien R, Cornelissen S, Aleman B et al. 

Genetic variants in TGFbeta-1 and PAI-1 as 

possible risk factors for cardiovascular disease 

after radiotherapy for breast cancer. Radiother 

Oncol 2011(in press) 

Hoebers F, Heemsbergen W, Moor S, 

Lopez M, Klop M, Tesselaar M, Rasch C. 

Reirradiation for head-and-neck cancer: 

delicate balance between effectiveness and 

toxicity. Int J Radiat Oncol Biol Phys 

2011;81:111-8 

Holt A, van Vliet-Vroegindeweij C, Mans 

A, Belderbos J, Damen E. Volumetricmodulated 

arc therapy for stereotactic body 

radiotherapy of long tumors: a comparison 

with intensity-modulated radiotherapy 

techniques. Int J Radiat Oncol Biol Phys 

2011;81:1560-7 

Hoving S, Heeneman S, Gijbels MJ, te 

Poele JA, Pol JF, Gabriels K, Russell NS, 

Daemen MJ, Stewart FA. Anti-infl ammatory 

and anti-thrombotic intervention strategies 

using atorvastatin, clopidogrel and 

knock-down of CD40L do not modify 

radiation-induced atherosclerosis in ApoE 

null mice. Radiother Oncol 2011;101:100-8 

Kappers I, Klomp HM, Koolen MGJ, 

Uitterhoeve LJ, KLoek JJ, Belderbos JSA, 

Burgers JA, Koning CCE. Concurrent 

high-dose radiotherapy with low-dose 

chemotherapy in patients with non-small cell 

lung cancer of the superior sulcus. Radiother 


Knegjens JL, Hauptmann M, Pameijer 

FA, Balm AJ, Hoebers FJ, de Bois JA, 

Kaanderen JH, van Herpen CM, Verhoef CG, 

Wijers OB, Wiggenraad RG, Buter J, Rasch 

CR. Tumor volume as prognostic factor in 

chemoradiation for advanced head and neck 

cancer. Head & Neck 2011;33:375-82 

Lamers-Kuijpers E, Heemsbergen W, van 

Mourik A, Rasch C. Sequentially delivered 

boost plans are superior to simultaneously 

delivered plans in head and neck cancer when 

the boost volume is located further away from 

the parotid glands. Radiother Oncol 

2011;98:51-6 

the targeting accuracy in CBCT by 0.5 mm. The staining results showed AuNPs 

are confi ned within the volume of the tumor. Radiation damage was shown to be 

centered in the tumor area (0.09 and 0.47 mm deviation, with and without AuNPs 

respectively). 

AuNPs are an effi cient and reliable contrast agent when injected directly in a 

tumor cells containing suspension, allowing their detection during the RT. CBCT 

of AuNPs in brain is a promising 3D addition to 2D bio-luminescence for tumor 

growth follow-up. The comparison of tumor and radiation damage positions 

demonstrates the high targeting accuracy of 0.1 mm of the μIGRT system. 

EPID DOSIMETRY 

Priscilla Camargo, Anton Mans, Igor Olaciregui-Ruiz, Raul Pecharromán-Gallego, Roel Rozendaal, 

Jan-Jakob Sonke, Hanno Spreeuw, Joep Stroom, Marcel van Herk, Ben Mijnheer 

In vivo dosimetry using an Electronic Portal Imaging Device (EPID) has been 

implemented in our institution for almost all high-energy photon treatments 

of cancer with curative intent. Lung cancer treatments were initially excluded, 

because the original back-projection dose-reconstruction algorithm does not 

account for tissue inhomogeneities accurately. The aim of this study was to test a 

new method, in aqua vivo EPID dosimetry, for fast dose verifi cation of lung cancer 

irradiations during actual patient treatment. The key feature of our method is the 

dose reconstruction in the patient from EPID images, obtained during the actual 

treatment, whereby the images have been converted to a situation as if the patient 

consisted entirely of water; hence the method is termed in aqua vivo. This is done 

by multiplying the measured in vivo EPID image with the ratio of two digitallyreconstructed 

transmission images for the unit-density and inhomogeneous 

tissue situation. For dose verifi cation, a comparison is made with the calculated 

dose distribution with the inhomogeneity correction switched off. The method 

was fi rst tested using inhomogeneous phantoms simulating a tumor in lung; 

subsequently IMRT and VMAT clinical lung cancer treatments were investigated. 

The improvements by applying the in aqua vivo approach are considerable. For 

instance, the percentage of γ values ≤1 increased on average from 66.2 to 93.1% and 

from 43.6 to 97.5% for 5 IMRT and 5 VMAT cases, respectively. The verifi cation 

results of the in aqua vivo method were statistically analysed for 751 lung cancer 

patients treated with IMRT. The results for this large group of patients had a mean 

γ of approximately 0.5, a percentage of γ values ≤1 larger than 89%, and a difference 

of the isocenter dose value less than 1%. From this study it can be concluded that 

with the in aqua vivo approach for the verifi cation of IMRT and VMAT lung cancer 

treatments, we can achieve results with the same accuracy as obtained during in vivo 

EPID dosimetry of sites without large inhomogeneities. 

Our current approach of EPID dosimetry does not give accurate results in wedged 

beams due to the resulting change in photon energy spectrum. We therefore 

modifi ed our back-projection dose reconstruction model to make it also applicable 

for wedged beams. This was done by inserting a single energy-dependent correction 

factor, α w, in the EPID pixel-to-dose relationship. α w takes into account the effect 

of changes in beam quality caused by the wedge on the dose reconstruction. To 

determine this parameter, wedge factors were measured at the position of the 

EPID with and without a polystyrene slab phantom in the beam, by means of an 

ionization chamber in a mini-phantom as well as the EPID. The resulting values for 

the α w parameter were 1.089 ± 0.004, 1.009 ± 0.004, and 1.003 ± 0.008 for the 6, 

10 and 18 MV photon beam, respectively. By applying these α w values, the EPIDreconstructed 

dose distributions in the phantom showed for a 10 x 10 cm 2 fi eld at 

10 cm depth generally agreement within 1% with those calculated by the treatment 

planning system, both in the wedged and non-wedged direction. The results of 

the γ-evaluation of the verifi cation of clinical cases showed on average for 8 breast 

treatments, treated with 6 and 10 MV beams, an increase in percentage of points 

with γ

This study showed that the insertion of a wedge-correction factor in the EPID pixelto-dose 

relationship provides an accurate method for pre-treatment and in vivo EPID 

dosimetry of treatments with wedged beams. 

Our current γ-evaluation for IMRT applies a 2D comparison of measured and 

planned dose distributions in a plane through the isocentre perpendicular to each 

IMRT beam. The criteria for IMRT verifi cation are based on the γ-index as well as 

the dose at the isocentre and differ for the various treatment sites. At present we 

are using the same criteria for the 3D dose comparison of VMAT checks. However, 

dose deviations in a 2D single beam or segment will be diluted in the total 3D dose 

distribution and serious errors in the dose calculation or in the position of the leaves 

may no longer be detected. Therefore, we are studying the use of stricter γ-criteria 

for 3D dose verifi cation in the clinic, such as 2%, 2 mm or 2%, 1.5 mm, also since 

identical criteria generally yield smaller γ-values in 3D dose verifi cation than in 2D. 

More clinical data are needed to assess optimal values for our gamma evaluation 

criteria for 3D verifi cation of VMAT treatments of various treatment sites. 

TREATMENT PLANNING 

Corine van Vliet-Vroegindeweij, Tomas Janssen, Zdenko van Kesteren, Emmy Lamers, Annemarie 

Lakeman, Angela Tijhuis, Linda Glaser, Peter de Ruiter, Amber Duijn, Roman Bohoslavsky, Marnix 

Witte, José Belderbos, Joost Knegjens, Jan-Jakob Sonke, Paula Elkhuizen, Eugène Damen 

Reducing inter-observer variation of boost-CTV delineation in breast conserving 

therapy using a pre-operative CT. Large inter-observer variation exist in the 

delineation of the boost-CTV in breast conserving therapy. The availability of a 

pre-operative CT scan (Preop-CT) might decrease this variation. Therefore, twentysix 

breast cancer patients underwent a CT scan prior to and after breast conserving 

surgery. Five observers delineated the boost-CTV according to guidelines defi ned by 

the observers prior to the study. At fi rst the boost-CTV was delineated on the Plan- 

CT without access to the Preop-CT (boost-CTV-1). After submission, the observers 

obtained the Preop-CT on which they delineated the GTV. Subsequently, the Plan- 

CT was registered with the Preop-CT and the boost-CTV was delineated again on 

the Plan-CT (boost-CTV-2). 

To study the impact of the Preop-CT on the delineation of the boost-CTV, three 

parameters were calculated: i) the delineated volume of both boost-CTV-1 and boost- 

CTV-2; ii) the conformity index (CI = (common volume) / (encompassing volume)); 

iii) the distance between the centres of mass (COMd). 

In the analysis, distinction was made between patients for whom consensus existed 

between the observers on the GTV (n=23), and patient for whom the observers did 

not agree on the GTV (n=3). 

The results for the three parameters are listed in the table. 

Whole population (n=26) Population with GTV 

consensus (n=23) 

Boost- Boost- p-value Boost- Boost- p-value 

CTV-1 CTV-2 

CTV-1 CTV-2 

Mean CI 0.36 0.36 n.s. 0.36 0.38 0.037 

Mean COMd (mm) 11.2 9.7

134 

RADIOTHERAPY 

Figure 4 


Rasch CRN, Hauptmann M, Balm AJM. 

Intra-arterial chemotherapy for head and 

neck cancer. Is there a verdict? Cancer 

2011;117:874-5 

Retel VP, van der Molen L, Hilgers FJ, 

Rasch CR, L’Ortye AA, Steuten LM, van 

Harten WM. A cost-effectiveness analysis of a 

preventive exercise program for patients with 

advanced head and neck cancer treated with 

concomitant chemo-radiotherapy. BMC 


Rit S, Nijkamp J, van Herk M, Sonke JJ. 

Comparative study of respiratory motion 

correction techniques in cone-beam computed 

tomography. Radiother Oncol 2011;100:356-9 

Roef M, Vogel WV. The effects of muscle 

exercise and bed rest on [18F]methylcholine 

PET/CT. Eur J Nucl Med Mol Imaging 

2011;38:526-30 

Ruskoné-Fourmestraux, Fischbach W, 

Aleman BMP, Boot H, Du MQ, Megraud F, 

Montalban F, Raderer M, Savio A, 

Wotherspoon A. EGILS consensus report. 

Gastric extranodal marginal zone B-cell 

lymphoma of MALT. Gut 2011;60:747-758 

uptake region of the primary tumour prior to treatment. A phase II PET-boost trial 

(NCT01024829) randomises patients between dose-escalation of the entire primary 

tumour (Arm A) or to the high FDG uptake region inside the primary tumour 

(>50% SUVmax) (Arm B), whilst giving 66 Gy in 24 fractions to involved lymph 

nodes. We analysed the planning results of the fi rst 20 patients for which both arm 

A and B was planned. 

Boost dose levels were escalated up to predefi ned normal tissue constraints with 

an equal mean lung dose in both arms. This also forces an equal mean PTV dose 

in both arms, hence testing pure dose-redistribution. Patients were randomised 

between arm A and B if dose-escalation to the primary tumour in arm A of at least 

72 Gy in 24 fractions could be safely planned. 

15/20 patients could be escalated to at least 72 Gy. Average prescribed fraction 

dose was 3.27±0.31 Gy [3.01-4.28 Gy] and 3.63±0.54 Gy [3.20-5.40 Gy] for arm A 

and B, resp. Average mean total dose inside the PTV of the primary tumour was 

comparable: 77.3±7.9 Gy vs. 77.5±10.1 Gy. For the boost region dose levels of on 

average 86.9±14.9 Gy were reached. An example of a dose distribution in arm A and 

B is given in the fi gure 4. 

No signifi cant dose differences between both arms were observed for the organs 

at risk. Most frequent observed dose-limiting constraints were the mediastinal 

structures (13/15 and 14/15 for arm A and B, resp.), and the brachial plexus (3/15 for 

both arms). 

From these results we conclude that dose-escalation using an integrated boost could 

be achieved to the primary tumour or high FDG uptake regions while keeping the 

pre-defi ned dose constraints. 

Clinically Relevant Pareto Fronts In evaluating a treatment plan, one typically 

has confl icting evaluation objectives (EO). Plans where one cannot improve on one 

EO, without worsening another, span up the Pareto front. In treatment planning 

studies, one typically only compares points from near the Pareto front. The choice 

of a point however is subjective and thus an observed difference may simply be due 

to different choices. Comparing the complete front however presents an objective 

way to compare techniques. For clinical relevance the fronts need to be constructed 

conform clinical practice, using a clinical treatment planning system, with the 

clinical planning objectives (PO) and the clinical EO. We therefore made an 

application to construct a clinically relevant Pareto front in the Pinnacle treatment 

planning system This application runs many optimizations with different PO 

for each run to steer the optimizer. New PO are generated by randomly changing 

parameters like the weight, or the dose level of a ‘master set’ of PO. From the plans 

the Pareto front for a set of EO is constructed using Matlab. To compare two fronts, 

for each EO a Pareto Similarity Index, Ψ, is defi ned as the average relative difference 

between the fronts of that EO. Ψ is normalized to lie between -1 and +1 such that two 

Pareto fronts are equal for Ψ = 0. 

As an example of the framework described, we have compared the Pareto fronts of 

VMAT with IMRT for three prostate cases. Pareto fronts were generated based on 

1000 plans for a set of EO consisting of the PTV homogeneity and the V75Gy and 

V65Gy of the rectal wall. Ideally all these EO are zero. 

Constructing and evaluating the Pareto fronts takes about 60 hours of CPU time 

per patient and about 15 minutes of human interaction. Ψ for the different patients 

and EO is given in the table. We see that for all patients, for all EO, Ψ is positive, 

implying in this case that the Pareto front for the VMAT plans lays lower. 

Patient Ψ (IMRT-VMAT) Ψ (IMRT-VMAT) Ψ (IMRT-VMAT) 

Homogeneity Rwall V65Gy Rwall V75Gy 

1 0.07 0.25 0.18 

2 0.01 0.11 0.01 

3 0.01 0.01 0.76 

From this work we conclude that implementation of a framework to construct and 

evaluate clinically relevant Pareto fronts using Pinnacle is possible. This framework 

allows for an objective comparison between planning techniques, without the

arbitrariness due to the choices of a planning RTT. As an example we compared 

VMAT with IMRT for prostate patients. We found that for the plans which are 

Pareto-optimal for the PTV homogeneity and the V75Gy and V65Gy of the rectal 

wall, the VMAT plans are superior for each EO. 

CLINICAL APPLICATION OF IMAGE GUIDED RADIOTHERAPY 

Jose Belderbos, Anja Betgen, Nabilla Bouzeya, Sanne Conijn, Johan de Boer, Paula Elkhuizen, Rick 

Haas, Joost Knegtjens, Danny Minkema, Jasper Nijkamp, Carmen Panneman, Coen Rasch, Peter 

Remeijer, Simon Rit, Maddalena Rossi, Roel Rozendaal, Eva Schaake, Christoph Schneider, 

Suzanne van Beek, Marcel van Herk, Simon van Kranen, Corinne van Vliet, Sandra. Vreeswijk, 

Maarten Wolfrat, Jan-Jakob Sonke 

HEAD AND NECK 

Improved tongue depressors for head-and-neck radiation therapy In our 

institute a tongue depressor is used to increase the distance between the tongue/ 

fl oor of mouth and the palate for head and neck cancer patients undergoing radiation 

therapy having a target volume close to the oral cavity. The purpose of this study was 

to assess the effect of a new tongue depressor on the reproducibility of surrounding 

patient anatomy. To that end, 374 CBCT scans of 47 patients (average of 8 scans per 

patient) were evaluated. For each patient, the distance between the upper jaw and 

tongue depressor (distance A) and the upper jaw and tip of the tongue depressor 

(distance B) where measured in the CBCT scans. Also bony anatomy registrations 

using three sub regions (upper jaw, lower jaw and cervical vertebrae were performed. 

The patients were divided in 3 groups: 1) patients with the old tongue depressor, 2) 

patients with a new tongue depressor normally attached and 3) patients with the new 

tongue depressor placed entirely below the mask for improved fi xation. We found no 

relevant differences in setup reproducibility of the tongue for the groups of patients. 

The group mean (M) differences in distances during treatment compared with the 

planCT ΔA/ΔB were for group 1: 0.01/-0.11 cm, group 2: -0.12/-0.16 cm and group 3: 

0.10/0.09 cm, respectively. The systematic deviation (Σ) for ΔA/ΔB were for group 

1: 0.25/0.66 cm, group 2: 0.36/0.40 cm and group 3: 0.17/0.22 cm, respectively. Σ 

was signifi cant between group 1 and 3 (p = 0.039). Group 3 also showed the smallest 

random errors, although this was not found statistically signifi cant. The random 

errors (σ) for distance ΔA/ΔB were for group 1: 0.25/0.51 cm, group 2: 0.25/0.35 

cm and group 3: 0.11/0.20 cm, respectively. No difference was found for the online 

local setup errors in the bony sub regions between the 3 tongue depressor groups. 

All online local setup errors also show that the M, Σ and σ are within 0.2 cm in all 

directions. In conclusion, Patients with the new developed tongue depressor placed 

below the fi xation mask had the best reproducibility and has become our clinical 

standard. 

Geometrical variability of parotid glands during radiotherapy measured with 

deformable Parotid shrinkage during radiotherapy (RT) of head and neck (H&N) 

cancer is frequently observed and associated with a shift of the gland into the high 

dose region, leading to increased toxicity. Plan adaptation could help to spare this 

organ at risk (OAR). 

The purpose of this study was to develop an automated method to routinely 

identify parotid geometrical variability during RT and to analyze daily geometrical 

variations of volume and position in order to derive objective criteria for plan 

adaptation. Daily CBCT scans of 11 patients with H&N cancer were non-rigidly 

registered with the planning CT using an in-house developed BSpline deformable 

registrations algorithm, based on mutual information and regularized on rigidity 

of bony anatomy and general smoothness of deformations. For each fraction 

volume and position of the lateral and contra-lateral parotid glands were measured 

by propagating the original contours according to the measured deformations. 

Deformable registration for all patients proofed robust and accurate. A volume 

reduction was seen for all patients over the full course of treatment: in the last week 

on average -19.4/-17.8% (lateral/contra-lateral, 7.3/8.0 % SD). Linear regression 

over parotid volume over time showed an average regression of -0.52 /-0.42 %/day 


135 

RADIOTHERAPY 

Scharpfenecker M, Floot B, Korlaar R, 

Russell NS, Stewart FA. ALK1 heterozygosity 

delays development of late normal tissue 

damage in the irradiated mouse kidney. 


Siedschlag C, Boersma L, van Loon J, 

Rossi M, van Baardwijk A, Gilhuijs K, 

Stroom J. The impact of microscopic disease 

on the tumor control probability in non-smallcell 

lung cancer. Radiother Oncol 

2011;100:344-50 

Smitsmans MH, de Bois J, Sonke JJ, 

Catton CN, Jaffray DA, Lebesque JV, van 

Herk M. Residual seminal vesicle 

displacement in marker-based image-guided 

radiotherapy for prostate cancer and the 

impact on margin design. Int J Radiot Oncol 

Biol Phys 2011;80:590-6 

Swellengrebel HA, Marijnen CA, Verwaal 

VJ, Vincent A, Heuf G, Gerhards MF, van 

Geloven AA, van Tets WF, Verheij M, Cats 

A. Toxicity and complications of preoperative 

chemoradiotherapy for locally advanced rectal 

cancer Br J Surg 2011;98:418-26 

Topolnjak R, de Ruiter P, Remeijer P, van 

Vliet-Vroegindeweij C, Rasch C, Sonke JJ. 

Image-guided radiotherapy for breast cancer 

patients: surgical clips as surrogate for breast 

excision cavity. Int J Radiat Oncol Biol Phys 

2011;81:187-95 

Van der Hage JA, Mieog JSD, van de 

Velde CJH, Putter H, Bartelink H, van de 

Vijver MJ. Impact of established prognostic 

factors and molecular subtype in very Young 

breast cancer patients: pooled analysis of four 

EORTC randomized controlled trials. Breast 

Cancer Res 2011;24:13:R68 

Van Herk M, Ploeger L, Sonke JJ. A novel 

method for megavoltage scatter correction in 

cone-beam CT acquired concurrent with 

rotational irradiation. Radiother Oncol 

2011;100:365-9 

Van der Molen L, van Rossum MA, 

Burkhead LM, Smeele LE, Rasch CRN, 

Hilgers FJM. A randomized preventive 

rehabilitation trial in advanced head and 

neck cancer patients treated with 

chemoradiotherapy: feasibility, compliance, 

and short-term effects. Dysphagia 

2011;26:155-70 

Van Kesteren Z, Olszewska A, Belderbos 

J, van Vliet-Vroegindeweij C. Letter to the 

editor: The distribution of brain metastases in 

the perihippocampal region. Radiother Oncol 

2011;98:143-44

136 

RADIOTHERAPY 


Van Kesteren Z, Belderbos B, van Herk 

M, Olszewska A, Lamers E, De Ruysscher 

D, Damen E, van Vliet-Vroegindeweij C. A 

Practical Technique to Avoid the 

Hippocampus in Prophylactic Cranial 

Irradiation for Lung Cancer. Radiother Oncol 


Van Lummel M, van Blitterswijk WJ, Vink 

SR, Veldman RJ, van der Valk MA, Schipper 

D, Dicheva BM, Eggermont AMM, ten 

Hagen TLM, Verheij M. Enriching lipid 

nanovesicles with short-chain 

glucosylceramide improves doxorubicin 

delivery and effi cacy in solid tumors. The 

Faseb Journal 2011;25:280-9 

Van Mourik A, van Kranen S, den 

Hollander S, Sonke JJ, van Herk M, van 

Vliet-Vroegindeweij C. Effects of setup errors 

and shape changes on breast radiotherapy. Int 

J Radiat Oncol Biol Phys 2011;79:1557-64 

Vogel WF, Guislain A, Kvistborg P, 

Schumacher TN, Haanen JB, Blank CU. 

Ipilimumab-induced sarcoidosis in a patient 

with metastatic melanoma undergoing 

complete remission. J Clin Oncol 2011(in 

press) 

Werkhoven E, Hart G, van Tinteren H, 

Elkhuizen P, Collette L, Poortmans P, 

Bartelink H. Nomogram to predict ipsilateral 

breast relapse base don pathology review from 

the EORTC 22881-10882 boost versus no boost 

trial. Radiother Oncol 2011;100:101-7 

Witte M, Shakirin G, Houweling A, 

Peulen H, van Herk M. Dealing with 

geometric uncertainties in dose painting by 

numbers: Introducing the ΔVH. Radiother 


(lateral/contra-lateral, 0.25/0.27 %/day SD). However, linear regression was not 

always appropriate: some patients only showed a single moment of volume reduction 

during treatment. Four out of 8 patients demonstrated a notable correlation in 

volume changes with the administration of chemotherapy around day 1, 22, 43. The 

center of mass of the glands had moved for all patients inwards during treatment: 

0.6, 1.6 and 2.3 mm in week 1&2, 2&3 and 4 to 7 relative to the planning situation 

(0.8, 0.8, 0.8 mm 1SD) . For the contra-lateral side, this was 0.9, 1.9 and 2.6 mm 

(0.9, 0.9, 1.2 mm 1SD) respectively. In conclusions, large inter-fraction and interpatient 

variability in parotid volume and position was observed, occasionally related 

to administration of chemotherapy. Therefore applying deformable registration to 

routinely assess daily geometrical variation of parotid glands is a feasible method to 

select patients that may benefi t from plan adaptation. 

BREAST 

Intra-fraction motion during partial breast irradiation A study concerning preoperative 

image guided accelerated partial breast irradiation (PAPBI) in early breast 

cancer patients has recently started in our institute. A marker placed during biopsy 

aids target volume delineation and image guidance using cone beam CT (CBCT). 

Intra-fraction movement is one of the residual uncertainties after image guidance. 

The purpose of this study was to quantify intra-fraction motion in patients treated 

with pre-operative PAPBI. For all 12 daily fraction in 8 patients CBCT scans were 

acquired prior to irradiation and registered to the bony anatomy fi rst, and next to 

the marker. Setup correction was performed based on the marker position. Post 

treatment another CBCT was acquired to determine intrafraction movement. When 

intra-fraction translations larger than 0.5cm were observer in any direction, an extra 

CBCT halfway the fraction was scheduled for this patient. Considerable intra-fraction 

motion was observed with a group mean of -0.09, -0.16 and -0.37 cm in the left-right 

(LR), cranial-caudal (CC) and anterior-posterior position respectively. Systematic 

position variability was 0.14, 0.09 and 0.26 cm while random intrafraction motion 

was 0.15, 0.17 and 0.27 cm respectively. In 50% of the patients an extra scan halfway 

each treatment fraction was made. In conclusion, large intrafraction motion was 

observed in the AP direction possibly due to the effect of gravity on the larger breast 

volumes. As such motion can have a severe effect on the delivered dose, methods to 

further minimize this source of geometrical uncertainties is warranted. 

LUNG 

4DCT – high quality imaging and contrast enhancement for 3D radiotherapy 

treatment planning The Mid-position CT is a 3D CT derived from a 4D CT through 

deformable registration with the anatomy in the time averaged mean position. The 

purpose of this study was to evaluate this new technique. To that end, ten patients 

were selected with a clearly visible tumour in the periphery of the lung. For all 

patients, a 4D-CT and an expiration breathhold (BH) CT were acquired. For 5 of 

these patients, contrast was given during acquisition of the BH-CT. Deformable 

registration was realized by applying an iterative multi-scale phase-based optical 

fl ow estimation procedure. For each patient 2 different 4D deformable registrations 

were performed. During the fi rst, the image set of maximum expiration was 

used as reference scan to which all other image sets were registered. This yields 9 

deformation vector fi elds (DVF) plus a reference DVF with motion 0. Subsequently, 

the average DVF over all image sets was subtracted from each DVF yielding all 

deformations with respect to the time weighted average positions. This DVF was 

applied to deform all image sets to the time weighted average position. Finally, the 

MidP-CT was calculated as median grey-value for each pixel over all 10 image sets. 

In the second warping procedure, the expiration BH-CT was taken as reference 

and an additional DVF from the BH-CT to the time-weighted average position was 

calculated. With the latter, a MidP-BH-CT was calculated by deforming the BH-CT 

to the time-weighted average position. Comparing the MidV-CT reconstructed from 

limited Ct-data with the MidP-CT, improvements were seen in the following : in 

the MidP-CT the frequency of artefacts due to irregular motion was clearly reduced. 

Also the signal to noise ratio increased. For those scans at which contrast was given, 

the contrast enhancement was for the MidP-CT increased to about 120 HU in vessels 

at the levels of tumours compared to MidV-CT. In conclusion, the use of deformable

egistration to derive a MidP-CT has proven to be robust and has been implemented 

clinically. Image quality has shown to be improved in reduced artefacts and noise. 

With this procedure more accurate and reproducible delineation of the tumour and 

involved lymph nodes can be expected (fi gure 5). 

Mediastinal lymph node position variability in Non-Small Cell Lung Cancer 

patients treated with radiotherapy Standard margins used often used for the 

pathological mediastinal lymph nodes in radiation therapy of lung cancer patients 

in the absence of detailed knowledge, on the position variability of these lymph 

nodes. The purpose of this study was to quantify mediastinal lymph nodes position 

variability relative to the bony anatomy (baseline variation) over the course of 

radiation therapy. Fiducials (0.35mm by 5mm golden markers) were placed in at 

least one lymph node station of non-small cell lung cancer (NSCLC) patients under 

Propofol sedation during a diagnostic trans-oesophageal endoscopic ultrasound 

fi ne needle aspiration (EUS-FNA) procedure. For fi ve patients a 4D planning CT 

and daily 4D CBCT scans were acquired during the radical radiotherapy course (66 

Gy/24 fractions). All CBCT scans were fi rst registered based on bony anatomy to the 

planning CT. The fi ducial marker in each scan was subsequently registered frame by 

frame to the planning CT. The range of motion of the fi ducial in the 4D CBCT scans 

was calculated to quantify the motion amplitude of the lymph node due to breathing. 

Differences between the bony anatomy- and the average 4D fi ducial registration 

were used to calculate the systematic and random baseline shifts. The peak-to-peak 

amplitude was largest in the cranial-caudal direction with an average (range) of 

0.51 (0.18 - 1.0) cm. The systematic baseline shifts were 0.14, 0.29 and 0.10 cm in 

the LR, CC and AP direction respectively. The random baseline shifts were 0.14, 

0.22 and 0.18 cm. In conclusion, substantial geometric uncertainties were observed 

for mediastinal lymph nodes during radical treatment of NSCLC patients using 

implanted fi ducial markers. These geometric uncertainties were predominantly in 

cranial-caudal direction, indicating the non-uniform margins could be benefi cial. 

A decision protocol for intra thoracic anatomical changes visualized on Cone 

Beam CT Substantial intra thoracic anatomical changes are regularly observed 

in CBCT scans routinely acquired for IGRT of lung patients. The purpose of this 

study is to develop a decision protocol to respond to such changes. From January 

2010 until November 2010, 180 patients were treated for lung cancer with a radical 

Intensity Modulated Radiotherapy (IMRT) plan. From the 83 patients, 7 CBCT’s 

with the most extreme intra thoracic anatomical changes of the normal tissues 

and/or tumor were selected. For every anatomical change the urgency of taking 

action was determined by our multi disciplinary team. Three urgency levels were 

classifi ed. Level 1 “immediate action” included the changes that affect the tumor 

and lymph nodes position with respect to the PTV. These changes can be caused 

by tumor growth, a large in- or decrease of atelectasis and focal increase of normal 

lung density, like post obstructive pneumonia. For level one changes the radiation 

oncologist needs to be consulted immediately, as a new planning CT or medication 

for the patient may be required. Urgency level 2 “No immediate action, inform 

radiation oncologist” includes minor in- or decrease of atelectasis and pleural 

effusion. Tumor reduction, without any other anatomical changes was determined 

to be in action level 3: “Positive change, no action”. For all urgency levels, except 

urgency level 1, the radiation oncologist was informed by email with a screenshot 

of the CBCT included, as no immediate decision by the radiation oncologist is 

required. The radiation oncologist responded before the next fraction. All RTT’s have 

now been trained in using the three urgency levels. 

Preliminary results of simultaneous CBCT acquisition during VMAT 

irradiation for SBRT in NSCLC patients Treatment times in SBRT lung patients 

have been reduced dramatically by replacing static IMRT with VMAT based 

treatment plans. VMAT based delivery, allows for simultaneous CBCT acquisition 

enabling verifi cation of the tumour position during the actual treatment. This 

purpose of this study was to evaluate preliminary results of this novel imaging 

opportunity. 16 patients receiving SBRT for NSCLC were setup according to 

departmental guidelines. 4D-CBCT scans simultaneously acquired during the fi rst 

Figure 5 

137 

RADIOTHERAPY

138 

RADIOTHERAPY 

arc were checked on completion before continuing with the second arc. Evaluation 

of the setup correction of the arc scans and any extra scans acquired during the arcs 

was performed. Group mean, SD and RMS were calculated between the arcs. In 

total 39 scans in 16 patients were analysed. Only in 2 cases was a setup correction 

actually performed as values exceeded departmental threshold values. (>2mm). 

Between the corrected scan and last arc, the greatest difference was observed in the 

Left/Right (LR) direction (1mm ± 3mm) for both the bony and tumour registration. 

In conclusion, the introduction of simultaneous (4D)CBCT acquisition during 

VMAT treatment in NSCLC patients allows tumour position verifi cation during 

treatment without the addition of extra time. Occasionally, intrafraction motion is 

corrected based on these scans. 

PROSTATE 

Collimator angle adjustments to account for rotational prostate and seminal 

vesicles misalignments during VMAT The largest rotations of the prostate and 

seminal vesicles occur around the left-right (LR) axis. Previously, an advanced 

correction strategy was developed based on collimator angle adjustments and 

validated for static IMRT treatments. Recently, however, volumetric modulated arc 

treatments have been introduced clinically for prostate cancer RT which includes 

a wide range of beam orientations and differs in modulation. The aim of this 

study was to validate this advanced correction strategy based on collimator angle 

adjustments for VMAT dose delivery. 

Inversely optimized single arc VMAT plans were created for ten prostate cancer 

patients. Portal images were acquired during treatment, binned for each 2° segment 

of the arc and subsequently the delivered 3D dose for each segment was calculated 

by our back-projecting based portal dose reconstruction algorithm. Collimator 

adjustments were simulated by rotating the reconstructed 3D dose of each segment 

around the collimator axis and compared to rotations of the 3D dose rotated around 

the LR axis. The total delivered 3D dose was calculated by accumulating over all 

segments. 

The difference between corrected D min and planned D min was generally smaller 

than 5%, except for large positive prostate rotations, where the tip of the seminal 

vesicles and apex rotate out of AP oriented beams for positive rotations, whereas 

they remain almost fully covered for negative rotations. The correction strategy 

signifi cantly showed better dose coverage for prostate rotations < -5° and > 15° (p < 

0.05). In conclusion, collimator angle adjustments safely correct LR rotations of the 

prostate and seminal vesicles for VMAT based treatment plans. 

Online palliative radiotherapy of bone metastases of the spine Many cancer 

patients develop symptomatic skeletal metastases, which may cause considerable 

morbidity such as pain, fractures, neurological complications and mortality. External 

beam radiotherapy (RT) plays an important role in the palliative management of 

these patients. Conventional simulators and CT based virtual simulation can be used 

to defi ne irradiation fi elds. In order to reduce the number of painful transfers from 

bed to simulator and treatment couch and to reduce the total treatment time for the 

patient, we developed a cone beam CT (CBCT) procedure that covers both simulation 

and treatment verifi cation for bone metastases. An in-house developed software 

program was developed to use a diagnostic CT, PETCT or MRI scan as a reference 

scan for the irradiation. In this application an isocenter is defi ned and standardized 

treatment fi elds (width of 10 cm and a variable length of 7-20 cm at intervals of 0.5 

cm) are chosen. Each treatment plan is designed to deliver 1x8Gy at a depth of 5 cm 

via a single PA fi eld. At the treatment machine preparations for the irradiation and 

image guidance are performed (i.e. importing the data in the Record & Verifi cation 

and image guidance systems). The patient is positioned on the treatment couch and 

a CBCT is acquired. This CBCT is registered to the diagnostic CT, PETCT or MRI 

scan. A couch shift is performed and as a second check an electronic portal image is 

acquired by administering 10MU’s. The patient can then be irradiated. This initial 

evaluation is based on 23 patients treated using this new application; all patients 

had metastases in the thoracic or lumbal vertebrae. In 9 patients the diagnostic 

CT-scan was used as a reference scan, in 5 patients the PETCT and in 8 patients 

the MRI scan. The preparation time (without patient) was about 45 minutes, which

includes preparation in the new application, treatment machine (Mosaiq) and the 

CBCT software (XVI). The total treatment time, which includes the positioning of 

the patient, acquiring a CBCT, registration to the reference scan and irradiation, was 

on average 23 minutes (range 13 – 45). In conclusion, a quick, reliable and patient 

friendly procedure was developed to treat patients with painful skeletal metastases 

using the CBCT for simulation and treatment verifi cation. 

BREAST CANCER 

Femke van der Leij, Marc van de Vijver (AMC, Department of Pathology, Amsterdam), Jelle 

Wesseling, Kenneth Gilhuijs, Hester Oldenburg, Claudette Loo, Adrian Begg, Wouter Vogel, Sandra 

Vreeswijk, Corine van Vliet-Vroegindeweij, Harry Bartelink, Paula Elkhuizen 

DEFINING RADIOTHERAPY SENSITIVITY 

A. Image guided Preoperative Accelerated partial Breast Irradiation (PAPBI) 

Radiotherapy is part of breast conserving therapy (BCT) and is known to reduce local 

recurrence rates in all patients by 60-70%. So far, no patient groups can be defi ned 

in whom radiotherapy would not be necessary. It is estimated that in approximately 

half of the patients whole breast radiotherapy is not necessary, while in others the 

tumor might be resistant to radiotherapy. If it would be possible to predict tumor 

response to radiotherapy, a more tailored treatment can be advised to individual 

patients (higher boost dose or primary mastectomy). To evaluate the in situ breast 

radiosensitivity, the image guided preoperative accelerated partial breast irradiation 

(PAPBI) is the best treatment strategy because of (i) low breast α/β ratio suggesting 

that breast cancer is more sensitive to high dose per fractions; (ii) very precise breast 

tumor irradiation; (iii) limited volume of irradiated area allowing larger fractions 

with no extra risk on late toxicities; (iv) short overall treatment time. 

This trial is directed at implementing pre-operatively given image guided accelerated 

partial breast irradiation without compromising local control in early breast cancer 

patients. By assessing tumor response to radiotherapy, the goal of the study is to 

develop a gene expression profi le that predicts the breast cancer radiosensitivity. 

This gene signature of breast radiosensitivity would further design optimal 

treatment strategies for individual breast cancer patients treated with BCT 

To qualify for the trial, patients must be 60 years or older, and have an unifocal cT1-2 

(

140 

RADIOTHERAPY 

B. Radiosensitivity breast cancer cell lines 

In addition with the clinical PAPBI study, 30 human breast cancer cell lines will be 

investigated for their radiosensitivity profi le. This will also be implemented in the 

study described in D. 

C. Case control study focusing local recurrence 

Genetic profi ling will be studied in 240 breast cancer patients (79 cases with 161 

matched controls). This study is in association with Institut Curie from France. 

The results will also be implemented in the study described in D. 

D. In the ongoing Young Boost Trial over 1200 patients under 50 years haven 

been randomized between normal boost dose vs. a higher boost dose after breastconserving 

therapy. From these patients frozen material as well as paraffi n 

embedded material is collected. Follow up is available for the fi rst years. The profi les 

found in the studies A-B to be related with radioresponse will be evaluated on this 

material whether a response profi le eventually is associated with local control. In 

addition, the results from the case-control study will be evaluated on this cohort. 

E. Cohort analysis breast conserving treatment in 10.000 patients treated between 

1979 and 2008. AL patients were radiated at the NKI-AVL, surgery was performed 

at the NKI-AVL or referring hospitals. Local relapse will be studied in relation to 

treatment year, patient characteristics and treatment factors. 

F. A new phase II study is in preparation in the Netherlands comparing the toxicity 

of 2 different pre-operative Partial Breast Irradiation schedules (10 x 4 Gy versus 

5 x 6 Gy). 

COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY / 

BIOLOGICALS 

Berthe Aleman, Harry Bartelink, José Belderbos, Andre Bergman, Jan Paul de Boer, Henk Boot, 

Michiel van den Brekel, Annemieke Cats, Frits van Coevorden, Ewout Courrech Staal, Luc Dewit, 

Johan Dikken, Ria Dubbelman, Rick Haas, Olga Hamming, Michel van den Heuvel, Simon 

Horenblas, Martijn Kerst, Edwin Jansen, Joost Knegjens, Maria Kuiper, Arash Navran, Floris Pos, 

Coen Rasch, Johanna van Sandick, Jan Schellens, Jurriën Stiekema, Anouk Trip, Vic Verwaal, 

Thelma Witteveen, Marcel Verheij 

Head and neck The CONDOR cooperative trial targets patients below 60 years 

with stage III/IV head and neck cancer and is open since 2008. Patients fi rst 

receive 3-4 courses of TPF chemotherapy followed by a randomization between two 

regimens of chemoradiation (CRT). Fifty-nine of the required 70 patients have been 

included so far. 

The fi nal report on the 5-year quality of life results for patients with CRT treated 

within the M99RAD trial was published. Results remain favorable compared to 

historical series with smaller tumors treated with surgery and radiotherapy. Quality 

of life before treatment and after one year is predictive for (disease specifi c-) survival. 

We also reported on an analysis of the tumor volume as prognostic factor in CRT 

for advanced head and neck cancer. Multivariate analysis in a series of 360 patients 

showed a signifi cant effect of tumor volume on local control. The hazard ratio for a 

local recurrence increased by 14% per 10 cc volume increase (95% CI, 8% to 21%). 

There was no signifi cant independent effect of T and N status on local control. 

The results of a randomized preventive rehabilitation trial (n=49) in advanced head 

and neck cancer patients treated with CRT were published. Preventive rehabilitation 

(regardless of the approach, i.e. experimental or standard) in head and neck cancer 

patients, despite advanced stage and burdensome treatment, is feasible, and 

compared with historical controls this approach seems helpful in reducing the 

extent and/or severity of various functional short-term effects of concurrent CRT. A 

cost-effectiveness analysis of this trial was also published demonstrating a higher 

probability of this endpoint by a preventive swallowing exercise program for CRT in 

advanced head and neck cancer when compared to standard care. 

