SCIENTIFIC ANNUAL REPORT 2002

Transcription

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2 SCIENTIFIC ANNUAL REPORT 2002

3 Patron HM The Queen Beatrix

4 SCIENTIFIC ANNUAL REPORT 2002 TH E N ETH ERLAN OS CANCER I NSTITUTE CANCER RESEARCH LABORATORY AN D CANCER HOSPITAL

5 COPYRIGHT Seientifie Annual Report 2002 Illustrations and unpublished data in these reports may not be used without permis sion of the author. Copyright : The N etherlands Caneer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan ex Amsterdam The N etherlands Phone II1 Fax

6 CONTENTS Board Members Research Divisions Introduction Education in Oncology I Division of Cell Biology 11 Division of Molecular Carcinogenesis 111 Division of Cellular Biochemistry IV Division of Immunology V Division of Molecular Biology VI Division of Tumor Biology VII Division of Molecular Genetics VIII Division of Experimental Therapy IX Division of Radiotherapy X Division of Medical Oncology XI Division of Surgical Oncology XII Division of Psychosocial Research and Epidemiology XIII Division of Diagnostic Oncology Biometrics Departrnent Research Facilities Clinical Trials Invited Speakers Projects Funding Personnel Index

7 Board of Directors AJM Berns, chairman and director of research S Rodenhuis, director clinical research and development W Van Harten, director organization and management BOARD MEMBERS International Scientific Advisory Board J R Be rt in 0, American Cancer Society Professor of Medicine and Pharmacology, Yale University School of Medicine, New Haven, USA RA Flavell, Professor ofimmunobiology, Yale University, New Haven, USA S Hellman, AN Pritzker Distinguished Service Professor, The University of Chicago, Chicago, USA WCJ Hol, Professor of Molecular Biology University of Washington, Biomolecular Structure Center, School of Medicine University of Washington, Seattle, USA J Mendelsohn, President, MD Anderson Cancer Center, The University oftexas, Houston, USA P Nurse, Professor of Microbiology, Director-General of Imperial Cancer Research Fund, London, UK R Nusse, Professor of Developmental Biology Stanford University; Investigator, Howard Hughes Medical Institute, Stanford, USA HL Ploegh, Edward Mallinckrodt, Jr. Professor of Immunopathology, Harvard Medical School, Boston, USA RA Weinberg, Professor of Biology Massachusetts Institute oftechnology, Whitehead Institute, Cambridge, USA C Weissmann, Professor of Molecular Biology University of Zürich, Institut für Molekularbiologie Zürich, Switzerland Board of Covernors WF Duisenberg, president HCJ Van der Wielen, vice-president PJ Kalff, treasurer D Sinninghe-Damsté JHM Temmink GNJ Tytgat PF Van der Heyden J Van der Meer GP Vooys MWM Vos-Van Gortel Scientific Advisory Council AJM Berns, president W Mooienaar, secretary J Neefjes S Rodenhuis H Te Riele L Van 't Veer National Scientific Advisory Board LA Aarden, Professor of Molecular Immunology, Amsterdam SWJ Lamberts, Professor ofintemal Medicine, Rotterdam B Löwen berg, Professor of Hematology, Rotterdam CJLM Meijer, Professor ofpathological Anatomy, Amsterdam CJ MMelief, Professor of Immunohematology, Leiden H M Pinedo, Professor of Clinical Oncology, Amsterdam FH Schröder, Professor ofurology, Rotterdam C N J Tytgat, Professor of Gastroenterology, Amsterdam AJ Van der Eb, Professor of Fundamental Tumor Virology, Leiden PC Van der Vliet, Professor ofphysiologïcal Chemistry, Utrecht

8 ,..' ". q... > _.' ".'. ~... '... j..... ' ~..'.... RESEARCH DIVISIONS I Cell Biology Ed Roos (head) J era Calafat J ohn Collard Kees Jalink Arnoud Sonnenberg 11 Molecular Carcinogenesis René Bernards (head) Roderiek Beijersbergen Anastassis Perrakis Titia Sixma I I I Cellular Biochemistry Wouter MooIenaar (head) Jannie Borst (until 01.IO.2002) Nullin Divecha Peter Ten Dijke Wim Van Blitterswijk Marcel Verheij IV Immunology Hergen Spits (head until 01.IO.2002) Jannie Borst (head from 01.IO.2002) John Haanen Marie Jose Kersten (until ) Ton Schumacher Florry Vyth-Dreese Kees Weijer V Molecular Biology Hein Te Riele (head) Piet Borst (honorary staff member) René Medema Henri Van Luenen Jan Wijnholds VI Tumor Biology Jacques Neefjes (head) Reuven Agami Maarten Fomerod J ohn Hilkens Rob Michalides Peter Peters Martin Van der Valk Rob Verstraeten (stationed at the Free University) VII I Experimental Therapy Adrian Begg (head) Petra Nederlof Jan Schellens Alfred Schinkel Fiona Stewart Marc Van de Vijver Bas Van Steensel Laura Van't Veer IX Radiotherapy Harry Bartelink Berthe Aleman J ose BeIderbos Anja Betgen Liesbeth Boersma Eugenè Damen RoeI De Boer J osine De Bois Katrien De Jaeger Luc Dewit Riek Haas Guus Hart Wilma Heemsbergen Edwin Jansen Joos Le besque Harry Masselink Ben Mijnheer Tom Minderhoud Luc Moonen Kenneth Pengel Coen Rasch Peter Remeijer Maddalena Rossi Nicola Russell Govert Salverda Christoph Schneider Bob Smulders Joep Stroom Bart Van Bunningen Peter Van de Ven Marcel Van Herk Marcel Verheij Fritts Wittkämper Lambert Zijp VII Molecular Genetics Peter Demant (head until ) Maarten Van Lohuizen (head from ) Anton Berns Paul Krimpenfort Margriet Snoek

9 X Medical Oncology Sjoerd Rodenhuis (head) Joke Baars Paul Baas Evert Bais Jos Beijnen Willem Boogerd Henk Boot Annemieke Cats Jan Paul De Boer Bert De Gast John Haanen Martijn Kerst Marie J ose Kersten Herman Neering Jan Schellens Jan Schornagel Babs Taal Wim Ten Bokkel Huinink Jaap Van der Sande Marchien Van der Weide Nico Van Zandwijk Ans Vielvoye-Kerkmeer XI Surgical Bin Kroon (board; head) Fons Balm (board) Omgo Nieweg (board) Axel Bex Dick Buitelaar Matthé Burger Myrtille De Vries Steven Gonggrijp Joris Hage Frans Hilgers Simon HorenbIas Hans Huitink Houke Klomp Frans Kroon Wim Meinhardt Hester Oldenburg May Ronday Emiel Rutgers Peter Schutte BingTan Adriaan Timmers Marc Van Beurden Frits Van Coevorden Michiel Van den Brekel Henk Van der Poel N ine Van der Vange Arnold Van Lindert Hans Visscher Leonie Woerdeman Frans Zoetmulder XI I I Diagnostic Oncology Marc Van de Vijver (head) Peter Besnard Hans Bonfrer Daphne De Jong Kenneth Gilhuijs Cees Hoefnagel Frans Hogervorst Elisabeth J oekes Wim Koops Robert Kröger Wolter Mooi Saar Muller Petra Nederlof Willem Nooijen Frank Pameijer Hans Peterse Leo Schultze Kool Ferida Sivro Renato Valdes Olmos Hester Van Boven OlafVan Tellingen Laura Van 't Veer Loes Van Velthuysen Senno Verhoef Biometncs Department Otilia Dalesio (head) Harm Van Tinteren HEADS OF GENERAL RESEARCH SERVICES Audiovisual Service Martin Lomecky Central Cancer Library Suzanne Bakker Financial Administration Bart Simmelink General Facilities Robert Clement Laboratory Animal Department Marco Breuer Rob Ten Berg Research Coordination Wouter MooIenaar, laboratory research coordinator Henri Van Luenen, laboratory research manager Egbert Vos, clinical research manager XI I Psychosocial Research and Epidemiology Neil Aaronson (head) Matti Rookus Frits Van Dam Floor Van Leeuwen

10 Director of Research Ton Berns INTRODUCTION I am pleased to present to you our Scientific Annual Report. It provides an overview of the scientific activities at the NKljAvL. Additional information may be found on the websites of the various divisions, as weli as at The Netherlands Cancer InstitutejAntoni van Leeuwenhoek Hospital (NKljAvL) is a Comprehensive Cancer Center, combining hospital and research laboratories under one roof in a single independent organization. The hospital comprises 180 beds, a large radiotherapy department and outpatient clinics. Facilities for patient research include a large patient database, clinical data management and active research groups in epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on mouse tumor modeis, mouse (reverse) genetics, celi biology, immunology and translational research requiring close collaboration between clinical and basic scientists. This scientific report deals mainly with the clinical and basic science in the NKljAvL. Information on patient care is described in our General Report. In 2002, our new hospital and radiotherapy unit began to take shape and the building activities are now moving from outside to inside. We are roughly on schedule which is an achievement in itself. We expect that this will also hold for the subsequent phases of the building activities. We expect to open the new hospital in the beginning of The renovated research facilities will become available several years later. This constitutes a major upgrade and will be a tremendous improvement for our patients and employees. The major deficit threatening our clinical activities in recent years has been successfully averted through renegotiation of our budget on the basis of the output of the hospital and acquisition of academie status. Shortage ofhospital personnel has prevented us from substantial growth in We have been forced to limit the number of patients admitted to prevent unacceptable delays between diagnosis and treatment. Although the job market is becoming less strained in general, this is not the case for hospital personnel. Hospitals in large cities are particularly hurt as they do not receive funding to compensate their personnel for more expensive housing or longer commutes. However, measures have been taken to make the NKljAvL a more attractive place to work. We expect that this wili allow us to reduce the shortage in personnel we are currently encountering. Fortunately, a shiny building is not a prerequisite for good research. Research can also thrive in an old overcrowded building which is now the case. I provide a selection of research highlights below. We are accelerating the development of our highly promising micro-array diagnosis for breast cancer patients. Micro-array analysis of tumors permits a considerably more accurate prediction of disease progression than has been possible so far. We are striving to make this technology available to patients as soon as possible. To achieve this, we have rented space from the nearby Slotervaart hospital. We envisage that this facility wili be transferred to a commercial entity that is better suited to further develop and offer such a service to cancer patients in and outside the NKljAvL. Two of our staff members will be actively involved in founding this company. Highlights The scientific output of the institute has been very substantial making it impossible to mention all the research conducted. Here follows a selection of items illustrating our progress. Ed Roos and colieagues (Division of Cell Biology) showed that the chemokine receptor CXCR4 is essential for the outgrowth of colon carcinoma metastases. Thus, existing CXCR4 antagonists may have potential for cancer therapy, in particular to suppress development of metastases from celis that have already disseminated. J ohn Coliard and colleagues, of the same division, found that Tiaml-knockout (Tiaml-/-) mice are resistant to the ab 1 Research funding NKljAvL from the different sources in period in million Euros Year Dutch Cancer Society Ministry of Health Project Grants Other income Total

11 development of carcinogen-induced skin tumors. Moreover, the few tumors produced in Tiam1-J- mice grew more slowly than tumors in wild-type mice. Although Tiam1-J-m ice developed very few tumors, a greater proportion of these progressed to malignancy, suggesting that TiamI-deficiency promotes malignant conversion. These in vivo data identify the Rac activa tor TiamI as a critical regulator of Ras-induced tumorigenesis. By using TiamI-deficient cells, Wouter MooIenaar and coworkers (Division of Cellular Biochemistry) established that TiamI is responsible for Rac activation by the lipid growth factor LP A; this solves the long-standing question ofhow LPA promotes Rac-mediated cell migration and invasion. The group of Peter Ten Dijke (Division of Cellular Biochemistry) found that TGF-~ in endothelial cells signals via two distinct receptors ( ALKI and ALK 5), one of which (ALK5) inhibits cell migration and proliferation whereas the other (ALKI) stimulates both processes. René Bemards and colleagues (Division of Molecular Carcinogenesis) successfully continued their in vitro screens and identified new genes that act in the RB and P53 tumor suppressor pathways. Daniel Peeper, until recently member of the same division, found that E2F repressors, established targets for the pi6 IN l<4a_prb pathway, are also critical targets for both PI9ARF and P53 during the induction of cell cyele arrest and senescence. These data predict that E2F-dependent transcriptional repression is deregulated in the vast majority ofhuman tumors. Thijn Brummelkamp and Reuven Agami (Division of Molecular Carcinogenesis and Division of Tumor Biology) developed a vector that mediates sta bie suppression of gene expression through persistent RNA interference. The construction of a set of some 25,000 'knockdown' vectors is in progress. This vector set will make it possible to carry out large-scale screens for recessive phenotypes in mammalian cells. René Medema and collaborators (Division of Molecular Biology) have identified, in collaboration with the group of Boudewijn Burgering (UMC, Utrecht), critical regulators that couple cell proliferation to protect against oxidative damage. This work demonstrated that tumor formation is coupled to changes in cellular metabolism and explains the glucose-dependency of tumors. This may help in developing new therapeutic strategies. The group of Hein Te Riele (Division of Molecular Biology) found that a single mismatch in a genetargeting vector can already activate mismatch repair directed antirecombination imposing an impediment to targeted gene modification. Following up on this observation, they found that in ES cells lacking the central mismatch repair protein MSH2, single-stranded DNA oligonueleotides can be used to site-specifically modify, dele te or insert a single or a few base pairs in the genome. 'Oligo targeting' was sufficiently effective to all ow elonal purification of modified cells using PCR based screening procedures and may therefore provide a novel route to the generation of mutant mouse strains. Rob Michalides (Division of Tumor Biology) showed that the effectiveness ofhormone receptor interactions can be accurately followed by FRET. This makes it possible to predict whether receptors will be refractory to tamoxifen treatrnent, but also provides a fast method to screen for compounds that retain downregulatory activity towards the estrogen receptor. Combined efforts of the groups of Ton Berns and Maarten Van Lohuizen (Division of Molecular Genetics) have illustrated the power of large scale insertional mutagenesis in mice with specific cancer predispositions, making optimal use of the now sequenced mouse and human genomes. HaraId Mikkers and coworkers (Berns) have applied 'complementation tagging' in Eu-Myc transgenic mice that are deficient for pimi and pim2 kinases, whereas Anders Lund and coworkers (Van Lohuizen) have performed insertional mutagenesis in mice deficient for the INl<4a/Arf cancer protection mechanism. The combined results will be made available through the Ensembl public domain database and will provide a new 'genomics' tooi for cancer gene discovery The group of Peter Demant (Division of Molecular Genetics) eloned SCCI (Susceptibility to colon cancer-i) gene of the mouse. It encodes a protein tyrosine phosphatase Ptprj. This is the first cancer Quantitative Trait Locus affecting tumorigenesis which has been positionallyeloned. It appears to be haplo-deficient in many human tumors, indicating that it is a tumor suppressor gene with a substantial contribution to the tumorigenic process. Bas Van Steensel and coworkers (Division of Experimental Therapy) used chromatin profiling to show that distinct heterochromatin complexes bind to specific chromosome locations in a developmentally controlled fashion. This is the first demonstration that these proteins indeed fulfill a function in the co-regulation of large sets of genes. Using BCRP/ABCG2 knockout mice Alfred Schinkel and colleagues (Division of Experimental Therapy) found that this A TP binding cassette drug transporter is an important factor in protecting against dietary toxins. For instance, BCRP / ABCG2 knockout mice are at least IOo-foId more sensitive to phototoxic damage caused by pheophorbide a, a breakdown product of chlorophyll that occurs to variabie extents in many plant-derived animal and human foods and in 'natural food supplements'. BCRP / ABCG2 efficiently prevents the uptake of pheophorbide a from the intestinal contents, illustrating the importance of this type of transporter in protection from environmental and dietary toxins. Hergen Spits and colleagues (Division of Immunology) succeeded in immortalizing human B cells by overexpressing a constitutive active mutant of the transcription factor STAT5b, or its direct target Bel-6. This opens up important possibilities for in vitro antibody production using antigen-specific B cells from human donors.

12 I-. " '-'.' -' "~~" ', - ] '.. _~ I Laura Van 't Veer and Marc Van de Vijver (Division of Diagnostic Oncology) continued their work on expres sion proflling ofbreast cancers. The power of the 70- gene classifier was confirrned in a larger group of patients, which now also included patients with tumorpositive axillary lymph nodes. The data support the notion that metastatic capacity is deterrnined by genetic lesions occurring early on in the tumorigenic process. The 7o-gene classifier is currently the strongest prognostic feature available in breast cancer and dramatically outperforms any other prognostic markers in current clinical use. This could eventually lead to a marked decrease in the number of early breast cancer patients who require adjuvant chemotherapy. The group of Jan Schellens (Division of Medical Oncology) has previously shown that gut epithelial BCRP (Breast Cancer Resistance Protein) is at least in part responsible for the poor absorption of topoisomerase inhibitor Topotecan from the gastrointestinal tract. The resulting low bioavailability precludes oral administration of this agent. However, the absorption can be greatly increased in patients if they receive the BCRP blocker Elacridar (GF ). In this way, oral administration oftopotecan becomes a practical and well-tolerated route of adrninistration. In 2002, the first complete analysis of the Dutch national trial ofhigh-dose chemotherapy in the adjuvant treatrnent ofbreast cancer was completed. This study was designed, coordinated and analyzed in our institute but it was conducted in all Dutch university hospitals and a single large regional hospital. lts ma in findings are (I) that high-dose chemotherapy is associated with a statistically significant improved relapse-free survival in patients with 10 or more axillary lymph node metastases, and (2) that patients with HER-2/neu positive tumors do not benefit from high-dose chemotherapy and may even have a worse outcome. Frans Zoetrnulder and colleagues (Division of Surgical Oncology) prepared the final analysis of the prospective randomized study in which the efficacy of HIPEC (aggressive surgery for peritoneal carcinosis followed by hyperthermic intraperitoneal chemotherapy) is compared with chemotherapy alo ne in colorectal cancer with peritoneal metastases. The progression-free survival in the experimental treatrnent group was significantly higher than that in the control group. One of the major innovations in oncologic surgery of the past five years is the sentinel node biopsy, which is now regarded as standard by many centers in the treatrnent ofbreast cancer. Sentinel node biopsy may be useful in several other tumor types as weil. The urology group was able to show that sentinel node biopsies can detect about 80% of occult lymph node metastases in penile cancer as weil. This could further improve the treatrnent results in this rare disorder and could prevent useless and of ten bilaterallymph node dissections in this patient group. Berthe Aleman (Division of Radiotherapy) demonstrated that involved field radiotherapy did not further improve the survival rates of patients with advanced Hodgkin's lymphoma in complete remission af ter chemotherapy. However, adding involved field radiotherapy to patients in partial remis sion resulted in similar survival rates as patients who obtained complete remis sion after chemotherapy. Rick Haas, of the same division, demonstrated that low dose radiotherapy for patients with follicular lymphomas was very effective in obtaining long lasting remissions. This unexpected effect oflow dose radiotherapy in these lymphomas is probably the result of irradiation-induced apoptosis. The synergy between research and clinical divisions was also demonstrated by the development of clinical applications in the physics section. Models have been developed for irradiating tumors while correcting for movement of the target area as a re sult of, for example, breathing or bladder ruling. Advanced IMRT techniques for treatrnent of prostate cancer, and ofhead and neck tumors have been developed, tested and clinicaily implemented. For the treatrnent ofleft-sided breast tumors, an IMRT class solution using predefined segments was designed, which resulted in a reduced late onset cardiac mortality. Quality of research The quality of research is monitored in several ways. Our scientific productivity as based on bibliometric parameters (citations and impact of scientific articles published by the NKI staft) has shown a steady increase since the beginning of the eighties. In the last years the number of citations and impact are leveling off (TabIe 2), with yearly fluctuations, indicating that we have reached b Short-term citations and impact of scientific articles published by the NKI research staff Publication year Citations Impact 1: II

13 a steady state. At the same time, the number of There were also changes in our staff. Hergen Spits, publications in high impact journals is rising, making it he ad of the Division of Immunology, left to become worthwhile to compare our bibliometrie parameters with Professor of Ceil Biology at the AM C in Amsterdam. those of other academie institutions. One specifk Peter Demant, head of the Division of Molecular illustration of the high impact of our research came Genetics, left to become staff member at Roswell Park, from a recent bibliometrie study on chemistry research. Buffalo, USA to continue his research on tumor Although only a fraction ofthe research atthe NKljAvL susceptibility genes. We wish both much success in their fails into the area of chemistry research, the NKI excels new positions. J annie Borst, staff member of the with more than a three-fold higher impact than the Division of Cellular Biochemistry has taken the position world subfield average. of Hergen Spits as he ad of the Division of Immunology and Maarten Van Lohuizen has been appointed as head Our competitiveness in obtaining grants is another of the Division of Molecular Genetics. Wouter measure of quality. We have been quite successful Mooienaar succeeded Ada Kruisbeek, who left at the during the last decade in obtaining grant support, scoring on average 2-3 fold better than our competitors as measured on the basis of Dutch Cancer Society grant Table 3 applications. For 2002 our scores in obtaining grants The number ofnkljavl articles published in journals from the Dutch Cancer Society has been above average with Impact Factor 2001 of 10 or higher over the last but not the 2-3 fold of previous years. Moreover, we have 10 years. won a number of major grants from other sources, including Pioneer (Titia Sixma), Program (Bas Van Steense!, Ton Berns and Maarten Van Lohuizen) and total VIDI grants (Daniël Peeper and Jos Jonkers) from NWO, '92 - Integrated Projects from the EU, and support from the IFoI Joumal '92 '98 '99 '00 'Ol 'Ol Netherlands Genomics Initiative. Our competitiveness is well reflected in a more than 25% increase in project 3 0 Nat Genet funding in 2002 as compared to 2001, reaching an all 22 Curr Opin Ceil Biol time high of 16-4 million Euro. 21 Gene Dev J Exp Med The third measure of quality is based on extern al site II Ann Intern Med visits, in which international leaders in a particular field 29 Cell of research review the work of a division or research 29 N Engl J Med I. 7 groups with a similar theme, on a quinquennial basis. 28 Nature These site visits require that group leaders prepare a 28 Nat Med report and reflect on their research activities. This by 23 Science itself appears very beneficial as it helps focus the 23 Adv Immunol research. Two site visits were held in The Division 19 Immunity of Cell Biology was visited by Benjamin Geiger 18 Trends Ceil Biol (Weizmann Institute, Rehovot), Richard Hynes (MIT, 18 JAMA Cambridge) and Alan Hall ( MRC, London). Martin 17 Nat Immunol Brown (Stanford University, USA), Adrian Harris 17 Mol Cell (Oxford, UK), and Branimir Sikic (Stanford University, 15 Nat Ceil Biol USA) evaluated the Division of Experimental Therapy. 14 Trends Biochem Sci The site visitors we re overall very positive ab out both 14 J N atl Cancer Inst divisions, but also indicated some weak spots and made 14 J Clin Invest a number of suggestions for improvement, that are 14 Curr Opin Genet Dev currently being implemented. 13 Lancet Gastroenterology Honors and appointments 13 J Cell Biol The NKljAvL cannot award university degrees. 12 EMBOJ However, many of our staff members hold special 12 Trends Genet part-time chairs at Dutch universities. This facilitates 12 Immunol Today the supervision and awarding of degrees to graduate II P Natl Acad Sci (USA) students receiving their training at The Netherlands II Curr Opin Struct Biol Cancer Institute. In 2002, Simon Horenbias was II Am J Hum Genet appointed Professor of Urology at the Free University in 10 Nat Struct Biol I Amsterdam. Piet Borst was awarded an honorary 10 Mol Cell Biol doctorate of the University of Leiden and I received the 10 BBA Rev Cancer prestigious AKZO-Nobel Science Award 2002 for my own work

14 beginning of 2002, as Laboratory Research Coordinator. We also recruited new staff. Heinz Jacobs joined the Division of Immunology as a tenured staff member. Bas Van Steense!, Daniël Peeper, and Jos Jonkers joined the institute as AvL fellows. Staff of the NKIjAvL fulfilled numerous functions in national and international organizations, on the scientific boards of scientific journais, as members of study sections, as organizer or co-organizer of scientific meetings, workshops and congresses. financial support of the government, the Dutch Cancer Society and of many individuals. Only with their help can we continue to develop new ideas that should lead to prevention, early detection, and more effective treatment of cancer. We hope they will feel encouraged by this report. Ton Berns Director of Research Outlook and acknowledgernents I am confident that research at the NKljAvL win continue to flourish, if adequate funding is assured. The cost of research rises steeply while at the same time we are facing a tight financial situation due to the current state of the economy. The shortage of qualified workers in the clinic and in research in general have evoked a substantial raise in wages and indirect costs not matched by an increase in our basic funding. Although our staff managed to acquire substantially more project funding, this funding rarely covers overhead cost. Consequently, the substantial increase in project funding augments rather than alleviates the pressure on our basic research budget. Therefore, we need to secure additional financial support. Funding from the Ministry of Health is especially lagging (see Table I). Although this is recognized and acknowledged by the Ministry, measures to correct this have not been taken as yet. A second concern deals with the recruitment of young talent to the institute. Recruiting post-docs in particular has become much more difficult. Although all academic institutions suffer from this problem, the NKIjAvL is affected more seriously as we have fewer staff to run our research programs and therefore rely more heavily on the expertise of post-docs. We will step up our PR efforts to help potential candidates for student and post-doc positions become more aware of the unique opportunities the NKljAvL offers for a career in research. Last but not least, we are slowed down by and have to assign increasing manpower to deal with unnecessary restrictive governmental regulations, and although there is an increasing awareness of this problem, there is little perspective of irnprovement. So far we have been unsuccessful in convincing our politicians that money is better spent on actual research than on 'red tape'. The Board of Directors will increase its efforts to negotiate for more appropriate funding parameters for our basic research. Both government and the public have an interest in a dedicated internationally renowned cancer institute and we hope to reach agreement on a funding system that meets the needs of an institute striving for excellence. Finally, we are much obliged to those who have supported us. For all our research we dep end on the

15 EDUCATION IN ONCOLOGY Theoretical and practical training in research are regular activities in the institute. Several senior staff members have joint appointrnents as professors at Dutch universities and contribute to the regular curriculum at these universities. In addition, many staff members teach courses for undergraduate and graduate students, either within the institute or at universities. The research departrnents attract graduate students from universities throughout the country, who contribute to ongoing scientific projects and receive in-house theoretical training. The NKI has a formal affiliation with the Science Faculty of the University of Amsterdam and is committed to contributing to courses for graduate and undergraduate students of this university. The institute participates in the Oncology Graduate School Amsterdam, together with the University of Amsterdam (UvA) and the Free University (VU). Most graduate students are assigned to this graduate school, for a four-year period. These graduate students make significant contributions to the scientific program. In addition they receive practical and theoretical training within the institute. The medical staff is actively involved in medical and surgical oncology fellowshipprograms, teaching programs for undergraduate medical students and (inter)national postgraduate courses. The departrnents of Surgical Oncology and Head & Neck Ta Course in Experimental Oncology, fall 2002 Part I Treatise on 'Cancer Biology' by RJ King R Michalides, J Hilkens Part 2 Will we cure cancer? Epidemiology Signal transduction'" Apoptosis* Cell adhesion* Cell cycle* Cell senescence>'< Genomic instability* Tumorigenesis'" Radiodiagnostics Gene array** Pathology**, Molecular Diagnostics* Surgery Conventional chemotherapy Radiothera py* o NA damage response Immunology, Immunotherapy>'< Rational drug design and delivery* Protein structure J Borst F Van Leeuwen W MooIenaar, P Ten Dijke J Borst E Roos R Bemards R Beijersbergen H Te Riele M Van Lohuizen, A Berns, J Jonkers SMuller R Kerkhoven o De Jong, M Van de Vijver, P Nederlof B Kroon J Schornagel M Verheij A Begg T Schumacher, J Haanen J Beijnen, A Schinkel T Sixma * including tutorial ** including tour T, e 2 Teaching committee President and Dean U ndergraduate School: General affairs: Publicity, tours, coordination HLO placements: Experimental Oncology course: Clinical teaching: Dean Oncology Graduate School Amsterdam (OOA): J Borst H Te Riele J Hilkens, J Collard J Borst, R Michalides, H Te Riele AJM Balm J Neefjes

16 I,'.:: '. '.',.:~~'.,.~, ',:.:.. ~':.~'- v:: ;:.",,:;;,.,,~.,'. '.~17<; '" Participants of the OOA retreat Oncology participate in the residency-training program of the Academic Medical Center Amsterdam. Lectures are read by staff members for nurses in training for their oncology certificate. Undergraduate students The undergraduate program in Experimental Oncology attracts students of all national universities in partial fulfillment of the obligations of their curriculum. Students have a background in (Medical) Biology, Health Sciences, Chemistry, Pharmacology, Medicine, or Psychology. The undergraduate program offers combined practical and theoretical training in various aspects of experimental oncology. Practical training includes participation in ongoing research projects for a minimum of four months, af ter which the student delivers or al and written accounts of the results obtained. In 2002, 29 university students completed a placement of 5-12 months at research divisions of the NKI. The majority of these students came from Amsterdam universities (UvA and VU) and Utrecht University; others from Wageningen, Groningen, Leiden and Maastricht. The institute also provides practical training opportunities to trainee technicians. In 2002, we opened our doors for the first time to a number of third year Medical Biology students of the UvA, for a 7-week rotation. Many of these enthusiastically chose to do a subsequent long-term placement in the institute. The co re element of theoretical training is the course in Experimental Oncology, given twice a year by staff members of the institute (Tabie 1). This course includes lectures and tutorials, as well as a treatise on the book 'Cancer Biology' by RJ King (especially for third year Medical Biology students of the UvA). The Teaching Committee (Tabie 2) supervises all educational activities. Specialized tasks include organization of the lecture course, guided tours to research departments, contact with technician training schools and promotional activities at universities. Graduate students Approximately 120 graduate students at the NKI participate in the Graduate School 'Onderzoekschool Oncologie Amsterdam' (OOA). The OOA is a collaboration between the NKI and the medical faculties of the Free University (VUMC) and the University of Amsterdam (AMC). The graduate students are scientists in-training who receive additional theoretical and practical education on various subjects related to cancer. Following a successful scientific training, graduate students can acquire their PhD degree at one of the national universities. The OOA organizes various activities. All graduate students are tutored by a committee comprised of the project leader, the head of the division and one or two academic staff members from other divisions. They meet once a year to evaluate scientific progress and problems. A graduate committee is installed to discuss graduate subject matters related to the educational program and scientific problems with the Dean of the OOA. As a result of these discussions, graduate students now host 'Special Seminars'. Moreover, a course on 'Writing and Presentation in English' has been organized and special courses for graduate students of the Division of Psychosocial Research and Epidemiology have been organized. The special seminars are organized such that graduate students first discuss a paper selected by the invited speaker, follow the seminar and finally have a small group discussion with the invited speaker. This year about 26 special seminars were organized. The OOA organizes a yeady retreat for graduate students, which they must attend three times during their graduation studies. First year graduate students usually present the goal and concept of their project in a poster session, graduate students oflater years will give an oral presentation. This year's retreat was organized on Texel, with 105 participating graduate students (80 from the NKI).

17 Table 3 Courses at the OOA 2002 April 2-5 Anatomy of the mouse Genomic instability in cancer WH Lamers H Te Riele June 12-14, In the footsteps of Antoni van Leeuwenhoek G Meijer, P Peters, RJC Van Noorden September October 19-II December 2,9 Quality of life assessment in clinical research and clinical practice Annual graduate student retreat Trends in Tumorimmunity N Aaronson J Neefjes, M Vd Velde RJ Scheper The OOA organizes various courses for graduate students. Graduate students must attend 3-4 courses during their PhD program. Courses are usually restricted to ab out graduate students to ensure optimal scientific interaction. Courses are advertized on the web page of the OOA and announced to every graduate student by .

18 I DIVISION OF CELL BIOLOGY ADH ESION M ECHAN ISMS IN M ETASTASIS The spread of tumor to distant sites is the main reason why cancer is such a deadly disease. Our aim is to elucidate the mechanisms underlying this process. Our research focuses on signaling pathways triggered by chemokine receptors and integrin adhesion molecules in lymphomas and carcinomas. These pathways regulate migration of tumor cens, required for invasion from the blood into tissues such as the liver or the lungs. For colon carcinoma, we have recently found that the chemokine receptor CXCR4 is required for outgrowth of metastases rather than invasion. This phenomenon is now being studied as wen. The chemokine receptor CXCR4 is essential for outgrowth of colon carcinoma micrometastases To assess a role for the chemokine receptor CXCR4 in CT-26 colon carcinoma cens, we transfected the CXCR4ligand SOF-I, extended with a KOEL sequence. The SOF-KOEL fusion protein is retained in the ER by the KOEL-receptor. There, it binds CXCR4 which is therefore also retained and never reaches the surface. This lack of surf ace CXCR4 almost completely prevented metastasis in the liver and lungs from cens injected into the spleen or the tail vein, respectively. In the spleen, the cens did grow. On control CT-26 cens, CXCR4levels in vitro we re very low, but greatly upregulated in vivo (see Fig. 1.1). This argues against a role for CXCR4 in the initial invasion because tail vein-injected cens do not express it. Moreover, inhibition of CXCR4 migration signals by transfected pertussis toxin, which bloeks lymphoma metastasis, had no effect on the colon carcinoma. Comparable numbers of control and CXCR4-deficient cens colonized the lungs and both remained quiescent for at least six days. Control cens expanded rapidly thereafter, whereas proliferation of CXCR4-deficient cens was very limited. We isolated cens from lung tissue by F ACS sorting for G FP. Two days af ter injection, 20% showed substantial CXCR4 expres sion, and 40% af ter seven days. Ex vivo, CXCR4 was downregulated within two days. Thus, the increase in CXCR4 was not due to selection of cens with constitutive expres sion. It is more likely that CXCR4 is induced by the in vivo environment, possibly by factors derived from host cens in lung and liver tissue (see Fig. 1.2). Synaptotagmin: role in chemotaxis? Lymphoid cens react rapidly to chemokines, probably within a second. We hypothesized that this involves rapid fusion ofvesicles with the plasma membrane, to insert the required signal machinery. In the nervous system, synaptotagrnins regulate calcium-triggered regulated fusion of docked synaptic vesicles. The isolated C2B domain of synaptotagmin can inhibit this fusion. T-Iymphoma cens express synaptotagmin-3, which is triggered by low calcium levels. Transfected C2B completely inhibited rnigration towards SOF-I as wen as SOF-I-dependent invasion into fibroblast monolayers. In contrast, a C2B mutant that still binds calcium but does not inhibit fusion, had no effect. We are presently attempting altemative approaches to confirm the involvement of synaptotagmin and are exploring its possible role in other cen types. C-protein and integrin signaling during Iymphoma metastasis: role of Jak2 Invasion by T-lymphoma cens is initiated by CXCR4 signals triggered by SOF-I, that cause polarization and movement of cens but also activation of the integrin adhesion molecule LFA-I. Interaction with the LFA-I ligands ICAM-I or -2 then elicits signals that are also required for invasion. The Jak kinase inhibitor AG490, but not aspecific Jak3 inhibitor, blocked invasion, indicating that Jak2 is involved. LFA-I-independent chemotaxis towards SOF-I is not affected by AG490. Thus, we found no role for Jak2 in the CXCR4 signal, in contrast to reports by others. Instead, J ak2 is involved in the LFA-I signal. Attempts to confirm this by stabie expression of constructs that should inhibit Jak2 failed because constitutive Jak2 activity is apparently required for survival and proliferation of the lymphoma cens. We have now circumvented this problem by expres sion of constitutively active variants ofpi3-kinase and STAT3. This should Division head, Group leader Ed Roos Ed Roos PhD Group leader Belén Alvarez Palomo PhD Post-doe Frank Opdam PhD Post-doe Peter Stroeken PhD Post-doe Ingrid Zeelenberg MSc Graduate student Rosalie De Bruijn Technical staff Marga Kamp Technical staff Lisette Ruuls-Van Stalle Technical staff Yvonne Wijnands Technical staff Natasja Borsje Secretary CT26 colon carcinoma in culture CT26 cells trom liver metastases ~~I;~ ''', 01 CT26 cells : :~,~ d'~ trom lung : '\ " \ metastases ~I ~ ~ t CXCR4 on surface Fig. 1.1: Expression ofthe chemokine receptor CXCI4 is low on cultured CnG colon carcinoma ceus, but strongly upregulated in vivo

19 Selected publications Zeelenberg IS, Ruuls-Van Stalle L, Roos E. The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res, (in press). enable us to test J ak2 inhibitors as wen as a possibly relevant substrate containing an ITAM which, upon phosphorylation by Jab, could bind ZAP-70. PI-3-kinase (p8s/pllo) is required for T-Iymphoma metastasis In neutrophils, PI3K (phosphatidylinositol-3-kinase)-gamma is essential for SOF-r-triggered chemotaxis, but PI3K inhibitors had no effect on the T-Iymphoma eens. In contrast, invasion was inhibited by -50%, both by PI3K inhibitors and by a transfected dominant-negative (ON) mutant ofthe p85 subunit ofpi3k-alpha. These reagents completely blocked Mn 2 +- or PMA-induced adhesion to surfaces coated with ICAM-r at low, but not at high ICAM-r density. Thius, p85/prro PI3K amplifies the LFA-r signal and this is only required when ICAM-I is limiting. The metastatic capacity of the cells transfected with ON-p85 was strongly reduced, suggesting that expres sion levels oficam-i and -2 in tissues such as liver and kidney are in fact limiting, so that the effect ofpi3k inhibition in vivo is more pronounced than in the fibroblast invasion assay in vitro. The lil integrin-binding protein ICAP-l interacts with Rho kinase: role in migration? We previously identified BI integrin mutants that could not mediate Br-dependent invasion. These mutants did not interact with the bi-binding protein ICAP-I, prompting us to study the role oficap-i in migration. Yeast two hybrid analysis revealed that ICAP-I binds to the Rho-associated kinase ROCK. This was confirmed by co-immune precipitation from transfected COS cells and colocalization in ruffles. In C2CI2 myoblast cells, overexpression oficap-i promoted serum- and HGF-induced migration, in particular on laminin. ICAP-I overexpression also led to reduced spreading on laminin. The HGF-induced migration is blocked by a ROCK inhibitor. These results suggest that the binding oficap-r to ROCK promotes ROCK-dependent migration. Currently, we are attempting to blo ek the interaction of ICAP-I with ROCK in cells by transfecting fragments ofkinase-dead ROCK containing the putative binding sites. This should provide further evidence for this hypothesis. Hypothesis: role SDF-1/CXCR4 in outgrowth carcinoma micrometastases Carcinoma cell CXCR/SDF-1 Carcinomaderived factors? ~ Stromal cell Induction of genes involved in growth and survival Fig. l.2: We propose that stromal cells produce factors that trigger synthesis of CXCR4 in carcinoma cells, perhaps after being stimulated by tumor cell-derived factors. Next, interaction ofthis CXCR4 with SDF-l, also produced by host cells, induces transcnption of genes involved in survival and prolifèration

20 GENETIC CONTROL OF INVASION AND METASTASIS The aim of our research is to identify and eharaeterize genes whieh play an essential role in the acquisition of an invasive and metastatic phenotype of tumorigenie eens. Insight into the signaling pathways involved may lead to the development of novel diagnostic tools or improved therapeutic strategies. Rho family proteins control dynamic cytoskeletal changes Rho-like GTPases control a wide range of biologie al processes. In partieular, they act as key molecules in signaling pathways that regulate the reorganization of the actin cytoskeleton in response to receptor stimulation and thereby determine the morphology, adhesion and motility of eens. Our recent studies implieate Rho-like GTPases in invasion and metastasis of tumor eens. Earlier we identified the invasion-inducing Tiamr gene, whieh encodes an activator (GEF) for the Rho-like GTPase Rac. Overexpression of Tiamr in fibroblasts causes extensive membrane ruffling, similar to the effects indueed in VI2RaCI-expressing cens. By pull-down assays, we eonfirmed that Tiamr regulates the aetivity ofrac. PI3-kinase aetivity is required for Tiamr-mediated Rae activation. Produets ofpi3-kinases, sueh as PIP3, bind to the N-terminal Pleekstrin Homology (PH) domain oftiamr, whieh is required for membrane localization of the protein. Only membrane-ioealized Tiamr is threonine-phosphorylated and is able to aetivate Rae, suggesting that phosphorylation events mayalso play a role in activation and/or loealization oftiamr. Tiamr also interacts direetly with aetivated Ras in a PI3- kinase independent way, providing an alternative link from activated Ras towards Rac. Group leader John Col/ard John Collard PhD Group leader Angeliki Malliri PhD Post-doe Cristina Olivo PhD Post-doe Leo Price PhD Post-doe Rob Roovers PhD Post-doe Saskia Van Es PhD Post-doe Amra Hajdo-Milasinovic MSc Graduate student Sander Mertens MSc Graduate student Stephan Huveneers Undergraduate student Kris Clark Technical staff Rob Van der Kammen Technical staff Rho-like GTPases and invasion of T-Iymphoma cells Similar to Tiamr, expres sion of eonstitutively aetive Vr2Rae induees an invasive phenotype in T-Iymphoma eens. Aetivated Vr2CdC42 also induees invasion oft-lymphoma eens, which is not eaused by CdC42-mediated activation of Rac. Aetivated Vr4RhoA potentiates invasion but fails to mimic Rae and CdC42. Expression oftiamr and Vr2RaCI promotes integrin-mediated adhesion to various substrates. Additional screens for genes involved in T-Iymphoma invasion revealed a number of novel genes implieated in integrin-mediated een-matrix adhesion or signaling by Rho-like GTPases. For these screens, retroviral edna libraries were used in combination with in vitro selection of invasive T-lymphoma eens. LPA in Rac and Rho signaling Lysophosphatidic acid (LPA) is a serum-borne lipid and is required for T-lymphoma invasion. In addition to stimulating Ras-mediated eeli proliferation, LPA also promotes Rac- and Rho- mediated signaling pathways involved in tumor een migration. We found that the prototypie LPAr/Edg2 receptor, in addition to transiently activating RhoA, also mediates prolonged activation ofrac. LPA-indueed Rac activation is inhibited by pertussis toxin and requires PI3-kinase aetivity. Epithelial and fibroblast eens that laek Tiamr fail to aetivate Rac in response to LP A, while enforced expres sion oftiamr restores LPA-indueed Rae activation. These data indieate that LPAr/Edg2 receptors eouple to a Gi-PI3K-Tiamr pathway that aetivates Rac. ~ 100 :::l 0 E 80.a ;; 60.~ CD t.) ë '#. ~ Tiam Tiam Weeks post initiation Tiaml-Rac signaling and invasion of epithelial cells Metastasis of carcinomas is often associated with redueed E-eadherin-mediated een-eell adhesion. Tiamr loealizes to adherens junctions and ectopie expression oftiamr in epithelial eells inhibits HGFindueed eeli seattering by inereasing E-eadherin-mediated adhesion. Inereased Tiamr- Ct rl V12Ras+ Myc Fig. IJ: Invasive growth in papilloma derived from a Tiaml-defieient mouse Fig. I+ Graph: Tiaml-defieient miee are resistant to Ras-indueed tumors; Photograph: Tiamldeficient MEFs are resistant to Ras-indueed foei formation

21 Selected publications Driessens MHE, Olivo C, Nagata K, Inagaki M,. Collard JG. B plexins activate Rho through PDZ-RhoGEF. FEBS Lett 2002; 529: Lambert JM, Lambert QT, Reuther GW, Malliri A, Siderovski OP, Sondek J, Collard JG, Der CJ. Tiaml mediates Ras activation ofrac by a PI{J)K-independent mechanism. Nat Cell Bio12002; 4: Malliri A, Ten Klooster JP, Olivo C, Collard JG. Determination ofthe activity of Rho-like GTPases in cells. Methods Mol Bio12002; 189: Malliri A, Van der Kammen RA, Clark K, Van Der Valk M, Michiels F, Collard JG. Mice deficient in the Rac activator Tiaml are resistant to Ras-induced skin tumours. Nature 2002; 41T Tsutsumi S, Gupta SK, Hogan V, Collard JG, Raz A. Activation ofsmall GTPase Rho is required for autocrine motility factor signaling. Cancer Res 2002; 62: Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Mooienaar W. Rac activation by lysophosphatidic acid LP Al receptors: a critical role for the guanine nucleotide exchange factor Tiam1. ] Biol Chem (in press). Van Wetering S, Van Buul JO, Quik S, Mul FP, Anthony EC, Ten Klooster JP, Collard JG, Hordijk Plo Reactive oxygen species mediate Rac-induced loss of celkeu adhesion in primary human endothelial ceus. ] Cell Sci 2002; 11 P Rac signaling inhibits invasion and migration of fibroblastoid Vr2Ras-transformed MDCK cells by restoring E-cadherin-mediated adhesions and inducing an epithelial phenotype. We found that oncogenic Ras signaling permanently downregulates Rac and upregulates Rho activity, which is accompanied by epithelial-mesenchymal transition. Oncogenic Ras provokes changes in Rac and Rho activity through sustained activation of the Raf/MAP-kinase pathway, which causes transcriptional downregulation oftiamr. Reconstitution of Rac activity by exogenous expression oftiamr decreases Rho activity and restores the epithelial phenotype in mesenchymal VI2Rasor RafCAAX-transformed cells. Direct proof for a role oftiamr in the formation and maintenance of E-cadherin-mediated cen-cen adhesions comes from studies using short interference RNA. Tiamr-specific sirna introduced in epithelial MDCK cells leads to loss of cell-cell adhesions, a mesenchymal phenotype, and migratory cells. Tiaml mutant mice in vitro studies have implicated Rho-like GTPases in processes such as cell migration, and Ras-induced focus formation, suggesting that these GTPases play a role in the formation and progression of tumors in vivo. We have generated Tiamr-deficient mice to investigate the consequences of changes in Tiamr/Rac signaling in tumorigenesis. Consistent with the role oftiamr as an activator of Rac, reduced Rac activity was detected in Tiamr-deficient embryonic stem cells and in primary keratinocytes derived from Tiam{/-mice. Although Tiamr is expressed during embryonic development, Tiam{/-mice develop, grow and reproduce normally. Tiamr is widely expressed in a great number of tissues. In mouse skin, Tiamr is present in basal and suprabasal keratinocytes of the interfollicular epidermis and in hair follicles. Therefore, skin tumors were initiated by application of a two-stage chemical carcinogenesis protocol. This protocol entails tumor initiation in epidermal keratinocytes by a single treatrnent with the carcinogen dimethylbenzanthracene (DMBA), which invariably induces oncogenic activation of the c-ha-ras gene. Subsequent repeated treatrnents with the tumor promoter phorbol ester TPA re sult in the outgrowth and progression of initiated cells. Papillomas appeared in wild-type mice within 7-10 weeks from DMBA initiation. Tiam{/-mice displayed a dramatic resistance to tumor formation and their number was on average reduced ten-fold during the promotion period. Tiamr-deficiency also affected tumor growth. The few tumors that developed in Tiam{/-mice were significantly smaller than the many tumors appearing in Tiaml+/+mice. From our data we conclude that Tiamr critically affects the efficiency of Ras-induced tumor initiation, as determined by tumor numbers, as well as TPA-induced tumor promotion, as reflected in tumor growth, by two independent signaling pathways affecting apoptosis and cell proliferation, respectively. With respect to tumor progression, DMBA/TPA-treated wild-type mice developed a variety of skin tumors, predominantly benign papillomas of which approximately 35% ultimately progressed to malignant tumors. In contrast, approximately 80% of the tumors that arose in Tiam{/-mice acquired a malignant phenotype. Thus, although the number of tumors was strongly reduced in Tiam{/ mice, Tiamr-deficiency increased the frequency of malignant conversion of Ras-induced skin tumors. Immunohistochemical analysis of wild-type tumors showed a correlation between the loss of the expression oftiamr and malignant progression. Thus, while Tiamr function appears to be essential for the initiation and promotion of Ras-induced skin tumors, the histological and biochemical data indicate that a subsequent loss oftiamr increases the malignant conversion of benign tumors. Taken together, our data illustrate an important role for the Rac activator Tiamr in different aspects of Ras-induced tumorigenesis in vivo. Plexins and Rho activation Plexins are receptors for the repulsive axon guidance molecules, semaphorins and signal directly to different Rho-like GTPases. Plexin-Br interacts directly with the activated form of Rac but the activation and clustering of plexin-br, causes the formation of stress fibers, indicating that Plexin-Br activates Rho and not Rac. Indeed, using yeast-two hybrid system we found that Plexin Br binds directly to the Rho-specific exchange factor, PDZ-RhoGEF, via the PDZ domain. Mutation ofthe carboxyterminal amine acids ofplexin-br, abrogates the binding of PDZ-RhoG EF and the ability to induce stress fiber formation. PDZ-RhoGEF thus plays a role in B-Plexin-mediated activation of Rho/Rho-kinase signaling, implicated in the regulation of axon guidance and cell migration.

22 RECEPTORS FOR MATRIX ADH ESION Integrins connect the extraceliular matrix with the cell interior and trans duce signals via interactions of their cytoplasmic domain with cytoskeletal and signaling molecules. In our group we study the mechanism by which integrins provide signals for the regulation of important cellular processes such as cell migration and proliferation. In particular, we are interested how integrins contribute to the process of transformation. Role of a6p4 in the formation of hemidesmosomes Hemidesmosomes are multi-protein complexes that promote stabie adhesion of epithelial cells to the underlying basement membrane. The integrin a6~4 plays a central role in the assembly ofhemidesmosomes. Loss of a6~4 due to mutations in the ~4 gene causes a distinct form of epidemolysis bullosa (JEB), associated with pyloric atresia, characterized by fragility and extensive blistering of the skin. A similar phenotype is seen in ~4 null mutant mice. Binding sites for the hemidesmosmal components plectin, BPr80 and BP230 have been identified on the ~4 cytoplasmic domain. We showed that the plectin actin-binding do ma in (ABD) interacts with ~+ This type of ABD is present in members of the spectrin family. However, ex cept for the ABD of dystonin, those ABDs do not bind ~4. We showed that two missense mutations in ~4 in patients with non-lethal JEB, render ~4 unable to interact with plectin. The mutations affected basic residues in the second FNIII repeat. We have now identified residues in the plectin ABD that are required for binding to ~4. With the exception of the dystonin ABD, these residues are not conserved in other spectrin family members. Group leader Amoud Sonnenberg Arnoud Sonnenberg PhD Group leader Stephanie Bessone PhD Post-doe Erik Danen PhD Post-doe Cecile Geuijen PhD Post-doe Karine Raymond PhD Post-doe Gertien Smits PhD Post-doe Quido Valent Post-doe Sandra van Wilpe PhD Post-doe Jan Koster MSc Graduate Student Sandy Litjens MSc Graduate Student Iman Van den Bout Graduate student Maaike Kreft Technical staff Ingrid Kuikman Technical staff Petra Sonneveld Techn ical staff Erwin Wijnands Technical staff Martina Bauer Undergraduate student Role of a6p4 in cell migration In addition to its role in adhesion, the a6~4 integrin has also been implicated in celi migration and invasion. Therefore, we have studied the effects on migration of both intraceliular and extracellular interactions of a6~+ We have developed a method to quantify keratinocyte migration on a defined ECM. Using PA-JEB cells that we re either ~4-deficient or expressed wild-type ~4, we showed that a6~4 slows down migration on laminin-s matrices that the celis deposit onto the substrate. In contrast, a6~4 stimulated migration on a pre-deposited laminin 5 matrix. Disruption of the a6~4-plectin interaction led to even faster migration. Live celi imaging showed additionally that cells migrating over a predeposited matrix gain an intrinsic directionality, caused by a longer persistence of movement. In cells that have deposited their own laminin-s matrix, a6~4 is extensively clustered, whereas it is diffusely distributed over the basal surface in cells migrating over the pre-deposited matrix. The interaction with plectin promoted this clustering. We hypothesize that the extent of clustering, induced either by intracellular or extracellular factors, determines the dynamic behaviour of a6~4, and that in the presence ofligand this more dynamic a6~4 stimulates migration. Generation of conditional P4 knockout mice Last year, we generated a conditional ~4 knockout mouse, in which we observed no obvious abnormalities. After crossing with transgenic keratin Kr4-Cre mice, the 'floxed' ~4 DNA segment was efficiently removed, resulting in the loss of a6~4 from the skin, and the formation of blisters reminiscent of those seen in ~4 nuli mice. From these mice we established immortalized keratinocyte celilines. Furthermore, we induced skin tumors in these mi ce using a two-stage carcinogenesis protocol, and established independent transformed keratinocyte ceillines from the resulting tumors. We will study their capacity, with or without ~4 (after transient expres sion of the Crerecombinase), to grow, differentiate and migrate as weil as to survive when they are deprived of interaction with ECM components. Furthermore, we will assess their tumorigenic potential af ter subcutaneous injection into immuno-deficient mice. Control of cell cycle progression by integrin-mediated adhesion Integrinmediated celi adhesion controls proliferation, differentiation and survival of cells. It also regulates the activity of Rho GTPases, a family of Ras-related small GTPases, which play important roles in the organization of the actin cytoskeleton. We have generated ceillines expressing either as~r or av~3 and observed that as~r-mediated Fig. 1.5: Model of a hemidesmosome illustrating the molecular interactions involved in its assembly

23 Selected publications Danen EH). Sonneveld P, Brakebusch C, Fässler R, Sonnenberg A. The fibronectinbinding integrins a5f31 and avf33 differentially modulate RhoA GTP-loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis. J Cell Biol (in press). EI Mourabit H, Poinat P, Koster j, Sondermann H, Wixler V, Wegener E, Laplantine E, Geerts D, Georges Labouesse E, Sonnenberg A, Aumailley M. The PDZ domain of TIP-2/ GIPC interacts with the C-terminus of the integrin a5 and a6 sub units. Matrix Bio12002; 21: Geuijen CAW, Sonnenberg A. Dynamics of the a6f34 integrin in keratinocytes. Mol Biol Cell (in press). Koster j, Geerts D, Favre B, Borradori L, Sonnenberg A. Analysis ofthe interactions between BP180, BP230, plectin and the integrin a6b4 important for hemidesmosome assembly. J Cell Sci (in press). Koster j, Kuikman I, Kreft M, Sonnenberg A. Two different mutations in the cytoplasmic domain of the integrin f34 subunit in non-lethal forms of epidermolysis bultosa prevents interaction of f34 with plectin. j. Invest Dermatol 2001; 11T Sterk LMT, Geuijen CAW, Van der Berg ). Claessen N, Weening Jj, Sonnenberg A. Association ofthe tetraspanin CD151 with the laminin-binding integrins a3f31, a6f31, a6f34 and a7f31 in ceus in culture and in vivo. J Cell Sci 2002; 115:1161-7]. adhesion to fibronectin supports high levels of RhoA activity, whereas av~3-mediated adhesion does not. Cells adhering via av~3 assembie focal contacts but they fail to generate the tension that is required for translocation of focal contacts to cellular protrusions and for fibronectin fibriuogenesis. In line with our previous finding that the stimulation of ceu cyele progression by fibronectin is RhoA-dependent, fibronectin stimulates Cl phase progression only in ceus expressing a5~i. Intriguingly, oncogenic transformation is often accompanied by an impaired fibronectin fibrillogenesis due to a loss of a5~i. Instead, high levels of av~3 are frequently found in many human cancers. The activity of the Src protein tyrosine kinase is often also increased in those cancers and we have recently discovered that transformation of epithelial ceus by constitutively activated Src Y5 2 9 F, but not by Ras GI2V, requires the expres sion of av~3 at high levels. Our current experiments are aimed at understanding I) how fibronectin controls RhoA activity in non-transformed ceus, 2) which proliferative signaling pathways are stimulated by a5bi-mediated ceu adhesion, and 3) how increased levels of av~3 can support oncogenic transformation by activated Src. Regulation of ge ne express ion by integrin signaling Last year, we reported that increased expres sion of av~3 in ~I-deficient ceus induces an epithelial-mesenchymal (EMT)-like transition comparable to that induced by expres sion of ~I. To understand the underlying mechanisms, gene expression profi.les induced by ~I and ~3 expression in ~I-deficient ceus we re compared. A smau number of genes was found to be differentiauy regulated by the expres sion of ~I or ~3. Currently, we are studying whether the differences in gene expression are also reflected at the protein level. If so, we will determine the relevance of their regulation in EMT-like transition. Role ofthe M ARCKS fam ily members in T cell activat ion In neuronal cells MARCKS and related proteins have been implicated in the regulation of actin dynamics during formation of ceu protrusions. The MARCKS effector domain (ED) can specifically bind to PIP 2 present at the plasma membrane, which prevents PIP 2 from binding to proteins that are essential for actin dynamics. MARCKS binding to PIP2 is regulated by phosphorylation of the MARCKS-ED by PKC, or by binding of activated calmodulin to the ED.T ceus that recognise their designated antigen on a specialised antigen-pres enting ceu (APC) create a large contact area with the APC. This contact area is referred to as the immunological synapse and involves alocal activation of actin dynamics that ultimately leads to the partial engulfment of the APC, to maximise ceu-ceu contact. Furthermore, certain PKCs are activated after T ceu activation and PKCQ. specificauy localises to the synapse area. Also, calcium levels that activate calmodulin are readily increased upon APC contact. We are currently investigating whether MARCKS family members play a key role in T ceu activation. Of particular interest are the potential functions in the regulation of local actin dynamics and PIP 2 availability to non-actin related molecules such as PI3-kinase and PLCy. Van der Flier A, Kuikman I, Kramer D, Geerts D, Kreft M, Takafuta T, Shapiro SS, Sonnenberg A. Dijferent splice variants of filamin-b affict myogenesis, subcellular distribution and determine binding to integrin f3 subunits. j. Celt Bio12002; 156:

24 BIOPHYSICS IN CELL SIGNALING Detailed understanding of signals that are short-lived or exist very locally within the cell requires techniques to study cellular processes with high spatiotemporal resolution in single living celis. To provide this resolution, we employ biophysical approaches, ranging from mass spectrometry and electrophysiology to confocal imaging and spectroscopy. Fluorescent detection of membrane phosphatidylinositol bisphosphate (PI P 2 ) levels in single living cells We have developed a PIP 2 assay that is based on fluorescence resonance energy transfer (FRET) between GFP-tagged, PIP 2 -binding pleckstrin homology (GFP-PH) domains in live celis. This assay allows prolonged monitoring of cellular PIP 2 kinetics with subsecond tempo ral resolution. FRET reveals receptor-specific differences in the dynamics of PIP 2 hydrolysis that are not uncovered by other assays. We employ FRET to study the mechanism of inactivation of G protein-coupled receptors by monitoring the kinetics of inactivation in C-terminal receptor mutants. Group leader Kees Jalink Kees Jalink PhD Group leader Michiel Langeslag MSc Graduate student Jacco van Rheenen MSc Graduate student Bas Ponsioen MSc Graduate student Distribution of PI P 2 along the plasma membrane of single cells It has been put forward that plasma membrane [PIP 2] may be a key regulator of cellular responses, induding ion channel gating, receptor internalization and cortical actin rearrangements. A key unanswered question is: how can a single pool of a diffusible messenger at the plasma membrane independently regulate different cellular responses, apparently with spatial precision? Microscopical studies of cells expressing GFP-PH as a PIP z probe reported the existence ofmicrometer-sized domains that appear enriched in this lipid (so-called PIP 2 patches). We undertook an in-depth analysis of the spatial distribution of this lipid along the plasma membrane. Detailed colocalization studies of GFP-PH fluorescence with various fluid-phase membrane markers indicated that PIP 2 distributes homogeneously along the plasma membrane by rapid lateral diffusion. Rather, we show by several independent approaches that PIP 2 patches are in fact due to subresolution membrane folds (Fig. 1.6). An altemative possibility is that PIP 2 is locally enriched in submicroscopical «5onm) lipid domains called rafts. Because enrichment in such small domains would likely escape detection by confocal microscopy, this possibility is addressed by fluorescence resonance energy transfer assays that have a resolution of a few nm. Role of the TRPM7 ion channel in cell adhesion TRPM7 is a non-selective cation channel with inherent kinase activity that is expressed in many cell types. Recently, Van Leeuwen and colleagues showed that in NrE-IIS neuroblastoma and several other celis, significant myosin heavy chain (MHC) phosphorylation is detected following activation oftrpm7 by agonists. This leads to dramatic changes in ceu adhesion and morphology. We studied TRPM7 function with electrophysiological (patch damp) and ion imaging techniques. TRPM7 was found to act as an agonist-operated Ca 2 + channel (Fig. 1.7), and that Ca z + influx is crucial for MHC-phosphorylation and morphological changes. The mechanism of this Image Z-stack Reconstruction Fig. I.6: Direct demonstration that apparent PIP 2 enrichments are submicroscopic membrane folds. Left: GFP-PH labeled basal membrane of a HEK293 cell showing brightly labeled patches; middle: Z-stack of 4onm-spaced images collected from the area in the white rectangle; right: High-resolution reconstruction of the surface of the region calculated from the Z-stack using a novel numerical approach.

25 Selected publications Van Rheenen J. Jalink K. Agonist-induced PIP 2 hydrolysis inhibits cortical actin dynamics: Regulation at a global but not at a micrometer scale. Mol Biol Cell 2002; 13:] Van Rheenen J. Jalink K. Visualizing the suiface profile of the basal membrane with high precision. Bioforum International 2002; 6: Ca 2 + influx dependence, as well as the signaling that leads to opening of the channel, are currently being addressed. Towards chemical imaging of cells Mass spectrometry (MS) is sensitive enough to detect just a few molecules. In a collaborative effort with the MS group led by R Heeren at AMOLF, we are aiming to develop approaches to exploit MS for chemical imaging with subcellular resolution. The limiting step is the generation of suitable ions from cells and tissue samples. This desorption-ionization process is being optimized using a number of approaches. Van Rheenen J, Jalink K. Reconstruction of the arial pointspread function: Imaging subresolution membrane folds Imaging and Microscopy 2002; ~ ro ~L U 305 Fig. 1.7: TRPM7 expression causes increased cell adhesion and Ca 2 + influxffi

26 IMMUNO-EM FACILITY Immuno-electron microscopy reveals a wealth of structural detail in the 2 to 100 nm range providing information that can be obtained in no other way. This and other techniques were used to study the localization and trafficking of molecules in the celi i.e. sorting and processing of proteins and localization of molecules during signal transduction events. Local ization and trafficking of molec ules in the cell In collaboration with Niels Borregaard of the National University Hospital, Copenhagen, Denmark, we have studied the subcellular localization of Cysteine-rich secretory protein 3 (CRISP-3) and NRAMPI proteins in human neutrophils. By double-labeling immunogold electron microscopy, CRISP-3 was found in a subset of granules with characteristics ofboth specific and gelatinase granules. In addition, CRISP-3 was found as a granule protein in eosinophilic granulocytes. The presence of CRISP-3 in peroxidase-negative granules of neutrophils, in granules of eosinophils, and in exocrine secretions indicates a role in the innate host defense. Mutations of NrampI cause susceptibility to infections with intracellular pathogens. NrampI is expressed most abundantly in neutrophils suggesting that NRAMPI plays an important role in the activity of these celis. Immunogold studies showed that most (75%) NRAMPI-positive granules are also positive for gelatinase, but also suggest further heterogeneity in this granule population. Presence of NRAMPI in tertiary granules is in agreement with the latestage appearance of NRAMPI mrna during neutrophil maturation in bone marrow. NRAMPI is recruited from tertiary granules to the phagosomal membrane on phagocytosis, supporting a role for NRAMPI in the antimicrobial defenses ofhuman neutrophils. In collaboration with Inge Olsson from Lund University, Sweden we studied the sorting and processing ofhuman neutrophil granule proteins. The aim was to understand the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Murine myeloid RBL and 32D celllines were transfected with cdna encoding human neutrophil granule proteins MPO, BPI and hcap-i8 and the non-myeloid normally secretory proteins LBP and m-microglobulin as well as mutants and chimeras. A propeptide-deleted MPO precursor was not processed to mature MPO but degraded or secreted, indicating that the propeptide is a prerequisite for final processing and targeting of prompo to granules. Chimeras between the MPO propeptide and secretory protein prolonged ER retention. Thus the propeptide may mediate the long ER-residence of prompo. The amino-terminal half of BPI was targeted for storage, whereas the carboxy-terminal half was not, but it increased the stability of BPI and chimeric proteins. The non-myeloid LBP and m-microglobulin were targeted to but unstable, in granules. These results indicate cell-specific retention mechanisms for sorting for storage in myeloid celis. Structural requirements were found to be important for sorting and stability of some proteins. Collaboration wi t h ot her gro ups fro m N Kl j AvL In collaboration with F Itoh (Division of Celiular Biochemistry) we have shown that the Smad anchor for receptor activation (SARA) is localized in early endosomes and that it is required for efficient TGF-~/Smad signalling. With R MuEmann (Division Molecular Biology) we demonstrated that the transferrin receptor (TfR) of Trypanosoma brucei is retained in the flagellar pocket by aspecific and saturable mechanism. In bloodstream form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface. Similar surface accumulation was observed when trypanosomes were starved for transferrin. With J Mulder (Division of Cellular Biochemistry) we have studied the function of the protein pn6 RiP. In the presence of pn6 Rip, F-actin was organized into thick bundies similar to those formed by the actin-bundling protein a-actinin. Moreover the N-terminal actin binding domain induced actin bundling similar to full-iength pn6 Rip, whereas no actin bundles we re observed after incubation off-actin with the C-terminal coiled-coil region. This shows that pn6 Rip functions as an actin-bundling protein and that the F-actin-binding domain resides in the N-terminal region. Group ~eader jero Ca~afat Jero Calafat PhD Group leader Hans Janssen Technical staff Seleeted publieations Bulow E, NauseefWM, Goedken M, McCormick S, Calafat j. Gullberg U, Olsson I. Sortingfor storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor. j Leukoc Bio12002; 71: Canonne-Hergaux F, Calafat j, Rieher E, Cellier M, Grinstein S, Borregaard N, Gros P. Expression and subcellular localization ofnramp1 in human neutrophil granules. Blood 2002; 100: Itoh F, Diveeha N, Broeks L, Oomen L, janssen H, Calafat J, Itoh S, Ten Dijke P. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-fJ jsmad signalling. Genes Cells 2002; 3J Udby L, Cal afat J, Sorensen O E, Borregaard N, Kjeldsen L. Identijication of human cysteine-rich secretory protein 3 (CRISP-3) as a matrix protein in a subset of peroxidase-negative granules of neutrophils and in the granules of eosinophils. j Leukoc Bio12002; 72:462-9.

27 11 DIVISION OF MOLECULAR CARCINOGENESIS REGUlATION OF THE MAMMALIAN CEll CYClE Division head, Group leader René Bernards René Bernards PhD Group leader Katrien Berns PhD Post-doe Luis Borlado PhD Post-doe Annette Dirac PhD Post-doe Michael Edel PhD Post-doe Pieter Eichhorn PhD Post-doe Tiffany van der Meer PhD Post-doe Liming Wang MD phd Post-doe Thijn Brummelkamp MSc Graduate student Menno Creyghton MSc Graduate student Mirjam Epping MSc Graduate Student Roderik Kortlever MSc Graduate Student Sebastian Nijman MSc Graduate student David Markusic Undergraduate student Marielle Hijmans MSc Technical staff Mandy Madiredjo Technical staff Herrie Winterwerp Technical staff Marlijn Sonne Secretary mrna ShoI1 interfering RNA RNase i The focus of this group is on the application of innovative functional genomics tools to identify novel genes having a role in carcinogenesis. We use both high-throughput gain-of-function genetic screens with retroviral cdna expres sion libraries and loss-of-function genetic screens with RNA interference libraries to identify novel components of cancer-relevant pathways. Gain-of-function genetic screens We use retroviral cdna expression libraries to identify novel genes that act in pathways, which are frequently deregulated in human cancer. In short, these genetic screens involve the infection of a cell population with a high-complexity retroviral cdna expression library, selection of cells with altered phenotype, followed by identification of the cdna responsible for the phenotype. In the past year, we have successfully identified novel genes that act in different biological processes: the P53 pathway, the prb pathway and chromatin remodeling. The plg ARF _M DM2-P53 pathway P53 functions as a transcription factor that can activate expres sion of a range of cellular genes involved in cell cycle inhibition and induction of apoptosis. P53 is not only activated during the cellular response to DNA damage, but also when primary celis undergo senescence during in vitro propagation. Upregulation of PI9ARF, leading to activation of PS3, is a critical early event in the senescence response as rodent celis lacking P19ARF escape from senescence and are immortal. To identify novel regulators of the P19ARF_MDM2-P53 pathway, we have generated a mouse fibroblast celiline conditionally immortalized by a temperature sensitive allele of SV 40 large Tantigen. Upon shift to the non-permissive temperature these cells undergo rapid senescence. Introduction of retroviral cdna expression libraries prior to temperature shift led to the identification of more than ten genes that allow escape from senescence, including BCL6. This gene was originally identified as the target of an oncogenic translocation in non-hodgkin's lymphoma. Our data indicate that BeLG overrides the senescence response downstream of P53 through a process that requires induction of cyclin Dl expression. Importantly, BCL6 also extends the life span of primary human B cells in culture, indicating that BCL6 has a similar mode of action in rodent fibroblasts and in human B cells. We have also identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-Box gene, which is found mutated in the human developmental disorder Ulnar-Mammary Syndrome. TBX-3 strongly represses expression ofboth mouse P19ARF and human Pl/RF. More recently, we have identified a G protein coupled receptor, a guanine nucleotide exchange factor, an immediate early response gene and ahorneobox transcription factor as senescence-inhibitory genes using the same approach. The interaction of these factors with the P19ARF_MDM2-P53 pathway is currently under investigation. Using a similar approach, we have identified DRILl, the human orthologue of the Drosophila Dead Ringer transcriptional regulator, as a gene that allows escape from RAS-induced premature senescence. Immortalization by DRILI correlates with induction ofe2f activity. As a re sult, DRILl expression leads to induction ofthe E2F target gene CYCLIN El, overexpression of which is sufficient to trigger escape from senescence. Thus, DRILl disrupts cellular protection against RAS-induced proliferation downstream of the PI9 ARF /P53 pathway. Stem loop precursor ~ i 1 Degradation of mrna Figure II.1: A vector that mediates sta bie suppression of gene expression. The ps UP ER vector contains the Hl-RNA promoter upstream of a gene-specific targeting sequence, which consists of a 19 nt sequence derived from the target transcript separated by a short spacer from the reverse complement of the same sequence, and jive thymidines as termination signal. The predicted secondary structure of the transcript is a hairpin (stem-loop), which is processed intracellularly to a double stranded RNA of 21 nucleotides. The sirna-like product can basepair with its complementary mrna transcript, which in turn recruits an RNA-Induced Silencing Complex (RISC) that degrades the mrna.

28 The p16 INK4A -cyciin D-pRB pathway The pr6 IN I<4A_cyclin D-pRB pathway is deregulated in virtually every form ofhuman cancer. The E2F transcription factor is the best-studied downstream target of this growth-regulatory pathway and its activation is sufficient to induce transition from Gr to S phase of the cell cycle. Over the past years, several key components of this pathway have been identified through biochemical and genetic studies. Nevertheless, it is likely that quite a few additional (as yet unknown) genes act in this cancer-relevant pathway. To identify novel components of this pathway, we have constructed a rat fibroblast celiline that expresses both a temperature sensitive mutant of SV 40 T antigen and a non-phosphorylatable mutant of prb. These eens proliferate at 32 C but enter into a prb-dependent growth arrest upon shift to the non-permissive temperature. We performed a screen with retroviral cdna expres sion libraries to identify cdnas that confer resistance to this prb-induced proliferation arrest. We found that the helixloop-helix transcription factor TFE3 not only overrides growth arrest induced by prb, but also overrides Gr arrest induced by the upstream regulator of prb, pr6 IN I<4A. In addition, TFE3 expres sion blocks the anti-mitogenic effects oftgfb. We found that TFE3 specifically inhibits prb-mediated suppression of the E2F-regulated gene CYCLIN E. These data demonstrate that TFE3 can render cells insensitive to anti-mitogenic signals emanating from the pr6 IN I<4A_cyclin D-pRB tumor suppressor pathway. Chromatin remodeling An important level of transcriptional control takes place at the level of chromatin structure. Histone Acetyl Transferases (HATs) and Histone DeACetylases (HDACs) catalyze the addition and removal of the acetyl group from the lysine residues in the N-terminal tails of nucleosomal co re histones, respectively. This results in alterations in chromatin structure, leading to changes in cellular gene expression. The activity ofboth HDACs and HATs is of ten deregulated in cancer. This provides an opportunity for therapeutic intervention. Indeed, HDAC inhibitors are a promising new class of anti cancer drugs, of which several have been entered into clinical trials. However, the molecular basis for their preferential growthinhibitory activity on cancer cens is presently unclear. To study the mechanism of action of HDAC inhibitors, we have used retroviral cdna expression libraries to search for genes that ren der cens insensitive to the antiproliferative actions of these compounds. To date, we have identified several cdnas that allow cells to proliferate in the presence of growth-inhibitory concentrations of HDAC inhibitors. One of them was found to encode PRAME (PReferential Antigen MElanoma). This protein was first identified as a tumor antigen recognized by Cytotoxic T Lymphocytes (CTL) in melanoma and, more recently, also found to be expressed in renal cell carcinoma, myeloma and acute leukemia. Interestingly, we recently found through DNA micro array analysis that PRAME is also one of the 23r genes that predict disease progression in breast cancer. Together, these data suggest that PRAME contributes to tumor progression by conferring a selective advantage to a number of cancer cens. The molecular mechanism by which PRAME confers this selective advantage is currently being investigated. Loss-of-function genetic screens Mammalian genetic approaches to study gene function have been hampered by a lack of tools for efficient generation of stable lossof-function phenotypes. A recent advance has been the finding that introduction of synthetic short interfering RNAs (sirnas) into mammalian cells can significantly suppress expression of specific genes through a process known as RNA interference. However, this reduction in gene expres sion is transient, which severely restricts its applications. To overcome this limitation, we have designed a mammalian expres sion vector (psuper, suppression of endogenous RNA) which directs the synthesis of short hairpin transcripts that get processed intracellularly into sirna-like molecules (Fig. I1.r). We found that this vector can stably inhibit gene expres sion in a highly specific fashion. We used psuper to target only the mutant K_RAS V12 allele in human pancreatic carcinoma, while leaving the wild type K-RAS allele untouched. In spite of the fact that pancreatic carcinoma cells have many genetic alterations, we found that loss of K_RAS V12 expression leads to loss of tumorigenicity, indicating that this oncogene is still required late in tumorigenesis to maintain an oncogenic phenotype. Selected publications Bernards R, Weinberg RA. Metastasis genes: A progression puzzle. Nature 2002; 418:823. Brummelkamp TR, Bernards R, Agami R. A system for stabie expression of short inteifering RNAs in mammalian cells. Science 2002; 296: Brummelkamp TR, Bernards R, Agami R. Stabie suppression of tumorigenicity by virus-mediated RNA interference. Cancer Ce1l2002; 2: Peeper DS, Shvarts A, Brummelkamp T, Douma S, Koh E, Daley GQ, Bernards R. Afunctional screen identifies hdrill as an oncogene that rescues RAS-induced senescence. Nature Cell Biology 2002; 4:148'53. Rowland DB, Denissov SG, Douma S, Stunnenberg HG, Bernards R, Peeper DS. E2F transcriptional repressor complexes are critical downstream targets ofp19arf/p53' induced proliferative arrest. Cancer Cell 2002; 2: Shvarts A, Brummelkamp T, Koh E, Daley GQ, Bernards, R. A senescence rescue screen identifies BCL6 as an inhibitor of antiproliferative P19ARF_P53 signalling. Genes Dev 2002; 16: Van de Vijver MJ, He YD, Van 't Veer Lj, Da i H, Hart AAM, Voskuil D, Schreiber GJ, Peterse jl, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ETh, Friend SH, Bernards R. A gene expression signature as a predictor of survival in breast cancer. New England ] Med (in press). Van 't Veer LJ, Dai H, Van de Vijver MJ, He YD, Hart AAM, Mao M, Peterse HL, Van der Kooy K, Marton MJ, Witteveen AT, Schreiber Gj, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling predicts clinicaloutcome ofbreast cancer. Nature 2002; 415:53,6.

29 BCL-6 DRIL-1 Stress + signals p19""f.", p53 } senescence TBX-3 Figure J1.2: Escape from senescence by newly-identified genes. Stress signais, such as in vitro culturing of primary rodent fibroblasts, induces both the P19ARF-P53 pathway and the p16/ Nf4A -cyclin D-pRB pathway. TBX-3, BCL-6 and DRIL-l interfere at different levels with senescence signaling. More recently we have used this vector to stably suppress expres sion of individual members of several gene families, ineluding histone aeetyl transferases (HATs), Protein Phosphatase 2A regulatory 'B' subunits, de-ubiquitinating enzym es (DUBs) and a large number of protein kinases. Using funetional screens, we have been able to plaee several of the members of these gene families in caneer-relevant pathways. We are currently using this vector system to generate a large collection of some 25,000 sirna vectors that each targets a single transcript. This collection of sirna vectors will allow us for the first time to perform genome-wide functional screens for loss-of-funetion phenotypes. We will use these sirna libraries to identify novel genes that are involved in control of cell cyele, responses to cytotoxic drugs and replicative senescenee.

30 CONTROL OF TElOMERASE ACTIVATION DURING IMMORTALIZATION AND TUMORIGENIC TRANSFORMATION Telomerase activation is a hallmark of the transition of a normal eell to a tumor eell. The extensive proliferative eapaeity observed in malignant tumor eells is direetly associated with the activation of telomerase. Understanding the meehanism of telomerase activation willlead to the identification of proteins or pathways, whieh are deregulated in human eaneer and possibly providing a therapeutic target. Normal human somatic eens, whieh generally laek telomerase aetivity, exhibit progressively shortened telomeres with repeated een divisions and enter seneseenee af ter a limited number of eell divisions. When the life span of norrnal eells is extended beyond seneseenee, life span terminates at crisis, a point at whieh telomere loss results in chromosome instability and eell death. However telomere shortening is stopped by the expres sion of htert, the eatalytie subunit of the telomerase enzyme, in those eells that have become immortal. htertmrna eannot be deteeted in most norrnal eelilineages but is present in a variety of tumor eelllines and is deteetable in 90% ofhuman tumors. The ectopie expres sion of htertis suffieient to restore telomerase aetivity and maintain or extend telomeres. The introduction of htertin several primary human eells prevents seneseenee, allowing them to bypass crisis and beeome immortal. In contrast, inaetivation of telomerase in imrnortal eens results in telomere shortening and eell death. Together these results indieate that htert expression represents the rate-limiting determinant that regulates the levels of telomerase enzyme aetivity in tumor eens and that the proeess of eell immortalization is linked to the expres sion of htert. We are studying the molecular meehanisms responsible for derepression of the htert gene that oecurs upon immortalization. We have generated a model system with very early passage primary human embryonic kidney eells transformed with SV 40 Large T. These eells remain telomerase negative for more than 80 population doublings af ter whieh they undergo massive eell death due to telomere erosion. At low frequeney, immortal een clones emerge that have aetivated telomerase. We have used this een system to identify genes involved in telomerase regulation by use of retroviral edna expres sion libraries, retroviral sirna libraries and retroviralmediated-mutagenesis. In addition we have eompared gene expres sion byedna micro-array analysis of 8 independent mortal clones and 8 immortal clones originating from the human embryonic kidney eells. Several up- or down-regulated ednas have been identified in all independent clones upon the activation of telomerase and immortalization. Work is in progress to verify the altered expression of these genes and their effect on htert gene expression. We areeontinuing to use the htert-reporter system whieh allows for the identification of proteins or pathways that ean either repress or activate htert expression. A large promoter fragment of the h TERT gene is used to direct the expression of G FP in telomerase positive eelilines. These reporter eelllines will be used in combination with TET indueible retroviral edna libraries or retroviral-mediated-mutagenesis with the aim to identify potential repressors of htert gene transcription. Group leader Roderiek Beijersbergen Roderiek Beijersbergen PhD Group leader Leone Carlee MSc Graduate student Wouter Nijkamp Technical staff Lennart Post Undergraduate student Johan Kuiken Undergraduate student The result of these studies may allow us to elucidate the molecular meehanism that enables ca neer eells to overcome two barriers, seneseenee and crisis, before they become immortal. The identification of the proteins or pathways that are involved in regulation of telomerase expres sion may have implications for interferenee with the proeess of immortalization and provide us with a molecular event, specific in the generation of tumor eells.

31 PROTEIN STRUCTURE AND FUNCTION Group leader Titia Sixma Titia Sixma PhD Group leader Gretel Buchwald PhD Post-doe Patrick Celie PhD Post-doe Joyce lebbink PhD Post-doe Valerie Notenboom PhD post-doe Puck Knipscheer Graduate student Meindert lamers Graduate student Ganesh Natrajan Graduate student Stephan Watkins Graduate student Pim van Dijk Technical staff Sari van Rossum-Fikkert Technical staff Herrie Winterwerp Technical staff Esther Frische Undergraduate student Development of cancer is generaliy due to errors that occur in cellular pathways. Structural biology can help in the understanding of these errors at the atomic level, by studying the proteins and the DNA involved. We use X-ray crystaliography as a tooi to provide three-dimensional structures. Subsequently we interpret the structural data using a variety ofbiochemical and biophysical techniques. These studies provide more insight in the molecular processes and they can also provide targets for drug design studies. In our group the focus is mainly on proteins involved in DNA stability (transposition, mismatch repair), transmembrane signaling and celi cyele control. DNA mismatch repair Mutations in DNA mismatch rep air genes predispose for one of the most prevalent type of familial cancers, hereditary non-polyposis colon carcinoma (HNPCC). This type of DNA rep air is specific for a single mis match or a smali stretch of unpaired bases in DNA. It involves a cascade of proteins highly conserved from bacteria to humans. The initial step of the repair is the recognition and binding of mismatched DNA by the MutS protein (E. Goli) or its heterodimeric MSHz/MSH6 or MSHz/MSH3 homologs (eukaryotes). This complex is then recognized by the MutL protein or its homologs. To understand how E. GoH MutS can recognize all the different mismatches, we have cocrystallized the MutS protein with different DNA mismatches. In a set of structures of 4 mismatches and one looped-out base, we have seen that the protein contacts to the DNA are always preserved. All the DNA oligomers are kinked similarly while the mismatched bases adapt their orientation to allow hydrogen bond forrnation to glutarnate 38 in the protein, involving the N3 in purines or N7 in pyrimidines. Thus smali adjustrnents in the DNA create a very conserved binding mode. In our initial crystal structure of E. Goli MutS the homodirner was structurally asymmetric, not only at the DNA recognition site, but also in the ATPase domains (Fig 11.3). Based on a series ofbiochemical assays we were able to show that the protein is asymmetric in solution as well and that the two domains alternate in ATP hydrolysis. The ATPase asymmetry is lost upon R697A mutation, as indicated by the loss of the high affinity site that is present in one monomer (Fig 11.3). Interestingly this mutant no longer shows ATP-induced DNA release, indicating that communication between mis match binding and ATPase domains may be dependent on structural asymmetry. Altogether our results indicate that a sequential mechanism of the ATPase domains is essential for MutS and its role in mismatch rep air. Acetylcholine binding protein (AChBP) We are studying a homolog to the nicotinic receptor ligand-binding domain, called Acetylcholine binding protein. It is a c Fig II.y Structural analysis of asymmetry in MutS ATPase domains. (A) Structure ofthe MutS dimer in complex with mismatched DNA. (B) Overview of the two ATPase domains as viewed from the arrow in (A). The side chains ofthe two Arg 697residues are located at the centre ofthe inteiface ofthe two ATPase domains. (C) Schematic representation ofthe asymmetric interactions ofthe ATPase domains of mono mer A and B.

32 water-soluble protein found in glial cells in the fresh-water snail, Lymnaea stagnalis. AChBP has high similarity (rs-28% sequence identity) to the extracellular domains of all pentameric ligand-gated ion channels, a superfamily which ineludes the nicotinie acetylcholine receptor, as well as the GABA A and GABA c, the glycine and the SHT3 serotonin receptors. Most of these are important drug targets. In 200r we solved the crystal structure of AChBP, the first high-resolution structure in this superfamily. In the resulting model, almost all residues shown to be involved in ligand binding in the nicotinie receptor are found in a pocket at the subunit interface. This pocket is lined with aromatic residues, and filled with a HEPES buffer molecule. The AChBP crystal structure explains many of the biochemical studies on the nicotinie acetylcholine receptors and has provided a model for many new studies. Now we have refined the structure at higher resolution (2.0 A), with which we were ab Ie to improve the orientation of the HEPES buffer molecule in the binding site. The critical cation-pi interaction between the positive nitrogen in the HEPES and the Trpr43 in the protein was unchanged in the new model (Sixrna & Smit, in press). Recently we have solved the crystal structure of AChBP complexed to nicotine (Fig II.4). Although the concentration of nicotine used was relatively low, the electron density is good and contacts can be analyzed. Our crystal structure serves as a basis for structure-based drug design targeting Alzheimer's disease, certain forms of schizophrenia and smoking. Ubiquitin dependent conjugation Ubiquitin conjugation processes are emerging as a general addressing system that is essential for cell stability, by controlling degradation of short-lived proteins, DNA repair and targeting to specific areas in the cell through endocytosis. Because of its importance for regulating cell cyele (e.g. PS3, MDM2, E2F'S, P27, cyelin D), apoptosis (e.g. ccbi, laps) and DNA rep air (e.g. Rad6, Radr8, Brcar) deregulation of ubiquitin-dependent processes often leads to cancer. The process of conjugation by ubiquitin(-like) proteins involves covalent linking of one or more 76-amino-acid ubiquitins to a target protein by an ErjE2jE3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, the atomie details have been lacking and the specificity determining factors are unelear. We are studying several E2jE3 complexes. We have created E. coli and baculovirus expres sion systems for the human SCF components Skpr, Cullin and several F-box proteins (e.g., Skp2, FbX4, Fb17) The E2/E3 complex of mammalian Rad6jRadr8 is involved in error-free postreplicative DNA rep air. In collaboration with J Hoeijmakers (Rotterdam) we have purified the complex from E. coli to homogeneity. The complex is stabie through a number of purification steps and is currently being submitted to crystallization trials. In collaboration with the group of A Muysers in Amsterdam we are analyzing the Rad6-Radr8 interactions by mass spectrometry (Back et al, 2002). Selected publications Back JW, Notenboom V, De Koning Lj. Muijsers AO, Sixma TK, De Koster CG, De Jong L. Fadle identification of crosslinked peptides for protein interaction studies using mass spectrometry and 180 labeling. Anal Chem 2002; 74: Brejc K, Van Dijk Wj. Smit AB, Sixma TK. The 2.7 A structure of AChBP, homolog of the ligand-binding domain of the nicotinic acetylcholine receptor. Novartis Found Symp 245, Ion channels: from atomie resolution physiology to functional genomics 2002; 245:22-]2. Sixma TK, Smit AB. Acetylcholine binding protein (A ChBP): a secreted glial protein that provides a high resolution model for the extracellular domain of pentameric ligandgated ion channels. Annu Rev Biophys Biomol Struct (in press). Fig II+ Electron density of nicotine bound to AChBP. Knowledge ofthe binding site mayaid design of anti-smoking compounds.

33 STRUCTURAl BIOLOGY Group leader Anastassis Perrakis Anastassis Perrakis PhD Group leader Serge Cohen PhD Post Doe Rebecca Persson PhD Post Doe Koen Verschueren PhD Post Doe Oliver Weichenrieder PhD Post Doe Kostas Repanas MSc Graduate student Nuno Rocha MSc Graduate student Evangelos Christodoulou Technical staff Mattheos Kakaris MSc Software Engineer Dunja Ferring Guest Michael Müller Guest 200r was the fitst year this group was in full operation. The interests, as envisaged, remain shared between performing structural biology research, predominantly in collaboration with in-house groups, and developing methods for X-ray crystallography. For the structural biology projects our major tooi for structural elucidation remains X-ray crystallography. The arrival of the new electron microscope lays the foundation for projects using single particle electron microscopy, which will start in Structural Biology The interests of the group are on understanding functionjstructure relationships in a variety ofbiological systems. The projects that are in areasonabie advanced state are briefly discussed below. Structural characterisation of the hu man L 1 retrotransposition machinery The Lr (LINE-r) retrotransposon is the most important human mobile element and shapes the genome in many ways. The Lr element is an autonomous non-ltr (nonlong-terminal repeat) retrotransposon that expands in nurnber by a copy-and-paste mechanism via an RNA intermediate. Lrs account directly or indirectly for about one third of the hurnan genome and it is likely that retrotransposons actually are responsible for creating more than half and perhaps even the bulk of the hurnan genome. Active Lr elements contain a S' untranslated region with an internal promoter, a r kb ORFr that encodes a protein with RNA binding capability, a 4 kb ORF2 that encodes a protein with endonuclease and reverse transcriptase activities, a short 3' UTR, and a poly(a) tail. Insertion into chromosomal DNA probably occurs by a process termed target-primed reverse transcription (TPRT); the endonuclease of ORF2 nicks a single strand of DNA, leaving a 3' OH that serves as the primer for reverse transcription with the Lr RNA as template. The ORF2 endonuclease (ORF2_EN) is an important determinant for Lr integration specmcity and very similar (23% identity) to hurnan apurinicjapyrimidinic endonuclease (APEr), a major player in DNA repair. In the past year we have cloned, expressed and purrned ORF2_EN to homogeneity. The protein is soluble and behaves like a monomer in gel Hltration chromatography. It is active in a DNA nicking assay, converting supercoiled plasmid DNA into the open circle form and this activity depends on the presence of magnesium. We were able to obtain two crystal forms of this protein from slightly different crystallization conditions. Crystals ofboth forms diffract to about 2 Aresolution. They diffract to beyond 2 Aresolution and contain two molecules per asymmetric unit. A number of diffraction datasets were collected at the synchrotron bearnlines at the EMBL in Hamburg and at the ESRF in Grenoble. Understanding the structure of Lr endonuclease and its exact specificity and mechanism of action may be important information for the engineering of an Lr-based gene delivery system that ins erts a single gene copy into a unique genomic location. Regulation of Iysosomal motility by RAB7/RI LP Intracellular vesicle trafficking is a tightly controlled process by the RAB family of proteins. These small GTPases localize to specific subcellular membrane compartments and interact with aspecific subset of effectors, enhancing the selectivity, as well as spatial and temporal regulation of vesicle trafficking. Knowledge of the structural basis by which RAB proteins selectively interact with their specific set of effectors is critical for understanding the specificity of mem bra ne trafficking. RAB7 has been identified to be involved in governing aggregation and function on late endocytic transport. The activated, GTP-bound form of RAB7 interacts specifically with at least with one effector protein, RILP (RAB-Interacting Lysosomal Protein, identified by I Jordens and J Neefjes, Division ofturnor Biology), which may be involved in vectorial vesicle transport by recruiting locally functional dynein-dynactin motors. Our research plan is to deterrnine the structure of the RAB7 jrilp complex by X-ray crystallography. Attempts to purify soluble recombinant RILP under native conditions (expressed in E. coli) failed due to aggregation and proteolysis. Celllysis in the presence of mild non-ionic detergents yield reasonable amounts of pure recombinant RILP. Very small crystals we re obtained from commercial

34 crystallization screens. We are currently developing a dicistronic expression system for coexpression in E. coli ofboth RAB7 and its effector protein RILP, doning various deletion constructs that will be used for functional and structural characterization and designing constructs for expression in insect cells. A monomeric transcription factor: the mitochondrial D-Ioop binding protein (mtdbp) Mitochondria contain their own DNA, separate from the cell's DNA and, in addition, they can create their own proteins and repro duce to form new mitochondria. Some mitochondrial components, however, are encoded by nudear genes and are imported into the mitochondrion; these indude the proteins necessary for regulation, replication and transcription of the mtdna. One of these nudear encoded components is the mitochondrial D-Ioop binding protein (mtdbp), a monomeric 40kDa transcription termination factor (348 residues as mature protein) which binds to two -25bp long homologous sequences on the mitochondrial DNA. We are attempting to determine the three-dimensional structure of the sea urchin mtdbp through X-ray crystallography. The protein is purified and concentrated up to I5-20 mg/mi and crystallization trials of the protein on its own and in the presence of specific oligonudeotides of different sizes representing the DNA binding site, have been set up. Some promising conditions in which the protein forms small crystals, have been found and these are currently being refined in order to obtain crystals suitable for X-ray diffraction. Selected publications Morris Rl. Perrakis A, Lamzin VS. ARP /warp's model-building algorithms. I. The ma in chain. Acta Crystallogr D Biol Crystallogr. 2002; 58: Ruivenkamp CA, van Wezel T, Zanon C, Stassen AP, Vlcek C, Csikos T, Klous AM, Tripodis N, Perrakis A, Boerrigter L, Groot PC, Lindeman J, Mooi WJ, Meijjer GA, Scholten G, Dauwerse H, Paces V, Van Zandwijk N, Van Ommen Gl. Demant P. Ptprj is a candidate for the mouse colon-cancer susceptibility locus SCCI and is frequently deleted in human cancers. Nat Genet. 2002; Structural basis of the regulation of CTLA4 activity by binding to cofilin Cytotoxic T-Iymphocyte antigen 4 (CTLA 4), an activation-induced integral membrane receptor, is a vital negative regulator oft-cell response. The cytoplasmic tail of CTLA 4 lacks intrinsic enzymatic activity but is known to interact with several other proteins. Using a biochemical screening method Q Valent (Division of Immunology) has identified cofilin, an essential regulator of actin dynamics, as one interaction partner of the cytoplasmic tail. Interactions only occur with the phosphorylated (on Ser3) form of cofilin, which maintains no actin-severing activity. To investigate the nature of interactions between the CTLA 4 cytoplasmic tail and cofilin, genetic constructs we re made for recombinant expression of the native sequence of cofilin and two mutants, Ser3~Asp and Ser3~Glu (in an effort to 'spatially mirnic' a phosphorylated state). The proteins were expressed in E. coli to high yields and purified using a three-step purification protocol in their native form, without purification tags. Using bacterial extra cts containing the recombinantly expressed cofilins it was shown that while the mutant forms did associate with the CTLA 4 cytoplasmic tail the native form did not, which is in analogy with the phosphorylated and non-phosphorylated forms of cofilin, respectively. Crystallization conditions have been established for the Ser3~Glu mutant in uncomplexed form and co-crystallization trials with the peptide corresponding to the CTLA 4 cytoplasmic tail are about to commence to allow for structural investigation of the complex. Future studies also indude deterrnination of binding characteristics using Biacore and ITC. Methods for X-ray crystallography 200I was marked by the release of ARP /warp version 6, after an 'incubation period' of nearly three years, which was a major breakthrough in the history of the ARP /warp software package. The diffraction data resolution requirements were decreased to around 2.5A and the rate of convergence to a complete model was tripled. Over 600 academic downloads and 40 commercial licenses were the best reward for our efforts. The main focus of our work in 200I has been the 'Side Trace' program of the ARP /warp suite. Scientific software is usually developed in a manner referred to as 'incremental programrning', where ideas and algorithms are tumed to computer code to achieve intermediate goals. The whole ARP /warp suite has evolved in such a way over the last decade. We decided to re-engineer the vital 'Side Trace' module utilising a state-of-the-art engineering approach and modern object-oriented programming methods. That is envisaged not only to extend the functionality of the program but also as a crucial step of enabling maintainability of the program. That project is also serving as a pilot since it is one of the pioneering efforts in creating X-ray crystallography software in a truly professional manner. Fig. II-J: Crystals ofli Endonuclease (top), mtdbp (middle) and Coffilin Ser~Glu mutant (bottom).