Based on our preclinical research on apoptosis-modulation, we have initiated a 

clinical phase I-II trial in locally advanced head and neck cancer combining standard

cisplatin-based CRT with dose escalating AT-101, a small molecule inhibitor of antiapoptotic 

Bcl-2/Bcl-XL. The trial is open since 2010 and to date, 12 patients have 

been included; no DLT has been observed. 

Gastroenterology – Esophageal cancer Over recent years a database was set up 

including data on all patients treated for esophageal cancer in our institute since 

1997 (approximately 1500 patients). As previously reported, we evaluated the toxicity 

and effi cacy of three different regimens of concurrent chemoradiation (CRT) in 94 

patients with esophageal cancer treated between 1997 and 2007. Furthermore, the 

database was used to study quality of care oesophageal cancer patients in a broader 

perspective. Between 2003 and 2008, 821 oesophageal cancer patients were referred 

to our institute. Indicators to measure quality of care (i.e., process and outcome 

measures) were defi ned and comparisons between two time periods were made. 335 

patients came for a second opinion only, 382 patients received palliative treatment 

and 104 (13%) patients underwent potentially curative treatment. The median time 

between the fi rst hospital visit and start of treatment decreased from 24 days in 

period I to 18 days in period II (p=0.03). Of patients who underwent potentially 

curative treatment, 81% in period I and 86% in period II were discussed during a 

weekly multidisciplinary meeting (p=0.54). Compliance with the national guideline 

was comparable in both periods (84% vs. 80%, p=0.27). There were no signifi cant 

differences in outcome. By evaluating different dimensions of health care quality, we 

have identifi ed which steps in the multidisciplinary care path need more attention in 

order to raise the whole level of care. Efforts for improvement should focus primarily 

on process measures rather than on outcome measures for which high-quality 

standards are already met. 

Gastroenterology – Gastric cancer In the US Intergroup 0116 study, a signifi cant 

survival benefi t in postoperative CRT was reported in gastric cancer. Based on these 

results we completed three adjuvant chemoradiotherapy (CRT) phase I-II trials in 

gastric cancer. From these trials we identifi ed a regimen for the experimental arm in 

the CRITICS study. In this international randomized phase III study patients with 

operable gastric cancer receive preoperative chemotherapy, surgery and postoperative 

chemotherapy, or preoperative chemotherapy, surgery and postoperative CRT. At this 

moment over 400 of the required 788 patients have been randomized. In addition 

to Sweden that joined the trial in 2008, Denmark participates as well since ultimo 

2010. Furthermore, we are running a phase I-II study together with the AMC and 

VUmc, in which neoadjuvant CRT with weekly paclitaxel and capecitabine is applied 

in patients with inoperable gastric cancer. So far, 10 patients have been included in 

this NARCIS trial. The results of this trial have been combined with those from the 

University Medical Center Groningen where a comparable study was carried out. 

At the moment we are investigating the late effect of CRT on kidney, spleen and liver 

function. Furthermore, as we found signifi cant interobserver variation in contouring 

a clinical target volume (CTV) for radiotherapy planning, we are prospectively and 

retrospectively performing quality assurance of radiotherapy treatment plans in the 

earlier mentioned CRITICS study. 

Gastroenterology – Rectal cancer Pre-operative capecitabine-based 

chemoradiotherapy (CRT) has become standard treatment in locally advanced rectal 

cancer. We evaluated acute toxicity and surgical complications in these patients 

following total mesorectal excision (TME) after preoperative CRT with capecitabine. 

Between 2004 and 2008, 147 consecutive patients with clinical tumour category (cT) 

3-4 (with a threatened circumferential resection margin or cT3 within 5 cm of the 

anal verge) or clinical node category 2 rectal cancer were treated with preoperative 

CRT (50 Gy, capecitabine 825 mg/m 2 twice daily, days 1-33). TME followed 6 weeks 

later. Toxicity was scored according to the CTC (version 3.0) and RTOG scoring 

systems. Treatment-related surgical complications were evaluated for up to 30 days 

after discharge from hospital using the modifi ed Clavien-Dindo classifi cation. The 

mean cumulative dose of capecitabine was 95% and 98% of patients received at least 

45 Gy. One patient died from sepsis following haematological toxicity. Grade 3-5 

toxicity developed in 32 patients (22%), in particular diarrhoea (10%) and radiation 

dermatitis (12%). There were no deaths within 30 days after surgery. Anastomotic 

141 

RADIOTHERAPY

142 

RADIOTHERAPY 

leakage and perineal wound complications developed after 13 of 47 low anterior 

resections and 23 of 62 abdominoperineal resections. Surgical reintervention was 

required in 30 patients. Twenty-seven patients (20%) of 138 patients who had a 

laparotomy were readmitted within 30 days after initial hospital discharge. We 

concluded that preoperative CRT with capecitabine is associated with acceptable 

acute toxicity, signifi cant surgical morbidity but minimal postoperative mortality. 

Lung - NSCLC In 2010 an increasing amount of inoperable non-small cell lung 

cancer (NSCLC) patients received concurrent chemoradiotherapy (CCRT) with daily 

cisplatin (100 patients up to December 1st). Dose-escalation using IMRT is studied 

by boosting the radiation dose within the primary tumor based upon biological 

activity on pre-treatment FDG-PET scan. In 2010 a randomized phase II ”PET-Boost 

study” (M09PBO) was initiated in close collaboration with prof. de Ruysscher in 

patients with inoperable stage II or III NSCLC. Patients are randomized to receive 

the standard 66 Gy in 24 fractions with a dose escalation to the primary tumor as 

a whole or to the volume with ≥50% SUVmax of the primary tumor (on the pretreatment 

PET-FDG scan). In both treatment arms, the patients are irradiated to the 

same maximum tolerated dose to the lung and receive concurrent chemotherapy. 

The primary objective of this study is local progression free survival at 1 year. 

Especially, recurrence patterns as a function of the local dose and volume will be 

studied. Until November 2011, 23 patients were randomized (12 patients from the 

NKI-AVL). 

In May 2011 the RADITUX trial (M07CCL) was closed after accrual was completed. 

A total of 93 patient3 from NKI-AVL were included. In this multi-center randomized 

phase II trial patients with inoperable locally advanced NSCLC were randomized 

to our CCRT regimen (66 Gy in 24 fractions and daily Cisplatin 6 mg/m 2 ) with 

or without the weekly addition of the EGFR monoclonal antibody Cetuximab. The 

addition of Cetuximab to our CCRT was well tolerated in a phase I trial. In the 

group of NSCLC patients treated with concurrent chemoradiotherapy using IMRT, 

we studied the esophagus toxicity using dosimetric parameters. Almost 23% of the 

185 patients studied treated with CCRT developed esophagitis grade 3 (CTC-3.0). 

The V50 was identifi ed as most accurate predictor of AET grade ≥3 (fi gure 6). A 

signifi cant higher incidence was observed, especially of grade 3 AET, in this patient 

group treated with IMRT and CCRT, compared to previous series of patients treated 

with sequential chemo-radiation or RT-only and 3D-conformal radiotherapy. This 

increased risk is probably due to the concurrent use of chemotherapy as well as the 

more inhomogeneous dose distributions typically prescribed with IMRT. 

Figure re 6 

Lung - SCLC Approximately 20% of malignant tumors of the lung are due to 

small cell carcinoma. In general, these patients carry a worse prognosis compared 

to NSCLC patients. For limited stage small cell lung cancer (LS-SCLC), the 

combination of chemotherapy and thoracic radiotherapy is the standard treatment. 

Two meta-analyses have shown that thoracic radiotherapy given concurrently with

chemotherapy improves both local control and survival. Nevertheless, several 

important questions including the optimal total radiation dose and radiation 

fractionation are still unanswered. To establish a standard chemo-radiotherapy 

regimen for LS-SCLC, an EORTC international, multicenter randomized phase III 

trial started in 2009 comparing twice daily radiotherapy with high dose radiation 

delivered once daily, both given concurrently with standard cisplatin and etoposide 

chemotherapy (CONVERT trial). To date, approximately 266 patients were treated 

within this trial (3 patients from the NKI-AVL). A comparable 3-arm RTOG trial has 

started accrual in 2008 (RTOG 0538). 

For extensive stage (ES-) SCLC, chemotherapy is the cornerstone of treatment. 

However, over 75% of patients have persisting intra-thoracic disease after initial 

chemotherapy, and about 90% manifest intra-thoracic disease progression at 1 year 

after completing initial chemotherapy. In the absence of promising systemic agents 

that can improve local response, the role of thoracic irradiation in patients with 

ES-SCLC is currently being evaluated in a multicenter phase III randomized trial 

initiated by the VUmc (CREST trial). The objective of this study is to investigate 

whether thoracic radiotherapy can improve 1-year survival, following a response to 

chemotherapy. A total of 284 patients have now been treated within this trial. At 

the NKI-AVL, patient accrual started in September 2009 and to date 32 patients 

have been included. Within the RTOG, a Phase II trial to determine the role of 

consolidation extracranial radiotherapy (thoracic and other extracranial metastatic 

sites) plus prophylactic cranial irradiation after a response to systemic chemotherapy 

has been activated (RTOG 0937). 

Urology – Bladder The standard treatment for muscle invasive bladder cancer is 

cystectomy. For those not eligible for this type of treatment, or for those wanting 

to preserve their bladder, the alternative treatment is radiotherapy preferably in 

combination with cisplatin-based chemotherapy. Although the reported results 

following cisplatin-based chemoradiotherapy compare reasonably well with those of 

cystectomy, there is substantial room for improvement. Two thirds of the patients 

achieve a complete response rate, but the reported 5-year survival rate varies between 

30 and 50%. Moreover, the combination of radiotherapy with cisplatin-based 

chemotherapy appears to be rather toxic. Therefore, we are currently looking for less 

toxic and more effective radiosensitizers. 

In recent years, new generations of novel biology-driven drugs have been developed. 

New molecular targeting agents can interfere with complex cell–cell interactions 

and micro-environmental factors in the tumor. Some of these drugs can selectively 

interfere with radiobiological mechanisms of tumor resistance, and thereby 

potentially increase the effi cacy of radiotherapy, for example EGFR-interfering agents. 

In bladder cancer, EGFR status is associated with poor outcome and seems to be 

linked to radiation sensitivity. Its inhibition might enhance the radio-responsiveness 

of bladder tumors and improve the outcome of radiotherapy for bladder cancer. Given 

the very high affi nity for EGFR in combination with the favorable toxicity profi le, 

the combination of radiotherapy with concurrent Panitumumab is very attractive 

to explore in bladder cancer. In close cooperation between the departments of 

Urology, Medical Oncology and Radiotherapy, we initiated a phase I trial evaluating 

the combination of radiotherapy with Panitumumab. A schedule of 66 Gy in 33 

daily fractions of 2 Gy is combined with weekly courses of Panitumumab in a dose 

of 2.5 mg/kg i.v. over 60 m to a total of 7 courses. For this trial a sample size of 31 

patients has been chosen. Primary endpoint of the study is the acute toxicity rate 

during radiotherapy with Panitumumab. Secondary endpoints are the complete 

response rate at 3 months, local control rate at 6, 12, and 18 months, and at 2 years, 

bladder preservation rate and any grade 3 or 4 adverse event during and within one 

month after completion of therapy. Exploratory endpoints are % EGFR expression, 

RAS mutational status, response rate in correlation with EGFR and RAS status and 

response rate in relation to treatment path. 

Urology – Prostate In a randomized, double-blind multicenter phase 3 trial we 

are comparing ipilimumab vs placebo following radiotherapy in patients with 

Castration Resistant Prostate Cancer (CRPC) who have received prior treatment 

with docetaxel. The hypothesis tested is that treatment with radiotherapy followed 

143 

RADIOTHERAPY

144 

RADIOTHERAPY 

by ipilimumab results in a signifi cant increased survival compared to radiotherapy 

followed by placebo in patients with CRPC, who have progressed during or following 

prior docetaxel treatment. The mechanism behind this is that the combination of 

radiotherapy to symptomatic bone metastasis with an agent able to stimulate T-cell 

response will amplify the immune response generated by radiotherapy and will 

result in systemic anti-tumor activity leading ultimately to an improved survival. 

Subjects receive radiotherapy to metastatic bone lesions at 8 Gy for 1 day with 2 

days of the fi rst dose of blinded study drug. The blinded study drug (10 mg/kg 

ipilimumab or placebo) will be given every 3 weeks for up to 4 doses. Until now 

7 patients have been included in the trial and at this moment 4 patients are in 

screening phase (December 1st, 2011). So far, no severe side effects are reported in 

this small group of patients. One patients continued treatment after the induction 

phase. 

Soft tissue sarcoma STS represent less than 1% of all newly diagnosed malignant 

tumors. Approximately 40% of STS occur in the extremity. For patients diagnosed 

with localized disease, the combination of surgery and radiation therapy achieves 

better outcomes than either treatment alone. In addition to its benefi t in reducing 

local relapse rates, a signifi cant advantage on limb preserving therapy is observed. 

Hence, the combination of radiotherapy and surgery is considered standard 

treatment in selected cases. Preoperative radiation might reduce tumor burden prior 

to resection, potentially allowing more conservative surgical therapy. Postoperative 

radiation allows histological examination of the tumor specimen, especially the 

margins, aiding in further treatment planning, and may be associated with less 

wound complications. In preoperative, as compared to postoperative radiation, lower 

doses (50 versus 60 to 66 Gy) and smaller fi eld sizes can be used, resulting in a 

reduced risk of late, often irreversible complications. Consequently, preoperative 

radiation is the preferred approach in many centers. 

Sarcomas with a myxoid histology (myxoid liposarcomas and myxoid fi brosarcomas) 

show a remarkable tumor necrosis after preoperative irradiation to 25 x 2 Gy and this 

phenomenon might be explained by a shut-down of the crowfeet-like vasculature. 

As the tumor vasculature is a target in the radiation treatment of sarcomas other 

than myxoid types, the combination with an anti-angiogenic compound should be 

pursued. Furthermore, small molecule vascular endothelial growth factor receptor 

tyrosine kinase inhibitors (VEGFR-TKIs) exert promising anti-tumor activity against 

several STS-subtypes. In the M09RTP phase I clinical study of a combined modality 

treatment of sarcomas of the extremities with radiotherapy and dose-escalation of 

Pazopanib, we aim to defi ne the recommended dose of this drug in combination 

with preoperative radiation. So far (n=3), no DLTs have been encountered. 

BRACHYTHERAPY 

Berthe Aleman, Marcel Steggerda, Baukelien van Triest, Thelma Witteveen, Simon Horenblas, 

Floris Pos, Luc Moonen 

Brachytherapy of the prostate Brachytherapy with low dose rate I-125 seeds has 

proven to be a highly effective treatment modality for both low- and intermediaterisk 

prostate cancer. Research in our group focuses on efforts to reduce the toxicity 

of this treatment. Mandatory is to indentify predictors for frequently occurring 

side effects such as lower urinary tract symptoms (LUTS) and erectile dysfunction. 

Previous research has identifi ed dose hot spots in the bladder as a predictor for 

LUTS. In an effort to gain more detailed information about the infl uence of the dose 

distribution on the occurrence of LUTS, the bladder was divided in the sections 

bladder wall and bladder neck on post-implant CT images. The bladder neck was 

defi ned as a structure including the urethral orifi ce plus a 5 mm margin in all 

directions. In a group of 108 patients the dose to 1 cm 3 (D 1cc) in the bladder neck as 

well as the prostate volume were found to be independent predictors for early LUTS 

(p

equired a transurethral resection (TURP) of the prostate to resolve persistent 

obstructive urinary symptoms. The median time interval between brachytherapy 

and TURP was 14 months. Prostate volume and D 1cc of the bladder appeared to 

be independent predictors (p=0.017 and p=0.042 resp.). Dividing the group in 

combinations of -equal or smaller- and -larger than- median values of the prostate 

volume and the D 1cc (41 cm 3 and 131Gy, resp.) resulted in the Kaplan-Meier diagram 

shown below (fi gure 7). 

Figure 7 

Preparations for the realization of focal brachytherapy, i.e. treating the dominant 

cancer lesion within the prostate only, are ongoing. The expectation is that, in a 

selected group of patients, focal brachytherapy will reduce morbidity substantially 

compared to whole gland brachytherapy, without compromising local control. 

Technical feasibility studies are ongoing and involve identifi cation and delineation of 

the dominant lesion and the planning target volume on combinations of functional 

and anatomical MRI images, the registration of pre-treatment MRI images and 

ultrasound images used for treatment planning on the OR, and the geometrical 

accuracy of I-125 seed placement. 

Brachytherapy of the cervix In locally advanced cervical cancer, the treatment of 

choice is a combination of EBRT, chemotherapy and brachytherapy. In recent years, 

Image Guided Brachy Therapy (IGBT) has been introduced in the NKI-AVL. Using 

IGBT for cervical cancer, dose distributions are customized for each individual 

patient and fraction, making use of 3D MRI information. MRI information is the 

key to accurate target and OAR delineation, due to its superior soft tissue contrast. 

Furthermore, the presence of 3D anatomical information opens up the possibility 

to calculate dose volume parameters of the combined radiotherapy treatment (EBRT 

and brachy). 

The EMBRACE study is an observational study to link clinical outcome to dosimetric 

treatment parameters in locally advanced cervical cancer. Although the NKI-AVL 

is not participating, our treatments are being adapted as much as possible to the 

EMBRACE study protocol. Consistency of target delineation and reporting of 

dosimetric parameters among the participating centres is an important aspect of the 

study. 

The dose prescription for the complete treatment (EBRT and brachy) was adapted 

from the Utrecht group (D90 of the HR-CTV >85 Gy, D2cc of rectum and sigmoid 

146 

RADIOTHERAPY 

geometric accuracy of MRI imaging of the needles, and performed a planning study, 

showing the benefi t of using needles. However, more experience has to be gained to 

fully exploit the possibilities that the needles offer for treatment individualisation. 

In summary, in 2011 signifi cant steps were made to implement state-of-the-art 

brachytherapy for cervical cancer in the NKI-AVL. 

MECHANISMS, MODULATION AND PREDICTION OF 

RADIATION-INDUCED CELL DEATH 

Maaike Alderliesten, Wim van Blitterswijk, Jannie Borst, Lília Cordeiro Pedrosa1 , Ben Floot, Albert 

van Hell, Gerben Koning1 , Dyane Martins, Rogier Rooswinkel, Baukelien van Triest, Conchita Vens, 

Shuraila Zerp, Marcel Verheij 

1Erasmus MC, Rotterdam, the Netherlands 

The basic and translational research performed within our group aims at (1) the 

identifi cation and preclinical testing of novel targets and agents to enhance the 

cytotoxic effect of radiation and on (2) the validation of new endpoints and clinical 

biomarkers to quantify and predict the effi cacy and toxicity of new combination 

therapies. The ultimate objective is to rapidly translate these strategies from the lab 

into the clinic. 

In close collaboration with several research groups within the NKI, new agents 

are identifi ed on the basis of their mechanism of action and subsequently tested 

for their ability to induce apoptotic cell death and to increase the cytotoxic effect 

of radiation in vitro and in vivo. Current research projects focus on synthetic 

alkyl-phospholipids (Edelfosine, Perifosine, Erucyl-PC), death receptor ligands 

(TRAIL, CD95L/FasL/APO010), small molecule inhibitors of Bcl-2 (Gossypol/AT- 

101, ABT-737) and DNA damage repair inhibitors (Olaparib, APO866). To monitor 

tumor response and predict treatment outcome, we explore new functional imaging 

modalities including in vivo imaging of apoptosis. 

In a separate project we investigate the patented concept of improved drug delivery 

by short chain sphingolipid-enriched liposomes in vitro and in vivo. 

Alkyl-phospholipids (APLs) APLs represent a fi rst group of compounds that has 

become available for clinical application along this translational approach. These 

synthetic anti-tumor agents are known to accumulate in sphingomyelin- (SM) and 

cholesterol-enriched plasma membrane microdomains, also known as “lipid rafts”. 

Once taken up via these membrane portals, APLs interfere with lipid metabolism, 

inhibit proliferative and survival signaling, affect cell cycle distribution and 

stimulate apoptosis induction in a variety of tumor cell systems. In combination 

with radiation, APLs cause a synergistic apoptotic effect and reduce clonogenic 

cell survival. The orally available APL perifosine has been tested in a randomized, 

double-blind, placebo-controlled multicenter phase II study to assess its effi cacy in 

combination with radiotherapy in patients with locally advanced NSCLC. The design 

of future clinical protocols will be based on the results of this trial. Meanwhile, we 

continue to explore underlying mechanisms of cellular uptake and sensitivity of 

these compounds and focus on other promising APL derivatives with potentially 

better bioavailability and radiosensitizing properties, like the i.v. compound Erucyl- 

PC (erucylphosphocholine). 

S49 mouse lymphoma cells undergo apoptosis in response to the APL edelfosine 

(1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), FasL (Fas ligand) and DNA 

damage. S49 cells made resistant to ALP (S49(AR)) are defective in sphingomyelin 

synthesis and ALP uptake, and also have acquired resistance to FasL and DNA 

damage. However, these cells can be re-sensitized following prolonged culturing 

in the absence of ALP. The resistant cells show sustained ERK (extracellularsignal-regulated 

kinase)/Akt activity, consistent with enhanced survival signaling. 

In search of a common mediator of the observed cross-resistance, we found that 

S49(AR) cells lacked the PtdIns(3,4,5)P(3) phosphatase SHIP-1 [SH2 (Src homology 

2)-domain-containing inositol phosphatase 1], a known regulator of the Akt survival 

pathway. Re-sensitization of the S49(AR) cells restored SHIP-1 expression as well as 

phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the

S49(AR) phenotype in terms of apoptosis cross-resistance, sphingomyelin defi ciency 

and altered phosphoinositide levels. Collectively, the results of the present study 

suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma 

cell death irrespective of the nature of the apoptotic stimulus. 

In collaboration with W Moolenaar (Division of Cell Biology I) we evaluate the 

antitumor and radiosensitizing properties of Erucyl-PC, a new and i.v. administrable 

APL. We found that expression of the membrane fl ippase complex CDC50a/ 

ATP8B1 plays an important role in cellular sensitivity towards this and related APLs, 

providing a potential biomarker for APL effi cacy in vivo. 

Other radiosensitizers in preclinical development (1) Pro-apoptotic death 

receptor agonists: In two separate projects with J Borst (Division of Immunology) 

we study other radiosensitizers. A long term line of research focuses on the 

interaction between death receptor ligands (TRAIL and MegaFasL/APO010) and 

DNA damaging regimens (radiation and etoposide). TRAIL induces apoptosis in a 

wide variety of tumor tissues, but lacks normal tissue toxicity in preclinical animal 

models. In several leukemic and solid tumor cell lines, we demonstrated combined 

(additive to synergistic) effects of TRAIL + radiation/etoposide. The effectiveness 

and acceptable toxicity of the combined treatment of TRAIL and radiation was 

subsequently demonstrated in vivo. These experiments have been expanded with 

another death receptor ligand (MegaFasL/APO010). Our current studies focus on 

the impact of TRAIL death receptor traffi cking on pro-apoptotic signaling. 

(2) Inhibitors of Bcl-2 family members: In collaboration with the University of 

Michigan and the division of Radiobiology at the Free University of Amsterdam, 

we have initiated preclinical effi cacy testing of small-molecule inhibitors of antiapoptotic 

members of the Bcl-2 family (AT-101 and ABT-737). Currently, the 

combination of AT-101 with cisplatin-based chemoradiotherapy is evaluated in a 

clinical phase I/II study in locally advanced head and neck cancer. So far, 13 patients 

have been included and no DLT has been observed. 

The novel anti-cancer drug ABT-737 mimics the action of BH3-only proteins, which 

induce apoptosis by binding to pro-survival Bcl-2 proteins. ABT-737 selectively 

binds to certain Bcl-2 family members in vitro, but its binding profi le in cells was 

not completely elucidated. We have determined the interaction of ABT-737 with 

all six pro-survival Bcl-2 proteins, as individually expressed in p53 wild-type or 

-mutant T-leukemic cell lines. Bcl-2, Bcl-xL and Bcl-w were targeted by ABT-737, 

to a differential degree, while Bcl-B, Bfl -1 and Mcl-1 were not. ABT-737 selectivity 

was refl ected in apoptosis-sensitivity and displacement of BH3-only protein Bim. 

Moreover, among the six pro-survival proteins, Bcl-2, Bcl-xL and Bcl-w conferred 

resistance to etoposide, which was overcome by ABT-737. To investigate which 

BH3-only proteins could overrule ABT-737 resistance, we inducibly expressed Noxa, 

Puma, Bim, or truncated Bid in Mcl-1-overexpressing cells and demonstrated that 

only Noxa evidently synergized with ABT-737 in cell death induction. Combined 

treatment with ABT-737 and the Noxa-inducer Bortezomib (Velcade) broke ABT-737 

resistance conferred by Bcl-B, Bfl -1 and Mcl-1, in both p53 wild-type and –mutant 

cells. These data identify Bcl-B, Bfl -1 and Mcl-1 as mediators of ABT-737 resistance 

and Noxa-inducing stimuli as optimal agents for combined modality treatment of 

ABT-737-resistant cancers. 

(3) DNA damage repair inhibitors: In collaboration with C Vens (Division of 

Experimental Therapy) we explore the interaction between radiation and inhibitors 

of DNA repair. A fi rst strategy targets NAD+ biosynthesis to enhance radiationinduced 

cell death. APO866 is a highly specifi c non-competitive inhibitor of 

nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the regulation 

of NAD+ biosynthesis. Inhibition of NAMPT reduces cellular NAD+ levels. 

Among NAD+ consuming enzymes are Poly(adenosine-diphosphate[ADP]-ribose) 

polymerases (PARPs) and Sirtuins. NAD+ is involved in numerous biochemical 

processes, including regulation of DNA repair, replication, transcription and 

apoptosis. We investigated the cytotoxic effect of APO866 alone and in combination 

with radiation on tumor cells in vitro and in vivo. APO866, in nanomolar 

concentrations, induces a time- and dose-dependent depletion of cellular NAD+ 

147 

RADIOTHERAPY

148 

RADIOTHERAPY 

levels in a panel of human tumor cell lines. In vitro, APO866-mediated reduced 

NAD+ levels resulted in enhanced cytotoxicity and, in combination with ionizing 

radiation, decreased clonogenic survival. In vivo experiments with PC3 xenografts 

showed that at NAD+ depleting doses, APO866/radiation combination was more 

effective than either treatment alone. 

Direct PARP inhibitors are also good candidates for combined use with DNA 

damaging agents. The main mechanism by which both radiation and cisplatin 

kill tumor cells is by an accumulation of un- or misrepaired DNA damage. PARP 

inhibitors increase radiation and chemotherapy (Cisplatin) response in preclinical 

studies. PARP inhibitors have been shown to specifi cally kill homologous 

recombination defi cient tumor cells as single agent. ATM mutations are expected 

to affect DSB repair and homologous recombination status therefore amplifying 

damage induced by the combined PARP inhibitor radiation (Cisplatin) treatment. 

Thus tumor targeted treatment and radio-chemosensitization could be achieved in 

the presence of frequently observed ATM gene mutations. We have designed 3 phase 

I-II studies evaluating the safety and tolerability of Olaparib in combination with 

(cisplatin-based chemo-) radiotherapy in locally advanced breast cancer, NSCLC and 

head and neck cancer. 

Improved in vitro and in vivo anti-tumor efficacy of glucosylceramideenriched 

liposomal doxorubicin Anti-cancer therapy is often suboptimal due 

to inability to deliver suffi cient levels of cytostatics into tumor cells. The cellular 

plasma membrane is an effective barrier for exogenous compounds to accumulate 

in the tumor cell. We identifi ed a well-defi ned class of sphingolipid analogs that 

effectively overcome this barrier, catalyzing drug-membrane traversal preferential 

for tumor cell membranes. A liposomal co-formulation of the short-chain lipid 

N-octanoyl-glucosylceramide (GC) and doxorubicin was applied in a genetically 

engineered mouse (GEM) breast tumor model (Wcre;Cdh1F/F;Trp53F/F). These 

tumors, which are resistant to a variety of conventional and biological antitumor 

therapies, responded to doxorubicin treatment when combined with the membrane 

modulation strategy. Co-administration of GC generated a sustained anti-tumor 

response and signifi cantly improved overall survival. Using a nuclear isolation 

procedure, we showed that the presence of GC enhanced the intracellular tumor 

accumulation in vivo by a factor 1.9 (p < 0.05). In contrast, the accumulation within 

normal heart tissue was not elevated. The data are in agreement with in vitro 

experiments using cultured cardiac myocytes. When compared to either clinically 

available formulations of doxorubicin (free or PEGylated liposomal doxorubicin) 

a favorable effi cacy, pharmacokinetic and toxicity profi le was obtained by coformulation 

of doxorubicin with GC. In order to elucidate the mechanism of action 

of the sphingolipid analogues, in vitro and model membrane studies and in silico 

molecular dynamics simulation experiments were designed. We demonstrated that 

short-chain sphingolipids act at the level of the plasma membrane, independently 

of lipid microdomain (raft) formation or membrane proteins. In artifi cial lipid 

membranes of well-defi ned compositions the short-chain sphingolipids enhanced 

doxorubicin-membrane traversal similar to cell membranes; N-octanoylglucosylceramide 

elevated the translocation rate of doxorubicin by a factor 1.93 (p 

< 0.05). Molecular dynamics simulations, in collaboration with RijksUniversiteit 

Groningen, revealed that the energetic barrier for the hydrophilic part of the 

doxorubicin to translocate to the opposite side of the membrane reduced by two-fold 

(p < 0.05) due to the presence of short-chain lipids. This phenomenon of facilitated 

traversal is explained by a transient hydrophilic gateway (hemifusion) through which 

amphiphilic drugs can traverse. 

Taken together, short-chain sphingolipids catalyze the transbilayer movement of 

amphiphilic cytostatic drugs by a transient drug-membrane gateway, and the shortchain 

lipid GC improves the therapeutic ratio in multi-drug resistant GEM breast 

tumors by enhancing the intracellular tumor accumulation of doxorubicin while 

limiting normal tissue exposure. Our data suggest that the therapeutic window of 

classical or newly developed cancer therapeutics can be widened by concomitant 

targeting of the cellular plasma membrane. Currently, extensive toxicology studies 

are carried out to prepare the translation of this concept in a clinical setting.

DIVISION OF SURGICAL ONCOLOGY 

IMAGE GUIDED SURGERY 

Theo Ruers, Marc van Beurden, Henk van der Poel, Michel Wouters, Germaine Relyveld, Jelle 

Wesseling, Sven Rottenberg, Danny Evers, Jarich Spliethoff, Ronni Wessels, Diederik Grootendorst, 

Breast Surgery group, Lung Surgery Group, Urology group, Head and Neck Oncology group 

This research line, which has started recently, is aiming at optimizing surgical 

procedures by better surgical guidance during operative procedures. To this end 

new imaging technologies are developed and tested to improve tumor mapping 

and staging pre- and intra-operative. These imaging and surgical guidance 

procedures should lead to more radical resections while sparing normal tissue 

and organ function. The research line is a strong collaboration between the NKI/ 

AVL, Technical University Twente and industrial partners. For the moment three 

projects are running. In the fi rst project we are developing a tool for optical biopsies 

by means of spectroscopy and fl uorescence techniques. An optical needle was 

developed together with industry. Ex vivo tissue specimens from more than hundred 

patients have been analyzed with an accuracy of over 90%. In vivo testing in breast 

cancer, liver metastases and lung tumors has been started in 2011 and results are 

awaited. In a second project, in cooperation with the group Biomedical Physics and 

Biomedical Photonics of AMC, optical coherence tomography is tested for improved 

tissue diagnosis in vulvar neoplasia (VIN), penis cancer and skin malignancies. 

For VIN lesions we were able to show that OCT images match well with defi nite 

histology, meaning that OCT can have a signifi cant role in the clinical workfl ow 

of this disease. A third project concentrates on the use of photoacoustic imaging 

for diagnosis of lymph node metastases and is running in close collaboration with 

the Biomedical Photonic Imaging group of the University of Twente. We recently 

showed that photoacoustic imaging is able to detect microscopic tumor foci within 

lymph nodes of melanoma patients. The research performed in the present projects 

leads to strong synerchy with the new innovative minimal invasive operating theatre 

complex planned to be build in 2014 and will result in a technology platform that 

can be used for further clinical studies. 

GYNAECOLOGY 

Gemma Kenter, Marc van Beurden, Willemien van Driel, Petra Biewenga, Jan Lange, Monique 

Brood, Pia Kvistborg 

The department focuses on innovative treatment for ovarian cancer, on interventions 

to improve quality of life for premature (iatrogenic) menopausal symptoms and for 

cervical carcinoma and immunotherapy for HPV related (premalignant) genital 

neoplasms. Research takes place in close cooperation with the other centers from the 

Center for Gynaecologic Oncology Amsterdam (CGOA) i.e. AMC and VUmc. 

Ovary In a randomized multicenter phase III clinical trial for stage III ovarian 

carcinoma coordinated by the NKI-AVL (v. Driel) the effect of secondary debulking 

surgery with or without hyperthermic intraperitoneal cisplatinum is being studied. 

Inclusion started March 2007 and currently runs in six centers in The Netherlands 

and one center in Belgium. Primary endpoint of this study is progression free survival. 

Up until now a total of 115 patients have been randomized (KWF CKTO2006-16). 

In a multicenter trial the effect of different hormonal replacement regimen on 

bone density, breast density and quality of life after prophylactic bilateral salpingo 

oophorectomy are examined in a randomized control trial (M05HIR Hirise). 

The effect of hormonal replacement therapy on menopausal complaints related 

to biochemical changes in surgically and naturally postmenopausal women is 

investigated in a prospective observational comparative study. (M06HRT Novaria). 

A multicenter randomized trial to investigate the effect of cognitive behavioral 

149 

SURGICAL ONCOLOGY 

Division head Theo Ruers 

Board 

Theo Ruers MD PhD Head 

Marc van Beurden MD PhD Academic staff 

Michiel van den Brekel MD PhD Academic staff 

General Surgical Oncology 

Emiel Rutgers MD PhD Head 

Arend Aalbers MD Academic staff 

Daphne Hompes MD Academic staff 

Frits van Coevorden MD PhD Academic staff 

Hester Oldenburg MD PhD Academic staff 

Houke Klomp MD PhD Academic staff 

Johanna van Sandick MD PhD Academic staff 

Jos van der Hage MD PhD Academic staff 

Koen Peeters MD Academic staff 

Marianne Piek-den Hartog MD Academic staff 

Marie-Jeanne Baas-Vrancken Peeters MD PhD 

Academic staff 

Michel Wouters MD Academic staff 

Omgo Nieweg MD PhD Academic staff 

Theo Ruers MD PhD Academic staff 

Vic Verwaal MD PhD Academic staff 

Martijn Stuiver Physiotherapist 

Ronnie Wessels Research assistant 

Ewout Courrech Staal MD Research physician 

Mila Donker MD Research assistant 

Rachel Numan PhD student 

Sjoerd Bruin MD Research physician 

Danny Evers MD Research physician 

Stella Mook MD Research physician 

Tjeerd Aukema MD Research physician 

Erik von Meyenfeldt MD Research physician 

Eva Schaake Research physician 

Jarich Spliethoff PhD student

150 


Head and Neck Oncology and Surgery 

Michiel van den Brekel MD PhD Head 

Corina van As-Brooks PhD Academic staff 

Alfons Balm MD PhD FRCS FACS Academic 

staff 

Maarten Borgemeester MD Research physician 

Cindy van den Boer MD Research physician 

Renee Clapham MSc PhD student 

Elin Derks MD Post-doc 

Amy Dohmen MD Research physician 

Paul van der Eerden MD Research physician 

Alexandros Fasolis MD Fellow 

Renske Fles MSc Researcher 

Ruth Hoffmans MD Resident 

Frans Hilgers MD PhD Academic staff 

Irene Jacobi PhD Phonetic scientist 

Quinten Kammeijer MD Resident 

Baris Karakullukcu MD Academic staff 

Martin Klop MD PhD Academic staff 

Cuna Knegt MD Resident 

Bart Knockaert MD Fellow 

Menno Krap DDS Academic staff 

Annemarijn Kreeft MD Research physician 

Michiel Lieshout DDS Academic staff 

Wouter Lodder MD Research physician 

Peter Lohuis MD PhD Academic staff 

Marlies Maatje MD Research physician 

Lisette van der Molen PhD Phonetic scientist 

Hester van Monsjou MD Research physician 

Saar Muller PhD Academic staff 

Jimmy Pramana MD Research physician 

Andrea Remmelts MD Resident 

Renske Scheenstra MD PhD Research physician 

Noortje Schwandt MD Phd Fellow 

Ludi Smeele MD DDS PhD Academic staff 

Fokko Smits MD Resident 

Rob van Son PhD Post-doc 

Sharon Stoker MD Resident 

Bing Tan MD PhD Academic staff 

Noortje Theunissen MD Research physician 

Jacqueline Timmermans MD Research 

physician 

Adriaan Timmers DDS Academic staff 

Caroline Verhagen MD Research physician 

Hidde Veenstra MD Research physician 

Lenka Vermeeren MD Research physician 

Maarten Wildeman MD Research physician 

Marloes Wondergem MD Research physician 

Volkert Wreesmann MD PhD Research 

physician 

Charlotte Zuur MD PhD Academic staff 

therapy (CBT) and physical exercise for climacteric symptoms in breast cancer 

patients experiencing treatment-induced menopause is fi nalized, the results of 

which are analyzed. (KWF project: NKI 2006-3470). 

The benefi cial effect of a laparoscopy in order to predict the operability in high stage 

ovarian carcinoma is being studied in a multicenter randomized trial coordinated by 

the CGOA-AMC. (ZON MW doelmatigheid 80-82310-97-11056). 

Vulva Participation in the GROINSS-V II study, an international multicenter 

observational study on patients with vulvar cancer. Patients with positive lymph nodes, 

diagnosed by sentinal node technic, undergo radiation without full lymphadenectomy. 

Endpoints of this study are recurrence free survival and quality of life. 

For patients with locally advanced carcinoma of the vulva an effi cacy study is 

ongoing during which patients are treated with induction chemoradiation and if 

necessary followed by surgery in order to reduce postoperative morbidity. (AMC 

locally advanced vulvar cancer effi cacy study). 

The benefi t of wearing therapeutic elastic stockings in order to prevent lymphedema 

for patients treated with inguinal lymph node dissection is conducted in a nonblinded, 

randomized controlled trial (M06PRO “Kousen” study). Inclusion was 

fi nalized and results will be published in 2012. 

Optical coherence tomography (OCT) is an emerging biomedical optical imaging 

technique that performs high resolution, cross sectional tomographic imaging 

generating pictures that resemble histopathological examination. We will investigate 

the potential role of OCT as “optical biopsy” device in patients with (pre)malignant 

vulvar disease. 

Patients with VIN III take part in a multicenter randomized trial to study the 

effect of vaccination with synthetic long HPV 16 E6/E7 peptides with or without 

imiquimod on the vaccination site. 

Cervix A multicenter trial is running to study the safety and immunogenicity of 

combined chemo-immunotherapy in high stage or recurrent carcinoma of the 

cervix. (van Meir, van Poelgeest, Kenter) 

Furthermore, preparations for a phase 1 trial with DNA HPV 16 E7 vaccination in 

patients with HPV related disorders of the genital region or head and neck region are 

being made. 

Making use of the large database from AVL, AMC and VUmc, a prognostic model 

study is going on in order to individiualize prognosis for survival in patients with 

early stage cervical cancer. (Biewenga et al). 

Together with the LUMC we started a project to study the needs assessment in 

patients and professionals on sexual complaints after treatment for cervical cancer. 

(KWF - Alpe D’huZes 00004760) (ter Kuijle, Stiggelbout, Kenter) 

BREAST CANCER 

Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters, Jos van der Hage, Mila Donker, 

Stella Mook, Caroline Drukker, Bas Koolen 

Mammographic screening has led to a proportional shift toward earlier-stage 

breast cancers at presentation. Mook et al confi rmed an independent association 

between screen-detection and good prognosis for overall and breast cancer-specifi c 

survival. It appeared that the method of detection provided prognostic information 

beyond stage of migration among patients with invasive breast cancer. The question 

remained whether these screen-detected carcinomas are also molecularly different 

from interval-carcinomas. Esserman et al. showed that the method of detection 

(screening) is associated with a higher likelihood of a biologically low risk tumor. 

In the near future we plan to start the MINDACT screening study. We will collect 

information about the method of detection of all Dutch MINDACT patients (2093) 

to see what the number of biologically low and ultralow tumors among the screendetected 

tumors. We hypothesize that the screen-detected tumors will mainly be low 

and even ultralow risk. A collaboration with the Dutch National Screening Facilities 

has already been established.

Another result of screening is the increased fi nding of ductal carcinoma in situ 

(DCIS). We have performed an update of the EORTC 10853 trial that investigated the 

role of adjuvant radiotherapy after a local excision for DCIS. We found that after a 

median follow up of 15 years, almost 1 in 3 women developed a recurrence after the 

local excision, half of these recurrences were invasive. Radiotherapy reduced this 

risk by 50%, equally divided over invasive or DCIS recurrences. 