35 III DIVISION OF CELLULAR BIOCHEMISTRY LYSOPHOSPHOLI PI 0 SIG NALI NG, CYTOSKELETAL REGULATION AND CELL-CELL COMMUNICATION Division head, group leader Wouter Mooienaar Wouter Mooienaar PhD Group leader Ben Giepmans PhD Post-doe laura Sayas PhD Post-doe Shula Grivell MSc Graduate student Jacqueline Mulder MSc Graduate student Francis Van Horck MSc Graduate student laurens Van Meeteren MSc Graduate student Agnes Van Rossum MSc Graduate student leonie Van Zeijl MSc Graduate student Floor Frederiks Undergradute student Shanti Gangaram-Panday Undergraduate student Dick Van den Boomen Undergraduate student Aafke Ariaens Techn ical staff Trudi Hengeveld Technical staff Paula Ruurs Technical staff c o Phosphodiesterase domain Somatomedin B-like domain ATX lysopld Proliferation Migration Figure IJI. l: Production ofbioactive LPA by autotaxinjlysopld. ATXjlysoPLD is a type-ij transmembrane glycoprotein that is proteolytically cleaved, as indicated, to yield a soluble exoenzyme. Soluble ATXjlysoPLD hydrolyzes carrierbound and membrane-associated LPC (and other lysophospholipids) to generate LP A Newly produced LPA acts on its own G protein-coupled receptors and thereby stimulates Ras-mediated cell proliferation and Rhoj Rac-regulated cell migration. Excess LPA is converted into monoacylglycerol (MAG) by membrane-bound lipid phosphatases. Lysophosphatidie acid (LPA) is serum-borne lysophospholipid that evokes a host of eeliular responses, ranging from rapid cytoskeletal remodeling to stimulation of eeli migration, proliferation and survival. LP A signaling has been implieated in wound healing, embryonie development and tumor progression. LP A is produeed extraeeliularly by a seereted lysophospholipase D, named autotaxin (ATX), previously identified as an 'autoerine motility factor'. LPA signals through three distinet G protein-eoupled reeeptors (GPCRs), whose individual properties remain to be eharaeterized. Sinee LP A drives tumor eeli migration, invasion and proliferation, whilst LP A levels are elevated in eaneer patients and its receptors as weli as ATX are found overexpressed in certain tumors, LPA receptors and ATX qualify as potential targets for therapy. Our studies explore the mode of aetion of ATX and how LPA reeeptors influence cytoskeletal remodeling, eeli migration and eeli-eeli communication. These studies should lead to novel ways of interfering with unscheduled LP A receptor signaling in eaneer eells and with LP A production in their microenvironment. The ins and outs of LPA receptor signaling A major question is how and where LP A is produced in vivo. It has reeently become clear that LP A can be generated by autotaxin (A TX), a motility factor secreted by tumor eells and recently identified as a serum lysophospholipase D whieh hydrolyzes circulating lysophosphatidylcholine (LPC) (Figure 111.1). Studies to examine the biosynthesis, subeeuular loealization and secretion of ATX are under way. Bacterial secreted (lyso)pld may serve as a useful model for mammalian ATX. We find that PLD from S. chromofuscus reproduces the multiple actions of LP A in many eeu types, but only if these cells express funetional LPA receptors and extraceliular LPC is not limiting. Exogenous PLD mimies LPA in provoking rapid intemalization of LPA reeeptors. Thus, bacterial PLDs may exploit LPA receptors to stimulate host eell proliferation. We have analyzed the effects of LP A receptor signaling on eeu behavior by expres sion of LPA r,2 (Edg2>4) in receptor-negative eelis. Af ter ligand addition, LPA receptors are rapidly intemalized as determined by live-eell imaging. Strikingly, LPA receptors mediate activation of the Rae GTPase in a Gi-mediated and PI3-kinase-dependent manner. LPA-indueed Rae activation is aeeompanied by myosin heavy chain phosphorylation and leads to lamellipodia formation, cell spreading and migration. Rac activation is accompanied by enhaneed GSK-3 protein kinase activity, whieh may may serve to modify the microtubular network. In eouaboration with the group of J CoUard (Division I), we have established that the invasion-inducing Tiaml guanine nucleotide exchange factor mediates LPA-induced Rae activation, with concomitant suppression of RhoA activation (Figure 111.2). Thus, LPA acts as a motility factor and chemoattractant by activation of a Gi-PI3kinase-Tiam1-Rac signaling pathway. Cytoskeletal regulation by plgorhogef, pl16rip and cortactin Rho family GTPases control eytoskeletal remodelling and are aetivated by guanine nucleotide exchange factors (GEFs). We previously isolated P19oRhoGEF, whieh specifically acts on RhoA and directly interacts with microtubules via its C-terminal region. Microtubule disruption promotes RhoA activation, similar to what is observed with P19oRhoGEF overexpression. Thus, P19oRhoGEF may provide a link between microtubule dynamics and RhoA signaling. To establish the in vivo role of P190RhoGEF, we are targeting its mrna in neuroblastoma eelis. Deletion of P19oRhoGEF is expected to modify cellular responses to microtubule-disrupting agents. We are also eharacterizing pn6rip, a putative seaffold protein containing two PH domains and a coiled-coil region. pn6rip loealizes to the aetin cytoskeleton; its N-terminal region binds directly to f-aetin and shows aetin-bundling activity in vitro. Overexpression of the actin-binding domain disrupts the actin cytoskeleton,

36 suggesting that pn6rip is essential for maintaining eytoskeletal integrity. Trough two-hybrid assays, we have identified pr30mbs, the regulatory subunit of myosin light ehain phosphatase, as a binding partner of pn6rip. Thus, pn6rip may serve to recruit essential eomponents of the RhoA-regulated eontraetile maehinery to the actin cytoskeleton. RNAi experiments to delete pn6rip in various eeli types are ongoing and should reveal the in vivo role of pn6rip. Another actin-binding protein, eortactin, is found overexpressed in mammary careinoma eelis and serves an established role in actin polymerization and (tumor) eeli migration. However, many questions remain about the precise physiological roles of eortaetin in eytoskeletal regulation. We have sueeessfuliy knoeked down cortaetin mrna in both normal and transformed eelis and are currently analyzing the phenotypie changes in eortaetin-deficient eelis. Cytoskeletal regulation by a Trp-related cation channel I M HC kinase Little is known about how LP A induees cytoskeletal relaxation, a prerequisite for eeli spreading and migration. LPA and other GPCR ligands induce a Ca 2 +-dependent phosphorylation of the myosin II heavy ehain (MHC), concomitant with Rae activation and disassembly of the eortical cytoskeleton. We have identified a candidate human MHC kinase that shows 30% identity to Dictyostelium heavy chain kinases. Intriguingly, the kinase domain is part of a transmembrane protein that belongs to the family of 'Trp-related' cation channels. This bifunetional protein is now known as TRP-PLIK, LTRPC7 or 'Channel Kinase' (ChaK), but its functions are largely unknown. We find that ectopie expression of ChaK promotes eeli spreading and celi-matrix adhesion and, furthermore, ChaK enhances Ca2+-influx in response to LPA (eoliaboration K Jalink, Division I). ChaK associates with actomyosin and ean phosphorylate myosin 11 heavy ehain, whieh correlates with an increase in bas al Ca 2 + levels. These results identify ChaK as a potential MHC kinase. Kinase-dead mutants of ChaK are currently being tested to substantiate the novellink between Ca 2 + influx, regulation of the cortical aetomyosin network and eeli spreading (coliaboration F Van Leeuwen, Nijmegen University). Cell-cell communication: connexin-43 Gap junctions are speeialized eeli-eeli junctions that mediate interceilular communication. They are composed of connexin proteins, whieh form transmembrane ehannels for small molecules. The C-terminal tail of eonnexin-43 (CX43), the most widely expressed eonnexin member, has been implicated in the regulation ofcx43 channel gating by LPA and other mitogens. The CX43 tail contains various protein interaction sites, but little is known about binding partners. We have ound that the CX43 tail binds directly the ZO-r protein, a 'tight junction' protein and to tubulin. We have identified a juxtamembrane region in the CX4 3 tail mediates rnierotubule binding. Immunofluorescence and electron microscopy studies reveal that mierotubules extend to CX43-based gap junctions in contacted eells. Sinee rnierotubules are dispensabie for the regulation of gap junctional communication, these findings suggest that, in addition to its weilestablished role as a ehannel-forrning protein, CX43 ean anchor mierotubule distal ends to gap junctions and thereby rnight influence the properties of mierotubules in eontacted ceils. To establish how CX43-based gap junctions are regulated by receptor stimulation, we have determined how expres sion of (mutant) eomponents of the phospholipase C pathway affects gap junctional communication. Our results support a model in which Gaq-mediated PIP2 hydrolysis, rather than second messenger production, provides the trigger for gap junction closure. Experiments using RNAi technology are underway to establish how CX4 3 deficiency affects cellular behavior. Figure HU: Fig. 5. LPA I receptor signalingpathways leading to activation ofrac and RhoA. Rac activation is mediated by G l and involves PI3K dependent activation ofthe Tiaml RacGEF. RhoA activation occurs in parallel via one or more G(alpha)12/1]-linked RhoGEF(s). Through an inhibitory cross-talk mechanism, Tiaml/Rac signaling suppresses RhoA activation. Coordinate regulation of Rac and RhoA activity thus controls LPA-induced cytoskeletal changes (cell spreading and rounding, respectively) and cell motility. Selected publications Baker DL, Morrison P, Milier B, Riely CA, Tolley B, Westermann AM, Bonfrer JM, Bais E, Mooienaar WH, Tigyi G. Plasma lysophosphatidic acid concentration and ovanan cancer. JAMA 2002; 287: Mooienaar WH. Lysophospholipids in the limelight: autotaxin takes center stage. J Cell Biol2002; 158: Mooienaar WH. The ins and outs of lysophosphatidic acid signaling. BioEssays (in press). Ruiter GA, Verheij M, Zerp SF, Mooienaar WH, Van Blitterswijk WJ. Submicromolar doses of alkyllysophospholipids induce rapid internalization, but not activation, of epidermal growth factor receptor and concomitant MAPK/ERK activation in A431 cells. Int J Cancer 2002; 102: Van Horck FP, Lavazais E, Eickholt BJ, Mooienaar WH, Divecha N. Essential role of type I phosphatidylinositol4-phosphate 5-kinase in neunte remodeling. Curr Biol 2002; 12: Van Leeuwen FN, Olivo C, Grivell S, Giepmans B, Collard JG, Mooienaar WH. Rac activation by lysophosphatidic acid LP Al receptors through the guanine nucleotide exchange factor Tiaml. J Biol Chem 2003; 278: PI3KB ; Tiam1 t Rac1 t cell spreading lamellipodia '- LPA 1 receptor ; \ Gj(By) GU 12 /13 ;? cell migration \ RhoGEF I t RhoA contraction /

37 LYMPHOCYTE ACTIVATION AND SURVIVAL Caspase-8 Group leader jannie Borst Jannie Borst PhD Group leader Annemieke De Melker PhD Post-doe Stephen Tait PhD Post-doe Yanling Xiao phd Post-doe Jenny Hendriks MSc Graduate student Anna Keiler MSc Graduate student Arlette Werner MSc Graduate student Gerda Van der Horst Technical staff Evert De Vries Technical staff Tamara Chessa Undergraduate student Martijn Kedde Undergraduate student CD95 CD27 Figure III.]: Mechanism of action oftnf receptor family members. + Bax + Bax + Bfl-1 tbid tbid tbid >< ---m- al ca mito...,~.iiii-cyt c sol mito sol I-Cyt c -Bax -Bfl-1 -tbid -Bax - Bfl-1 -tbid Figure III+ Bfl-l abrogates synergism between tbid and Bax or Bak in induction of Cyt c release. Isolated mouse liver mitochondria incubated with increasing concentrations of in vitro transcribedjtranslated proteins. Cytochrome c release is assayed by immunoblotting, presence of 35S-radiolabeled recombinant proteins in mitochondrial and soluble fractions by autoradiography. We are interested in the molecular basis oflifejdeath decisions taking pi ace in activated lymphocytes and the consequences of these decisions for lymphocyte expansion and the immune response. Our work is inspired by the desire to improve immunotherapy of cancer by rational interventions directed at sustaining survival of lymphocytes upon their activation by tumor antigens. This is expected to improve tumor immunity by enlarging the tumor-specific lymphocyte pool and by enhancing long-term maintenance of tumor-specific lymphocytes. The second aim of our work is to contribute to the design of novel therapies aimed at killing lymphoid tumors by activating apoptotic pathways. TN F receptor family members and the control of Iymphocyte expansion An adequate immune response depends on the numerical expansion of antigen-specific lymphocytes. The Go to Gr transition in lymphocytes is induced by antigen receptors, while subsequent proliferation is driven by cytokines. Throughout the expansion phase, lymphocytes acquire migratory and effector functions, allowing them to reach the site of antigenic challenge and to combat it. Resting T lymphocytes are thought to require two signals foe proliferation: one via the antigen receptor and one via a co-stimulatory receptor, of which CD28 is the prototype. Various members of the Tumor Necrosis Factor (TNF) receptor family have now moved to center stage as alternative co-stimulatory receptors. We have a long-term interest in CD27, one of these receptors. In the past year, we have compared the role of CD28 and CD27 in generation of the antigen specific Tand B cell pools. U sing influenza virus infection of mice lacking CD27 orjand CD28, we found that CD27 acts complementary to CD28 and is the principal determinant for generation of a virus-specific CD8+ T cell pool and its maintenance at the effector site (the lung). In vitro, both CD27 and CD28 rescued activated T cells from death. While CD28 promoted cell cycle entry and progression, CD27 did not. However, by stimulating the survival of activated T cells throughout successive rounds of cell division, CD27 greatly increased the yield oflive effector cells, even in the absence of CD28 signais. Adoptive transfer of fluorescently labeled T cells visualized this mechanism of CD27 action in lung-draining lymph nodes and emphasized its critical contribution to accumulation of virus-specific T cells in the lung. This work not only emphasizes the importance of CD27 in control of lymphocyte expansion, but also highlights that counteracting apoptosis is as important as stimulation of proliferation for generation of an adequate effector T cell pool. We also compared the role of CD28 and CD27 in the B cell response to influenza virus. Contrary to current dogma, CD28-/- mice could make germinal centers (GC), albeit of reduced size and frequency. Upon additional deletion of CD27, the B cell response was completely abrogated, indicating that CD27 supported GC formation in the absence of CD28. This was surprising, since we had earlier not found a defect in virus-specific Ig production in CD2i/- mice. However, detailed examination revealed a kinetic difference in GC formation between wild-type and CD2i/- mice. Adoptive T- and B cell transfer experiments revealed that this complementary pathway involves interaction between CD27 on T cells and its ligand, CD70, on B cells. In addition, we found that B cell expansion can be facilitated by CD27 on B cells. Future experiments are directed at examining the role of CD27 and its close relatives, OX-40 and 4-1BB in generation of immunological memory. This is do ne under conditions of defective or enhanced receptor function. Furthermore. in collaboration with R Van Lier at the Academic Medical Center, Amsterdam, we will identify CD27-regulated gene products and exarnine their impact on lymphocyte survival in adoptive transfer experiments. Pro- and anti-apoptotic signaling in Iymphocytes While CD27 and its relatives signal via the Traf adaptor family and in this way promote celi survival, pro-apoptotic TNF receptor family memhers (death receptors) can recruit Fadd and other adaptors, which allow them to induce apoptosis. In lymphocytes, death receptors are thought to play a role in downregulation of the immune response and selection of memory cells. We are focusing on molecular events involved in mitochondrial activation during

38 ~ ~~~ ol.,i~nli~~j~,!. :t~~~.i~ili~~.:&.~~ ~ apoptosis signaling. Induction of mitochondrial permeability and the consequent release of cytochrome care critical for the life death/decision in many ceil types in response to many stimuli. Permeability of the mitochondrialouter membrane is brought about by the collaborative action of proapoptotic BH3 domain-only Bcl-2 family members, such as Bid, and their partners Bax or Bak, which are thought to multimerize into a transmembrane pore. Earlier, we had selected variant ceils of the Jurkat T celiline for resistance to CD95-induced apoptosis. An in vitro cytochrome c release assay, using mouse liver mitochondria and recombinant proteins, revealed that resistance is due to a cytosolic factor that prevents truncated Bid from inducing mitochondrial permeability. Surprisingly, the Trail death receptor could bypass this resistance. Trail presently attracts attention as potential anti-cancer drug, because it kills a great variety of (chemo- or radioresistant) tumor ceils and tissues, while it is non-tmac for normal tissues in mouse and monkey pre-clinical modeis. We found that the Trail receptor signals to mitochondria via Fadd, Caspase-8, Caspase-cleaved Bid and Bax and in this way is indiscernible in its mechanism of action from CD95. However, the Trail receptor could bypass the cytosolic factor that prevents CD95 from activating mitochondria. l<'unctional screens have been initiated, using the introduction of retrovirallibraries into Jurkat cells, which wiil ailow the identification of selective regulators of the CD95 and Trail receptor pathways and may illuminate the partially divergent mechanism of action of these receptors. In a yeast two-hybrid screen, we found Bfl-I/AI as a Bid-interacting protein. Bfl-I is an apoptosis-inhibitory member of the Bcl-2 family, which is induced by NF-kB signals downstream from Traf-linked TNF receptor family members. We expect it to be a CD27-regulated gene product. Confocal microscopy demonstrated that Bfl-I is constitutively associated with mitochondria. It appeared to selectively bind to truncated Bid, not Bax or Bak, and to abrogate Bid-induced cytochrome c release both in vitro and in vivo. Our findings favor a model in which Bcl-2 family members function by sequestering BH3 domain-only members and preventing their interaction with Bax or Bak. We are currently testing this model using a variety of BH3 domain-only proteins and Bfl-r and Bcl-2 as inhibitory members. Since different apoptosis pathways employ different BH3 domain-only proteins, inhibitory Bcl-2 family members may be selective in their capacity to block these pathways. For example, etoposide-induced apoptosis can hardly be inhibited by Bfl-I, while the death receptor pathways are abrogated. Selected publications Tesselaar K, Arens R, van Schijndel GMW, Baars PA, Van der Valk M, Borst J, Van Oers MHJ, Van Lier RAW. Chronic costimulation via CD27jCD70 interactions exhausts the naive T cell compartment and results in lethal immunodeficiency. Nature Immunol (in press). Tesselaar K, Xiao Y, Arens R, Van Schijndel G, Schuurhuis D, Mebius R, Borst J, Van Lier RAW. Expression ofthe murine CD27ligand CD70 in vitro and in vivo. J Immunol (in press). Van Blitterswijk WJ, Verheij M, Veldman RJ, Van der Luit A, Borst J. Ceramide: second messenger or modulator of membrane structure and dynamics? Biochem J (in press). Werner AB, De Vries E, Tait S, Bontjer I, Borst J. Bcl-2family member Bjl-ljAl sequesters truncated Bid to inhibit its collabo rat ion with pro-apoptotic Bak or Bax. J Biol Chem 2002; 27T Werner AB, De Vries E, Tait SWG, Bontjer I, Borst J. TRAIL receptor and CD95 signal to mitochondria via FADD, Caspase-8jlO, Bid and Bax, but differentially regulate events downstream from truncated Bid. J Biol Chem 2002; 27T4 76o-7 Regulation of Iymphocyte activation The threshold for lymphocyte activation is set by antigen dose and the amount of antigen receptors. The Cbl family of scaffold proteins negatively regulates membrane expres sion of antigen receptors and various other tyrosine kinase-coupled receptors. We found that the Ring finger domain of c-cbl interacts with an E2-type ubiquitin-conjugating enzyme. Using the EGF receptor as a model, we are investigating the role of ubiquitination in receptor trafficking. Our finding that receptor ubiquitination occurs at the celi surface prior to receptor intemalization begged the question whether ubiquitination might regulate endocytosis. Using c-cbl and mutants, we have discovered that the ubiquitination function of c-cbl is required to recruit activated receptors into clathrin-coated pits. We found that a ubiquitin-receptor chimera constitutively traffics from the biosynthetic route via the plasma membrane to endosomes, consistent with ubiquitin serving as a signal for receptor irtternalization.

39 LlPID METABOLISM IN SIGNAL TRANSDUCTION '0 """ )( E.g Group leader WJ Van Blitterswijk Wim van Blitterswijk PhD Group leader Marcel Verheij MD PhD Academic staff Jurgen Van Baal PhD Post-doc Arnold Van der Luit PhD Post-doc Robert Veldman PhD Post-doc Alrik Los MSc Graduate student Gerald Ruiter MSc Graduate student Marianne Budde Technical staff John De Widt Technical staff Shuraila Zerp Technical staff 3H-ALP Fraction PC M LPC A B Figure HI.5: Sphingomyelin (SM) and ALP, but not lysophosphatidylcholine (LPC) accumulate in lipid rafts, separated by sucrose density gradient centrijugation. Rafts equilibrate at fractions 3 to 5. Lipids were labeled with 14C-choline. Lipid metabolism, membrane vesicular trafficking and signal transduction are tightly connected, interdependent processes_ Membrane sub-domains such as lipid rafts, being enriched in sphingolipids and cholesterol, are centers for cellular signaling and carriers of endocytic vesicular trafficking. Enzymatic breakdown and resynthesis of phospholipids is physically important, e.g. for raft coalescence and vesicle formation. It also serves the transient formation of second messengers, which recruit and activate specific enzymes_ We are investigating how certain unnaturallysophospholipids interfere with signaling and trafficking processes and can thereby act as apoptotic and anti-tumor agents. We are also modulating the physical properties of the plasma membrane to enhance uptake of anti-cancer drugs. Anti-tumor alkyl-iysophospholipids (ALPs): inducers of apoptosis Synthetic alkyl-iysophospholipids (ALPs) such as r-o-octadecyl-z-o-methyl-glycero-3- phosphocholine (Edelfosine), hexadecylphosphocholine (HePC; Miltofosine) and its piperidine analog D-zI266 (Perifosine) induce apoptosis in many tumor cens and are used as anti-cancer agents in the clinic. ALPs act on lipid-mediated signaling in cen membranes, thereby inhibiting proliferative (ERKjMAPkinase) and survival (AktjPKB) pathways, and stimulating stress-activated (JNK) pathways. We have found that ALP (Edelfosine) rapidly ins erts in the outer leaflet of the plasma membrane lipid bilayer, and becomes enriched in lipid rafts. Via these rafts, ALP is internalized by endocytosis and inhibits the de novo biosynthesis of phosphatidylcholine (PC) at its rate-determining enzyme, CTP:phosphocholine cytidylyltransferase. Continual synthesis of PC and ceramide is essential for the metabolic flux, at the Golgi, towards sphingomyelin, diacylglycerol and glycosphingolipid, which drives lipid raft formation and vesicular trafficking. Compromising this vital route by ALP will initiate apoptosis. cens can be rescued from ALP-induced apoptosis by providing them an alternative way to generate PC, that is, to let the cens acylate lysophosphatidylcholine (LPC), added exogenously. We are currently investigating precisely how and where in the cen this occurs. Contrary to ALP, the structurany similar LPC does not accumulate in lipid rafts (figure IILS), and internalizes by flipping in the fluid region of the plasma membrane. We compared the uptake of ALP in a mouse lymphoma cellline, S49, with an ALP-resistant variant, S49 AR, which showed cross-resistance to other ALPs and, intriguingly enough, also to other forms of cenular stress, such as H and ionizing radiation. S49 AR cens were defective in the endocytosis of ALP. They also displayed a dramatic reduction in the biosynthesis of sphingomyelin and, to a les ser extent, other complex sphingolipids. This suggests a defect in raft formation at the Golgi, and anomalous routing to the plasma mem brane and further endocytic vesicular trafficking. (More aspects of anti-cancer activities of ALP are reported by M Verheij, Division IX, Radiotherapy) ECF receptor internalization by low doses of ALPs While apoptosis is induced at micromolar concentrations of ALPs, we unexpectedly found that, at nanomolar doses, ALPs activated the MAPKjERK pathway in Ä431 cells without stimulating cell proliferation. Strikingly, ALPs also triggered rapid clustering and internalization of the epidermal growth factor receptor (EGFR) in these cens (Figure IIL6). Tyrphostin AGr478, an EGF receptor tyrosine kinase inhibitor, blocked ALP-induced MAPKjERK activation but not EGFR internalization. We have found no evidence for ALPs acting via G protein-coupled receptors andjor transactivation ofegf receptors, as determined by calcium mobilization, EGF receptor phosphorylation and Grbz binding assays. Since ALPs readily intercalate into the plasma membrane and accumulate in lipid rafts, our data suggest that ALPs induce subtle changes in the lipid micro-environment of the EGFR, resulting in clustering and internalization of the EG FR and concomitant ERK activation. N-hexanoyl-sphingomyelin potentiates cellular uptake and toxicity of doxorubicin Anticancer drugs generally act by binding to intracellular targets, which implicates a transport step over the plasma membrane. For amphipatic agents such as the anthracyclin doxorubicin this is presumed to occur by passive diffusion, a

40 process that is highly susceptible to the lipid composition of the membrane involved. We investigated whether pre-insertion of exogenously applied lipid analogues can improve cellular drug influx. N-hexanoyl sphingomyelin (C6-SM) was identified as a potent enhancer of doxorubicin influx in cultured endothelial cells and various tumor celllines. Whereas C6-SM itself was non-toxic, low micromolar amounts significantly enhanced doxorubicin cytotoxicity in these cell types. Importantly, the sensitizing effect of C6-SM was much less pronounced in cultured cardiac myocytes. Given that cardiotoxicity is a major dose-limiting factor in doxorubicin-based chemotherapy, co-administration of C6-SM might enlarge the therapeutic window of the drug. As to the mechanisms involved, a role for the endocytic machinery, induction of membrane leakage or ABC-transporter-mediated efflux could be excluded. Instead, we hypothesize that membrane insertion of C6-SM transiently impairs lipid packing, possibly within microdomains such as rafts or caveolae, thereby facilitating drug insertion into the bilayer. Oiacylglycerol kinases Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to yield phosphatidic acid. Since DAG activates protein kinase C (PKC), DGK may regulate PKC activation by extemal stimuli. We are currently investigating the physiological functions of the DGK-theta and-~ isotypes. In A43Icarcinoma cells, a GFP-DGKtheta fusion protein was rapidly translocated to and activated at the plasma membrane, following stimulation of purinergic P2Y receptors with ATP or UTP. DGKtheta translocation could also be evoked by phorbol ester, suggestingy that PKC exerts an inhibitory feedback mechanism by activating DG Ktheta. Translocation was independent of catalytic activity and was blocked by PKC inhibitors or by a point mutation (T42IA) in the DG Ktheta PH domain. Our data suggest that PKCepsilon is responsible for phosphorylation and translocation of DG Ktheta after purinergic receptor stimulation. The DGK~ isotype is localized in the nucleus, where it binds the retinoblastoma protein, prb. This 'pocket protein' is a key regulator of the E2F-mediated transcription and cell division. Both the nuclear localization of DG K~ and its binding to prb is negatively regulated by PKC-mediated phosphorylation of a distinct Ser residue in its MARCKS-homology domain. The function of DG ~ and other prbbinding lipid (PIP) kinases in the nucleus remains to be clarified. Selected publications Van der Luit AH, Budde M, Ruurs P, Verheij M, Van Blitterswijk WJ. AlkyUysophospholipid accumulates in lipid rafts and induces apoptosis via raftdependent endocytosis and inhibition of phosphatidylcholine synthesis. ] Biol Chem 2002; 27T Ruiter GA, Verheij M, Zerp SF, Moolenaar WH, Van Blitterswijk WJ. Submicromolar doses of alkyllysophospholipids induce rapid internalization, but not activation, of epidermal growth factor receptor and concomitant MAPKjERK activation in A431 ceus. Int] Cancer 2002; 102:] Ruiter GA, Zerp SF, Bartelink H, Van Blitterswijk Wl. Verheij M. Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol3-kinase - AktjPKB survival pathway. Anti-Cancer Drugs (in press). Van Blitterswijk Wl. Van der Luit AH, Veldman Rl. Verheij M, Borst J. Ceramide: second messenger or modulator of membrane structure and dynamics? BiochemJ (in press). Figure III.6: Low doses of ALP (Edelfosine) induce rapid (3 min) internalization ofthe EGF receptor in A431 ceus. The EGF receptor was immunostained and visualized by confocal microscopy.

41 SIGNALING THROUGH INOSITOL PHOSPHOLIPIDS Group leader Nullin Divecha Nullin Divecha PhD Group leader jonathan Halstead PhD Post-doe David jones PhD post-doe jurgen Van Baal PhD Post-doe David Weinkove PhD Post-doe Alrik los MSc Graduate student Yvette Bultsma Technical staff Satwinder Singh Undergraduate student Eukaryotic cells are constantly under the threat of oncogenesis induced by both cellular and environmental factors that lead to changes in the genetic makeup of the cello For example, viral transmission of oncogenes or DNA damage (induced by UV, X ray irradiation, cellular reactive oxygen species, etc) can lead to tumorigenesis. Normal cells, ho wever, have mechanisms (checkpoints), which sense the consequences of these changes and activate signalling pathways in order to counteract them. For example, cellular stress induced by DNA damage, leads to cell cyele arrest and rep air of damaged DNA or if the damage is too great, the elimination of the cell via apoptosis. Individuals that are compromised in these checkpoints show an increased incidence of cancer. The development of cancer is a consequence of the inhibition of checkpoint-stimulated pathways together with the up-regulation of pathways, which activate proliferation, block apoptosisjsenescence and increase motility of cells. Many of the pathways upregulated in cancer are regulated by changes in phosphoinositides, a group oflipid second messengers. Whether phosphoinositides are also involved in checkpoint-stimulated pathways is not known. Therefore our recent work has concentrated on investigating three key points: (i) Are levels of phosphoinositides regulated by cellular stress? (ii) Are the enzymes that can regulate phosphoinositides levels modulated by cellular stress? (iii) What are the potential downstream targets for phosphoinositides and are they linked to stress pathways? Our labo ra tory is focussing on Type I and Type 11 PIPkinases because in recent years we have found evidence that both these enzymes are involved in cellular responses to stress. The Type I PIPkinase is responsible for the generation of the major pool of the phosphoinositide, Ptdlns(4,5)P2' and has been implicated in numerous cellular processes, while no functional pathway regulated by the Type 11 PIPkinase has been defined (see Figure 111.7). Oxidative stress pathway Q. ~9-.. ~ ),lpki"m' ~..,... P/J-"7 ~ (31 \.(--.~ '"'- +U TI PIPkimlse J (I) ~ (5) (6) Til PIPkilJose Growth factor pathway ~4) Figure IIJI Type I PIPkinase regulates the levels of PtdIns(4,5)P 2 (1) through phosphorylation of PtdIns(4)P on the 5-position. Type I PIPkinase also generates PtdIns(3,4,5)P J (3) in vivo by phosphorylating PtdIns(j,4)P 2 (2) on the 5-Position, a pathway that is activated by oxidative stress. PtdIns(4,5)P 2 can also be synthesised by phosphorylation of PtdI ns (5) P (4) on the 4 position by a type IJ PIPkinase, although the major route of synthesis for this lipid is via the type I PIPkinase. PtdI ns (4,5) P 2 is also the substrate for a receptor activated phospholipase c which generates two new second messengers, DAG (6) and inositol(1-4-5)trisphosphate (5). Type I PI Pkinase is a target for oxidative stress We have demonstrated that exposure to oxidative stress regulates a nov el pathway leading to increased levels of Ptdlns(3A,5)P 3 synthesised by a Type I PIPkinase (see Figure 111.7). However, we have also shown that prolonged exposure to oxidative damage eventually leads to a decrease in both the Ptdlns(3A,5)P 3 and Ptdlns(4,S)P 2 levels (figure 111.8) and an attenuation of PKB signaling. Our data suggest that these changes in phosphoinositides occur through two distinct mechanisms acting on Type I PIPkinase. Firstly, Type I PIPkinase is catalytically inactivated by oxidative stress. We have identified a critical cysteine residue which, when mutated, generates an enzyme that is insensitive to oxidative stress and appears to be constitutively active when expressed in vivo. This suggests that cellular reactive oxygen species may be an important physiological regulator of Type I PIPkinases. Secondly, oxidative stress causes the enzyme to dissociate from the plasma membrane (where its substrate resides). Prolonged oxidative stress will eventually lead to the onset of apoptosis which we show can be attenuated by overexpression of Type I PIPkinase. Studies have demonstrated that malignant tissues show enhanced PIPkinase activity, which may be a mechanism by which tumor cells, which are of ten exposed to a variety of stresses, avoid decreases in phosphoinositides and thus the induction of cell death. Phosphoinositides in the nucleus Our group, together with others, have demonstrated that phosphoinositides in the nueleus are modulated in a different way than are their plasma membrane counterparts. We have developed and utilized sensitive mass assays to show that the levels of nuelear phosphoinositides change as cells progress from Gl to S-phase of the cell cyele and in response to cellular stress induced by UV, X-ray and oxidative damage. How these changes in phosphoinositides occur is not elear. However, we have discovered that the retinoblastoma protein (prb), which controls Gl to S-phase transition and is required for stress-mediated cell cyele arrest, interacts with and regulates the activity ofboth a Type I and Type 11 PIPkinase. We have also found that prb interacts with the 1; isoform of the diacylglycerol (DAG) kinase family, an enzyme that modulates the levels of intranuelear DAG, and is implicated in the regulation of cell cyele pro gres sion.

42 o PKB...L. Cell survival via PKB is downregulated APOPTOSIS TI PIPkinase gplns(3,4)p2 gplns(4,5)p2 gplns(3,4,5)p3 Figure III.8: J2 P-orthophosphate labelled cells were stimulated with H 2 0dor the indicated times (minutes). After extraction the lipids were deacylated and chromatographed on a PEl cellulose plate. Acute exposure to oxidative stress leads to the synthesis of Ptdlns (j,4,5) Py however, prolonged exposure leads to a decrease in both Ptdlns(4,5)P 2 and Ptdlns (j,4,5) P 3 levels. The model suggests that increased reactive oxygen (ROS) leads to both catalytic inhibition of the type I PIPkinase and its dissociation from the membrane. The decreases in the levels of Ptdlns(4,5)P 2 and Ptdlns(3,4,5)P 3 would lead to the induction of apoptosis. We have also identified a number of proteins with known nuelear functions that interact with phosphoinositides, and thus are likely to represent potential downstream targets. Future studies will de fine the relationship between cellular stress, nuelear phosphoinositides and the function of these proteins. Type 11 PIPkinase regulates oxidative stress signalling in vivo In mammais, there are three Type land three Type II PIPkinase isoforms, whereas C. elegans contain only one isoform of each, making it an ideal system to study PIPkinase function genetically. In order to define a functional role for Type II PIPkinase we have isolated a deletion mutant in C.elegans (ppk-2). The ppk-2 mutant is more sensitive to developmental arrest induced by oxidative stress (H Z 02) compared to wild type larvae. Genetic analysis showed that ppk-2 regulates responses to oxidative stress through the Forkhead transcription factor (daf16) and can cooperate with the PI-3-kinase pathway to regulate DAF-r6 function. The exquisite neuronallocalisation ofppk-2 (Figure III.9) suggests that it operates in a neuronal pathway that regulates responses to stress. In collaboration with R Medema (Division V) and B Burgering (UM C Utrecht) we are studying the mechanisms of F orkhead regulation by Type II PIPkinases in mammalian cells. Forkhead activity can lead to cell cyele arrest and apoptosis and is subject to negative regulation by the oncogenic PI-3-kinasejPKB pathway. Our data from C.elegans would suggest that the status of Type II PIPkinase may be important in defming Forkhead activity in tumor cells. Selected publications Oivecha N, Roefs M, Los A, Halstead j. Bannister A, O'Santos C. Type I PIPkinases interact with and are regulated by the retinoblastoma susceptibility gene product pre. Curr Bio12002; 12: Itoh F, Oivecha N, Brocks L, Oomen L, Janssen H, Calafat J et al. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-bjSmad signalling. Genes Cells 2002; 7:] Jones OR, O'Santos CS, Merida I, Oivecha N. T lymphocyte nuclear diacylglycerol is derived from both de nova synthesis and phosphoinositide hydrolysis. Int] Biochem Cell Bio12002; 34: Muris 0, Verschoor T, Oivecha N, Michalides R. Constitutive active GTPases Rac and Cdq2 are associated with endoreplication in P AE cells. EurJ Cancer 2002; 38:1775. Van Horck FP, Lavazais E, Eickholt BJ. Mooienaar WH, Oivecha N. Essential role of type I (alpha) phosphatidylinositol 4-phosphate 5-kinase in neurite remodeling. Curr Bio12002; 12: Ventral nerve cord Figure 111.9: C.elegans werefixed and immunostained using aspecific antibody to type I I PIPkinase. The enzyme appears to be localised at the plasma membrane and is expressed in all neurones including those of the nerve ring and in the ventral nerve cord.

43 TGF-~ SIGNAL TRANSDUCTION Division head Peter Ten Dijke Peter Ten Dijke PhD Group leader Sylviane Dennier PhD Post-doe Marie-José Goumans PhD Post-doe Susumu Itoh PhD Post-doe Olexander Korchynskyi PhD Post-doe Franck lebrin PhD post-doe Michiru Nishita PhD Post-doe Ester Piek PhD Post-doe Bernard Roeien PD Post-doe Fumiko Itoh MSc Graduate student Gudrun Valdimarsdottir MSc Graduate student Midory Thorikay MSc Technical staff Maarten Van Dinther Techninal staff Floor Frederiks Undergraduate student Jingyuan Fu Undergraduate student Transforming growth factor-~ (TGF-~) family members, which include TGF-~s, activins and bone morphogenetic proteins (BMPs), regulate a broad spectrum of biological responses on a large variety of celi types. TGF-~ family members have critical roles during embryogenesis and in maintainlng tissue homeostasis during adult life. Deregulated signaling by TG F-~ family members has been implicated in multiple developmental disorders and in various human diseases, including cancer, fibrosis and auto-immune diseases_ Our goals are to elucidate the molecular mechanisms by which TGF-~ family members elicit their celiular effects and to generate animal models for human diseases that are caused by perturbed TGF-~ family signaling. TGF-~ family members trans duce their signals across the plasma membrane by inducing the formation of specific heterorneric complexes of type I and type II serine/threonine kinase receptors (Figure IIl.ro). The type I receptor, also known as activin receptor-like kinase (ALK), acts downstream of type II receptor, and has been shown to determine signaling specificity within the receptor complex. The activated type I receptor propagates the signal by phosphorylating specific receptor regulated (R)-Smads. Whereas Smadz and Smad3 act downstream oftgf-~ and activin type I receptors, Smadr, Smads and Smad8 are phosphorylated by BMP type I receptors. Phosphorylated R-Smads form complexes with the common partner (Co)-Smad, i.e. Sma<4, which translocate into the nucleus, where they participate in regulation of gene expression (Figure IIl.ro). Smad activation and subceliular distribution are intricately regulated and many Smad interaction partners have been identified by us and others. Inhibitory (1-) Smads, i.e. Smad6 and Smad7, form a distinct subclass among Smads by acting opposite from the signal transducing R- and Co-Smads. i i e ~n Smad lnteracting.. ::"pöon GV ----~ G0 ~ - ~ Figure lii.10: TGF-f3family members induce heteromeric complexformation between type land type I I serinejthreonine kinase receptars. The activated type I receptor phosphorylates R-Smads that farm heterameric complexes with Co-Smad and translocate into the nucleus. Within the nucleus the heterameric Smad complexes can directly, or through transcriptional partners, bind to specific sequences in the promaters of target genes. TG F -13 family members have also been reported to activate smalt GTPases and MAP kinases. H owever, the (patho-) physiological significance of these Smad-independent pathways downstream of type I receptor are unclear. Abbreviations: JNK, c-jun N-terminal kinase, TAB, TAK-l bindingprotein, TAK, TGF-f3 activated kinase; XIAP, X-linked inhibitor of apoptosis protein. TGF-~ receptor signal transduction Escape from TGF-~/Smad-induced growth inhibition and apoptosis is frequently observed in tumors. Certain Smads have been found to be mutated in specific types of cancer and gene ablation of particular Smads in rnice has revealed increased rate in tumorigenesis. In late stage tumors, TGF-~ has been shown to function as tumor promoter. TG F-~ may mediate these effects directly on tumor celis via subverted Smad-dependent and/or Smad-independent pathways (Figure IIl.ro). To obtain more insight into TGF-~ receptor-initiated intraceliular responses that are distinct from Smad pathway, we have analyzed the activation of smali GTP-binding proteins and MAP kinases by TGF-~. We observed that TGF-~ can activate Rac and ERK, JNK and P38 MAP kinases in certain celi types. The rapid kinetics and the TGF-~-induced activation of these components in celis deficient of Sma<4 support the notion that these effects occur in a Smadindependent manner. In addition, we have generated a mutant TGF-~ type I receptor defective in Smad activation and characterized its signaling properties. The T~R-I-induced extracellular matrix induction, growth inhibition and epithelial to mesenchymal trans-differentiation were found to be dependent on the Smad pathway, but JNK MAP kinase activation did not require the Smad pathway. Further studies are ongoing to determine the (patho-) physiological importance of Smad independent signaling using the Smad defective T~R-I mutant and by transcriptional profiling on cens that lack certain R-Smads or Sma<4. TGF-~ family members and angiogenesis TGF-~ family members are potent regulators of proliferation and migration of endothelial cens and smooth muscle celis, the two celi types that from blood vessels. Moreover, genetic studies in humans and mice have revealed the importance oftgf-~ family members in vascular morphogenesis. In most cen types TGF-~ signals via T~R-I/ALKS. However, we have found that in endothelial celis TGF-~ can bind and trans duce signals via two distinct type I receptors, i.e. ALKr and ALKS. The widely expressed ALKS induces the phosphorylation of Smadz and Smad3, while ALKr, which is predominantly expressed in endothelial cens stimulates Smadr and Smads phosphorylation. Interestingly, the TGF-~/ALKS and TGF-~/ALKr were found to have oppposite effects on endothelial behaviour; ALKS inhibits EC migration and proliferation while ALKr stimulates both processes. We have identified genes that are induced

44 specifically by TGF-B-mediated ALKI versus ALK5 activation (Figure IILn). The activation state of the endothelium may be dependent on the level oftgf-b-induced ALKlor ALK 5 activation. Our results provide a framework to understanding previously conflicting reports in which pro- and anti-angiogeneic properties were ascribed to TGF-B. t Id11 Activation phase TGF-~ I \. ALK-1 ALK-5 P~~~~~i~n + I..-T_u_b_e_f_o_rm_a_t_io_n... 1 PAI-1 t Resolution phase Figure III.1l: TGF-f3 regulates the state of the endothelium via a balance between ALKI and ALK5 signaling. Idl was found to mediate the TGF-f3jALKI-induced migration, while induction of plasminogen activator inhibitor-l (PAl-I) by activated ALK5 may contribute to the TGF-b-induced maturation ofblood vessels. The ratio oftgf-f3 signals via ALKI versus ALK 5 determines whether TG F-f3 wil! have a pro- or antiangiogenic effict. Immunohistochemical analysis of tissue sections showed that BMP receptors are expressed in endothelial cells in vivo. BMP receptor activation was found to stimulate endothelial cell migration and tube formation in vitro (Figure ). IdI was identified as important BMP jsmad target gene; IdI is sufficient and necessary for BMP-induced endothelial cell migration. These results provide important new insights into the molecular mechanisms that underlie cardiovascular phenotypes in mi ce and humans with perturbed BMP jsmad signaling pathways. -BMP-6 +BMP-6 Selected publications Blokzijl A, Ten Dijke P, Ibanez C. GATA3-mediated recruitment ofsmad3 to target promoters allows TGF-f3 regulation of cytokine expression. Curr Biol 2002; 12J5-45 Goumans M-l, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, Ten Dijke P. Balancing the activation state of the endothelium via two distinct TG F -f3 type I receptors. EMBO J 2002; 21: He W, Li AG, Wang D, Han S, Zheng B, Goumans M-J, Ten Dijke P, Wang Xl. Overexpression ofsmad7 results in severe pathological alterations in multiple epithelial tissues. EMBO J 2002; 21: Korchynskyi 0, Ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Idl promoter. J Biol Chem 2002; 27T Rosendahl A, Pardali E, Speletas M, Ten Dijke P, Heldin C-H, Sideras P. Activation ofbone morphogenetic proteinjsmad signaling in bronchial epithelial cells during airway injlammation. Am J Respir CeU Mol Bio12002; 2p60-9. Ten Dijke P, Goumans M-l, Itoh F, Itoh S. Regulation of ceu proliferation by Smad proteins. J Cell Physiol2002; 191:1-16. Ten Dijke P, Korchynskyi 0, Valdimarsdottir G, Goumans M-l. ControUing cell fate by BM Preceptors. Mol Cell Endocrinol (in press). Figure III.12: BMP-6 stimulates formation of cord-like structures on Matrigel. Controlling mesenchymal cell fate by BM Ps Depending on their extracellular stimuli, mesenchymal cells can differentiate into muscle, fat, bone or cartilage. BMPs have important roles in directing mesenchymal cell fate choices. The expression of CbfaIjRullX2 and IdI were found to be potently induced upon BMP receptor activation in C2CI2 cells. We found that these two genes cooperate in BMP-induced osteoblast differentiation and inhibition of myoblast differentiation. IdI was identified as a direct target ofbmps, but not TGF-B. We identified three distinct sequence elements in the IdI promoter, i.e_ Smad binding elements (SBEs), GGCG and CAGC sequence motifs with pivotal role in the activation of this promoter by BMP_ Smads were found to be part of transcription factor complexes that specifically bind to SBEs and CCGCCC palindromic sequence in response to BMP but not TGF-B. Multimerization of all three distinct sequence motifs is needed to generate a highly sensitive and BMP jsmad dependent specific enhancer. We have found that clusters of all three sequence elements are also important for BMP-induced activation of other BMP target genes. Valdimarsdottir G, Goumans M-J, Rosendahl A, Brugman M, Itoh S, Lebrin F, Sideras P, Ten Dijke P. Stimulation of Idl expression by bone morphogenetic protein Is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial ceus. Circulation 2002; 106:

45 IV DIVISION OF IMMUNOLOGY DEVELOPMENT AND FUNCTION OF LYMPHOID CELLS Division head, group leader Hergen Spits Hergen Spits PhD Group leader Kees Weijer DVM PhD Academic staff Bianca Blom PhD Post-doc Ramon Gimeno PhD Post-doc Rosalie Luiten PhD Post-doc Marianne Naspetti PhD Post-doc Wendy Dontje MSc Graduate student Suzanne Ligthart MSc Graduate student Ferenc Scheeren MSc Graduate student Remko Schotte MSc Graduate student Maho Nagasawa Technical staff Arie Voordouw Technical staff Pauline Weder Technical staff Marie Anne van Halem Secretary Our research has two aims: one is to elucidate the mechanisms underlying development ofhuman lymphocytes, in particular those oft celis, from common lymphoid progenitor celis. The second aim is to generate immortalized cultures of primary human T and B cens and to explore their use for therapeutic applications. Regulation of Iymphoid development by transcription factors and cytokines In the thymus, multipotent hematopoietic stem cens develop not only into T cens but also into various dendritic cell (OC) subsets, macrophages and NK celis. The mechanisms of cen fate determination of thymic progenitor cens are as yet incompletely understood. Over the past years, several 'master' genes essential for development of certain celilineages have been identified. Such master genes direct differentiation in part by shutting off programs for other, competing, lineages. One example is the helix loop helix factor Id2, which is required for NK cell development and inhibits T, Band plasmacytoid OC development. Human plasmacytoid OC (poc), also called type 2 OC precursors or natural IFN-producing cens, represent a cen type with distinctive phenotypic and functional features. These cells are present in peripheral blood and various organs including the thymus. Recently, we found that poc in the periphery and thymus develop independently from each other. Thymic poc probably shares a common precursor with Tand NK celis. In an effort to identiry genes that control poc development, we have searched for genes of which the expression is restricted to human poc using a cona subtraction technique with activated monocyte-derived OC (Mo-OC) as competitor. We identified the transcription factor Spi-B to be expressed in poc but not in Mo-Oe. Spi-B belongs to the Ets family of transcription factors and is highly homologous to PU.I. Spi-B expression in poc was maintained upon in vitro maturation of poe. Spi-B was expressed in early C034 +C038- hematopoietic progenitors and in C034+COra- thymic precursors. Spi-B expression is downregulated when uncommitted C034+COra- thymic precursors differentiate into committed C034+COra+ pre T celis. Overexpression ofspi-b in hematopoietic progenitor cens resulted in inhibition of development oft celis both in vitro and in vivo. In addition, development of progenitor cells into Band NK cells in vitro was also inhibited by Spi-B overexpression. Our results indicate that Spi-B is involved in the control of poc development by limiting the capacity of progenitor celis to develop into other lymphoid lineages. GATA-3, a zinc finger transcription factor, was shown to be essential for T-cell development in the mouse. Using a GATA-3 RETROSUPER RNAi vector, we have demonstrated that inactivation of GATA-3 in fetalliver progenitor celis impaired their capacity to develop into T cells. Interestingly, overexpression of GATA-3 in precursor celis strongly inhibited development into poc and B celis and impaired NK-cen development. Moreover, T-cell development was stimulated by overexpression of GATA-3. These data identify GATA-3 as an important regulator ofhuman T-cell development. Early TCRaB lineage cells with functional TCRB rearrangements are selected in a process called B-selection. Cells with non-functional TCRB rearrangements are eliminated during B-selection. We have found evidence for an involvement of the phosphatase PTEN in this process. The lipid phosphatase PTEN is a negative regulator ofthe PI-3K pathway, which regulates survival ofvarious cen types. PTEN is a tumor suppressor frequently mutated in a remarkable variety of tumors. By dephosphorylating phosphoinositoi3,4,5 triphosphate (PIP3) it counteracts PI-3K, which catalyzes the reverse reaction. To investigate aspecific role of PTEN during T-cell differentiation, the Cre-IoxP system was used to generate mice in which Pten was disrupted in T-lineage cens in collaboration with P Krimpenfort (Oivision of Molecular Genetics). Analysis of these mice revealed that T-cell specific deletion of Pten results in perturbation in early TCRaB cen development around the B-selection checkpoint. Importantly, deletion of Pten in mice deficient of C03Y, which have a

46 r ~i. ~I~1.J~l.l~.~TÄ_~~ r ~ small thymus due to incomplete ~-selection, resulted in a numerical reconstitution of the thymus size and a selective survival of CD4 +CD8+ thymocytes negative for TCR~ protein. Strikingly, these mice develop lymphomas within the thymus at I.S-3 months of age. Alllymphoma's expressed high levels of the TCR/CD3 complex and were mostly CD4 and CD8 double positive. Our fmdings suggest a link between disturbance of ~-selection by Pten deletion and lymphomagenesis. T eell-mediated immunity against melanoma; immortalizing tumor-speeifie CTL for application in therapies Studies in various mouse models for example for prostate cancer, liver tumors, and brain tumors have demonstrated that adoptive therapies are superior to vaccination in the induction of anti-tumor immunity. A problem with application of adoptive transfer of tumor-specific CTL clones is their limited replicative life span that prevents large scale in vitro expansion of CTL to numbers required for adoptive transfer. We reported previously that overexpression of wild type human telomerase reverse transcriptase (htert) immortalizes both CD4+ and CD8+ T cells without loss of function, antigen-specificity and growth characteristics. A number of immortalized CTL clones we re tested together (with N Verra, this division) for their capacity to eliminate tumor eells in vivo. We demonstrated that htert immortalized Mart-r/MelanA-speeific CTL clones partly reduced melanomas which grew in immunodeficient mice. As expeeted CTL clones specific for influenza virus epitopes were unable to attaek the melanoma eelis, however, these latter CTL clones were very effective in removing the same melanoma eells which overexpressed the viral epitope recognized by these clones. We found that the influenza virus specific CTL clones are much more aetive than the Mart-r-speeific CTL. The relatively low efficiency of the Mart-r-specific CTL in attacking melanoma cells in vivo is probably related to the lower avidity of these self-reaetive CTL clones for the melanoma eells. Immortalization of human B eells A desirabie goal in B-cell biology is to establish long-term cultured lines of normal human B cells in vitro. B cells can be cultured in vitro following engagement of CD40 and the IL-2 or IL-4 reeeptors. Such eells initially proliferate vigorously but have a limited replicative life-span preventing application of sueh cells for production ofhuman antibodies with desired specifics. The meehanisms responsible for the limitation of replieative life-span are poorly understood. We have examined the role of the signal transdueer of activation and transcription (STATS) in proliferation ofhuman B eelis, sinee STATS molecules are activated by IL-2 and IL-4, both B cell growth factors. We found that introduction of eonstitutive active mutants (CA) ofstatsb into human B eells enhaneed their proliferative activity and greatly extends their replicative life span. We have constructed a fusion product of CA-STATSb and a mutated estrogen receptor (ER) that allowed regulation of CA-STATSb function by the horrnone 4-hydroxytamoxifen (4-HT). Following introduction of this fusion product by retroviral transduction, B eelllines were obtained that proliferated in response to CD40L and IL-2 or IL-4 in a 4-HT-dependent way. Such B cells can be maintained in vitro for extended periods of time. The cultured CA-STATSb-ER transduced B eells expressed cell surf ace immunoglobulin, around half of the cells express kappa and the other lambda light chains. We also demonstrated that STATSb directly controls expression of BCLG, which encodes a transcriptional repressor, frequently activated by chromosomal translocation in non-hodgkin's lymphoma and essential for the formation of germinal centres. A functionallink between STATSb and BCLG is indieated by our observation that BCLG expression in human B eells also immortalizes them. The observation that proliferation, replicative life-span and differentiation can be manipulated by genetic switches, as exemplified by the inducible STATSb construct, opens the perspective of generating monoclonal antibodies from human B cells either of exposed or deliberately immunized individuals. Selected publications Blom BR, Schotte R, Ligthart S, Spits H. Development ofhuman pre-dc2. Human Immunol (in press). Schotte R, Rissoan MC, Bendriss-Vermare N, Bridon JM, Duhen T, Weijer K, Briere F, Spits H. The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T, B, NK and T cell development. Blood (in press). Shvarts A, Brummelkamp T, Scheeren F, Koh E, Daley GQ, Spits H, Bernards R. A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative P19ARF_P53 signaling. Genes Dev 2002; 16: Spits H. Development oftcraf3 T cells in the human thymus. Nature Rev Immunol 2002; Van Montfrans C, Hooijberg E, Sol Rodrigez Pena M, De Jong EC, Spits H, Te Velde AA, Van Deventer S. Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer. Gastoenterology (in press). Weijer K, Uittenbogaart C, Voordouw A, Couwenberg F, Seppen J, Vyth-Dreese FA, Blom B, Spits H. Intrathymic and extrathymic development ofhuman plasmacytoid dendritic cell precursors in vivo. Blood 2002; 99:

47 DISSECTING VIRUS AND TUMOR SPECIFIC IMMUNITY The aim of oui research is straightforward I) to design novel technologies that can be used to examine and modify protein interactions that control T-cell immunity; 2) To use these tools to umavel and manipulate the molecular processes underlying immune recognition by T lymphocytes. Group leader Ton Schumacher Ton Schumacher PhD Group leader Nathalie Brouwenstijn PhD Post-doe Anna Calogero PhD Post-doe Arne Bakker MSc Graduate student Miriam Coccoris MSc Graduate student Karin De Visser MSc Graduate student Helmut Kessels MSc Graduate student Koen Schepers MSc Graduate student Monika Wolkers MSc Graduate student Jeanine Joling Technical staff Gitte Sotthewes Technical staff Erwin Swart Technical staff Mireille Toebes Technical staff Jos Urbanus Technical staff Marly Van den Boom Technical staff Sonja Noback Trainee technician Tumor-specific cytotoxic T-cell immunity In a subset ofhuman tumors such as melanoma, significant tumor-specific T-cell responses can be observed in patients (together with J Haanen, this division). There has been a long-standing debate on the mechanism through which the immune system is alerted to such solid tumors which, at least initially, grow outside the lymphoid organs. It has been postulated that induction of cytotoxic T cells against tumor antigens OCCUIS through direct interaction of CD8+ T cells and tumor cells that have migrated to the draining lymph nodes. Alternatively, evidence has been obtained for cross-presentation of tumor antigens as the relevant mechanism, in which host antigen pres enting cells take up antigens and present these antigens to tumor-specific T cells in the lymphoid organs. To address the relative merits of these two processes, we have previously set up a murine tumor model in which tumor-specific T-cell responses can be directly visualized by MHC tetramer technology. We have used this model to demonstrate that both direct priming and cross-priming can be efficient mechanisms for the induction of tumor-specific T-cell immunity. The crucial issue that has remained is under which conditions direct and cross-presentation can or cannot OCCUI. Recent experiments by the groups ofj Neefjes (Division of Tumor Biology) and J Yewdell (NIH, Bethesda, USA) have provided evidence for the notion that a major fraction of the antigenic peptides that are presented by MHC class I via the endogenous pathway is derived from newly synthesized proteins. Using a series of constructs that contain two T-cell epitopes, one in the signal peptide and one in the mature protein part, we have now provided evidence that whereas direct presentation correlates with synthesis rate, cross-presentation correlates with accumulation of the protein from which the epitope is derived (Figure IV.I). These data reveal a fundamental difference between the exogenous and endogenous MHC class I pathway and this antigen bias in cross-presentation affects the outcome of vaccination strategies that rely on this process. s(np)-gfp-hpv NPCTL epitope GFP HPVCTL epitope s(hpv)-gfp-np HPVCTL epitope GFP NPCTL epitope NP-specific T cell response ( cross-priming) HPV -specific T cell response ( cross-priming) s(np)-gfp-hpv 10 Day s(np)-gfp-hpv s(hpv)-gfp-np 10 Day s(hpv)-gfp-np 10 Day 10 Day Figure IVx Antigen bias in cross-presentation. To examine differences in antigen requirements between direct presentation and cross-presentation tumor cells were transduced with vectors that encode T-cell epitopes in the signal peptide and mature protein part ofgfp. Conventional direct presentation is indistinguishable for epitopes encoded at either site (not shown). In contrast, T cell responses induced through cross-presentation are heavily biased towards the antigen encoded in the mature protein part.