Furthermore we evaluated the identifi cation rate of the sentinel node and further 

nodal involvement in patients with a multifocal tumor as compared to a unifocal 

tumor in the EORTC 10981-22023 AMAROS trial. The sentinel node was more often 

positive in patients with a multifocal tumor, but further nodal involvement after a 

positive axillary SN was similar in both groups. With a high detection rate of 95.8% 

and similar distribution patterns, the sentinel node procedure seems to be adequate 

in patients with multifocal breast cancer. 

As opposed to patients with early-stage breast cancer who are in general treated 

with primary surgery, patients with locally advanced tumors are usually treated 

with neoadjuvant chemotherapy (NAC). Since distant metastases at diagnosis 

are more frequently seen in patients with large tumors or axillary lymph node 

metastases, it is standard practice to perform a search for distant disease prior to 

the start of NAC. We have shown that the FDG PET/CT has a higher sensitivity for 

the detection of distant metastases and new primary malignancies as compared 

with conventional staging procedures (bone scintigraphy, ultrasound of the liver 

and chest radiography). In 154 patients, no metastases were missed with the PET/ 

CT and in 16 (80%) of 20 patients with proven metastases, one or more lesions were 

exclusively found with PET/CT. This resulted in a change of treatment in 13 (8%) of 

154 patients. Current research regarding the use of PET/CT in breast cancer focuses 

on stage II/III breast cancer patients with emphasis on the classifi cation of the 

primary tumor, detection of (extra-)axillary lymph nodes and monitoring response 

of the primary tumor to NAC. 

In order to be able to perform breast-conserving surgery after NAC, preoperative 

localization of the original tumor bed is crucial. We assessed the feasibility of two 

methods for this purpose: Radioguided Occult Lesion Localization with technetium 

(ROLL- 99m Tc) and Radioactive Seed Localization (RSL) with a 125-iodine seed. With 

relative small excision volumes, both techniques were comparable in terms of tumor 

free margins. An advantage of RSL is the fact that no radiological localization is 

needed anymore prior to surgery and thereby it reduces scheduling confl icts. 

GASTRO INTESTINAL CANCER 

Vic Verwaal, Theo Ruers, Johanna van Sandick, Frits van Coevorden, Marie-Jeanne Vrancken 

Peeters, Arend Aalbers, Ewout Courrech Staal, Danny Evers, Loes Velthuizen, Jarich Spliethoff, 

Jurriën Stiekema, Sera de Leeuw, Sabrine Kol 

Oesophageal and gastric cancer In February 2011, Ewout Courrech Staal 

succesfully defended his thesis ‘Improvement of the multimodality treatment of 

oesophageal cancer’. Jurriën Stiekema continued to work on both oesophageal 

and gastric cancer research. Results of a literature review on the prognostic and 

predictive value of microarray analysis in oesophageal cancer indicated that although 

promising, current data are insuffi ciently validated for clinical application (paper 

submitted). A prospective study on gene expression profi ling in oesophageal 

cancer is ongoing. Together with fellow research physician Johan Dikken (LUMC), 

a systematic literature search on quality indicators for gastric cancer surgery 

was performed to defi ne an evidence-based framework for registration and 

benchmarking (paper in preparation). A database of all patients who underwent 

surgery for gastric malignancy in the NKI-AvL since 1995 has been constructed 

and will be the basis for further clinical and translational research, with focus on 

the biology and treatment outcome of diffuse gastric cancer and gastro-intestinal 

stromal cell tumours. 

Hipec The most eye catching study performed by the HIPEC group is a study in which 

a new classifi cation of peritoneal metastases is made based on cell atypia, mitosis index 

151 


Urologic Oncology 

Simon Horenblas MD PhD FEBU Head 

Axel Bex MD PhD Academic staff 

Wim Meinhardt MD PhD Academic staff 

Henk van de Poel MD PhD Academic staff 

Richard Meijer MD Academic staff 

Bas van Rhijn MD PhD Academic staff 

Gynaecology 

Gemma Kenter MD PhD Head 

Jan Lange MD Academic staff 

Marc van Beurden MD PhD Academic staff 

Monique Brood MD Academic staff 

Petra Biewenga MD Academic staff 

Willemien van Driel MD PhD Academic staff 

Christianne Lok MD Academic staff 

Plastic and Reconstructive Surgery 

J Joris Hage MD PhD Head 

Leonie Woerdeman MD PhD Academic staff 

Marieke van der Berg MD Academic staff 

Martine van Huizum MD Academic staff 

Brigitte Drost MD Academic staff 

Anesthesiology 

Peter Schutte MD Head 

Dirk Buitelaar MD Academic staff 

Katina Efthymiou MD Academic staff 

Christoph Hahn MD PhD Academic staff 

Sandra Huissoon MD Academic staff 

Lenie Hulshoff MD Academic staff 

Michael Šrámek MD PhD Academic staff 

Julia ten Cate MD Academic staff 

Ingeborg Vergouwe MD Academic staff 

Karin Ariese MD Academic staff 

Dermatology 

Wietze van der Veen MD PhD Head 

Germaine Relyveld MD PhD Head 

Inka Nieuweboer-Krobotova MD Academic staff 

Biljana Zupan-Kajcovski MD Academic staff 

Marianne Crijns MD PhD Academic staff

152 


Publications 

Ackerstaff AH, Rasch CRN, Balm AJM, 

de Boer JP, Wiggenraad R, Rietveld DH, 

Gregor RT, Kröger R, Hauptmann M, 

Vincent A, Hilgers FJM. Five-year quality of 

life results of the randomized clinical phase 

III (RADPLAT) trial, comparing 

comcomitant intra-arterial versus intravenous 

chemoradiotherapy in locally advanced head 

and neck cancer. Head Neck, Online, 2011 

Ahmed AKJ, Hahn D, Hage JJ, Bleiker E, 

Woerdeman LAE. Temporary banking of the 

nipple-areola complex in 97 skin-sparing 

mastectomies. Plast Reconstr Surg 

2011;127:531-539 


Rutgers EJ, Gilhuijs KG, Vrancken Peeters 

MJ. Radioactive Seed Localization of Breast 

Lesions: An Adequate Localization Method 

without Seed Migration. Breast J. 

2011;17:594-601 

Alkhateeb SS, Neill M, Bar-Moshe S, 

Rhijn BV, Kakiashvili DM, Fleshner N, Jewett 

M, Petein M, Schulman C, Hanna S, 

Bostrom PJ, Roumeguere T, Shariat SF, 

Rorive S, Zlotta AR. Long-term prognostic 

value of the combination of EORTC risk 

group calculator and molecular markers in 

non-muscle-invasive bladder cancer patients 

treated with intravesical Bacille Calmette- 

Guérin. Urol Ann. 2011;3:119-26 

Auprich M, Bjartell A, Chun FK, de la 

Taille A, Freedland SJ, Haese A, Schalken J, 

Stenzl A, Tombal B, van der Poel H. 

Contemporary role of prostate cancer antigen 

3 in the management of prostate cancer. Eur 

Urol. 2011;60:1045-54 


Tinteren H, Horenblas S, Haanen J. A phase 

II study of presurgical sunitinib in patients 

with metastatic clear-cell renal carcinoma 

and the primary tumor in situ. Urology. 

2011;78:832-7 

Bex A, Clarke N. Targeted caval 

cytoreduction: solid foundations or shifting 

sands? Eur Urol. 2011;59:919-20 

Bex A, Haanen J. Tilting the AXIS 

towards therapeutic limits in renal cancer. 

Lancet. 2011 

Bex A, Larkin J, Blank C. Non-clear cell 

renal cell carcinoma: how new biological 

insight may lead to new therapeutic 

modalities. Curr Oncol Rep. 2011;13:240-8 

Review 

Bex A, Powles T. The sequence of 

cytoreductive nephrectomy: glass half empty 

or glass half full? Ann Oncol. 2011;22:1691 

and percentage of mucous on one side and survival on the other side. PM could be 

categorized into four groups: low-grade, well-differentiated mucinous tumor (DPAM); 

intermediated-grade mucinous carcinoma (PMCA-i); high-grade mucinous carcinoma 

(PMCA); and high-grade nonmucinous carcinoma (PCA). Multivariate analysis showed 

that histological classifi cation, gender, number of segments affected, completeness 

of cytoreduction, and HIPEC as primary treatment were signifi cant related to OS and 

DFS. The 5-year OS was 64% in the DPAM group, 36% in the PMCA group, and 24% 

in the PCA group. Of PM originating from an appendix tumor, 29% were of non- 

DPAM type. Of primary colorectal tumors, 37% resulted in mucinous PM, and another 

26% of PM of colorectal origin had partly mucinous histology. The conclusion of this 

study was that histology is a signifi cant predictive factor of OS and DFS in PM treated 

with surgical cytoreduction and HIPEC. Due to collaboration between the HIPEC 

centres in the Netherlands a large epidemiologic study (3356 patients) is performed 

on the change in survival of metastasized colon cancer over the last decades. This 

study showed that the median survival of patients with liver metastasis changed from 

34 (1995 – 2000) to 51 weeks (2001 – 2006) where as the survival of patient with PM 

stayed the same (35 in 1995 – 2000 and 30 weeks in 2001 – 2006). A second fi nding 

was that the most patients with peritoneal carcinomatosis were still treated with 

systemic chemotherapy only, in stead of with HIPEC. 

Colorectal liver metastases Research is focused on imaging and local tumour 

destruction. The results of an international randomized study on the use of RFA 

for unresectable colorectal liver metastases were presented as an oral at SSO 2011. 

Further research is directed at immunological cell death and immunostimulation 

after local tumour destruction (e.a. HIFU) within a CTMM consortium. In addition, 

a European multicentre study was initiated to evaluate anti MUC-1 vaccination 

in patients treated pre-operatively with radiotherapy. Moreover, together with the 

Technical University Twente gold nanoshells, in combination with NIR light, are 

used for tumour detection and ablation of resection margins. In collaboration with 

industrial partners the use of spectroscopy for tumour diagnosis and guidance of 

minimal invasive procedures is investigated. The research line is funded by KWF, 

the UTwente, EORTC and industrial partners. 

MELANOMA 

Anton Bunschoten, Jos van der Hage, Bin Kroon, Omgo Nieweg, Hidde Veenstra, Ronnie Wessels, 

Michel Wouters 

The research interest of the melanomologists concerns the anatomy and physiology 

of the lymphatic system, implications for dissemination, implementation of new 

forms of imaging techniques and other new forms of diagnostic and staging 

methods, sentinel node biopsy, aspects of inguinal node dissection, in transit 

metastases and aspects of regional isolation perfusion. 

Lymphoscintigraphy can visualize sentinel nodes before the operation. One issue 

hampering lymphatic mapping is the observation that a sentinel lymph node often 

does not accumulate all of the 99m Tc-nanocolloid that is administered at the tumor 

site. Some of it passes through downstream to lodge in second- or higher-echelon 

nodes. The multitude of radioactive nodes hampers the identifi cation of the sentinel 

node on the lymphoscintigrams and particularly in the operating room, since the 

gamma-ray detection probe cannot discern one from the other. 

To address this problem, the specifi cs for a new tracer were outlined in cooperation 

with the Division of Diagnostic Oncology. The ability of human serum albumin 

(HSA) to form non-covalent self-assembled complexes with peptides via NIR cyanine 

dyes was explored. FRET quenching interactions between HSA-Cy5 and the noncovalently 

bound fl uorescent molecules ICG, IR783-CO 2H and three IR783-labeled 

targeting peptides were used to monitor complex assembly and disassembly. The hostguest 

interactions between HSA and IR783-labeled peptides enabled the formation 

of (bio)nanoparticles that are coated with peptides that may target α vβ 3-integrins, the 

chemokine receptor 4 (CXCR4), and somatostatin receptors. The potential of CXCR4targeted 

(bio)nanoparticles in sentinel lymph node procedures was demonstrated in

mice. It was concluded that by non-covalently binding NIR-dye labeled peptides to an 

already clinically approved HSA-scaffold targeted bionanoparticles were created. 

LIPOSARCOMA STUDY GROUP 

Frits van Coevorden, Daphne de Jong, Petra Nederlof, Rick Haas, Harm van Tinteren, 

Ronald de Vreeze 

We analyzed our liposarcoma database of 325 patients (1977-2007) and subjected the 

material to additional tests for improved classifi cation and grading. Adding clinical 

information of these cases created a set of data for further study and analysis. The 

occurrence of combined patterns of well-differentiated liposarcoma (WDLS) and 

myxoid/roundcell liposarcoma(MRLS) designated as mixed-type liposarcoma pose 

a conceptual problem as this feature may have consequences for treatment choice 

and prognosis. We have dissected the molecular relation of tumor components 

in cases of mixed-type liposarcoma. Based on heterogeneous preoperative MRI 

features, 8 cases were selected. Preoperative biopsies and resection specimens 

were analyzed including molecular and immunohistochemical analysis on all 

components. As controls, cases with homogeneous MRI features and uniform 

aspects of 10 MRLS and 5 WDLS were studied. All patients with heterogeneous MRI 

features showed morphological components of MRLS and WDLS. RTPCR showed 

FUS-DDIT3 fusion in both components in 5/8 cases in the absence (0/5) of MDM2 

and CDK4 amplifi cation. In 3/8 patients, MDM2 and/or CDK4 were overexpressed, 

and amplifi cation was shown by MLPA in the absence of MRLS translocations. All 

control patients showed a molecular pattern consistent with their morphological 

features. Therefore, mixed-type liposarcomas should not be regarded as collision 

tumors, but as an extreme variant of the morphological spectrum within a single 

biological entity, explaining the biological contradiction of mixed-type liposarcoma. 

For treatment stratifi cation, detailed classifi cation including molecular support 

should be performed in tumors with heterogeneous MRI features. 

The classifi cation of multifocal myxoid/round cell liposarcoma (MRLS), which is 

defi ned as tumor presentation in at least two separate sites before manifestation in 

the lungs, as either metastasis or as a second primary tumor, has essential clinical 

consequences. MRLS is characterized by t(12;16)(q13;p11) or t(12;22)(q13;q12), and 

various exon fusion transcripts are described with varying incidences, which permits 

their use as markers for clonality. Moreover, in solid tumors, analysis of loss of 

heterozygozity(LOH) is valuable for clonality analysis. Therefore, fi fteen multifocal 

MRLS-patients with 2-5 metachronous (n=12) or synchronous (n=3) localizations 

were investigated. Using RT-PCR, the detailed molecular characteristics of the FUS- 

CHOP and EWS-CHOP breakpoints were determined. LOH-analysis at twelve loci 

was then used to further analyze clonal relationships. In all patients, tumor sites 

showed identical FUS-CHOP fusion products. In six patients, identical rare fusion 

transcripts were found, supporting a clonal relationship. Nine patients had the 

common exon5-FUS/exon2-CHOP fusion transcript, and two of these were identifi ed 

as clonally related by LOH-analysis. In all other patients, LOH-analysis was highly 

suggestive of a clonal relationship, and no evidence for interpretation of a second 

primary tumor was found. This study supports the metastatic nature of apparent 

multifocal myxoid/round cell liposarcoma. 

THORACIC CANCER SURGERY 

Houke Klomp, Michel Wouters, Johanna van Sandick, Erik von Meyenfeldt, Eva Schaake, 

Tjeerd Aukema 

Local treatment is an accepted treatment option for oligometastatic pulmonary 

disease of various origins. Percutaneous Radio Frequency Ablation (RFA) has 

been reported as an alternative to surgery (possibly with less morbidity and less 

negative side effects). Results of RFA for local control of pulmonary metastases and 

(progression-free) survival were evaluated. 

153 



Bex A, Vermeeren L, Meinhardt W, 

Prevoo W, Horenblas S, Valdés Olmos RA. 

Intraoperative sentinel node identifi cation 

and sampling in clinically node-negative renal 

cell carcinoma: initial experience in 20 

patients. World J Urol. 2011;29:793-9 

Bex A. Translating translational 

repression: evolving possibilities in urooncology. 

Eur Urol. 2011;59:682-3 

Biewenga P, van der Velden J, Mol BWJ. 

et al. Prognostic Model for Survival in 

Patients With Early Stage Cervical Cancer 

CANCER 2011;117:4:768-776 

Bipat S, van den Berg RA, van der Velden 

J, et al. The role of magnetic resonance 

imaging in determining the proximal 

extension of early stage cervical cancer to the 

internal os European Journal of Radiology 

2011;78:1:60-64 

Boone J, Bex A, Prevoo W. Percutaneous 

Radiofrequency Ablation of a Small Renal 

Mass Complicated by Appendiceal 

Perforation. Cardiovasc Intervent Radiol. 


Borggreven PC, Braunius W, Lohuis 

PJFM. Maligniteit in de nasofarynx bij het 

kind. Ned Tijdschr KNO-Heelk. 2011;17:202-6 

Boström PJ, Alkhateeb S, Trottier G, 

Athanasopoulos PZ, Mirtti T, Kortekangas 

H, Laato M, van Rhijn B, van der Kwast T, 

Fleshner NE, Jewett MA, Finelli A, Zlotta 

AR. Sex differences in bladder cancer 

outcomes among smokers with advanced 

bladder cancer. BJU Int. 2011 

Bronkhorst IH, Ly LV, Jordanova ES, 

Vrolijk J, Versluis M, Luyten GP, Jager MJ . 

Detection of M2-macrophages in uveal 

melanoma and relation with survival. Invest 

Ophthalmol Vis Sci. 2011;52:643-50 

Brouwer OR, Valdés Olmos RA, 

Vermeeren L, Hoefnagel CA, Nieweg OE, 

Horenblas S. SPECT/CT and a portable 

gamma-camera for image-guided laparoscopic 

sentinel node biopsy in testicular cancer. J 


Bunschoten A, Buckle T, Kuil J, Nieweg 

OE, van Leeuwen FWB. Targeted noncovalent 

self-assembled nanoparticles based 

on human serum albumin. Biomaterials, 2011 

Chade DC, Shariat SF, Cronin AM, 

Savage CJ, Karnes RJ, Blute ML, Briganti A, 

Montorsi F, van der Poel HG, Van Poppel H, 

Joniau S, Godoy G, Hurtado-Coll A, Gleave 

ME, Dall’Oglio M, Srougi M, Scardino PT, 

Eastham JA. Salvage radical prostatectomy 

for radiation-recurrent prostate cancer: a 

multi-institutional collaboration. Eur Urol. 

2011;60:205-10

154 



Cibula D, Abu-Rustum N, Dusek L, Zikan 

M, Zaal A, Sevcik L, Kenter G, Querleu D; 

Jach R, Bats A-S. Prognostic signifi cance of 

low volume sentinel lymph node disease in 

early-stage cervical cancer. Gyncol Oncol 2011 

Courrech Staal EFW, Aleman BMP, van 

Velthuysen MLF, Cats A, Boot H, Jansen 

EPM, van Coevorden F, van Sandick JW. 

Chemoradiation for esophageal cancer: 

institutional experience with three different 

regimens. Am J Clin Oncol 2011;34:343-9 

Courrech Staal EFW, Smit VT, van 

Velthuysen MLF, Spitzer-Naaykens JMJ, 

Wouters MWJM, Mesker WE, Tollenaar 

RAEM, van Sandick JW. Reproducibility and 

validation of tumour stroma ratio scoring on 

oesophageal adenocarcinoma biopsies. Eur J 


Crijns MB. Psychologische aspecten van 

psoriasis capitis. Huid 2011;9:33-6 

De Bonilla-Damiá A, Roberto Brouwer O, 

Meinhardt W, Valdés-Olmos RA. Lymphatic 

Drainage in Prostate Carcinoma assessed by 

Lymphoscintigraphy and SPECT/CT: Its 

importance for the Sentinel Node Procedure. 

Rev Esp Med Nucl. 2011 

De Bruin-Visser JC, Ackerstaff AH, 

Rehorst H, Retèl VP, Hilgers FJM. 

Integration of a smoking cessation program in 

the treatment protocol for patients with head 

and neck and lung cancer. Eur Arch 

Otorhinolaryngol. 2011 (in press) 

De Rooij KA, Borggreven PA, Tromp 

Meesters RC, Godefroy WP, Lohuis PJFM. 

Coniotomie bij acute bovenste 

luchtwegobstructie tijdens intubatie. Ned 

Tijdschr KNO-Heelk. 2011;17:143-8 

De Ru JA, Schellekens PP, Lohuis PJ. 

Corrugator supercilii transection for headache 

emanating from the frontal region: a clinical 

evaluation of ten patients. J Neural Transm. 

2011;118:1571-4 

de Vreeze RS, van Coevorden F, 

Boerrigter L, Nederlof PM, Haas RL, Bras J, 

Rosenwald A, Mentzel T, de Jong D. 

Delineation of chondroid lipoma: an 


biological analysis. Sarcoma. 

2011;2011:638403 

de Vreeze RS, de Jong D, Koops W, 

Nederlof PM, Ariaens A, Haas RL, van 

Coevorden F. Oncogenesis and classifi cation 



analysis. Int J Cancer. 2011;128:778-86 

Delfos NM, Anten S, Crijns MB, Rothbart 

M, Lambregts MMC. Cowpox BMJ 

2011;343:d4430 

Primary tumour origin was colorectal cancer in 30%, sarcoma in 26%. Lesion size 

was < 2cm (69%), 2-3 cm 16%) > 3cm (15%). As expected, RFA was associated with 

pneumothorax in 34% of procedures (chest drain in 25%). Major complications 

occurred after 6%. Median follow-up after RFA was 22 months [range 2-65]. 

Analysis per procedure showed 1- and 3-year progression free survival rates of 33% 

and 11%. For patients (n=36) with all lesions treated, 1- and 3- year progression 

free survival rates were 49% and 20% respectively. Evaluation per lesion showed 

local progression in 25/90 (Kaplan Meier-estimate 22% at 1 year, 35% at 2 and 3 

years). Tumour size larger than 30mm was associated with a higher risk of local 

progression (47 vs. 24%). Overall survival at 3 years was 69%. 

Notwithstanding its relatively low morbidity, longer follow-up after RFA for 

pulmonary metastases shows a considerable rate of local progression. An overall 

survival rate of 69% after three years is surprisingly good, emphasizing the fact 

that patients with (even recurrent) oligometastatic disease have a relatively good 

prognosis. Therefore, this technique should be further improved, and hopefully new 

developments such as microwave ablation will be able to induce tumour necrosis 

more effi ciently. Further studies are needed to defi ne the role of local ablation 

techniques, stereotactic radiotherapy, and surgery. 

DEPARTMENT OF HEAD AND NECK SURGERY AND ONCOLOGY 

Alfons Balm, Michiel van den Brekel, Frans Hilgers, Baris Karakullukcu, Martin Klop, Peter Lohuis, 

Ludi Smeele, Bing Tan, Charlotte Zuur 

The department is a national referral center and one of the larger clinical departments 

in this fi eld treating about 600 new patients annually. The department is active in 

clinical and translational research. Currently, 5 full professors are part of the department 

and the staff members have part-time appointments at the Academic Medical 

Center (AMC) of the University of Amsterdam. Being a multidisciplinary fi eld, there 

are many clinical and research connections within the institute and also with the AMC. 

Translational research In 2011 the head and neck department was involved in several 

translational research projects. Together with Adrian Begg and Conchita Vens the 

Fanconi pathway in oral cancer samples as well as in cell lines was studied. So far 

over 100 patient samples are deep-sequenced. The fi ndings are currently analysed. A 

clinical trial using PARP inhibitors in combination with radiotherapy is initiated. In 

a collaborative project with Marcel Verheij on the BCL2 inhibitor gossypol, a clinical 

phase II study is conducted in patients treated with cisplatin-based chemoradiation. 

In an international study lead by Coen Rasch (Artforce) cetuximab will be combined 

with radiotherapy and several expression profi le studies are attached. Together with 

Jacques Neefjes and the department of pulmonology short term cultures are explored. 

These can be used for testing chemo-sensitivity pre-treatment and also for testing the 

chemical library of Huib Ovaa looking for new targets. Early detection and monitoring 

treatment outcome of nasopharyngeal cancer using (anti-)Epstein-Barr Virus (EBV) 

based tumor markers is studied in the Netherlands, UI-Jakarta, UGM-Yogyakarta 

together with the VUmc. With the same group a project on lytical induction therapy in 

recurrent nasopharyngeal cancer has been approved by the KWF. 

Photodynamic therapy The department is active in research on photodynamic 

therapy and several grants have been obtained (VENI, ZonMw, NWO, KWF). 

Currently the focus of this research is on interstitial PDT. In several projects 

treatment planning and dosimetry for interstitial PDT is studied by means of 

developing pre-treatment planning tools and intraoperative imaging guidance. In a 

national study with participation of all academic medical centers in the Netherlands, 

the cost effectiveness of Foscan PDT for incurable recurrent head and neck cancers 

is being studied. Monitoring of tissue oxygen saturation and Foscan concentration 

during PDT to optimize treatment parameters is another project. 

Rehabilitation Rehabilitation research focuses on several topics. One concerns 

prevention of swallowing and communication problems in patients treated with

chemo-radiation for advanced head and neck cancer. Research on postlaryngectomy 

vocal and pulmonary rehabilitation, in cooperation with Atos Medical Sweden, 

has resulted in the development of a new surgical instrument for voice prosthesis 

implantation, and in new heat-and-moist-exchangers. Mucociliary transport in the 

trachea after laryngectomy is another fi eld of interest. Together with the Amsterdam 

Centre for Language and Communication (ACLC) of the University of Amsterdam, 

the possibilities are explored to use automatic speech analyzers for the assessment 

of intelligibility of pathologic voices, e.g. after laryngectomy or chemoradiation. Also 

with ACLC (and with Neil Aaronson) a project on physician-patient communication 

is in preparation. A head and neck rehabilitation center implementing an evidencebased 

rehabilitation program developed in the NKI has been set up in 2011 and 

within this setting several strategies are studied. 

Clinical research Clinical research is quite diverse. Fields of interest are fl uorescent 

imaging of sentinel nodes, impact of sentinel node surgery in melanoma, automatic 

tumor volumetry, neck node imaging, clinical trials using induction chemotherapy 

regimens and clinical and biological aspects (HPV) of head and neck cancer in 

young patients. Prediction of inoperability and development of virtual surgery for 

oral function to predict morbidity after surgery are conducted in cooperation with 

the Technical University Twente and VUmc. 

UROLOGIC ONCOLOGY 

Simon Horenblas, Axel Bex, Wim Meinhardt, Henk van de Poel, Bas van Rhijn, Bin Kroon 

Research in urologic oncology has been centred around the following themes: 

Improved staging of urologic tumours, organ and function sparing, fundamental 

research in prostate, kidney and penis cancer. 

Improved staging of urologic tumours Research has been focussed on the role 

of sentinel node biopsy in kidney, prostate, testicular and penile cancer. In all 

above mentioned tumours, apart from kidney cancer, sentinel node biopsy has 

proven to be extremely useful in minimizing the morbidity of surgery of the lymph 

nodes and at the same time maximizing the detection rate of occult lymph node 

metastases. The collaboration with the department of nuclear physics has been very 

fruitful. Especially the use of SPECT/CT scans and intra-operative imaging with a 

mobile gamma camera (Sentinella ® ) has proven to be a clinical useful adjunct. In 

prostate cancer no false negative fi ndings were observed. The sentinel node strategy 

together with the Sentinella was effective in localizing sentinel nodes in a variety of 

anatomical locations, otherwise not removed during lymph node dissection. 

The scope of research has been widened by collaboration with the department of 

clinical physics. A comparative trial was started, comparing patent blue, ICG and 

ICG with technetium. Using a fl uorescence camera ICG is seen after i.v. injection. 

Integration of the fl uorescence camera in the robot camera is being investigated. 

In testicular cancer also no false negative fi ndings have been observed. Accrual in 

this clinical study is very slow unfortunately, mainly because of the fact that stage I 

germ cell tumours are mostly being treated outside of NKI-AVL. 

In penile cancer the false negative rate dropped from 20% to an acceptable 5.1%. 

For renal cancer we continue with the sentinel node study, approved in 2008, 

with the aim of elucidating the lymphatic pathways of renal cancer and the 

immunological effect of renal cancer in the sentinel nodes. The fi rst 20 patients 

were assessed and published recently. 

Organ and function sparing The role of cytoreductive surgery in metastatic renal 

cancer is being investigated in a randomized EORTC study comparing immediate 

versus deferred nephrectomy for patients with synchronous metastatic renal cell 

carcinoma and the primary tumour in situ. This study (E 30073) coordinated by 

Axel Bex, followed the experiences of the phase II study with neo-adjuvant Sutent 

(tyrosine kinase inhibitor). 

In bladder cancer we continue to expand our experiences with the so called 

155 



Dikken JL, Van Sandick JW, Swellengrebel 

HAM, Lind PA, Putter H, Jansen EPM, Boot 

H, Van Grieken N, Van de Velde CJH, 

Verheij M, Cats A. Neo-adjuvant 

chemotherapy followed by surgery and 

chemotherapy or by surgery and 

chemoradiotherapy for patients with 

resectable gastric cancer (CRITICS). BMC 


Donker M, Hage JJ, Woerdeman LA, 

Rutgers EJ, Sonke GS, Vrancken Peeters MJ. 

Surgical complications of skin sparing 

mastectomy and immediate prosthetic 

reconstruction after neoadjuvant 

chemotherapy for invasive breast cancer. Eur J 

Surg Oncol. 2011 

Duijts SF, Faber MM, Oldenburg HS, van 

Beurden M, Aaronson NK. Effectiveness of 

behavioral techniques and physical exercise 

on psychosocial functioning and healthrelated 

quality of life in breast cancer patients 

and survivors-a meta-analysis. 

Psychooncology. 2011;20:115-26 

Duijts SF, Stolk-Vos AC, Oldenburg HS, 

van Beurden M, Aaronson NK. 

Characteristics of breast cancer patients who 

experience menopausal transition due to 

treatment. Climacteric. 2011;14:362-8 

Early Breast Cancer Trialists’ 

Collaborative Group (EBCTCG), Davies C, 

Godwin J, Gray R, Clarke M, Cutter D, Darby 

S, McGale P, Pan HC, Taylor C, Wang YC, 

Dowsett M, Ingle J, Peto R. Relevance of 

breast cancer hormone receptors and other 

factors to the effi cacy of adjuvant tamoxifen: 

patient-level meta-analysis of randomised 

trials. Lancet. 2011;378:771-84 

Escudier B, Osanto S, Ljungberg B, Porta 

C, Wagstaff J, Mulders P, Gore M, Bex A, 

Bellmunt J, Bracarda S, Franklin A, Honoré 

PH, Ravaud A, Steijn JV, Aziz Z, Akaza H. 

Multidisciplinary management of metastatic 

renal cell carcinoma in the era of targeted 

therapies. Cancer Treat Rev. 2011 


M, Retèl VP, Mook S, Glas AM, Moore DH, 

Linn S, van Leeuwen FE, van ‘t Veer LJ. 



cancers. Breast Cancer Res Treat. 2011 

Gooden M, Lampen M, Jordanova ES, 

Leffers N, Trimbos JB, van der Burg SH, 

Nijman H, van Hall T.HLA-E expression by 

gynecological cancers restrains tumorinfi 

ltrating CD8+ T lymphocytes.Proc Natl 

Acad Sci U S A. 2011;108:10656-61 

Gooiker GA, van der Geest LG, Wouters 

MW, Vonk M, Karsten TM, Tollenaar RA, 

Bonsing BA. Quality improvement of 

pancreatic surgery by centralization in the 

western part of the Netherlands. Ann Surg 

Oncol. 2011;18:1821-9

156 



Gooiker GA, van Gijn W, Wouters MW, 

Post PN, van de Velde CJ, Tollenaar RA; 

Signalling Committee Cancer of the Dutch 

Cancer Society. Systematic review and 

meta-analysis of the volume-outcome 

relationship in pancreatic surgery. Br J Surg. 

2011;98:485-94 

Goossens-Laan CA, Gooiker GA, van 

Gijn W, Post PN, Bosch JL, Kil PJ, Wouters 

MW. A systematic review and meta-analysis 

of the relationship between hospital/surgeon 

volume and outcome for radical cystectomy: 

an update for the ongoing debate. Eur Urol. 

2011;59:775-83 

Goossens-Laan CA, Visser O, Hulshof 

MC, Wouters MW, Bosch JL, Coebergh JW, 

Kil PJ. Survival after treatment for carcinoma 

invading bladder muscle: a Dutch populationbased 

study on the impact of hospital volume. 

BJU Int. 2011 


HH, van Werkhoven E, Kerst JM, Horenblas 




management. J Urol. 2011;185:888-93 

Graafl and NM, Teertstra HJ, Besnard AP, 

van Boven HH, Horenblas S. Identifi cation 

of high risk pathological node positive penile 

carcinoma: value of preoperative 

computerized tomography imaging. J Urol. 

2011;185:881-7 

Grootendorst, D Jose J, Van der Jagt P, 

Van der Weg W, Nagel K, Wouters M,.Van 

Boven H, Van Leeuwen TG Steenbergen W, 

Ruers T and Manohar S. Initial experiences 

in the photoacoustic detection of melanoma 

metastases in resected lymph nodes. SPIE, 

Photons Plus Ultrasound: Imaging and 

Sensing 2011 Volume 7899 

Hage JJ, van Beurden M. Reconstruction 

of aquired perineo-vulvar defects: A proposal 

of sequence Semin Plast Surg 2011;25:148-54 

Hazewinkel MH, Gietelink L, van der 

Velden J, Burger MP, Stoker J, Roovers JP. 

Renal ultrasound to detect hydronephrosis: 

A need for routine imaging after radical 

hysterectomy? Gynecol Oncol. 2012;124:83-6 

Heusinkveld M, Welters MJ, van 

Poelgeest MI, van der Hulst JM, Melief CJ, 

Fleuren GJ, Kenter GG, van der Burg SH. 

The detection of circulating human 

papillomavirus-specifi c T cells is associated 

with improved survival of patients with deeply 

infi ltrating tumors. Int J Cancer. 

2011;128:379-89 

Hew MN, Baseskioglu B, Barwari K, 

Sexuality Preserving Cystectomy and Neobladder (SPCN). In a recent publication 

excellent continence and potency rates were seen without any danger of increased 

recurrences. Despite international scepsis we continue to advocate this procedure in 

well selected patients. In line with the wish to decrease the morbidity of the surgery 

robotic assisted cystectomy was started in 2010. 

We are involved in the active surveillance study for prostate cancer (Prias, initiated 

by Rotterdam) and have entered more patients than any one else. 

We are partners together with Rotterdam and Groningen in the PCMM consortium 

regarding prostate cancer and prostatectomy. 

Another modality will be investigated: the use of photodynamic therapy in localized 

prostate cancer. A randomized study is launched comparing active surveillance and 

photodynamic therapy (Tookad study). 

TRANSLATIONAL RESEARCH IN PROSTATE, KIDNEY AND 

PENILE CANCER 

Prostate cancer The role of Pim-1 expression in prostate cancer progression is 

investigated. Pim-1 was earlier shown to enhance the growth response of prostate 

cancer cells in vitro to low levels of DHT. A possible explanation for this may be 

the downregulation by Pim-1 of the 17-beta-hydroxysteroid-dehydrogenase type 2 

gene product (HSD17B2). HSD17B2 is involved in the enzymatic down regulation 

of DHT and testosterone in breast and prostate cancers. Its down regulation by 

Pim-1 overexpression may aid prostate cancer cells to survive in low testosterone 

environments, such as in castrate resistant prostate cancer (CRPC). 

The role of mTOR inhibition in chemotherapy is being assessed in a phase I study in 

collaboration with Andre Bergman, medical oncologist. In this study the combined 

use of everolimus (mTOR inhibitor) and cyclophosphamide in CPRC will be tested. 

In collaboration with prof T Schumacher the role of T-cell receptors in prostate 

cancer model vaccination studies was addressed. 

Penis cancer The role of HPV in penis cancer was further investigated in close 

collaboration with the department of pathology of the Free University medical 

centre. Using molecular and serological analyses for a wide range of HPV types and 

comparing serological fi ndings with age-matched male controls. Primarily HPV 16 

infection is directly involved in penile carcinogenesis. Based on earlier micro-array 

results a renewed analysis was started comparing HPV+ and HPV- tumors. 

Kidney cancer In collaboration with the department of immunology, the 

department of angiogenesis of Arjan Griffi oen at the Free University medical centre 

and Eric Jonasch, MD Anderson Cancer Center, Houston, kidney cancer tissue is 

analysed, specifi cally looking into neo-vascularization and immune modulation. 

Also together with the department of immunology analysis of rapid expansion of 

TIL cells was started. 

Improved quality control of treatment results Using prospective data collection, 

almost all uro-surgical treatments at the NKI-AVL can be analysed now. Extensive 

use of these databases was made for prostate, bladder and penile cancer. Quality 

of life analysis was done of in patients with localized prostate cancer managed by 

brachytherapy or robot assisted laparoscopic prostatectomy (RALP). 

Prospective data are analysed for risk estimation of treatment or astein-komen 

13-11-1943ctive surveillance for prostate cancer within the framework of the IMPACTstudu, 

funded by ZonMW 

ANESTHESIOLOGY AND INTENSIVE CARE MEDICINE 

Peter Schutte, Dirk Buitelaar, Katina Efthymiou, Christoph Hahn, Sandra Huissoon, Lenie Hulshoff, 

Michael Šrámek, Julia ten Cate, Ingeborg Vergouwe 

The principal aim of the Department of Anesthesiology and Intensive Care Medicine

is to deliver the highest standard of anesthesiological and intensive care. 

Presently our research focuses mainly on the role of peripheral nerve and central 

neuraxial blockades as an adjuvant to general anesthesia. The purpose of these 

blockades is threefold: a reduction in perioperative opioid consumption, the 

prevention of chronic pain and shortening of length of hospital stay. 

In collaboration with the Departments of Surgical Oncology, Head and Neck Oncology 

and Urological Oncology, innovations are/have been implemented. E.g. superfi cial 

cervical plexus blockade in patients undergoing head and neck surgery, fi eld blockade 

and paravertebral blockade in breast surgery and transversus abdominis plane 

blockade (TAP-block) in, for instance, robot assisted laparoscopic prostatectomies. 

The results of the N07TAP–trial are evaluated and submitted for publication. 

Furthermore two more study protocols are currently being developed. One study 

focuses on perioperative pain treatment in patients undergoing laryngectomies 

and commando resections. In this study the benefi cial effects of intravenous S(+)- 

Ketanest and lidocaïne will be studied. The other study focuses on the intraoperative 

position of patients undergoing thoracotomies in relation to the prevention of 

postoperative ipsilateral postthoracotomy shoulder pain. 

Future focus of interest is the role of regional and general anesthesia in the 

prognosis of surgically treated oncologic patients. 

PLASTIC AND RECONSTRUCTIVE SURGERY 

J Joris Hage, Brigitte Drost, Carolien Wever, Leonie Woerdeman, Marieke van den Berg, Marije 

Hoornweg, Martine van Huizum 

The research of the Department of Plastic and Reconstructive Surgery is focused 

predominantly on innovative reconstructive techniques after ablative surgery by 

other specialists. Additionally, ongoing vascular and functional research is being 

done in collaboration with the Department of Plastic Surgery at the University 

Medical Centre Utrecht (prof. dr. M Kon) and with the Faculty of Human Movement 

Sciences of the Vrije Universiteit, Amsterdam (prof dr HEJ Veeger) 

Impact of skin sparing mastectomy and immediate prosthetic breast 

reconstruction on the pectoralis major muscle To prevent cranial displacement 

of the sub-pectorally implant and to allow for a natural shape of the reconstructed 

breast, the origin of the muscle is partially detached from the lower costal arch 

and the caudal part of the sternum. A pre- to post operative comparative study was 

conducted in 22 women with a mean age of 36 years (range, 26-54; SD, 8.6) who had 

bilateral preventive breast surgery to assess the subjective and objective functional 

loss induced by such detachment. Furthermore, it was assessed whether synergistic 

function of other shoulder gird muscles could compensate for such loss. 

Although the postoperative end score (mean, 5.73; SD, 7.51) of the Disability of the 

Arm, Shoulder and Hand (DASH) questionnaire was statistically higher (p =< 0.01) 

than the preoperative end score (mean, 0.83; SD,1.49), this difference is irrelevant 

for ADL activities. This is in line with the observed postoperative lack of restrictions 

of range of motion in any of the six shoulder movements as compared to the 

preoperative measurements. The mean postoperative maximum force measured 

by dynamometry in four directions refl ective of the pectoralis major muscle 

function (endorotation, 90 0 anteversion, 90 0 retroversion, and 90 0 adduction) was 

7.13 N, or 3.8%, lower than the preoperative force (p = 0.079) whereas the mean 

postoperative maximum force in the two contra-movement directions (45 0 abduction 

and exorotation) was 15.5 N, or 14%, higher than the pre-operative force (p = 0.04). 

Electromyographically, both parts of the deltoid muscle pre- and postoperatively 

showed the same peaks during dynamometry in the six directions. Contrastingly, 

the clavicular part of the major pectoral muscle postoperatively showed a statistically 

signifi cant increase of peak of muscle activity in three of the four directions 

refl ective of major pectoral function (endorotation (p = 0.04), 90 0 retroversion 

(p = 0.03), and 90 0 adduction (p < 0.01)) and in both contra-movement directions 

(45 0 abduction (p = 0.04) and exorotation (p = 0.04)), indicating some compensatory 

activity. 

157 



Axwijk PH, Can C, Horenblas S, Bex A, 

Rosette JJ, Pes MP. Critical appraisal of the 

PADUA classifi cation and assessment of the 

R.E.N.A.L. nephrometry score in patients 

undergoing partial nephrectomy. J Urol. 

2011;186:42-6 

Hinnen KA, Schaapveld M, van Vulpen 

M, Battermann JJ, van der Poel H, van Oort 

IM, van Roermund JG, Monninkhof EM. 

Prostate Brachytherapy and Second Primary 

Cancer Risk: A Competitive Risk Analysis. J 

Clin Oncol. 2011 

Hompes D, Prevoo W, Ruers T. 

Radiofrequency ablation as a treatment tool 

for liver metastases of colorectal origin.Cancer 

Imaging. 2011;11:23-30 

Hompes D, W Prevoo, Ruers T. Review: 

incidence and clinical signifi cance of 

Bevacizumab-related non-surgical and 

surgical serious adverse events in metastatic 

colorectal cancer.Eur J Surg Oncol. 

2011;37:737-46 

Horenblas S. Editorial comment. J Urol. 


Horenblas S. Editorial comment. J Urol. 

2011;186:1307 

Horenblas S. Lymphadenectomy in penile 

cancer. Urol Clin North Am. 2011;38:459-69 

Horenblas S. Words of Wisdom. Re: 

variations in treatment policies and outcome 

for bladder cancer in The Netherlands. Eur 

Urol. 2011;59:300 

Hulshoff L, Rood E, ten Cate J, Bosman 

RJ, van der Voort PHJ. Telemedicine in the 

ICU: a review. Neth J Crit Care 2011;1:9-12 

Jerjes WK, Upile T, Wong BJ, Betz CS, 

Sterenborg HJ, Witjes MJ, Berg K, van Veen 

R, Biel MA, El-Naggar AK, Mosse CA, Olivo 

M, Richards-Kortum R, Robinson DJ, Rosen 

J, Yodh AG, Kendall C, Ilgner JF, Amelink A, 

Bagnato V, Barr H, Bolotine L, Bigio I, Chen 

Z, Choo-Smith L6, D’Cruz AK, Gillenwater 

A, Leunig A, MacRobert AJ, McKenzie G, 

Sandison A, Soo KC, Stepp H, Stone N, 

Svanberg K, Tan IB, Wilson BC, Wolfsen H, 

Hopper C. The future of medical diagnostics: 

review paper. Head Neck Oncol. 2011;3:38 

Johannsen M, Staehler M, Ohlmann CH, 

Flörcken A, Schmittel A, Otto T, Bex A, Hein 

P, Miller K, Weikert S, Grünwald V. Outcome 

of treatment discontinuation in patients with 

metastatic renal cell carcinoma and no 

evidence of disease following targeted therapy 

with or without metastasectomy. Ann Oncol. 

2011;22:657-63 

Jose J, Grootendorst DJ, Vijn TW,

158 



Wouters M, van Boven H, van Leeuwen TG, 

Steenbergen W, Ruers TJ, Manohar S. Initial 

results of imaging melanoma metastasis in 

resected human lymph nodes using 

photoacoustic computed tomography. J 

Biomed Opt 2011;16:096021 

Kakiashvili DM, van Rhijn BW, Trottier G, 

Jewett MA, Fleshner NE, Finelli A, Azuero J, 

Bangma CH, Vajpeyi R, Alkhateeb S, Hanna 

S, Kostynsky A, Kuk C, Van Der Kwast TH, 

Zlotta AR. Long-term follow-up of T1 

high-grade bladder cancer after intravesical 

bacille Calmette-Guérin treatment. BJU Int. 

2011;107:540-6 

Kappers I, Klomp HM, Koolen MG, 

Uitterhoeve LJ, Kloek JJ, Belderbos JS, 

Burgers JA, Koning CC. Concurrent high-dose 

radiotherapy with low-dose chemotherapy in 

patients with non-small cell lung cancer of the 

superior sulcus. Radiother Oncol. 

2011;101:278-83 

Kappers I, Vollebergh MA, Van Tinteren 

H, Korse CM, Nieuwenhuis LL, Bonfrer 

JMG, Klomp HM, Van Zandwijk N, Van Den 

Heuvel MM. Soluble epidermal growth factor 

receptor (sEGFR) and carcinoembryonic 

antigen (CEA) concentration in patients with 

non-small cell lung cancer: correlation with 

survival after erlotinib and gefi tinib treatment 

Ecancer 2010;178 

Karakullukcu B, De Boer JB, Van Veen R, 

Wegman J, Tan IB. Surgical debulking 

combined with photodynamic therapy to 

manage residual extramedullary 

plasmacytoma of the nasopharynx. 

Photodiagnosis Photodyn Ther. 2011;8:264-6 

Karakullukcu B, Kanick SC, Aans JB, 

Sterenborg HJ, Tan IB, Amelink A, Robinson 

DJ. Clinical feasibility of monitoring 

m-THPC mediated photodynamic therapy by 

means of fl uorescence differential path-length 

spectroscopy. J Biophotonics. 2011;4:740-51 

Karakullukcu B, Van Oudenaarde K, 

Copper MP, Klop WMC, Van Veen R, 

Wildeman M, Tan IB. Photodynamic therapy 

of early stage oral cavity and oropharynx 

neoplasms: an outcome analysis of 170 

patients. Eur Arch Otorhinolaryngol. 

2011;268:281-8 

Klop WMC, Balm AJM, Van der Veen 

JPW, Nieweg OE, Van der Hage JA, Lohuis 

PJFM. Het cutaan hoofd-halsmelanoom: 

diagnostiek en behandeling. Ned Tijdschr 

KNO-Heelk. 2011;17:183-9 

Klop WMC, Veenstra HJ, Vermeeren L, 

Nieweg OE, Balm AJ, Lohuis PJ. Assessment 

of lymphatic drainage patterns and 

implications for the extent of neck dissection 

in head and neck melanoma patients. J Surg 

Oncol. 2011;103:756-60 

Knegjens JL, Hauptmann M, Pameijer 

DERMATOLOGY 

Wietze van der Veen, Inka Nieuweboer-Krobotova, Biljana Zupan-Kajcovski, Germaine Relyveld, 

Marianne Crijns 

Optical Coherence Tomography in melanocytic lesions Optical coherence 

tomography (OCT) is an emerging biomedical optical imaging technique that 

performs high resolution, cross sectional tomographic imaging generating pictures 

that resemble histopathological examination. The images are generated by scanning 

an optical beam across the tissue and measuring the echo time delay and intensity 

of backscattered light. The image penetration depth of OCT is determined by optical 

scattering and is up to 2 mm in tissue. We use (in collaboration with the Biomedical 

Engineering and Physics department of the AMC) the Santec Inner Vision 2000 

with a 10 μm axial resolution, 11 μm lateral resolution, and light with a wavelength 

around 1300 nm. This high resolution OCT functions as a type of ‘optical biopsy’, 

providing cross sectional images of tissues in analogy to histopathology but without 

removal or staining tissue. 

With regard to tissue diagnosis: we are focusing on the use of OCT in melanocytic 

lesions. We would like to determine the possible value of OCT in distinguishing 

benign nevi from malignant melanoma in patients with suspicion of malignant 

melanoma in comparison with histopathology as the gold standard, by viewing OCT 

images and by determining the attenuation coeffi cient of the B-scan OCT images. 

At this moment, we are including and imaging patients with one or more suspicious 

lesions with OCT. 

Local immunotherapy by the synergism of monobenzone and imiquimod 

cream (MI) for cutaneous metastases in stage III-IV melanoma patients 

Melanoma is a good candidate for treatment with immunotherapy, during 

which vitiligo development is a favourable prognostic sign. At the department 

of Dermatology of the AMC, we have developed a new therapy for melanoma, 

based on the potent vitiligo-inducing effect of monobenzone combined with the 

immunostimulatory adjuvants imiquimod (MI therapy) and/or cytosine-guanine 

oligodeoxynucleotides (CpG) (MIC therapy). MI and MIC therapy both induced 

strong melanoma-reactive immunity, which effectively eradicated established 

melanoma in mice (van den Boorn al, PLoS one 2010). MIC treatment also induced 

a protective memory response that conferred long-term tumor-free survival, and 

protected against secondary tumor growth. 

Based on these data, we started a phase II (proof of concept) clinical trial of 

monobenzone-imiquimod (MI) therapy in stage III-IV melanoma patients at 

the NKI-AVL. The trial is open for patient accrual since March 2011. Twentyone 

melanoma patients with cutaneous metastases will be treated locally with 

monobenzone and imiquimod during 12 weeks. Study objectives are: 1) to study 

the clinical effi cacy of local MI treatment on cutaneous metastases, 2) to study the 

induction of local tumor-specifi c immunity by MI treatment, as measured by the 

accumulation of melanoma/melanocyte specifi c T-cells at the treatment site. In 

addition, potential systemic immunity will be measured in the peripheral blood. 

The MI regimen is a low-cost, simple therapy, which is applicable in broad range 

of patients regardless of HLA-haplotype. It does not require elaborate patientspecifi 

c in vitro cultures nor non-myeloablative lymphodepletion, reducing patient 

treatment burden. The MI compounds have each already been used in humans, 

making it readily available and easily applicable in the clinic. This trial will provide 

information on the effi cacy of monobenzone and imiquimod to raise melanocyte/ 

melanoma-specifi c (auto)immunity, which may be effective for the treatment of 

melanoma.


FA, Balm AJM, Hoebers FJ, de Bois JA, Kaanders 

JH, van Herpen CM, Verhoef CG, Wijers OB, 

Wiggenraad RG, Buter J, Rasch CR. Tumor 

volume as prognostic factor in chemoradiation for 

advanced head and neck cancer. Head Neck. 

2011;33:375-82 

Kolfschoten NE, Marang van de Mheen PJ, 

Gooiker GA, Eddes EH, Kievit J, Tollenaar RA, 

Wouters MW; Dutch Surgical Colorectal Audit 

group. Variation in case-mix between hospitals 

treating colorectal cancer patients in the 

Netherlands. Eur J Surg Oncol. 2011;37:956-63 

Koolen BB, Vegt E, Rutgers EJ, Vogel WV, 

Stokkel MP, Hoefnagel CA, Fioole-Bruining A, 

Vrancken Peeters MJ, Valdés Olmos RA. 


cancer patients: a PET/CT case series. Ann Nucl 

Med. 2011 

Koolen BB, Vrancken Peeters MJ, Aukema TS, 

Vogel WV, Oldenburg HS, van der Hage JA, 

Hoefnagel CA, Stokkel MP, Loo CE, Rodenhuis S, 

Rutgers EJ, Valdés Olmos RA. 18F-FDG PET/CT 

as a staging procedure in primary stage II and III 

breast cancer: comparison with conventional 

imaging techniques. Breast Cancer Res Treat. 2011 

Krabbe PF, Tromp N, Ruers TJ, van Riel PL. 

Are patients’ judgments of health status really 

different from the general population? Health 

Qual Life Outcomes 2011 

Kreeft AM, Tan IB, Leemans CR, Balm AJ. The 

surgical dilemma in advanced oral and 

oropharyngeal cancer: how we do it. Clin 

Otolaryngol. 2011;36:260-6 

Kroon MW, Wind BS, Meesters AA, 

Wolkerstorfer A, van der Veen JP, van der Wal 

AC, Beek JF. Non-ablative 1550 nm fractional laser 

therapy not effective for erythema dyschromicum 

perstans and postinfl ammatory 

hyperpigmentation: a pilot study. J Dermatolog 

Treat. 2011 

Kroon W.M. and Wind B.S., Beek J.F., Van der 

Veen J.P.W., Nieuweboer-Krobotová L., Bos J.D., 

Wolkerstorfer A. Non-ablative 1550-nm fractional 

laser therapy versus triple topical therapy for the 

treatment of melasma: a randomised controlled 

pilot study. J Am Acad Dermatol 2011;64:516-23 

Lawindy SM, Rodriguez AR, Horenblas S, 

Spiess PE. Current and future strategies in the 

diagnosis and management of penile cancer. Adv 

Urol. 2011;593751 

Linthorst Homan MW, Spuls PI, Nieuweboer- 

Krobotova L, de Korte J, Sprangers MA, Bos JD, 

Wolkerstorfer A, van der Veen JP. A randomized 

comparison of excimer laser versus narrow-band 

ultraviolet B phototherapy after punch grafting in 

stable vitiligo patients. J Eur Acad Dermatol 

Venereol 2011 

Lodder WL, Teertstra HJ, Tan IB, Pameijer FA, 

Smeele LE, Van Velthuysen ML, Van den Brekel 

MWM. Tumour thickness in oral cancer using an 

intra-oral ultrasound probe. Eur Radiol. 

2011;21:98-106 

Lodder WL, Van den Brekel MWM. 

Extracapsular tumor extension in cervical lymph 

nodes: reconciling the literature and seer data. 

Head Neck (in press) 

Lohuis PJ, Godefroy WP, Baker SR, Tasman 

AJ. Transposition fl aps in nasal reconstruction. 

Facial Plast Surg Clin North Am. 2011;19:85-106 

Lok CAR, Donker M, Massuger LFAG, 

Thomas C, Ansink AC. The psychological impact 

of follow-up after low-risk Gestational 

Trophoblastic Disease. J Reprod Medicine 

2011;56:47-52 

Lok CAR, Poynter C, Tait J. Life-Threatening 

Complications of Operative Hysteroscopy: A Case 

Report and Review of the Literature. J Gyn Surg 


Lok CAR, Snijder K, Nieuwland R, Van der 

Post JAM, de Vos P, Faas MM. Microparticles of 

preeclamptic patients activate endothelial cells via 

monocytes in-vitro. Am J Reprod Immunol 2011 

Lok CAR, van der Post, JAM, Sturk A, Sargent 

IL, Nieuwland R. The functions of microparticles 

in preeclampsia. J Pregn Hypert 2011;1:59-65 

Luteijn MC, Boonstra HE, Casteelen G, Ever 

AWM, de Korte J, Spillekom-van Koulil S, van der 

veen JPW, Crijns MB. Psychodermatologie in de 

Nederlandse dermatologische praktijk. Ned 

Tijdschrift Dermatol 2011 (in press) 

Meinhardt W, van der Poel HG, Valdés Olmos 

RA, Bex A and Horenblas S. Laparoscopic sentinel 

lymph node dissection for prostate cancer: the 

relevance of locations outside the extended 

dissection area. Prostate Cancer 2011; Hindawi 

Publishing Corporation 

Mensink G, Karagozoglu KH, Strackee SD, 

van Teeseling RA, Smeele LE, Becking AG. 

Autotransplantation of two maxillary premolars in 

a free vascularized fi bula reconstructed mandible. 

Int J Oral Maxillofac Surg. 2011;40:219-21 

Mérol JC, Charpiot A, Langagne T, Hémar P, 

Ackerstaff AH, Hilgers FJM. Randomized 

controlled trial on postoperative pulmonary 

humidifi cation after total laryngectomy: External 

Humidifi er versus Heat and Moisture Exchanger. 

Laryngoscope (in press) 

Mhallem Gziri M, Han SN, Van Calsteren K, 

Heyns L, Delaere P, Nuyts S, Van den Heuvel F, 

Cheron AC, Fossion E, Van Den Weyngaert D, 

Lok CAR, Amant F. Tongue cancers during 

pregnancy: case reports and review of literature. 

Head and Neck 2011 

Mittempergher L, de Ronde JJ, Nieuwland M, 

Kerkhoven RM, Simon I, Rutgers EJ, Wessels LF, 

Van ‘t Veer LJ. Gene expression profi les from 

formalin fi xed paraffi n embedded breast cancer 

tissue are largely comparable to fresh frozen 

matched tissue. PLoS One. 2011;6:e17163 

159 



Mook S, Van ‘t Veer LJ, Rutgers EJ, 

Ravdin PM, van de Velde AO, van Leeuwen 

FE, Visser O, Schmidt MK. Independent 

prognostic value of screen detection in invasive 

breast cancer. J Natl Cancer Inst. 

2011;103:585-97 

Nachabé R, Evers DJ, Hendriks BH, 

Lucassen GW, van der Voort M, Rutgers EJ, 

Peeters MJ, Van der Hage JA, Oldenburg 

HS, Wesseling J, Ruers TJ. Diagnosis of 

breast cancer using diffuse optical 

spectroscopy from 500 to 1600 nm: 

comparison of classifi cation methods. J 

Biomed Opt. 2011;16:087010 

Nachabé R, Evers DJ, Hendriks BH, 

Lucassen GW, van der Voort M, Wesseling J, 

Ruers TJ. Effect of bile absorption coeffi cients 

on the estimation of liver tissue optical 

properties and related implications in 

discriminating healthy and tumorous 

samples. Biomed Opt Express. 2011;2:600-14 

Nierkens S, den Brok MH, Garcia Z, 

Togher S, Wagenaars J, Wassink M, Boon L, 

Ruers TJ, Figdor CG, Schoenberger SP, 

Adema GJ, Janssen EM. Immune adjuvant 

effi cacy of CpG oligonucleotide in cancer 

treatment is founded specifi cally upon TLR9 

function in plasmacytoid dendritic cells. 


Nieweg OE, Hoekstra HJ. 

Gerandomizeerd onderzoek verschaft meer 

duidelijkheid over sentinel-nodebiopsie bij 

melanoom. Ned Tijdschr Oncol (in press) 

Nieweg OE, Veenstra HJ. False negative 

sentinel node biopsy in melanoma. J Surg 


Nunnink CJ, de Vries RR, Meinhardt W, 

van der Poel HG, Bex A, Horenblas S. 

Pregnancy following sexuality-preserving 

cystectomy for bladder carcinoma. Ned 

Tijdschr Geneeskd. 2011;155:A2820 

Oudejans JJ, Tuut MK, Engelbrecht MRW, 

Van Es R, Kummer JA, Morreau J, Marres 

HAM, Klomp HM, van de Wouw AJ. 

Richtlijn primaire tumor onbekend (Clinical 

guideline: Unknown Primary Tumor) 2011 

Paes EC, Schellekens PPA, Hage JJ, van 

der Wal MBA, Bleys RLAW, Kon M A cadaver 

study of the vascular territories of dominant 

and non-dominant internal mammary artery 

perforators Ann Plast Surg 2011;67:68-72 

Patard JJ, Pignot G, Escudier B, Eisen T, 

Bex A, Sternberg C, Rini B, Roigas J, 

Choueiri T, Bukowski R, Motzer R, Kirkali Z, 

Mulders P, Bellmunt J. ICUD-EAU 

International Consultation on Kidney Cancer 

2010: treatment of metastatic disease. Eur 

Urol. 2011;60:684-90

160 


Publications (continued) Publications (continued) 

Powles T, Blank C, Chowdhury S, 

Horenblas S, Peters J, Shamash J, Sarwar N, 

Boleti E, Sahdev A, O’Brien T, Berney D, 

Beltran L, Nathan P, Haanen J, Bex A. The 

outcome of patients treated with sunitinib 

prior to planned nephrectomy in metastatic 

clear cell renal cancer. Eur Urol. 2011;60:448- 

54 

Powles T, Chowdhury S, Jones R, Mantle 

M, Nathan P, Bex A, Lim L, Hutson T. 

Sunitinib and other targeted therapies for 

renal cell carcinoma. Br J Cancer. 

2011;104:741-5 

Powles T, Kayani I, Blank C, Chowdhury 

S, Horenblas S, Peters J, Shamash J, Sarwar 

N, Boletti K, Sadev A, O’Brien T, Berney D, 

Beltran L, Haanen J, Bex A. The safety and 

effi cacy of sunitinib before planned 

nephrectomy in metastatic clear cell renal 

cancer. Ann Oncol. 2011;22:1041-7 

Rasch CRN, Hauptmann M, Balm AJM. 

Intra-arterial chemotherapy for head and 

neck cancer: is there a verdict? Cancer. 

2011;117:874-5 

Retel VP, Van der Molen L, Hilgers FJM, 

Rasch CRN, L’Ortye AA, Steuten LM, Van 

Harten WH. A cost-effectiveness analysis of a 

preventive exercise program for patients with 

advanced head and neck cancer treated with 

concomitant chemo-radiotherapy. BMC 

Cancer. 2011;11:475 

Ruers, T; Review: incidence and clinical 

signifi cance of Bevacizumab-related 

non-surgical and surgical serious adverse 

events in metastatic colorectal cancer. Eur J 

Surg Oncol. 2011;37:737-46 

Russnes HG, Kuligina ESh, Suspitsin EN, 

Voskresenskiy DA, Jordanova ES, Cornelisse 

CJ, Borresen-Dale AL, Imyanitov EN Paired 

distribution of molecular subtypes in bilateral 

breast carcinomas. Cancer Genet. 

2011;204:96-102 

Rutgers E, Piccart-Gebhart MJ, Bogaerts 

J, Delaloge S, Veer LV, Rubio IT, Viale G, 

Thompson AM, Passalacqua R, Nitz U, 

Vindevoghel A, Pierga JY, Ravdin PM, 

Werutsky G, Cardoso F. The EORTC 10041/ 

BIG 03-04 MINDACT trial is feasible: 

Results of the pilot phase. Eur J Cancer. 2011 

Rutten MJ, Gaarenstroom K, Arts HJG, 

Bossuyt PM, ter Brugge H, van Gorp T, 

Hermans RHM, Kenter GG, Opmeer BC , 

Pijnenborg H, Schreuder HWR, Schutter E, 

Spijkerboer AM, Wensveen C, Zusterzeel P, 

Mol BWJ, Buist MR. Laparoscopy to predict 

the result of primary cytoreductive surgery in 

advanced ovarian cancer patients (LapOvCatrial): 

a multicenter randomized controlled 

study. 

Rijkaart DC, Berkhof J, Rozendaal L, van 

Kemenade FJ, Bulkmans NWJ, Heideman DAM, 

Kenter GG, Cuzick J, Snijders PJF, Meijer CJLM. 

Human papillomavirus testing for the detection of 

high-grade cervical intraepithelial neoplasia and 

cancer: fi nal results of the POBASCAM 

randomised controlled trial. Lancet Oncol 2011 

Sadeghi R, Gholami H, Zakavi SR, Kakhki VR, 

Tabasi KT, Horenblas S. Accuracy of Sentinel 

Lymph Node Biopsy for Inguinal Lymph Node 

Staging of Penile Squamous Cell Carcinoma: 

Systematic Review and Meta-Analysis of the 

Literature. J Urol. 2011 

Santegoets LA, van Baars R, Terlou A, 

Heijmans-Antonissen C, Swagemakers SM, van 

der Spek PJ, Ewing PC, van Beurden M, van der 

Meijden WI, Helmerhorst TJ, Blok LJ. Different 

DNA damage and cell cycle checkpoint control in 

low- and high-risk human papillomavirus 

infections of the vulva. Int J Cancer. 2011 

Scheenstra RJ, Muller SH, Hilgers FJM. 

Endotracheal temperature and humidity in 

laryngectomized patients in a warm and dry 

environment and the effect of a heat and moisture 

exchanger. Head Neck. 2011;33:1285-93 

Scheenstra RJ, Muller SH, Vincent A, 

Ackerstaff AH, Jacobi I, Hilgers FJM. A new heat 

and moisture exchanger for laryngectomized 

patients: endotracheal temperature and humidity. 

Respir Care. 2011;56:604-11 

Scheenstra RJ, Muller SH, Vincent A, Hilgers 

FJM. Heat and moisture exchange capacity of the 

upper respiratory tract and the effect of 

tracheotomy breathing on endotracheal climate. 

Head Neck. 2011;33:117-24 

Schellekens PPA, Paes EC, Hage JJ, van der 

Wal MBA, Bleys RLAW, Kon M Anatomy of the 

vascular pedicle of the internal mammary artery 

perforator (IMAP) fl ap as applied for head and 

neck reconstruction JPRAS 2011;64:53-57 

Schilder CMT, van Dijk SC, Meinhardt W, Van 

Dam FSAM, Schagen SB Cognitief functioneren 

van prostaatkankerpatiënten die hormonale 

therapie ondergaan. Neuropraxis 2011;15:20-26 

Schrevel M, Gorter A, Kolkman-Uljee SM, 

Trimbos JB, Fleuren GJ, Jordanova ES . Molecular 

mechanisms of epidermal growth factor receptor 

overexpression in patients with cervical cancer. 

Mod Pathol. 2011;24:720-8 

Terlou A, Kleinjan A, Beckmann I, Heijmans- 

Antonissen C, van Seters M, Santegoets LA, van 

Beurden M, Helmerhorst TJ, Blok LJ. Nonsteroidal 

anti-infl ammatory drugs do not interfere with imiquimod 

treatment for usual type vulvar intraepithelial 

neoplasia. Int J Cancer. 2011;128:2463-9 

Terlou A, Santegoets LA, van der Meijden WI, 

Heijmans-Antonissen C, Swagemakers SM, van 

der Spek PJ, Ewing PC, van Beurden M, 

Helmerhorst TJ, Blok LJ. An Autoimmune 

Phenotype in Vulvar Lichen Sclerosus and Lichen 

Planus: A Th1 Response and High Levels of 

MicroRNA-155. J Invest Dermatol. 2011 

Terlou A, van Seters M, Ewing PC, Aaronson 

NK, Gundy CM, Heijmans-Antonissen C, Quint 

WG, Blok LJ, van Beurden M, Helmerhorst TJ. 

Treatment of vulvar intraepithelial neoplasia with 

topical imiquimod: seven years median follow-up of 

a randomized clinical trial. Gynecol Oncol. 

2011;121:157-62 

Trommel van N, Lok CAR, Thomas C, Bulten 

J, Massuger LFAG. Clinical aspects of Placental 

Site Trophoblast Tumors in the Netherlands. 

AJRM (accepted) 

Upile T, Jerjes WK, Sterenborg HJ, Wong BJ, 

El-Naggar AK, Ilgner JF, Sandison A, Witjes MJ, 

Biel MA, Van Veen R, Hamdoon Z, Gillenwater 

A, Mosse CA, Robinson DJ, Betz CS, Stepp H, 

Bolotine L, McKenzie G, Barr H, Chen Z, Berg K, 

D’Cruz AK, Sudhoof H, Stone N, Kendall C, 

Fisher S, MacRobert AJ, Leunig A, Olivo M, 

Richards-Kortum R, Soo KC, Bagnato V, 

Choo-Smith LP, Svanberg K, Tan IB, Wilson BC, 

Wolfsen H, Bigio I, Yodh AG, Hopper C. At the 

frontiers of surgery: review. Head Neck Oncol. 


Van den Brekel MWM, Balm AJM, Lohuis 

PJFM, Van der Veen JPW. 10th international 

symposium on diagnosis and treatment of head 

and neck cancer: skin cancer of the head and neck. 

Expert Rev Anticancer Ther. 2011;11:1013-5 

Van den Tillaart SA, Trimbos JB, Dreef EJ, 

Jordanova ES, Fleuren GJ Patterns of parametrial 

involvement in radical hysterectomy specimens of 

cervical cancer patients. Int J Gynecol Pathol. 

2011;30:185-92 

Van der Heiden-Van der Loo M, de Munck L, 

Visser O, Westenend PJ, Van Dalen T, Menke 

MB, Rutgers EJ, Peeters PH. Variation between 

hospitals in surgical margins after fi rst breastconserving 

surgery in the Netherlands. Breast 

Cancer Res Treat. 2011 

Van der Houwen EB, Van Kalkeren TA, Post 

WJ, Hilgers FJM, Van der Laan BF, Verkerke GJ. 

Does the patch fi t the stoma? A study on peristoma 

geometry and patch use in laryngectomized 

patients. Clin Otolaryngol. 2011;36:235-41 

Van der Molen L, Van Rossum MA, Burkhead 

LM, Smeele LE, Rasch CR, Hilgers FJ. A 

randomized preventive rehabilitation trial in 

advanced head and neck cancer patients treated 

with chemoradiotherapy: feasibility, compliance, 

and short-term effects. Dysphagia. 2011;26:155-70 

Van der Molen L, Van Rossum MA, Jacobi I, 

Van Son RJJH, Smeele LE, Rasch CRN, Hilgers 

FJM. Pre- and posttreatment voice and speech 

outcomes in patients with advanced head and neck 

cancer treated with chemoradiotherapy: expert 

listeners’ and patient’s perception. J Voice (in 

press)


Van der Ploeg APT, Van Akkooi ACJ, 

Rutkowski P, Nowecki Z, Michej W, Mitra A, 

Newton-Bishop J, Cook M, Van der Ploeg IMC, 

Nieweg OE, Van Hout MFCM, Van Leeuwen 

PAM, Voit C, Cataldo F, Testori A, Robert C, 

Hoekstra HJ, Verhoef C, Spatz A, Eggermont 

AMM. Prognosis in patients with sentinel 

node-positive melanoma is accurately defi ned by 

the combined Rotterdam tumor oad and Dewar 

topography criteria. J Clin Oncol 2011;29:2206- 

2214 

Van der Poel HG, Buckle T, Brouwer OR, 

Valdés Olmos RA, Van Leeuwen FW. 

Intraoperative laparoscopic fl uorescence guidance 

to the sentinel lymph node in prostate cancer 

patients: clinical proof of concept of an integrated 

functional imaging approach using a multimodal 

tracer. Eur Urol. 2011;60:826-33 

Van der Post JA, Lok CA, Boer K, Sturk A, 

Sargent IL, Nieuwland R. The functions of 

microparticles in pre-eclampsia. Semin Thromb 

Hemost. 2011;37:146-52 

Van der Veen JPW. Provoking factors in vitiligo. 

Ned Tijdschr Dermatol 2011;21;20-21 

Van der Velden Jacobus; Fons Guus; Lawrie 

Theresa A Primary groin irradiation versus 

primary groin surgery for early vulvar cancer. 

Cochrane database of systematic reviews 2011;5 

Van Geel AN, Wouters MW, Lans TE, Schmitz 

PI, Verhoef C. Chest wall resection for adult soft 

tissue sarcomas and chondrosarcomas: analysis of 

prognostic factors. World J Surg. 2011;35:63-9 

Van Geel N, Speekaert R, Taieb A, Picardo M, 

Böhm M, Gawkrodger DJ, Schallreuter K, 

Bennett DC, van der Veen JP, Whitton M, Moretti 

S, Westerhof W, Ezzedine K, Gauthier Y; VETF 

members. Koebner’s phenomenon in vitiligo: 

European position paper. Pigment Cell Melanoma 


Van Kalkeren TA, van der Houwen EB, Duits 

MA, Hilgers FJM, Hebe A, Mostafa BE, Lawson 

G, Martinez Z, Woisard V, Marioni G, Ruske D, 

Schultz P, Post WJ, Verkerke BJ, van der Laan BF. 

Worldwide, multicenter study of peristomal 

geometry and morphology in laryngectomees and 

its clinical effects. Head Neck. 2011;33:1184-90 

Van Leersum NJ, Kolfschoten NE, Klinkenbijl 

JH, Tollenaar RA, Wouters MW. ‘Clinical 

auditing’, a novel tool for quality assessment in 

surgical oncology Ned Tijdschr Geneeskd. 

2011;155:A4136 

Van Leeuwen AC, Buckle T, Bendle G, 

Vermeeren L, Valdés Olmos R, van de Poel HG, 

van Leeuwen FW. Tracer-cocktail injections for 

combined pre- and intraoperative multimodal 

imaging of lymph nodes in a spontaneous mouse 

prostate tumor model. J Biomed Opt. 

2011;16:016004 

Van Monsjou H, van Velthuysen M, van den 

Brekel M, Jordanova E, Melief C, Balm A. HPV 

status in young HNSCC patients. Int J Cancer 


Van Monsjou HS, van Velthuysen ML, van 

den Brekel MWM, Jordanova ES, Melief CJ, Balm 

AJM. Human papillomavirus status in young 

patients with head and neck squamous cell 

carcinoma. Int J Cancer (in press) 

Van Oeveren J, de Ruiter M, Jesse T, van der 

Poel H, Tang J, Yalcin F, Janssen A, Volpin H, 

Stormo KE, Bogden R, van Eijk MJ, Prins M. 

Sequence-based physical mapping of complex 

genomes by whole genome profi ling. Genome Res. 

2011;21:618-25 

Van Rhijn BW, van der Kwast TH, Liu L, 

Fleshner NE, Bostrom PJ, Vis AN, Alkhateeb SS, 

Bangma CH, Jewett MA, Zwarthoff EC, Zlotta 

AR, Bapat B. The FGFR3 Mutation is Related to 

Favorable pT1 Bladder Cancer. J Urol. 2011 

Van Rhijn BW, van der Kwast TH, Alkhateeb 

SS, Fleshner NE, van Leenders GJ, Bostrom PJ, 

van der Aa MN, Kakiashvili DM, Bangma CH, 

Jewett MA, Zlotta AR. A New and Highly 

Prognostic System to Discern T1 Bladder Cancer 

Substage. Eur Urol. 2011 

Van Rhijn BW. Prospective trial to identify 

optimal bladder cancer surveillance protocol: 

reducing costs while maximizing sensitivity. BJU 

Int. 2011;108:1123-4 

Van Vugt HA, Roobol MJ, van der Poel HG, 

van Muilekom EH, Busstra M, Kil P, Oomens EH, 

Leliveld A, Bangma CH, Korfage I, Steyerberg 

EW. Selecting men diagnosed with prostate cancer 

for active surveillance using a risk calculator: a 

prospective impact study. BJU Int. 2011 

Veenstra HJ, Vermeeren L, Valdés Olmos RA, 

Nieweg OE. The additional value of lymphatic 

mapping with routine SPECT/CT in unselected 

patients with clinically localized melanoma. Ann 

Surg Oncol ahead of press 

Veenstra HJ, Wouters MJWM, Kroon BBR, 

Valdés Olmos RA, Nieweg OE. Less false-negative 

sentinel node procedures in melanoma patients 

with experience and proper collaboration.J Surg 

Oncol 2011;104;454-457 

Verleye L, Ottevanger PB, Kristensen GB, 

Ehlen T, Johnson N, Van der Burg MEL, Reed NS, 

Verheijen RHM, Gaarenstroom KN, Mosgaard B, 

Seoane JM, Van der Velden J, Lotocki R, Van der 

Graaf W, Penninckx B, Coens C, Stuart G, 

Vergote I; Quality of pathology reports for 

advanced ovarian cancer: Are we missing essential 

information? An audit of 479 pathology reports 

from the EORTC-GCG 55971/NCIC-CTG OV13 

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Vermeeren L, Valdés Olmos RA, Klop WMC, 

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Vermeeren L, Valdés Olmos RA, Meinhardt 

W, Horenblas S. Intraoperative imaging for 

sentinel node identifi cation in prostate carcinoma: 

its use in combination with other techniques. J 


161 



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Buning-Kager JC, Kors e CM, Hauptmann 

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162 

BIOMETRICS DEPARTMENT 

Head of department Otilia Dalesio 

Otilia Dalesio MSc Head 

Harm van Tinteren PhD Academic staff 

Andrew Vincent PhD Academic staff 

Erik van Werkhoven MSc Academic staff 

Tinie Benraadt MD Academic staff 

Jolanda Appelman Technical staff 

Robin Arens Technical staff 

Danny Baars Technical staff 

Nathalie Barbier Technical staff 

Frauwkje Bessels Technical staff 

Judith Boot Technical staff 

Henk Botma Technical staff 

Audi Boucher Technical staff 

Monique Carreno Technical staff 

Saskia Cooke Technical staff 

Gerda de Jong Technical staff 

Marjolijn de Waal MSc Technical staff 

Ineke de Wit Technical staff 

Brigitte Dufournij Technical staff 

Marlou Eilers MSc Technical staff 

Ernesto Fernandez Salcedo MSc Technical 

staff 

Yvonne Groot Technical staff 

Christiane Hagenaars Technical staff 

Song-Hieng Hau MSc Technical staff 

Caroline Heemskerk Technical staff 

Roos Hennink Technical staff 

Annelies Hiemstra Technical staff 

Paula Hoekstra MSc Technical staff 

Eduard Ivanov Technical staff 

Figure 1: Number of trials in TENALEA 

from 2007 to the close of the EU-funded 

initial deployment phase in mid 2011. 

Figure 2: Number of trials registered and 

published in each year since 1980 (based 

on the year of publication of articles in 

the Cochrane Central Register of 

Controlled Trials and the year of 

registration of studies in trials registries 

available through the World Health 

Organisation’s International Clinical 

Trials Registry Platform). 


ICT PROJECTS 

During the last 20 years we have developed tools to assist and support us and the 

participating medical staff in the running of clinical trials. These developments 

have often been partly funded within EC projects. 

The most extensive system we developed is ALEA, which is a comprehensive system 

to register, randomize and automatically distribute notifi cations of the randomization; 

and to allow the development of applications for studies, by which entry of data 

from the participating centers can be done directly into the Data Center databases 

(electronic case record forms or eCRF system). The eCRF system allows detection of 

inconsistencies at the source resulting in less data queries at later times and speeding 

up the statistical analysis and reporting. The system includes an audit trail in 

compliance with the requirements of the International Committee for Harmonization 

and produces standard based export facilities (CDISC, ODM and SDTM). 

Since 2006, we have been leading a European Union funded project (TENALEA 

Initial Deployment) to deploy ALEA in several European centers. ALEA makes 

it easier for academic researchers around the world to conduct clinical trials to 

the highest standards. For example, ALEA includes a secure way to protect the 

concealment of the allocation up until the point that a patient enters the study; 

thereby ensuring the integrity of the randomization. We have worked with partners 

in other cancer research groups across Europe, as well as with the UK Cochrane 

Centre based in Oxford throughout the initial deployment of the project. The funded 

project came to a successful conclusion in June 2011, with 32 active data centres 

and nearly 400 clinical trials in the system (fi gure 1), 65 of which were pending and 

more than 250 were open. Our Institute has made agreements with a new start up 

company, FormsVision, to take care of the marketing and future development of 

ALEA. Like this, the primary long term goal of the project that the Services could 

continue to be available to Academics after the completion of the TENALEA Initial 

Deployment project at a moderate marginal cost, seems to have been achieved. 

The last year of enhancements to ALEA saw considerable new developments, 

including the implementation of several novel features to meet the needs of current 

and future users; as well as the adoption of the system by several innovative new trials, 

and the identifi cation of its considerable potential for researchers around the world.

Initially, the main focus of TENALEA was on a randomization service, which would 

support the conduct of clinical trials comparing different interventions, and we 

prepared a variety of statistical tools to do this. Over time, the need to extend the 

service to allow registration in other types of study and to support the conduct of 

paperless or paper-minimal trials was met through the addition of new features. 

Many trials now rely on electronic case report forms (eCRF), and ALEA makes 

it easier to create these forms, to complete them online, to share the information 

quickly and accurately by electronic means, and to use the data they contain to 

trigger changes to a patient’s care, to report progress on the trial, etc. An ever 

increasing proportion of NKI trials rely exclusively, or almost exclusively, on the 

eCRF, having moved away from paper forms, leading to improvements in both the 

quality and effi ciency of the clinical trial process. 

The eCRF usually focus on clinical outcomes, but the emphasis in health research is 

increasingly moving towards patient-reported outcomes and 2011 saw considerable 

progress within TENALEA in this area. A facility to use the system for electronic 

patient-reported outcomes (ePRO) was added, allowing patients to log in and provide 

information about their own symptoms and health status. This has already been 

implemented within a European trial on women with breast cancer. TENALEA will 

be used for a special investigation in this study, with more than 2000 women in The 

Netherlands using the ePRO facility to complete a lengthy questionnaire when they 

fi nish the study. 

Another new element in TENALEA allows the results of tests on a patient to be 

connected to their record within the data fi le. This integration allows images and 

measurements from laboratory investigations to be brought into TENALEA through 

a new data exchange which also supports asynchronous PACS exchange. 

One of the recently opened clinical trials made more feasible by TENALEA is a 

placebo-controlled study of calcium supplementation before and during pregnancy. 

This study is being run by the World Health Organisation and will recruit 1400 

women who developed pre-eclampsia or eclampsia in previous pregnancies. The 

calcium and placebo treatment packs weigh several kilograms each and are shipped 

to the four sites in South Africa and Zimbabwe in batches of 64, stacked on a pallet. 

The drug supply features of TENALEA have made it much easier to cope with the 

complexities of preparing these pallets, supplying them to the sites and ensuring 

that it is easy to obtain the relevant, uniquely-coded pack when it has been allocated. 