48 Induction and maintenance of CD4+ T-cell immunity To provide a better understanding of the development and regulation of CD4 + T-cell immunity, we have developed strategies for the generation of mouse and human MHC class 11 tetramers. In collaboration with F Ossendorp and C Melief (LUMC, Leiden), we have first used the murine-specific MHC class 11 reagents to determine the relationship between the development ofcd4+ and CD8+ T-cell immunity in a retrovirus-induced sarcoma model. In recent experiments, we have used human influenza antigenspecific MHC class 11 tetramers in collaboration with E Wiertz (LUMC, Leiden) to track the adverse effect of the Epstein Barr Virus gp42 protein on the induction of CD4+ T-cell immunity. TCR gene therapy Data from human allogeneic trans plant studies and animal experiments provide very strong evidence that circumvention of self-tolerance is crucial for the induction of strong tumor-specific T-cell immunity. Indeed, allogeneic transplantation, which is based on minor histocompatibility antigen differences between host and recipient, is currently the only accepted form oft-cell based tumor immunotherapy. While minor histocompatibility antigen differences can result in tumor regression, such differences are also the cause of Graft-versus Host disease, and the development of more selective forms of adoptive therapy would be desirabie. In the past years, our group has pioneered the retroviral introduction of antigen-specific T-cell receptors into peripheral T cells as a means to induce virus and tumor-specific immunity in vivo. In this strategy, autologous or donor-derived T-cell populations are equipped with a TCR of defined reactivity in short-term ex vivo procedures, and re-infusion of the redirected cells is used to supply T-cell reactivity against defined antigens. These experiments have revealed that T cells redirected by TCR gene transfer expand dramatically upon viral infection of mice and efficiently home to effector sites. Furthermore, very small numbers of TCR-transduced T cells can promote the rejection of antigen-expressing tumors in vivo. IfTCR gene therapy can be developed into a viabie clinical strategy it should have a number of important advantages. TCR gene transfer would allow the instantaneous generation of defined T-cell immunity. Furthermore, TCR gene therapy would allow the introduction oftcrs with specificities not present naturally, thereby providing a strategy to circumvent the limitations of the endogenous Tcell repertoire. In collaboration with K De Visser and A Kruisbeek, we have analyzed 'the shape' of the hole in the T-cell repertoire that is formed by the presence of selfantigens. These data demonstrate that self-tolerance has little impact on the T-cell repertoire for structurally related antigens, aresult that argues in favor of the use of TCR optimization approaches to gather a collection of desirable self-specific T-cell receptors. Several groups are currently aiming to test the value oftcr gene transfer in clinical trials, but a number of issues may require prior preclinical evaluation. A particular issue is formed by the extensive polymorphism of MHC genes. T-cell receptors are normally selected against reactivity with self-mhc molecules complexed with endogenous peptides during thymic development. However, T-cell receptors transferred into a new genetic background will be confronted with novel MHCjself-peptide combinations and it is currently unclear whether cross-reaction of the introduced TCR with non-donor MHC products could form a significant risk factor for the development of autoimmunity. A major effort in our lab in the past year has therefore been to determine the feasibility oftcr gene transfer in settings where the 'TCR recipient' is matched with the 'TCR donor' for the relevant MHC allele (the restriction element), but is mismatched for other MHC alleles. While this is still 'work in progress', the results obtained to date are encouraging. Selected publications Dickinson AM, Wang XN, Sviland L, Vyth-Dreese FA, Dunn J, Jackson GH, SchumacherTNM, Haanen JBAG, Mutis T, Goulmy E. In situ dissection of Graft-versus-Host activities of cytotoxic T ceus specific for minor histocompatibility antigens. Nature Med 2002; 8: Kessels HWHG, Ward AC, Schumacher TNM. Specificity and affinity motifs for Grb2 SH2-ligand interactions. Proc Natl Acad Sci USA 2002; 99: Kessels, HWHG, Wolkers MC, Schumacher TN M. Adoptive transfer of T ceu immunity. Trends Immunol 2002; 23: Kessels HWHG, Wolkers MC, Van den Boom M, Van der Valk M, Schumacher TNM. Immunotherapy through TCR gene transfer. Nature Immunol2001; 2: Schepers K, Toebes M, Vyth-Dreese F, Dellemijn TAM, MeliefCJM, Ossendorp F, Schumacher TN M. Di1ferential kinetics of antigen-specific CD/CD8+ T ceu responses in the regression of retrovirus-induced sarcoma. ] Immunol2002; 169:] SchumacherTNM. Tcel! receptor gene therapy. Nature Rev Immunol2002; 2: Wolkers MC, Stoetter G, Vyth-Dreese FA, Schumacher TN M. Redundancy of direct priming and cross-pnming in tumor-specific CD8+ T ceu responses. ] Immunol2001; 16T Wolkers MC, Toebes M, Okabe M, Haanen JBAG, SchumacherTNM. Optimizing the efficacy of epitope-directed DNA vaccination. ] Immunol2002; 168:

49 IMMUNOTHERAPY I The primary aim of this group is to develop cytokine-based immunotherapies and advanced technologies for characterization of immune responses in peripheral blood and at the tumor site. Group leader Eert De Gast Bert De Gast MD PhD Group leader Marie-Jose Kersten MD Academic staff Florry Vyth-Dreese PhD Academic staff Natascha Verra MSc Graduate student Trees Dellemijn Technical staff Johan Sein Technical staff Willeke Van de Kasteele Technical staff Paul Van den Berk Technical staff Adoptive transfer of htert transduced human CTL in mice bearing autologous human melanoma lung tumors To test the effect ofhuman CTLs against the autologous tumor, in collaboration with K Weijer, H Spits and R Luiten (this division) a model has been developed using Rag-2jcommon y chain double knockout mice, lacking T, Band NK cells, to facilitate transplantation ofhuman cells. In this model, a human melanoma cellline is used to generate lung metastases and the anti-tumor activity of two autologous CTL dones, both recognizing the same Mart-I peptide in HLA-A2 was tested, in comparison with two control dones recognizing an influenza peptide in HLA-A2. All dones had been immortalized by transduction with htert to enable large-scale expansion of the cells. Upon intravenous injection of MelAKR melanoma cells, anti-tumor effects ofctl, their number found in the lungs as wen as the number and totalload of the metastases, were determined by immunohistochernistry. Single injection of Mart-I specific CTL had a limited effect on the number and total tumor load in the lungs. Repeated injections induced significant reduction of 30-50% of the number oflung microtumors and 50% of total tumor load. Injection of influenza specific CTL had no effect on tumor growth. In condusion, human CTL recognizing their autologous tumor can exert in vivo anti-tumor activity in this model. However, total tumor eradication was not achieved. Higher expression of the antigen on the tumor might enhance recognition of the tumor cells by CTL and may therefore lead to stronger anti-tumor activity. To test this, a melakr variant overexpressing Mart-I and influenza epitopes has been generated. Surprisingly, Mart-I specific dones, which lysed these tumor cells in vitro, showed no in vivo tumor reduction. The influenza specific dones showed similar in vitro toxicity towards the transduced melanoma cens as the Mart-I dones. When these cens were injected into tumor-bearing mice, tumor reduction was found between 30% and 85% after a single injection. Currently, we are investigating these four dones for differences that can explain the divergent results found in the in vitro and in vivo experiments. Effect of peri-operative immunotherapy in peripheral blood and at the tumor site in renal cell cancer and head and neck cancer patients Renal cen carcinoma (RCC) is an immunogenic tumor. The current treatrnent for metastatic RCC is immunotherapy with the cytokines IL-2 and IFNa. IL-2 stimulates T cells, whereas IFNa enhances MHC dass I expres sion. GM-CSF potentially enhances immunotherapy by generating and differentiating dendritic cens (DC) necessary to initiate specific immune responses. To test this combination of cytokines, perioperative immunotherapy from day - 3 till day + 5 af ter the operation was given to RCC patients (in conaboration with S Horenblas and A Bex, division XI and 0 De Jong, division XIII) in a phase I dose-escalation study, where radical surgery can remove alllocally visible tumor. The peri-operative setting has been chosen for two reasons. First, immunotherapy is thought to counteract the immune suppressive effect of the surgical procedure itself. Seeondly, in a situation of minimal residual disease, the immune system, if activated, should be able to attack this minimal amount of tumor and so may prevent the outgrowth of micro-metastases. The maximal tolerable dose was 2.5 Ilgjkg GM-CSF, I MIU jm 2 IL-2 and 3 MIU IFNa. Higher doses of IL-2 indueed proteinacious fluid aecumulation in the contralateral pleural cavity and peritoneally after the operation. Compared to patients with nephreetomy without immunotherapy post-operative immune depression in peripheral blood was eompletely inhibited with the highest dose of GM-CSF. Immunohistology revealed an increase of activated CD8+ T cells in the tumor (median 3.8x; infiltrating in between tumor cells) and ofsioo+ and CD83+ dendritic eens (2-20X increase) (Figure IV.2). Accumulation of these cells was not seen in normal renal tissue. Quantification was performed by computer-assisted measurement of the area of staining in microscopic slides. Subsets of CD8+ T cens and DC will be further characterized by 3-color confoeallaser scanning rnicroscopy

50 Selected publications Figure IV.2: Infiltration oft ceus in primary renal ceu carcinoma in a patient treated with cytokine immunotherapy and in a non-treated control patient. (CLSM) to determine their activationjmaturation state, effector function and cytokine expres sion in situ. The treatment is now given as pre-operative therapy (day - 9 till- 2), which is better manageable in a clinical setting and may in duce a more sustained effect towards the renal tumor. De Gast GC, Vyth-Dreese FA, Nooijen W, Van den Boogaard CjC, Sein J, Holtkamp M, Linthorst G, Baars JW, Schornagel JH, Rodenhuis S. Re-infosion of autologous lymphocytes with GM-CSF induces rapid recovery ofcdl and CD8+ T ceus after high dose chemotherapy for metastatic breast cancer. ] Clin Oncol 2002; 20: Dickinson AM, Wang XN, Sviland L, Vyth-Dreese FA, Dunn J, Jackson GH, Schumacher TNM, Haanen JBAG, Mutis T, Goulmy E. In situ dissection of Graft-versus-Host activities of cytotoxic T ceus specific for minor histocompatibility antigens. Nature Med 2002; 8: In situ analysis of human thymic T-cell and antigen presenting cell development In an ongoing conaboration with the group ofh Spits (this division), in vivo development and maturation of precursor populations oft cens and DC, injected into Rag 2jcommon y chain double knockout mice, was fonowed in situ. Analyses including the development, activation and maturation status of subsets of both lymphoid and myeloid DC lineages have been published. In situ analysis of antigen presenting cell subsets in inflammatory lesions: activation/maturation and cytokine profiles Inflammatory lesions of patients suffering from inflammatory bowel disease we re studied for expres sion of antigen presenting cens (in conaboration with the group of S Van Deventer and A Te Velde, AMC, Amsterdam). The CLSM technique proved very useful in discriminating subsets of antigen presenting cens (DC and macrophages) in colon lesions of patients with Crohn's Disease as wen as in non-affected colon tissues ofhealthy donors or unrelated patients. Using multicolor analysis, insight was gained in the state of activationjmaturation and cytokine potential of these cen types; strong expres sion ofil-r2, IL-r8 and TNFa was found, pointing to the Thr character of this disease. These studies may have direct implications for future immunotherapy approaches and form the basis for analysis of tumor lesions. Detection of antigen specific T Iymphocytes in experimental and human tissues Immunohistological techniques have been developed to visualize tetrameric peptidejmhc complexes binding antigen specific and virus specific CD8+ as wen as CD4+ T lymphocytes. In situ analysis of (subsets of) immune cens and accessory cens has added to out understanding of effector mechanisms operational in different modeis, a retrovirus induced sarcoma model and a P-Mel transgenic mouse melanoma model; in the latter, in situ localization of specific effector T cens nicely related to vitiligo effects as wen as specific tumor attack. In addition, the technique has been successfully applied for the detection of minor antigen specific T cens in a human skin explant model of Graft versus Host reactivity in a conaborative study with the group of E Goulmy (LUMC, Leiden). Ongoing studies indicate that this technique anows detection of antigen specific T cells, not only in fresh, but also in cryopreserved tissues and allows visualization of the functional status of cens in situ as wen. This novel immunohistochemical approach will provide a tooi for further detailed in situ studies ofhuman T-celljtumor cen interactions, the unravelling of t.~e Graft versus Host reaction in human skin and the monitoring of immunotherapy triéils. Schepers K, Toebes M, Vyth-Dreese F, Dellem ijn TAM, MeliefCjM, Ossendorp F, Schumacher TNM. Differential kinetics of antigen-specific CDlcD8+ T ceu responses in the regression of retrovirus-induced sarcoma. ] Immunol2002; 169:] Te Velde AA, Van Kooyk Y, Hommes D, Dellemijn T, Slors JFM, Vyth-Dreese FA. Characterization of dendritic ceus in the colon ofcrohn's disease patients: subsets of dendritic ceus with different IL-12 and IL-18 production. EurJ Immunol (in press). Weijer K, Uittenbogaart CH, Voordouw A, Dellemijn TAM, Couwenberg F, Seppen J, Blom B, Vyth-Dreese FA, Spits H. Thymus dependent and independent development of human plasmacytoid dendritic ceus in vivo. Blood 2002; 99:

51 IMMUNOTHERAPY 11 The main objective of this line of research is the development of novel T-cell immunity based strategies for the treatrnent of patients suffering from solid tumors, especially melanoma and renal cell carcinoma. Group leader John Haanen john Haanen MD PhD Group Leader Willem Overwijk PhD Post-doe Adriaan Bins MD Graduate student Annelies jorritsma MSc Graduate student Esmeralda De jong Technical staff Raquel Gomez Technical staff Felicia Tirion Technical staff Spontaneous T-cell immunity in advanced-stage melanoma patients In a large group ofhla-a* 020r-positive stage IV melanoma patients, the presence of spontaneous melanoma-specific CD8+ T-cell immunity is studied systematically in close collaboration with the group ofb De Gast (this division). Peripheral blood samples, taken before the start of chemotherapy-based treatrnent are analyzed using MHC tetramer technology. In collaboration with the group oft Schumacher (this division), soluble multimers ofhuman MHC class land 11 molecules complexed with melanoma antigen-derived peptides have been developed. At the moment, a large set ofmhc tetramers containing peptides from either melanocyte differentiation antigens MART-r, tyrosinase and gproo or products from cancerjtestis genes NY-eso-r and MAGE family members is available for detection of melanoma-specific T cells. Naturally occurring T-cell immunity against MART-r and tyrosinase is more common in these patients than any other antigen tested so far, amounting to respectively 40% and ro%. The frequency of these cells in peripheral blood is highly variabie but frequencies of> ro% of the total peripheral blood CD8+ T-cell pool have been observed (see Figure IV.3). Whereas especially MART-r-specific T cells can be detected in healthy individuals, the melanoma-specific T cells found in these patients are higher in number and display a memory phenotype, indicating recent antigen encounter and expansion. Interestingly, the clinicaloutcome of patients with high frequencies of melanomaspecific peripheral CD8+ T cells is significantly worse than that of patients without detectable melanoma-specific T cells. Furthermore, the frequency of patients with melanoma-specific T-cell immunity amongst stage 111 disease patients appears to be much lower than we find amongst stage IV disease patients. These results suggest that the presence of melanoma-specific T-cell immunity is a reflection of the tumor load and spread of the disease and therefore is oflittle clinical benefit. Thus, strategies aiming at amplification of melanoma-specific T-cell immunity should be developed for patients with minimal residual disease. Melanoma-speeifie TeelIs in the blood from patients..,. + ~ ~ C') MART-l Tyrosinase Gpl00... ~ 'Ot~ ~ ~ I (\j (\j (\jo < ~ ~,...;j ::r:: ~~ ~~ ~o ~ 0 0 0,.. ~ ~ ~ Q,j e.....,. =,.. 'Ot~... ~ ~ Q,j... M < C')~ C') C') ~ ~ < (\j~ C\I "b,...;j ~ ::r:: ~~ ~~ ~~ ~ ~ ~ CD 4, 13, 14, 16 and 19 Figure IV.]: Peripheral blood mononuclear ceus from 3 HLA-N~o201-positive (top) or HLA-A *o201-negative (bottom) melanoma patients were stained with a panel of monoclonal C') antibodies against lineage markers and HLA-A*020I tetramers complexed with MART-I, tyrosinase and gploo peptides and subsequently analyzed by flow cytometry. In the upper lejt quadrant of each dot plot from HLA-A*0201-positive melanoma patients MDA-specific CD8+ T ceus are visualized. HLA-A*020I negative samples were used as negative controls. 1

52 In vivo requirements for T-cell mediated regression of established tumors Tumors are notoriously po or inducers oft-cell immunity. Vaccination can give rise to specific immune responses, yet only results in durable tumor regres sion in a small fraction of cancer patients. We have developed vaccination strategies against a nonmutated 'self antigen, gproo, expressed on melanoma and normal melanocytes. Using tetramer analysis, we have found that gproo-specific transgenic CD8+ T cells were neither activated nor deleted in mice bearing the gproo+ melanoma BI6. Upon vaccination with gproo peptide, we have seen a small, therapeutically ineffective increase in gproo-specific T cells in blood and spleen. Vaccination with an altered gpioo peptide with higher affinity for MHC Class 1 molecules has induced strong T-cell responses that modestly inhibited tumor growth (see Figure IV.4). Administration of the T-cell growth factor interleukin-2 (IL-2) greatly improved the anti-tumor effect of vaccination, leading to regression and even complete cure of I4-day, 10 mm solid tumors. Histological analysis (in collaboration with F Vyth-Dreese and T Dellemijn, this division) revealed greatly enhanced T-cell accumulation and tumor destruction in tumors from mice receiving IL-2, whereas F ACS analysis showed that inhltrating T cells from IL-2 treated animals exhibited spontaneous IFNy production directly ex vivo. Together, these results define in vivo requirements for regression of established tumors: i) adoptive transfer of tumorspecific T cells, ii) powerful vaccination with altered peptide ligand, iii) administration of IL-2. This work was initiated in the former group of A Kruis beek. Development of vaccination strategies as adjuvant treatment for high risk melanoma patients Peptide-based vaccines are designed for clinical application in advanced-stage melanoma patients. Helper epitopes and adjuvants, such as GM-CSF, are added for amplification of peptide-induced T-cell responses. Furthermore, a GMP DNA vaccination unit is being built for the production of naked DNA vaccines for clinical application. In a humanized mouse model, both DNA- and peptide-based vaccines are being validated before clinical use. Selected publications Dickinson AM, Wang XN, Sviland L, Vyth-Dreese FA, Dunn J, jackson GH, Schumacher TNM, Haanen jbag, Mutis T, Goulmy E. In situ dissection of Graft-versus-Host activities of cytotoxic T ceus specific for minor histocompatibility antigens. Nature Med 2002; 8: Schumacher TNM, Haanen jbag. Ex vivo and in situ detection of tumor-specific T ceu immunity. In: Stauss, HJ, Kawakami Y, Parmiani G, (Editors). Tumor antigens recognized by T ceus and antibodies with MHC tetramers. Tumor Immunology Series, Vol. In Harwood Publishers (in press). Wolkers Me, Toebes M, Okabe M, Haanen jbag, Schumacher TNM. Optimizing the efficacy of epitope-directed DNA vaccination. J Immunol2002; 168: N" 150 E.s GI. ~ E 2 50 vacc. days after tumor inoculation ex u '0 60 ~ 50 ex u 30 E ~ vacc. days after tumor inoculation Figure IV+ Persistenee and Junction of gp100 reactive ceus after adoptive tranifer, vaccination and administration of IL-2 in mice bearing subcutaneous B16 tumors. a, C57BL/6 mice bearing subcutaneous B16 tumors treated with lx10 7 CD8+ pmel-l T ceus + vaccination with lx10 7 pju recombinant vaccin ia virus (rvv) and 100,000 IU ril-2 twice daily for 5 days. pmel-l + rvvhgploo + IL-2 (_), pmel-l + rvvhgploo (0 ), pmel-l + rwlacz + IL-2 (.), or no treatment (X). b, Tetramer analysis of PBLfrom mice described in (a). CDS+Vfhl ceus are depicted as % oftotal CD8+ ceus.

53 V DIVISION OF MOLECULAR BIOLOGY MOUSE MODELS FOR HEREDITARY CANCER Division head, group leader Hein Te Riele Hein Te Riele PhD Group leader Jan-Hermen Dannenberg PhD Post doe Jacob Hansen PhD Post-doe Marije Scholte PhD Post-doe Camiel Wielders PhD Post-doe Nanna Claij MSc Graduate student Floris Foijer MSc Graduate student Marjolein Sonneveld MSc Graduate student OlafVan den Broek Undergraduate student Jaco Van der Torre Undergraduate student Tinke Vormer Undergraduate student Oliver Wiebenga Undergraduate student Conny Brouwers Technical staff Marleen Dekker Technical staff Sandra De Vries MSc Technical staff Valerie Doodeman Technical staff Elly Van Riet Technical staff Anja Van der Wal Technical staff EB ~ ~~~ )C'C... r-- Figure V.l: Model for mismatch-repair-directed antirecombination Genetic instability and deregulated cell cycle control are hallmarks of human cancer. Research in our group involves both aspects focusing on (I) cancer predisposition by loss of DNA mismatch repair functions and (2) the role of the retinoblastoma gene family in cell cycle control and tumor suppression. The principle tools include gene inactivation in embryonic stern (ES) cells and analyses of the phenotypic consequences in homozygous, heterozygous and chimeric knockout mice and in cell lines derived thereof. DNA mismatch repair Cancer predisposition in HNPCC, the non-polyposis form of hereditary colorectal cancer, is mainly caused by a defect in one of the DNA mis match rep air proteins, hmsh2 or hmlhr. While the primary function of the mis match rep air (MMR) system, the correction of mis- or unmatched nucleotides that can arise by erroneous DNA replication, is well-established, its role in two other mutation-avoidance mechanisms is less well-studied. These are suppression of recombination between homologous but not identical DNA sequences and the cellular response to certain types of DNA damage. To study the role of mismatch recognition proteins in the different MMR functions, we have generated ES celllines carrying disruptions in the central MMR genes Msh2, Msh6 and Msh3. To study MMR-dependent anti-recombination, we have constructed a recombination reporter consisting of a neg gene that was rende red inactive by either a two base-pair insertion in the open reading frame or a base-pair substitution in the start codon. We found that both types of mutations could be effectively restored by targeting vectors that correct the mutated neo sequence, but that the efficiency of gene correction in Msh2 ḻ cells was 10- to 5o-foId higher than in wildtype cells. Thus, a single base-pair difference is already sufficient to activate MMRdependent anti-recombination (Fig. V.I). This prompted us to investigate whether MMR deficiency would allow gene modification mediated by small synthetic oligonucleotide sequences. We indeed found that single-stranded oligonucleotides consisting of deoxyribonucleotides could be used to delete, insert or substitute one or a few base pairs. While in Msh2-deficient cells a frequency was obtained of 2-4 per 10 5 ceils, the presence ofmsh2 in wild-type cells reduced the efficiency of oligonucleotide-mediated gene correction up to 300-fold. The efficiency of 'oligo targeting' in Msh2-deficient cells was sufficiently high to identify and clonally purify modified cells by PCR-based procedures. In this way, we have substituted a single codon in the retinoblastoma suppressor gene Rb. However, we realise that the general application of this oligo targeting protocol may be hampered by the mutator phenotype associated with Msh2 deficiency leading to inadvertent genetic alterations above the desired oligonucleotide-mediated modification. Therefore, we are currently developing procedures to transiently inactivate MSH2. We expect that under such conditions, oligo targeting may occur sufficiently effective while the accumulation of spontaneous mutations remains below an acceptable level. The role of MMR in mediating the toxicity of methylating agents is weil established and has been attributed to the processing of 06-methylguanine lesions in DNA. We have found that in mice, Msh2 deficiency strongly potentiates the oncogenic potential of an ethylating agent, N-ethyl-N-nitrosourea (ENU). This suggests that mismatch-repair deficiency and ENU-induced mutagenesis synergistically collaborate in inducing tumorigenesis. We recently found that MSH2-deficient ES were highly sensitive to ENU-induced mutagenesis. Mutation analysis of Hprt mutants resulting from the combination ofmmr deficiency and ENU-induced DNA damage, revealed that base substitutions occurred predominantly at AT base pairs rather than GC base pairs. These results indicate that MMR modulates the processing of ethylation damage at AT base pairs.

54 r- ~Il ~! ~ :t~jq.~ia ~IL ~ The retinoblastoma gene family We have previously analyzed the response of poeket-protein-deficient primary mouse embryonie fibroblasts (MEFs) to a number of growth-inhibiting conditions and found that ablation of ali three pocket proteins fully alieviated eeu eyele arrest imposed by serum withdrawal, eeu-eeu contact and DNA-damage. To determine the eapaeity of single pocket proteins to elieit anormal response, we eompared eeli eyele profiles of MEF cultures of ali three double knoekout combinations under growth-inhibiting eonditions. The eeli eyele profiles of growing cultures were not the same for all genotypes. The ratio of G 1 over S-phase eeus eould be ranged as fouows: wildtype=prb+>plo7+pi30+=plo7+>pi30+>tko. At conjluency, this ratio inereased approximately four-fold for wild-type and Plo7/PI30 expressing eens and 2 to 3-fold for eaeh of the single pocket protein expressing genotypes while TKO eens did not arrest at all. Thus, eaeh of the three pocket proteins was effeetive in implementing a growth arrest upon eeli-eeli contact, albeit somewhat less effeetively than wild-type eelis. A somewhat different picture emerged upon serum withdrawal. Cen cyele profiles we re determined af ter I, 2 and 3 days of eulturing under low serum. The ratio of G 1 over S-phase eeus inereased maximany in wild-type and prb-expressing eeus. In contrast, eens expressing only PlO7 or PI30 showed a modest inerease in the GI/S ratio, although they elearly responded better than TKO eelis. CeUs expressing both PlO7 and PI30 performed better than the single expressors. Finally, we measured the response of eens to ionizing radiation. The inerease of the GdS ratio was highest in wild-type and prb-expressing eelis, only modest in PlO7- and Plo7/pI3o-expressing eelis and virtually absent in eens devoid of pocket proteins or expressing only PI30. The results of these experiments are qualitatively summarized in Figure V.2. While eaeh single pocket protein ean mediate eeu eyele arrest upon eeli-eeu contact, prb is the eritieal mediator of een eyele arrest upon growth factor deprivation and irradiation. Plo7 ean partially eompensate for the absence of prb under both eonditions. PI30 ean mediate a modest response upon serum withdrawal whieh is additive to that of PlO7, but does not play a role in the response of eens to ionizing radiation. The striking similarity between the behaviour of Rb/PlO7- and Rb/pI30-defieient MEFs is further underseored by the finding that both genotypes eaused immortality and reversed the growth-inhibitory effect ofras vi2 into a proliferative stimulus. This similarity is also seen in vivo: ehimerie miee generated from Rb/plO7-defieient or Rb/PI30 defieient ES eeus, developed retinoblastoma with eharaeteristies of the amaerine eeli eompartment of the retina. By labeling the ES eens with a LaeZ marker, we we re able to identify lesions in the developing retina of ehimerie mice showing enhaneed and ectopie incorporation of Brd U, but also elevated levels of apoptotie eeu death. This suggests that simultaneous loss of prb and either PlO7 or PI30 sustained prolonged eeu turn over that may prove to be essential for retinoblastoma development later in life. Our eurrent work is foeusing on the identification of additional genetic events that eouaborate with loss of the Rb gene family in oneogenie transformation. Selected publications Claij N, and Te Riele H. Methylation tolerance in mismatch repair proficient cel!s with low MSH2 protein level. Oncogene 2002; 21: Kohonen-Corish M RJ, Daniel JJ, Te Riele H, Buffinton GD, Dahlstrom JE. Susceptibility of Msh2 deficient mice to inflammation-associated colorectal tumors. Cancer Res 2002; 62: Q in J, Baker S, Te Riele H, Liskay RM, and Arnheim N. Evidencefor the lack of mismatch-repair directed antirecombination during mouse meiosis. ] Heredity 2002; 9Y201-5 Rayman JB, Takahashi Y, Indjeian VB, Dannenberg JH, Catchpole S, Watson RJ, Te Riele H, Dynlacht BD. E2F mediates cel! cycle-dependent transcriptional repression in vivo by recruitment ofan HDAC1/mSin3B corepressor complex. Genes Dev 2002; 16: Van den Broek W, Nelen MR, Wansink DG, Coerwinkel MM, Te Riele H, Groenen PJTA, and Wieringa B. Somatic expansion behaviour ofthe (CTG )n-repeat in myotonic dystrophy knock-in mice is differential!y affected by Msh3 and Msh6 mismatch-repair proteins. Human Mol Genet 2002; 11: Yoshino M, Nakatsu Y, Te Riele H, Hirota S, Kitamura Y, Tanaka K. Additive roles ofxpa and MSH2 genes in UVB induced skin tumorigenesis in mice. DNA Repair 2002; 1: Geno- Cell/eell low Irra- sene- RAS VI2 PI9 ARF Retinotype contact serum diation scence blastoma Wild type no prb no P /- +/- +/- yes PI /- +/- yes P107+PI3O / /- no TKO ND Figure V.2: Pocket protein requirement for cel! cycle arrest and tumour suppression. (++ strong arrest; + arrest; +/- moderate arrest; - no arrest; ND not determined)

55 ANTIGENIC VARIATION IN TRYPANOSOMES Group leader Piet Borst Piet Borst MD PhD Group leader Henri Van luenen PhD Academic staff Jan Wijnholds PhD Academic staff (honorary) Henk De Vries PhD Post-doc Rainer Mussmann PhD Post-doc Nobuhito Ono MD PhD Post-doc Glen Reid PhD Post-doc Peter Wielinga PhD Post-doc Cristiane Bentin Toaldo MSc Graduate student Paul-André Genest MSc Graduate student Sebastian Ulbert MSc Graduate student Zhong Yu MSc Graduate student Noam Zeker MSc Graduate student Sietske Bakker Undergraduate student Mira Franken Undergraduate student José Koppes Undergraduate student Marcel De Haas Technical staff Rudo Kieft Technical staff Annemieke Kuil Technical staff liesbeth Van Deemter Technical staff Ingrid Van der Heijden Technical staff linda Römer Secretary Bartie Van Houten Secretary Selected publications Borst P. Antigenie variation and allelic exclusion. Ce1l2002; 109."5-8. Borst P, Oude Elferink R. Mammalian ABC transporters in health and disease. Annu Rev Biochem 2002; 71: The Mrican trypanosome Trypanosoma brucei can freely multiply in the bloodstream of its mammalian host. It escapes complete destruction by the host immune system by antigenic variation of its surf ace coat. We are studying the molecular mechanisms of antigenic variation in strains of T. brucei that are not infectious in humans and that can readily be grown in rnice and in vitro culture. Our recent work has focussed on two issues: antigenic variation of the transferrin receptor of T. brucei and the biosynthesis and function of the unusual DNA base J. Base J (B-glucosyl-hydroxymethyluracil), which we discovered in trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993; 7S:II29-36), is a minor base present in all kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in DNA and is predominantly located in repetitive sequences, such as telomeric repeats. Base J is synthesized by modification of thymine in DNA, but we have not yet found the enzymes involved. However, we have characterized a J-binding protein (JBP1) that binds with high specificity to J-containing duplex DNA (Cross et al. EMBO J 1999; 18: ). A JBPl gene knock-out in T. brucei has the remarkable effect of reducing the J content of trypanosome DNA to 5% of wild type, without any detectable physiological consequences. In the related kinetoplastid Leishmania, a JBP1 KO is lethal and we are studying why. The biosynthesis of J involves hydroxymethyluracil (HMU) as an intermediate. This is also a product of oxidative DNA damage and many organisms contain one or more DNA glycosylases that take out HMU. We have found that such enzymes are lacking in trypanosome extracts, as one would expect. Indeed, introduction of an inducible gene for such an enzyme (SMUG1) kills the trypanosome through DNA fragmentation caused by extensive DNA repair. We are testing whether the absence of this class of DNA repair enzym es makes trypanosomes hypersensitive to some DNA damaging agents. Transferrin receptors To satisry its need for iron the trypanosome takes up transferrin (Ti) from its mammalian host by means of a heterodimeric transferrin receptor (Tf-R). We have shown that T. brucei can make about 20 different Tf-Rs differing slightly in amino acid sequence. This diversity in Tf- Rs allows the trypanosome to cope with the diversity in Tfs in the large range of mammals that it can colonize (Bitter et al., Nature 1998; 391: ). We have found that the trypanosome can use several methods to produce sufficient amounts of a suitable 7 6 A twd active ~ 7' 6' A' mln 0 I expression site inactive expression site Burg D, Wielinga P, Zelcer N, Saeki T, Mulder GJ, Borst P. Inhibition ofthe M ultidrng Resistance Protein 1 (M RP1) by Peptidomimetic Glutathione-Conjugate Analogs. Mol Pharmacol2002; 62: Cross M, Kieft R, Sabatini R, Dirks-Mulder, A, Chaves I, Borst P. J binding protein increases the level and retention of the unusual base J in trypanosome DNA. Mol Microbiol (in press). in situ switch - o express other Tf R and VSG altered expression ' increased RNA stability 'higher expression of the active site 'higher expression from the inactive sites -. twd - I tv,.., DI express more and different Tf Rs Tf R gene convers ion tv..., - I ~ WO 0 I express different Tf R Figure V.j: Transferrin receptor (TfR) variants oft. brncei. The figure shows a simplified scheme of a telomeric expression site for Variant Surface Glycoprotein (VSG) genes, A is the VSG gene and 7 and 6 are the genes for the Tf R sub-units in the active expression site. The flag is the promoter and the broken arrow indicates the extent of transcription. A', 7' and 6' are the corresponding genes of one of the nineteen inactive expression sites.

56 Tf-R variant, as schematically indicated in Fig. V-3- We are especially interested in mechanisms allowing overproduction oftf-rs, which appear to be regulated by changes in RNA processing. MULTIDRUG RESISTANCE OF CANCER CELLS We are interested in mechanisms of drug resistance in cancer cells and focus on resistance caused by increased ATP-dependent transport of drug out of the cello We have isolated the genes for these transporters and are characterizing their substrate specificity and sensitivity to inhibitors in transfected cells. We make extensive use of the baculovirus system to produce insect cell membrane vesicles containing high amounts of transporter protein and suitable for vesicular transport studies. We also use transfected polarized kidney cell monolayers in which the transporters either route to the apical or the basolateral membrane, allowing the study of vectorial transport through the celilayer. Drug transporters causing chemotherapy resistance in tumors are present in (some) normal tissues, so we are also interested in their physiological nmction in metabolism and defence of the body against drugs and xenotoxins. We have therefore inactivated several genes for drug transporters by targeted gene disruption in mice. Initially, we looked at P-glycoproteins; most recently we have studied the Multidrug Resistance Protein family members MRP3, 4, and 5, and have just begun to study MRP8 (collaboration with Ira Pastan, Bethesda, USA) and MRP9 (collaboration with Toshi Ishikawa, Tokyo, Japan). M RP3 We have shown that this transporter is an organic anion transporter, like other MRPs, but only marginally associated with drug resistance. Some of the major bile salts are low-affinity substrates for MRP3 (collaboration with R Oude Elferink), but we have recently found that glucuronidated hydroxylated bile salts, formed in the liver during cholestasis, are high affinity substrates (collaboration with HU Marschall, Stockholm). In the Mrp3 KO mouse, generated in our lab, we are now testing the hypothesis that Mrp3, located in the basolateral membrane of the hepatocyte, helps the liver get rid of toxic organic anions, such as bile salts, when bile flow is blocked. M RP4 and M RP5 We have shown that MRP5 is an organic anion transporter with the unusual ability to transport nucleotide analogs. Similar results were obtained with MRP 4, a close relative of MRP5 (collaboration with J Schuetz, Memphis). Other groups have found that MRP4 and MRP5 can transport the cyclic nucleotides cgmp and camp and have suggested that both transporters might help limit the intracellular concentration of these second messengers. U sing intact transfected cells and vesicular transport assays, we have found transport by MRP 4 andjor MRP5 of several nucleotide analogs used in cancer or antiviral chemotherapy, but the affmity ofboth transporters for these compounds is low, making a major role in drug resistance in patients doubtful. Very low affinity was also found for cgmp and camp, casting doubt on the importance of cmrp4 and 5 in cyclic nucleotide disposition. This scepticism appears justified by our recent finding that MRP 4 is able to transport some conjugated bile acids and steroids, such as dehydroepiandrosterone-3-sulfate, with high affinity, providing a plausible physiological function for this transporter (Fig. VA). We are still searching for a highaffinity substrate for MRP5 and our Mrp5 KO mouse has no phenotype thus faro Selected publications Icontinued) Sabatini R, Meeuwenoord N, Van Boom JH, Borst P. Recognition ofbase i in duplex DNA by i-binding protein. i Biol Chem 2002; 27T Sabatini R, Meeuwenoord N, Van Boom JH, Borst P. Site-specific interactions ofibp with base and sugar moieties in duplex i-dna. Evidence for both major and minor groove contacts. i Biol Chem 2002; 27T Ulbert S, Cross M, Boorstein Rj, Teebor GW, Borst P. Expression ofthe human DNA glycosylase hsmug1 in Trypanosoma brucei causes DNA damage and interferes with i biosynthesis. Nucleic Acids Res 2002; 30: Wielinga PR, Reid G, Challa EE, Van der Heijden I, Van Deemter L, De Haas M, Mol C, Kuil Aj, Groeneveld E, Schuetz J D, Brouwer C, De Abreu RA, Wijnholds j, Beijnen JH, Borst P. Thiopurine metabolism and identification ofthe thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney ceus. Mol Pharmacol 2002; 62: i I o.oso J! i.{l [DHEAS) (~Mr' o [DHEAS] (~M) Figure V+ Vesicular transport of dehydroepiandrosterone-sulfate (DH EAS) by membrane vesicles from insect (Sf9) ceus expressing a baculovirus M RP 4 construct. The va/ues are corrected for the (low) transport by vesicles from wild-type Sf9 ceus.

57 CEll CVClE CHECKPOINTS Group leader René Medema René Medema Group leader Alexandra Bras Post-doe Jamila laoukili Post-doe Susanne lens Post-doe Marc Schmidt Post-doe Rob Wolthuis Post-doe Marie Stahl Graduate student Barbara Van de Weerdt Graduate student Marcel Van Vugt Graduate student Marieke Rozendaal Undergraduate student Gerben Vader Undergraduate student Jos Kauw Technical staff Rob Klompmaker Technical staff Cell division of eukaryotic cells is a highly regulated process. Cells can respond to a variety of extracellular signais, which together dictate cellular behaviour, ineluding the decision to proliferate, differentiate, or undergo apoptosis. Cell proliferation is controlled by multiple growth-regulatory pathways acting together to ensure proper cell division. At the late G 1 restriction point (R point) the cell weighs the activity of positive and negative regulatory signais. Part of our research is focused on the mechanisms by which growth factors, through their cognate receptors, modulate the activity of cell cyele regulatory proteins that control passage through G r. After passage through the R point, mitogenic growth factors are no longer required for cells to complete division and cells become refractory to growth inhibitory signais. Instead, cells come to rely upon the intrinsic regulators of the cell cyele machinery for orderly progression through the remainder of the cell cyele. Several checkpoints have been defined that monitor progress through the various stages and can block the initiation of the next cell cyele phase if errors are detected. For example, DNA damage checkpoints arrest cell cyele progression at different stages so that the damage can be repaired before cell division. In addition, the spindle checkpoint prevents separation of sister chromatids before all chromosomes have attained bipolar attachment to the spindle. This checkpoint ensures accurate separation of the sister chromatids to the daughter cells. As such, these checkpoints are essential for genomic stability. Selected publications Barnouin K, Dubuisson ML, Child ES, Fernandez De Mattos S, Glassford j, Active PKB/Akt Active FoxO, -.. "..0(:..' Medema RH, Mann Dj, Lam EW. H induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21cip1 expression. ] Biol Chem 2002; 27Tl Kops GJPL, Dansen TB, Polderman PE, Saarloos I, Wirtz KWA, (offer Pj, Huang T-T, Bos JL, Medema RH, Burgering BMT. The Forkhead transcription factor FOX03a/FKHR-L1 protects quiescent cells from oxidative stress. Nature 2002; 419J Kops Gj, Medema RH, Glassford j, Essers MA, Dij kers PF, (offer PJ, Lam EW, Burgering BM. Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol Cell Bio12002; 22: Mackus Wj, Lens SM, Medema RH, Kwakkenbos MJ, Evers LM, Oers MH, Lier RA, Eldering E. Prevention of B cell antigen receptor-induced apoptosis by ligation of CD40 occurs downstream of cell cycle regulation. Int Immunol2002; 14: Medema JP, Medema RH. Chromosome instability: lessonsfrom HIV. Nat Cell Biol 2002; 4: Figure V.5: PKB/Akt-induced proliferation is coupled to a highly glucose-dependent state, while FoxOinduced quiescence causes cells to become less dependent on glucose. Tumor cells with constitutive active PKB/Akt will be unable to make the metabolic switch necessary for survival under conditions oflow glucose-availability. Indeed, many tumors are highly dependent on glucose (Warburg effect), and die very rapidly upon removal of glucose from the culture medium. Our results have demonstrated that the activation of FoxO is sufficient to protect cells from death induced by glucose-deprivation, and that this involves transcriptional regulation ofthe anti-oxidant enzyme MnSOD. Forkhead transcription factors and cel! cycle regulation In elose collaboration with a number of groups at the University Medical Center in Utrecht, we have studied the function of the FoxO subfarnily of forkhead transcription factors in regulating cell cyele progression. FoxO transcription factors are negatively regulated by protein kinase B (PKB/Akt). We have shown that FoxO factors positively regulate transcription of the cdk-inhibitor P27kipI, but in addition, cause the repression of cyelin Dr/D2 expres sion. As such, activation of FoxO factors is sufficient to force cells into a state of quiescence, as we have shown in a variety of cell types. We have recently been able to demonstrate that FoxO-induced quiescence is tightly coupled to an altered metabolic state of the quiescent cells. We could show that FoxO directly regulates the expres sion of the anti-oxidant enzyme MnSOD, and that this induction is responsible for protection against oxidative stress under conditions oflow glucose availability. These data combined sketch a picture in which PKB/Akt forces cells into a glucose-dependent proliferative state, while on the other hand FoxO factors drive cells into a state of quiescence that can survive under conditions oflow glucose availability. Finally, in sharp contrast to our observations in fibroblasts, we have shown that the activation of FoxO in lymphocytes results in programmed cell death and the activation of apoptosis-promoting Bcb-related proteins. Future experiments will be directed at a better understanding ofhow the same pathway can cause such very distinct cellular responses in different cell types. In parallel, we have studied the role of another Forkhead transcription factor, FoxMr/Trident. We have shown that FoxMr is involved in checkpoint control that prevents DNA re-replication during G2. We are currently investigating the

58 mechanism by which FoxMr ensures normal alternation between S-phase and mitosis, and attempt to identiry FoxMr target genes by cd NA-array analysis that could play a role in this. These projects are performed in collaboration with B Burgering (Laboratory for Physiological Chemistry, UMC Utrecht), P Coffer (Departrnent of Pulmonary Diseases, UMC Utrecht) and H Clevers (Department ofimmunology, UMC Utrecht). Cell cycle checkpoints in G 2 and mitosis During the last three years we have concentrated on checkpoint functions in G 2 and mitosis. Firstly, we have studied the role of p2r and Polo-like kinase-r (Plkr) in DNA damage responses during these phases of the cell cyele. We have shown that p2r blocks the activating phosphorylation of CdC2 on ThrrGr and shown that Plkr is inhibited in an ATMjATRdependent fashion by DNA damage in G 2 and in mitosis, which constitutes a novel mechanism by which DNA damage can prevent activation of CdC2SC. Our data provide important extensions to the existing models on DNA damage checkpoints. Our data with p2r demonstrate the existence of multiple DNA damage activated pathways that inhibit CdC2 activity. In addition, we have shown that Plkr is also inhibited in cells that have passed the G 2 jm transition, providing the first example of a mammalian kinase that is inhibited in mitosis by DNA damage. These data indicate that a DNA damage checkpoint is also active in mitosis in mammalian cells, similar to what has been described in yeast. We are currently studying the diverse roles ofplkr in mitosis. Many distinct functions have been ascribed to PIkr, and the combination of the available data suggests that PIkr-functionality must somehow be modified as cells progress through mitosis. We would like to understand how those different functions ofpikr are separated throughout mitosis. To this end we have established inducible celllines that express a variety of PIkr-mutants, in which we are studying the effect of such mutants on mitotic entry, APC-activation and spindle checkpoint function. In addition, we are using RNAi-mediated knock-down of PIkr to follow mitotic progression of cells depleted of PIkr by time-lapse microscopy. These combined approaches will hopefully help us understand the multiple roles of Plkr and how cells have developed mechanisms to keep those functions separated in time and space. Last but not least, we are studying the role of survivin in cell cyele regulation versus programmed cell death. I t has been proposed that survivin functions at the interface between mitotic progression and apoptosis. We have developed RNAi-vectors that successfully target survivin expres sion. These vectors are currently being used to allow time-lapse microscopy of survivin-depleted cells as well as a variety of flow cytometric assays that we have developed for a comprehensive analysis of mitotic processes. Selected publications (continued) Schmidt M, Fernandez de Mattos S, Klompmaker R, Kops GJPL, Lam E W-F, Burgering BMT, Medema RH. FoxO forkhead transcription factors induce cel! cycle arrest by downregulation of D-type Cyclins. Mol Cel! Biol2002; 22: Stahl M, Dijkers PF, Kops GJPL, Coffer PJ, Burgering BMT, Medema RH. The Forkhead transription factor Fox03 regulates transcription of P27kip1 and Bim in response to IL 2. J lmmunol 2002; 168: Van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Battle E, Coudreuse 0, Haramis A-P, Tjon-Pon Fong M, Moerer P, van den Born M, Soete G, Pais S, Eilers M, Medema RH, Clevers H. The f3.cateninjtcf-4 complex controls the proliferationjdifferentiation switch in colon epithelium through c-myc mediated repression of p21cip1jwaft. Cel! 2002; 111: Figure V.6: Localization of survivin during the different stages of mitosis as determined by confocal analysis. Survivin (arrows indicate bright staining) localizes to the kinetochoresjcentromeres in prometaphase, but translocates to the spindle midzone during anaphase and ends up in the midbody in telophase. (DNA is light grey in these pictures).

59 VI DIVISION OF TUMOR BIOLOGY ANTIGEN PRESENTATION SY MHC CLASS land 11 MOLECULES Division head, group leader Jacques Neejjes Jacques Neefjes PhD Group leader Carla Herberts PhD Post-doe Eric Reits PhD post-doe Alex Griekspoor MSc Graduate student Ingrid Jordens MSc Graduate student Coen Kuyl MSc Graduate student Marije Marsman MSc Graduate student Joost Neijssen MSc Graduate student Marcel Van lith MSc Graduate student Martijn Van der Velde Undergraduate student Wilbert Zwart Undergraduate student Lennert Janssen Technical staff Marieke Van der Velde Secretary José Overwater Secretary peptide peptidase Figure VI. l Left: Confocal image of a living Mû Juso cell expressing the peptide transporter TAP tagged with GFP. Right: Although peptides diffuse rapidly through the cell, most wilt not encounter TAP but wilt be degraded by cytosolic peptidases (Eric Reits). Our lab is studying the molecular basis of the specific immune response. The critical molecules generating specificity and controlling the immune response are MHC class land II molecules. These molecules are instrurnental in a successful anti-tumor and anti-viral immune response. Understanding the principles and specificity of the individual components involved in this immune response wiil be critical in the development of novel vaccination strategies and the use of MHC molecules in combating tumor ceils. For more details see: Antigen presentation by M He class I molecules MHC class I molecules are designed to present fragments from proteins degraded in the cytosol or nucleus by the proteasome. MHC class I enables the immune system to spot hidden (that is intracellular) pathogens, although in the form of small fragments (peptides). The components shown to be involved in this process are: the proteasome that cleaves proteins into peptide fragments, a peptide pump called TAP to translocate peptides over the membrane of the ER, a chaperone called tapasin to stabilize MHC class I molecules and MHC class I molecules themselves. MHC class I molecules are retained in the ER lumen until successful peptide binding occurs. These molecules are subsequently transported to the plasma membrane. Defects in any of these components will re sult in a failure to mount a proper MHC class I response as is clear from the fact that various virus es target these to prevent their presentation to the immune system. The specificity of the degradation by the proteasome, the translocation by TAP and the binding of peptides by MHC class I molecules is known and already allows some prediction of the peptides presented by MHC class I molecules from a linear sequence. However, these predictions are far from perfect. We have studied the fate of peptides in living cells combining biochemistry, chemistry and biophysics. Peptides with a fluorescein-quencher couple were used. Degradation will uncouple the fluorescence from the quencher allowing fluorescence to appear. The appearance of fluorescence was followed af ter microinjection in living cells. Peptides we re found to be rapidly degraded by amino peptidases (an in vivo half-life of 4-6 seconds was measured). We have shown by FRAP analysis that peptides moved about one time through a cell during this period. Since we have determined both the rate of degradation and of motility, this suggests that many peptides we re degraded in the cytosol before encountering the peptide transporter TAP. This was experimentally verified in biochemical and biophysical experiments. Peptide degradation was fast and complete. Only a fraction of peptides escaped this rapid degradation by entering the nucleus and interacting with chromatin structures. We have shown that TAP is not located at the inner face of the nuclear envelope. Therefore, these peptides must leave the nucleus through the nuclear pore before contacting TAP. These experiments imply an important role for cytosolic peptidases in the class I immune response. We have subsequently introduced systematic variations in our model peptides and tested their degradation in living cells. The obtained peptide degradation rate pointed to a clear size selectivity and no sequence specificity. These studies are now being pursued to validate the results with minigene constructs and to test specific combinations of amino acids in various cell types including dendritic cells. Combining single cell biochemistry with specific inactivation of peptidases by RNAi may elucidate the contribution of individu al peptidase on the rate of peptide degradation in single cells. We have previously shown that the mobility of the peptide transporter TAP depends on its activity and we have used this observation to visualize the intracellular peptide pool in living cells. We have generated cells containing various YFP and CFPcontaining subunits of the MHC class I loading complex. Using FRET, we aim to follow conformational changes in this complex as the re sult of peptide transfer. We have used the mobility ofgfp-tagged TAP to visualize the intracellular peptide pool

60 ~ ~~' II,AI.~(.)~.~~I~ ~~ af ter mimicking various elinically relevant conditions. y Irradiation markedly affected the peptide pool. We have identified four peptides that were presented by MHC elass I molecules only after irradiation. Three peptides were derived from DNA repair proteins. The fourth protein is unknown and appears to be a mitochondrially-expressed protein with unknown function. Electronmicroscopy revealed that this protein is expressed in the intercistemea of mitochondria. We have made RNAi constructs and GFP-tagged constructs to study the function of this protein during y irradiation. We have made various attempts to isolate T cells from healthy individuals using MHC elass I tetramers loaded with the isolated peptides. The low affinity T cells isolated by this procedure could not be propagated and we are now continuing these studies in HLA-A2 transgenic mice. Antigen presentation by M HC class 11 molecules Like MHC elass I molecules, MHC elass 11 molecules present peptides to the immune system. However, unlike MHC elass molecules, these peptides are generated in the endocytic pathway. MHC elass 11 molecules therefore use another set of proteins for peptide generation (Cathepsines) and for support in loading. The latter is supported by a unique lysosomal chaperone called HLA-DM that is co-expressed with MHC elass II molecules. In B cells and in some B-cell tumors another molecule, HLA-DO, is expressed controlling the activity of HLA-DM. HLA-DO interacts strongly with HLA-DM. In doing so HLA-DO is altering the ph dependency ofhla-dm and restricting peptide loading of HLA-elass 11 molecules to late endocytic structures. HLA-DO is retained in the ER in the absence of HLA-DM (like MHC elass I H chains in the absence of B2m). However, we have shown that HLA-DO a novel can form protein complex in association with the elass I H-chains. This complex ofhla-do and elass I H-chains is expressed at the plasma membrane ofb cells. Thus, two molecules that would have been retained individually in the ER are transported to the plasma membrane when associated. It is predicted that this complex will be expressed on immature B cells and particular B celilymphomas. To study the function ofhla-do in further detail, we have sequenced the genes encoding for HLA-DOA and -DOB from control, autoimmune patients and patients with B cell lymphomas. Only one mutation was found. Expression of this HLA-DO mutant in celllines revealed that it did not affect the function ofhla-do. Whether HLA-DO is uniquely controlled and thus affects the elass 11 immune response is currently being studied. MIIC traffic MHC elass 11, HLA-DM and -DO are de live red to lysosomal-like structures called MIIC before they are transported to the plasma membrane. These MIIC are transported by the mictotubule-based motor proteins kinesin and dyneinjdynactin in a bidirectional manner and in a stop-and-go fashion. We have shown that the small GTPase Rab7 is critical in the regulation of this transport. We have identified a novel Rab7 effector called RILP that, when overexpressed, recruits the dynein-dynactin motor to MIICs and effectively blocks their transport. Using Rab7 mutants we have also show that for dynein-mediated transport, the GTPase cyele of Rab7 is critical. Using FRAP experiments with GFP-Rab7 expressing cells we showed that the Rab7 cyele is controlled by extracellular stimuli like EGF. We have defined two pathways controlling activation of the Rab7 cyele and are in the process of eloning additional candidate regulatory genes. The control of motility and transport of a critical molecule (MHC elass II) in the immune response remains a challenging field of investigation. Selected publications Gromme M, Neefjes j. Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways. Mol Jmmunol2002; 39: Reits E, Griekspoor A, Neefjes, j. Herpes viral proteins manipulating the peptide transporter TAP. Curr Top Microbiol Jmmunol2002; Reits E, Griekspoor A, Neijssen J, Groothuis T, jalink K, Van Veelen P, janssen H, Calafat J, Drijfhout jw, Neefjes j. Peptides in Living CeUs: DiJfusion, Protection and Degradation in Nuclear and Cytoplasmic Compartments before Antigen Presentation by MHC Class J. Jmmunity (in press). Van Lith M, Van Ham M, Neefjes j. Novel polymorphisms in HLA-DOA and HLA-DOB in B eeu malignancies. Jmmunogenetics 2002; 54." Villalba M, Bi K, Hu J, Altman Y, Bushway P, Reits E, Neefjes J, Baier G, Abraham RT, Altman A. Translocation of PKC[theta] in T eeus is mediated by a nonconventional, PJ3-K- and Vavdependent pathway, but does not absolutely require phospholipase C. J CeU Biol 2002; 15T253-63'

61 DNA DAMAGE CHECKPOINTS AND TUMORIGENESIS Group leader Reuven Agami Reuven Agami PhD Group leader Mathijs Voorhoeve PhD post-doe Anja Duursma MSc Graduate student Carlos Le Sage MSc Graduate student Bart van Leeuwen Technical staff Most human tumors harbor multiple genetic alterations that activate oncogenes, inhibit tumor suppressors and induce genornic instability. It is still unclear which of the genetic events are continuously required and which, when inactivated, may inhibit tumorigenesis. We have developed a vector-based system that mediates suppression of gene expres sion through RNA interference. With this system we demonstrate persistent inactivation of a dominant RAS oncogene that results in loss of tumorigenicity. In the year to come we intend to use our gene inactivation system to study the importanee of tumor suppressors and DNA-damage checkpoint and repair genes in protecting humans from cancer. For more details see: jwww.nki.nljnkidepjagami. Studying tumorigenesis using RNA interferenee tools for human eells For a long period, the lack of tools to efficiently generate stabie loss-of-function phenotypes has hindered mammalian genetic approaches to study gene function_ Recently, we have developed a novel vector system, named psuper, which directs the inhibition of gene expres sion through RNA interference (RNAi). With it we have demonstrated persistent suppression of gene expres sion that allows the analysis ofloss-of-function phenotypes that develop over longer periods of time (Figure VI.2). A The psuper system B Immunofluorescence insert o Northern P53, sirnas 19- prsprs p53 prs c p53.. Immunoblot prs prs -p53 ::> Red : Actin prs p53 5S rrna loading control....._ -----' Figure VI.2: A retroviral vector that mediates RNA inteiference. A: A schematic drawing of retroviral pretrosuper RNA interference vector (prs). B: Human U2-0S ceus were infected with prs and prs-p53 retrovirus and selcted for one week with puromycin. Polyclonal populations of puromycinresistant ceus were immunostainedfor P53. C: Whole ceu extracts were madefrom the same infected polyclonal populations ofu2-0s ceus as in B, separated by SDS-polyacrylamide gel electrophoresis (PAGE, and immunoblotted to detect P53 protein. D: Northern blot analysis with total RNAfrom the same injected ceu population described in B was peiformed and probed with the sense 19 nucleotide targeting P53 sequence. We have also found that this system is sensitive to mutations to the extent that a single nucleotide mismatch in the targeting sequence abrogates its ability to suppress gene expres sion. This feature has shown us opportunities for developing new therapy approaches by targeting disease-derived transcripts (e.g. oncogenes) with dominant activating mutations. One such example is the occurrence of mutated RAS alleles in many human tumors. To examine the role of RAS mutants in tumor ma in tena nee we have developed a psuper tooi to specifically target oncogenic K_RAS VI2 allele without affecting its wild type counterpart. We have shown that this tooi is powerful enough to inhibit the tumorigenicity of cancer cell-lines that harbor this exact type of genetic alteration.

62 As K_RAS VI2 is a frequent event in human cancer, the psuper-k-ras vi2 and similar expres sion vedors can now be used to identify essential gene tic events in human cancers and also may possibly serve as genetic tools for cancer therapy. In a complementary approach, we are using the psuper system to target putative tumor suppressor and DNA damage checkpoint genes in order to identify and define combinations of genetic events that are capable of converting primary human cells to cancerous cells. The data extracted from such study enables us to understand the function of various tumor suppressors in tumor progression and to identify new critical genes required for cancer development in humans. Cellular DNA damage responses Probably all tumors harbor a certain degree of genomic instability, underlying the tight connection between DNA damage responses (rep air and checkpoint) and tumorigenesis. In the past, we have shown that the DNA damage response to ionizing radiation is composed of two processes: initiation and maintenance (Figure VI.3). We have found that DNA damage causes a fast Cr-arrest by rapidly degrading cyclin-di. Interference with cyclin-di destruction prevented the initiation of Cl-arrest and rendered cells more susceptible to DNA damage, indicating that cyclin-di is an essential component of the cellular response to genotoxic stress. Later, this initial Cr-response to DNA damage is maintained and further strengthened by the stabilization of P53, leading to the induction of p2rcïpi. Intriguingly, both genes function in tumorigenesis, P53 is a tumor suppressor and cyclin-dr is an oncogene. Currently, we are using RNA interference tools to uncover genes essential for cyclin-dr destruction and P53 stabilization and other events that function in response to DNA damage. We would like to further test the role of these genes in DNA-damage rep air and checkpoint response and to link these functions to the development of cancer. Selected publications Agami R. RNAi and related meehanisms and their potential use for therapy. Curr Opin Chem Genet (in press). Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian eells. Seienee 2002; 296:55-3. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenieity by virus-mediated RNA inteiferenee. Caneer Cell 2002; ]: Voorhoeve MP, Agami R. Knoekdown stands up. Trends Bioteehnol (in press). DNA damage Cyclin-Dl ps3 No lreat. initiation Gl arrest Figure VIT Initiation and maintenanee of Gl arrest in response to DNA damage. A sehematie model showing the initiation and maintenanee proeesses leading to the Gl arrest in response to DNA damage. Irradiated eells initiate rapid destruction of cyclin Dl protein, eausingfast but transient arrest in the Gl phase ofthe cell cycle. Independently, P53 is aeeumulating to maintain and Jurther strengthen this Gl arrest.

63 NUClEOCYTOPlASMIC TRANSPORT AND THE ROlE OF THE NUClEAR PORE COMPLEX Group leader Maarten Fomerod Maarten Fornerod PhO Group leader Helen Pickersgill PhO Post-doe Rafael Bernad-Fernandez MSc Graduate student Oieuwke Engelsma MSc Graduate student Jolita Hendriksen MSc Graduate student Heila Van der Velde Teehnieial staff In our research group we study macromolecular transport between the nucleus and the cytoplasm, and investigate how this fundamental process is used in the regulatory circuits of the cello Eukaryotes are characterized by a separation of their transcription and translation machineries in different subcellular compartrnents, the nucleus and the cytoplasm. Because translation products are required for transcription and transcription products for translation, this topology requires a large amount of traffic between the nucleus and the cytoplasm. Apart from this, nucleocytoplasmic transport also allows a regulation of nuclear and cytoplasmic activities that is both rapid and easily reversible. Indeed, it has become clear in the last years that many essential cellular processes, such as signal transduction, cell cycle progression and apoptosis are co-regulated on the leve of nuclear transport. For more details see: jwww.nki.nljnkidepjmaartenjfornerod.htrn. N uclear transport The cell has several mechanisms to maintain the nuclear and cytoplasmic identities in interphase. First, passage to or from the nucleus is selectively allowed or blocked by nuclear pore complexes (NPCs, Figure VI.4), the channel forming units between the nucleus and the cytoplasm. These 125 MDa complexes re strict free diffusion of molecules or particles with a diameter larger than 9 nm, corresponding to the si ze of a globular protein of kd. However, complexes of up to 25 nm in diameter (e.g. ribosomal subunits of several megadaltons) are efficiently transported, provided they carry a nuclear import or export signal. With the identification of transport signals, transport receptors and Ran, the specificity of cargo recognition is relatively weli-understood. However, specificity within the nuclear pore complex remains largely unknown. Figure VI+ Nuclear pore complexes in Xenopus laevis oocyte nuclear envelopes visualised by scanning electron microscopy from the cytoplasmic (upper) or nuclear (lower) side. Arrows point to cargoes caught in transit (images courtesy of Terry Allen, Paterson Cancer Research Institute, Manchester, UK). Identification of specific transport pathways though the nuclear pore complex using in vitro nuclear reconstitution In both lower and higher eukaryotes, the NPC is made up of -30 different proteins that are collectively named nucleoporins, and occupy distinct positions within the NPC. Nuclei and pronuclei form efficiently in vitro from crude rnitotic or meiotic cytosol up on addition of chromatin and an energy regenerating system. These (pro)nuclei have a sealed nuclear envelope with nuclear pore complexes, and are functional in nuclear import and export. In vitro nuclear assembly allows us to biochemically remove specific nucleoporins from NPCs and study their function in transport and ultrastructural morphology. This way we aim to contribute to a functional map of the nuclear pore complex. In the past year we have characterized the function and composition of two of the three known cytoplasmically oriented proteins of the NPC, RanBP2jNup358 and CANjNup214, in collaboration with the laboratories ofiain Mattaj (EMBL Heidelberg, Germany), Terry Allen (paterson Cancer Research Institute, Manchester, UK) and Volker Cordes (Karolinska Institiute, Stockholm, Sweden). Removal of RanBP2jNuP358 resulted in an NPC that lacked cytoplasmic filaments, elements that had long been assumed to play important roles in nuclear protein import (Figure VI.5). Surprisingly, such 'shaven' pores imported like wild-type, compelling us to consider the way cargo is imported into nuclei. We have also performed initial studies on the third known cytoplasmically oriented nucleoporin, Nup88. Immuno-electron microscopic studies and RNA interference suggest that this nucleoporin is localized at the base of the cytoplasmic fliaments and may bridge the interaction between those and the core-npc. Role of the nuclear pore complex in ~ catenin nuclear signalling B catenin has a dual role in cell adhesion and signal transduction. It is an essential mediator in the Wnt signalling pathway, which regulates cell proliferation during embryogenesis and is a major oncogenic target in humans. Mutations in B catenin are strongly connected with colon cancers and melanomas. Necessary for B catenin's oncogenic

64 activity is its translocation to the nucleus, where it coactivates a celi proliferative genetic program in conjunction with the transcription factor TCF flef. The mechanism of nuclear import or export of ~ catenin is at least in part independent of conventional transport pathways and dependent on the nuclear pore complex. Structural similarities between b catenin and nuclear transport receptors suggest a direct binding to the NPC. By investigating the role of nucleoporins in ~ catenin nucleocytoplasmic transport we aim to de fine a new level of regulation in this signal transduction pathway. In the past year we have identified RanBP3 as a ~ catenin interacting factor, in coliaboration with Francois Fagotto (MPI Tuebingen, Germany). RanBP3 is a nuclear RanGTP binding protein that shares characteristics with nucleoporins, and functions as a nuclear export cofactor for CRMlfexportinr. ~ catenin was found to physically interact with RanBP3 in a RanGTP-stimulated way, independent of the general export receptor CRMlfexportinr. Depletion of RanBP3 by RNAi caused a nuclear accumulation of ~ catenin (Figure VI.6A) and an increased transactivating capacity (Figure VI.6B). In addition, microinjection of RanBP3 mrna in early Xenopus embryos inhibits ~ catenin mediated dorsoventral axis duplication (Figure VI.6C). These data suggest that RanBP3 stimulates the export of ~ catenin from the nucleus to the cytoplasm, thereby downregulating the Wnt signalling pathway. Selected publications Fornerod M, Ohno M. Exportin-mediated nuclear export of proteins and ribonuleoproteins. Res Probl Cell Diff 2002; J5: Walther TC, Pickersgill HS, Cordes VC, Goldberg MW, Allen TD, Mattaj IW, Fornerod M. The cytoplasmic filaments of the nuclear pare complex are dispensible for selective nuclear import. ] Cell Biol2002; 158:6J-77 A c l., t ' /1-catenin B.' f ~,.~ 1i1f: ' ~' RANBP3 uninjected ~-catenin Figure VI.5: Shaving the pare. Nuclei were reconstituted in vitro from wild-type extracts (upper panel) or extracts lacking RanBP2jNup214 and CANjNup214 (lower panel). Cytoplasmic filaments, that appear as blobs on the ring-like NPC, are absent in the RanBP2 and CANdeficient NPCs. However, these NPCs import as wild-type. Bar, 100 nm. /H:atenin + RanBP3 TCFILEF + FRAT _ psuper-ranbp psuper empty TCFILEF lucifirase reporter mutant wild-type Figure VI.6: RanBPJ negatively influences the Wnt signalling pathway. A. Downregulation of RanBPJ by RNAi (right, note ceus with decreased nuclear levels) leads to increased nuclear localisation of f3 catenin in the presence of Wnt signalling (left). B. It also leads to an increased transactivating activity as measured by a TCF JLEF responsive luciferase reporter in the presence of Wnt signalling (TCF jlef+frat). C. RanBPJ prevents f3 catenin induced dorsoventral axis duptication in Xenopus embryos. Embryos were injected with RanBPJ andjor f3 catenin mrna. As expected, f3 catenin overexpression effect dorsoventral axis duplication (middle panel). However, co-injection of RanBPJ mrna inhibits this effict (lower panel).

65 GENES INVOLVED IN BREAST CANCER PROGRESSION AND METASTASIS Group leader john Hilkens John Hilkens PhO Group leader Hubert De Leeuw PhO Post-doe Melanie Kimm MSc Graduate student Vasso Theodorou MSc Graduate student Mandy Boer Teehnieial staff Sandra De Haan Undergraduate student Oaniela Bernardini M Se Guest Selected publications Vinall LE, King M, Novelli M, Green CA, Daniels G, Hilkens J, Sarner M, Swallow DM. Altered expression and allelic association ofthe hypervariable membrane mucin MUC1 in Helicobacter pylori gastritis. Gastroenterology 2002; 12Y41-9. Metastasis is the main eause of death of most eaneer patients. However, the biological processes leading to the metastatic phenotype are poody understood. We aim to identify novel genes involved in breast cancer pro gres sion and metastasis by insertional mutagenesis and to understand the biological nmction of eeli surf ace associated mucins, notably episialinjmuc1, which have been implicated in breast cancer pro gres sion. Identification of novel genes involved in mammary tumor progression and metastasis by M MTV tagging During recent years we have set up a mouse model system that can be used to identify novel metastasis genes in a mouse mammary tumor virus (MMTV) infected BALBjc substrain by retroviral tagging. Almost au of the breeders of this strain develop mammary tumors, which often give rise to micrometastases in the lung, that are, however, too smali for gene tic analysis. We have found that subcutaneous (s.c.) transplantation oflung tissue from tumor bearing animals develop into a tumor if metastases of the mammary carcinoma were present. These s.c. tumors provided us with a source of mammary tumor metastases derived tissue for genetic analysis, which was previously not available. We have searched for novel MMTV targeted genes in the primary tumors and lung metastasis derived from these tumors. We have analyzed more than IOO integration sites of which ten were 'common' (i.e. found more than once). Most common sites are already described, but in addition we have found two genes of the Wnt and Fgffamily that have not been shown previously to be targeted by MMTV (Wnt-JA) or never shown to be involved in mammary tumorigenesis (Fg{-lO). Our results demonstrated that Wnt-I; -2, -J, JA and lob are frequently activated in mammary tumors. RT-PCR studies have revealed that in all of 43 MMTV induced BALBjc mammary tumors one of these genes was aetivated suggesting that all mammary tumors need to have the WNT-pathway activated. Additionally, Fgf genes are frequently activated as has been described. Membrane-associated mucins in breast cancer Membrane associated mucins are frequently upregulated in various types of cancers. Severallines of evidence indicate that these mucins promote metastasis and are of prognostic significance, however their exact biological function is unknown. Episialin (also designated EMA, CA 15-3 antigen, CD 227), eneoded by the MUCI gene, is a paradigrn of this class of mucins. We have shown that episialin reduces celi-ceu and ceu-matrix adhesion and promotes metastasis as a re sult of shielding the adhesion receptors by its extremely elongated extra celiular domain. The function of episialin in normal ceus has not yet been established. We have found that episialin is localized in the trailing edge of migrating epithelial celis and fibroblasts, often in uropod-like structures, whereas in non-migrating ceus it is mainly confined to microvilii (Figure VI.7). Moreover, episialin is present in cholesterol-sphingolipid rich lipid rafts. We are presently investigating whether raft localization is responsible for the trailing edge localization and whether the presence of episialin affects celi migration. Figure VI. T EpisialinjM UCl is present in the trailing edge of migrating cells. The picture shows xy (top panels) and xz (lower panels) confocal slices of a migrating (left) and non-migrating (right) BT-549 breast cancer cello The first panel shows staining with a monoclonal antibody against episialin (indirectly labeled with FITC). The second panel shows actin directly labeled with Alexa Fluor 568 conjugated Phalloidin. Episialin in non-migrating cells is mainly confined to microvilli, whereas episialin relocates to the trailing edge in migrating ceus. Bar is 10 J1.m.

66 ULTRASTRUCTURAL STUDIES ON ENDOCYTOSIS AND SIGNALLING Recent evidence from literature predicts a role for endocytic proteins in pathological conditions where the balance between stimulation and attenuation of signaling is subverted; the first and foremost of which is cancer. We continue to investigate membrane traffic in cells and how this may relate to disease states. The focus is on understanding the molecular machinery and organization of molecular sorting within the endomembrane system of a cello We concentrate our work on the vesicular transport mechanism within the endocytic pathway and the link to pathogenesis. Cryo immunogold-electron microscopical methods are our main techniques, which allow the subcellular detection of gene products at the highest resolution. The research is divided in three projects. ARF6 project Our previous work has shown that the Ras-related GTPase, ARF6, regulates the recycling of endosomes and their subsequent fusion with the plasma membrane. A growing body of evidence has accrued in support of the notion that membrane insertion at defined sites on the cell surface may play a role in cellular processes that require cell surface remodeling, such as phagocytosis, antigen presentation, cell migration and perhaps invasion. Therefore, we have focused our efforts on understanding the cellular basis of ARF6-regulated endosomal recycling. With VW Hsu of the Harvard Medical School in Boston US, we have further characterized the ARF6 endocytic coated vesicles. These vesicles are enriched for the v-snare cellebrevin that functions in transport from the recycling endosome to the plasma membrane. Currently our group is evaluating the knockdown of the ARF6 molecules in cell culture using RNA interference to study its effect on endocytosis. In collaboration with C D'Souza-Schorey (Notre Dame University, USA) we have demonstrated that expression of arfaptin 2 (a ARF6 interacting protein) in cultured cells induces the formation of pericentriolar and nuclear aggregates which morphologically resembie mutant huntingtin aggregates characteristic of Huntington's disease. Endogenous arfaptin 2 localizes to aggregates induced by expres sion of an abnormal amino-terminal fragment ofhuntingtin that contains polyglutamine (polyq) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cellfree biochemical assays, we have shown that arfaptin 2 inhibits proteasome activity. Finally, we have shown that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function. Group leader Peter Peters Peter Peters PhD Group leader Alexander Mironov MD PhD Post-doe Nicole Van der Wel PhD Post-doe Tom Groothuis MSc Graduate student Gerty Schreibelt Undergraduate student Martijn Romeijn Undergraduate student Erik Bos MSc Technical staff COl project The maturation of dendritic cells is accompanied by the redistribution ofmhc class 11 molecules from the lysosomal MHC class 11 compartment (MIIC) to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CDr molecules that mediate presentation of lipid antigens. This novel class of antigen pres enting molecules presents mycobacteriallipid antigens either added exogenously or derived from late endosome-resident live bacteria to the classical T cell antigen receptor oft cells. We previously found that the subcellular localization between the CDr isoforms differs and is dependent on a cytoplasmic targeting signal. This year, in collaboration with M Brenner of the Harvard Medical School Boston US, we have found that the pathway CDr and MHC class 11 molecules follow a different course during dendritic cell maturation. We have found that in human monocyte-derived dendritic cells (DCs), unlike MHC class 11, the steady state distribution oflysosomal CDrb and CDrc isoforms was unperturbed in response to LPS-induced maturation. However the lysosomes in these cells underwent a dramatic reorganization in membrane structure and in the distribution oflysosomal molecules, and matured into a novel and morphologically unique compartment, here termed mature dendritic celllysosome (MDL). Furthermore, we have shown that clathrin-mediated endocytosis of CDrb, as well as of transferrin was still functional. Therefore, the constitutive endocytosis of CDrb molecules and the selective sorting of MHC class 11 from lysosomes explain the separation of peptide and lipid antigen

67 Selected publications Cao X, Sugita M, Van Der Wel N, Lai J, Rogers RA, Peters PJ, Brenner MB. CD1 Molecules Efficiently Present Antigen in Immature Dendritic Cells and Traffic Independently ofmhc Class IJ During Dendritic Cell Maturation. J Immunol. 2002; 169: Peters PJ, Ning K, Palacios F, Boshans RL, Kazantsev A, Thompson LM, Wood man B, Bates G P, D'Souza-Schorey C. Arfaptin 2 regulates the aggregation of mutant huntingtin protein. Nat Cell Biol. 2002; ]:24-5 Figure VI.8: Pr pc labeled by immunogold is concentrated in the cytosol in a population of neurons. The space indicated by the opposing arrows shows the lumen of the endoplasmic reticulum (er). Bars represents 200 nm. pres enting molecules during dendritic cell maturation. Consistent with this, DCs efficiently presented CDlb-restricted lipid antigens (Ags) to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by COl may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells. Prion project Prion diseases are fatal neurodegenerative disorders. Biochemical purification of the infectious material and transmission studies employing transgenic and PrP-ablated (Pmpojo) mi ce have shown that transmission and neurodegeneration are mediated by the abnormally folded prion protein (PrP Se ). PrPSe is mostly an insoluble, partially protease-resistant glycoprotein, which accumulates in affected brains. PrPSe is forrned from a glycosylphosphatidyl inositol (GPI)-anchored, protease-sensitive cellular protein, PrP C, by a post-translational refolding process. During refolding, regions of the mainly alpha-helical Prpc molecules are converted into beta-sheet structures. Localization of normal prion protein (Prp c ) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructurallocalization of Prpc still remains enigmatic. We have performed, in collaboration with S Prusiner, University Califomia San Francisco, a quantitative study of the ultrastructurallocalization of Prpc in the mouse hippocampus using cryoimmunogold electron microscopy. Prpc follows the standard biosynthetic trafficking pathway with a preferentiallocalization in late endosomal compartrnents and on the plasma membrane of neuron al body and processes. Prpc is found with the same frequency in the synaptic specialization and perisynaptically, but is almost excluded from synaptic vesicles. Remarkably, Prpc is expressed also in the cytosol (Figure V1.8) in subpopulations of neurons in the hippocampus, neocortex and thalamus. The cytosolic prion protein may have altered susceptibilty to aggregation suggesting that these neurons may play a significant roie in the pathogenesis of prion diseases, in particular those with a genetic predisposition.

68 CEll CVClE CONTROL GENES, ESTROGEN RECEPTOR AND BREAST CANCER Estrogen independent activation of the estrogen receptor (ER), is most likely a mechanism of tumor progression in breast cancer and of anti-estrogen resistance in treatrnent ofbreast cancer. Understanding and manipulating these, would be directly relevant for clinical application. We followed two strategies to visualize the effects of ER activation. Graup leader Rob Michalides Deregulation of cyclins and t herapy We have demonstrated that the estradiolindependent activation of ER by cyclin DI rendered ER mediated transcription of reporter genes partly insensitive to antiestrogens tammcifen and ICI Proliferation oft47d and MCF7 breast cancer cells also became insensitive to these antiestrogens when high levels of cyclin DI, but also of cyclins A and E, were transiently expressed. Using various cyclin DI mutants, we have demonstrated that the latter was due to a shift in occupancy of cdk-inhibitors p2i/p27 from cyclin E-A/cdk2 to cyclin DI/C<ik4 complexes. Retrospective clinical studies showed that cyclin A is an independent predictor of recurrence of early stage breast cancer and can as such be used as a marker for response in patients treated with tamoxifen. We have recently found that overexpression of cyclin DI also modifies sensitivity of cells towards taxol. The ability of cyclin DI to stimulate expression of cyclin kinase inhibitor p21 is responsible for the increased sensitivity of cells towards taxol that, in concentrations applied in the clinic, acts in G2/M via p2i. Single cell assessment of estrogen receptor activity In collaboration with Alexander Griekspoor, Jacques Neefjes (Departrnent of Tumor Biology) and Kees Jalink (Departrnent of Cell Biology), we have visualized ER activity at the single ceillevei. The conformation of a helix-i2 domain in the ligand binding domain of ER determines the ability to recruit and interact with the components of the transcriptional machinery and thus to elicit an estrogen-sensitive response by ER. At the molecular level, multiple mechanisms may be responsible for resistance. To study this in further detail, we developed GFP labelled-er molecules that allowed us to study inactivation of the ER by antiestrogens and the effect of estrogenindependent activation in single cells. Using FRAP (fluorescence recovery after photobleaching), we have determined that a pure antiestrogen ICI completely immobilizes the ER, whereas the partial antagonist tamoxifen causes a reduction in ER mobility, as compared to estrogen activated ER. Using ER-recombinant constructs with YFP at the N terminus and CFP at the C-terminus in FRET (Fluorescence Resonance Energy Transfer) experiments, inactivation of ER by antiestrogens resulted in a positive FRET signa!, which indicated an intramolecular change in the conformation of ER. Inactivation of ER by antiestrogens resulted in an irreversible alteration in FRET signaling (Figure VI.9). Estrogen-independent activation of ER by camp prevented tamoxifen associated FRET, whereas the ICI associated FRET was only prevented by the combined action of camp, cyclin DI and SRC-I (steroid receptor co-factor). This indicated that resistance to tamoxifen is mediated by camp alone, whereas the resistance towards ICI requires additional factors. These findings compare with ER transactivation studies using ERE-reporter assays. They show that activation/inactivation of the ER can be directly measured in single cells and provide a direct way of evaluating SERMs (selective estrogen receptor modulators) and putative inhibitors of these. These studies visualize the molecular events associated with tamoxifen resistance. ~ ~ ~1 2 CFP ~-- ~ --~ ~-- ~ time (s) x l 0 J Figure VI.9: FRET analysis of U20S ceus transfected with YFP-ER-CFP. ExGÏtation ofcfp was at 425 nm, and emission ofyfp at 530 and CFP at 475 nm was measured. Energy transfer {rom CFP to YFP and altered conformation ofthe ER resulted in an increased emission at 530nm and a consequent alteration in the emission ratio 530/475. An example is given of an altered FRET signauing (positive FRET) after treatment ofthe ceus with tamoxifen (i). Rob Michalides PhD Group leader Astrid 8alkenende Technical staff Desirée Verwoerd Technical staff Selected publications Bindels E, Laiiemand F, Balkenende A, Verwoerd D, Michalides R. Involvement of G1/S cyclins in estrogen-independent prolijeration of estragen/receptor-positive breast cancer ceus. Oncogene 2002; 21: Michalides R, Tiemessen M, Balkenende A, Verschoor T, Coco Martin J. Overexpression of cyclin Dl enhances taxol-induced mitotic death in MCF7 ceus. Breast Cancer Res and Treatment 2002; 74: Michalides RJAM, Van den Brekel M, Balm F. Defects in Gl-S cel! cycle contral in head and neck cancer, a review. Head and Neck 2002; Michalides R, Van Tinteren H, Balkenende A, Vermorken J B, Benraadt J, Huldij J, Van Diest P. Cyclin A is a prognostic indicator in early stage breast cancer with and without tamoxifen treatment. Br ] Cancer 2002; 86: Muris DFR, Verschoor T, Divecha N, Michalides RJAM. Constitutive active GTPases Rac and CdC42 are associated with endoreplication in P AE ceus. EurJ Cancer 2002; 38: Van Diest P, De Jong J, Baak J, Michalides R, Van der Veld E. Pragnostic value of pralijeration and apoptosis in breast cancer_ In: Predictive and pragnostic factors in breast cancer. Rosemary Walker (editor) (in press).

69 VII DIVISION OF MOLECULAR GENETICS ROlE OF MOUSE POlYCOMB-GROUP GENES IN TRANSCRIPTIONAl REPRESSION AND TUMORIGENESIS Group leader Maarten Van Lohuizen Maarten Van lohuizen PhD Group leader Maria Hernandez PhD Post-doe Anders lund PhD Post-doe Inhua Muyrers-Chen PhD Post-doe Michela Prudenziati PhD post-doe Erwin Boutsma Msc Graduate student Merellingbeek Msc Graduate student Konstantin Matentzoglu Msc Graduate student Panthea Taghavi MSc Graduate student Petra Van der Stoop Msc Graduate student Florence Van Tienen Undergraduate student Danielle Huisman Technical staff Jan Paullambooij Technical staff Ellen Tanger Technical staff Els Verhoeven Technical staff Tammy Philippo Secretary Katinka Van den Berg Secretary Marie Anne Van Halem Secretary Probes: p 19ARF p16 + I I + I I Bmi I status: ~ "I- '+ ~"I- '+ ElF U.IJ Myc "- 1 Bmil I \ F ail-safe against ~tion / Cell cycje progression 1 jjj~l Figure VII.l: Northem blots illustrating that Bmil-deficient MEFs show derepression of P19ARF and p16ink4a (top). Bmil-containing Pc-G protein complexes act as repressors of the INK4a/ARF fait-safe mechanism, which is activated by oncogenes such as Myc and RAS in pnmary ceus (Bottom). We are studying the mechanism of stabie inherited transcriptional repression by Polycomb-group (Pc-G) protein complexes, and the effects of deregulation of Pc-G genes on Homeobox (Hox) gene expres sion, development, cell cycle control and cancer formation. In addition, we are performing large-scale genetic screens in primary cells and in cancer-predisposed mice to identify cancer-relevant combinations of collaborating oncogenes and tumor-suppressor genes. Functional characterization of Pc-G protein complexes Repressive Pc-G proteins and counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are involved in maintenance of proper gene expres sion pattems during development at the level of chromatin structure. As such, these factors constitute a crucial 'cellular memory' mechanism of transcriptional states. Important targets include the Hox gene clusters but also critical cell cycle regulatory genes (see below). At least two biochemical distinct evolutionary highly conserved Pc-G protein complexes can be distinguished. The first contains EnxrjEnx2 (SET domain proteins acting as Histone H3 methylases), Su(z)r2, Eed and histone deacetylases and is required during early development when Pc-G repression is initiated. The second large complex encompasses BmirjMelr8, M33jMPC2, MphrjMph2 and RingrbjRingra together with other proteins yet to be characterized and is required throughout development to maintain proper target gene repression. Unlike in Drosophila, in mammals most Pc-G genes are represented as highly related gene pairs (such as Bmil and Me118). To study Pc-G function we are focusing on representative me mb ers of these groups: Bmil, Me118, M33, Ringlb and Eed, respectively in gain- and loss-of-function studies in mice. New insights have come from our recent analysis of Ringlb-deficient mice. Unlike other single Pc-G gene knockout mice (which display various defects but are viabie, see below), Ringlb null mutant mice die shortly after gastrulation. This may relate to the central positioning of the Ringlb protein in the Pc-G complex, and in addition suggests that the closely related Ringla gene cannot compensate for loss of Ringlb. This severe phenotype resembles that of the Eed null mutant mi ce (the only Pc-G homolog known as yet to be represented by a single gene), and suggests that loss of Ringlb results in complete loss of Pc-G maintenance function. This phenotype has been studied in detail by in situ hybridization with early developmental markers (In collaboration with BRoeIen and J Deschamps, Hubrecht Lab, Utrecht). In addition, we have generated Ringlbconditional knockout ES celllines and mice, which are being used to study Pc-G function in relation to differentiation and development in a timed and cell-typespecific manner. To study the molecular mechanism ofpc-g repression we are characterizing local changes in chromatin structure as a function of changes in the expression levels of Pc-G proteins, using Chromatin Immunoprecipitation (CHIP) and chromatin prohling using Ecoli dam methylase (DAMid, in close collaboration with B van Steense!, Division of Experimental Therapy). The main focus of these studies is on the currently best-characterized in vivo relevant cell cycle target gene: Cdkn2A (p16ink4ajp19arf) and on Hox cluster genes. Connections between Pc-G gene repression, cell cycle regulation and tumorigenesis We originally identified Bmil as an oncogene that cooperates with the cmyc oncogene in the induction of Band T cellleukemia in mice, underscoring the connection between deregulation of Pc-G repression and cancer. In line with this, Bmil-overexpressing transgenic mice have a high incidence of B- and T-cell lymphomas. In contrast, Bmil knockout mice show severe proliferation defects and increased apoptosis oflymphoid and myeloid cells, resulting in severe lymphopenia. In addition, also primary embryo fibroblasts (MEFs) and neurons in the cerebellum of Bmil knockouts show such defects. We have demonstrated that these defects are due to increased levels of the INK4ajARF-encoded tumor suppressors p16ink4a and

70 P19ARF, that in turn are critical regulators of the RB and the P53 tumor suppressor pathways. Our recent results indicate that P19ARF is the most critical Bmil target in mice, whereas p16ink4a is more important in primary human celis. Conversely, overexpression of bmil facilitates immortalization, and neoplastic transformation by hyper-repressing p16ink4a and P19ARF, and in certain human mammary epithelial celis can contribute to activation ofhtert, the catalytic sub unit of telomerase (collaboration with G Dimri, Boston). These results connect Pc-G repression to cell cyde control and control of the senescence 'fail-safe' governed by these tumor suppressors (Figure VII.1), in primary celis ofboth mouse and human origin. This connection also explains the strong cooperativety between Bmil and cmyc: Bmir represses the P19ARF 'fail-safe' which is induced by cmyc. The relevanee of these findings for human cancer is underscored by the arnplification of BMI! in Mantle celilymphomas and the overexpression of BMI1 in various tumor types induding non-small celilung cancer. In vivo and in vitro genetic screens to identify new groups of collaborating oncogenes As loss of the INK4ajARF fail-safe is common to all cancers, we have performed a genetic screen in INK4ajARF-deficient mice, using MuLV-insertion mutagenesis on a genome-wide scale. Sequences flanking the proviral insertions have been obtained in a high-throughput manner using efficient PCR methods, and have been mapped using the mouse genome databases, yielding 17 known and 37 new common insertion sites involved in lymphoid and myeloid leukemia. These results combined with screens performed in the Berns and Copeland labs underscore the power of large-scale insertional mutagenesis in specific cancer predisposed mice. In dose collaboration with A Berns (this division) we are now extending these types of screens to other cancer relevant models such as breast cancer. We have also started two types of in vitro high-copy suppressor screens. The first is to bypass premature senescence of Bmil knockout MEFs using tumor-derived retroviral cdna library transduction (Figure VII.2). Since the INK4ajARF locus is intact in these MEFs, this screen has the potential to uncover both new upstream regulators and downstream effectors of the INK4ajARF 'fail-safe' pathways. This is illustrated by the identification of the T-box member TBX2 as a potent immortalizing gene that acts by directly repressing P19ARF transcription through a variant T-binding site in the mouse and human Arf promoter. In collaboration with the Department of Pathology, we found TBX2 to be amplified in a subset of primary human breast cancers, suggesting a potential oncogenic role. A second type of screens is aimed at identifying genes contributing to tumor progression. Hereto, combinations of predisposing oncogenes are introduced in MEFs or conditionally immortalized primary epithelial celis prior to retrovirallibrary transduction. Transformed dones are subsequently selected in semi-solid medium, and the relevant genes are isolated using efficient PCR strategies. The role and mechanism of action of these new putative oncogenes or tumor suppressors, and their interference with adhesion and invasion of primary mouse and human celis is under investigation. Selected publications Brummelkamp TR, Kortlever RM, Lingbeek M, Trettel F, MacDonald ME, van Lohuizen M, Bernards R. TBX-3, the gene mutated in Ulnar-Mammary Syndrome, is a negative regulator of P19ARF and inhibits senescence. ] Bid Chem. 2002; 277: Dimri GP, Martinez JL, Jacobs j), Keblusek P, Itahana K, Van Lohuizen M, Campisi J, Wazer DE, Band V. The Bmi-l oncogene induces telomerase activity and immortalizes human mammary epithelial cells. Cancer Res. 2002; 62: Itahana K, Zou Y, Itahana Y, Martinez JL, Beausejour C, Jacobs J). van Lohuizen M, Band V, Campisi). Dimri GP. Control of the replicative lift span ofhuman fibroblasts by the Polycomb protein Bmi-l and p16. Mol Cell Biol (in press). Jacobs j), Van Lohuizen M. Polycomb repression: from cellular memory to cellular proliferation and cancer. Biochim Biophys Acta. 2002; 1602: Lingbeek ME, Jacobs J). Van Lohuizen M. The T-box repressors TBX2 and TBX3 specifically regulate the tumor suppressor gene plifarf via a variant T-site in the initiator. ] Biol Chem. 2002; 277: Lund AH, Turner G, Trubetskoy A, Verhoeven E, Wientjens E, Huisman D, RusselI R, DePinho RA, Lenz J, Van Lohuizen M. Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice. Nature Genet. 2002; 32: Lund AH, Van Lohuizen, M. RUNX: a trilogy of cancer genes. Cancer Cello 2002; 1: Passage: pi p2 p3 p4 ~_9X_~ * ~ 18_X ~.buik~ "", Tumor-derived - retroviral cdna Iibrary.'-,.,.." TBX2 ~ are senescent..,/'. ~ _ ~ -wait for immortal c10nes to appear ~-----~ + isolate relevant oncogemc cdnas Figure VIl.2: In vitro senescence-bypass high-copy suppressor screen. Bmil-deficient MEFs have a de-repressed P19ARF gene, due to loss of Pc-G repression. Consequently, these cells enter a tight premature senescence arrest (see insert: large flat senescent cells). Transduction of early passage Bmil-j- MEFs with human tumor-derived retroviral cdna libraries at passage 2,Jollowed by passaging until most cells become senescent. CDNAs in colonies that escaped arrest and were immortal (see insert) are isolated, and were found to contain the TBX2 gene.

71 MOUSE MODELS FOR CANCER Group leader Ton Berns Ton Berns PhD Group leader Paul Krimpenfort PhD Academic staff Johan Jongsma PhD Post-doc Jos Jonkers PhD Post-doc Jaap Kool PhD Post-doc Xiaoling Liu PhD Post-doc Scott Lyons PhD Post-doc Ralph Meuwissen PhD Post-doc Martijn Nawijn PhD Post-doc Andrej Alendar MSc Graduate student Nicole Feldmann MSc Graduate student Ate Loonstra MSc Graduate student Carla Martins MSc Graduate student Haraid Mikkers Msc Graduate student Renée Van Amerongen MSc Graduate student Erwin Van Montfort MSc Graduate student Rahmen Bin Ali Technical staff Joost De Moes Technical staff Loes Rijswijk Technical staff Vedrana Tabor Technical staff Marcelle Treur-Mulder Technical staff Fina Van de Ahé Technical staff Hanneke Van der Gulden Technical staff John Zevenhoven Technical staff Selected publications Berns A. Seneseenee: a eompanion in ehemotherapy? Caneer Ce1l2002; 1:] Hwang H.C, Martins c.p, Bronkhorst Y, Randel E, Berns A, Fero M, Clurman BE. Identifieation of oneogenes eollaborating with P27Kip110ss by insertional mutagenesis and high-throughput insertion site analysis. Proe Natl Aead Sei USA. 2002: 99: Jonkers J. Berns A. Conditional mouse models of sporadie eaneer. Nature Rev Caneer 2002; 2: The mouse is used as a model organism for establishing the role of oncogenes and tumor suppressor genes in tumor development. Using Cre-recombinase mediated switching, multiple oncogenes and tumor suppressor genes are mutated within a defined window of time in a subset of the cells. This permits more accurate mimicking of tumorigenesis as it occurs in man. Specific genotype-phenotype correlations can be established in this fashion. These models are also suited to test prevention and intervention strategies when combined with sensitive in vivo imaging techniques. Furthermore, the models permit us to identify new oncogenes and tumor suppressor genes involved in tumor progression using a variety of techniques, among which array comparative genome bydridization, expression profiling and proviral insertional mutagenesis. Functional analysis of tumor suppressor genes We are utilizing mice carrying conditional tumor suppressor gene alleles to model a range of tumors. Cre recombinase is used to activate or inactivate the conditional alleles. We have introduced Cre-ERT2, an improved version of Cre-ERT, into the Rosa26 locus from which widespread expres sion is achieved. This mouse line will be used to con trol Cre expression in a spatiotemporal fashion in locations that can be exposed effectively to tamoxifen. In addition, we are using Cre transgenes with distinct tissue specific expres sion pattems and somatic gene transfer of Cre via adenovirus to target expression to specific locations. These methods allow us to concomitantly switch the various oncogenes and tumor suppressor genes. This allows us to model a number of different tumor types with high incidence and short latency period. Focus is on combinations oflesions that are found in the corresponding tumors in man. Mammary tumors Inheritance of one defective BRCAl or BRCA2 allele predisposes humans to breast cancer. We have established a mouse model for BRCAr and BRCA2-associated breast ca neer. We have shown that Cre-mediated loss of Brcal or Brca2 and p53leads to a high incidence of mammary tumors. We have determined the expres sion profile of these tumors to identify the signatures that are specific for BRCAr or BRCA2 loss. In addition, the effects ofloss of E-cadherin on mammary tumor incidence and tumor progression is being explored. Lung tumors Mutations in K-Ras play a pronounced role in human non-small cell lung cancer, whereas loss of Rb and P53 are more predominant in small celllung cancer. We have developed a mouse lung tumor model in which K-Ras is sporadically activated through Cre-Iox mediated somatic recombination. Adenoviral mediated delivery of Cre recombinase in lung epithelial cells gives rise to rapid onset of tumorigenesis, yielding pulmonary adenocarcinomas with roo% incidence af ter a short latency. In order to follow the onset and progression of these tumors we have combined the conditional Ki-Ras allele with a conditional Luciferase transgene. With this tooi we can follow lung tumor development in time by in vivo imaging (see Figure VIL3). This permits us to follow growth kinetics of these tumors from early on and test the response of tumors to intervention protocols. When Rb and P53 were inactivated by AdenoCre injection, primarily small-cell-iung-cancer (SCLC) was found. Both the loss of Rb and P53 we re required for the development of SCLC (Figure VIL4). Interestingly, the tumors not only closely resembied SCLC in man but also showed a similar metastatic behavior. Mesotheliomas Very little is known about the genetic lesions contributing to the development of mesotheliomas. In a subset of mesotheliomas in man, inactivation of Figure VII.j: Lung tumors. Tumor progression in time in {J-Aetin ki_ral 1ox ; {J-Aetin-Luciferas/ 1ox miee. Intensity of signal is a measure for the tumor mass. Three eonseeutive measurements with 2 wk intervals.