Other recent trials include the TULP study comparing two surgical procedures for 

hernia repair, which started in September 2010 and has already recruited more than 

750 patients; and a randomized trial of the effects of a mousse for re-mineralizing 

teeth, which is being coordinated by the internationally renowned oral health 

research group at the University of Manchester in England. Among the many Dutch 

trials using ALEA, NVALT12 is testing the addition of nitroglycerin patches to 

paclitaxel-carboplatin-bevacizumab in patients with stage IV non-squamous-nonsmall 

cell lung cancer, and making extensive use of the eCRF features of the system. 

The potential for TENALEA is well illustrated by the growth in clinical trials over 

the last three decades, and the increasing recognition of the need to ensure that the 

quality of these trials is suffi cient to infl uence future health care. More than 25,000 

reports of trials are published annually and the number of studies registered for 

the fi rst time each year is approaching this number (fi gure 2). We are continuing to 

work with colleagues in the MRC Network of Hubs for Trials Methodology Research 

on these aspects of the history and epidemiology of clinical trials, highlighting the 

need to strengthen further the research base and to provide the tools needed to 

identify reliably the effects of different interventions and actions. 

CLINICAL STUDIES AND OTHER COLLABORATIONS 

We have developed collaborations with several cooperative groups; academic groups, 

industry and clinical research organizations, and we function as partner for clinical 

studies and clinical trials, being involved from the generation of the idea, protocol 

setting, the planning, and providing project management, randomization services, 

163 


Marissa Jansen Technical staff 

Aarti Jibodh Technical staff 

Irene Jonkers Technical staff 

Joop Jonkman MSc Technical staff 

Josien Kant Technical staff 

Lies Kolmschate Technical staff 

Marianne Mahn MSc Technical staff 

Ingrid Mandjes MSc Technical staff 

Carla Modder Technical staff 

Pietje Muller Technical staff 

Elvira Nuijten Technical staff 

Lindsay Ongering Technical staff 

Cecile Paulus van Pauwvliet Technical staff 

Loes Pronk MSc Technical staff 

Harriet Rehorst MSc Technical staff 

Vera Reijnders Technical staff 

Anneke Reinders Technical staff 

Marielle Roskam Technical staff 

Martin Ryan Business advisor 

Vincent Scuric Technical staff 

Dea Storm Technical staff 

Beata Sznajder PhD Technical staff 

Ria Tilgenkamp Technical staff 

Alex Torres Acosta Technical staff 

Ludy Valkenet MD Technical staff 

Digna van den Broek Technical staff 

Marjolijn van den Haak MSc Technical staff 

Emile van der Donk Technical staff 

Tony van de Velde Technical staff 

Gabry van Netten Technical staff 

Wil van Waardenberg Technical staff 

Steven Vanhoutvin Technical staff 

Anneke Wals Technical staff 

Lidwina Wever Technical staff 

Yvonne Wijnands Technical staff 

Els Willemse Technical staff 

Jeroen Zandbergen MSc Technical staff

164 


Publications 

Ackerstaff AH, Rasch CR, Balm AJ, 

de Boer JP, Wiggenraad R, Rietveld DH, 

Gregor RT, Kroger R, Hauptmann M, 

Vincent A, Hilgers FJ. Five-year quality of 

life results of the randomized clinical phase 

III (RADPLAT) trial, comparing 

concomitant intra-arterial versus 

intravenous chemoradiotherapy in locally 

advanced head and neck cancer. Head 

Neck 2011;10 


Tinteren H, Horenblas S, Haanen J. A 

phase II study of presurgical sunitinib in 

patients with metastatic clear-cell renal 

carcinoma and the primary tumor in situ. 

Urology 2011;78:832-7 

Biesma B, Wymenga AN, Vincent A, 

Dalesio O, Smit HJ, Stigt JA, Smit EF, 

van Felius CL, van Putten JW, Slaets JP, 

Groen HJ. Quality of life, geriatric 

assessment and survival in elderly patients 

with non-small-cell lung cancer treated 

with carboplatin-gemcitabine or 

carboplatin-paclitaxel: NVALT-3 a phase III 

study. Ann Oncol 2011;22:1520-7 

Boekhout AH, Vincent AD, Dalesio OB, 

van den Bosch J, Foekema-Tons JH, 

Adriaansz S, Sprangers S, Nuijen B, 

Beijnen JH, Schellens JH. Management of 

hot fl ashes in patients who have breast 

cancer with venlafaxine and clonidine: 

a randomized, double-blind, placebocontrolled 

trial. J Clin Oncol 2011;29:3862-8 

Bruin SC, He Y, Mikolajewska- 

Hanclich I, Liefers GJ, Klijn C, Vincent A, 

Verwaal VJ, de Groot KA, Morreau H, van 

Velthuysen ML, Tollenaar RA, van ‘t Veer 

LJ. Molecular alterations associated with 

liver metastases development in colorectal 

cancer patients. Br J Cancer 2011;105:281-7 

Darby S, McGale P, Correa C, Taylor C, 

Arriagada R, Clarke M, Cutter D, Davies 

C, Ewertz M, Godwin J, Gray R, Pierce L, 

Whelan T, Wang Y, Peto R. Effect of 

radiotherapy after breast-conserving surgery 

on 10-year recurrence and 15-year breast 

cancer death: meta-analysis of individual 

patient data for 10,801 women in 17 

randomised trials. Lancet 2011;378:1707-16 

Davies C, Godwin J, Gray R, Clarke M, 

Cutter D, Darby S, McGale P, Pan HC, 

Taylor C, Wang YC, Dowsett M, Ingle J, 

Peto R. Relevance of breast cancer hormone 

receptors and other factors to the effi cacy of 

adjuvant tamoxifen: patient-level metaanalysis 

of randomised trials. Lancet 

2011;378:771-84 

de Boer JP, Raderer M, van Tinteren H, 

Aleman BM, Boot H, de Jong D. Treatment 

of extranodal marginal zone lymphoma of 


development of (electronic) case record forms, data handling, monitoring and quality 

assurance, and statistical expertise. In addition, the tools that we have developed 

within our ICT projects are attracting new associations. 

From 1616 patients of the EORTC 22881-10882 (boost versus no boost) clinical trial 

on patients with breast cancer, centrally reviewed pathology data were collected and 

were used to construct a model to predict the probability of ipsilateral breast relapse. 

A spline curve was used to allow for a non-linear effect of age. Figure 3 shows the 

age effect estimated by the model, where the hazard ratio on vertical axis has been 

graduated according to the log scale. The relative hazard has been set to one at 

the reference age of 50 years. Other variables in the model were tumor diameter, 

histological grade, presence or absence of DCIS, chemotherapy, tamoxifen, and boost 

treatment. The model 

itself was published in the 

form of a nomogram and 

a website is now under 

construction (http:// 

research.nki.nl/ibr/) to 

make the model easily 

accessible for clinicians. 

Figure 3: Age effect estimated 

by the model, where the 

hazard ratio on vertical axis 

has been graduated according 

to the log scale 

The statistical 

team works with the Renal Tumor Study Group of the International Society of 

Pediatric Oncology (SIOP), which has its origin in the late sixties when European 

pediatricians joined forces in the treatment of children with nephroblastoma 

(Wilms’ tumors). In addition to carrying out trials the group is collecting data 

in a register that now includes about 7000 nephroblastomas and other renal 

malignancies, such as renal cell carcinomas, clear cell carcinomas and rhabdoid 

tumors. A project is being presented to the EC to fund the maintenance and further 

utilization of the data to answer questions and defi ne possible prognostics factors. 

NVALT CANCER STUDIES 

The Department functions as Data Center for the cancer studies of the Dutch 

Chest Physician Association (NVALT) and we collaborate in planning, designing 

and running of their clinical trials in lung cancer and mesothelioma. More than 75 

hospitals in the Netherlands participate in the clinical trials. 

Support for data management and statistical analysis has been obtained from the 

KWF for 8 studies of the 14 trials that have or are being run by the group. 

Accrual to the NVALT12, a randomized study of docetaxel, cisplatin, bevacizumb 

with or without nitroglycerine patches in patients with stage IV non squamous non 

small cell lung cancer, started in 2011 and already 85 patients have been randomized 

by 14 participating centers. Accrual to the NVALT14, a randomized trial comparing 

longstanding indwelling pleural catheters with pleurodesis as a frontline treatment for 

malignant pleural effusion, also started in 2011 with 34 patients entered by 4 centers. 

Final analysis of the NVALT 5, a randomized phase III trial of the antiangiogenic 

agent Thalidomide in patients with malignant pleural mesothelioma after fi rst line

chemotherapy, was completed in 2011 and the publication has been submitted. 

The NVALT4 trial, placebo-controlled study of docetaxel/carboplatin with celecoxib 

or placebo in patients with locally advanced or metastatic non-small cell lung cancer, 

has been published this year (Groen HJ et al, J Clin Oncol 2011;29:4320-6). 

The accrual to NVALT 10 continued at a consistent pace and 224 patients had already 

been randomized by 18 participating centers in this study comparing erlotinib 

vs erlotinib and chemotherapy combination in pretreated patients with advanced 

NSCLC. The accrual will be completed end of 2011 and fi nal analysis and publication 

is expected in 2012. 

In the NVALT11 study the value of prophylactic cranial irradiation (PCI) versus 

observation is studied in radically treated patients with stage III non-small cell lung 

cancer. It is done as a cooperation of the NVALT, the Dutch Lung Cancer Research 

Group (DLCRG) and the National Platform for Radiotherapy in Lung Tumors 

(LPRL). A total of 116 patients have been included in this study. Collaboration with 

the Italian and with the Danish Groups is being organized to increase the rate of 

accrual which is still lower than expected. 

OTHER STUDIES 

Collaboration of the statistical team with the Dutch Colorectal Cancer Group 

(DCCG) had resulted in several large phase III randomized studies in patients with 

advanced colorectal cancer previously untreated. In 2011 a new confi dential interim 

analysis of the CAIRO 3 study was prepared to be discussed by the Independent 

Data Monitoring Committee. The study compares maintenance treatment with 

capecitabine and bevacizumab versus observation after induction treatment with 

capecitabine, oxaliplatin, and bevacizumab as fi rst-line treatment. In excess of 500 

patients have been already randomized in the study by 64 centers. 

Collaboration with the Department of Nuclear Medicine of the Vu has resulted in 

several publications and new trials are being prepared in collaboration with the lung 

physicians of the VU. 

We collaborate with medical departments in our Institute in carrying out, handling 

data and SAEs and providing statistical support for the multi center clinical trials 

they coordinate. We coordinate with the radiotherapy department a large randomized 

study in young women with early breast cancer (Young Boost trial). In excess of 2400 

patients have already been entered by participating centers in the Netherlands, France 

and Germany and accrual will be completed end of the year. Tumor material is being 

collected for translational research analyses. Cosmetics results are being evaluated 

with pictures taken in series. New studies are currently being developed. 

With the surgeons and medical oncologists we collaborate in several multicentric 

studies, like the OVHIPEC, which is a study of intraperitoneal chemotherapy and 

hyperthermia in ovarian cancer, the Matador in breast cancer and the TRAIN 

study, a neoadjuvant study in breast cancer, the Tyvtax in head and neck cancer and 

the Raditux and the PetBoost studies in lung cancer. We also collaborate with the 

pharmacology department as statistical center for 2 randomized studies. One of 

them is a randomized double blind study of treatment of hot fl ashes in breast cancer 

patients in menopause following chemotherapy for their breast cancer. The fi nal 

report was published this year (Boekhout AH et al, J Clin Oncol 2011;29:3862-8). 

The second study is a double blind randomized study on cardio-protection of breast 

cancer patients treated by Herceptin for which our statistical team has produced this 

year the 2nd confi dential interim analysis report to be discussed by the independent 

data monitoring committee. 

The statisticians have collaborated with other departments of the institute and other, 

academic and not academic institutes in the region in a variety of studies many of 

which resulted in co-authorships as can be seen in the publication list. 

165 



fl udarabine: effect on tumor microenvironment. 

Leuk Lymphoma 2011 

de Langen AJ, van den Boogaart V, 

Lubberink M, Backes WH, Marcus JT, 

van Tinteren H, Pruim J, Brans B, Leffers 

P, Dingemans AM, Smit EF, Groen HJ, 

Hoekstra OS. Monitoring response to 

antiangiogenic therapy in non-small cell 

lung cancer using imaging markers derived 

from PET and dynamic contrast-enhanced 

MRI. J Nucl Med 2011;52:48-55 



Guchelaar HJ, Smits PH, Beijnen JH, 

Punt CJ, Schellens JH, Cats A. 

Relationship between single nucleotide 

polymorphisms and haplotypes in DPYD 

and toxicity and effi cacy of capecitabine in 

advanced colorectal cancer. Clin Cancer Res 

2011;17:3455-68 

Dingemans AM, de Langen AJ, van 

den Boogaart V, Marcus JT, Backes WH, 

Scholtens HT, van Tinteren H, Hoekstra 

OS, Pruim J, Brans B, Thunnissen FB, 

Smit EF, Groen HJ. First-line erlotinib 

and bevacizumab in patients with locally 

advanced and/or metastatic non-small-cell 

lung cancer: a phase II study including 

molecular imaging. Ann Oncol 2011;22: 

559-66 

Doeksen A, Bakx R, Vincent A, 

van Tets W, Sprangers M, Gerhards M, 

Bemelman W, van Lanschot J. J-pouch 

versus side-to-end coloanal anastomosis 

after preoperative radiotherapy and total 

mesorectal excision for rectal cancer: 

a multicenter randomized trial. Colorectal 

Dis 2011;10-1318 

Evers DJ, Smeenk RM, Bottenberg PD, 

van Werkhoven ED, Boot H, Verwaal VJ. 

Effect of preservation of the right gastroepiploic 

artery on delayed gastric emptying 

after cytoreductive surgery and HIPEC: 

a randomized clinical trial. Eur J Surg 


Frings V, de Langen AJ, Smit EF, van 

Velden FH, Hoekstra OS, van Tinteren H, 

Boellaard R. Repeatability of metabolically 

active volume measurements with 18F-FDG 

and 18F-FLT PET in non-small cell lung 

cancer. J Nucl Med 2010;51:1870-7 

Gadiot J, Hooijkaas AI, Kaiser AD, 

van Tinteren H, van Boven H, Blank C. 

Overall survival and PD-L1 expression in 

metastasized malignant melanoma. Cancer 

2011;117:2192-201 

Gast MC, Zapatka M, van Tinteren H, 

Bontenbal M, Span PN, Tjan-Heijnen VC, 

Knol JC, Jimenez CR, Schellens JH, 

Beijnen JH. Postoperative serum proteomic 

profi les may predict recurrence-free survival

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in high-risk primary breast cancer. J Cancer 

Res Clin Oncol 2011;137:1773-83 

Giesen E, Mager A, van Tinteren H, 

Rodenhuis S, Kerst JM. An alternative 

treatment regimen of advanced seminoma 

with carboplatin, etoposide, and bleomycin 

instead of cisplatin-based therapy. Urol 

Oncol 2011 

Godzinski J, van Tinteren H, de Kraker 

J, Graf N, Bergeron C, Heij H, von 

Schweinitz D, Fuchs J, Cecchetto G, 

Audry G, Gauthier F, Sandstedt B. 

Nephroblastoma: does the decrease in tumor 

volume under preoperative chemotherapy 

predict the lymph nodes status at surgery? 

Pediatr Blood Cancer 2011;57:1266-9 


HH, van Werkhoven E, Kerst JM, 

Horenblas S. Inguinal recurrence following 

therapeutic lymphadenectomy for node 

positive penile carcinoma: outcome and 

implications for management. J Urol 

2011;185:888-93 

Groen HJ, Sietsma H, Vincent A, 

Hochstenbag MM, van Putten JW, van 

den Berg A, Dalesio O, Biesma B, Smit HJ, 

Termeer A, Hiltermann TJ, van den Borne 

BE, Schramel FM. Randomized, Placebo- 

Controlled Phase III Study of Docetaxel Plus 

Carboplatin With Celecoxib and 

Cyclooxygenase-2 Expression As a 

Biomarker for Patients With Advanced 

Non-Small-Cell Lung Cancer: The NVALT-4 

Study. J Clin Oncol 2011;29:4320-6 

Hompes D, Boot H, van Tinteren H, 

Verwaal V. Unresectable peritoneal 

carcinomatosis from colorectal cancer: a 

single center experience. J Surg Oncol 

2011;104:269-73 

Joerger M, Burgers SA, Baas P, Smit 

EF, Haitjema TJ, Bard MP, Doodeman VD, 

Smits PH, Vincent A, Huitema AD, 

Beijnen JH, Schellens JH. Germline 

polymorphisms in patients with advanced 

nonsmall cell lung cancer receiving fi rst-line 

platinum-gemcitabine chemotherapy: A 

prospective clinical study. Cancer 2011;10 

Korse CM, Taal BG, Bonfrer JM, 

Vincent A, van Velthuysen ML, Baas P. An 

elevated progastrin-releasing peptide level in 

patients with well-differentiated 

neuroendocrine tumours indicates a primary 

tumour in the lung and predicts a shorter 

survival. Ann Oncol 2011;22:2625-30 

Korse CM, Taal BG, Vincent A, van 

Velthuysen ML, Baas P, Buning-Kager JC, 

Linders TC, Bonfrer JM. Choice of tumour 

markers in patients with neuroendocrine 

tumours is dependent on the histological 

grade. A marker study of Chromogranin A, 

Neuron specifi c enolase, Progastrin-releasing 


peptide and cytokeratin fragments. Eur J Cancer 


Lips EH, Mulder L, de Ronde JJ, Mandjes IA, 

Vincent A, Vrancken Peeters MT, Nederlof PM, 

Wesseling J, Rodenhuis S. Neoadjuvant 

chemotherapy in ER+ HER2- breast cancer: 

response prediction based on immunohistochemical 

and molecular characteristics. Breast Cancer Res 

Treat 2011 

Nijkamp J, Doodeman B, Marijnen C, 

Vincent A, Vliet-Vroegindeweij C. Bowel 

exposure in rectal cancer IMRT using prone, 

supine, or a belly board. Radiother Oncol 2011 

Palma DA, Senan S, Haasbeek CJ, Verbakel 

WF, Vincent A, Lagerwaard F. Radiological and 

clinical pneumonitis after stereotactic lung 

radiotherapy: a matched analysis of threedimensional 

conformal and volumetric-modulated 

arc therapy techniques. Int J Radiat Oncol Biol 

Phys 2011;80:506-13 

Palma DA, Sornsen de Koste J, Verbakel WF, 

Vincent A, Senan S. Lung density changes after 

stereotactic radiotherapy: a quantitative analysis 

in 50 patients. Int J Radiat Oncol Biol Phys 

2011;81:974-8 

Phernambucq EC, Palma DA, Vincent A, 

Smit EF, Senan S. Time and dose-related changes 

in radiological lung density after concurrent 

chemoradiotherapy for lung cancer. Lung Cancer 

2011;74:451-6 

Richard W, Timmer F, van Tinteren H, de 

Vries N. Complications of hyoid suspension in the 

treatment of obstructive sleep apnea syndrome. Eur 

Arch Otorhinolaryngol 2011;268:631-5 


Ackerstaff AH, Jacobi I, Hilgers FJ. A new heat 

and moisture exchanger for laryngectomized 

patients: endotracheal temperature and humidity. 

Respir Care 2011;56:604-11 


Hilgers FJ. Heat and moisture exchange capacity 

of the upper respiratory tract and the effect of 

tracheotomy breathing on endotracheal climate. 

Head Neck 2011;33:117-24 

Smets AM, Tinteren HV, Bergeron C, 

Camargo BD, Graf N, Pritchard-Jones K, Kraker 

JD. The contribution of chest CT-scan at diagnosis 

in children with unilateral Wilms’ tumour. Results 

of the SIOP 2001 study. Eur J Cancer 2011 

Swellengrebel HA, Marijnen CA, Vincent A, 

Cats A. Evaluating long-term attachment of two 

different endoclips in the human gastrointestinal 

tract. World J Gastrointest Endosc 2010;2:344-8 

van den Heuvel-Eibrink MM, van Tinteren 

H, Rehorst H, Coulombe A, Patte C, de 

Camargo B, de Kraker J, Leuschner I, 

Lugtenberg R, Pritchard-Jones K, Sandstedt B, 

Spreafi co F, Graf N, Vujanic GM. Malignant 

rhabdoid tumours of the kidney (MRTKs), 

registered on recent SIOP protocols from 1993 to 

2005: a report of the SIOP renal tumour study 

group. Pediatr Blood Cancer 2011;56:733-7 


Wessels LF, Schmidt MK, van Beers EH, 


Vries EG, Schrama JG, Wesseling J, van de 

Vijver MJ, van Tinteren H, de Bruin M, 

Hauptmann M, Rodenhuis S, Linn SC. An 

aCGH classifi er derived from BRCA1-mutated 

breast cancer and benefi t of high-dose platinumbased 

chemotherapy in HER2-negative breast 

cancer patients. Ann Oncol 2011;22:1561-70 

Werkhoven E, Hart G, Tinteren H, 

Elkhuizen P, Collette L, Poortmans P, Bartelink 

H. Nomogram to predict ipsilateral breast relapse 

based on pathology review from the EORTC 

22881-10882 boost versus no boost trial. Radiother 


Wildeman MA, Zandbergen J, Vincent A, 

Herdini C, Middeldorp JM, Fles R, Dalesio O, 

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setting? Trials 2011;12:190

CLINICAL TRIALS 

Type of Study 

cancer study coordinator Activated 

(nick name) Title in NKI-AVL Phase (closed) 

ALL SITES 

M07KUC A phase I, open-label, study to assess the safety and tolerability of 

KU-0059436 in combination with Carboplatin, KU-0059436 in 

combination with Paclitaxel/Carboplatin doublet and KU-0059436 

in combination with Paclitaxel in the treatment of patients with 

advanced solid tumours 

Schellens, JHM I 25-6-2007 

M07MKC A phase I dose escalating study evaluating MK-1775 in both 

monotherapy and in combination with Gemcitabine, Cisplatin or 

Carboplatin in adult patients with advanced solid tumors 


M07OTS A phase I study of oral topotecan in subjects with cancer and 

impaired renal function 


M07PFU Pharmacogenomic and pharmacokinetic safety and cost-saving Schellens, JHM other 16-5-2007 

analysis in patients treated with fl uoropyrimidines (6-12-2011) 

M08HYT A phase I open-label study of the safety, tolerability and 

pharmacokinetics of two schedules of oral topotecan in combination 

with pazopanib in subjects with advanced solid tumors 


M09BGJ A phase I open-label, multicenter, dose escalation study of oral 

BGJ398, a pan FGF-R kinase inhibitor in adult patients with 

advanced solid malignancies 


M09DAZ A phase I open label multicenter study to assess the safety and 

tolerability, pharmacokinetics, preliminary anti-tumor activity 

of ascending doses of AZD4547 in patients with advanced solid 

malignancies 


M09GDC A phase Ib open label, dose-escalation study of the safety and 

pharmacology of GDC-0941 in combination with erlotinib with 

advanced solid tumours 


M09GSK Phase I open label, dose-escalation study of the phosphoinositide Schellens, JHM I 31-3-2010 

3-kinase inhibitor (GSK2126458) in subject with solid tumor 

or lymphoma 

(18-11-2011) 

M09LAP An open label phase Ib continuation study of lapatinib monotherapy Schellens, JHM I 26-11-2009 

(E111767) or lapatinib in combination with other anti-cancer treatment in 

patients with solid tumors 


M09NIB The NIB-Cohort study, therapeutic drug monitoring of tyrosine 

kinase inhibitors 

Schellens, JHM other 9-6-2009 

M09RGD A phase II, open label, non-randomized, multicenter, pilot, effi cacy 

study of [F-18]RGD-K5 Positron Emission Tomography (PET) as 

a tool to monitor response to an anti-angiogenic drug 

Ruers, TJM II 20-5-2010 

M10AZD A phase I open-label, multicenter study to assess the safety, 

tolerability, pharmacokinetics and preliminary anti-tumor activity of 

ascending doses of AZD5363 under adaptable dosing schedules in 

patients with advanced solid malignancies 


167 

CLINICAL TRIALS

168 


Type of Study 



M10BBP A phase Ib multi-center, open-label, 4 arm dose-escalation study Steeghs, N I 14-3-2011 

(CBEZ235A2118) of oral BEZ235 and BKM120 in combination with weekly paclitaxel 

in patients with advanced solid tumors and weekly paclitaxel/ 

trastuzumab in patients with HER2+ metastatic breast cancer 

M10BEZ A phase I/Ib, multi-center, open-label study of BEZ235 administered Schellens, JHM I 17-2-2011 

(CBEZ235A2101) orally on a continuous daily dosing schedule in adult patients with 

advanced solid malignancies including patients with advanced 

breast cancer 

M10FES An open-label, dose escalation, pharmacodynamic, pharmacokinetic Schellens, JHM I 5-4-2011 

(LIFE-110) and effect of food phase 1 study of E7820 to determine the maximum 

tolerated dose following twice daily oral administration in subjects 

with unresectable solid tumours 

M10IPC A randomized trial comparing longstanding indwelling pleural catheters Heuvel van den, MM III 31-1-2011 

(NVALT 14) with pleurodesis as a frontline treatment for malignant pleural effusion 

M10PKS Use of individual PK-guided sunitinib dosing: A feasibility study in Steeghs, N other 14-3-2011 

patients with advanced solid tumors 

M11BYL A phase 1a, multicenter, open label dose-escalation study of oral 

BYL719, in adult patients with advanced soild malignancies, whose 

tumors have a PIK3CA gene 

Schellens, JHM I 28-7-2011 

M11GLP An open-label dose escalating phase 1b study for the assessment of Schellens, JHM I 27-9-2011 

(GALAPAGOS) safety, tolerability, pharmacokinetics and pharmacodynamics of 

multiple intravenous doses of GLPG0187 in subject with solid tumors 

M11LDK A phase I, multicenter, open-label dose escalation study of LDK378, 

administered orally in adult patients with tumors characterized by 

genetic abnormalities in anaplastic lymphoma kinase (ALK) 

Steeghs, N I 5-4-2011 

M11MSC A multicentre, open label, Phase I trial of the MEK inhibitor Schellens, JHM I 4-7-2011 

(20862) MSC1936369B given orally to subjects with solid tumours 

M11RCE Phase I open label multicenter dose-escalation study to evaluate, Schellens, JHM I 17-11-2011 

(huMab HER3) safety pharmacokinetics and activity of RO5479599, a glycoengineerd 

antibody against HER3, administered as IV infusion either alone or 

in combination with Cetuximab or in combination with Erlotinib in 

patients with metastatic and/or locally advanced malignant HER3positive 

solid tumors of epithelial cell origin 

M11TBB An open-label, phase I/IIa dose escalating study of 2B3-101 in 

patients with solid tumors with brain metastases 

Brandsma, D I/II 6-7-2011 

M11TCO A phase I open label two stage randomized cross over comparative 

(single dose) pharmacokinetic and safety study of two formulations 

of CO-1.01 for injections in patients with advanced solid tumors 


M11TWE A phase I Multiple Ascending Dose (MAD) study of RO5458640, a Schellens, JHM I 5-7-2011 

(TWEAK) Humanized Monoclonal Antibody against the TNF-like weak inducer 

of Apoptosis (TWEAK) Ligand, in patients with advanced solid tumors 

N06PFB Pleural fl uid bank Heuvel van den, MM other 27-9-2006 


N07DOW Weekly administration of oral Docetaxel in combinaton with Ritonavir Schellens, JHM I 14-11-2007

Type of Study 



N07NEX Phase I study of gemcitabine and carboplatin plus sorafenib in 



N08CTC Validation of circulating tumor cells (CTC) detection techniques in 


Schellens, JHM other 31-7-2008 

N08EDO Phase I interaction study of docetaxel with supplementation of 

St. John’s wort or Echinacea 


N08NPM NVALT Palliative care Protocol on malignant pleural effusion Heuvel van den, MM other 13-3-2008 

N10BOM Weekly administration of (bi-) daily Oral Docetaxel in combination 

with Ritonavir 


N10CRC Proof of principle and pharmacological phase 0 crossover study with Marchetti, S other 17-11-2011 

(ModraCape001) controlled release capecitabine (ModraCape001) 

N10EFP feasibility of ejection fraction measurement with gallium-68 citrate Vogel, WV other 11-6-2010 

PET/CT (EF-PET) (6-12-2011) 

N10MOP Development and clinical activity of low dose metronomic 

chemotherapy with oral paclitaxel 


N10MTD Phase I interaction study of docetaxel and tolbutamide with 

supplementation of Milk Thistle 


BRAIN / CNS 

M10BEL Randomized phase II study of bevacizumab versus bevacizumab plus Brandsma, D II 27-9-2010 

(BELOB) lomustine versus lomustine in patients with recurrent glioblastoma, 

the BELOB trial 


N05THB Totale hersenbestraling met een eenmalige boost bij patienten met Dewit, LGH other 7-4-2005 

solitaire hersenmetastase van een epitheliale tumor 

- een registratiestudie 


BREAST 

E10041 Micro-array in node-negative disease may Avoid Chemotherapy: a Rutgers, EJTh III 2-1-2007 

(MINDACT) prospective randomized study comparing the 70-gene signature with 

The common clinical-pathological criteria in selecting patients for 

adjuvant chemotherapy in node-negative breast cancer 


E22051 SUPREMO, an MRC phase III randomised trial to assess the role of Russell, NS III 27-2-2007 

(SUPREMO) adjuvant chest wall irradiation in ‘intermediate risk’ operable breast 

cancer following mastectomy 

M03RBC Radiation dose intensity study in breast cancer in young women: Bartelink, GMM III 29-3-2004 

(YOUNG BOOST) a randomized phase III trial of additional dose to the tumor bed 

(“young boost”) 

M04MAT Microarray Analysis in breast cancer to Tailor Adjuvant Drugs Or Linn, SC III 26-4-2004 

(MATADOR) Regimens (MATADOR) 

M05BRI Long term risk of breast cancer following treatment of Hodgkin’s Russell, NS other 5-1-2006 

(BRIGHT) disease 

169 

CLINICAL TRIALS

170 


Type of Study 



M05HIR Hormonal substitution after prophylactic adnectomy in women Beurden, M van other 31-5-2005 

(HIRISE) with an increased risk for breast- and ovarian cancer due to a 

genetic predisposition: HIRISE (High-Risk women and hormonal 

Substitution Exposure) 

M06HER Prospective randomized pharmacological intervention study; Schellens, JHM III 18-6-2007 

(CANDY) evaluating the effect of angiotensin II-receptor (AT1) blocker (23-2-2011) 

candesartan versus placebo in prevention of trastuzumab-associated 

cardiotoxicity in patients with primary breast cancer treated with 

trastuzumab 

M06TM2 A. Multicentre, prospective phase II trial investigating the effi cacy of Linn, SC III 26-9-2006 

(TEAM II) neoadjuvant hormonal therapy with exemestane for six months 

B. Randomised, multicentre, prospective, phase III trial investigating 

the effi cacy and safety of the addition of ibandronate to adjuvant 

hormonal therapy in postmenopausal women with hormone receptor 

positive early breast cancer 

M07CBE Late effects of chemotherapy on brain functioning in the elderly Schagen, SSB other 1-7-2008 

M07LET IDEAL: Investigation on the duration of extended adjuvant letrozole Rutgers, EJTh III 16-11-2007 

(IDEAL) treatment. An open lable, randomized phase III trial comparing 2,5 

year duration of letrozole (Femara) treatment with 5 year duration in 

patients previously treated for endocrine sensitive early breast cancer 


M08BCP Prospective and Retrospective register study of the German Adjuvant Linn, SC other 27-3-2008 

(BOOG 2003-04) Cancer Study Group (GABG) for diagnosis and treatment of breast 

cancer in pregnancy 

M08CPE A two-arm randomized open label phase 2 study of CP-751,871 in Linn, SC II 27-1-2009 

combination with exemestane versus exemestane alone as fi rst line 

treatment for postmenopausal patients with hormone receptor 

positive advanced breast cancer 


M08HAT A randomized phase II study of concomitant trastuzumab, 

bevacizumab with paclitaxel versus trastuzumab and bevacizumab 

followed by the combination of trastuzumab, bevacizumab and 

paclitaxel at progression as fi rst-line treatment of patients with 

metastatic breast cancer with H er2-neu overexpression 

Linn, SC II 20-4-2009 

M08MAM An exploratory clinical study for initial validation of a novel small Valdes Olmos, RA other 29-4-2009 

(MARI) ring device for positron emission mammotomography 

M08MUL Multicenter feasibility study of the sentinel node procedure in Oldenburg, HSA other 16-4-2009 

(MULTISENT) patients with multiple breast tumors (MULTISENT) 

M08PBI Image guided Preoperative Accelerated partial Breast Irradiation Elkhuizen, PH other 1-10-2009 

(PAPBI) (PAPBI): defi ning radiotherapy sensitivity 

M08TRA Trastuzumab in a neoadjuvant regimen for Her2+ breast cancer – Sonke, GS II 18-9-2008 

(TRAIN) the TRAIN study (11-8-2011) 

M09MLA An open label study to examine the effects of low-fat and high-fat Schellens, JHM I 26-11-2009 

(EGF111582) meals on the pharmacokinetics of orally administered lapatinib in 

metastatic Erb2 positive breast cancer patients 


M09SRB Sentinal Node and recurrent breastcancer; regional staging and Rutgers, EJTh other 27-10-2009 

(SNARB) registration (SNARB)

Type of Study 



M09TNM Randomized phase II/III study of individualized neo-adjuvant Rodenhuis, S II/III 7-1-2010 

(Neo-TN) chemotherapy in triple negative breast tumors 

M10ATR A phase Ib/ II, multi-center, open-label study to evaluate the effi cacy Steeghs, N 28-2-2011 

(CAUY922A2109) of AUY922 in combination with Trastuzumab in patients with locally 

advanced or metastatic HER2-positive breast cancer, that has pro- 

gressed after or during at least one Trastuzumab-containing regimen 

M10DCI A randomized phase III study of radiation doses and fractionation Russell, NS III 9-6-2010 

Schedules for ductal carcinoma (DCIS) of the Breast 

M11FAM Breast density as indicator for the use of mammography or MRI to Rutgers, EJTh other 30-11-2011 

(FaMRIsc) screen women with familiar risk for breast cancer: a RCT 

N04POM Tailored preoperative chemotherapy in stage II or III breast cancer 

with either a primary tumor over 3 cm in size or a clinically 

tumor-positive axilla 

Rodenhuis, S II 21-3-2005 

N04RTB Effecten van bestraling op bloedvaten Russell, NS other 5-10-2004 

N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis Valdes Olmos, RA other 7-6-2005 

and prediction of tumour respons to chemotherapy and/or 

radiotherapy in cancer patients 


N06GLB Phase I study of gemcitabine plus lapatinib (GW572016) in women 

with advanced breast cancer 


N06VNI Meting van de functie van de arm en de bijdrage daaraan van de 

grote borstspieren voor en na de tweezijdige implantatie van een 

endoprothese ten behoeve van een borstreconstructie die direct 

aansluitend aan de huidsparende borstoperatie wordt uitgevoerd 

Hage, JJ other 26-9-2006 

N07BOS Genetic determinants of survival and second breast cancer Rutgers, EJTh other 12-12-2007 

(BOSOM) development in premenopausal breast cancer patients 

N07MAN Randomized phase II/III study of second-line endrocrine treatment Rodenhuis, S II/III 3-4-2008 

(Mandjes studie) followed by capecitabine versus capecitabine followed by endrocrine 

treatment in patients with metastatic ER positive breast cancer 

N08AFT A randomized prospective trial of 2-6 weeks pre-operative hormonal Linn, SC II 4-8-2008 

(AFTER) treatment for hormone receptor positive breast cancer: 

Anastrozole +/- fulvestrant or tamoxifen exposure - response in 

molecular profi le (AFTER-study) 

N08RMB Tumorresponse monitoring in patients with breast cancer treated Vrancken Peeters, other 23-9-2008 

with primary systemic therapy: towards predicting response in 

both the primary tumor and in axillary lymph nodes 

MTFD 

N09PRF Analgesia and nerve function following pulsed radiofrequency for Lukas, A other 2-6-2010 

(PRF4PMPS) postmastectomy pain 

N10BES Biomarker Evaluation and Selection of Targeted Therapy Schellens, JHM other 15-11-2010 

(BEST-Rx) (15-9-2011) 

N10BMT Effect of biphosphonates on telangiectasia in irradiated breast cancer 

patients (biphosphonate modulation of telangiectasia) 

Russell, NS other 24-6-2011 

N10RDA Pilot study to determine the utility of a Likert-like scale to assess 

patients’ experience of radiation dermatitis during radiotherapy for 

breast cancer 

Russell, NS pilot 4-8-2011 

171 

CLINICAL TRIALS

172 


Type of Study 



N10RFS Observational study into specifi c in vivo human discrimination Ruers, TJM other 25-10-2010 

(OpSpect) between benign and malignant tissue using a combination of diffuse 

refl ectance and fl uorescence spectroscopy 

N11ISN Monitoring of the “healthy” immune response in the sentinel Rutgers, EJTh other 27-7-2011 

GASTRO INTESTINAL 

lymph node of patients undergoing a prophylactic mastectomy 

M05RAX Pre-operative chemoradiotherapy regimen with capecitabine and Cats, A II 20-3-2006 

(RAX) bevacuzimab in locally advanced rectal cancer. A feasability study (RAX) (26-4-2011) 

M06CRI A multicenter randomized phase III trial of neo-adjuvant chemotherapy Cats, A III 11-1-2007 

(CRITICS) followed by surgery and chemotherapy or by surgery and 

chemoradiotherapy in resectable gastric cancer 

(CRITICS-study: ChemoRadiotherapy after Induction chemo Therapy 

In Cancer of the Stomach) 

M06SCR A multicenter phase III randomised trial comparing total mesorectal Cats, A III 9-5-2006 

(SCRIPT) excision with pre-operative radiotherapy with or without post-operative 

oral capecitabine in the treatment of operable primary rectal cancer 

M07CBO Maintenance treatment with capecitabine and bevacuzimab versus Cats, A III 4-9-2007 

(CAIRO3) observation after induction treatment with capecitabine, oxaliplatin and 

bevacuzimab as fi rst-line treatment in patients with advanced colorectal 

carcinoma, a randomised phase III study (CAIRO3) 

M07HBT Feasibility study of external beam radiation therapy followed by Triest, B van I/II 25-7-2007 

(HerBerT) high-dose rate endorectal brachytherapy (HDBRT) in inoperable rectal 

cancer patients 

M07NAR Neo-Aduvant Radiotherapy-Chemotherapy In Stomach Cancer. Jansen, E I/II 28-1-2008 

(NARCIS) Induction therapy with carboplatin, paclitaxel and radiotherapy in 

patients with locally advanced gastric cancer. The “NARCIS” study 

M07RBV Clinical pilot study: radiolabeled bevacizumab as a tracer of VEGF Ruers, TJM I 12-2-2008 

expression in patients with colorectal liver metastases. Selecting patients (5-12-2011) 

who may benefi t from anti-VEGF therapy and visualizing the response 

M08ACL Accelerated growth of synchronous colorectal liver metastases: effects Ruers, TJM II 21-2-2008 

(SILENT) of neo-adjuvant therapy 

M08CEL Capsule endoscopy in Lynch Syndrome for small intestinal tumor Cats, A other 13-7-2010 

(CELSIUS) screening: the CELSIUS study 

M08DCS Tumor destruction and DC activation in situ: towards in vivo loaded DC Ruers, TJM pilot 18-9-2008 

Vaccines 

M08GPO 

(PROFOC) 

Genetic and protein profi ling in patients with oesophageal cancer Sandick JW van other 1-9-2008 

M08LYN 

(CHROMOLYNCH) 

Chromoendoscopy in Lynch syndrome patient Cats, A other 13-7-2010 

M08MEK Open-label, multicenter, dose-escalation phase I study with extension to 

evaluate safety, pharmacokinetics and activitiy of RO4987655, a MEK 

inhibitor, administered orally as monotherapy in patients with advanced 

tumors 

Schellens, JHM I 4-2-2009

Type of Study 



M09OCB A pilot evaluating response to induction chemotherapy with oxaliplatin, Verwaal, VJ pilot 25-3-2010 

capecitabine and bevacizumab in patients with extensive peritoneal 

carcinomatosis of colorectar origin 

M10BDO Phase II study of docetaxel, oxaliplatin, capecitabine with bevacizumab Cats, A II 8-2-2011 

(B-DOCT) and trastuzumab in case of human epidermal growth factor receptor 2 

(HER2)-positivity in patients with locally advanced or metastatic gastric 

cancer or adenocarcinoma of the gastro-oesphageal junction 

(B-DOCT study) 

M10STE A comparison of two expandable metal stents for the palliation of Boot, H other 10-8-2010 

(STEMA-STUDIE) malignant esophageal disease (5-1-2011) 

M11BIO Feasibility study of biomarker development for response prediction by Steeghs, N other 7-12-2011 

(CPCT-01) large scale DNA mutational analysis of metastatic lesions 

M11SOM A multicenter, randomized, blinded effi cacy and safety study of 

pasireotide LAR vs octreotide LAR in patients with metastatic carcinoid 

tumors whose disease-related symptoms are inadequately controlled 

by somatostatine analogues. CSOM230C2303 

Tesselaar, M III 17-11-2011 

N05STP Serum and tissue protein profi ling and tumour genetic analysis in 

patients with potential premalignant conditions or colorectal cancer 

Cats, A other 19-1-2006 

N08ICG Pilot study on the use of fl uorecence imaging of lymph nodes during 

colorectal lymphadenectomy using indocyanine green 

Ruers, TJM pilot 30-12-2008 

N08RCT Deformation of target volume during radiotherapy for rectal cancer, Verheij, M other 14-1-2009 

a repeat CT study (18-4-2011) 

N10ICG Pilot study on the use of fl uorescence imaging of lymph nodes during 

colorectal lymphadenectomy, using indocyanine green 

Ruers, TJM pilot 16-2-2011 

N10RFS Observational study into specifi c in vivo human discrimination between Ruers, TJM other 25-10-2010 

(OpSpect) benign and malignant tissue using a combination of diffuse refl ectance 

and fl uorescence spectroscopy 

N11BMT Bevacizumab modulation of telangiectasia in irradiated rectal cancer 

Patients 

Russell, NS other 8-12-2011 

GYNAECOLOGICAL 

M05HIR Hormonal substitution after prophylactic adnectomy in women with an Beurden, M van other 31-5-2005 

(HIRISE) increased risk for breast- and ovarian cancer due to a genetic 

predisposition: HIRISE (High-Risk women and hormonal Substitution 

Exposure) 

M05PPO Proteomic patterns in blood and tissue of ovarian cancer patients Driel van, WJ other 12-1-2006 

M05SNV GROningen International study on sentinel nodes in vulvar cancer Driel van, WJ other 26-3-2007 

(GROINSS-V II) (GROINSS-V) II 

M06HRT The effect of hormonal replacement therapy on menopausal complaints Korse, CM other 25-9-2006 

(NOVARIA) related to biochemical changes in surgically and naturally postmenopausal 

women. A prospective observational comparative study 

M06OVH Phase III randomised clinical trial for stage III ovarian carcinoma Driel, WJ van III 4-1-2006 

(OVHIPEC) randomising between secondary debulking surgery with or without 

hyperthermic intraperitoneal chemotherapy (OVHIPEC-1) 

173 

CLINICAL TRIALS

174 


Type of Study 



M06RTE Randomised phase III trial comparing concurrent chemoradiation and Triest, B van III 28-3-2007 

(PORTEC-3) adjuvant chemotherapy with pelvic radiation alone in high risk and 

advanced stage endometrial carcinoma: PORTEC-3 

M07RCV Phase II study of defi nitive radiochemotherapy for locally advanced 

squamous cell cancer of the vulva: an effi cacy study 

Driel, WJ van II 26-6-2007 

M10MKO Phase II and pharmacological study with WEE-1 inhibitor MK-1775 

combined with carboplatin in patients with p53 mutated epithelial 

ovarian cancer 

Schellens, JHM II 8-7-2010 

M10MKT A two Part, phase I-IIa study evaluating MK1775 in combination with Schellens, JHM I/II 26-5-2010 

Topotecan/Cisplatin in adult patients with cervical cancer (22-2-2011) 

M11LOC Laparoscopy to predict the result of primary cytoreductive surgery in Driel, WJ van other 5-7-2011 

(LapOvCa) advanced ovarian cancer patients 

N05CGO Randomized clinical pharmacological dose-escalation study to explore 

both safety and preliminary effi cacy and pharmaco-kinetics, -dynamics 

and -genomics of fi xed dose rate gemcitabine and 30-minute standard 

gemcitabine infusion both administered in combination with carboplatin 

as second-line treatment in patients with adv ovarian ca 


N10OCT Optical vulvar biopsy by optical coherence tomography (OCT) Beurden, M van other 24-8-2010 

HEAD AND NECK 

M07CMD Treatment of myogenic cranio mandibular dysfunction (CMD): Hilgers, FJM III 29-8-2007 

a prospective randomised clinical trial, comparing a mechanical 

stretching device (Therabite) with standard physiotherapy 


M08CON A randomized study of docetaxel/cisplatin/5-fl uorouracil (TPF) as Boer, JP de II 2-2-2009 

(CONDOR) neoadjuvant chemotherapy followed by concomitant chemoradiotherapy 

(CRT) with conventional radiotherapy (RT) versus concomitant CRT 

with accelerated RT in patients with locally advanced head and neck 

squamous cell cancer (HNSCC) in good condition. (CONDOR) 

M08DDL Docetaxel versus Docetaxel and Lapatinib in recurrent or metastatic Boer, JP de II 12-2-2009 

(TYVTAX) squamous cell carcinoma of the head and neck (SCCHN); an open 

label multicenter randomized phase II study. 