72 Genotype mjce Iwn2r Loss Lox alleles 1Qt!. RbLoX/LoX;pS3LOX/LoX SCLC Cre-meeliated NA ps3 LoX / LoX NSCLC Cre-mediated NA Rb LO X/LoX;pS3LoX/wt SCLC Cre-meel iated LOH ps3 RbLOX/Wt; p53lox/lox SCLC Cre-meel iated LOH Rb NSCLC Cre-meel iated No LOH rb Figure VIl+ Adeno-Cre induced lung tumors in several genetic backgrounds. Note the requirement for loss of Rb and P53 in SCLC. Footnote: LOH = loss ofheterozygosity; NA = not applicable; SCLC = smalt ceu lung cancer; NSCLC = non smau ceu lung cancer. NF2 and INK4a has been reported. We have used these observations as a basis for the development of a mesothelioma mouse model and have inactivated Nf2 in combination with either Rb, Ink4ajAif, or P53 by Adeno-Cre infection of mesothelial celis of the thoracic and intraperitoneal cavity. Mesotheliomas were found at low incidence upon loss of Nf2. Concomitant loss of Ink4ajAif or P53 but not Rb strongly accelerated the development of mesotheliomas. We are in the process of analyzing the tumors by array CG Hand expres sion prohling to gain access to additionallesions involved in the progression of these tumors. In addition, we are exploring retroviral insertional mutagenesis in this model to identify genes involved in mesothelioma progression. Ink4a loss confers susceptibility to various tumors We have previously shown that inactivation of Ink4a does not lead to a marked spontaneous tumor predisposition in mi ce or altered growth characteristics of mouse embryo fibroblasts derived from these mice. However, Ink4a-deficient mi ce do show increased susceptibility to various tumors when these mice are chalienged with carcinogens or when mice are hemizygous for P19Arf. Ink4a loss also synergizes with other tumorpredisposing lesions. In Ink4a-deficient mice simultaneously hemizygous for P53, an increased level of spontaneous T lymphomas is observed. In Ink4a-deficient mice expressing a low level of Ki- Ras in melanocytes a high incidence of melanomas is observed, whereas in the Ink4a-proficient mice, carrying this bi-ras allele, only naevi are observed indicating that both in man and mice the same signaling pathways are involved in melanoma development. (Figure VII.S). This should make these mice particularly useful for exploring various intervention strategies. Identification and characterization of new oncogenic pathways Proviral insertional mutagenesis in transgenic and compound mutant mi ce can mark genes acting in specific signal transduction pathways. Emphasis in this program is on the identification of oncogenes, on the unraveling of the biochemical function of the encoded proteins, on their physiological role and on their specific contribution to the tumorigenic process. We have set up genetic screens to identify genes that can complement or substitute signaling pathways important for cancer. In one line of research, we are identifying genes that can substitute for PIM oncoproteins in lymphomagenesis. Pim oncogenes we re found earlier as one of the most potent oncogenes coliaborating with Myc in lymphomagenesis. We have generated compound mice carrying the E!!-myc transgene on a Pimr/ Pim2 double knockout background. In these mice, lymphomas have been induced by Murine Leukemia Virus infection. In a high throughput screen, we have identified a number of genes that can compensate for PIM deficiency. We are currently characterizing these genes. We have also generated triple Pim KO mice. Currently, the phenotypic aberrations in these mice are being analyzed. We expect these approaches to give insight into the signaling pathway of PIM proteins. Fratr-3 is the second gene family being studied. Fratr has been cloned as a tumor progression gene using retroviral transposon tagging. The FRA T proteins bind to GSK3 and are therefore intimately connected with the b-catenin pathway. The three members of this protein show substantial overlap in expression. Inactivation ofboth Fratr and Frat2 gene does not lead to any obvious developmental aberrations in mice. We are now generating and analyzing phenotypic aberrations in triple knockout mice. Furthermore, we are studying the capacity offrat proteins to impair GSK3B activity under various conditions in celi culture. Selected publications (continued) Marino S, Krimpenfort P, Leung C, Van Der Korput HA, Trapman J, Camenisch I, Berns A, Brandner S. PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebeuum. Development 2002; 129: Martins CP, Berns A. Loss Ofp27(Kip1) but not p21(cip1) decreases survival and synergizes with MYC in murine lymphomagenesis. EMBOJ 2002; 21: Mikkers H, Allen J, Berns A. Proviral activation of the tumor suppressor E2a contributes to T ceu lymphomagenesis in Ef-lMyc transgenic mice. Oncogene 2002; 21: Mikkers H, Allen J, Knipscheer P, Romeyn L, Hart A, Vink E, Berns A. Highthroughput retroviral tagging to identify components of specific signaling pathways in cancer. Nature Genet ]2; 2002:153-9 Vooijs M., Jonkers J, Lyons S, Berns A. Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 2002: 62: Vooijs M, Te Riele H. Van Der Valk M., Berns A. Tumor formation in mice with somatic inactivation of the retinoblastoma gene in interphotoreceptor retinol binding protein-expressing ceus. Oncogene 2002; 21: Mikkers H, Berns, A. 2002; Retroviral insertional mutagenesis: Tagging cancer pathways. Adv. Cancer Res (in press). Figure VIlT Skin phenotype in f3-actin Ki-Ras and f3-actin Ki-Ras;Ink4a;~/* mice. The thickening represents a melanoma, to the right a typical naevus is visible.

73 CELLULAR PROTECTION AGAINST ONCOGENIC TRANSFORMATION Group leader Daniel Peeper Daniel Peeper PhD Group leader Theo van Laar PhD Post-doe Cristina Martinez Muiioz PhD Post-doe Chrysiis Michaloglou Bsc Graduate student Benjamin Rowland MSc Graduate student Liesbeth Vredeveld MSc Graduate student Sirith Douma Technical staff Vector HA-Dril1 Figure VII.6: DRILl bypasses normal and RAS VI2 -induced senescence in primary mouse fibroblasts. MEFs were (co-)infected with retroviruses expressing cdnas as indicated and photographed two weeks later, at 40x magni.fication. In our recently established group we are studying mechanisms by which mammalian ceus are protected against oncogenic transformation, with a focus on signaling mediated by the RAS VI2 oncogene product. Primary murine and human ceus require multiple genetic events to undergo complete oncogenic transformation. This implies that various pathways controling critical processes including proliferation, ceu-ceu contact, apoptosis and differentiation must be deregulated for cancer to occur. For example, rather than proliferating indefinitely upon overexpression ofras VI2, untransformed ceus undergo ceu cycle arrest. Apparently, such an inadequate, constitutive and, in principle, highly mitogenic signal elicits an anti-mitogenic response, and additional genetic events are required to bypass this arrest. We are using both functional genomic and classical approaches to identify networks that contribute to cancer upon deregulation. Funetional genomic sereens to identify novel genes that proteet against oneogenie transformation RAS VI2 -induced arrest resembles 'senescence', given its seemingly irreversible character and the upregulation of senescence-associated markers including acidic ~-galactosidase. This ceu cycle arrest depends on established tumor suppressor genes like P19ARF, P53 and, as we have shown recently in a couaborative study with H te Riele (Division of Molecular Biology), the retinoblastoma (Rb) family. Genetic inactivation of each couaborates with RAS VI2 in stimulating proliferation, contributing to cancer. Not only tumor suppressor gene deficiency, but also (overexpression of) oncogene products like c-myc or adenovirus ErA couaborate with RAS VI2 in transformation. These observations help explain why immortalizing events can disrupt the normal anti-oncogenic response to excessive RAS VI2 signaling, and argue that senescence may rnimic a tumor-suppressing mechanism that protects ceus against oncogenic transformation by RAS VI2 and other oncogene products. To identify novel ceuular genes that allow escape of primary mouse embryo fibroblasts (MEFs) from RAS V I2 -induced senescence, we have designed a sensitive cell system to perform an unbiased, retroviral cdna library screen. A functional genomic approach has important advantages to classical approaches, including its function-based and genome-wide character. We have generated conditionally immortalized MEFs, co-expressing a temperature-sensitive SV 40 Large T (LT) mutant and RAS VI2 These 'BTR' cells proliferated indefinitely at 32 C, but became rapidly senescent at 39.5 C, due to both the disappearance of LT antigen and the presence of RAS VI2. Thus, we screened for proliferating colonies amidst a population ofras vi2 -expressing MEFs that had undergone premature senescence. Mter infection with one of several high-complexity retrovirus cdna libraries and subsequent processing and analysis, we have isolated 250 colonies. At present, we have isolated four cdnas: the relatives GKLF/KLF4 and EKLF/KLF1, LRF and DRILl. The latter cdna is the human ortholog of the mouse Bright and Drosophila Dead Ringer genes. The murine ortholog of DRILl, 'Bright', encodes ab cell-specific transcription factor that binds to the matrix-associated region (MAR) of the IgH locus. The product of the Drosophila ortholog of DRILl, 'dri', is required for recruitment of Groucho to Dorsal, a potent transcriptional repressor, and is essential for viability. We have observed that DRILr rendered primary murine fibroblasts unresponsive to RAS VI2 -induced, anti-proliferative signaling by pr9 ARF jp53jp2r CIP1, as weu as by pr6 IN l<4 a. Moreover, DRILrjRAS VI2 -expressing fibroblasts were oncogenic in nude, athymic mice. Furthermore, DRILr immortalized primary MEFs, in the presence ofhigh levels of pr6 IN l<4 a. Immortalization by DRILr, whose product binds the prb-controlled transcription factor E2Fr, correlated with induction of E2Fr activity. Consistent with this observation, DRILr induced the E2Fr target CYCLIN Er, overexpression of which was sufficient to trigger escape from senescence. Thus, our first characterization indicates that DRILr disrupts ceuular protection against RAS VI2 -induced proliferation downstream of the pr6 IN l<4 a and pr9arf jp53 pathways. Future studies will address the mechanistic details of DRILr-mediated oncogenicity. Currently, we are investigating the mechanism by which GKLF jklf4, EKLF jklfr and LRF bypass RAS VI2 -induced senescence. Moreover, we are in the process of

74 analyzing the remainder of the 250 dones obtained in the screen. Finally, we have begun to design functional genomic screens that are aimed at identifying genes products that not only bypass RAS VI2 -induced senescence, but cause oncogenic transformation. We anticipate that these approaches will broaden our understanding of a network of gene products that protects against oncogenic transformation. Senescence signaling and cell cycle control The pi6 IN l<4a/prb/e2f and ARF /PS3 tumor suppressor pathways are disrupted in most human cancers. It is currently thought that E2F transcriptional repressor complexes are important and downstream components specifically of the pi6 IN l<4a_prb pathway. Both PI9ARF and PS3 are required for the induction of senescence in primary MEFs, but little is known about their downstream targets. We have found that E2F repressors are also critical targets for both the PI9ARF and PS3 tumor suppressors during the induction of proliferative arrest and senescence. Disruption of E2F-mediated transcriptional repression in MEFs caused a sharp increase in the expression of E2F target genes, induding P19ARF. We have detected no contribution of E2F-mediated transactivation in this setting, indicating that a predominant role of endogenous E2F in primary MEFs is to repress its target genes. Unexpectedly, in spite of de-repressing P19ARF, disruption of E2F-mediated repression prevented proliferative arrest induced by both PI9ARF and PS3- Moreover, relief of transcriptional repression by E2F rendered MEFs resistant to both spontaneous and oncogenic RAS vi2 -induced senescence, without disrupting either pi6 IN l<4a, PI9ARF or PS3- We condude that E2F transcriptional repressor complexes are critical downstream mediators of anti-proliferative PI9ARF /PS3 signaling. Thus, the pi6 IN I<4A and PI9ARF pathways converge at the level of E2F. As this transcriptional repressor is controlled by these two major tumor suppressor pathways, our data predict that E2F-dependent transcriptional repression is deregulated not only as a re sult of a mutant pi6 IN l<4a_prb pathway, but also upon mutation of the ARF /PS3 pathway, i.e., in the vast majority ofhuman tumors. Selected publications Peeper DS, Mooi W. Pathogenesis of melanocytic naevi - growth arrest linked with cellular senescence? Histopathology 2002; 41S: Rowland BD, Bernards R, Peeper DS. E2F repressor complexes are critical for P19ARF /P53-induced replicative arrest. Cancer Cell2002; 2: Peeper DS, Shvarts A, Brummelkamp T, Douma S, Koh EY, Daley GQ, Bernards R. AJunctional screen identijies hdril1 as an oncogene that rescues RAS-induced senescence. Nature Cell Biol2002; 4: Senescence-like proliferative arrest in melanocytic naevi as a potential melanoma-suppressing mechanism Melanocytic naevus ofthe skin is an extremely common, small, benign tumor of cutaneous melanocytes. Most arise in childhood and adolescence and after a phase of gradual enlargement caused by proliferation of the lesional melanocytes, proliferation stops completely. This largely unstudied phenomenon may be of relevance in understanding the continued proliferation of the malignant counterpart of melanocytic naevus: cutaneous melanoma. In a collaborative study with W Mooi (Division of Diagnostic Oncology), we have hypothesized that the proliferative arrest in naevi may be caused by senescence, sirnilar to premature replicative senescence seen after either explantation and in vitro culturing of primary cells or the introduction of activated RAS VI2 into non-neoplastic cells. It is conceivable that such 'in vivo premature senescence' can also be initiated when cells migrate to sites providing a non-physiological microenvironment, such as the dermal mesenchyme and the lymph nodal tissue in case of human epidermal melanocytes. We hypothesize that replicative senescence may act as a genuine tumor-suppressing mechanism in vivo, at least in melanocytic naevi. This hypothesis is supported by the presence of activating RAS mutations in some naevi, as weil as by recent evidence that a critical effector of RAS, B-RAF (which like RAS VI2 induces premature senescence in vitro), is activated by mutation in a high proportion of melanomas. Recently, we have begun to test our hypothesis by determining the level of expres sion in naevi of a number of proteins known to play a role in in-vitro senescence. In particular, we are addressing whether B-RAF activation may act as the initiator in the onset of naevus formation. This work is accompanied by the culturing of primary human and murine melanocytes to study the regulation ofboth normal and oncogeneinduced senescence of these cells in vitro. Finally, we will use a functional genomic approach to identify melanocyte-specific pathways whose deregulations contribute to melanoma. Figure VII.T E2F repressor complexes are required for P19ARF-induced cell cycle arrest. (A) Overexpression of P19ARF causes cell cycle arrest in control cells, as revealed by the intense P19ARF staining (white dots in the nucleoli) in a large, arrested cell and the absence of P19ARF staining in proliferating cells. (B) Loss of E2F-mediated transcriptional repression allows ceus to proliferate in the presence ofhigh levels of P19ARF.

75 CANCER GEN ETICS Group leader Peter Demant Peter Demant MD PhD Group leader Margriet Snoek PhD Academic staff Tamas Csikos PhD Post-doc Marcel Van Kooy phd Post-doc Claudia Ruivenkam MSc Graduate student Carlo Zanon MSc Graduate student Koen De Groot Technical staff Elly Delzenne-Goette Technical staff Joost De Moes Technical staff Anita Klous Technical staff Marcelle Treur-Mulder Technical staff Huub Van Vugt Technical staff The familial cancer syndromes caused by germ-line mutations in tumor suppressor or mismatch repair genes like APC, Rb, NF2, MSH2 comprise only a small part of all cancers. The largest proportion of cancers is of non-familial, sporadic, type. However, also in these apparently non-inherited cancers the host genetic factors play an important role. In contrast to the germ-line mutations of oncogenes or tumor suppressor and mis match rep air genes, the genes affecting development and progression of sporadic cancers are multiple and have smaller effects. Therefore, they are difficult to detect in classical family and population studies, and they have to be analyzed in experimental animals, which offer the advantage of defined genetic constitution, the possibility of producing informative crosses and standardized tumorinduction procedures. We have used recombinant congenic strains of mice to map and identiry lung and colon cancer susceptibility genes in mi ce and subsequently to search for their homologs in humans. This group has also been dedicated to the development of a high trough-put SNP (single nucleotide polymorphism) genotyping system. Colon cancer susceptibility The cloning of cancer susceptibility genes is becoming a subject of major interest for cancer geneticists. The first reported success in this respect is the SCC! (Susceptibility to colon cancer I) gene, identified as the Ptprj (Protein tyrosine phosphatase receptor-type J). Ptprj has one intracytoplasmatic enzyme domain, a transmembrane part and extracellular chain consisting of fibronectin III - like domains. lts expression in cancer celllines has been shown to increase upon differentiation, and its transfection into non-differentiated celllines causes growth retardation and differentiation. The Ptprj products of the susceptible and resistant strain differ at several amino acids in their fibronectin type III domains. Most of these amino-acid substitutions reside in the extracellular portion of the molecule. Similarly, the sequence of the co ding regions of the homologous human gene (PTPRJ), which has been established in a large number ofhuman DNAs, revealed several amino-acid replacements in extracellular regions. Sequence alignment, secondary structure prediction and homology modeling predicted with high confidence levels that the substitutions in the mouse as well as in humans occur in the exposed regions of the molecule that are available for interactions with ligands or other proteins, and thus could affect the signaling process. A large proportion of colon, breast and lung cancers exhibit loss ofheterozygosity (LOH) at the short PTPRJ-bearing segment of chromosome IIPII with their minimal shared segment containing PTPRJ. We detected one colon cancer in which the PTPRJ is the only gene deleted, supporting the hypothesis that PTPRJ is a tumor suppressor gene, whose haplodeficiency can lead to selective advantage. In order to investigate the specific pathway and tumor progression stage, at which this gene may operate, we have examined the possible correlation of the LO H in colon cancers with other somatic alterations. This analysis revealed a correlation between the LOH at PTPRJ and the deletion of the chromosomal segment 18q12-21 in progressed adenomas, indicating that haplodefficiency of PTPRJ is an early event in colon carcinogenesis and that its loss, in possible synergy with a deficiency of annother gene on chromosome 18q, supports the transition of early adenomas into a more progressed stage. Lung cancer susceptibility Lung cancer is the leading cause of cancer deaths worldwide. lt exhibits familial clustering both in smokers and non-smokers, indicating the involvement of presently unknown genes. However, no such genes have been identified as yet in humans. Using mice as a model for study of genetics of lung cancer, the comparison of a number of mouse inbred strains revealed large differences in susceptibility to both spontaneous and chemically induced lung tumors and resulted in the mapping of several susceptibility loci. We have shown that the total number of Sluc genes (Susceptibility to lung cancer) probably exceeds 60. Thirty of them have been mapped in the crosses of OcBjDem RC strains, in which the genomes ofthe strain 020 and B are segregated. OcBjDem RCS have each only a random -12.5% subset of genes derived from the resistant strain Bro.020, and the rest from the strain 020. In the past period we have used these strains to study the genetic control oflung cancer phenotype, because their respective parents, 020 and Bro.020 produce lung tumors with different biological properties.

76 The microscopic appearance of cancer has been the major predictor of its clinical behavior. In order to study the genes affecting lung tumor progression and correlate them with gene expression patlems, we have used a new method for qualitative assessment of mouse lung tumors that provides a numerical description of the 3-D shape of a tumor. Eight new lung tumor shape determining (Ltsd) loci have been detected that influence the overall three dimensional (3D) shape of a tumor. We have established the specific effects of these loci on presence of focal or regional regions of tumor progression (Figure VII.8). These studies lay the basis for a more detailed study of the mechanisms of genetic control of tumor pro gres sion. These findings open the way for expres sion profiling studies to establish the correlation between the host genotype tumor phenotype, and tumor gene expres sion patlern. Mouse genome profiling Genetic background plays an important role in the phenotypic outcome of a studied genetic difference. Most characteristics of a trait are under multigene control. Modifier loci and complicated interactions can alter the efficiency of protein nmction and modify the pathways in which they are involved. In designing mouse models for cancer it is of importance to 'control' the genetic background. For practical reasons most transgenic and mutant mouse models in the Netherlands Cancer Institute have mixed background genes, which may be interfering with the experimental outcome. U sing marker assisted selection protocols 'speed-congenic' mouse strains can be generated in order to obtain the transgene or mutant on a desired gene tic background. For genotyping we will use SNPs (single nucleotide polymorphisms). The first generation SNP map of the mouse is freely available at the website of the Whitehead Institute and includes data on about 1000 loci of commonly used laboratory strains, however no information is available on the in-house used FVB and 129/01a strains. We are setting up a method for high throughput micro-array based SNP analysis. The method uses single base extension (SBE) using bifunctional primers carrying a unique sequence tag in addition to a locus specific sequence. Microarrays are generic and primers complementary to the unique sequence tags are spotled on the glass. SNPs are genotyped in multiplex reactions, hybridized to tag-matching spots and visualized by different dyes. It is of interest to investigate how the micro-array technology can be used to make chips for complete mouse genome profiling and how well hybrids can be recognized. Genome profiling is not only applicable to marker assisted breeding, but is of importance for linkage studies as well as for the genetic control of the colony. A. 8. Selected publications Bodnar JSS, Chatterjee A, Castellani LW, Ross DA, Ohmen J, Cavalcoli J, Wu C, Da ins KM, Catanese J, Chu M, Sheth SS, Charugundia K, Demant P, West DV, DE Jong P, Lusis AJ. Positional cloning of the combined hyperlipidemia gene Hyplip 1. Nature Genet. 2002; 30: Ruivenkamp CAL, Van Wezel T, Zanon C, Stassen APM, Vlcek C, Csikos T, Klous A, Tripodis N, Perrakis A., Boerrigter L, Groot PC, Lindeman J, Mooi WJ, Scholten G, Dauwerse H, Paces, Van Zandwijk N, Van Ommen GJB, Demant P. Ptprj is a candidate for the mouse colon cancer susceptibility locus, SCcl, and is frequently deleted in human cancers. Nature Genet 2002; 31: Badalova J, Svobodova M, Havelkova H, Vladimirov V, Vojtiskova j, Engova J, Pilcik T, VoIfP, Demant P, Lipoldova M. Mapping of multiple genes that control IgE level in Laishmania major injècted mice. Genes Immunity 2002; 3: Lipoldova M, Svobodova M, Havelkova H, Krulova M, Badalova J, Nohynkova E, Hart AAM, Schlegel D, VolfP, Demant P. Mousegenetic modelfor clinical and immunolgical heterogeneity of leishmaniasis. Immunogenetics 2002; 54: Tripodis N, Demant, P. Genetic analysis of 3 D shape of mouse lung tumors reveals 8 Lung tumor shape determining (Ltsd) loci, that are associated with tumor heterogeneity and symmetry. Cancer Res (in press). Ruivenkamp C, Hermsen M, Postma C, Klous A, Baak J, Meijer G, Demant P. LOH of PTPRJ occurs early in colorectal cancer. Oncogene (in press). Figure VII.8: A) Schematic representation ofheterogeneous asymmetrical tumour showingfocal pattems of alveolar (pink) and papiuary (red) growth. The box represents the approximate place that is shown in the photo below, showing clear papillary jèatures and more intensely stained ceus on the left and alveolar structures...vith higher nuclear pleiomorphism on the right (200X magnification). B) Schematic representation ofheterogeneous symmetrical tumour with peripheral (pink) alveolar and ii.temal secondary papillary (red) structures. The box represents the approximate place that is shown in the photo, showing peripheral alveolar adenoma structures, while Jurther in typical papillary projections can be seen (200X magnification).

77 VIII DIVISION OF EXPERIMENTAL THERAPY PREDICTION AND MODIFICATION OF TREATMENT OUTCOME <IJ 3l 50 <IJ <l> > <l> 40 > ~ g> 30 c:: ûî <l> o I 10 ~ Division head, group leader Adrian Begg Adrian Begg PhD Group leader Gerard Blommestijn PhD Academic staff Frank Hoebers MD Radiation Oncologist Conchita Vens PhD Post-doc Hilde Janssen MD Graduate student Gaby Rumping MSc Graduate student Christie VermeuJen MSc Graduate student lise Graversen Undergraduate student Ingrid Hofland Technical staff Debbie Sprong Technical staff Manon Verwijs Technical staff Thea Eggenhuizen Secretary 60 r , H&N gliomas % IUDR negative vessels r=o.94 p<o.001 Figure VIII.l: Correlation between Hoechst negative blood vessels and IUdR-negative vessels (no tumor celllabeling surrounding vessel) in human tumor xenografts. Each point represents a separate tumor line. These data support the use of IUdR as a perjusion marker in human cancer. Collaboration with group of A Van der Kogel, Nijmegen Our lab has a long-standing interest in developing ways to predict outcome after radiotherapy, in order to select patients for more individualized and efficient treatment. This has included prediction of repopulation, intrinsic radiosensitivity and hypoxia. In parallel, we are focusing on genes and pathways affecting radiosensitivity, attempting to use this information to eventually modulate radiosensitivity for therapeutic benefit. Role and prediction of tumor hypoxia Tumor hypoxia measured is a negative prognostic factor for patients undergoing radiotherapy, chemotherapy or surgery. We are studying rapid and simple ways to measure both chronic and acute hypoxia in human head and neck cancer using pimonidazole (a bioreductive hypoxic marker drug), IUdR (iododeoxyuridine; S-phase marker), and endogenous markers. These will be assessed for the ability to select patients at high risk of treatment failure. In a collaboration with Leuven (K Haustermans, P Delaere), Nijmegen (J Kaanders, H Marres, A Van der Kogel) and Brussels (P Mahy, V Gregoire), we have now accrued 83 head and neck cancer patients who were given pimonidazole and IUdR the night before primary surgery. We are analyzing vasculature, pimonidazole staining, IUdR-negative but Ki67-positive tumor cells around individual vessels (a measure of perfusion deficit), and expression of HIF-IU (Hypoxia Inducible Factor) as an endogenous hypoxia marker. Of 44 patients analyzed so far, no correlations have been seen between pimonidazole fraction and either T-stage, N-stage, differentiation grade or tumor diameter, and no correlation between primary tumor and lymph node metastases. Pimonidazole staining (chronic hypoxia) therefore appears to be independent of other clinical prognostic factors. First correlations with outcome will be carried out at the end of the year. Further validation of IUdR as a perfusion marker was carried out in collaboration with the Nijmegen group (A Van der Kogel and colleagues). A series ofhuman head and neck tumor xenografts and a series ofhuman glioma xenografts we re labeled with both Hoechst 33342, a direct perfusion marker, and IUdR. There was a strong correlation between the fraction of IUdR-negative vessels (no labeled tumor cells in the vicinity) and the fraction of Hoechst-negative vessels, supporting the use of IUdR-negative vessels as a measure of perfusion in individual vessels (Figure VIIl.I). This may therefore provide a simple measure of the extent of acute hypoxia (further studies are necessary for confirmation); no such assay has as yet been described for this potentially important parameter for use in humans. Sharp and unusual transitions to pimonidazole staining is often seen in areas of differentiationjkeratinization in head and neck tumors, raising the question of whether this is hypoxia-related. We hypothesized that a possible cause could be increased enzyme levels (for nitro-reduction) in these areas, leading to staining even if not hypoxic. We employed a nitrotetrazolium salt (NBT) which yields a blue color on reduction, indicating levels of nitroreductive enzymes. Cells overexpressing P450 reductase (I-electron reduction; hypoxia dependent) or DT-diaphorase (2-electron reduction, partially hypoxia independent) were used to test staining. Higher NBT staining was seen under oxic conditions for both P450 and DT-diaphorase overexpressing lines. Frozen sections ofhead and neck tumors were incubated with either pimonidazole or NBT under oxic or hypoxic conditions. More pimonidazole staining was always seen under hypoxic than oxic conditions, as expected. However, under oxygen, areas of pimonidazole staining we re seen around some areas of keratinization, but these did not correlate with NBT staining. We conclude that oxicrelated staining can occur, although high concentrations of nitroreductive enzymes is not the likely cause. For accurate application as an hypoxia marker, developing ways to assess non-hypoxia-related staining remains a worthwhile goal.

78 ~ ~I~S~~~:.ILil~~.'~ ;.=1.1.~.1~~" ;~1.~1~L ~ Mechanisms and modulation of radiosensitivity We showed that expression of the DNA binding domain of DNA polymerase ~ (pol~dn), an enzyme involved in base excision rep air (BER), radiosensitized two human celllines, presumably by dominant negative action resulting in successful competition for binding sites with the wildtype protein. This implicates BER as an important factor in determining radiosensitivity. In order to exclude effects of the pol~dn on cell survival unrelated to BER, we analyzed further the response to DNA damage. No sensitization of the polbdn expressing cells was found with UVC irradiation, producing predominantly thymine dimers, indicating that nucleotide excision rep air (NER) is not inhibited by this dominant negative. It also demonstrates that sensitization by pol~dn to ionizing irradiation does not result from some secondary DNA response that is common to both pathways, i.e. P53 activation or apoptosis signaling pathways. Moderate although variabie sensitization by the dominant negative was seen with cells treated with H These data are again consistent with an inhibitory effect on repair of oxidative damage (base damage and single strand breaks). We further analyzed cell cycle blocks induced by irradiation using IUdR and flow cytometry, showing that the GrjS and G2jM blocks were similar to those in nonpol~dn-expressing cens. This exdudes an effect on checkpoints and is consistent with our hypothesis of an effect on DNA repair. We also analyzed double strand break (DSB) induction and repair using constant field gel electrophoresis and did not find any differences between polbdn - expressing and vector control cens. There is therefore apparently no effect, through undiscovered mechanisms, on DSBs. However, since it is known that unrepaired single strand breaks can lead to the production ofdsbs, we plan to confrrm these studies using more sensitive methods. To look in more detail at the role of DNA polymerase ~, we established several single cen dones of polymerase ~ knockout and wildtype murine cens expressing either the pol~dn or empty vector. These will be ideal tools to use together with a plasmid assay which we have set up this year to monitor BER efficiency. Extracts from the cens will be used to rep air engineered damage in the plasmid, from which we can elucidate the contribution of the long versus short patch pathway in repair after ionizing radiation. In surnmary, these data continue to support a significant role for base excision rep air in determining the cell's survival chance after ionizing radiation. Selected publications Awwad HK, Lotayef M, Shouman T, Begg AC, Wilson G, Bentzen SM, Abd EI-Moneim H, Eissa S. Accelerated hyperfractionation (AH F) compared to coventional fractionation (CF) in the postoperative radiotherapy oflocally advanced head and neck cancer: injluence of proliferation. Br J Cancer 2002; 86: Begg AC, Sprong D, Balm A, Coco Martin jm. Premature chromosome condensation and cell separation studies in biopsies from head and neck tumors for radiosensitivity prediction measurements; Radiother Oncol2002;62:j Girard PM, Riballo E, Begg AC, Waugh A, jeggo PA. NbSl promotes ATM dependent phosphorylation events including those required for GIjS arrest. Oncogene 2002;21: Hoebers FPl. janssen H LK, Valdes Olmos RA, Sprong D, Nunn AD, Balm Aj, Hoefnagel CA, Begg AC, Haustermans KMG. Phase 1 study to identify tumor hypoxia in patients with head and neck cancer using TC-99m BRU 59-21, Eur J Nucl Med Mol Imaging 2002;29: Hofland I, Ramakers B, Begg AC, Yens C. Rapid Jluorescence ratio assay for detecting changes in treatment sensitivity. Radiat Res 2002;15T734-9 janssen HLK, Haustermans KMG, Sprong D, Blommestijn G, Hofland I, Raleigh JA, Semenza GL, Varia MA, Balm Al. Van Velthuyzen L, Delaere P, Sciot R, Begg AC. HIF-w, pimonidazole and iododeoxyuridine to estimate hypoxia and perfusion in human head and neck tumors. Int J Rad Oncol Biol Phys, (in press). Yens C, Mooren E, Verwijs-janssen M, Begg AC. The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells. Nucleic Acid Res 2002:]0:

79 VASCULAR MEDIATED TISSUE DAMAGE AND THERAPY The focus of our lab is on vascular damage related to cancer therapy. Group leader Fiona Stewart Fiona Stewart PhD Group leader Paul Baas MD PhD Academic staff Liesbeth Boersma MD PhD Academic staff Nicola RusselI MD PhD Academic staff Maurice Aalders PhD Post-doc Jacqueline Kruse PhD Post-doc Martijn Triesscheijn MSc Graduate student Saida Zeamari MSc Graduate student Ben Floot Technicial staff Marjan Ruevekamp Technicial staff Johannes Te Poele Technicial staff Selected publications Cooper MP, Tan BI, appelaar H, Ruevekamp MC, Stewart FA. mediated photodynamic therapy in early stage squamous ceu carcinoma ofthe head and neck. Arch Otolaryngol Head Neck Sur; (in press). Cramers P, Ruevekamp M, appelaar H, Dalesio 0, Baas P,Stewart FA. uptake and disctribution in relation to photodynamic therapy. Br J Cancer (in press). Kuin A, Kruse )J, stewart FA. Proteinurea and vascular changes after renal irradiation: the role of reactive oxygen species (ROS) and vascular endothelial growthfactor (VEGF). Radiat Res (in press). schouwink H, appelaar H, Ruevekamp M, Van der Valk M, Hart G, Rijken P, Baas P,Stewart FA. Oxygen depletion during and after mthpc mediated photodynamic therapy in RIFI and H-MESO 1 tumors. Radiat Res (in press). Radiation induced vascular damage Cancer patients treated with radiotherapy are at risk for the development oflate radiation-induced damage to normal tissues, much of which is related to vascular injury. In large vessels radiation-induced injury is manifest as atherosclerosis, resulting in severe thrombo-embolic events like stroke. In smaller vessels it manifests as telangiectasia, causing cosmetic and functional problems in the skin and more severe problems, e.g. excessive bleeding, in rectal or bladder mucosa. We have previously identified several thrombotic and inflammatory changes that precede functional impairment in irradiated tissue. However, the interaction between deregulated pathways and their functional significance is still unclear. The aim of the current work is to identify molecular changes that are directly related to the development of radiation telangiectasia, and which may be suitable targets for intervention. Histological specimens from irradiated human skin and rectum, and from irradiated mouse rectum and kidney, all showed the presence of abnormal, telangiectatic blood vessels at late times after irradiation (from about 20 weeks). Micro-array experiments were performed using amplified RNA isolated from irradiated mouse kidney and rectum, and from age matched, unirradiated controls, at r to 30 weeks after treatrnent. Expression profiles were compared and differentially expressed genes identified after normalization procedures, using self-self and color reverse control experiments. The extent of overlap in expres sion profiles for kidney and rectum during the phase of vascular damage was also examined. The mouse kidney showed differential expression of a limited number of transcripts «105 upregulated and <230 downregulated) over the period weeks af ter irradiation. Several genes were persistently differentially expressed during the period of developing telangiectasia; these were of particular interest in terms of their known involvement in inflammatory response and vascular injury (e.g. Jagged-r, TSAr and Spi-4). Irradiated mouse rectum had 73 upregulated and 2r downregulated genes at 20 weeks. Only five of the upregulated genes we re common to both telangiectatic tissues. One of these, was Jagged-r, a Notch ligand known to play a role in angiogenesis. Micro-array analysis of irradiated mouse tissue has therefore revealed new genes of interest, which may be involved in the vascular mediated damage. Similar experiments are ongoing to compare telangiectatic and normal human skin and rectum from breast and prostate cancer patients previously treated with radiotherapy. Photodynamic Therapy (PDT) involves systemic delivery of a photo-sensitive drug and local tumor activation by illumination with light of aspecific wavelength. This generates free radicals and singlet oxygen, which leads to local tissue damage. Clinical protocols for PDT are based on the assumption that optimum intervals between photosensitizer administration and illumination are times of maximum differential between drug uptake in tumor and surrounding normal tissue. PDT mediated tumor destruction can, however, occur both via direct cell killing and via vascular mediated damage. The hypothesis to be tested in this project is that drug exposure of endothelial cells in vessels feeding the tumor, and the resultant vascular damage, are more important determinants of PDT response than the tumor drug levels. This could have a major influence on the design of optimal clinical protocols. In vitro experiments were carried out to compare the toxicity of PDT in human tumor cells, endothelial cells and fibroblasts, for illumination at various times after exposure to the photosensitizer Foscan. These experiments demonstrated that short drug exposure times (3h) were sufficient to induce maximum phototoxicity in microvascular endothelial cells (HMVECs), whereas much longer times (r2-24h) we re required in tumor cells (HMESO-r, HNXOE) and fibroblasts. In vivo studies demonstrated a major discrepancy between times of maximum photosensitizer levels in HMESO-r or HNXOE tumors grown in nude mice and optimum drug-light intervals for PDT response (tumor cure and growth delay). There was a much closer correlation between plasma drug levels and PDT response; with maximum effect seen for drug-light intervals of r-3h. This supports our

80 hypothesis of a major influence of vascular (endothelial cell) mediated PDT response. We are also investigating the relationship between PDT response and photosensitizer uptake and distribution in patients with multiple basal cell carcinomas. A total of 189 tumors, in five patients, have been treated so far. Pharmacokinetic profiles for Foscan showed delayed plasma peak at hours af ter bolus i.v. injection, with exponential clearance thereafter. Tumor control for illumination at 24 hours after photosensitization was %, depending on tumor location, whereas only 33-80% CR was obtained for drug-light intervals of hours. These results are being correlated with pharmacokinetic profiles for Foscan, to determine whether plasma levels give a better prediction of PDT effect than tumor drug levels, as was found in animal studies. A novel technique, optical coherence tomography (OCT), has recently been developed that allows non-invasive imaging of structural and functional changes in superficial tissues. We are currently using this technique to monitor early PDT changes in normal tissues and tumors in mice. Prelirninary results indicate that PDT initially targets the capillary vascular layer, followed by a more generalized vascular shutdown. These studies will be extended to determine whether vascular mediated changes in tumors and normal tissues can be quantified, in real time, after PDT. This technique may eventually have clinical application for monitoring the efficacy of PDT. Inhibition of tumor cel! seeding in surgical wound sites Peritoneal surgery for abdominal cancer disturbs the normally non-adhesive and non-thrombotic peritoneum and tumor cell seeding in surgical scar tissue is a common cause of early recurrence. The purpose of this study is to identify growth factors and adhesion molecules released at peritoneal wound sites, which are involved in tumor seeding and growth. At a later stage, this information will be used to design and test intervention strategies. A reproducible model for peritoneal granulation tissue was established by implantation of small pieces of gelatin sponge in the mesenteric fan of nude mice. This model was used to investigate the invasion of tumor cells into a wound in relation to the expression of specific growth factors. Intraperitoneal inoculation of tumor cells during the first three days after sponge implantation induced tumor seeding and rapid growth in 100% cases, whereas inoculation after >1Od resulted in only limited growth in <33% of granulation matrixes. These results suggest that there are factors released at early stages in the development of granulation tissue which stimulate tumor cell inhltration and growth. PCR analysis of mrna isolated from granulation tissue showed increased expres sion oftgf~, VEGF and LPA receptors I and 2 from 14 days after initiation. However, expres sion of these growth factors was not measurably increased in the early granulation tissue. Expression prohles of early (3d) and late (28d) granulation tissue we re subsequently compared using micro-array analysis. These experiments identified 334 genes that were significantly upregulated in early granulation tissue, and ISO genes that were significantly downregulated. These include several possible candidates for anti-tumor seeding therapies (e.g. CXCR-4, Jagged-I and TIMP-2). In future experiments we will test specific inhibitors of factors expressed in early granulation tissue for their efficacy in preventing peritoneal tumor cell seeding. To quantify peritoneal tumor load in a non-invasive way, we have transfected both human (HT29) and rat (CCS3I) colon carcinoma cells with luciferase. Transfected cells injected i.p. in nude mi ce or rats gave rise to disseminated tumor nodules in the peritoneum. Small tumors (I-2mm in mice) were detectable by extemal imaging ofbioluminescence, and peritoneal tumor growth could be followed in time (Figure VIII.2). These models will be used to evaluate specific anti-tumor cell seeding therapies in future experiments. Selected publications (continued) Stewart FA, Te Poele JAM, Van der Wal AF, Oussoren YG, Van Kleef EM, Kuin A, Verheij M, Dewit LGH. Radiation nephropathy: the link between Junctional damage and vascular mediated injlammatory and thrombotic changes. Acta Oncologica 2 1;4 : : o 20 Figure VII I.2: Sequential extemal imaging of nude mice, at 1 to 3 weeks ajter intraperitoneal (i.p.) injection of 10 6 luciferase-transftcted CC531 ceus, demonstrates increasing signal intensity (top panels), which is quantitatively related to tumor load. Bottom panel shows increasing i.p. tumor growth with time in a group of 4 mice (mean +j- SD). o 2 3 Time after inoculation (weeks)

81 GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG RESISTANCE AND PHARMACOKINETICS Group leader Alfred Schinkel Alfred Schinkel PhD Group leader Maarten Huisman MSc Graduate student Johan Jonker MSc Graduate student Antonius Van Herwaarden MSc Graduate student Ellen Boischer Technical staff Marije Buitelaar Technical staff Corina Van der Kruijssen Technical staff Els Wagenaar Technical staff out in Figure VlIIj: Structure of a prototypic ABC drug transporter. Our research focuses on genes and proteins that cause (multi-)drug resistance in tumors, and/or influence the pharrnacological behavior of anti-cancer, anti HIV/AIDS, and many other drugs. Insight into these systems may improve current and novel chemotherapy approaches for cancer and HIV/AIDS, as weil as pharmacotherapy in a broader sense. 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. CeIllines obtained from these mi ce are further used as tools to characterize drug resistance genes. Active drug transporters affecting HIV protease inhibitor drugs Mdn-type P-glycoproteins (P-gps) are plasma membrane proteins of the ATP binding cassette (ABC) family of proteins (Figure VIII.3). They actively export a wide range of anticancer, anti-hiv/aids, and many other drugs from ceils. This ATP-dependent drug extrusion can cause multidrug resistance (MDR) in tumor ceils. P-gps localize to the apical membrane of polarized epithelial ceils, resulting in vectorial transport of drug substrates. Experiments in Mdna/rb knockout mi ce indicated that the P-gps can protect an organism against exogenous toxins and drugs by limiting penetration of substrates into brain, testis, and fetus, by restricting uptake of oraily administered substrates, and by mediating excretion of substrates via liver and intestine. P-gp limits the brain and fetal penetration, and the oral uptake of the HIV protease inhibitor (HPI) saquinavir. The low penetration of HPIs into pharmacological sanctuary sites may ailow continued replication of HIV in these tissues, thus lowering the chance of complete viral eradication. The low oral bioavailability of HPIs results in high 'pill burden' for patients, making strict therapy adherence much more difficult. Using a pharmacological P-gp inhibitor, we demonstrated the feasibility of in vivo P-gp inhibition to enhance the oral uptake and sanctuary site penetration of saquinavir, although we also observed some toxic side effects. This principle, when applied cautiously, might be tested for improvement of HIV/AIDS therapy. Recent findings by others indicate that the multidrug resistance protein 2 (MRP2 or ABCC2) has similar pharmacological roles as P-gp. We demonstrated that the commonly used HPIs saquinavir, ritonavir and indinavir are all transported substrates for MRP2. Despite extensive efforts, we have not been able to identify suitable inhibitors of this MRP2-mediated HPI transport. However, we did find that the uricosuric drugs probenecid and sulfinpyrazone can markedly stimulate the transport of these HPIs. We are currently investigating whether this stimulation phenomenon can also play a role in the in vivo handling of HPIs, as this could decrease HPI plasma levels. Wild-type Analysis of the BCRP/Bcrpl multidrug transporter Like P-gp, the Breast Cancer Resistance Protein (BCRP/ABCG2) is an active ABC drug transporter for a range of anticancer and other drugs, it is apically located in epithelial cells, and it is present in intestinal epithelium, bile canalicular membrane, and placental trophoblasts. U sing the BCRP inhibitor G Fr209r8 we found indications that murine Bcrpr can limit the oral bioavailability of topotecan, mediate its hepatobiliary excretion, and can shield the fetus from topotecan in the matemal circulation. To validate these results, and to study the normal physiological functions of Bcrpr, we generated Bcrpr knockout mice. Bcrp1-1 - Figure VIII+ BCrpl knockout mice are extremely sensitive to the dietary chlorophyll catabolite pheophorbide a, resulting in severe phototoxic damage to the ear. Mice lacking Bcrpr look overall normal, but they are extremely sensitive to the dietary chlorophyll breakdown product pheophorbide a, resulting in severe, sometimes Ie thai phototoxic lesions on light-exposed skin (Figures VIII.2 and VIII.3). Pheophorbide a is a porphyrin that occurs in various plant-derived foods and food supplements. Bcrpr transports pheophorbide a and is highly efficient in lirniting its uptake from ingested food. Furthermore, as predicted from previous in vivo inhibition experiments, Bcrpr knockout mi ce demonstrated a 6-fold increased oral uptake, and 2-fold increased fetal penetration of topotecan. Bcrpr knockout mice also displayed a novel type of protoporphyria: Erythrocyte levels of the heme precursor and

82 a ~ 5 80 i' c::::::::::j Wild-type I/) c.=; _ Bcrp1 (-1-) 0 X 4 "iii 0:: * ~ a.. C'O 4) 3 4) >. (.) (.) 40 'x c Ö Ö 20 - Bcrpr '- 20% ~..c w -0- a.. Bcrpr'- 10% -0- Wild-type 20% time (days) b * * Normal Phototox. Synth. Figure VIII.5: (a) Incidence of phototoxic ear lesions in wild-type and BCrpl knockout mice on diets containing various levels of alfalfa, the dietary source of pheophorbide a. (b) Bcrp knockout mice have increased protoporphyrin IX (PPIX) levels in erythrocytes (protoporphyria), independent ofthe amount of pheophorbide a in the various diets. phototoxin protoporphyrin IX were IO-fold increased. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity, and pro vide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents. The data imply that prolonged inhibition of BCRP by administration of BCRP inhibitors to patients should be monitored carefully for unexpected side effects. The identification by others of mutations at arginine 482 (R482) in human BCRP in drug-selected celllines explained some discrepancies observed in the BCRP-associated cross resistance profiles. We found that three mouse celllines independently selected for resistance to the anthracycline doxorubicin also acquired mutations exclusively at R482 of mouse BcrpI. Although the Bcrpr amino acid substitutions (M or S) are distinct from those seen in human BCRP (G or T), they all have similar consequences: greater resistance to anthracyclines and lower resistance to topotecan. The ready selection of R482X mutations seen in vitro might also occur in tumors treated with anthracyclines. It is noteworthy that the efficacy of Bcrpr inhibitors was not markedly affected by the mutations. The Bcrpr mutations occurred af ter prior amplification and overexpression of the wild-type gene, indicating that wild-type Bcrpr also mediates resistance to anthracyclines. This was confirmed in Bcrpr-transduced celllines. These observations emphasize the importance of the arginine at amino acid 482 for substrate specificity of BCRP jbcrpi. They also indicate that wild-type Bcrpr remains a potential source of resistance to anthracyclines, and a potential factor in anthracycline pharmacokinetics. The same is most likely true for human BCRP. CYP3A transgenic and knockout mice The cytochrome P450 3A (CYP3A) complex is a major factor in the metabolic breakdown of most drugs used today. As its activity shows great inter- and intra-patient variability, it has a profound influence on variable drug behavior and drug toxicity. Moreover, its drug substrates overlap extensively with those of the drug transporters P-gp, BCRP and MRP2. To study this important system and its interactions with drug transporters in well-defined modeis, we have started a project to generate CYP3A transgenic and knockout rnice. We have already generated several intermediate strains, but additional strains, currently under construction, are needed to begin informative studies on drug behavior. Diet Selected publications Allen jd, jackson SC, Schinkel AH. A mutation hot-spot in the BCrpl (Abcg2) multidrug transporter in mouse celllines selected for doxorubicin resistance. Cancer Res 2002; 62: Allen jd, Van loevezijn A, lakhai jm, Van der Valk M, Van Tellingen 0, Reid G, Schellens jhm, Koomen G-j, Schinkel AH. Potent and specijic inhibition ofthe breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of Jumitremorgin C. Mol Cancer Ther 2002; 1: Allen jd, Schinkel AH. Multidrug resistance and pharmacological protection mediated by the breast cancer resistance protein. Mol Cancer Ther 2002; 1: Huisman MT, Smit jw, Crommentuyn KMl, Zelcer N, Wiltshire HR, Beijnen jh, Schinkel AH. Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs. AIDS (in press). Huisman MT, Sm it jw, Wiltshire HR, Beijnen jh, Schinkel AH. Assessing sajèty and efficacy of directed P-gp inhibition to improve the pharmacokinetic properties of saquinavir used in combination with ritonavir. ] Pharmacol Exp Ther (in press). jonker jw, Buitelaar M, Wagenaar E, Van der Valk MA, Scheffer Gl, Scheper Rj, Plösch T, Kuipers F, Oude Elferink RPj, Rosing H, Beijnen jh, Schinkel AH _ The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria. Proc Natl Acad Sci USA (in press). Schinkel AH, jonker jw. Mammalian drug elflux transporters of the ATP binding cassette (ABC) family - an overview. Adv Drug Deliv Rev (in press). Wang D-S, jonker jw, Kato Y, Kusuhara H, Schinkel AH, Sugiyama Y. Involvement of organic cat ion transporter 1 in the hepatic and intestinal distribution of metjormin. ] Pharmacol Exp Ther 2002; 302:

83 CLINICAL AND PRECLINICAL PHARMACODYNAMICS OF ANTICANCER DRUGS Group leader jan ScheUens Jan Schellens MD PhD Group leader Jos Beijnen PhD Academic staff Alfred Schinkel PhD Academie staff Karin Mohrmann PhD Post-doe Natalie Appels MSc Graduate Student Pauline Breedveld MSc Graduate student Suzanne Bocxe Undergraduate student Özgür Sönmezer Undergraduate student Dick Pluim Technical staff Monique Van Eijndhoven Technical staff Selected publications Baker SD, Verweij J, Rowinsky EK, Donehower RC, Schellens JH, Grochow LB, Sparreboom A. Role of Body-Surface Area of Investigational Anticancer Agents in Adults: j Nat Cancer Inst (in press ). Crul M, De Klerk GJ, Swart M, Van 't Veer LJ, De Jong D, Boerrigter L, Palmer PA, Bol CJ, Tan H, De Gast GC, Beijnen JH, Schellens JHM. Phase I clinical and pharmacologic study of chronic oral administration of the famesyl protein transjerase inhibitor R in advanced cancer. j Clin Oncol 2002; 2 0 : Crul M, Van Waardenburg RCAM, Bocxe S, Van Eijndhoven MA). Pluim D, Beijnen JH, Schellens JHM. DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin. Biochem Pharmacol (in press). Diestra JE, Scheffer GL, Catala 11, Maliepaard M, Schellens JH, Scheper R). Germa-Lluch JR, Izquerdo M. Frequent expression ofthe multidrug resistance-associated protein BCRP/ MXR/ABCP/ ABCG2 in human tumors detected by the BXP-21 monoclonal antibody in paraffin-embedded matenal. j Pathology 2 002; 198: Generation of M RP2-deficient mice and compound M RP2/M RP" M RP2/P-glycoprotein and M RP2/BCRP, knockout mice MRP2 shows a considerable overlap in drug substrates with P-glycoprotein (P-gp), Multidrug Resistance Protein r (MRPr) and Breast Cancer Resistance Protein (BCRP). To establish the role of MRP2 and its functional overlap with other transporters we will generate constitutive Mrp2-deficient mice. By using homologous recombination we already exchanged exons 4-6 of mrp2 by a hygromycine cassette in ES cells. When mrp2-deficient mice have been generated we will use crossbreeding with the existing knockout strains (Mrpr-, Bcrp- and p-gp- deficient strains) to generate double knockout strains. These models will enable to study the functional role of MRP2 in vivo. In addition, the double and multi-knockouts are considered excellent models to explore the pharmacological behavior of substrate drugs that show overlap in affmity for MRPr, MRP2, P-gp and Bcrp. Regulation of tissue specific expression of the m ultidrug resistance transporter BCRP Breast resistance protein (BCRP IABCG2) is expressed in organs involved in uptake, metabolism and excretion of xenobiotics. The level of BCRP expression is variabie between different cell types and tissues. Extemal factors, like drug treatrnent and food compounds, may influence BCRP expres sion and thereby the uptake and excretion of anti-cancer drugs. In order to investigate the regulation of BCRP expression in more detail we tested several human celllines such as HepG2 (liver) and CaC02 (colon), Hek-293 (kidney) for BCRP expression. Furthermore, we tested the capacity of several compounds to induce BCRP expres sion. Until now we have not found a compound that is able to induce BCRP expres sion in these cell lines. Members of the nuclear receptor superfamily ofligand-activated transcription factors play an important role in processes like cholesterol homeostasis, bile acid biosynthesis and transport and xenobiotic metabolism. We will be investigating the possible role of several known transcription factors in the regulation of BCRP expression. Role of BCRP and M RP2 in variability in clinical pharmacokinetics of anticancer drugs Based on clinical observations we are exploring whether BCRP and MRP2 expres sion play a role in the clinical pharmacokinetics of camptothecin analogs. We showed that carbamazepine significantly increased the tissue expres sion of Bcrpr in mouse kidney and liver 1.5 to 2-fold and Mrp2levels in liver 2-fold. Currently, we are investigating whether this upregulation correlates with increased clearance of topotecan in P-gp ko mice (Mdna/rb -1-) after treatment with carbamazepine or dexamethasone. Topotecan has high affinity for BCRP and moderate affinity for P-gp. To elucidate whether topotecan is also a substrate drug of MRP2 we have performed an in vitro transport study using MDCK celllines, parental and stably transfected celllines with MRP2 or Bcrp1. In the Bcrpr-transfected MDCK cellline the transport of topotecan was very efficient, as expected. Preliminary results reveal that topotecan is not efficiently transported by MRP2. MRP2 is known to play a role in methotrexate resistance. For methotrexate, many clinically relevant drug interactions are known, for example with omeprazole, pantoprazole, ciprofloxacin, oxacillin, piperacillin and probenecid. We have therefore investigated whether MRP2 and BCRP play a role in these clinically reported interactions. In order to test this, we performed in vitro transport studies using MDCK celllines (parental, MRP2-transfected and Bcrpr-transfected). Preliminary results reveal that omeprazole and pantoprazole, which are proton-pump inhibitors that are widely used in the treatrnent of peptic ulcers, inhibit the Bcrpr-mediated basolateral to apical transport of topotecan effectively. This inhibition is concentration-dependent. Furthermore, we observed in the MRP2-transfected MDCK cellline that the MRP2-mediated transport of vinblastine, a substrate drug of P-gp and MRP2, can be stimulated by pantoprazole and omeprazole. Therefore, we have also performed an in vitro transport study using membrane vesicles prepared from

84 r ~IIS.~.~.I,~,.~II~~~I~I~~.'~~~:r:)~ ~,~.~~~ ~ Sf9 insect cells transfected with MRP2 expres sion vector (in collaboration with the group of Prof. P. Borst). Preliminary results revealed a concentration-dependent 2 to 6-fold stimulation of transport of the MRP2 substrate drug estradioi2-17-~-dglucuronide by omeprazole. The clinical relevance of these inhibitory and stimulatory effects will be explored in selected in vivo models and later in patients. We have also studied whether ET743, a new anticancer agent derived from the marine organism Ecteinascidia turbinata, is a substrate drug of BCRP andjor MRP2. An in vitro transport study using MDCK celilines (parental, MRP2-transfected and BcrpI-transfected), showed that ET743 is efficiently transported by MRP2 and is not transported by BcrpI. Additional in vivo studies will therefore be performed to examine the role ofmrp2 in the pharmacokinetics ofet743. Other experimental drugs that are under investigation to test their substrate specificity for BCRP are the mitoxantrone derivative CI3II, amonafide and epothilone D. Support of clinical studies We are continuing to support clinical trials with the cisplatin-gemcitabine combination. This combination is being explored in a schedule-intensive combination in advanced non-small ceillung cancer (NSCLC). In addition, it is the basis for combination trials that we are performing with LY317615, a PKC inhibitor, and zarnestra, a farnesyltransferase inhibitor. We have continued the support of the phase I trial with the combination of carboplatin-topotecan. We have determined the pharmacokinetics of both drugs and pharmacodynamic endpoints, especially platinum DNA adducts, topoisomerase Iexpression and activity in white blood cells and tumor tissue. We are also supporting the clinical mass balance trial with 14C-Iabeled E7070, a sulphonurea derivative currently in clinical development. The main metabolic pathway is glucuronidation. Selected publications (continued) Faneyte IF, Kristel PM, Maliepaard M, Scheffer GL, Scheper RL, Schellens JHM, Van de Vijver. Expression ofthe breast cancer resistance protein in breast cancer. Clin Cancer Res 2002; 8:1068'74. Kruijtzer CMF, Beijnen JH, Rosing H, Ten Bokkei Huinink WW, Schot M, Jewell RC, Paul EM, Schellens JHM. Complete oral bioavailability of topotecan in combination with the Breast Cancer Resistance Protein (BCRP) and P glycoprotein (P-gp) inhibitor GF ] Clin Oncol2002; 20: Schoemaker NE, Schellens JHM, ten Bokkei Huinink WW, Beijnen JH. A phase land pharmacokinetic study of MAG-CPT, a water soluble polymer conjugate of camptothecin. BrJ Cancer 2002; 8T Zamboni WC, Gervais AC, Egorin MJ, Schellens JHM, Hamburger OR, Delauter BJ, Grim A, Zuhowski EG, Joseph E, Pluim 0, Potter DM, Eiseman J L. Inter- and Intra-tumoral Disposition of Platinum in Solid Tumors after Administration of Cisplatin. Clin Cancer Res 2002; 8:

85 MOLECULAR PATHOLOGY Group leader Laura Van 't Veer Laura Van 't Veer PhD Group leader Petra Nederlof Ph 0 Academ ic staff Floor Van Leeuwen PhD Academic staff Sjoerd Rodenhuis MD PhD Academic staff Nicola RusselI MD PhD Academic staff Senno VerhoefMD PhD Academic staff Annegien Broeks PhD Post-doc Erik Van Beers PhD Post-doc Dorien Voskuil PhD Post-doc Britta Weigeit MSc Graduate student Alina Vrieling MSc Graduate student Lotte De Wit MSc Research assistant Elke De Grouw Undergraduate student Astrid Bosma Technical staff Arno Floore Technical staff Angelina Huseinovic Technical staff Siegina Klaver Technical staff Ben Nota Technical staff Tibor Van Welsem Technical staff ArM heterozygous germline mutations contribute to breast cancer susceptibility Epidemiological studies have indicated that female heterozygous carriers of an ataxia-telangiectasia mutated gene (ATM) have an increased risk of breast cancer (RR 3-9). The purpose of our study is to determine the contribution of ATM heterozygosity to the risk of (radiation-induced) breast ca neer. The frequency of ATM gerrnline mutations is examined in three case-control studies of women who developed breast ca neer subsequent to exposure to various levels of ionizing radiation. One of these studies now includes 258 breast cancer patients who were treated by radiotherapy and revealed four truncating ATM gerrnline mutations among 58 cancer patients who subsequently developed a contralateral breast cancer and four among 200 unilateral breast cancer control patients. The proportion of ATM heterozygotes among contralateral breast cancer patients (7%) is significantly higher compared to a: the ATM carrier frequency in the general population, approximately r%, and b: the ATM carrier frequency among the unilateral controls (2%; P=0.05, Fisher's exact test). Our results indicate that unilateral breast cancer patients who are heterozygous for an ATM germline mutation have an RR of 3.6 to develop bilateral breast cancer at young age and an RR of 5.7 (95%CI ; P=0_05) for a contralateral breast cancer. To exarnine the separate and joint effects of radiation exposure and ATM germline mutations on breast cancer development we are now conducting a case-control study with cases defined as incident contralateral breast cancer cases and selected regardless of treatrnent history. Our case-control study on breast cancer patients following radiation treatrnent for Hodgkin's disease where approximately 90% of these tumors are estimated to be radiation-induced, did not reveal any ATM germline mutations. However, comparative genomic hybridization, does show us that these breast cancers have a specific pattern of genomic alterations. In a small related study we investigated whether ATM heterozygous mutations are involved in familial breast cancer. Three out of II5 high-risk breast ca neer families carry an ATM gerrnline mutation. Co-segregation of the mutation with the occurrenee ofbreast cancer is suggestive though not yet conclusive in these families. The understanding and identification of cancer susceptibility genes and genotypes that predispose women for radiation-induced breast cancer win provide the possibility to identify genetically radiation susceptible individuals. Molecular profiling for disease staging and therapy response in breast cancer We have established by gene expres sion profiling an expres sion pattern of 70 marker genes in tumors predictive of a short interval to distant metastasis ('prognosis signature') in young lymph node negative breast ca neer patients (see Division XIII). In addition, we are now evaluating whether profiles determined in the primary tumors of patients that later metastasize are still present in these distant metastases. This knowledge is important for the use of the initial profile in determining adjuvant treatrnent selection. The next relevant question to answer is the prediction of treatrnent response. In patients treated in the context of a tamoxifen trial we are currently establishing a tamoxifen response profile. Prospective clinical trials have been started to evaluate short-term clinical responses (e.g., doxorubicin, taxanes, aromatase-inhibitors). We have identified a range of genes that we re expressed in breast cancer, but were absent in the profiles ofblood or bone marrow cells. Quadratic discriminant analysis using re al-time quantitative PCR for four marker genes prb, ps2, CKr9 and EGP2 provided us with a discriminant value with 30% positivity in blood of the breast cancer patient group and no false positives among the healthy volunteers. The positive value is seen in a subgroup of patients that were known to have bone marrow metastases at the time of blood sampling and is absent in patients with metastatic disease in other organs. Moreover, the MRD detection in peripheral blood seems indicative for a shorter survival interval. The Insulin-like Growth Factor {IGF} system in relation to breast cancer and colorectal carcinogenesis Large prospective epidemiological studies have recently confirmed that the risk of several cancers (e.g. breast, colorectal, prostate, lung) is

86 increased in individuals with relatively high serum concentrations ofigf-r. Experimental evidence shows that at the tissue level, IGF-r is a potent mitogenic and anti-apoptotic factor. Both lines of evidence point towards the IGF-system as a potential target for cancer prevention studies. The purpose of this project is to evaluate the effects of several dietary strategies on the circulating IGF-system, and to characterize the association between the circulating IGF-system and breast and colorectal tissue IGF systems, using bioassays and molecular techniques. In a series of70 female BRCA1/BRCA2 gene carriers who had undergone a prophylactic mastectomy, we performed preliminary analyses oflevels ofigf-system components in serum and tissue. IGF-r and IGF-binding protein 3 (IGFBP-3) serum concentrations were assessed by radioimmunoassays. IGF-1, IGF-2, IGF type r receptor, and IGF type 2 receptor mrna levels were quantitatively determined by re al-time PCR. In the small series of normal breast tissue and serum collected within one year of mastectomy, the expres sion levels of tissue IGF-r and IGF type r receptor mrnas were positively correlated with the serum levels ofigf-i. In the first six months of the project, ethical approval for a pilot intervention study has been conducted (65% participation rate) and has provided information on logistics, use of intervention and placebo pills, clarity of the protocol, sample handling and serum concentration measurements. The proposed research is expected to provide a better fundamental understanding of whether and how the IGF-system could be the target of cancer preventive measures. Ultimately, dietary intervention strategies will provide means of decreasing the risk of cancer development. Selected publications Bosma Aj, Wei geit B, Lambrechts AC, Verhagen OJHM, Pruntel R, Rodenhuis S, Va n 't Veer LJ. Detection of ciculating breast tumor cells by differential expression of marker genes. ] Clin Can Res 2002; 8:1871'7. Broeks A, Urbanus j, De Knijff P, Devilee P, Nicke M, Klopper K, Dork T, Floore A, Van 't Veer LJ. IVSlO-6T>G: an ancient ATM germline mutation associated with breast cancer. Human Mutation (in press). Wessels LFA, Hart AAM, Van 't Veer Lj, Reinders MJT, Nederlof PM. Molecular classification ofbreast carcinomas by CGH: aspecific somatic genetic profile for BRCAl tumors.. Cancer Research (in press). GENOME-WIDE ANALYSIS OF CHROMOSOME INSTABILITY IN BREAST CANCERS Genetic predisposition for breast cancer Several population-based studies have shown that IO-r5% of the breast cancer patients have a positive family history for the disease and it has been estimated that approximately 5-10% ofbreast cancer can be attributed to hereditary factors. Germline mutations in the two breast cancer susceptibility genes BRCAI and BRCA2, explain approximately 2-3% of all breast cancer cases and about 50% of all hereditary cases. In about 25% of the families fulfilling the criteria for DNA mutation analysis for BRCAr and BRCA2 (breast cancer RR >3) that are investigated in the Netherlands (n=2200 families tested) a mutation was determined in one of these genes. In the remaining cases (75%) no mutation was identified. Moreover, missense mutations and so-called unclassified sequence alterations have been identified for which the proof is lacking that these are disease-causing mutations. We are using a new strategy to identify possible BRCAr germline mutation carriers on the basis of genomic alteration present in their breast carcinomas. The hypothesis is that BRCAr tumors develop by a distinct pathway involving specific chromosomal changes. We aim to determine both common as wen as unique rearrangements for specific breast cancer classes (BRCAr, BRCA2, BRCAX). This should be instrumental in elucidating the various mechanistic bases of disease, and hopefully be useful for diagnostic screening of potential germline mutation carriers. We are employing both classic comparative genomic hybridization (classic-cgh) on metaphase chromosomes and Bacterial Artificial Chromosome (BAC) array CG H to assess somatic genetic changes (profiles) of primary breast tumors > " 2S 26' 2S.J 2' " ' 35 Construction of a BRCAl classifter We have applied statistical methods to develop a BRCAr classifier that discriminates BRCAr tumors (28) from non-brcar carriers (42) on basis of their CG H profile. We evaluated possible classifiers (Bayesian classifier, penalized logistic regres sion and nearest neighbor classifier) and optimized the classification performance by selecting an appropriate resolution (arms, bands or channels) and representation (continuous or discrete). The Simple Bayesian Classifier (SBC) appeared the best classifier, with a performance of 84% using the fluorescence ratios of only three chromosomal regions 3 r, 3-5 and 5.2 (see Figure VIII.6). Figure VIII.6: Schematic representation ofthe BRCA1-Classifier, which discriminates BRCA1 carriers from non-carriers on basis of classic-cg H. The arrows indicate the bands that are included in the classifier. Note that although more bands show significant deviations from controls, the above classifier performs best in discriminating the BRCA1 tumors from the controls (Wessds et al. Cancer Res. in press)

87 Currently, we are applying the same approach to determine a BRCA2 classifier with classic-cgh. The BRCA2 samples were in part obtained through collaboration with P Devilee, C Cornelisse, and R Van Oldenburg (LUMC, Leiden). Preliminary results show differences compared to controls as well as to the BRCAr tumors. In addition, we will analyze high-risk families with an as yet negative test result in order to identify potential BRCAr/2 carriers or aspecific genetic profile for BRCAX. To fine map the gene tic changes we have analyzed breast tumors on BAC-arrays containing> 500 BACs known to be involved in cancer (collaboration with J Gray, UCSF, San Francisco). r9 BRCAr samples and r8 non-brcar samples were analyzed and comparison to our CG H results of the same tumors showed good correlation (0.877). Differences can be explained by the lower resolution of the CGH compared to BAC array experiments.

88 MOLECULAR ANALYSIS OF BREAST CANCER Genetic alterations in ductal carcinoma in situ and invasive carcinoma of the breast The aim of this work is to identify genetic alterations in breast cancer and, more particularly, the changes that are involved in the progression of carcinoma in situ to invasive breast cancer. Until now, no specific invasion-associated genetic alterations have been found. Of r2 invasive breast carcinomas with a relatively large in situ component, we compared the genetic alterations in the invasive and in situ component of the same tumor by comparative genomic hybridization (CG H) using chromosome spreads. In a collaboration with the laboratory 00 Gray (UCSF, San Francisco, USA), we have also used BAC arrays to perform CG H on 20 pairs of in situ and invasive carcinoma from the same tumor. In some tumors, we 0 bserved a few distinct differences between otherwise identical genome profiles ofboth components suggesting that the number of genetic alterations involved in breast tumor progression is limited. By additional fluorescence in situ hybridization (FISH) analysis, we found in one tumor high-level amplification of C-MYC in the invasive component only (Figure VIII.7). To validate this correlation, we identified from a panel of r88 invasive carcinomas r8 cases with C-MYC amplification, nine of which had an adjacent in situ component. Using FISH, C-MYC signals >5 per nucleus were found in seven and C-MYC/CEP8 ratios >4 were found in five of nine invasive components but not in any associated in situ component. With probes of three BAC clones derived from chromosome 8q the minimal amplified region was defined at 8q24.r-8qter. C-MYC amplification was correlated with overexpression of C-MYC and two target genes, TERTand FBL. Thus, high-level C-MYC amplification is the first identified genetic alteration that is strongly associated with progression from the in situ to the invasive stage ofbreast carcinomas. Group leader Marc Van de Vijver Marc Van de Vijver MD PhD Group leader Harry Bartelink MD PhD Academic staff Hans Peterse MD Academ ic staff Els Robanus Maandag PhD Post-doc Juliane Hannemann MSc Graduate student Bas Kreike MSc Graduate student Cathy Bosch Technical staff Hans Halfwerk Technical staff Petra Kristel Technical staff Selected publications Faneyte IF, Kristel PM, Maliepaard M, Scheffer GL, Scheper RJ, Schellens JH, et al. Expression of the breast cancer resistance protein in breast cancer. Clin Cancer Res 2002;8: Genetic expression profiling in breast cancer to predict clinical behaviour Gene expres sion profiling has been performed using RNA from 295 breast carcinomas. A detailed description of this work is presented in the section on the Division of Diagnostic Oncology. In brief, RNA from each tumor was amplified and erna was fluorescently labeled and hybridized to a micro-array containing -25,000 human genes synthesized by inkjet technology. Analyzing fluorescence intensities of scanned images, it was found that -5,000 genes were significantly altered across the group of samples, i.e., there was at least a two-fold difference with a significant p value ofless than o.or in more than five tumors. We are now exploring whether gene expression profiles can be identified, corresponding to: Specific genetic alterations in the DNA of these breast carcinomas, histologie features of the tumors (including type and grade), patterns of the formation of distant metastases, response to specific forms of systemic therapy, locoregional recurrenee of disease. Schrama JG, Faneyte IF, Schornagel JH, Baars JW, Peterse JL, Van de Vijver Ml. et al. Randomized trial ofhigh-dose chemotherapy and hematopoietic progenitor-cell support in operable breast cancer with extensive lymph node involvement: final analysis with 7 years of follow-up. Ann Oncol 2002;1]: Van 't Veer Ll. Dai H, Van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome ofbreast cancer. Nature 2002;415: Van de Vijver MJ, He YD, Van 't Veer LJ, Dai H, Hart AAM, Voskuil DW, Schreiber Gl. Peterse J L, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ETh, Friend SH, Bernards R. A gene expression signature as a predictor of survival in breast cancer. New Engl J Med (in press). Figure VIII.7: Breast carcinoma analyzed using FISH with a C-MYC probe and a CEP8 probe. The ductal carcinoma in situ component (DCI S) shows anormal C-MYC copy number; the invasive ductal carcinoma component (IDC) shows >10 C-MYC copies; the CEP8 centromere probe shows two copies in both components.

89 CHROMATIN GENOMICS Group leader Bas van Steensel Bas van Steensel PhD Group leader Romeo Lascaris PhD Post-doe Marike Feenstra MSc Graduate student Frauke Greil MSc Graduate student Maart je Vogel MSc Graduate student The main goal of our laboratory is to understand how gene expres sion programs in higher eukaryotes are regulated. In particular, we are focusing on mechanisms of chromatin-mediated gene silencing. It is becoming increasingly clear that gene silencing is essential for the establishment and maintenance of cellular identity, and that aberrant gene silencing can contribute to cancer. Using Drosophila and mammalian cells as model systems, we are developing and applying new whole-genome approaches to study the mechanisms of gene silencing. Chromatin profiling of gene silencing complexes Last year, we published a novel technique (named 'chromatin profiling') for the genome-wide mapping of in vivo target loci of chromatin proteins (Van Steensel et al (ZOOI) Nature Genet. zt304-8). This micro-array based method allows for rapid screening of genomic sequences for in vivo binding of achromatin protein or transcription factor of interest. We have now substantially improved this technique: by incorporating a PCR-based amplification strategy, we have managed to scale down the method by almost three orders of magnitude. This will enable us to map target loci of chromatin proteins in small numbers of cells, in single flies, or even in dissected tissues. In collaboration with J Delrowand S Henikoff (Fred Hutchinson Cancer Research Center, Seattle, USA), we have performed a detailed analysis ofheterochromatin, a specialized type of chromatin involved in gene silencing. We have recently completed 6,000-gene binding profiles of three heterochromatin proteins in Drosophila: HPI/HPla, HPIC, and Su(var)3-9. This has resulted into the identification of at least one hundred target genes for each protein. Comparative analysis of these binding patterns has revealed the existence of different types ofheterochromatin with distinct chromosomal distributions. We have found that some of these heterochromatin types are preferentially associated with distinct networks of developmentally co-regulated genes. Understanding the molecular signals that direct the assembly and function ofheterochromatin is one of our main goals in the future. We are currently testing and adapting the chromatin profiling technology for use in mammalian cells. Bioinformatics Targeting of chromatin complexes to specific sets of genes must be guided at least in part by cis-regulatory elements. To find sequence elements that may mediate the recruitrnent of achromatin protein, we have initiated an extensive collaboration with bioinformaticist Dr. H Bussemaker (Columbia University, New York, NY). We have successfully tested and optimized the REDUCE algorithm for analysis of our whole-genome protein binding maps. This algorithm enables us to identify the preferred sequence context for in vivo binding of a given chromatin protein. For example, we found by REDUCE analysis that HPla preferentially binds 8 pericentric heteraramat;n 7 0> c '6 c :0 T""" 0... I <D > ~ ~ Figure VIII. 8: Map of HP1 binding sites for 1765 probed genes on chromosome 2 in Drosophila. Each verticalline represents one gene. A value of -1 indicates background signal; higher values indicate binding of HP1. o 2L 2R

90 ~ I,~~~~~~I~I~~.r~1 ~~ ~.~I~ ~ to loci that are enriched in specific AT-rich motifs, whereas HPIC binding correlates with the occurrence of a distinct palindromic motif. The possibility that these motifs play a direct role in recruiting specific types of heterochromatin will be subject of further studies. We have also started to develop bioinformatics tools for automated linking of our data to existing biological knowledge databases. In particular, we are integrating our whole-genome protein binding maps with gene annotation and gene expression databases. This allows for the unbiased identification of gene networks (groups of functionally related genes) that are controlled by gene silencing complexes. Selected publications Schübeler 0, Scalzo 0, Kooperberg C, Van Steensel B, Delrow J, Groudine M. Genome-wide DNA replication profile for Drosophila melanogaster: A link between transcription and DNA replication. Nature Genet 2002; ]2: Tompa R, McCalium CM, Delrow J, Henikoff JG, Van Steensel B, Henikoff S. Genome-wide profiling of DNA methylation reveals transposon targets 0fCHROMOMETHYLASE3. Curr Biol 2002; 12:65-8.

91 IX DIVISION OF RADIOTHERAPY Division head, group leader Harry Bartelink c~ c o c 5 X Artignanl, A Betgen, T Bortfeld 2, B Brand, J de Bois, N Dekker, K Deurloo, J Duppen, M Frenay, K Gilhuijs, H Lotz, D Jaffray3, SMuller, P Nowak 4, LS Ploeger, e Rasch, P Remeijer, e Schneider, M Smitsmans, JJ Sonke, R Steenbakkers, L Zijp, JV Lebesque, M van Herk Berthe Aleman MD Academic staff Harry Bartelink MD PhD Academic staff Jose Beiderbos MD Academic staff Nina Bijker MD PhD Academic staff liesbeth Boersma MD PhD Academic staff Eugenè Damen PhD Academic staff Roei De Boer PhD Academic staff Patricia De Haan MD Academic staff Katrien De Jaeger MD Academic staff Luc Dewit MD PhD Academic staff Riek Haas MD Academic staff Cuus Hart MSc Academic staff Wilma Heemsbergen MSc Academic staff Frank Hoebers MD Academic staff Edwin Jansen MD Academic staff Bas Kreike MD Academic staff Joos Lebesque MD PhD Academic staff Harry Masselink MD Academic staff Ben Mijnheer PhD Academic staff Luc Moonen MD PhD Academic staff Dimitry Nuyten MD Academic staff Floris Pos MD Academic staff Coen Rasch MD PhD Academic staff Peter Remeijer PhD Academic staff Nieola RusselI MD PhD Academic staff Covert Salverda MD Academ ic staff Christoph Schneider PhD Academic staff Roei Steenbakkers MP Academic staff Joep Stroom PhD Academic staff Caroline Tissing-Tan MD Academic staff Fred Ubbels MD Academic staff Bart Van Bunningen MD Academic staff Marcel Van Herk PhD Academic staff Corine van Vliet-Vroegindeweij PhD Academic staff Marcel Verheij MD PhD Academic staff Conny Vrieling MD PhD Academic staff Fritts Wittkämper Academic staff Luc Bos PhD Post-doc Kirsten Deurloo PhD Post-doc Martijn Engelsman MSc Post doc Mischa Hoogeman PhD Post-doc Rob Louwe PhD Post-doc Anne Saarnak PhD Post-doc Yvette Seppenwoolde PhD Post doc Jan-Jakob Sonke PhD Post-doc Niels Dekker MSc Graduate student Leah McDermott Ba/Bs (Hons) Graduate student Agnieszka Olszewska MSc Graduate Student Lennert Ploeger MSc Graduate student Marco Schwarz MSc Graduate student Monique Smitsmans MSc Graduate student Anja Betgen Technical staff The analysis and reduction of geometrical uncertainties in radiotherapy treatrnents is the major research area of the image-processing group of the radiotherapy department. Starting with the development of verification methods for radiotherapy delivery (portal imaging acquisition and analysis), the group now investigates all geometrical aspects of radiotherapy, i.e., tumor delineation, organ motion and setup error. Projects include a study of the delineation uncertainty and development of simulation tools to study the effects of errors on tumor control. Currently, we are equipping linear accelerator with a kilo-volt source and a flat-panel imaging device, to allow cone-beam CT imaging on the treatrnent machine. Using this device will introduce highly accurate image guided radiotherapy of prostate and bladder. Application of video imaging for improvement of patient set-up For radiotherapy of pro state cancer, the patient is positioned in the left-right (LR) direction based on alignment of a single marker on the skin to a room laser, resulting in a po or setup. We tested the use of a room-mounted video camera (on the ceiling) to improve patient setup using 127 video images of 22 patients. The set-up error derived from matching video images with rendered CT was retrospectively compared to the set-up error derived from portal images. The standard deviation (SD) of the systematic and random components of the set-up errors derived from the portal images in the LR direction were I.5 and 2.1 mm, respectively. When the set-up of the patients was retrospectively adjusted based on the video mages, the SD of the systematic and random errors decreased to I.I and I.3 mm, respectively. The technique will be implemented clinically. Respiration - correlated CT of lung cancer patients Respiration limits the accuracy of treatrnent planning and delivery in lung cancer patients. We developed a respiration correlated CT scanning technique that allows reconstruction of CT data in an arbitrary phase of the breathing cycle. A small thermometer placed under the nose-monitored respiration during scanning. CT scans in arbitrary phases reconstructed interpolating pairs of slices around the requested phase. The raw CT scans con sist of about 3 0 slices. For each selected phase, scans with about 40 slices remain with irregular slice spacing. By repeating this procedure for many phases, high quality movie loops obtained showed diaphragm and tumor motion in 3-D. Tumor motion analyzed automatically registered a region of interest around the tumor in one phase with all other reconstructed phases. The resulting tumor displacement curves show the precise 3D path of the tumor (Figure IX.I). Byapplying the measured tumor displacement to each slice, the movement-corrected scan obtained is much more accurate than the original scan (Figure IX.2). Automatic prostate localization for image-guided radiotherapy of the prostate To deal with prostate motion during radiotherapy, on-line image guided protocols are being developed based on markers, ultrasound and CT. Because pressure of the ultrasound probe on the abdomen shifts the prostate (we measured typical displacements of 3 mm in volunteers), contact-less imaging is preferred. We will use kv co ne beam CT acquired on the treatrnent machine to localize the prostate. We developed a method for high-speed automatic localization of the 1 ESTRO-EDRO fellowship (Ee Project SI ) 2 N PTe Boston 3 PMH Toronto 4 DDHK Rotterdam

92 1- _. L-R - S-I -A-P ,-... ;:: Notmatched Matched on tumor region Ê 0.4 ~ , Ë 0 ~ i -0.2 lil ëi -0.4 _ I "'<-~" Phase (one cycle) Matched for 32 phases Figure IX.l: Measurement of respiration motion using a respiration-correlated CT scan. A region of interest (ROl) is selected around the tumor visible in the first reconstructed phase. Next, this ROl is used to register the subsequent phases to the first phase, thereby measuring the tumor motion, displayed in the graph. prostate in CT data, to allow immediate correction for organ movement. Pro state is performed Gray-value registration of a region of interest defined in the planning CT performed a pro state localization. No delineation of the cone-beam CT is required. Automatic prostate localization was compared with manuallocalization using delineated prostate contours for 250 CT scans of 19 patients. The differences we re small: 0.7 mm SD translation and 1.5 degrees SD rotation, and a part of this difference is due to delineation variability. However, the failure rate is about 15% and needs improvement. In conclusion, fast automatic prostate localization in CT is feasible with reasonable reliability and accuracy. In case the automated method fails, a method based on reduced delineation will be used to localize the prostate. Bob Brand Technical staff Josine De Bois Technical staff JooP Duppen Technical staff Michel Frenay Technical staff Corinne Goedbloed Technical staff Tom Minderhoud Technical staff Pietje Muller Technical staff Kenneth Pengel Technical staff Maddalena Rossi Technical staff Bob Smulders Technical staff René Tielenburg Technical staff Peter Van de Ven MSc Technical staff lambert Zijp Technical staff Xavier Artignan MD Guest Werner Bär MSc Guest John Cho MD Guest Carol McGibney MD Guest Patricia Haye-Fewer Secretary Image guided radiotherapy of the bladder Recent years demonstrated the feasibility of bladder preserving multi-modality approaches in selected patients with invasive bladder cancer. Optimization of radiotherapy can further increase their success. However, for the bladder, extreme variations in shape and position occur due to variations in bladder and rectum filling, which significantly limits the accuracy of external beam radiotherapy. Even in image-guided radiotherapy, there are inevitabie time delays between CT scanning and radiotherapy, in which bladder shape changes will occur. We are currently studying shape variability of the bladder to all ow correction for these shape changes. The shape of the bladder was described in polar maps and measured in 250 CT scans ofr9 prostate cancer patients. The shape variation can be weu described using a linear relation between the local Planning CT Corrected CT Figure IX.2: When the slices of the original scan data are corrected for the measured tumor motion, the shape ofthe tumor is shown without the enormous distortion that occurred in the original planning CT scan. These images are a surface rendering of the tumor from the CT scan.

93 Selected publications Ackerstaff AH, Tan IB, Rasch CR, Balm Aj, Keus RB, Schornagel JH, Hilgers FJ. Quality-ofLife Assessment after supradose selective intra-arterial cisplatin and concomitant radiation (RAD PLAT) for inoperable stage IV head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2002; 128: bladder radius in the polar maps and the bladder and rectum volume. The model works very weil, as illustrated by an example where the bladder shape of a volunteer was predicted based on an MRI scan made more than one hour earlier (Figure IX.3). U sing this model, a correction for bladder shape change during the time delay between imaging and treatment will be made. Bartelink H, Horiot JC, Poortmans P. Recurrence rates after treatment ofbreast cancer with standard radiotherapy with or without additional radiation. N EnglJ Med 2002; 346:863-4, Bartel ink H, Schellens JH, Verheij M. The combined use of radiotherapy and chemotherapy in the treatment of solid tumors. EurJ Cancer 2002; 38: Review. Figure IX.y Bladder shape predicted based on a generic bladder shape model (green) applied to a scan made one hour previously displayed on an MRI scan made one hour later (grey value image). The errors are very small (less than 3 mm), even though the bladder volume has tripled over this period. Bartelink H, Van den Bogaert W, Horiot JC, Jager j, Van Glabbeke M. Concomitant cisplatin and radiotherapy in a conventional and modified fractionation schedule in locally advanced he ad and neck cancer. A randomized phase TI EORTC trial. EurJ Cancer 2002; 38:667-7]. Bos Lj, Damen EM, De Boer RW, Mijnheer BJ, McShan DL, Fraass BA, Kessler M L, Lebesque Jv. Reduction of rectal dose by integration of the boost in the large-field treatment plan for prostate irradiation. Int] Radiat Oncol Biol Phys 2002; 52: Simulation ofthe combined effect of delineation variation, setup error and organ motion Many groups are performing simulations of the effect of geometric error on outcome. It is unknown, however, how to correctly simulate delineation variation. The purpose of this study is to simulate the combined effect of preparation errors (setup error, delineation variation, organ motion) and execution errors (setup error, organ motion). Three observers outlined the pro state for 18 patients. For each patient, six treatment plans we re created. Random and systematic geometrical errors were simulated to compute probability distributions of several dose parameters. The influence of observer variation was measured by combining CTVs and treatment plans of different observers. The reduction of the overall TCP is small indicating that a PTV margin of 10 mm is adequate. Next, simulations were done with varying error levels, treating delineation errors as if they we re translations only. The results show that intra-observer delineation variation can be described by a simple translation with a normal distribution. However, this is not true for inter-observer variation. Bos Lj, Danciu C, Cheng CW, Brugmans Mj, Van der Horst A, Minken A, Mijnheer BJ. Interinstitutional variations of sensitometric curves of radiographic dosimetric films. Med Phys 2002; 29: Cho BC, Hurkmans CW, Damen EM, Zijp Lj, Mijnheer BJ. Intensity modulated versus non-intensity modulated radiotherapy in the treatment of the left breast and upper intemal mammary lymph node chain: a comparative planning study. Radiother Oncol2002; 62: De Bree E, Van Coevorden F, Peterse J L, RusselI NS, Rutgers EJ. Bilateral angiosarcoma of the breast after conservative treatment ofbilateral invasive carcinoma: genetic predisposition? EurJ Surg Oncol2002; 28:]92-5. Biological and physical fractionation effects of random geometrical errors The purpose of this study is to investigate biological and physical fractionation effects of random geometrical errors and respiration. Random errors drawn from anormal distribution were used to displace a dose distribution for each simulated fraction. The displaced dose distributions we re summed to obtain the total dose. To simulate biological effects of fractionation, the physical dose was converted to a biologically effective dose, then summed and converted back to physical dose for comparison. The combination of random errors and a finite number of fractions in radiotherapy leads to a residual geometrical error with the SD of the random displacement divided by the square root of the number of fractions. The total biological dose distributions are slightly wider than the total physical dose distributions: 0A mm for an alphajbeta ratio of I Gy and a random error SD of 3 mmo Respiration motion causes a deviation in the shape of the total dose distribution compared with Gaussian blurring. With a random error SD of 3 mm and respiration amplitude of I cm or less «}6 mm SD), this effect is negligible. Comparison of CT and M RI delineation for 3-0 conformal treatment planning of the prostate The purpose of this study is to determine the influence of MRI versus CT based prostate delineation on the dose to the target and organs at risk during external beam radiotherapy. Three observers delineated the pro state on both modalities. The PTV was generated from the delineated prostates with a 3D mar gin of 10 mmo A three-field treatment plan with a prescribed dose of 78 Gy to the ICRU point was automatically generated. A significant difference in position between CT

94 and MRI rectum was observed, probably due to the rounded tabletop of the MRI scanner. Therefore, the mean CT rectal wall EUD difference between treatments plans based on the CT delineated pro state and treatment plans based on the MRI delineated prostate was 5.1 Gy. For the MRI rectal wall the EUO difference between treatment plans was 3-6 Gy. Allowing the same damage to the rectal wal!, the prescribed dose to the PTV could be raised by 10% when using the MRI delineated prostate for treatment planning. The mean dose difference received by the bulb of the penis between treatments plans based on the CT delineated pro state and treatments plans based on the MRI delineated pro state was n.6 Gy. In conclusion, the dose delivered to critical structures is significantly reduced with the treatment plans based on the MRI delineated pro state compared to CT delineated prostate. OOSI M ETRY J Cho, EMF Damen, R Louwe5, L McDermott 6, M Van Herk, BJ Mijnheer An algorithm has been developed for 3D-dose reconstruction using portal images obtained with an electronic portal-imaging device (EPID) in patients irradiated with high-energy photons. For this purpose the algorithm for 20-dose reconstruction, which was previously developed in our institution, was adapted. The accuracy of the algorithm was determined by irradiating two anthropomorphic breast phantoms. The dose values derived from portal images were compared with data obtained with ionization chamber and film dosimetry, as well as with results from 30-dose calculations. It was found that by applying contour information, for the total dose resulting from all treatment beams, the average deviation is only -0.1% ± 1.7% (lsd). If contour information is not available, the differences increase up to ± 20% for the individual beams. If the irradiated volume is enclosed by planes less than 5 cm distant from the isocenter plane, then the average deviation of the 30-reconstructed dose from the calculated dose is only 0.5% ± 3-4% (lsd). It can be concluded that this new algorithm for 30-dose reconstruction using EPIO images, allows an accurate verification of the midplane dose and the dose-volume histogram of the irradiated volume during breast cancer treatment MU/min & 320MU MU/min & 160MU MU/m in & 80MU 61 MU/min & 40MU Selected publications (continued) Dorresteijn LD, Kappelle AC, Boogerd W, Klokman Wj. Balm AJ, Keus RB, van Leeuwen FE, Bartelink H. Inereased risk of ischemie stroke after radiotherapy on the neek in patients younger than 60 years. ] Clin Oneol2002; 20: Erridge SC, Seppenwoolde Y, Muller SH, Van Herk M, De Jaeger K, Beiderbos JSA, Boersma Lj. Lebesque Jv. Portal imaging to assess set-up errors, tumor motion and tumor shrinkage during conformal radiotherapy of non smalllung ca neer. Radiother Oneol (in press). Hoebers Fj. Janssen HL, Olmos AV, Sprong D, Nunn AD, Balm Aj. Hoefnagel CA, Begg AC, Haustermans KM. Phase 1 study to identify tumor hypoxia in patients with head and neck eaneer using technetium-99m BRU EurJ Nucl Med Mol Imaging 2002; 29: Hoogeman MS, Van Herk M, Yan D, Boersma Lj. Koper PCM, Lebesque Jv. A model to simulate day-to day variations in rectum shape. Int] Radiat Oneol Biol Phys 2002; 54: Hurkmans CW, Cho BC, Damen E, Zijp L, Mijnheer BJ. Reduction ofeardiae and lung eomplieation probabilities after breast irradiation using conformal radiotherapy with or without intensity modulation. Radiother Oneol2002; 62: ) 10 0 ; \"'''-'~~~,~~,.. _---=--~ time after beam otf(s) Figure IX+ 'Ghosting effect' ofthe a-si EPID for 4 dose-rates. Different doses were given to maintain a constant beam time. Kitamura K, Shirato H, Seppenwoolde Y, Onimaru R, Oda M, Fujita K, Shimizu S, Shinohara N, Harabayashi T, Miyasaka K. Three-dimensional intrafraetional move ment of prostate measured during realtime tumor tracking radiotherapy in supine and prone treatment positions. Int] Radiat Oncol Biol Phys 2002; 5]: With the advent of flat panel electronic portal imaging device (EPIO) technology for dosimetric purposes, new challenges have arisen to obtain accurate and time effective verification of patient dose distributions. The Elekta iview-gt amorphous silicon (a-si) EPIO (Elekta Oncology Systems, Crawley, UK) is currently being examined for radiotherapy dosimetry in our institution. A lmm copper plate over the phosphor layer is used to provide additional build-up material. We started to investigate the dose-response relationship of the EPIO under a wide range of treatment conditions, the effects of image persistence (ghosting) and the requirement of an additional build-up layer for dosimetry. The long-term stability and temperature dependence Kummer E, Rasch CR, Keus RB, Tan IB, Ba I m AJ. T stage as prognostic factor in irradiated loealized squamous eell carcinoma ofthe nasal vestibule. Head Neck 2002; 24:268-7]. McKenzie A, Van Herk M, Mijnheer B. Ma rgi ns for geometrie uncertainty around organs at risk in radiotherapy. Radiother Oncol2002; 62:299' Funding: Dutch Cancer Society (NKI ) 6 Funding: Dutch Cancer Society (NKI 00'2255)

95 Selected publications (continued) Mechalakos j, Mageras G, Zelefsky M, Lyass 0, Van Herk M, Kooy H, Leibel S, Ling C. Time trends in organ position and volume in patients receiving prostate threedimensional conformal radiotherapy. Radiother Oncol 2002; 62:261. Meijer Gj, Rasch C, Remeijer P, Lebesque Jv. 3D anisotropic margins for bladder, based on 3D analysis of delineation errors, setup errors and organ motion. Int J Radiat Oncol Bial Phys (in press). Ploeger LS, Smitsmans MH, Gilhuijs KG, Van Herk M. A methodforgeometrical verification of dynamic intensity modulated radiotherapy using a scanning electronic portal-imaging device. Med Phys 2002; 29: Rasch C, Eisbruch A, Remeijer P, Bos L, Hoogeman M, Van Herk M, Lebesque Jv. Irradiation of paranasal sinus tumors, a delineation and dose comparison study. Int J Radiat Oncol Biol Phys 2002; 52:120-7 Remeijer P, Rasch C, Lebesque JV, Van Herk M. Marginsfor translational and rotational uncertainties: a probability based approach. Int J Radiat Oncol Biol Phys 2002; 5]: Ruiter GA, Verheij M, Zerp SF, Mooienaar WH, Van Blitterswijk WJ. Submicromolar doses of alkyl-lysophospholipids induce rapid intemalization, but not activation ofthe EGF receptor and concomitant MAPK/ERK activation in A431 ceus. lnt J Cancer 2002; 102,"] Seppenwoolde Y, Engelsman M, De Jaeger K, Muller SH, Baas P, McShan DL, Fraass BA, Kessler ML, Beiderbos JS, Boersma Lj, Lebesque Jv. Optimizing radiation treatment plans for lung cancer using lung perfusion information. Radiother Oncol2002; 6]: Seppenwoolde Y, Lebesque JV, De Jaeger K, Beiderbos JSA, Boersma Lj, Schilstra C, Henning GT, Hayman JA, Martel MK, Ten Haken RK. Comparing different NTCP models that predict the incidence of radiation pneumonitis. Int J Radiat Oncol Biol Phys (in press). of the dose-response for 4, 6, 8, and 18MV photon beams we re also investigated. The relative amount of image persistence depends mostlyon the number of irradiated frames and recovery time, while dose and dose rate have a much smaller effect. (Figure IX.4) By regularly updating the dark field correction used for calibration of each image, a standard deviation ofless than 0.5% was achieved in the response behavior for four energies, for a period up to 5 months. With a well-defined response under a wide range of treatment conditions, adequate build up material and verified long-term stability, the a-si-type EPID has strong potential for applications in clinical dosimetry. Vnderstanding set-up uncertainty effects on dose distributions is an important clinical problem but difficult to model accurately due to their dep enden ce on tissue inhomogeneities and changes in the surface contour. We therefore performed a study of which the aims were: (I) to evaluate and quantify effect of set-up uncertainties on target DVHs; (2) to propose a method to interpolate DVHs. A lung cancer patient was presented to estimate the significance of set-up uncertainties in target DVHs. The RMS deviation in the integral DVHs' relative dose between the invariant calculation and full calculation was 5.8%, and between interpolated curves and full calculation 2.5%. For the EVD these figures were 0.01 Gyand Gy, respectively. Since a 'worst-case' example was selected, we conclude that, in the majority of clinical cases, of contour changes and tissue inhomogeneities on EVD are negligible. Interpolation is avalid, efficient method to approximate DVHs. [ c W Bär, JSA Beiderbos, LJ Bos, J Cho, K De Jaeger, M Engelsman, BA Fraass, JV Lebesque, DL McShan, TJ Minderhoud, CRN Raseh, AE Saarnak, CJ Schneider, M Schwarz, B. Smulders, C van Vliet-Vroegindeweij, BJ Mijnheer, EMF Damen General Three main topics have been investigated in the field of treatment planning and quality control of intensity-modulated radiotherapy (IMRT). Firstly, we investigated if and how inaccuracies in the dose calculation influence the overall dose distribution and the results of the optimization process. Secondly, for prostate and head and neck irradiation we investigated if the current clinically used technique, utilizing a limited number of user-defined beam segments (forward planning), might be improved by an automated optimized segment creation (inverse planning). Thirdly, we investigated whether the effect of set-up uncertainties on the dose distribution in cases with a changing patient contour and varying tissue density, can be approximated by interpolation of only a few do se-volume histograms (DVHs). Oose calculation for IMRT In 'step-and-shoot' IMRT, the total dose is delivered by a large number of small beam segments. As a result, a large part of the total dose in normal tissues and even in tumor may be composed of dose contribution from outside segment edges. We investigated the effect of inaccuracies in the calculation of the dose outside the segment edges on the overall dose distribution and on the optimization process of IMRT treatment plans. Dose calculations of two treatmentplanning systems (TPS) were compared with measurements in a water phantom. Successively, ten treatment plans for patients with prostate and head and neck tumors were calculated in both systems. The calculations we re compared in selected points as well as in combination with volumetric parameters conceming the Planning Target Volume (PTV) and organs at risk. For the clinical IMRT plans used for prostate, the dose to the PTV is predicted accurately by both TPS, since the plan consists of relatively few segments and large segments that cover the entire target volume deliver a major fraction of the dose. DVHs of the rectal wall differ below 60 Gy, due to differences in dose calculation outside the segments. However, since this dose region is not used in the plan optimization, this difference does not influence the optimization results. For the head and neck cases, the dose calculation in the two TPS may result in different target coverage (up to 22% difference), since a considerable fraction of the target dose originates from outside segments. Similarly,

96 ; 5 :g 4 Cl TPS1 Measurements O~~~~LL------~U-~~--~~LL~~ Segment number Figure IXT Comparison of calculated and measured dose at a point in a head and neck treatment geometry with 12 segments. This point is directly irradiated only by segment #10. lts measured total dose is 22.6% ofthe dose at the isocenter. The total dose values calculated by the two TPS are 19.0% and 28.1%, respectively. for normal tissues like the parotid glands, differences in mean dose ranging from 1.0 to 3.0 Gy are observed (Figure IX.5). In addition, the results ofthe optimization are influenced by the choice of the dose calculation algorithm. From these results, we conclude that the accuracy of the dose calculation outside the segment edges is important for the determination of the dose to both organs at risks and target volumes and for a correct outcome of the optimization process. This aspect should therefore be of major concern in the commissioning of a TPS intended for use in IMRT. Selected publications (continued) Seppenwoolde Y, Shirato H, Kitamura K, Shimizu S, Van Herk M, Lebesque JV, Miyasaka K. Precise and real-time measurement of3d tumor motion in lung due to breathing and heartbeat, measured during radiotherapy. Int] Radiat Oncol Biol Phys 2002; 53 : Smulders B, Bru invis IAD, Mijnheer BJ. Monitor unit calculations for wedged asymmetrie photon beams. Phys Med Biol 2002; 47: Van der Lu it AH, Budde M, Ruurs P, Verheij M, van Blitterswijk WJ. Alkyllysophospholipids induce apoptosis via raft-mediated endocytosis and inhibition of phosphatidylcholine synthesis. ] Biol Chem 2002; 27T Forward versus inverse planning for IMRT In forward planning ofimrt, segments are created manually in beam's-eye-view, based on the experience of the planning technician. The relative weight of these segments is computer-optimized by minimization of a score function. In inverse planning, only beam directions are predefined and for each beam, the fluence distribution is computer-optimized using a score function. Subsequently, the optimized fluence is approximated by a number ofbeam segments using a computerized sequencing method. In theory, the latter method results in better dose distributions in terms of target coverage and sparing of normal tissues, since it has a higher degree of freedom in segment shapes and number of segments. We investigated the benefit of inverse planning versus forward planning for prostate and he ad and neck tumors. Dose distributions produced by inverse planning using the Hyperion system developed by the University of Tübingen were compared to the clinically used forward plans for a number of patients. Van Herk M, Remeijer P, Lebesque Jv. Inclusion of geometrie uncertainties in treatment plan evaluation. Int] Radiat Oncol Biol Phys 2002; 52: Van 't Veer L). Dai H, Van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton M). Witteveen AT, Schreiber Gj, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling prediets clinical outcome ofbreast ca neer. Nature 2002; 41Y53o-6. For prostate plans it was concluded that inverse treatrnent planning resulted in a similar PTV coverage as the clinically used five-field multi-segment class solution. Inverse planning reduced the dose to the rectal wall compared to forward planning slightly, resulting in lower NTCP values. The magnitude of this reduction in NTCP depended on the amount of overlap between PTV and rectal wall and ranged from 3.3% for large overlap to 0.5% for small overlap. The number of segments created with forward planning ranged from 9 to 13, and with inverse planning from 33 to 52. Based on these results we concluded that inverse treatrnent planning is only beneficial for patients that have a large overlap between PTV and rectal wall. However, this conclusion may depend on the details of the score nmction used in the optimization process. More work is needed to investigate this aspect. For he ad and neck tumors we concluded that inverse treatrnent planning is beneficial for both target coverage and sparing of the parotid glands (Figure IX. 6). U sing the same beam directions as the clinically used forward plan, a reduction of the mean dose by 3 - II Gy for at least one parotid gland could be achieved. The same was true for inverse plans that used nine equidistant beam directions. The number of segments in the inverse plans increased on ave rage by a factor of three compared to the forward plans. ;R := (1) ~ S "0 40 > Figure IX.6: DVHs ofthe PTV and parotid glands for a head and neck IMRT treatment plan resulting from the inverse and forward approach.