M08EBV Standardized early detection of primary and recurrent nasopharyngeal 

carcinome (NPC) using (anti-) EBV based tumor markers in 

The Netherlands 

Tan, IB other 29-4-2009 

M08MSH Measuring shoulder disability after neck dissection; a comparison of 

4 self report scales 

Stuiver, M other 25-3-2009 

M09HZT Effi cacy of adding hyperbaric oxygen therapy to the treatment of late Smeele, LM III 23-11-2009 

(HBOT) radiation damage of the lower jaw (osteonecrosis) 

M09NST Clinical feasability of a new surgical tool for primary or secondary Hilgers, FJM other 3-12-2009 

tracheoesophageal puncture and voice prosthesis insertion for 

prosthetic voice rehabilitation after total laryngectomy 


M09OSA Prospective cohort study on the prevalence of obstructive sleep apnea Brekel, M van de other 2-12-2009 

in patients with head and neck cancer treated wither either radiotherapy, 

chemoradiation or surgery 

(28-7-2011)

Type of Study 



M09PDT A multi centre cost-effectiveness evaluation of a novel treatment option Tan, IB other 6-8-2009 

in the Netherlands: photo dynamic therapy with temoporfi n for the 

treatment of advanced incurable head and neck cancers, for whom 

prior conventional treatments have failed 

M11ART Adaptive and innovative radiation treatment for improving cancer Hamming-Vrieze, O II 20-12-2011 

(ARTFORCE) treatment outcome 

M11ELN Diagnostic accuracy of an electronic nose for the detection of early Balm, AJM other 29-8-2011 

endobronchial squamous cell cancer after prior history of head and 

neck cancer or lung cancer 

M11FOR FORECAST: Functional Outcome of Radiotherapy and Laser in Early Klop, WMC III 25-10-2011 

(FORECAST) Glottic Carcinoma 

N04RTB Effecten van bestraling op bloedvaten Russell, NS other 5-10-2004 

N05HME De korte termijn invloed van een Heat and Moisture Exchanger op de 

endotracheale temperatuur en luchtvochtigheid bij 

gelaryngectomeerden 

Hilgers, FJM other 1-9-2005 

N07CRH Phase I/II study of combined treatment with AT-101, cisplatin and Verheij, M I/II 16-2-2010 

(gossypol) radiotherapy in patients with locally advanced head and neck cancer 

N07MCP Microcirculatory changes during photodynamic therapy (PDT) in 

squamous cell carcinoma of the oral cavity and oropharynx 

Copper, MP other 21-5-2007 

N07VIN Transoral resection of stage I-II carcinomas of the oropharynx by 

robot-assisted surgery: a feasability study 

Brekel, M van de other 12-3-2007 

N08HHF Validation of hypoxia imaging of cancer in the head and neck area 

with FAZA-PET 

Vogel, WV II 22-10-2008 

N08PTR Phase I study of radiotherapy dose escalation on the FDG-PET Hamming-Vrieze, O I 28-1-2010 

(PET01RAD) avid region of the primary tumor in locally advanced oropharynx and 

oral cavity tumors eligible for concurrent chemoradiation 


N09BIO Prospective study of changes in the oral biofi lm and the composition 

of salivary proteins in patients, being irradiated for a tumour in the 

head-and-neck area 

Balm, AJM other 25-3-2010 

N10BAS Clinical feasibility of a new adhesive base plate (placeholder for stoma Hilgers, FJM other 17-5-2010 

appliances) for rehabilitation after total laryngectomy (5-12-2011) 

N10GVV Phase I-II study of gemcitabine and valproic acid plus valganciclovir in Boer, JP de I/II 15-2-2011 

(EAT) patients with advanced nasopharyngeal carcinoma 

N10OPT Optimising photodynamic therapy for the treatment of head and 

neck cancer 

Karakullukcu, B other 1-11-2010 

N10VMO Evaluation of tumor variability with MRI during radiotherapy treatment in Hamming-Vrieze, O 

patients with an oropharyngeal or oral cavity carcinoma 

other 8-11-2010 

N11HME Short term effect of heat and moisture exchanger on tracheal mucociliary Hilgers, FJM 

clearance in laryngectomized individuals 

11-10-2011 

175 

CLINICAL TRIALS

176 


Type of Study 



LEUKAEMIA / MDS 

M10OMB Ofatumumab maintenance treatment versus no further treatment in Kerst, M III 10-11-2010 

(HOVON 101) relapsed CLL responding to induction therapy 

M11APO Protocol apoptose regulatie van CLL Baars, JW other 29-9-2011 

LUNG 

C11AFA Named patient use of Afatinib for patients with advanced stage NSCLC: Quispel-Jansen, J 

evaluation of response and toxicity profi le 

other 12-5-2011 

E08072 Concurrent Once-daily Versus twice-daily RadioTherapy: a 2-arm Knegjens, JL III 11-11-2009 

(CONVERT) randomised controlled trial of concurrent chemo-radiotherapy 

comparing twice-daily and once-daily radiotherapy schedules in 

patients with limited stage small cell lung cancer (SCLC) and good 

performance status (CONVERT) 

E08092 Double blind randomized phase III study of maintenance Pazopanib Baas, P III 23-8-2011 

(MAPPING) versus placebo in NSCLC patients non progressive after fi rst line 

chemotherapy. MAPPING, an EORTC Lung group study 

M06NEL Effi cacy of neoadjuvant erlotinib in patients with clinical stage I/II Klomp, HM I/II 8-11-2006 

(NEL) non-small cell lung cancer (NSCLC) 

M07CCL Open-label, randomised multi-center study investigating Cetuximab, Heuvel, MM van den I/II 13-3-2008 

(Raditux) in combination with concurrent chemo-radiotherapy in locally 

advanced non-small cell lung carcinoma (RADITUX) 


M07OSM A phase III randomized, double blind, placebo controlled trial of oral Baas, P II/III 9-10-2007 

(OSM) suberoylanilide hydroxamic acid (L-001079038) in patients with 

advanced malignant pleural mesothelioma previously treated with 

systemic chemotherapy 


M07STN A multi-center phase III randomized, double-blind placebo-controlled Heuvel, MM van den III 13-12-2007 

(START) study of the cancer vaccine Stimuvax (L-BLP25 or BLP25 liposome 

vaccine) in non-small cell lung cancer (NSCLC) subjects with 

unresectable stage III disease 


M08MEK Open-label, multicenter, dose-escalation phase I study with extension 

to evaluate safety, pharmacokinetics and activitiy of RO4987655, 

a MEK inhibitor, administered orally as monotherapy in patients with 

advanced tumors 


M09CRE Randomized trial on chest irradiation in extensive disease small cell Knegjens, JL III 19-3-2009 

(CREST) lung cancer 

M09LST Evaluation of molecular sputum test diagnostics for lung cancer Burgers, JA other 18-5-2009 

M09N10 A randomized phase II study of erlotinib compared to single agent Burgers, JA II 11-9-2009 

(NVALT 10) chemotherapy-erlotinib combination in pretreated patients with 

advanced NSCLC (NVALT10 study) 

M09PBO Dose escalation by boosting radiation dose within the primary tumor Belderbos, JSA II 26-11-2009 

(PET-BOOST) on the basis of a pre-treatment FDG-PET-CT scan in stage Ib, II and III 

NSCLC: a randomised phase II trial (PET-BOOST trial) 

M09PCI Prophylactic Cranial Irradiation(PCI) versus observation in radically Belderbos, JSA III 20-10-2009 

(NVALT 11) treated patients with stage III non-small cell lung cancer: a phase III 

randomized study (NVALT 11)

Type of Study 



M09ROS A randomized clinical trial of radiosurgery ( stereotactic radiotherapy) Belderbos, JSA III 28-4-2009 

(ROSEL) or surgery in patients with IA NSCLC who are fi t to undergo primary (5-12-2011) 

Resection 

M10LUC Registration phase III study of LucanixTM( Belagenpumatucel-L) in 

advanced non-small cell lung cancer: an international multicenter, 

randomized, double-blind, placebo-controlled study of LucanixTM 

maintenance therapy for stages III/IV NSCLC subjects who have 

responded to or have stable diseasefollowing one regimen of front-line 

platinum-based combination chemotherapy 

Heuvel, MM van den III 30-6-2010 

M10N12 A randomized phase II study of paclitaxel-carboplatin-bevacizumab Burgers, JA II 10-3-2011 

(NVALT12) with or without nitroglycerin patches in patients with stage IV 

non-squamous-non-small cell lung cancer (NVALT12) 

M10SON A phase II study of sorafenib in patients with locally advanced and/or Burgers, JA II 8-7-2010 

metastatic (stage IIIb or IV) non small cell lung cancer (NSCLC) with 

a K-RAS mutation 


M11ELN Diagnostic accuracy of an electronic nose for the detection of early 

endobronchial squamous cell cancer after prior history of head and 

neck cancer or lung cancer 

Balm, AJM other 29-8-2011 

M11GDT A phase II Open label, multicenter, randomized study to assess the 

effi cacy and safety of GSK1120212 compared with docetaxel in 2nd 

line subjects with targeted mutations (KRAS,NRAS,BRAF,MEK1) 

in locally advanced or metastatic non-small cell lung cancer 

(NSCLC stage IIIb-IV) 

Baas, P II 6-12-2011 

M11VOL Treatment of larger tumor volumes or 2 lung metastases simultaneously Peulen, H I/II 29-9-2011 

(VOLUMES) in lung cancer patients using SBRT in a mean-lung dose escalation 

study 

N04LSN Feasibility of lymphoscintigraphy and sentinel node biopsy in patients 

with non-small lung cancer 

Klomp, HM pilot 8-9-2004 

N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and Valdes Olmos, RA other 7-6-2005 

prediction of tumour respons to chemotherapy and/or radiotherapy in 



N07NRA A phase I/II study with NAMI-A, a Novel Ruthenium Anticancer Agent, 

and gemcitabine combination second-line therapy in NSCLC patients 

Schellens, JHM I/II 15-3-2011 

N08CPA A randomized phase I/II study of standard chemotherapy (cisplatin 

and pemetrexed) with or without Axitinib in patients with malignant 

mesothelioma: interim biopsy analysis to determine effi cacy 

Baas, P I/II 22-5-2009 

N09MLN Quantifi cation of mediastinal lymph node position variability by 

implanted fi ducial markers using cone beam computer tomogram in 

non-small cell lung cancer patients treated with radical irradiation 

Belderbos, JSA other 8-4-2010 

N10ILM Designing and testing new intervention therapies for lung cancer and 

Mesothelioma 

Baas, P other 30-8-2010 

N10RFS Observational study into specifi c in vivo human discrimination between Ruers, TJM other 25-10-2010 

(OpSpect) benign and malignant tissue using a combination of diffuse refl ectance 

and fl uorescence spectroscopy 

177 

CLINICAL TRIALS

178 


Type of Study 



LYMPHOMA - HODGKIN’S DISEASE 

E20051 The H10 EORTC/GELA randomized Intergroup trial on early PET-scan Baars, JW III 23-11-2006 

(H10) guided treatment adaptation versus standard combined modality (24-6-2011) 

treatment in patients with supradiaphragmatic stage I/II Hodgkin’s 

lymphoma 

LYMPHOMA - NON-HODGKIN’S 

M07H80 Phase II study on the feasability and effi cacy of R-DHAP-MTX combined Baars, JW II 10-9-2007 

(HOVON 80) with i.t. rituximab and autologous SCT in patients with a recurrent (4-11-2011) 

aggressive B-cell NHL with CNS localisation 

M07H84 Randomized phase III study on the effect of early intensifi cation of Baars, JW III 22-1-2008 

(HOVON-84) rituximab in combination with 2-weekly CHOP chemotherapy followed 

by rituximab maintenance in elderly patients with DLBCL 

M09ORC Ofatumumab versus Rituximab salvage chemoimmunotherapy Baars, JW III 13-7-2010 

(ORCHARRD) followed by ASCT in relapsed or refractory DLBCL 

M09TAM Treatment induced alterations in microenvironment in follicular Jong, D de other 27-5-2009 

(TAMIL) lymphoma. A multicenter descriptive study 

M10EXI Exist: Physical exercise to improve fi tness and combat fatigue in Baars, JW 3-2-2011 

(Exist) patients with multiple myeloma and (non-)Hodgkin’s lymphoma treated 

with high dose chemotherapy and autologous stem cell transplantation 

M10H105 Rituximab in primary central nervous system lymphoma. Baars, JW III 23-11-2010 

(HOVON 105) A randomized HOVON/ALLG intergroup study. 

N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and Valdes Olmos, RA other 7-6-2005 

prediction of tumour respons to chemotherapy and/or radiotherapy in 



MELANOMA / SKIN 

E18071 Adjuvant immunotherapy with anti-CTLA-4 monoclonal antibody Haanen, JBAG III 27-4-2010 

(ipilimumab) versus placebo after complete resection of high-risk III 

melanoma: a randomized, double-blind phase 3 trial of the EORTC 

Melanoma Group. 


M05MSL A phase III multicenter randomized trial of sentinel lymphadenectomy Nieweg, OE III 11-9-2006 

(MSLT-II) and complete lymph node dissection versus sentinel lymphadenectomy 

alone in cutaneous melanoma patients with molecular or 

histopathological evidence of metastases in the sentinel node 

M07DNA Intradermal naked DNA vaccination for mounting tumor-specifi c 

immunity in stage IV melanoma patients: a phase I clinical study 

Haanen, JBAG I 9-1-2009 

M08MEK Open-label, multicenter, dose-escalation phase I study with extension 

to evaluate safety, pharmacokinetics and activitiy of RO4987655, 

a MEK inhibitor, administered orally as monotherapy in patients with 

advanced tumors 


M10BRA A phase III randomized open label study comparing GSK2118436 to Blank, C III 31-3-2011 

(BRF113683) DTIC in previously untreated subjects with BRAF mutation positive 

advanced (stage III) or metastic (stage IV) melanoma

Type of Study 



M11BRI An open-label, multicenter expanded access study of RO5185426 in Blank, C IV 30-3-2011 

(MO25515) patients with metastatic melanoma 

M11MME A phase II, open-label study to assess the safety and effi cacy of oral Blank, C II 30-3-2011 

(CMEK162X2201) MEK162 in adults with locally advanced and unresactable or 

metastatic malignant cutaneous melanoma, harbouring BRAFV600E 

or NRAS mutations 

N03LAM Longitudinal analysis of melanoma-specifi c immunity in stage III and IV Haanen, JBAG 

melanoma patients 

other 22-8-2003 

N06TIS Integrated analyses of melanoma-T cell interactions; relevance for 

immunotherapy 

Haanen, JBAG other 29-8-2006 

N10BIO Novel targets for medication in melanoma patients who are progressive 

after treatment with BRAF inhibitor RO5185426 or dacarbazine 

Haanen, JBAG other 12-7-2010 

N10MMI Local immunotherapy by the synergism of monobenzone and 

imiquimod cream (MI) for cutaneous metastases in stage III-IV 

melanoma patients 

Veen, JPW van der II 24-1-2011 

N10MSN Pilot study on the use of fl uorescence imaging of lymph nodes during 

melanoma sentinel node procedure, using indocyanine green 

Wouters, M pilot 20-12-2010 

N10TCR Donor leukapheresis for T-cell receptor gene therapy for treatment 

of metastatic melanoma (skin cancer) 

Haanen, JBAG pilot 5-8-2010 

N10TIL Randomized phase II study using a non-myeloablative lymphocyte Haanen, JBAG II 29-3-2011 

(TIL) depleting regimen of chemotherapy followed by infusion of tumor 

infi ltrating lymphocytes and interleukin-2 in metastatic melanoma 

MISCELLANEOUS 

M09BOU The role of microparticles bearing active tissue factor in cancer and Tesselaar, M other 11-2-2010 

(BOUILLAUD) thrombosis: “the Bouillaud-study” (8-12-2011) 

M09XLT An international, randomized, double-blinded, phase III effi cacy study Boer, JP de III 8-7-2009 

of XL184 versus placebo in subjects with unresectable, locally 

advanced or metastatic medullary thyroid cancer 


M10RTW 

(return to work) 

To enhance return-to-work in cancer patients - a randomised trial Driel, WJ van other 14-9-2010 

M11COM Phase III randomized double blind cross-over trial of supersaturated Haanen, JBAG III 12-12-2011 

(COMTT) calcium-phosphate rinse (Caphosol)versus NACL 0,9% in the relief 

of oral mucositis in renal cell carcinoma, hepatocellular carcinoma 

andgastrointestinal stromal tumor patients receiving Targeted Therapy 

N03THY Therapeutic management of thymoma and thymic carcinoma: 

- a prospective registration study based on preoperative risk 

assessment of local failure 

Dewit, LGH other 25-11-2003 

N09DRF Intraoperative real time imaging with a dual radioactive/fl uorescence 

modality for sentinel node localization. A feasibility study including 

reproducibility of multimodality lymphatic mapping with a cocktail 

tracer containing 99mTc nanocolloid/ICG 

Valdes Olmos, RA other 8-6-2009 

179 

CLINICAL TRIALS

180 


Type of Study 



SOFT TISSUE / OSTEOSARCOMA 

E62063 A phase III randomized study evaluating surgery of residual disease Coevorden, F van III 18-2-2009 

(SURGIST) in patients with metastatic gastro-intestinal stromal tumor responding (11-3-2011) 

to Imatinib mesylate 

M09RTP Phase I clinical study of a combined modality treatment of sarcomas Haas, RLM I 24-6-2010 

(PASART-1) of the extremities with radiotherapy (RT) and dose-escalation of 

Pazopanib (PASART-1) 

M10ETD An observational, multicenter, open-label study of the management Kerst, M IV 17-2-2011 

(ET-D-010-10) of patients with advanced soft tissue sarcoma after failure of 

anthracyclines and/or ifosfamide or atients unsuited to receive these 

drugs 




andgastrointestinal stromal tumor patients receiving Targeted Therapy 

N10DMY 

(DOREMY) 

Dose reduction of preoperative radiotherapy in Myxoid liposarcomas Haas, RLM II 15-12-2010 

N10PPS Perfusion PET sarcoma; feasability of tumor perfusion quantifi cation 

with Gallium-68-citrate PET/CT in sarcoma and radiotherapy. 

Vogel, WV other 3-12-2010 

URO-GENITAL 

C11ABI Abiraterone compassionate use programma Bergman, A IV 1-11-2011 

E22043 Post-operative external radiotherapy combined with concomitant and 

adjuvant hormonal treatment versus post-operative external radiotherapy 

alone in pathological stage pT3a-b R0-1 / N0M0 / pT2R1 N0M0, 

Gleason score 5-10 prostatic carcinoma. A phase III study. 

Pos, F III 29-12-2010 

E30072 A phase III randomised double blind study comparing sorafenib with Kerst, M III 21-7-2009 

placebo in patients with resected primary renal cell carcinoma at high 

or intermediate risk of relapse 


E30073 Randomized phase III trial comparing immediate versus deferred 

Nephrectomy in patients with synchronous metastatic renal cell 

carcinoma 

Bex, A III 17-3-2010 

M06LAN Late Adverse effects in Dutch testicular cancer survivors: a Nationwide Kerst, M other 12-2-2007 

(LANCE) case-control study on Cardiovascular Events 

M06SIL Phase I dose-escalation trial for the combination of sorafenib with 

interleukin-2 treatment in patients with clear cell renal carcinoma 

Haanen, JBAG I 20-11-2006 

M07PGC Phase II trial of paclitaxel, gemcitabine and cisplatin in patients with Kerst, M II 8-1-2008 

relapsing germ cell cancer after fi rst line chemotherapy (5-9-2011) 

M08PZD A phase III, placebo-controlled, double-blind study to assess the effi cacy Poel, HG van der III 9-3-2009 

(M15) and safety of once-daily orally administered ZD4054 10 mg in 

non-metastatic hormone-resistant prostate cancer patients 


M09PRG Evaluation of PROTEX absorbable injectable hydrogel when used to Triest, B van other 20-7-2009 

(protex) maintain space between rectum and prostate in men undergoing 

radiation therapy for stage T1-T2 prostate cancer: a non-randomized 

single-arm clinical study 

(21-1-2011)

Type of Study 



M10AZP A phase I, open-label, multicenter study to assess the safety, tolerability, Schellens, JHM I 23-8-2010 

(SARAFTip) pharmacokinetics and preliminary anti-tumour activity of ascending 

doses of AZD3514 in patients with castration-resistant prostate cancer 

M10IPI A randomized, double-blind, phase III trial comparing ipilimumab vs Bergman, A III 14-12-2010 

(CA 184-043) placebo following radiotherapy in subjects with castration resistant 

prostate cancer that have received prior treatment with docetaxel 

M10PCM Prostate cancer molecular medicine (PCMM) Poel, HG van der other 17-2-2011 

(PCMM) 

M10TAD A randomised, double-blind, placebo-controlled study to evaluate Poel, HG van der IV 22-2-2011 

the effect on unassisted erectile function of the early use of 

Tadalafi l 5 mg once a day and Tadalafi l 20 mg on demand treatment 

for 9 months in subjects undergoing bilateral nerve-sparing radical 

prostatectomy 





and gastrointestinal stromal tumor patients receiving Targeted Therapy 

M11DPC A randomized phase 3 study comparing standard fi st-line Docetaxel/ 

Prednisone to Docetaxel/Prednisone in combination with Custirsen 

(OGX-011) in men with metastatic castrate resitant prostate cancer 

Bergman, A III 21-7-2011 

M11PRC Impact of new approaches to pharmacological management of patients Horenblas, S other 18-8-2011 

(PERCEPTION) with renal cell carcinoma: a population-based study of process 

outcomes in The Netherlands 

M11TOO A European randomised phase 3 study to assess the effi cacy and 

safety of TOOKAD Soluble for low risk localised prostate cancer 

compared to active surveillance 

Poel, HG van der III 4-8-2011 

M94SAL Salvage regimen incorporating repeated ablative chemotherapy with 

autologous PSCT, a phase II study 

Rodenhuis, S II 4-7-1994 

N06MPM Samarium-153-EDTMP (QUADRIMET) versus docetaxel for multiple Poel, HG van der II 14-3-2007 

(QUADRIMET) painful oseous metastases in prostate cancer 

N08API Analysis of prostate-specifi c immunity in stage III and IV prostate cancer Haanen, JBAG 

patients 

other 22-1-2009 

N08SNR Site and distribution of sentinel lymph nodes in renal cell carcinoma, 

a phase II study 

Bex, A II 19-3-2009 

N09IGF Pilot study on the use fl uorescence imaging of lymph nodes during 

laparos copic pelvic sentinel node dissection for prostate cancer 

using indocyanine green 

Poel, HG van der pilot 14-7-2009 

N09QPB Quantative FDG-PET/CT of primary bladder cancer Vogel, WV pilot 8-6-2009 


N10BPA Phase I trial evaluating combined image guided radiotherapy with 

Panitumumab (Vectibix) in patients with muscle invasive transitional 

cell carcinoma of the bladder 

Bergman, A I 26-1-2011 

181 


CLINICAL TRIALS

182 

INVITED SPEAKERS 


David Adams, Cambridge, UK 

Finding and validating cancer genes in the mouse 

Jean-Christophe Andrau, Marseille, France 

From enhancer of transcription to enhancer transcription 

Avi Ashkenazi, San Francisco, CA, USA 

Pro-apoptotic receptor agonists as a strategy for cancer therapy 

Keith Baggerly, Houston, TX, USA 

The importance of reproducibility in high-throughput biology: 

case studies in forensic bioinformatics 

Chris Bakkenist, Pittsburgh, PA, USA 

ATM kinase inhibition does not phenocopy ATM protein disruption 

David Barford, London, UK 

Structural basis for the subunit assembly of the anaphase promoting 

complex 

Amy Berrington de González, Bethesda, MA, USA 

Projected cancer risks from current levels of diagnostic radiation exposure 

Cédric Blanpain, Bruxelles, Belgium 

Stem cells and skin cancers 

Luc Brunsveld, Eindhoven, The Netherlands 

Inducing and blocking protein interactions with designed molecules; 

caspases and nuclear receptors as targets 

Sandeep Burma, Dallas, TX, USA 

DNA double-strand break repair and the glioblastoma connection 

Jason Carroll, Cambridge, UK 

Estrogen receptor genomics 

Young-Tae Chang, Singapore 

Development of bioimaging probes for in vitro, in vivo and clinical 

applications by Diversity Oriented Fluorescence Library Approach 

(DOFLA) 

Hans Clevers, Utrecht, The Netherlands 

Wnt signaling, Lgr5 stem cells and cancer 

Gerald Cohen, Leicester, UK 

Bcl-2 family, function and targeting 

Steve Elledge, Boston, MA, USA 

Adventures in human genetics 

Wolfram Goessling, Boston, MA, USA 

Fishing for novel regulators of liver development and cancer 

Eyal Gottlieb, Glasgow, UK 

Metabolism controls cell fate 

Piet Gros, Utrecht, The Netherlands 

Molecular mechanisms underlying complement activation and regulation 

Robert Grosse, Marburg, Germany 

Signal regulation of Formin-mediated actin assembly 

Nir Hacohen, Boston, MA, USA 

Reconstructing circuits of the innate immune system 

Ron Heeren, Amsterdam, The Netherlands 

Pathway Imaging Mass Spectrometry: Multiplexed label-free detection 

of molecular signals on biological surfaces 

Uulke van der Heide, Utrecht, The Netherlands 

Application of MRI in radiotherapy for tumor delineation 

and characterization 

Phil Hodgkin, Melbourne, Australia 

Tracking clonal cell division, death and differentiation fates reveals 

a statistical model for the cell 

Wim Hol, Seattle, USA 

Structural biology and drug design for tropical diseases 

David Holden, London, UK 

Exploitation of mammalian cells by Salmonella 

Nancy Hynes, Basel, Switzerland 

Targeting signaling pathways in breast cancer 

Krystian Jazdzewski, Warsaw, Poland 

The role of microRNAs in predisposition to thyroid cancer 

Peter Jones, Los Angeles, CA, USA 

The cancer epigenome 

Jan Karlseder, La Jolla, CA, USA 

Telomere function and dysfunction during the cell cycle 

Harry de Koning, Glasgow, UK 

The role of transporters in chemotherapy 

Jan Korbel, Heidelberg, Germany 

Detecting large-scale genetic variations in humans with second generation 

sequencing data 

Paul Lehner, Cambridge, UK 

Viral regulation of plasma membrane proteins – new approaches to dissect 

host-pathogen interactions 

Gustavo Leone, Columbus, OH, USA 

Pten and p53 Pathways in the tumor microenvironment 

René van Lier, Amsterdam, The Netherlands 

With a little help of co-stimulation: selection of (sub)dominant T cell 

clones during immune reactions 

Sabine Linn, Amsterdam, The Netherlands 

Outlooks for treatment of breast cancer 

Vivek Malhotra, Barcelona, Spain 

Mechanism of protein secretion: conventional and unconventional

Michael Marks, Philadelphia, PA, USA 

Lysosome-related organelle biogenesis: 

What do dendritic cells and pigment cells have in common? 

Jean-Claude Martinou, Geneva, Switzerland 

Membrane remodeling by Bcl-2 family members to trigger apoptosis 

Maria Masucci, Stockholm, Sweden 

Highjacking of Ub and UbL signaling in Epstein-barr virus infection 

Ultan McDermott, Hinxton, Cambridge, UK 

The genomics of drug sensitivity in cancer 

Pascal Meier, Leicester, UK 

Caspase activation and inhibitor of apoptosis proteins 

Andrew Mouland, Montréal, Canada 

At the crossroads: Expanding roles of endosomal membranes in infectious 

disease and cancer 

Debayan Mukherjee, Dundee, UK 

Untargeted effect of ionising irradiation as a cause for genomic instability 

in murine haematopoietic tissue 

Patricia Muller, Glasgow, UK 

Mutant p53 drives RCP-mediated recycling of integrins and growth factor 

receptors to enhance invasion 

Joseph Nadeau, Boston, MA, USA 

Transgenerational genetic effects on phenotypic variation and disease 

Wojciech Niedzwiedz, Oxford, UK 

FANCM safeguards the genome against replicative stress 

Christopher Pearson, Toronto, Canada 

Repeat instability, DNA repair, epigenetics, and human disease 

Lorenza Penengo, Novara, Italy 

Ubiquitination and genome stability: how the ubiquitin ligase RNF168 

regulates the DNA damage response 

Roland Rad, Hinxton, Cambrid ge, UK 

Cancer gene discovery in mice using PiggyBac transposon mutagenesis 

Christian Reinhardt, Cologne, Germany 

Exploiting defects in the DNA damage response for personalized cancer 

Carlos Reis, Groningen, The Netherlands 

Computational design of TNF ligands 

Antoni Ribas, Los Angeles, CA, USA 

TCR engineering and BRAF targeted therapy for melanoma 

Kevin Ryan, Glasgow, UK 

Autophagy in cell death and cancer 

Ton Schumacher, Amsterdam, The Netherlands 

Dissecting T cell immunity in mice and men 

Yosef Shaul, Rehovot, Israel 

Mechanisms and metabolic regulation of protein degradation by default 

Yang Shi, Boston, MA, USA 

Histone methylation dynamics: mechanisms and link to human 

Ilya Shmulevich, Seattle, WA, USA 

Molecular networks in cancer and innate immunity 

Jane Skok, New York, NY, USA 

Chromosome dynamics in immune diversifi cation 

183 


Quentin Smith, Amarillo, TX, USA 

Improving drug delivery to brain for the treatment of brain metastases of 

breast cancer 

Holger Stark, Göttingen, Germany 

Dynamic macromolecular complexes: The ribosome in motion 

Fraser Symmans, Houston, TX, USA 

Developing, testing and implementing molecular approaches to tailor 

treatment of breast cancer patients 

Stephen Tait, Memphis, TN, USA 

Mitochondrial regulation of cell death and survival 

Shunichi Takeda, Kyoto, Japan 

Analysis of DNA damage response using the chicken DT40 cell line 

Stavros Taraviras, Patras, Greece 

Differential role of Geminin in progenitor T cells and haematopoietic 

stem cells 

Roman Thomas, Cologne, Germany 

Lung cancer genomics 

Marc Timmers, Utrecht, The Netherlands 

Dynamic regulation of transcription and chromatin 

Frank Uhlmann, London, UK 

Temporal ordering of progression through cell division 

Emil Unanue, St. Louis, MO, USA 

Antigen presentation and peptide selection in autoimmune diabetes 

Peter Vandenabeele, Gent, Belgium 

Cutting both ways: How caspases promote and prevent cell death 

Ashok Venkitaraman, Cambridge, MA, USA 

Chromosome stability mechanisms in tumor suppression and cancer 

therapy 

Christophe Verlinde, Seattle, WA, USA 

Forging a human prenylation inhibitor into a T.cruzi ergosterol 

biosynthesis blocker that cures mice from acute Chagas disease 

Peter Verrijzer, Rotterdam, The Netherlands 

Undercover transcription factors in development and disease 

Rob Wolthuis, Amsterdam, The Netherlands 

Mechanisms of Cell Division: the Be All and the End All 

Jerry Workman, Kansas City, MO, USA 

Protein complexes that modify chromatin for transcription

184 

PROJECTS 

PROJECTS SUPPORTED 

BY THE DUTCH CANCER SOCIETY 

Project leader 

Number of 

project Title Started 

Aaronson, Neil KWF 2006-3470 Cognitive behavioral therapy (CBT) and physical exercise 

for climacteric symptoms in breast cancer patients 

experiencing treatment-indiced menopause 

Aaronson, Neil KWF 2009-4299 A-CaRe Project 2: Effectiveness of physical exercise 

during chemotherapy to improve physical fi rness and 

reduce fatigue: A randomized trial 

Agami, Reuven KWF 2007-3881 Role of miRNA genes in etiology of malignant germ cell 

tumors 

Ended/ 

ends 

9/1/2006 3/1/2012 

8/1/2009 8/1/2013 

3/1/2007 3/1/2011 

Agami, Reuven KWF 2009-4469 Role of RNA binding proteins in cancer 3/1/2010 3/1/2014 

Agami, Reuven KWF 2009-4498 Bromodomain containing proteins in cancer 4/1/2010 4/1/2014 

Belderbos, José KWF 2010-4675 Bestralingsdosis verhoging door het geven van een 

boost op de longtumor gebaseerd op een PET-scan bij 

patiënten met een stadium II of III niet klein-cellig 

longcarcinoom 

Bergman, Andre KWF 2009-4356 Investigating the Role of Infl ammation in Prostate 

Cancer Development 

Bernards, Rene KWF 2008-4027 Understanding resistance to HER2-targeting therapy in 

human breast cancer through functional genetics 

Bernards, Rene KWF 2008-4042 Identifi cation of enzymes involved in regulatory 

ubiquitination in TNF a--stimulated signalling by lossof-function 

screens 

Bernards, Rene KWF 2009-4337 Identifi cation of genetic modifi ers of sensitivuty to 

mTOR pathway inhibition in breast cancer 

Bernards, Rene KWF 2009-4496 Determinats of the response to retinoic acid in 

neuroblastoma 

Berns, Ton KWF 2008-4027 Designing and testing new intervention therapies for 

lung cancer and mesotheliomas 

Berns, Ton KWF 2008-4253 Designing and testing new intervention therapies for 

lung canccer and mesotheliomas 

Blank, Christian KWF 2008-3988 Blockade of PD-1/D-L1 interaction to improve T cell 

mediated immunotherapy of cancer 

Bleiker, Eveline KWF 2008-4016 Identifi cation of psychosocial problems and perceived 

need for support in cancer genetics 

Borst, Jannie KWF 2008-4028 T cell programming at the dendritic cell interface: 

impact on anti-tumor immunity 

3/25/2010 3/25/2014 

8/1/2009 8/1/2015 

11/1/2008 11/1/2012 

11/1/2008 11/1/2012 

8/1/2009 8/1/2013 

1/1/2010 1/1/2014 

1/1/2008 1/1/2015 

5/1/2009 5/1/2015 

1/1/2008 1/1/2012 

1/1/2008 1/1/2013 

2/1/2008 2/1/2013


Number of 


Borst, Jannie KWF 2008-4110 Impact of TRAIL death receptor traffi cking on proapoptotic 

signaling. 

185 PROJECTS 

Ended/ 

ends 

3/1/2008 3/1/2013 

Collard, John KWF 2007-3753 The par complex in cell polarity and tumor progression 7/1/2007 7/1/2011 

Dalesio, Otilia KWF 

Datamanagement 

Dannenberg, Jan- 

Hermen 

Driel, Willemien 

van 

KWF Datamanagement 1/1/1982 1/1/2012 

KWF 2007-3978 Genetic analysis of class I HDACs in development and 

treatment of cancer 

KWF 2006-4176 Phase III randomised clinical trial for stage III ovarian 

carcinoma randomising between secondary debulking 

surgery with or without hyperthermic intraperiotoneal 

chemotherapy (OVHIPEC-1) 

Elkhuizen, Paula KWF 2009-4389 Properative accelerated partial breast irradiation 

(PAPBI): defi ning radiotherapy sensitivity 

Haanen, John KWF 2007-3943 HPV 16 E6/E7 DNA prime and E6/E7 long peptide 

boost vaccination for multifocal Vulvar Intraepithelial 

Neoplasia (VIN) 

Haanen, John KWF 2011-5162 TIL therapy for melanoma: development of a simplifi ed 

TIL production process using a novel bioreactor 

Harten, Wim van KWF 2010-4854 A-CaRe 2: ICT supported patient empowerment, returnto-work, 

telerehabilitation and implementation of Cancer 

Rehabilitation Programs 

Herk, Marcel van KWF 2007-3751 High precision image-guided radiotherapy for bladder 

cancer 

Herk, Marcel van KWF 2007-3895 Probability based treatment planning with biological 

objectives for high-dose high-precision radiotherapy of 

prostate cancer 

Jacobs, Heinz KWF 2008-4112 A cancer genome atlas of the activation-induced cytidine 

deaminase: novel insights into the pathogenesis and 

prognosis of b cell lymphoma. 

1/1/2008 1/1/2012 

3/14/2007 9/14/2011 

1/1/2010 1/1/2014 

1/1/2008 1/1/2012 

10/1/2011 10/1/2013 

11/1/2010 11/1/2014 

1/1/2008 1/1/2012 

11/1/2007 5/1/2012 

3/1/2008 3/1/2012 

Jacobs, Jacqueline KWF 2007-3907 Dissecting the telomere damage response 8/1/2008 8/1/2012 

Jalink, Kees KUN 2007-3733 TRMP7, a novel regulator of cytoskeletal tension: 

implications for cancer progression, invasion and 

metastasis. 

Jalink, Kees KWF 2010-4626 Calcium and phosphoinositides in the control of 

podosome formation and tumor cell invasion 

Jonkers, Jos KWF 2006-3486 Dissection of the role of E-cadhering loss-of-function in 

breast cancer development and metastasis 

9/1/2007 9/1/2011 

10/1/2010 10/1/2014 

2/13/2006 2/12/2011 

Jonkers, Jos KWF 2006-3715 KWF 2006-3715 1/1/2007 1/1/2011

186 

PROJECTS 


Number of 


Jonkers, Jos KWF 2007-3772 Preclinical validation of chemical inhibitors of poly 

(ADP-ribose) polymerase (PARP) in conditional mouse 

models for BRCA-associated breast cancer 

Jonkers, Jos KWF 2008-4116 Impact of BRCA muntations on breast tumorigenesis 

and treatment response: functional analysist of defi ned 

truncation mutants and unclassifi ed variants 

Jonkers, Jos KWF 2011-5232 The effect of the tumor microenvironment on 

radioresistance of breast cancer 

Leeuwen, Fijs van KWF 2009-4344 Targeted CXCR4 with multimodal imaging agents as a 

means to improve the effi cacy of breast cancer surgery 

via combined pre-, intra- and postoperative imaging 

Leeuwen, Floor van KWF 2006-3631 Long-term risk of cancer after ovarian stimulation for in 

vitro fertilization 

Leeuwen, Floor van KWF 2008-3994 Cardiovascular morbidity and mortality in breast cancer 

survivors 

Leeuwen, Floor van KWF 2010-4720 Assessment of late effects of treatment for Hodgkin’s 

lymphoma 

Leeuwen, Fred van KWF 2009-4511 Role of the histone methyltransferase Dot1 in gene 

expression and leukemic transformation 

Linn, Sabine KWF 2006-3706 Towards patient-tailored systemic therapy in breast 

cancer: a combined approached of translational research 

and mouse model systems 

Lohuizen, Maarten 

van 


van 


van 

KWF 2007-3803 Cancer stem cells and breast cancer: a polycomb 

connection 

KWF 2007-3877 Epigenetic regulation by the deubiquitinating enzyme 

USP3: impact on genome stability and tumorgenesis 

KWF 2010-4757 Deciphering the role of Bmi1 and Ezh2 polycomb group 

genes in prostate carcinogenesis and metastatis 

Ended/ 

ends 

2/1/2007 2/1/2012 

1/1/2008 1/1/2012 

11/1/2011 11/1/2013 

9/1/2009 10/1/2011 

4/1/2007 4/1/2012 

10/27/ 

2008 

10/27/ 

2012 

1/1/2011 1/1/2016 

9/1/2010 9/1/2014 

1/1/2007 1/1/2013 

4/1/2007 4/1/2011 

3/1/2007 3/1/2012 

11/1/2010 11/1/2014 

Medema, Rene UU 2010-4706 Building Sister Chromatid Cohesion 10/1/2011 10/1/2014 

Medema, Rene UU 2011-5103 Cohesin loading and stripping: two fundamental 

principles for the maintenance of genomic stability 

Moolenaar, Wouter KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase, 

as a therapeutic target 

Nederlof, Petra KWF 2007-3749 Specifi c chromosomal imbalance in breast carcinomas 

as a marker for breast cancer susceptibility 

Neefjes, Jacques KWF 2007-3883 Anti-cancer drugs and their effects on the ubiquitin 

cycle 

Ovaa, Huib KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase, 


10/1/2011 10/1/2017 

8/1/2010 8/1/2014 

8/15/2007 8/15/2012 

4/1/2007 4/1/2011 

8/1/2010 8/1/2014


Number of 


Peeper, Daniel KWF 2007-3957 Towards improved melanoma treatment: gene 

identifcation, in vivo modeling and drug target discovery 

Peeper, Daniel KWF 2009-4552 Dissecting the essential contribution of Fra-1 to human 

tumor cell metastasis 

187 PROJECTS 

Ended/ 

ends 

7/1/2008 7/1/2014 

1/1/2010 1/1/2014 

Peeper, Daniel KWF 2011-5247 Dissecting the mammalian non-canonical Wnt-pathway 10/15/2011 10/15/2013 

Perrakis, Anastassis KWF 2010-4781 Validation of autotaxin, a metastasis-enhancing exophosphodiesterase, 


Remeijer, Peter KWF 2008-4024 Image guided radiotherapy for breast cancer 10/20/ 

2008 

Riele, Hein te KWF 2006-3589 Signifi cance of the Fanconi anemia caretaker pathway in 

development and treatment of cancer 

Riele, Hein te KWF 2007-3790 Role of the G2 restriction point in tumor development 

and behavior 

Riele, Hein te KWF 2009-4477 Characterization of unclassiefi ed allelic variants of DNA 

mismatch repair genes to improve genetic counseling 

Rodenhuis, Sjoerd KWF 2009-4593 Randomized phase II/III study of individualized 

neoadjuvant chemotherapy in triple negative breast 

tumors 

Rookus, Matti KWF 2007-3756 Heterogeneity of risk of breast and ovarian cancer in 

BRCA1 and BRCA2 mutayion carriers. 

Rottenberg, Sven KWF 2009-4303 Analysis of tumor recurrence in a mouse model for 

hereditary breast cancer: how do tumor-initiating cells 

escape eradication by chemotherapy 

Schagen, Sanne KWF 2009-4284 Structural, biochemical and functional indices of 

chemotherapy-induced cognitive defi cits in cancer 

patients 

Schagen, Sanne KWF 2010-4829 Molecular and cellular mecanisms of cognitive 

impairment following chemotherapy for cancer 

Schagen, Sanne KWF 2010-4876 An online testing approach to assess cognitive problems 

associated with cancer and cancer treatment 

Schagen, Sanne KWF 2010-4894 The application of neuroimaging techniques in studying 

cognitve impaiments and complaints after chemotherapy 

Schinkel, Alfred KWF 2007-3764 OATP1A/1B (SLCO1A/1B) drug uptake transporters in 

anticancer drug pharmacokinetics and toxicity risks; 

possible strategies for therapy optimization. 

Schmidt, Marjanka KWF 2007-3839 Genetics determinants of survival and second breast 

cancer development in premenopausal breast cancer 

patients 

8/1/2010 8/1/2014 

10/20/ 

2012 

10/1/2006 10/1/2011 

4/1/2007 4/1/2012 

2/1/2010 2/1/2014 

1/7/2010 1/7/2015 

11/15/2007 11/15/2011 

10/1/2009 10/1/2013 

1/1/2010 1/1/2014 

2/1/2011 2/1/2015 

7/1/2011 7/1/2015 

1/1/2011 1/1/2013 

12/1/2007 12/1/2012 

1/1/2007 1/1/2013 

Schmidt, Marjanka KWF 2009-4363 Breast cancer outcome: genetic destiny 7/1/2009 7/1/2015

188 

PROJECTS 


Number of 


Schmidt, Marjanka KWF 2009-4535 The Tissue Issue: towards a uniform consent procedure 

for research with excised (cancer) tissue 

Schumacher, Ton KWF 2006-3530 Generation of human tumor specifi c T cells with high 

affi nity T cell receptors 

Schumacher, Ton KWF 2009-4282 Preclinical development of TCR gene therapy for 

prostate carcinoma 

Ended/ 

ends 

8/1/2010 8/1/2014 

12/1/2006 12/1/2011 

12/1/2009 12/1/2013 

Schumacher, Ton KWF 2009-4581 UV-gevoelig MHC peptide uitwisselbare streptameer. 7/1/2009 12/1/2011 

Schumacher, Ton UL 2007-3825 SNP-based genome-wide identifi cation of hematopoiesisrestricted 

minor histocompatibility antigens. 

Sixma, Titia KWF 2006-3476 Structural and functional analysis of a novel E3 ubiquitin 

ligase consisting of the polycomb proteins Bmi1 and 

Ring 1b (RNF2) 

7/1/2007 7/1/2011 

8/1/2006 8/1/2011 

Sixma, Titia KWF 2008-4014 Comparative study of Ubiqutin-specifi c proteases 2/1/2008 2/1/2013 

Sonke, Jan-Jakob KWF 2006-3545 Imaged guided correction strategies to optimize the 

precision of radiotherapy 

Sonke, Jan-Jakob KWF 2009-4568 Optimization of stereotactic image guided radiotherapy 

for lung cancer through detailed toxicity assessment 

Sonke, Jan-Jakob KWF 2005-3378 High precision radiotherapy in the presence of 

anatomical changes 

Sonnenberg, 

Arnoud 

Sonnenberg, 

Arnoud 

KWF 2008-3996 A mouse stem cell model for skin cancer: Dual role of 

the integrin a6B4 in tumor initiation and progression 

KWF 2009-4485 Dissection of the regulation of hemidesmosome 

disassembly in tumor cell invasion 

Stewart, Fiona KWF 2008-3993 The role of endoglin in induction and repair of 

cardiovascular damage after radiation alone or in 

combinatione with trastuzumab (Herceptin) 

Stewart, Fiona KWF 2009-4480 Modulation of vessel repair to prevent radiation-induced 

microvascular damage 

8/1/2006 8/1/2011 

1/1/2010 1/1/2014 

11/14/2005 11/14/2010 

8/1/2008 8/1/2012 

9/1/2010 9/1/2014 

6/16/2008 6/16/2012 

1/1/2010 1/1/2014 

Tan, Bing KWF KWF Ontwikkelingssamenwerkingsprogramma 1/1/2008 1/1/2012 

Tan, Bing KWF 2008-4233 Early detection of primary and recurrent nasopharyngeal 

carcinoma (NPC) using (anti-)EBV based tumor markers 

and options for using photodynamic therapy (PDT) in 

the treatment of local disease 

Veer, Laura van ‘t KWF 2007-3839 Genetics determinants of survival and second breast 

cancer development in premenopausal breast cancer 

patients 

9/1/2008 9/1/2013 

1/1/2007 1/1/2011 

Veer, Laura van ‘t UU 2010-4893 Benchfee S. Elias KWF-Fellow 1/1/2010 1/1/2012 

Vens, Conchita KWF 2010-4877 Tumor-specifi c radiosensitization by exploiting base 

excision repair defi ciencies 

6/1/2011 6/1/2015


Number of 


Verheij, Marcel KWF 2007-3939 Improvement of chemoradiation response in head and 

neck cancer by (-)Gossypol (AT-101), a small molecule 

inhibitor of Bcl-XL/Bcl-2 

Verheij, Marcel KWF 2008-4113 Short-chain sphingolipids for enhanced cellular uptake 

of amphiphilic anti-cancer drugs 

189 PROJECTS 

Ended/ 

ends 

2/1/2008 2/1/2010 

11/1/2008 11/1/2012 

Verwaal, Vic KWF Pixels tegen darmkanker 8/1/2011 8/1/2012 

Visser, Karin de KWF 2011-5004 Cancer cell-intrinsic and -extrinsic regulation of breast 

cancer metastasis formation; implications for adjuvant 

therapy 

Vogel, Wouter KWF 2011-5024 Recurrent differentiated thyroid cancer: personalized 

treatment based on evaluation of tumor characteristics 

with PET (THYROPET) 

Wolthuis, Rob KWF 2007-3789 Targeting Mitotic Protein Destruction as an Approach 

for Cancer Therapy 

Wolthuis, Rob KWF 2008-4135 Divergent Control of Cyclin B1-Cdk1 in Cancer Cells: a 

Key Role in Therapy 

Zwart, Wilbert KWF 2009-4435 In kaart brengen van actieve Estrogen Receptor binding 

sites bij tamoxifen en aromatase inhibitor resistente 

cellijnen en borsttumoren, om zo prodictieve markers te 

defi niëren. 

5/1/2011 5/1/2015 

10/1/2011 10/1/2015 

9/1/2007 9/1/2011 

9/1/2008 9/1/2012 

11/1/2009 11/1/2013

190 

PROJECTS 

PROJECTS SUPPORTED 

BY OTHER ORGANISATIONS 

Project leader Sponsor Title Started 

Aaronson, Neil Stichting Achmea 

Gezondheidszorg 

Ended/ 

ends 

A-CaRe subprogram Clinical Research 5/1/2009 5/1/2013 

Aaronson, Neil EORTC Methods and measures for assessing the HRQL of mid to long term 

survivors of testicular and prostate cancer 

5/1/2009 3/1/2012 

Aaronson, Neil IKA Onco-Move Paces studie 3/1/2010 1/1/2013 

Aaronson, Neil Stichting Nuts Ohra Spoed - erfelijkheidsonderzoek bij borstkanker: invloed op 

behandelingskeuzes en welbevinden / Behavioral and psychosocial 

effects of rapid genetic counseling and testing in newly diagnosed 

breast cancer patients: a multicenter study 

Aaronson, Neil Stichting Achmea 

Gezondheidszorg 

2/18/2008 11/30/2012 

A-CaRe 1/1/2010 7/31/2012 

Agami, Reuven CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach 

Agami, Reuven Dr. Josef Steiner 

Krebsstiftung 

1/1/2009 1/1/2014 

Dr Josef Steiner Cancer Award 2007 5/1/2007 5/1/2011 

Agami, Reuven EU-ERC Indentifying novel regulatory mechanisms of miRNA functions. 10/1/2008 10/1/2013 

Agami, Reuven NWO Combined miRNA profi ling and genetic screens to identify and 

characterize cancer-related miRNAs 

Agami, Reuven ZonMw MicroRNAs and RNA Binding Proteins involved in regulation of 

KNA damage responses 

10/1/2007 10/1/2012 

3/15/2010 7/15/2011 

Agami, Reuven ZonMw VICI 6/1/2011 6/1/2016 

Amendola, Mario EMBO Gene regulation by chromatin associated long non-coding RNA 1/1/2011 12/31/2012 

Beijersbergen, 

Roderick 

Beijersbergen, 

Roderick 

Inte RNA 

Technologies BV 

Identifi cation of miRNA’s that control response to Trastuzumab in 

Breast Cancer 

12/1/2009 1/1/2012 

NWO-ALW Cancer Systems Biology Centre 9/1/2010 9/1/2015 

Belderbos, José EU Adaptive and innovative Radiation Treatment FOR improving Cancer 

patients treatment Outcome 

Bernards, Rene AstraZeneca Identifi cation and validation of new targets for colorectal cancer 

through molecular redefi nition of the disease 

Bernards, Rene CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

3/1/2011 3/1/2016 

1/1/2011 1/1/2013 

1/1/2009 12/31/2013 

Bernards, Rene CGC CGC 2008-2012 8/1/2008 1/1/2013 

Bernards, Rene EU COLTHERES Modelling and predicting sensitivity to targeted 

therapies in colorectal cancers 

1/1/2011 1/1/2015


191 1 

Ended/ 

ends 

Bernards, Rene EU Rubicon 1/1/2006 6/30/2011 

Bernards, Rene EU-ERC Functional Genomics Dissecting genetic dependencies in cancer 6/1/2010 6/1/2015 

Bernards, Rene EU EUROCANPLATFORM A European Platform for Translational 

Cancer Research 

Bernards, Rene EU RATHER Rational Therapy for Breast Cancer: Individualized 

Treatment for Diffi cult-to-Treat Breast Cancer Subtypes 

1/1/2011 1/1/2016 

1/1/2011 1/1/2016 

Bernards, Rene NWO Spinozapremie 9/26/2005 12/31/2012 

Bernards, Rene NWO-ALW CSBC-Bernards 9/1/2010 9/1/2015 

Bernards, Rene Overig Therapie op maat door mutatieanalyse bij kanker 7/1/2011 7/1/2015 

Bernards, Rene TI Pharma Kinases in cancer 7/1/2007 7/1/2011 

Berns, Ton CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

1/1/2009 12/31/2013 

Berns, Ton CGC CGC 2008-2012 1/1/2008 1/1/2012 

Berns, Ton CGC Mutagenesis strategies to identify new oncogenes and Tumor 

suppressor genes 

Berns, Ton CTMM Identifi cation of biomarkers for SCLC and NSCLC using mouse 

models 

Berns, Ton EU EUROCANPLATFORM - A European Platform for Translational 


1/1/2010 1/1/2013 

1/1/2009 1/1/2014 

1/1/2011 1/1/2016 

Berns, Ton TI Pharma Kinases in Cancer (TI Pharma) 7/1/2007 7/1/2011 

Blank, Christian MedImmune Ltd. Characterization of a new inducible melanoma model. Collaborative 

Research Agreement 2947 

Blank, Christian Melanoma Research 

Alliance 

Development of combined molecular/immunotherapy regimens for 

human melanoma 

Bleiker, Eveline Pink Ribbon Long-term psychosocial and medical impact of breast cancer genetic 

counselling offered soon after diagnosis 

Borst, Jannie NWO A novel type of ubiquitination regulates apoptosis signaling by BH3only 

protein Bid 

Borst, Jannie NWO-ALW Impact of CD27-CD70 co-stimulation on effector development of 

CD4 T cells 

1/1/2011 1/1/2013 

5/1/2011 5/1/2014 

2/1/2011 1/31/2013 

11/1/2008 11/1/2012 

2/15/2010 2/15/2011 

Borst, Jannie TI Pharma TNF ligands in cancer 3/1/2006 7/1/2012 

Borst, Piet EU CHEMORES Molecular mechanisms underlying chemotherapy 

resistance, therapeutic escape, effi cacy and toxicity 

Borst, Piet ZonMw Characterization of the physiological roles of Multidrug Resistance 

Protein (MRP) 1-6 by in vivo screening for their substrates 

PROJECTS 

2/1/2007 2/1/2012 

1/1/2008 1/1/2012 

Boven, Hester van Astra Neoadjuvante registratiestudie BOOG 1/1/2006 1/1/2013

192 

PROJECTS 


Brandsma, Diete 2BBB technologies 

BV 

Brekel, Michiel 

van den 

Brekel, Michiel 

van den 

An open-label, phase I/IIa dose escalating study of 2B3-101 in 

patients with solid tumors with brain metastases. 

ATOS Development and evaluation of a third generation ProvoxTM voice 

prosthesis 

Ended/ 

ends 

8/22/2011 2/22/2013 

6/1/2003 7/1/2015 

Verwelius BV Fanconi anemia pathway defects in head and neck cancer. 10/1/2010 10/1/2013 

Broeks, Annegien EU EUROCANPLATFORM - A European Platform for Translational 


Brummelkamp, 

Thijn 

Brummelkamp, 

Thijn 

Brummelkamp, 

Thijn 

1/1/2011 1/1/2016 

CGC CGC 2008-2012 12/1/2010 12/31/2012 

NIH The role of the HIppo pathway on mammalian organ size, progenitor 

cells and cancer 

ZonMw VIDI Tissue size control and the regulation of Hippo pathway activity 

in mammals 

Burgers, Sjaak CTMM AIR FORCE Personalized chemo-radiation of lung and head and 

neck cancer 

1/1/2009 1/1/2014 

4/1/2011 4/1/2016 

10/1/2008 10/1/2013 

Collard, John EU An integrated concept of tumor metastasis: implications for therapy. 4/1/2008 4/1/2012 

Dalesio, Otilia AVL Fonds Ondersteuning datamanagement voor het NKI-AVL neoadjuvante 

chemotherapieprogramma 

Dalesio, Otilia EU “eTEN” Programme - Call identifi er: 05/1 ETEN-029334 - 

“TENALEA-ID” 

11/15/2010 11/15/2014 

11/1/2006 6/1/2011 

Dalesio, Otilia IKA IKA kankerregistratie 9/1/1990 1/1/2012 

Dalesio, Otilia NVALT Statistical Center NVALT Clinical Trials in oncology 5/1/2004 1/1/2012 

Dannenberg, Jan- 

Hermen 

NWO-ALW The role of class I HDACs in normal physiology, tumorigenesis and 

transcriptional regulation 

8/1/2007 8/1/2012 

Elsakkers, Peter AMC Poli Familiare Tumoren 1/1/2008 1/1/2012 

Elsakkers, Peter LSCA Valorisation 

Fund 

Elsakkers, Peter Ministerie van 

VROM 

Filippo, Ronald Koninklijk 

Nederlands 

Genootschap voor 

Fysiotherapie 

(KNGF) 

Clinical validation of a Phase-sensitive Gamma Detector 7/1/2010 7/1/2012 

weerstandverhoging 12/1/2008 12/1/2013 

ontwikkeling KNGF-standaard Beweeginterventie oncologie 10/1/2009 7/1/2012 

Gilhuijs, Kenneth CTMM Breast Care 2/1/2009 2/1/2014 

Gilhuijs, Kenneth CTMM BREASTCARE Neoadjuvant drug treatment for breast cancer - 

response-prediction and response monitoring 

2/1/2009 2/1/2014 

Gilhuijs, Kenneth SenterNovem Image guided intervention and therapy IGIT4health 7/17/2009 1/16/2011


Graaf, Carolyn de EMBO Cis-acting determinants of DNA-Nuclear Lamina interactions in 

mammalian cells. 

Haanen, John Bristol-Myers 

Squibb Company 

High-throughput analysis of Ipilimumab-induced Cytotoxic T cell 

responses: An analysis of blood samples from metastatic melanoma 

patients treated with ipilimumab 

193 1PROJECTS 

Ended/ 

ends 

9/1/2011 8/31/2013 

7/1/2011 1/1/2013 

Haanen, John NanoNextNL Drug Delivery 7/18/2011 7/18/2012 

Haanen, John ZonMW 9/1/2006 7/1/2012 

Harten, Wim van CTMM Breast Care 2/1/2009 2/1/2014 

Harten, Wim van EU EUROCANPLATFORM - A European Platform for Translational 


Hauptmann, 

Michael 

EU Epidemiological study to quantify risks for paediatric computerized 

tomography and to optimise doses 

1/1/2011 1/1/2016 

2/1/2011 2/1/2016 

Herk, Marcel van CTMM AIR FORCE 10/1/2008 10/1/2013 

Herk, Marcel van Elekta Elekta XVI 3/29/2005 12/31/2014 

Horenblas, Simon Stichting 

J.C. van Veen 

Bijdrage onderzoek urologie 1/1/2011 1/1/2016 

Innocenti, Metello CGC CGC 2008-2012 5/1/2009 12/31/2012 

Jacobs, Jacqueline NWO VIDI 2/1/2007 2/1/2012 

Jalink, Kees SenterNovem MEM-FLIM: Modulated Electron-Multiplied all-solid-state camera for 

Fluorescence Lifetime Imaging Microscopy 

3/1/2008 3/1/2012 

Jalink, Kees STW Labeling strategies for nanoscopy of complex biological specimens 9/1/2011 9/1/2015 

Jonkers, Jos AstraZeneca Intervention studies with AZD2281, AZD2461 and AZD7762 in 

mouse mammary tumor model 

Jonkers, Jos AstraZeneca Targeting the P13K network in genetically engineered mouse models 

of invasive lobular breast cancer 

1/1/2010 1/1/2015 

11/1/2008 11/1/2012 

Jonkers, Jos CTMM BREAST CARE 2/1/2009 2/1/2014 

Jonkers, Jos EU EUROCANPLATFORM - A European Platform for Translational 


1/1/2011 1/1/2016 

Jonkers, Jos EU Eurosystem 3/1/2008 3/1/2012 

Jonkers, Jos EU The role of Polycomb genes in normal stem cell and breast cancer 

stem cell self-renewal and maintenance 

3/1/2009 3/1/2011 

Jonkers, Jos NWO-ALW CSBC 9/1/2010 9/1/2015 

Jonkers, Jos TI Pharma Kinases in cancer 7/1/2007 7/1/2011 

Jonkers, Jos ZonMw Ultra-high throughput analysis of insertional mutations to study 

collaborating cancer genes and genetic networks in mouse tumors 

7/1/2010 3/1/2012 

Kerkhoven, Ron CBG Deep Sequencing project CBG 1/1/2009 1/1/2013

194 

PROJECTS 


Kind, Jop NWO Tracing and mapping of genome - nuclear lamina interactions in 

single cells. 

Klomp, Houke Roche Roche HQ Neo adjuvant Tarceva: Tumor remission rate with 

neoadjuvant erlotinib in operable patients with clinical stage I/II 

non-small cell lung cancer (NSCLC) 

Leeuwen, Fijs van CTMM MUSIS Intra-operative Multi-Spectral Imaging Systems for radical 

tumor resection 

Leeuwen, Fijs van CTMM BREAST CARE Neoadjuvant drug treatment for breast cancerresponse-prediction 

and response monitoring 

Leeuwen, Fijs van EU HYPERImage: Hybrid PET-MR system for concurrent ultra-sensitive 

imaging 

Ended/ 

ends 

9/1/2011 8/31/2014 

7/1/2006 1/1/2012 

4/1/2010 10/1/2011 

2/1/2009 10/1/2011 

4/1/2008 10/1/2011 

Leeuwen, Fijs van STW Reversible noncovalent assemblies for in-vivo imaging applications 4/1/2011 10/1/2011 

Leeuwen, Floor 

van 


van 


van 


van 


van 


van 


van 


van 


van 


van 

Leeuwen, Fred 

van 

Leeuwen, Fred 

van 

Leeuwen, Fred 

van 

CTSU Heart-breast and heart-Hodgkin case-control studies 11/1/2009 1/1/2013 

CTSU Oxford Cardiovascular disease (CVD) after breast cancer and CVD after 

Hodgkin case-control studies 

1/1/2010 1/1/2013 

EU Collaborative Oncological Gene-environment Study 5/1/2009 5/1/2013 

Erasmus Medisch 

Centrum 

Long-term risk of endometrial cancer after ovarian stimulation for 

in-vitro fertilization. A case-cohort analysis. 

1/1/2011 1/1/2013 

LVN BETER 7/1/2010 8/31/2011 

Lymfklierkanker 

Vereniging 

Screening van overlevenden van Hodgking Lymfoom (BETER) 7/1/2010 7/1/2011 

NIH Prediction Model: Breast cancer in women irradiated for a pediatric 

malignancy. 

NIH Risk and prognosis of uterine corpus cancer after tamoxifen 

treatment for breast 

Pink Ribbon Voorlichting over en screening op mammacarcinoom bij jonge 

vrouwen die met radiotherapie zijn behandeld voor Hodgkin 

lymfoom 

Vlissingen 

Lymfoomfonds 

4/1/2010 1/1/2014 

9/25/2007 9/1/2011 

2/1/2011 2/1/2013 

Van Vlissingenfonds: polikliniek overlevers Hodgkin 8/1/2009 8/1/2012 

NPC Chromatin dynamics and epigenetic changes 1/1/2009 1/1/2014 

NWO The role of histone methylation in silencing and transcriptional 

memory. 

NWO CW Finding readers that can decipher the epigenetic language of the 

chromatin core 

2/1/2005 1/19/2011 

9/1/2011 1/15/2015


Linn, Sabine A Sister’s Hope Validation of the BRCA-likeCGH test to predict sensitivity to 

intensifi ed alkylating chemotherapy 

Linn, Sabine A Sister’s Hope Development of a breast cancer BRCA2-like classifi er for treatment 

benefi t of DNA-DSB inducing agents. A Sister’s hope. 

Linn, Sabine EU EUROCANPLATFORM - A European Platform for Translational 


Linn, Sabine LSCA Valorisation 

Fund 

Linn, Sabine LSCA Valorisation 

Fund 

Linn, Sabine Pink Ribbon/A 

Sister’s Hope 

Translation of a BAC array-besed chip into an oligo array-based with 

chip to identify hereditary breast cancers with BRCA1 mutatioans 

and sporadic breat cancers with a defi cient BRCA1 singnaling 

pathway 

Translation of a test for BRCAness in breast cancer into a 

standardized test ready to use in daily clinical practice 

195 1 

Ended/ 

ends 

7/1/2011 7/1/2012 

1/1/2011 1/1/2015 

1/1/2011 1/1/2016 

7/1/2010 7/1/2012 

6/1/2010 7/1/2012 

Clinical evaluation of anti-estrogen resistance in breast cancer 11/1/2007 1/31/2012 

Linn, Sabine TI Pharma Applied Breast Cancer Diagnostics 8/15/2011 12/15/2013 

Linn, Sabine TI Pharma Applied breast cancer diagnostics 1/1/2012 1/1/2015 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

Lohuizen, 

Maarten van 

CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

1/1/2009 1/1/2014 

CGC CGC 2008-2012 10/1/2010 1/1/2013 

EU Eurosystem 3/1/2008 3/1/2012 

EU FGCMOG Functional genetic characterization of a mouse model of 

Glioma 

8/1/2010 8/1/2012 

Merck Merck LKR 6/2/2006 2/1/2012 

NIRM NIRM Epigenetic profi ling and lineage tracing of normal and cancer 

stem cells: Safeguarding normal regenerative stem cells in cancer 

1/1/2010 1/1/2016 

NPC Properties of pluripotency in mouse ESCs and iPS 1/1/2009 1/1/2014 

NWO-ALW Maintenance of genome integrity by histone (de)ubiquitinating 

enzymes. 

Michalides, Rob TI Pharma Nuclear receptors in targeted cancer therapy: improved methods for 

candidate selection 

Moolenaar, 

Wouter 

Moolenaar, 

Wouter 

CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

NWO CW Autotaxin, a secreted phosphodiesterase with diverse roles in disease: 

structural and functional studies 

PROJECTS 

11/1/2008 11/1/2013 

11/1/2006 1/1/2012 

1/1/2009 1/1/2011 

2/1/2011 2/1/2017 

Mukherjee, Sach NWO-ALW CSBC-Junior 9/1/2010 9/1/2015

196 

PROJECTS 


Neefjes, Jacques CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

Neefjes, Jacques EU ERC MHC Class II-omics’ Towards understanding and manipulation of 

MHC class II antigen presentation. 

Neefjes, Jacques EU Systems biology of phagosome formation and maturation - 

modulation by intracellular pathogens. 

Ended/ 

ends 

1/1/2009 12/31/2013 

9/1/2010 9/1/2015 

11/1/2008 5/1/2012 

Neefjes, Jacques NPC Kinome and phosphatasome knock-down libraries 1/1/2009 1/1/2014 

Neefjes, Jacques NWO Manipulating the MHC class II system to control immune responses 

in autoimmunity 

10/1/2007 10/1/2012 

Neefjes, Jacques NWO Rubicon 12/1/2010 12/1/2011 

Neefjes, Jacques NWO The molecular mechanism of the degradation of large molecular 

complexes 

Neefjes, Jacques NWO-ALW Using high-throughput screening of chemical libraries to identify 

factors involved in cross-presentation 

Oldenburg, 

Hester 

1/1/2010 1/1/2013 

5/1/2010 5/1/2011 

Pink Ribbon Werkhervatting en seksualiteit na borstkanker 9/1/2008 9/1/2012 

Ovaa, Huib CTMM Design and use of enhanced T cell antigens and T cell detection 

technology for human disease 

10/1/2010 10/1/2014 

Ovaa, Huib EU Construction of Well-defi ned Ubiquitin Conjugates SP3 People 10/15/2008 10/15/2011 

Ovaa, Huib EU Decoding the ubiquitin code 11/1/2011 11/1/2016 

Ovaa, Huib EU EU-OPENSCREEN Europenan Infrastructure of Open Screening 

Platforms for Chemical BIology 

11/1/2010 11/1/2013 

Ovaa, Huib EU Protein Stabilization by chemistry: total synthesis of an MHC class I 1/1/2011 1/1/2013 

Ovaa, Huib EU UPStream-European Resarch Training in the Ubiquitin Proteasome 

System Marie Curie 

Ovaa, Huib NGI An Integrated approach to immunomodulatory agents and T-cell 

tumor vaccines. 

Ovaa, Huib NGI Combinatorial unnatural epitope libraries coupled with MHC 

exchangeligand enrichment for rapid defi nition of T-cell vaccine 

canidates 

Ovaa, Huib NGI Commercial development of ubiquitinated peptides and screening 

assays produced by high throughput robotic peptide synthesis 

11/1/2011 11/1/2014 

10/1/2011 10/1/2015 

8/1/2010 8/1/2011 

1/1/2010 1/1/2012 

Ovaa, Huib NPC Tyrosine phosphatase inhitors, baits and ABPs 1/1/2009 1/1/2014 

Ovaa, Huib NPC NPC V1 Commercial development of synthetic Nedd8 and SUMO 

conjugates 

Ovaa, Huib NWO Development of conditional protein-ligand exchange applied to 

immune technology; Onderzoeksprogramma: Integration of 

Biosynthesis & Organic Synthesis (IBOS) 

1/1/2009 1/1/2014 

9/1/2008 9/1/2012


Ovaa, Huib NWO How proteoglycan receptors affect antigen cross-presentation and 

immunity 

Ovaa, Huib NWO CW Pan- Dub Inhibitors to delineate DUB dependent biology and tools to 

study the ubiqultinated proteome 

197 1 

Ended/ 

ends 

10/1/2010 10/1/2013 

8/1/2009 8/1/2013 

Ovaa, Huib NWO-ALW Post-translational transpeptidation and immunity 10/1/2009 10/31/2012 

Ovaa, Huib STW Development and Commercialization of High Throughput Assays for 

Drug Discovery in the Ubiquitin Field 

Ovaa, Huib STW Large scale production 4-thiolysine to enable commercial production 

of ubiquitin and ubiquitin-like conjugates 

7/1/2011 1/1/2014 

9/1/2010 3/1/2011 

Ovaa, Huib ZonMw Druggable DUBs in trypanosomes 8/1/2010 10/1/2011 

Peeper, Daniel A Sister’s Hope Screening and in vivo validation of pharmalogical inhibitors of 

human breast cancer metastasis 

Peeper, Daniel NWO Synthetic lethality: a novel tactic for selective anticancer drug target 

discovery 

4/1/2011 4/1/2013 

6/1/2007 6/1/2012 

Peeper, Daniel Overig The role of oncogene-induced senescene in long-latency leukemia 6/30/2010 6/30/2014 

Peeper, Daniel SFN Melanoma Xenografting Platform 11/1/2011 11/1/2012 

Perrakis, 

Anastassis 

Perrakis, 

Anastassis 

Perrakis, 

Anastassis 

NWO The NKI Protein Facility: Indentifi cation, Production, 

Characterization and interactions 

NWO CW Autotaxin, a secreted phosphodiesterase with diverse roles in disease: 

structural and functional studies 

NWO CW PDB_REDO: procedures for rebuilding and refi nement of 

macromolecular models in X ray crystallography 

Peters, Peter Aeras Global TB 

Vaccine Foundation 

1/1/2009 9/30/2011 

2/1/2011 2/1/2017 

12/1/2011 11/30/2014 

DGIS Grant 1/1/2011 12/31/2014 

Peters, Peter EU NOTOX Predicting long-term toxic effects using computer models 

based on systems characterization of organotypic cultures Grant 

Agreement Number 267038 

Peters, Peter EU Protecting the food chain form prions: shaping European priorities 

through basic and applied research 

1/1/2011 1/1/2016 

10/1/2009 10/1/2013 

Peters, Peter Leprastichting How mycobacteria lyse the phagosomal membrane 1/1/2011 1/1/2015 

Peters, Peter MIN OCW E-cadhering and cadhering-11 and their role in cancer migration” 

binnen het SmartMix programma NIMIC. 

1/1/2009 7/1/2013 

Peters, Peter NIH Pathways of Antigen Presentation by CD1 7/1/2010 6/30/2015 

Peters, Peter STW FES HTSM ‘Towards a Sustainable and Open Innovation Ecosystem’ 1/1/2010 12/31/2016 

Poel, Henk van 

der 

CTMM PCCM Prostate Cancer Molecular Medicine 12/1/2009 12/1/2014 

Rasch, Coen EEG-CEC / EU Adaptive and innovative Radiation Treatment FOR improving Cancer 


PROJECTS 

3/1/2011 3/1/2016

198 

PROJECTS 


Riele, Hein te MAAG LEVER 

DARM 

Ended/ 

ends 

Modifi ers of tumor development in Lynch syndrome 11/1/2010 11/1/2012 

Riele, Hein te NWO Horizon 050-71-051 12/1/2006 12/1/2011 

Riele, Hein te NWO Sister chromatid cohesion: lessons from yeast, mice and men 5/1/2011 5/1/2014 

Riele, Hein te NWO-ALW A novel procedure for enzymatic production of low-complexity RNAI 

Libraries to identify coding- and non-coding turmor suppressor 

genes 

Riele, Hein te ZonMw Oligonucleotide-directed gene modifi cation in human induces 

pluripotent stem cells 

Rodenhuis, Sjoerd CTMM BREAST CARE Neoadjuvant drug treatment for breast cancer - 

response prediction and response monitoring 

Rodenhuis, Sjoerd SK Foundations Randomized phase II/II study of individualized neoadjuvant 

chemotherapy in triple negative breast tumors. 

Rookus, Matti BBMRI-NL HEBON - Plating of 10.000 DNA samples of women tested for BRCA 

1/2 mutations 

8/1/2009 8/1/2012 

6/1/2011 6/1/2015 

2/1/2009 2/1/2014 

1/7/2010 1/7/2015 

5/1/2010 5/1/2012 

Rookus, Matti EU Collaborative Oncological Gene-environment Study 5/1/2009 5/1/2013 

Rookus, Matti Pink Ribbon Website voor families met een hoog risico op borst- en/of 

ovariumkanker 

Rookus, Matti Universiteit Utrecht Nightingale Study: Shift work and risk of breast cancer, a prospective 

cohort study among Dutch nurses 

Rookus, Matti Universiteit Utrecht Shift work and risk of breast cancer, a prospective cohort study 

among Dutch nurses 

Rookus, Matti ZonMw HEBON research facility for individuals tested for BRCA 1/2 

mutations 

Rottenberg, Sven CTMM BREAST CARE Neoadjuvant drug treatment for breast cancer - 


Rottenberg, Sven ZonMw Predicting and targeting primary chemotherapy resistance in 

conditional mouse models of breast cancer 

Ruers, Theo CTMM Non-invasive treatment of cancer by MRI-guided high-intensity 

focused ultrasound ablation 

Ruers, Theo Philips Research Collaboration Agreement for Data Collection - Ex-vivo 

human tissue study 

9/1/2010 9/1/2012 

9/1/2010 9/1/2014 

9/1/2008 9/1/2016 

10/23/ 

2009 

10/23/ 

2013 

2/1/2009 2/1/2014 

1/1/2011 12/31/2015 

12/1/2009 12/1/2014 

10/12/ 

2009 

1/1/2011 

Ruers, Theo Philips Research collaboration Philips 4/1/2010 1/1/2012 

Sandick, Johanna 

van 

Sandick, Johanna 

van 

Vrolijk Lab slokdarmkankeronderzoek Stichting Vrolijk 9/1/2009 1/1/2012 

Vrolijk Slokdarmkankeronderzoek Stichting Vrolijk 1/1/2008 5/1/2013 

Schellens, Jan aCBG Conv. NKI-aCBG (Geneesm.) 8/1/2006 1/1/2012


Schellens, Jan Astra Preclinical in vivo testing of AZD1152 to determine the effect of 

the ABC-drug transporters P-glycoprotein (Pgp, Mdr1) and BCRP 

(Bcrp1, Abcg2) on tissue distribution and clearance of AZD1152. 

199 1 

Ended/ 

ends 

9/1/2007 1/1/2011 

Schellens, Jan Merck Merck OncoNet Member Agreement 12/1/2009 1/1/2012 

Schellens, Jan Roche Support Roche clinical research associate Candersartan study 

M06HER 

Schmidt, 

Marjanka 

Schmidt, 

Marjanka 

Schmidt, 

Marjanka 

BBMRI-NL Breast cancer risk polymorphisms in familial, non-BCRA 1/2 breast 

cancer patients (for BOSOM) 

BBMRI-NL Regenboogproject 6 Towards a joint strategy for the return of results 

and optimal communication with biobank donors 

2/1/2009 1/1/2012 

12/1/2010 12/1/2011 

1/1/2012 1/1/2014 

EU CAncer Risk and INsulin analoGues 11/1/2011 11/1/2015 

Schumacher, Ton ACTS (NWO) Development of conditional protein-ligand exchange applied to 

immune technology 

Schumacher, Ton Centre for Cancer 

Immune Therapy 

(CCIT) 

Development and clinical evaluation of new strategies for adoptive 

cell transfer (ACT) in the treatment of cancer. 

Schumacher, Ton CTMM Design and use of enhanced T cell antigens and T cell detection 

technology for human disease 

Schumacher, Ton EU ATTRACT - Advanced Teaching and TRaining for Adoptive Cell 

Therapy 

Schumacher, Ton EU Barcoding approach for dendritic cells differentiation (BARDIF) 

Marie Curie 

9/1/2008 9/1/2012 

1/1/2009 1/1/2014 

10/1/2010 10/1/2014 

10/1/2009 10/1/2013 

4/1/2011 4/1/2013 

Schumacher, Ton EU Life-his-T - Tracing the life histories of T cells 5/1/2011 5/1/2016 

Schumacher, Ton EU Mapping the life histories of T cells 5/1/2011 5/1/2016 

Schumacher, Ton EU SPHINX - Spontaneous clearance in Patients acutely infected with 

HCV - Immune profi ling, Novel biomarkers and X-omics approaches 

Schumacher, Ton Landsteiner 

Stichting 

Graft versus Host disease by TCR-engineered T cells: mechanisms 

and means for prevention 

11/1/2010 11/1/2013 

1/1/2009 1/1/2012 

Schumacher, Ton LLS Career Development Program. The Leukemia & Lymphoma Society 7/1/2010 7/1/2013 

Schumacher, Ton ZonMw Intravital imaging and computational modeling of skin immunity 3/1/2011 3/1/2015 

Schumacher, Ton ZonMw T cell receptor gene therapy of metastatic melanoma: a phase I 

clinical trial. 

Sixma, Titia CBG Identifi cation of cancer-relevant genes using a functional genetic 

approach. 

4/1/2005 2/1/2013 

1/1/2009 1/1/2014 

Sixma, Titia EU Rubicon 1/1/2006 6/30/2011 

Sixma, Titia EU Mismatch2model-Characterization and quantitative modeling of 

DNA mismatch repair and its role in the maintenance of genomic 

stability and cancer avoidance. 

PROJECTS 

11/1/2008 11/1/2012

200 

PROJECTS 


Ended/ 

ends 

Sixma, Titia EU Neurotransmitter Cys-loop recptors: structure, function and disease. 2/1/2008 2/1/2012 

Sixma, Titia EU The balance of ubiquitin conjugation and deconjugation 2/1/2010 2/1/2015 

Sixma, Titia NWO CW Initiation of DNA mismatch repair 8/1/2011 8/1/2016 

Sixma, Titia NWO CW The regulatory role of Ubl domains in ubiquitin specifi c protease 

function 

Sixma, Titia TI Pharma New approaches for Ligand-Gated Ion Channel (LGIC) drug 

discovery 

Sonke, Jan-Jakob CTMM BREAST CARE Neoadjuvant drug treatment for breast cancer - 


Sonke, Jan-Jakob EU HYPERImage: hybrid PET-MT system for concurrent ultra-sensitive 

imaging 

Sonnenberg, 

Arnoud 

Sonnenberg, 

Arnoud 

Sonnenberg, 

Arnoud 

Sonnenberg, 

Arnoud 

10/1/2010 10/1/2015 

1/1/2007 7/1/2011 

2/1/2009 2/1/2014 

4/1/2008 10/1/2011 

DEBRA Studying Kindler Syndrome in an in vivo model system 10/1/2011 10/1/2013 

Nierstichting Function of tetraspanin-integrin complex Cd151- a3B1 in development 

and maintenance of the glomerular fi ltration barrier. NSN project. 

NWO-ALW Controlling hemidesmosome dynamics by phosphorylation of 

residues on the integrin b4 subunit. 

10/1/2008 10/1/2012 

3/1/2007 3/1/2011 

NWO-ALW Mechanical control of chromatin organization and gene expression 9/1/2011 9/1/2014 

Steensel, Bas van NGI Chromatin Protein Discovery Project 2/1/2008 2/1/2012 

Steensel, Bas van NWO-ALW Nuclear lamina genome interactions in mammalian cells 7/1/2009 7/1/2014 

Steensel, Bas van NWO-ALW Veni Jop Kind 9/1/2011 9/1/2014 

Stewart, Fiona EU Cardiorisk - The mechanisms of cardiovascular risks after low 

radiation doses 

2/1/2008 7/1/2011 

Tan, Bing Biolitec The cost-effectiveness study 1/1/2009 1/1/2012 

Tan, Bing ZonMw A multi-centre cost-effectiveness evaluation of a novel treatment 

option in the Netherlands: Photo Dynamic Therapy with temoporfi ne 

for the treatment of advanced incurable head and neck cancers, for 

whom prior conventional treatments have failed. 

Tan, Bing ZonMw New therapy for Epstein-Barr virus driven tumours by targeting the 

virus itself 

Tellingen, Olaf 

van 

Valdes Olmos, 

Renato 

1/1/2009 1/1/2012 

1/1/2011 1/1/2013 

Stop Hersentumoren Promising drug combinations in the treatment of high-grade glioma 12/1/2011 12/1/2013 

EU Mammography with molecular imaging (MAMMI) Specifi c Targeted 

project 

1/1/2007 1/1/2011 

Veer, Laura van ‘t BBMRI-NL BBMRI NL Project CP45 10/1/2010 12/1/2011 

Veer, Laura van ‘t CGC CGC 2008-2012 8/1/2008 1/1/2013


201 2 

Ended/ 

ends 

Veer, Laura van ‘t EU Breast Cancer Somatic Genetics Study 7/1/2010 7/1/2014 

Veer, Laura van ‘t EU Collaborative Oncological Gene-environment Study 5/1/2009 5/1/2013 

Veer, Laura van ‘t EU TRANSBIG Translating molecular knowlegde into early breast cancer 

management 

3/1/2004 3/1/2011 

Veer, Laura van ‘t TI Pharma Predicting drug responses in breast cancer 7/1/2007 7/1/2011 

Verheij, Marcel AstraZeneca Olaparib-Radiotherapy combination trials: optimization, normal 

tissue toxicity evaluation and biomarkers for toxicity and tumour 

response 

Verheij, Marcel EU Adaptive and innovative Radiation Treatment FOR improving Cancer 


1/1/2012 1/1/2014 

3/1/2011 3/1/2016 

Verheij, Marcel Elekta EOS II 8/1/2010 8/1/2020 

Verheij, Marcel EOS / voorheen 

Philips 

Verheij, Marcel LSCA Valorisation 

Fund 

Vijver, Marc van 

de 

Framework Research Agreement between Elekta and NKI/AvL 8/1/2010 8/1/2020 

Dose-fi nding study for a novel liposomal doxorubicon formulation 7/1/2010 7/1/2012 

CTMM BREAST CARE Neoadjuvant drug treatment for breast cancer - 


Visser, Karin de AICR Tumor-associated macrophages as therapeutic targets for metastatic 

breast cancer 

Visser, Karin de AstraZeneca Functional assessment of CSF-1 receptor signalling in de novo breast 

cancer development and metastatis information 

Visser, Karin de EU BMDCMET - Innate and adaptive immune cell contribution to the 

pre-metastatic niche 

Visser, Karin de Roche Anti-cancer effi cacy of a murinized antibody against the CSF-1 

receptor in a conditional mouse model for de novo invasive and 

metastatic breast cancer 

2/1/2009 2/1/2014 

6/1/2011 6/1/2014 

11/1/2009 12/31/2012 

6/1/2011 6/1/2013 

1/13/2010 1/13/2011 

Visser, Karin de Roche Research Agreement Roche / NKI Amsterdam Combichemo 5/1/2011 5/1/2013 

Visser, Karin de ZonMw The infl ammatory tumor microenvironment and its impact on breast 

cancer development and therapy 

1/1/2009 1/1/2014 

Wesseling, Jelle AMC HER2 NVVP Kwaliteitscontrole Project 1/1/2009 7/1/2011 

Wesseling, Jelle ZonMw Automated histology slide imaging and biomarker and biomarker 

recognition for standardized, quantitative and high-throughput 

tissue evaluation. 

Wessels, Lodewyk AstraZeneca Identifi cation and validation of new targets for colorectal cancer 

through molecular redefi nition of the disease 

Wessels, Lodewyk AstraZeneca Pathway-based tumour characterisation & alignment of cell panels 

against tumour samples 

PROJECTS 

9/1/2011 9/1/2015 

1/1/2011 12/1/2013 

2/1/2010 2/1/2012

202 

PROJECTS 


Wessels, Lodewyk CTMM Neoadjuvant drug treatment for breast cancer - response-prediction 

and response monitoring (breast care); SP (sub-workpackage) 10: 

Biomarkers informatics 

Ended/ 

ends 

2/1/2009 2/1/2014 

Wessels, Lodewyk EU Collaborative Oncological Gene-environment Study 5/1/2009 5/1/2013 

Wessels, Lodewyk EU EuroTARGET 3/1/2011 3/1/2016 

Wessels, Lodewyk NBIC NBIC / BioAssist / HTHC Programmeur 1/1/2008 9/1/2011 

Wessels, Lodewyk NBIC BioRange 10/1/2009 12/31/2013 

Wessels, Lodewyk NGI NCSB 11/1/2010 11/1/2013 

Wessels, Lodewyk NWO-ALW CSBC 9/1/2010 9/1/2015 

Wessels, Lodewyk ZonMw Signaling pathways and gene regulatory networks responsible for 

th17 cell differentiation 

Wolthuis, Rob HFSPO Cycle-Quant: Defi ning Cell Cycle Progression and Responses to 

Perturbations 

Wolthuis, Rob Nefkens Foundation Advancing Automated Cell Biology and Optogenetics at the NKI 1/1/2011 

5/1/2010 5/1/2013 

10/1/2010 10/1/2013 

Wolthuis, Rob NWO Bridging the gap between Genomics and Cell Biology 1/1/2011 12/31/2011 

Wolthuis, Rob NWO-ALW Coordination of cell division by regulated protein destruction 1/1/2007 12/31/2011

PERSONNEL INDEX 

Aalbers, Arend 149 

Aaronson, Neil 95 

Abdallah, Abdallah 35 

Abrao Possik, Patricia 84 

Achachah, Mohamed 103 

Adriaansz, Sandra 116 

Afanasyev, Pavel 35 

Agami, Reuven 48 

Ait Moha, Daoud 103 

Ajouaou, Abderrahim 103 

Akthar, Waseem 80 

Albers, Harald 33 

Alderliesten, Maaike 26 

Alderliesten, Tanja 128 

Aleman, Berthe 90, 127 

Alendar, Andrej 82 

Alexi, Xanthippi 47 

Amendola, Mario 50 

Amore, Alessia 33 

Angelopoulos, Nicos 72 

Anirudhan, Gireesh 84 

Appelman, Jolanda 162 

Appels, Marije 115 

Arens, Robin 162 

Argenzio, Elisabetta 26 

Ariotti, Silvia 58 

Arkies, Hester 102 

Aukema, Tjeerd 149 

Baank, Saskia 102 

Baars, Danny 162 

Baars, Jessica 99 

Baars, Joke 115 

Baas, Paul 37, 115 

Baas-Vrancken Peeters, 

Marie-Jeanne 149 

Babala, Nikolina 54 

Badhai, Jitendra 82 

Bakker, Jeroen 31 

Bakker, Martine 102 

Bakker, Sandra 115 

Bakker, Sietske 62 

Balague Ponz, Olga 102 

Balm, Alfons 150 

Banishki, Nikola 64 

Barbier, Nathalie 162 

Bartelink, Harry 127 

Batteau, Lukas 104 

Beekman, Chantal 104 

Beelen, Karin 68, 116 

Beijersbergen, Roderick 76 

Beijnen, Jos 41, 115 

Belderbos, José 97, 127 

Bendle, Gavin 56 

Benraadt, Tinie 162 

Bentin Toaldo, Cristiane 47 

Bergman, André 89, 115 

Berkers, Celia 33 

Berkhof, Marianne 95 

Berlin, Ilana 31 

Bernards, René 74 

Berns, Anton 82 

Berns, Katrien 74 

Bes-Gennissen, Annemiek 74 

Besnard, Peter 103 

Bessels, Frauwkje 162 

Betgen, Anja 128 

Bies, Laura 56 

Bijos, Dominika 50 

Bin Ali, Rahmen 82 

Blank, Christian 61, 115 

Bleiker, Eveline 99, 103 

Bloemers, Monique 127 

Blom, Marie-José 90 

Blom, Marleen 80 

Blomen, Vincent 22 

Boekel, Naomi 90 

Boekhout, Annelies 116 

Boekhout, Michiel 78 

Boer, Mandy 86 

Boerrigter-Barendsen, Lucie 103 

Boessenkool-Pape, Juliet 90 

Bombardelli, Lorenzo 82 

Boogerd, Willem 97, 115 

Boon, Ute 66 

Boot, Henk 115 

Boot, Judith 162 

Borgemeester, Maarten 150 

Borst, Gerben 127 

Borst, Jannie 54 

Borst, Piet 64 

Bos, Arnold 48 

Boshuizen, Rogier 115 

Bosma, Astrid 74 

Bot, Ilja 115 

Botma, Henk 162 

Boucher, Audi 162 

Boutmy-de Lange, Majella 103 

Bouwman, Peter 66 

Brandenburg, Alexander 43, 90 

Brandsma, Dieta 115 

Braumuller, Tanya 66 

Braunschweig, Ulrich 50 

Broeks, Annegien 43, 90, 102 

Brohet, Richard 90 

Brouwer, Oscar 102 

Bruin, Natascha 102 

Bruin, Sjoerd 149 

Bruining, Annemarie 103 

Brummelkamp, Thijn 22 

Bucholtz, Karina 116 

Buckle, Tessa 104 

Buikhuisen, Wieneke 115 

Buil, Levi 102 

Buning-Kager, Marian 102 

Bunschoten, Anton 103 

Burgers, Sjaak 115 

Burylo, Artur 41 

Cadot, Sidney 72 

Caillat, Christophe 20 

Canisius, Sander 72 

Cantelli, Erika 62 

Carreno, Monique 162 

Cats, Annemieke 99, 115 

Chen, Chun 128 

Chen, Shih-Cheng 48 

Chen, Wei 104, 128 

Chin, Patrick 103 

Ciampricotti, Metamia 66 

Citterio, Elisabetta 80 

Clapham, Renee 150 

Clerici, Marcello 18 

Clijsters, Linda 78 

Coffelt, Seth 66 

Cooke, Saskia 162 

Coquet, Jonathan 54 

Cordeiro Pedrosa, Lília 39 

Cornelissen, Paulien 80 

Cornelissen, Sten 43 

Courrech Staal, Ewout 149 

Dalesio, Otilia 162 

Dannenberg, Jan-Hermen 53 

de Benedictis, Cinzia 78 

de Boer, Jan Paul 115 

de Boer, Johan 128 

de Boer, Roel 127 

de Bois, Josien 128 

de Cortie, Karin 37 

de Graaf, Carolyn 50 

de Groot, Dirk-Jan 24 

de Haan, Rosemarie 127 

de Haas, Marcel 64 

de Jong, Annemieke 33 

de Jong, Daphne 102 

de Jong, Gerda 162 

de Jong, Jeroen 102 

de Jong, Johann 72 

de Jong, Monique 127 

de Jong, Rianne 128 

de Jong, Trees 58 

de Lange, Judith 90 

de Leeuw, Renée 47 

de Lint, Klaas 76 

de Punder, Karin 35 

de Ronde, Jorma 45, 72 

de Ruiter, Michiel 97 

de Visser, Karin 66 

de Vries, Evert 54 

de Vries, Hilda 82 

de Vries, Kim 127 

de Vries, Nienke 80 

de Vries, Sandra 62 

203 

PERSONNEL INDEX

204 


de Waal, Marjolijn 162 

de Wit, Ineke 162 

Deenen, Maarten 116 

Dees-Ribbers, Hermine 128 

Dekker, Marleen 62 

Dekker, Rob 62 

Delzenne-Goette, Elly 62 

Derissen, Ellen 116 

Derks, Elin 150 

Devriese, Lot 41, 116 

Dewit, Luc 127 

Dirac, Annette 74 

Dohmen, Amy 150 

Donker, Mila 149 

Doodeman, Barry 128 

Doornebal, Chris 66 

Dorlo, Thomas 116 

Douma, Sirith 84 

Doumont, Gilles 66 

Droog, Marjolein 47 

Drost, Jarno 48 

Drost, Rinske 66 

Drukker, Caroline 43 

Dubbelman, Anne Charlotte 116 

Dubbelman, Ria 116 

Dufournij, Brigitte 162 

Duppen, Joop 128 

Durmus, Selvi 70 

Eijzenga, Willem 95, 99 

Eilers, Marlou 162 

Ekkebus, Reggy 33 

El Atmioui, Dris 33 

El Oualid, Farid 33 

Elkhuizen, Paula 127 

Elkon, Rani 48 

Elouarrat, Dalila 26 

Elshof, Lotte 104 

Engelhardt, Ellen 43, 90 

Evers, Danny 149 

Faesen, Alex 18 

Fan, Lin 102 

Fasolis, Alexandros 150 

Feenstra, Christel 102 

Feenstra, Heleen 97 

Fernandez Salcedo, Ernesto 162 

Filion, Guillaume 50 

Fish, Alex 18 

Fles, Renske 150 

Floot, Ben 37 

Foekema, Joke 117 

Frederix, Geert 41, 116 

Frijns, Evelyne 24 

Gadiot, Jules 61 

Gallenne, Tristan 84 

Gambino, Valentina 74 

Gapp, Bianca 22 

Gargiulo, Gaetano 80 

Garstka, Gosia 31 

Gasparini, Alessia 128 

Gebretensae, Abadi 116 

Gerlach, Carmen 56 

Gerritsen, Bram 72, 76 

Gerritsma, Miranda 90, 95 

Geumann, Ulf 54 

Geurink, Paul 33 

Geutjes, Ernst-Jan 74 

Gilhuijs, Kenneth 103 

Gisler, Santiago 80 

Godsave, Sue 35 

Goey, Andrew 116 

Gorsira, Amber 103 

Gort, Eelke 68 

Greig, Kylie 84 

Grernrum, Wipawadee 74 

Groen, Wim 101 

Groenendijk, Floris 74 

Groot, Harmke 90 

Groot, Yvonne 162 

Groothuizen, Flora 18 

Guislain, Aurelie 61 

Gundy, Chad 95, 97, 99 

Guyader, Charlotte 64 

Haanen, John 58, 115 

Haas, Rick 127 

Hagenaars, Christiane 162 

Hahn, Daniela 99, 103 

Halonen, Pasi 76 

Hameed, Dharjath 33 

Hamming-Vrieze, Olga 127 

Hanegraaf, Maaike 82 

Haramis, Anna-Pavlina 88 

Harinck, Femme 99 

Harinck, Nieke 127 

Haringhuizen, Annebeth 115 

Harms, Emmy 117 

Harmsen, Tim 62 

Hau, Song-Hieng 162 

Hau, Tissee 66 

Hauptmann, Michael 94 

Haussmann, Jens 20 

Heemsbergen, Wilma 94, 127 

Heemskerk, Bianca 58 

Heemskerk, Caroline 162 

Heidebrecht, Tatjana 20 

Heideman, Richard 53 

Heijmink, Stijn 103 

Helgason, Helgi 116 

Hendrikx, Jeroen 70, 116 

Henneman, Linda 28, 66 

Hennink, Roos 162 

Heynen, Guus 74 

Hibbert, Rick 18 

Hiemstra, Annelies 162 

Hijmans, Marielle 74 

Hilgers, Frans 150 

Hilkens, John 86 

Hilkmann, Henk 33 

Hill, Steven 23 

Hillebrand, Michel 116 

Hinnen, Karel 127 

Hoefnagel, Cornelis 102 

Hoekstra, Paula 162 

Hoffmans, Ruth 150 

Hogenbirk, Marc 60 

Hogervorst, Frans 90, 102, 103 

Hollenstein, Andreas 56 

Hollmann, Birgit 127 

Holtkamp, Marjo 117 

Hölzel, Michael 74 

Hompes, Daphne 149 

Honnef, Joeri 128 

Hooijkaas, Anna 61 

Hoppes, Rieuwert 33 

Houben, Anna 26 

Houlleberghs, Hellen 62 

Hoving, Saske 37, 103 

Huang, Sidong 74 

Huijbers, Ivo 82 

Huitema, Alwin 115 

Hulsman, Danielle 80 

Ikink, Gerjon 86 

Innocenti, Metello 30 

Iskit, Sedef 84 

Isogai, Tadamoto 30 

Iusuf, Dilek 70 

Ivanov, Eduard 162 

Jacobi, Irene 150 

Jacobs, Bart 41 

Jacobs, Heinz 60 

Jacobs, Jacqueline 87 

Jae, Lucas 22 

Jager, Nynke 116 

Jalink, Kees 28 

Jan, Sabrina 64 

Jansen, Edwin 127 

Jansen, Hans 35 

Jansen, Marissa 163 

Jansen, Robert 64 

Janssen, Esther 90 

Janssen, Lennert 31 

Janssens, Tine 116 

Jaspers, Janneke 66 

Jastrzebski, Kathy 76 

Jeanson, Kiki 43, 90, 103 

Jenal, Mathias 48 

Jibodh, Aarti 163 

Jongsma, Maikel 26 

Jongsma, Marlieke 31 

Jonker, Marcel 128 

Jonkers, Irene 163 

Jonkers, Jos 45, 66 

Jonkman, Joop 163 

Joosten, Krista 20 

Joosten, Robbie 20 

Kaiser, Andrew 61 

Kalisvaart, Robin 128 

Kammeijer, Quinten 150 

Kant, Josien 163 

Kaplon, Joanna 84 

Karakullukcu, Baris 150 

Kas, Sjors 66 

Kasiem, Mobien 103 

Kedziora, Kasia 28

Keessen, Marianne 117 

Kelderman, Sander 56 

Kemper, Kristel 84 

Kemperman, Myrle 97 

Kersbergen, Ariena 64 

Kerst, Martijn 115 

Kersten, Kelly 66 

Kersten, Kim 90, 97 

Ketema, Mirjam 24 

Kieffer, Jacobien 95, 97 

Kind, Jop 50 

Kist, Jacob 102 

Klarenbeek, Jeffrey 28 

Klarenbeek, Sjoerd 66 

Kleinsmit, Gerke 101 

Klijn, Christiaan 66, 72 

Klomp, Houke 149 

Klop, Martin 150 

Klous, Marjolein 115 

Kluijt, Irma 99, 103 

Klü mpen, Heinz Josef 116 

Knegjens, Joost 127 

Knegt, Cuna 150 

Knijnenburg, Theo 72 

Knockaert, Bart 150 

Knol, Cora 90 

Kobus, Monica 97 

Koenen, Annemieke 117 

Kolmschate, Lies 163 

Koning, Gerben 39 

Kooijman, Karen 90 

Koolen, Bas 102 

Koolen, Stijn 116 

Koopman-Kroon, Ciska 116 

Koops, Wim 103 

Koornstra, Rutger 68 

Koppelmans, Vincent 97 

Koppens, Martijn 80 

Korse, Tiny 102 

Kort, Anita 116 

Kouwenberg, Hanneke 102 

Krap, Menno 150 

Kreeft, Annemarijn 150 

Kreft, Maaike 24 

Kregel, Eva 66 

Krewinkel, Han 127 

Krewinkel, Hans 128 

Krijger, Peter 60 

Krimpenfort, Paul 82 

Kröger, Robert 103 

Kromdijk, Wiete 116 

Kruis, Matthijs 128 

Kuenen, Marianne 90, 95, 97 

Kuijpers, Wilma 101 

Kuiken, Johan 76 

Kuikman, Ingrid 24 

Kuil, Joeri 103 

Kuiper, Maria 117 

Kujala, Pekka 35 

Kumar, Prasanth 74 

Kvistborg, Pia 58 

Kwint, Margriet 128 

Kwon, Min-chul 82 

Lambooij, Jan Paul 82 

Lammens, Chantal 95 

Lammerts van Buren, Alicia 35 

Lancini, Cesare 80 

Lange, Charlotte 103 

Lankheet, Nienke 116 

Laval, Severine 24 

Lebesque, Joos 127 

Leestemaker, Yves 33 

Leijen, Suzanne 41, 116 

Lenain, Christelle 84 

Léveillé, Nicolas 48 

Leyton Puig, Daniele 28 

Lieftink, Cor 76 

Lieshout, Michiel 150 

Linders, Dorothé 102 

Linn, Sabine 68, 97, 115 

Linnemann, Carsten 56 

Lips, Esther 45 

Liu, Zhen 30 

Loayza, Fabrizio 48 

Lodder, Wouter 150 

Lohuis, Peter 150 

Loo, Claudette 103 

Lopez, Marta 94 

Lucas, Luc 116 

Lukas, Anne 115 

Lutkenhaus, Lotte 104 

Maatje, Marlies 150 

Mahn, Marianne 163 

Majewski, Ian 74 

Maletta, Massimiliano 35 

Mallo, Henk 117 

Mandjes, Ingrid 163 

Mans, Anton 128 

Marchetti, Serena 41, 115 

Margadant, Coert 24 

Matas-Rico, Elisa 26 

Mattiroli, Francesca 18 

Meijerman, Irma 41 

Meijnen, Philip 127 

Meissl, Katrin 84 

Melis, Jacoline 95 

Melo, Carlos 48 

Mencarelli, Angelo 128 

Menendez, Victoria 31 

Menning, Sanne 97 

Merkx, Remco 33 

Merqui-Roelvink, Marja 116 

Meuleman, Wouter 50, 72 

Meulenaar, Jelte 116 

Meulendijks, Didier 41 

Meulepas, José 94 

Mewes, Janne 101 

Mezzadra, Riccardo 56 

Michalak, Ewa 66 

Michaut, Magali 72 

Michels, Samira 58 

Mijnheer, Ben 127 

Miltenburg, Nienke 115 

Minderhoud, Tom 128 

Miquel Cases, Anna 101 

Mittemperger, Lorenza 74 

Modder, Carla 163 

Moes, Johannes 116 

Mooij, Thea 90 

Mook, Stella 127, 149 

Moolenaar, Wouter 26 

Moonen, Luc 127 

Moore, Hayley 48 

Morris, Ben 76 

Mueller, Judith 84 

Mukherjee, Sach 23 

Mulder, Lennart 45 

Muller, Pietje 163 

Muller, Saar 102, 103, 150 

Muris, Jettie 102 

Mylvaganan, Chelvi 102 

Nacerdine, Karim 80 

Nagtegaal, Tanja 99 

Naik, Shalin 56 

Nan-Offeringa, Lianda 116 

Navran, Arash 127 

Nederlof, Petra 45, 102, 103 

Neefjes, Jacques 31 

Nieweg, Omgo 149 

Nijkamp, Jasper 128 

Nijkamp, Wouter 76 

Nijwening, Jeroen 76 

Nol, Annemarie 117 

Nooijen, Willem 102 

Nowee, Marlies 127 

Nuijen, Bastiaan 115 

Nuijten, Elvira 163 

Numan, Rachel 149 

Oates, Chris 23 

Obernosterer, Gregor 22 

Ogink, Janneke 78 

Oldenburg, Hester 95, 97, 149 

Olszewska, Agnieszka 128 

Onderwater, Suzanne 117 

Ong, Nico 35 

Ongering, Lindsay 163 

Oosterhuis, Koen 58 

Oosterkamp, Rianne 74 

Opdam, Mark 68 

Opstal-van Winden, Annemieke 90 

Oude Vrielink, Joachim 48 

Ouwens, Gabey 90 

Ovaa, Huib 33 

Paape, Anita 104 

Paauwe, Madelon 54 

Pagie, Ludo 50 

Pang, Baoxu 31 

Panneman, Carmen 128 

Pasca, Edoardo 103, 128 

Paul, Petra 31 

Paulus van Pauwvliet, Cecile 163 

Pawlitzky, Inka 80 

Pecharroman Gallego, Raul 128 

205 

PERSONNEL INDEX

206 


Peeper, Daniel 84 

Peeters, Koen 149 

Pelders, Saskia 90 

Pengel, Kenneth 104, 128 

Peric-Hupkes, Daniel 50 

Perie, Leila 56 

Perrakis, Anastassis 20 

Peters, Carolien 128 

Peters, Peter 35 

Peulen, Heike 127 

Peuscher, Marieke 87 

Pevenage, Philip 103 

Philips, Daisy 56 

Piek-den Hartog, Marianne 149 

Pieterse, Mark 66 

Pijpe, Anouk 90 

Pindyurin, Alexey 50 

Ploeger, Lennert 128 

Plug, Rob 103 

Pluim, Dick 41 

Pluister, Yvonne 102 

Pool, Bert 102 

Ports, Michael 24 

Pos, Floris 127 

Postel, Ruben 24 

Prahallad, Anirudh 74 

Pramana, Jimmy 150 

Prevoo, Warner 103 

Pritchard, Colin 82 

Pronk, Loes 163 

Proost, Natalie 82 

Pruntel, Roelof 103 

Qi, Wen 104 

Quispel, Josine 115 

Radhakishun, Nalini 116 

Rajan, Abinaya 101 

Rana, Anas 23 

Rasch, Coen 127 

Raspe, Marcel 28 

Rebers, Susanne 43, 90 

Rehorst, Harriet 163 

Reichgelt, Babs 127 

Reijnders, Vera 163 

Reinders, Anneke 163 

Remeijer, Peter 127 

Remmelts, Andrea 150 

Retèl, Valesca 101 

Rhodius, Robert 115 

Rigaill, Guillem 72 

Rodenhuis, Sjoerd 45, 115 

Rodenko, Boris 33 

Rohr, Jan 56 

Roodbergen, Rita 97 

Rookus, Matti 90 

Rooswinkel, Rogier 54 

Rooze, Lyandra 102 

Rosado, Arantxa 50 

Rosenberg, Efraim 102, 103 

Rosing, Hilde 115 

Roskam, Marielle 163 

Rossi, Maddalena 104, 128 

Rottenberg, Sven 64 

Rozendaal, Roel 128 

Rucktooa, Prakash 18 

Ruers, Theo 149 

Ruijs, Marielle 103 

Ruijter-Schippers, Henrique 103 

Rumpf, Cornelia 78 

Russell, Nicola 37, 90, 127 

Rutgers, Emiel 90, 95, 149 

Ryan, Martin 163 

Sachs, Norman 24 

Sadaka, Charlotte 31 

Sahtoe, Danny 18 

Salm, Liesbeth 102 

Salomon, Izhar 31 

Salverda, Govert 127 

Sanders, Joyce 103 

Sani, Musa 35 

Sapthu, Sunny 64 

Saveur, Lisette 117 

Sawicki, Emilia 116 

Scanu, Tiziana 31 

Schaake, Eva 149 

Schaapveld, Michael 90 

Schagen, Sanne 97 

Scharpfenecker, Marion 37 

Scheenstra, Alize 128 

Scheenstra, Renske 150 

Scheepbouwer, Roy 128 

Scheerman, Esther 103 

Schellens, Jan 41, 115 

Schinkel, Alfred 70 

Schlicker, Andreas 72 

Schmidt, Marjanka 43, 90 

Schmitz, Alexander 104 

Schmitz, Annemarie 104 

Schneider, Christoph 127 

Schol, Joke 116 

Schot, Margaret 117 

Schotte, Remko 56 

Schouten, Philip 68, 116 

Schrier, Mariette 48 

Schrijver, Lieske 90 

Schumacher, Ton 56 

Schwandt, Noortje 150 

Scuric, Vincent 163 

Secades, Pablo 24 

Seemann, Ingar 37, 104 

Seigers, Riejanne 97 

Serresi, Michela 80 

Severson, Tesa 68 

Sidharta, Grace 95, 99 

Siedschlag, Christian 104 

Sinaasappel, Michiel 102, 103 

Sivro-Prndelj, Ferida 102 

Sixma, Titia 18 

Smeele, Ludi 150 

Smit, Judith 18 

Smit, Marjon 84 

Smits, Fokko 150 

Snoek, Margriet 82 

Sol, Wendy 64 

Sombroek, Cherita 90 

Somer-Kristel, Petra 45 

Sommeijer, Dirkje 115 

Sonke, Gabe 95 

Sonke, Gabe 115 

Sonke, Jan-Jakob 127 

Sonneborn, Mariska 102 

Sonnenberg, Arnoud 24 

Soueidan, Hayssam 72 

Spaan, Mandy 90 

Spaapen, Robbert 31 

Sparmann, Anke 80 

Spliethoff, Jarich 149 

Spreeuw, Hanno 128 

Städler, Nicolas 23 

Staiger, Christine 72 

Staring, Jacqueline 22 

Steeghs, Neeltje 115 

Steuten, L 101 

Stewart, Fiona 37 

Stoker, Sharon 150 

Stokkel, Marcel 102 

Storm, Dea 163 

Stornaiuolo, Mariano 18 

Stouthard, Jacqueline 115 

Stroom, Joep 127 

Stuiver, Martijn 95, 149 

Stulemeijer, Iris 52 

Stuurman, Rik 116 

Sun, Chong 74 

Sutherland, Kate 82 

Sznajder, Beata 163 

Taal, Babs 115 

Talhout, Wendy 50 

Tan, Bing 150 

Tang, Seng Chuan 70 

Tanger, Ellen 80 

te Poele, Johannes 37 

te Riele, Hein 62 

Teertstra, Jelle 103 

ten Hoeve, Jelle 72 

ter Brugge, Petra 45, 66 

ter Riet, Bas 78 

Terweij, Marit 52 

Tesselaar, Margot 115 

Teunissen, Bas 116 

Theunissen, Noortje 150 

Thijssen, Bas 116 

Tibben, Matthijs 116 

Tielen, Ivon 103 

Tielenburg, René 128 

Tilgenkamp, Ria 163 

Timmermans, Jacqueline 150 

Timmers, Adriaan 150 

Toebes, Mireille 56 

Tolhuis, Bas 80 

Tomasoa, Brenda 127 

Topolnjak, Rajko 128 

Torres Acosta, Alex 163 

Trip, Anouk 127

Tripathi, Pankaj 64 

Tulner, Linda 116 

Uckelman, Michael 18 

Urbanus, Jos 56 

Uyterlinde, Wilma 117 

Valdés Olmos, Renato 102 

Valkenet, Ludy 163 

van ’t Veer, Laura 90 

van Amerongen, Renee 84 

van Arensbergen, Joris 50 

van As-Brooks, Corina 150 

van Bemmel, Joke 50 

van Berkel, Peter 101 

van Beurden, Marc 95, 149 

van Boven, Hester 102 

van Burgsteden, Johan 89 

van Buuren, Marit 56 

van Coevorden, Frits 149 

van de Kasteele, Willeke 58 

van de Kooij, Bert 54 

van de Noll, Ruud 116 

van de Velde, Tony 163 

van de Ven, Marieke 66 

van de Vrugt, Henri 62 

van de Wetering, Koen 64 

van de Wiel, Bart 102 

van de Ahé, Fina 82 

van Deemter, Liesbeth 64 

van Delft, Foke 115 

van den Belt-Dusebout, Sandra 90 

van den Berk, Paul 60 

van den Boer, Cindy 150 

van den Brekel, Michiel 149, 150 

van den Broek, Bram 28 

van den Broek, Digna 163 

van den Broek, Sandra 43, 90 

van den Haak, Marjolijn 163 

van den Heuvel, Michel 115 

van den Hoven, Jolanda 116 

van der Burg, Eline 45, 66 

van der Donk, Emile 163 

van der Eerden, Paul 150 

van der Gulden, Hanneke 66 

van der Hage, Jos 149 

van der Heijden, Ingrid 66 

van der Heijden, Iris 116 

van der Heijden, Michiel 74, 115 

van der Kammen, Rob 30 

van der Kant, Rik 31 

van der Kolk, Lizet 103 

van der Laan, Elsbeth 117 

van der Leij, Femke 127 

van der Maas, Martin 58 

van der Molen, Lisette 150 

van der Sar, Jana 117 

van der Steeg, Wim 116 

van der Torre, Jaco 87 

van der Velden, Sophie 99, 103 

van der Velden, Yme 88 

van der Wal, Anja 62 

van der Weide, Marchien 115 

van der Wel, Nicole 35 

van Deventer, Sjoerd 31 

van Diessen, Judi 127 

van Dijk, Pim 18 

van Dongen, Miranda 103 

van Dyk, Ewald 72 

van Eggermond, Anja 90 

van Esch, Anita 70 

van Geer, Michael 115 

van Gerwen, Suzan 103 

van Gijn, Roel 115 

van Haaften, Gijs 48 

van Harn, Tanja 62 

van Harten, Wim 101 

van Hasselt, Coen 116 

van Heeteren, Jane 89 

van Hell, Albert 39 

van Herk, Marcel 127 

van Kasteren, Sander 33 

van Kouwenhove, Marieke 48 

van Kranen, Simon 128 

van Leerdam, Monique 115 

van Leeuwen, Fijs 37, 102, 103 

van Leeuwen, Flora 90 

van Leeuwen, Fred 52 

van Leeuwen, Marieke 95 

van Lent, Wineke 101 

van Lohuizen, Maarten 80 

van Luenen, Henri 64 

van Meerbeeck, Jan 115 

van Miltenburg, Martine 66 

van Monsjou, Hester 150 

van Mourik, Anke 128 

van Netten, Gabry 163 

van Nimwegen, Rianne 90 

van Pel, Renee 102 

van Piggelen, Hans 128 

van Rens, Anja 99, 103 

van Rooij, Nienke 56 

van Rossum-Schornagel, Quirine 115 

van Sandick, Johanna 149 

van Son, Rob 150 

van Steensel, Bas 50 

van Tellingen, Olaf 97, 102 

van Thienen, Hans 116 

van Tilburg, Gabrielle 33 

van Tinteren, Harm 162 

van Triest, Baukelien 39, 127 

van Veen, Michiel 26 

van Velthuysen, Loes 102 

van Vliet, Mariska 116 

van Vliet-Vroegindeweij, Corine 127 

van Vugt, Huub 80 

van Waardenberg, Wil 163 

van Waart, Hanna 95 

van Welsem, Tibor 52 

van Werkhoven, Erik 162 

van Winden, Annemieke 116 

van Wouwe, Merian 90 

van Zeeburg, Hester 89 

van Zeijl, Leonie 20 

van Zon, Maaike 35 

van Zweeden, Annette 116 

Vanhoutvin, Steven 163 

Vanneste, Ben 127 

Vargas, Mark 18 

Veenstra, Hidde 150 

Vegt, Erik 102 

Vendel, Brian 127 

Vens, Conchita 39 

Veraar, Elise 54 

Verhagen, Caroline 39, 150 

Verheij, Marcel 39, 127 

Verheus, Martijn 90 

Verhoef, Senno 90, 95, 99, 103 

Verhoeven, Els 80, 103 

Verloop, Janneke 90 

Vermeeren, Lenka 150 

Vermeulen, Eric 90 

Verwaal, Vic 149 

Verwijs, Manon 39 

Verzijlbergen, Kitty 52 

Vidal-Rodriguez, Jordi 76 

Vincent, Andrew 162 

Visser, Daan 28 

Visser, Dick 117 

Visser, Nils 37, 84 

Vissers, Joep 80 

Vlaming, Hanneke 52 

Voets, Erik 78 

Vogel, Celia 84 

Vogel, Wouter 102, 127 

Vollebergh, Marieke 68 

von Castelmur, Eleonora 20 

von Meyenfeldt, Erik 149 

Voncken, Francine 127 

Vos, Matthijn 35 

Vreeswijk, Sandra 128 

Vrijaldenhoven, Suzan 116 

Vroonland, Colinda 102 

Vyth-Dreese, Florry 58 

Wagenaar, Els 70 

Wals, Anneke 163 

Wals, Kim 33 

Wang, Liqin (Bruce) 88 

Wesseling, Jelle 45, 102 

Wessels, Lodewyk 45, 72 

Wessels, Ronnie 149 

Westerman, Bart 80 

Westerveld-de Zwart, Ingrid 103 

Wever, Lidwina 163 

Wevers, Marijke 95, 99 

Weyts, Kathleen 102 

Wielders, Camiel 62 

Wielders, Eva 62 

Wientjens, Ellen 66 

Wieringa-Ariaens, Aafke 103 

Wijdeven, Ruud 31 

Wijnands, Yvonne 163 

Wildeman, Maarten 150 

Willemse, Els 163 

Wilting, Roel 53 

207 

PERSONNEL INDEX

208 


Winter, Marcel 43 

Winter-Warnars, Gonneke 103 

Winterwerp, Herrie 18 

Wit, Niek 60 

Witte, Marnix 127 

Witteveen, Thelma 127 

Wittkämper, Frits 127 

Woerdeman, Leonie 99 

Wojciechowicz-Grzadka, Kamila 62 

Wolthuis, Rob 78 

Wondergem, Marloes 150 

Wouters, Michel 149 

Wouterse, Sanne 128 

Wreesmann, Volkert 150 

Wu, Amy 31 

Xiao, Yanling 54 

Xu, Guotai 64 

Xu, Ning 70 

Xue, Zheng 74 

Yanover, Eva 53 

Yazdi, Amir 103 

Zandbergen, Jeroen 163 

Zander, Serge 64 

Zerp, Shuraila 39 

Zevenhoven, John 82 

Zhang, Hua 128 

Zijp, Lambert 128 

Zimmerman, Marion 117 

Znabet, Anass 33 

Zuur, Charlotte 150 

Zuurmond, Kirsten 104 

Zwart, Wilbert 47, 68

THE 

NETHERLANDS 

CANCER 

INSTITUTE 

SCIENTIFIC 

ANNUAL 

REPORT 2011 



Plesmanlaan 121 

1066 CX Amsterdam 

The Netherlands

Scientific Anual Report 2011 - NKI / AvL

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