97 STUDIES OF RADIATION RESPONSE IN NORMAL TISSUE JSA Beiderbos, LJ Boersma, W Boogerd, K De Jaeger, L Dorrestein, C Goedbloed, M Hoogeman, PCM Koper 7, P Muller, K Pengel, M Rossi, Y Seppenwoolde, M Verheij, H Bartelink, JV Lebesque With the advent of computer optimization of radiotherapy plans, detailed knowledge of the radiation response of organs at risk, as a nmction of the 3D dose distribution is essential. Therefore these responses are being studied for a nurnber of normal tissues. Lung: radiation pneumonitis and NTCP models Data from 382 breast cancer, malignant lymphoma and inoperable non-small celllung cancer patients from two centers were studied. Radiation pneumonitis was scored using the SWOG criteria. Dose-volurne histograms (DVHs) of the lungs were calculated from the dose distributions that were corrected for dose per fraction effects. The DVH of each patient was reduced into a single parameter using different local dose effect relationships. Examples of single parameters were the mean lung dose (MLD) and the volume oflung receiving more than a threshold dose (VDth). Parameters for the different NTCP models were fit to patient data using a maximum likelihood analysis. ~ 0.6 :ë Cl! e 0.4 a.. (a) Bleeding : superior 75 % 0.8 " " 0.2 The best fit resulted in a linear local dose-effect relationship, with the MLD as the resulting single parameter. The relationship between MLD and NTCP could be described with a TD 50 of 30.8 Gy and a steepness parameter m of The best fit for the relationship between the VDth and the NTCP was obtained with a Dth of 13 Gy. The MLD model was found to be significantly better than the VDth model (P<0.03). However, for 85% of the studied patients, the difference in NTCP calculated with both models is less than 10%, because of the high correlation between the two parameters. For dose distributions outside the range of studied DVHs, the difference in NTCP, using the two models could be higher than 35%. For arbitrary dose distributions, an estimate of the uncertainty in the NTCP could be determined using the probability distribution of the parameter values of the Lyman Kutcher-Burman model. 0.8 ~ 0.6 :ë Cl! e 0.4 a Area (%) (b) Spotting: inferi or 25 % "---''------'~ '~----L~ '_~ '_~ ' '_' o Mean dose (Gy) Figure IX. 7: (a)the bars denote the actuarial incidence ofbleeding at three yearsfollow up as a function of the area that received a dose higher than 60 Gy for the superior 75% of the map. The solid line is the estimated incidence ofbleeding within three years according to the proportional hazard regression model. The dashed lines correspond to the 95% confidence limits. (b) The results for spotting as a function of the mean dose to the inferior 25% ofthe map. Rectum: rectum dose maps and complications We have developed a method to 'unfold' the delineated rectal wall and project the received dose to the outer rectal surface onto a 2D angular map. These dose maps contain the full spatial information of the received dose to the outer rectal wall. Therefore, dose maps are ideal to study dose-effect relationships for distinct parts of the rectal wall surface. We used the dose maps to analyze late rectal toxicity of 199 pro state cancer patients, who we re treated with 66 Gy in a trial at the Erasmus MC-Daniel den Hoed Cancer Center in Rotterdam, The Netherlands (PCM Koper). Randomization took place between 3D conformal and conventional fields. We used these data to study two patient complaints, namely rectal bleeding and spotting, and their relationship with dose map parameters. A proportional hazard regres sion model was applied to estimate the probability of a complaint within three years, as a fimction of several dose map parameters. The dose map parameters we re extracted from different regions of the dose map. In addition, the Kaplan Meier method was used to determine the actuarial incidence of the complaints within defined dose bins. The analysis of the 75% most superior part of the dose map (excluding the anal region) showed a significant relation between the irradiated area enclosed by the 60-Gy dose line and the probability of any bleeding within three years, as shown in Figure IX.7 (p=0.00i5). The irradiated area of the 25% inferior part of the dose map (anus region) did not significantly relate with the probability of rectal bleeding. For the spotting, we observed a different pattem. A significant relation was found for dose parameters received by the most inferior 25% of the dose map (Figure IX.7b), while no significant relations were found between for the 75% most superior part. These results demonstrated that different complaints correlate with dose parameters of different regions of the gastrointestinal tract. Therefore, combining different 7 Erasmus MC-Da niel Hoed den Ca neer Center in Rotterdam, The Netherlands

98 complaints or different dose regions, as often seen in clinical studies, might mask do se-volume-effect relationships. late vascular damage To estimate the risk ofischemic stroke in patients irradiated, the incidence of ischemie stroke was determined in 367 patients with head and ne ck tumors. Fourteen cases of stroke occurred (expected, 2.5; RR, 5.6; 95% confidence interval [Cl], 3.1 to 9-4): eight in patients with laryngeal carcinoma (expected, 1.56; RR, 5.1), four in pleiomorphic adenoma patients (expected, 0.71; RR, 5.7), and two in parotid carcinoma patients (expected, 0.24; RR, 8.5). Five of six strokes in patients irradiated for a parotid tumor occurred at the ipsilateral side. Analysis of other risk factors for cerebrovascular disease showed hypertension and DM to cause an increase of the RR after RT. After more than ten years' follow-up, the RR was 10.1 (95% Cl, 4-4 to 20.0). The 15-year cumulative risk of stroke after RT on the neck was 12.0%. Consequently, a prospective study will be started in order to quantify systematically the increase of intima thickness and to design strategies to prevent late vascular damage. (see Section VIII) TENSITY OD ATED RADlOT E P (I MRT) SA Beiderbos, LJ Boersma, R De Boer, JJP De Goede, K De Jaeger, WD Heemsbergen, M Hoogeman, EA Lamers, G Meijer, P Muller, C Raseh, P Remeijer, M Van Herk, HP Vijlbrief, JV Lebesque High-dose conformal ra dia ti on treatrnents, including IMRT, were carried out for 300 patients. For 2003 we have planned to increase our capacity for this type of treatrnent. This will be achieved by increasing the number of IMRT treatrnents for both head & neck patients and breast cancer patients. Within the context of IMRT treatrnents, dose escalation trials for prostate and lung cancer are being performed. Furthermore, different aspects of tumor treatrnent margins are investigated. Pro state cancer: the dose escalation trial The dose escalation trial (68 Gy vs. 78 Gy) is still ongoing. In cooperation with other radiotherapy institutes, over 600 patients have been recruited now, balanced over the treatrnent arms. The inclusion of study patients will be discontinued in J anuary The first group of 600 randomized patients will be subject of statistical analyses in the near future; toxicity as well as disease free survival will be studied in this first evaluation of efficacy. Prostate cancer: clinical strategies to reduce the impact of prostate motion Prostate mobility in the planning phase of radiotherapy of prostate ca nc er leads to an uncertainty of its localization during treatrnent. A safety margin is used to minimize the effect of the geometrie uncertainty on the total received dose, despite the fact that it leads to an increase in the dose to normal tissues. We studied strategies to re duce this systematic error and the safety margin for planning and treatrnent uncertainties. Data of 19 pro state cancer patients, who each received el even repeat CT scans, was used. We found that the rectum volume in the planning CT scan relates with a dis placement of the prostate in the Anterior-Posterior (AP) direction and with a rotation around the Left-Right (LR) axis. A correction of the prostate position and orientation based on this rectum volume reduces the standard deviation of this systematic error by 7% to 19% (AP translation, center-of-mass - tip seminal vesicles) and 28% (LR-axis rotation). For this strategy, no extra CT scans are needed. A further reduction of the systematic error can be achieved by using n repeat CT scans in the beginning of the treatrnent to estimate the mean prostate position and orientation during treatrnent. With three repeat CT scans the systematic error can be reduced with 53% (AP translation) and 61% (LR-axis rotation). Both strategies were also evaluated by simulating prostate treatrnents and comparing Tumor Control Probabilities (TCP). A TCP loss of 6% (4-mm margin) could be reduced to 2-3% by applying the correction strategies Bladder cancer: treatment margins for irradiation Pelvic CT images we re obtained for 10 male bladder patients. Apart from the initial planning CT scan, three follow-up scans we re made for each of the patients. The bladder volumes in planning

99 CT scan were consistently outlined by seven radiation oncologists. Generally small variations occurred (1.5 mm - 3 mm, 1 SD), although in 50% of the patients larger discrepancies were observed in discriminating the bladder from the base of the prostate. The setup errors derived from portal imaging showed setup errors up to 3 mm (1 SD). Organ motion was the predominant geometrical uncertainty in the radiotherapy process (5 mm, 1 SD, at the cranial side of the bladder), although nine out of ten patients were able to pres erve a reproducible bladder volume during the complete treatment course. As aresult, anisotropic margins between the CTV and PTV are needed in conformal radiotherapy of the bladder. Especially at the cranial side of the bladder, larger margins are needed because of the impact of bladder shape variation. Lung cancer: the dose escalation trial The ma in eligibility criteria of the trial are histopathologically proven in opera bie NSCLC, ECOG performance status 0 or 1, weight loss < 10% and no chemotherapy < 6 weeks prior to radiotherapy. No elective nodal irradiation was given. Patients are treated five days a week with 2.25 Gy per fraction and a 6 weeks overall treatment time. Five risk groups were defined according to the relative mean lung dose (rmld). Within each group the do se is escalated with three fractions per step (6.75 Gy). The next dose levelopens after a toxicity free follow-up of six months in three patients. The maximum tolerabie dose is reached if two out of six patients experience a dose limiting toxicity (pneumonitis 2: grade 3 (SWOG), grade 3 early and grade 2 late esophagus toxicity or any other grade 314 (RTOG) complications). Since October 1998, 80 patients have been entered. The results of the first 55 patients were analyzed. Tumor stage was IIII in 47%, lila in 33% and IIIB in 20%. The majority of the patients received a dose of74-3 Gy (n=17) or 81.0 Gy (n=23). Radiation pneumonitis occurred in seven patients: in four grade 2, two grade 3 and one grade 4. Esophagus toxicity was mild. In 50 patients, tumor response at three months follow-up was scored. In six patients a complete response was recorded, in 38 a partial response, five stabie disease and one patient experienced progressive disease. Only one patient developed an isolated failure in an uninvolved no dal area. So far the radiation dose was safely escalated to 94.5 Gy in group 1 (lowest rmld), 87.8 Gy in group 2 and 3 and 81.0 Gy in group 4. Lung ca neer: patient set-up, tumor movement and shrinkage In 97 patients, electronic portal images (EPIs) were acquired and corrected for set-up using an off-line correction protocol based on a shrinking action level. Without correction, 41% of patients had a set-up error of more than 5 mm vector length, but with the setup correction protocol this was reduced to 1%. For 25 selected patients, the orthogonal EPIs (taken at random points in the breathing cycle) throughout the six to seven week course of treatment were assessed to establish the tumor position in each image using both an overlay and a delineation technique. The position of the tumor in the digitally reconstructed radiograph (DRR) was compared to the average position of the lesion in the EPIs. The mean amplitude of tumor motion was 7.3 mm, 12.5 mm and 9.4 mm in the three main directions, respectively. Tumor motion was greatest in the cranio-caudal direction and in particular for lower lobe tumors. In 40% of the patients, the projected area of the tumor regressed by more than 20% during treatment in at least one projection. In 16 patients, it was possible to define the position of the center of the tumor in the DRR. There was a mean difference of 6 mm vector length between the tumor position in the D RR and the average position in the portal images. BREAST CANCER LBoersma, J (ho, E Damen, B Mijnheer, N RusselI, ( Vrieling, H Bartelink In the EORTC 'boost versus no boost' trial, patients with early stage breast cancer we re randornized between no boost and a 16 Gy boost. An analysis of patients older

100 Prognostic score T----~IC: ~-~ ~~.=-::.::v.:~ ~ ~ :.; :.::.,:.:- :.:... :.;... :.:.:.:.:.:....:.:.:,.:: r-..., At 5 years: Good: Intermediate: Poor: Overall Logrank test: p = % ( ) 95.5% ( ) 91.4% ( ) , , , , ,years N Number ot patients at risk: (good) (poor) Figure IX.B: Local control rate of patients older than 50 years by prognostic score (intennediate) than SO years of age was performed to evaluate the influence of the boost on local control in different prognostic subgroups. Overall, patients in the no boost group had a s-year local recurrence rate of 4.1% compared with 3.0% for patients in the boost group (p = 0.02). Multivariate analysis showed that performance status, excision volume and pathological tumor size were significantly related to local control. According to these factors a prognostic score was defined, leading to three different prognostic groups with a s-year local recurrence rate of 2.5%, 4.S% and 8.6%, respectively. The influence of the boost on local control was assessed for the different groups separately, but the boost did not improve local control significantly in any of the prognostic groups. This is in contrast to the findings in patients younger than SO years of age, for whom the boost did improve local control significantly. (Figure IX.8) As a result of the EORTC trial with two dose levels of radiation (H Bartelink NEJM 2001) a prospective trial is now designed for patients less than 50 years where again randomization takes place between two dose levels. In this prospective study material will be collected for micro-arrays (See Section XIII). I M RT of breast ca neer For the treatrnent ofbreast tumors we designed an intensitymodulated radiotherapy (IMRT) class solution using predefined segments (IMC) and compared this technique with a 3-D conformal (CRT) and a full-fluence IMRT (IMF) approach. Twenty left-sided breast cancer patients were planned with these three different techniques using the same tangential beam orientations and normalized to the planning target volume (PTV) mean dose and equivalent uniform dose (EUD). Plan evaluation consists of dose-volume histogram analysis and normal tissue complication probability (NTCP) estimates for late excess cardiac mortality and radiation pneumonitis. The IMC and IMF plans have a small volume of underdosage. When normalized to iso-euds with different a-values, the CRT's cardiac NTCPs are always greater than the other plans. (Figure IX.9) It can be concluded that for similar clonogen celi kill the IMC plans result in cardiac NTCP sparing comparable to the IMF plans but are simpier to implement. All plans have low lung NTCPs. A NTCP for Ex ce ss Late cardiac Mortality 60r ~ ~ ~crt D m: ~ 30 Om PatîenUD B ~ (.)!z NTCP tor Radiation Pneumonitis ~ D lmc D imf MECHANISMS AND MODUlATION OF RADIATION-INDUCED CEll DEATH o De Jong, R Haas, GA Ruiter, RA Valdes, WJ Van Blitterswijk, S Vink, SF Zerp, H Bartelink, M Verheij Our line of research continues to focus on developing pharmacological strategies to increase the cytotoxic effect of radiation. A stepwise program of sequential in vitro, PatientlO Figure IX.9: Histogram ofthe normal tissue complication probability (NTCP) for late excess cardiac mortality (A) and radiation pneumonitis ( B) ranked by patient and plan technique sorted by descending CRT plan values.

101 A B Figure IX. 10: Anti-angiogenic effects of ALPs. Human microvascular endothelial cells were grown on fibrin matrices. In the presence of VEGFjbFGF and TNF?, tubular structures are formed invading the matrix (A). ALP inhibits tube formation (B). H«<.E stained cross sections (magnification: 400X; bar = 40 f.-lm). preclinical and clinical screening tests has been set up to identify novel promising drugs for combined modality treatment. Alkyl-lysophospholipids (ALPs, such as Et-I8-0CH 3 jedelfosine, HePCjMiltefosine and 0-2I266jPerifosine) represent a first group of compounds that have become available for clinical use along this approach. These synthetic anti-tumor agents are known to accumulate in cellular membranes where they disturb the lipid microenvironment and interfere with lipid-mediated signaling. We have previously demonstrated that ALPs inhibit MAPKjERK and PI3K-AktjPKB signaling and activate the SAPK/JNK stress pathway. In combination with radiation, ALPs cause a synergistic apoptotic effect and re duce clonogenic cell survival. Besides these radiosensitizing properties, ALPs show potent anti-angiogenic effects in vitro (Figure IX.ro). In two other projects (in collaboration with W Van Blitterswijk, Oivision 111) we are investigating mechanisms of celluiar ALP uptake and transport, studying the functional implications of an altered membrane lipid composition on celluiar drug uptake and radiosensitivity. Based on the above-mentioned favorable in vitro properties, ALPs were tested in a mouse xenograft solid tumor model. Following oral administration of Perifosine, maximal plasma concentrations we re measured af ter four hours and significant intratumoral drug concentrations were detected from two to six hours onwards. Recently, a clinical phase I study was started in our institute, testing the feasibility and tolerability of combined modality treatment consisting of oral administration of Perifosine and radiotherapy in patients with locally advanced inoperable solid tumors. Perifosine proved to be tolerabie at a dose of 50-roo mgjday given concurrently with fractionated radiotherapy. Patient recruitment continues, as the Maximal Tolerated Oose has not yet been reached. Apoptosis as a mechanism of radiation-induced cell death Follicular lymphomas, frequently characterized by a t(i4;i8) translocation and overexpression of the anti-apoptotic bcl-2 gene product, are highly radioresponsive even to very low doses. The HORA-I phase 11 study investigated the role oflow dose involved field radiotherapy up to 4 Gy in 133 patients with recurrent indolent B-cell non-hodgkin lymphoma (NHL) in the period Eligible were patients with follicular NHL (n=i06), chronic lymphocytic leukemia (n=i6), marginal zone NHL (n=9), and immunocytomas (n=2). This new regimen proved to be highly effective to palliate locoregional symptoms in an otherwise incurable patient population. The overall response rate was 90% (58% CR, 32% PR) with a median time to progression of 13 months. The response of patients achieving a CR lasted up to 77 months with a median time to progression of 25 months. For PR patients, the response lasted up to 28 months with a median time to progression of 9 months. Toxicity was minimal. These data compare very favorably with systemic chemotherapy. To test the hypothesis that radiation-induced apoptosis is the predominant mode of cell death in these malignancies, we introduced a novel in vivo apoptosis imaging technique. This 99 m Tc-Annexin V scintigraphy (TAVS) is based on the exposure of phosphatidylserine (PS) at the outer leaflet of the plasma membrane lipid bilayer during the early phase of apoptosis. The human endogenous protein annexin V has a high affinity for PS, providing a detection method for apoptotic cells in vitro and in vivo. In seven patients a baseline and 24 hours post-radiotherapy TAVS we re performed. In addition, fine needle aspiration cytology (FNAC) was carried out before and af ter radiation for cytomorphological confirmation. In six out of seven the results ofthe TAVS and FNAC were concordant; both positive for apoptosis in five and both negative in one patient. There was one false-negative result (cytology positive but scan negative for apoptosis). Whether TAVS can be used as a test to predict treatment response win be evaluated not only in lymphomas, but also in solid tumors. (Figure IX.lI) CO BI AT ON OF RADIOTHERAPY AND CHEMOTHERAPY BAleman, jsa Beiderbos, EPM jansen, M Verheij, H Bartelink, C Rasch The increased local control and improved survival obtained by using the concomitant radiotherapy and cisplatin in advanced solid tumors has led us to explore several

102 ways of increasing this treatment regimen. Alternative fractionation schedules of irradiation have been tested as weil as the administration of intra-arterial cisplatin in order to achieve higher drug doses at the time of irradiation. A phase I trial was started with aral Perifosine concomitant with radiotherapy in locally advanced tumors, as mentioned earlier. This regimen is based upon our pre clinical studies to identify novel promising drugs for this concomitant use. Intra-arterial cisplatin and irradiation A total number ofr45 patients have been entered in the Phase 111 radplat trial (M99RAD), comparing 70 Gy radiation + intraarterial cisplatin or intravenous cisplatin for inoperable head and neck carcinoma. The trial is ongoing; a total number of 240 patients are planned. Interim analysis after 80 treated patients was performed. The trial data monitoring committee has advised to proceed with the trial. The data of patients with head and neck carcinoma treated with conventional radiation and daily cisplatin infusion were analyzed. The compliance with the schedule was below expectation; more than 50% the patients we re unable to complete the schedule. The main toxicity was due to the combination of ren al damage as a consequence of the cisplatin infusion and poor intake caused by the radiation mucositis. For this group of patients, not eligible in the M99RAD trial, a new chemoradiation schedule was designed. Concomitant Lv. cisplatin and multiple irradiations per day Fifty-three patients were entered in a randomized phase 11 trial where radiotherapy was given with conventional one fraction per day during seven weeks, or multiple fractions per day (MFD) in week 1,4 and 7, keeping the same overall treatment time and total dose. Cisplatin was given with a daily dose of 6 mgjm 2 or 10 mgjm 2, respectively. No difference was observed in acute and late toxicity in both treatment arms, while a similar or even better tumor response has been obtained with MFD. We concluded that a 67% higher daily dose of cisplatin concomitant with irradiation could be given in this three week multiple fractionations per day schedule, compared to the cisplatin dose given in the conventional daily fractionation schedule of seven weeks with the same total radiation dose. Involved field radiotherapy in patients with advanced Hodgkin's Iymphoma: an EORTC Lymphoma Group randomized controlled trial (#20884) The role of radiotherapy is still controversial in patients with advanced Hodgkin's Lymphoma (HL). In a multicenter randomized trial involved field radiotherapy (IF-RT) was compared to no further treatment (no-rt) in previously untreated stage IIljlV HL patients in complete remis sion (CR) after MOPPjABV (mechlorethamine, vincristine, procarbazine, prednisonej doxorubicine, bleomycin and vinblastin) hybrid chemotherapy. Patients in partial remis sion (PR) after six cycles of MOPP jabv received IF-RT. Figure IX.ll: In vivo apoptosis imaging by 99m Tc annexin V scintigraphy in a patient with low grade NHL in the right parotid gland (arrow). A,B,C: baseline scan; D,E,F: post treatment scan (24 hours ajter 2X2 Cy). A,D:frontal; B,E: transaxial; C,F: sagittal view. Of739 patients, 421 (57%) achieved CR; 161 patients were randomized to no further treatment, 172 to IF-RT and 88 we re not randomized for various reasons. Among randomized patients, af ter a median follow-up of 6.5 years, no significant differences were observed in relapse-free survival, event-free survival or overall survival. Among the 250 PR patients af ter chemotherapy, the 5-year event-free survival and overall survival rates we re comparable to the rates of patients in CR after chemotherapy. In conclusion, involved field radiotherapy did not improve the clinical outcome in advanced stages Hodgkin's lymphoma patients in complete remis sion af ter MOPP jabv chemotherapy. In patients with a partial remis sion treated with involved field radiotherapy, event-free survival and overall survival were similar to that of patients in complete remission.

103 X DIVISION OF MEDICAL ONCOLOGY Division head, group leader Sjoerd Rodenhuis Sjoerd Rodenhuis MD PhD Head Joke Baars MD PhD Academic staff Paul Baas MD PhD Academic staff Evert Bais MD Academic staff Jos Beijnen PhD Academic staff Willem Boogerd MD PhD Academic staff Henk Boot MD PhD Academic staff Metin Bülbül MD Academic staff Annemieke Cats MD Ph Academic staff Jaul Paul De Boer MD PhD Academic staff Bert De Gast MD Ph Academic staff Emine Göker MD Academic staff John Haanen MD PhD Academic staff Helgi Helgason MD Academic staff Alwin Huitema PhD Academic staff Martijn Kerst MD PhD Academic staff Marie José Kersten MD PhD Academic staff Mariëlle Kruijtzer MD Academic staff Philomeen Kuijer MD Academic staff Sabine Linn MD PhD Academic staff Herman Neering MD ph Academic staff Rianne Oosterkamp MD Academic staff Chris Rikers MD Academic staff Jan Schellens MD PhD Academic staff Jan Schornagel MD PhD Academic staff Jolanda Schrama MD Academic staff Babs Taal MD PhD Academic staff Wim Ten Bokkei Huinink MD Ph Academic staff Jaap Van der Sande MD PhD Academic staff Marchien Van der Weide MD PhD Academic staff laurence Van Warmerdam MD PhD Academic staff Nico Van Zandwijk MD PhD Academic staff Ans Vielvoye-Kerkmeer MD PhD Academic staff Jantien Wanders MD Academic staff Hanneke Zuetenhorst MD Academic staff Karin Mohrmann Post-doc Natalie Appels Graduate student Heleen Bardelmeijer Graduate student Jan Hendrik Beumer Graduate student Tessa Bosch Graduate student Marjan Bouma Graduate student Kristel Crommentuyn Graduate student Miriam Crul Graduate student Milly De Jonge Graduate student Monique De Maat Graduate student Monique Den Brok Graduate student Bregt Kappelhof G raduate student Marleen Kemper Graduate student Marjolein Klous Graduate student Isa Kuppens Graduate student Wandena lakhai Graduate student Mirthe Malingré Graduate student CLI N ICAl PHARMACOlOCY Jan Schellens, Jos Beijnen, Wim Ten Bokkei Huinink, Paul Baas, Evert Bais, Sjoerd Rodenhuis, Jan Schornagel, Nico Van Zandwijk Main research interests Research themes include the modulation of oral bioavailability through inhibition of drug transport proteins (especially P-glycoprotein and BCRP and drug metabolizing cytochrome P450), phase land pharmacological studies, modeling of sparse pharmacokinetic and pharmacodynamic data using NONMEM and studies to unravel the metabolism of anticancer drugs. We have started two new research activities: chemo-radiotherapy using ether lipids as radiosensitizers and pharmacogenotyping of patients. Phase I studies and drug scheduling The number of active studies is the same as last year. At present, 18 phase I/II and pharmacologic studies are being conducted, of which 13 studies are open for accrual. One of the major translational research activities continues to be the development of oral treatment schedules for paclitaxel and docetaxel in combination with oral cyclosporin A (CsA). We have shown that gut epithelial BCRP plays an important role in the absorption of oral topotecan. Follow up studies have been activated to determine the minimal effective dose of the BCRP blocker elacridar (GFI209I8) that results in a maximal enhancement of the oral bioavailability of topotecan. Studies were finalized with zosuquidar (LY335979), a strong inhibitor ofpglycoprotein, which is administered in phase I in combination with i.v. paclitaxel. Zosuquidar is given orally in two doses of 450 mg, the first one hour prior to paclitaxel (175 mg/m 2 given, q three weeks) and the second 12 hours later. Dose-limiting toxicity is reversible ataxia and visual disturbances. In the phase I study with the farnesyltransferase inhibitor zamestra (RII5777) in combination with cisplatin and gemcitabine, dose-limiting toxicity was reached at the level of 100 mg/m 2 cisplatin day one, 1000 mg/m z gemcitabine day one and eight and zarnestra 200 mg day 1-7, q three weeks. Main toxicities are myelosuppression, nausea and vorniting. A new pharmacological study with zarnestra was started in patients with mild or moderate liver dysfunction. Patient inclusion was discontinued because of unpredictable liver toxicity. The two-center phase land pharmacologic study with oral CPTII plus capecitabine daily times 14 is ongoing. Toxicities are as expected and consist mainly of diarrhea and nausea. In a further phase I/I! study in colorectal cancer we explore the combination of i.v. CPTII on day I plus capecitabine daily for 14 days, q day 2I. The randomized phase I study with weekly or two-weekly gemcitabine and cisplatin in patients with NSCLC has recruited 83 patients to date. The dose-intensity was highest in the two-weekly schedule and was established at 105 mg/m 2 of cisplatin plus 15 0 mg/m 2 of gemcitabine. The dose-intensity of cisplatin is 50% higher than in the standard schedule of this combination. Currently, we are exploring the reversed sequence of administration. The study to unravel novel metabolites ofe7070 is ongoing. We have identified new metabolites in the mass balance study with 14C-Iabeled E7070. One of the major pathways is glucuronidation. The phase I study with Kahalalide F (KF), a novel dehydroaminobutyric acidcontaining peptide isolated from the Hawaiian herbivorous marine species of mollusk Elysia rufescens, is ongoing in androgen resistant pro state cancer. Twenty-one patients have been recruited thus far and doses could be increased rapidly from 20!-tg/m2/day on a daily times 5 schedule q three weeks to 930!-tg/m 2 /day. This level was considered DLT (dose limiting toxicity) and showed reversible grade 4 transaminase increase. PSA marker reduction was seen in four patients coinciding with meaningful clinical improvement in one patient during six months. The activity ofkf is now being explored at the level of700!-tg/mz/day times 5, q day 2I. The combination study of carboplatin and topotecan is still ongoing. Tumor biopsies have been collected in 20 of 22 recruited patients to measure platinum-dna adducts

104 and the catalytic activity and expression of topoisomerase I. Dose-limiting toxicity is myelosuppression. The phase I study with the organic ruthenium compound NAMI-A has been completed. The schedule is daily times S q 3 weeks. Doses could be rapidly escalated from the starting level of 12 mgjm 2 jcourse to 2S00 mgjm 2 jcourse in only 12 steps. The pharmacokinetics of total and unbound ruthenium are linear over the wide dose range. Dose-limiting toxicity is blister formation and general malaise. The recommended dose for phase 11 studies is IS00 mgjm 2 over S days, q day 21. No objective responses were seen in the phase I study. The two-center phase I study with the polymer platinum compound APS280 was finalized. Doses were rapidly increased from the starting dose-ievel of 90 mgjm 2 to 4SoO mgjm 2 Dose-lirniting toxicity is vomiting. Despite the high doses of platinum, DNA-adduct levels in tumor cells were not higher than those obtained after standard carboplatin dosing. The phase I study with topotecan and ifosfamide has reached dose-limiting toxicity at a dose of topotecan of 1.4 mgjm 2 and ifosfamide of 1.2 gjm 2, both given at a daily times 3, q 3 weeks schedule. This study was finalized. The phase I study with paclitaxel and carboplatin in NSCtC has rapidly recruited 21 patients. The aim is to individualize the dose and infusion duration of paclitaxel in order to reach the target level of 0.1!J.M during a time-period of at least 16 hours. Carboplatin is dosed at an AUC (area under the curve of drug concentration plotted against time) of S. The targeted exposure to paclitaxel could be achieved during course 2. A further phase I study has been initiated in advanced breast cancer employing the combination of ora} vinorelbine on days land 8 and cyclophosphamide on days Main toxicity is myelosuppression. Thus far, oral tolerability is good. A two-center phase I study was initiated with the protein kinase C inhibitor LY31761S in combination with cisplatin and gemcitabine. Cisplatin is given on day one and gemcitabine on days land 8 of a 21-day schedule. Oral LY3I761S is given daily without interruption. The study includes assessment of the pharmacokinetics of all three drugs and pharmacodynamic measurements of circulating endothelial cells and soluble angiogenic factors. A new phase I study was initiated with the combination of the radiosensitizer perifosine and radiation. Perifosine is an ether lipid previously explored by us in a phase I study. The current dose is 100 mg per day in combination with standard dosed radiation. Phase 11 and pharmacologic studies Recruitrnent in three of the four phase 11 studies exploring the activity of oral paclitaxel plus CsA in advanced breast, gastric cancer and NSCLC and docetaxel plus CsA in breast cancer has been completed. Paclitaxel (60 mgjm 2 ) and CsA (10 mgjkg) are given orally bidaily on a weekly schedule. The overall response rate of 23% in stage IIIbjIV NSCLC is promising. The phase 11 study in second line ofbreast cancer with oral docetaxel plus CsA revealed a high response rate of 4S%. The oral safety of this schedule is good. Docetaxel is given as a flat dose of 100 mg. The dose of CsA is IS mgjkg. The partial response rate in first line of advanced gastric cancer is 33%. Jeany Rademaker-lakhai Graduate student Liesbeth Rook Graduate student Nadja Schoemaker Graduate student Ellen Stokvis Graduate stuent Desirée Van den Bongard Graduate student Sabien Van der Schoot Graduate student Charlotte Van Kesteren Graduate student Esther Boerhorst Techn ical staff Michel Hillebrand Technical staff Sindy lansen Technical staff Ciska Koopman-Kroon Techn ical staff Lianda Nan-Offeringa Technical staff Bastiaan Nuyen Technical staff Mariët Ouwehand Technical staff Hilde Rosing Technical staff Bas Thijssen Technical staff Mathijs Tibben Technical staff Selma Van Dam Technical staff Roei Van Gijn Technical staff Rianne Van Maanen Technical staff Diane Batchelor Nurse practitioner Ria Dubbelman Nurse practitioner Marjo Holtkamp Nurse practitioner Marianne Keessen Nurse practitioner Henk Mallo Nurse practitioner Margaret Schot Nurse practitioner Martha Swart Nurse practitioner Yvonne Arts-Blom Secretary Mariëlle De Kwant Secretary Feedback = (CirclCircF ) Proliferation E drug --v- ' ~ kcirc(=ktr) MIT Figure X.l: Schematic representation ofthe semi-physiologic model of E7070' explaining the extent and time course of dose-limiting neutropenia and thrombocytopenia.

105 Selected publications Clinical pharmacology Baker SD, Verweij J, Rowinsky EK, Donehower RC, Schel lens JHM, Grochow LB, Sparreboom A. Role ofbodysurface area of investigational anticancer agents in adults: ] Natl Cancer Inst 2002; 94: Crul M, De Klerk GJ, Swart M, Van 't Veer LJ, De Jong 0, Boerrigter L, Palmer PA, Bol CJ, Tan H, De Gast GC, Beijnen JH, Schel lens JHM. Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transjerase inhibitor R in advanced cancer. ] Clin Onco12002; 20: Kruijtzer CMF, Beijnen JH, Rosing H, Ten Bokkei Huinink WW, Schot M, Jewell RC, Paul EM, Schel lens JHM. Complete oral bioavailability of topotecan in combination with the Breast Cancer Resistance Protein (BCRP) and P glycoprotein (P-gp) inhibitor GF ] Clin Onco12002; 20: Studies within the framework of Biomed2 are ongoing. Final population analysis is being performed. The aim is to determine pharmacokinetic-response and -toxicity relationships with the combination of doxorubicine-cyclophosphamide in first line of advanced breast cancer and carboplatin-paclitaxel in first line of advanced ovarian cancer. Population pharmacokinetics and dynamics Population analysis of sparse data with NONMEM is extensively being applied to support the Biomed2 project and phase 11 studies with ET743 and E7070. We have developed a semi-physiological model to explain the myelosuppression ofe7070 (Figure X.I). Population analysis of E7070 is now being applied in phase 11 studies. We have developed a model for the pharmacokinetics of oral paclitaxel using NONMEM. A population pharmacokinetic model was developed for methotrexate. Clinical Pharmacogenetics We embarked on a new research line with the aim to determine the genetic profile of patients for drug oxidizing and conjugating enzyme systems and drug transporters. The genetic profile will be correlated with exposure levels of anticancer drugs and toxicity and efficacy parameters in patients. This approach may identify patients at risk for severe overexposure or undertreatrnent. We currently determine single nucleotide polymorphisms (SNPs) of drug metabolizing and transporting systems (Figure X.2). This methodology is currently being applied in two clinical trials in which patients are treated with docetaxel or CPTII. Kru ijtzer CMF, Schel lens JHM, Mezger J, Scheulen ME, Keilholz U, Beijnen JH, Rosing H, Math6t RM, Marcus S, Van Tinteren H, Baas P. A phase IJ and pharmacological study of weekly oral paclitaxel (Paxoral ) plus cyclosporin A (CsA) in patients with advanced non-small celllung cancer (NSCLC). ] Clin Oncol 2002; 20: Raymond E, Ten Bokkei Huinink WW, Taïeb J, Beijnen JH, Faivre SH, Wanders J, Ravic M, Fumoleau P, Armand JP, Schellens JHM. Phase land pharmacokinetic study of E7070' a novel chloroindolyl-sulphonamide inhibiting the activation of cdb and cyclin E, administered as a 1-hour injusion every 3 weeks in patients with advanced cancer. ] Clin Onco12002; 20J Figure X.2: PCR-RFLP analysis of polymorphic CYP2C19 '~2 in 19 subjects illustrating homozygous wildtype (bands at 109 and 212 bp), heterozygotes (bands at 1 9,212 and 312 bp) and homozogous mutants (one band at 321 bp). PHARMACY Jos Beijnen, Jan Schellens, Wim Ten Bokkei Huinink The research programs of the Department of Pharmacy and Pharmacology concern the pharmaceutical formulation and drug level monitoring of (investigational) anticancer drugs to support pre clinical (see Division XIII) and clinical pharmacologic research. The investigations are conducted in the setting of a foundation (NLADF), which is a collaboration between The Netherlands Cancer Institute and Slotervaart Hospital. The department holds GMP (Good Manufacturing Practice) and GLP (Good Laboratory Practice) licenses, GLP in the field of analytical chemistry. This allows us to manufacture investigational anticancer drugs for (international) clinical trials and to provide laboratory data to be used for drug registration purposes (Figure X.3).

106 .:.."..." Selected publications (continued) Pharmacy Crul M, Mathöt RAA, Giaccone G, Punt CA, Rosing H, Hillebrand MJX, Ando Y, Nishi N, Tanaka H, Schellens JHM, Beijnen JH. Population pharmacokinetics of the novel anticancer agent KRN7ooo. Cancer Chemother Pharmacol2002; 49: Figure x.j: Preparation of experimental chemotherapy for administration. Formulation The following investigational cytotoxic compounds have been formulated in the past year: aplidine, kahalalide F, ES-28S.HCI, epothilone 0, E09 (Neoquin ), the ruthenium complex NAMI-A and the HPMA(hydroxypropyl methacrylamide)-polymer with peptide spacer bound platinum compounds AP-S28o and AP-S346. We manufactured vials for both preclinical (E09, ES-28S.HCI) and clinical research (aplidine, NAMI-A, AP-p80, kahalalide F, E09). An oral formulation is now being developed for epothilone o. Aplidine 2mg vials were manufactured because phase I studies resulted in higher MTOs than anticipated requiring larger vial sizes. The formulation of AP-S346 is in its development stage. This also includes its quality control and stability research with techniques such as platinum-nmr and size-exclusion chromatography. Our formulation research has a long tradition in the use of freeze-drying. This technique is applied, in general, for unstable, water soluble compounds. Many new anticancer drugs are chemically unstable, but also poorly water-soluble. This has stimulated us to investigate alternative ways to lyophilize drugs from non-aqueous vehicles. We found that it is possible to lyophilize ES-28S.HCl and epothilone 0 after inclusion into 2-hydroxypropyl-~-cyclodextrin in combination with t-butanol. The freeze-dried product is sta bie and soluble in an aqueous solvent. Experimental design methods have been implemented to select the most optimal formulation conditions. Stokvis E, Rosing H, L6pez-Lázaro L, Rodriguez I, Jimeno JM, Supko JM, Schellens JHM, Beijnen JH. Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry. ] Mass Spectrom 2002; 37: Van Kesteren Ch, Mahöt RAA, Raymond E, Armand JP, Ditlrich Ch, Dumez H, Roché H, Droz JP, Punt C, Ravic M, Wanders J, Beijnen JH, Fumoleau P, Schellens JHM. Population pharmacokinetics of the novel anti-cancer agent E7070 duringfour phase I studies: model building and validation. ] Gin Onco12002; 20: Villalona-Calero MA, Eckhardt SG, Weiss G, Hidalgo M, Beijnen JH, Van Kesteren C, Rosing H, Campbell E, Kraynak M, Lopez-Lazaro L, Guzman C, Von Hoff DD, Jimeno J, Rowinsky EK. A phase land pharmacokinetic study of ecteinascidin-743 on a daily x 5 schedule in patients with solid malignancies. Clin Cancer Res. 2002; 8: Bioanalysis and clinical pharmacology Bioanalytical services have been delivered for numerous preclinical and clinical trials in- and outside the institute. This concerns the following compounds and metabolites (selection): topoisomerase-i inhibitors (irinotecanjsn-38, topotecanjn-desmethyltopotecan), gemcitabine, mitomycin C, melphalan, taxoids (paclitaxel, docetaxel), platinumjruthenium compounds, marine compounds (ET-743, ES-28S, kahalalide F) and the CTC (cyclophosphamide, thiotepa, carboplatin) combination. Most assays are performed by the use of coupled (miniaturized) liquid chromatography with tandem mass spectrometric detection (LCjMSjMS). Oue to the large number of samples, we are also exploring the possibility of assay automatization e.g. by the use of 96-wells autosampler plates and direct sample injection with on-line sample pretreatrnent. All method validations are executed according to current FOA guidelines. An LCjMSjMS assay has been developed for the simultaneous quantification of cyclophosphamide, 4-hydrOxycyclophosphamide, 2-dechloroethylcyclophosphamide, 4-ketocyclophosphamide, thiotepa and TEPA. Blood samples are stabilized with semicarbazide. For therapeutic drug monitoring and dose individualization during a course of a patient treated with the high-dose CTC regimen drug levels in plasma are required within 24-hour af ter sampling. With the new assay this requirement is fulhlled.

107 Topotecan 15 mg/kg --Cl- Lipotecan 5 mg/kg 0, Time(h) Figure X+ Plasma concentrations of topotecan in rats with (lipotecan) or without liposomal encapsulation. To support preclinical pharmacokinetic research of topotecan formulated in polymeric micelles, liposomes (lipotecan) and immunoliposomes we have adapted and validated our topotecan HPLC assay for these applications. The dramatic effect ofliposomal encapsulation on topotecan pharmacokinetics is exhibited in figure X+ Kahalalide F levels in plasma are measured under basic conditions by HPLC coupled to electrospray ionization (ESI) MS/MS. In the beginning of the phase I study, dose escalations were performed per patient and on the basis of pharmacokinetic outcome. A rapid turn-around of analytical results is pivotal in such studies and appeared feasible with the developed assay. ES-285 ((2S,3R)-2-amino-3-hydroxy-octadecane) is a novel investigational anticancer agent isolated from the marine clam Spisula polynoma. In addition to its pharmaceutical formulation, we have developed an LC/MS/MS assay for this compound in animal and human plasma. Preclinical pharmacokinetic studies have been finalized using this assay and will support the forthcoming phase I study. A validated ion-pair HPLC assay with UV detection was set up for the analysis of gemcitabine-triphosphate in leucocytes to support gemcitabine clinical studies. As part of several multicentre phase 11 studies with the cell cycle inhibitor E7070, also in combination with other cytotoxic drugs, we have measured plasma samples. The LC/ESI/MS/ MS assay developed in our laboratory also includes the metabolite MI (IA-benzenesulphonamide). Sample pretreatrnent is performed by solid-phase extraction with Oasis cartridges. Elacridar (GF ) is a BCRP blocker and tested for its potency to enhance the oral bioavailability of topotecan. An LC/MS/MS assay was developed and validated for the measurement of elacridar in biological matrices and implemented in clinical research with the drug. An LC/MS/MS assay, with liquid-liquid extraction to clean up the samples, was designed for the novel, orally active, tubulin poison D-2485I. The limit of quantitation is 1 ng/ml. The method will be implemented in the forthcoming phase I study with the drug in our institute. The pharmacokinetics of A (ABT 518), a novel matrix metalloproteinase inhibitor, have been investigated in a phase I trial. Plasma samples were analyzed by an LC/MS/MS method we developed previously. This analysis revealed that the drug is extensively metabolized. At least six metabolites were found. The proposed metabolic breakdown of the molecule in humans is depicted in Figure X.5. ~ / ~ NH~,Ii,o S, o +-0 R A ~ Formamtde kelal ~ A AminckClal ~ / ~;C)(o HcJ T - R OH A Amidcdiol 1 o,s/o HJC/ 'R o 1\ /OH N 0 0 ~ 1 ~s,li Hó' / 'R OH A Activediol A Methyl sulfonc.~ Figure X.5: Proposed metabolism ofthe matrix metalloprotease inhibitor A (ABT 518) in humans. A new paclitaxel LC/MS/MS assay for paclitaxel, ten-fold more sensitive than HPLC-UV, has been developed and can be used for drug analysis in mouse plasma and brain tissue to support pre clinical studies investigating P-glycoprotein modulation and paclitaxel penetration in brain/tumor tissue.

108 +- ~~I~~(.~.J~L~.J~.~[". ~I~~ ~ An LC/MS assay is being developed for (non-)prenylated RAS proteins. We are pursuing the following strategy. Af ter protein isolation by ultrahltration, the RAS protein is digested to yield a specific (non-) prenylated terminal tetrapeptide which is quantified by LC/ESI/MS/MS. Mitomycin C is measured to support HIPEC (Hyperthermie Intraperitoneal Chemotherapy) (Division XI, Surgical Oncology) and melphalan is measured to study liver up take of arterial drug infusion during retrograde rat liver perfusion (bioanalytical service for investigators in Leiden, LUMC). To support cellular transport and metabolic studies (group Piet Borst) in MRP 4 and MRP5 transfected celis, we quantified several purines and pyrimidines with an accurate HPLC-assay with photo-diode array detection. Assays to measure probenecid and pheophorbide in biological matrices were set up to support MRP2 transport studies and plasma analysis of BCRP knock-out mice, respectively (group Alfred Schinkel). Our MS group facilitates the quality control of peptides synthesized in the Institute (Henk Hilkrnan). The department also has research programs ongoing with the bioanalysis and clinical pharmacology of opiates and antiretroviral drugs. IMMUNOTHERAPY Bert De Gast, John Haanen, Willem Boogerd, Martijn Kerst, Marie josé Kersten Selected publications (continued) Immunology De Gast GC, Vyth-Dreese FA, Nooijen W, Van den Bogaard -CJC, Sein J, Holtkamp MMJ, Linthorst GAM, Baars jw, Schornagel jh, Rodenhuis S_ ReinJusion of autologous lymphocytes with granulocyte-macrophage colony-stimulating factor induces rapid recovery OfCD4+ and CD8+ T ceus after high-dose chemotherapy for metastatic breast cancer_ ] Clin Oncol 2002; 20: De Gast GC, Batchelor D, Kersten Mj, Vyth Dreese FA, Sein J, Van de Kasteele WF, Nooijen Wj, Nieweg OE, De Waal M, Boogerd W. Temozolomide fouowed by combined immunotherapy with CM-CSF, low dos IL-2 and IFNa in patients with metastatic melanoma. Brit] Cancer (in press). Metastatic melanoma Following a phase 11 study of sequential Temozolomide and combined immunotherapy, a phase 111 multicenter study oftemozolomide ± immunotherapy was started in stage IV melanoma. A phase 11 study of neo-adjuvant and adjuvant chemo-immunotherapy around lymphadenectomy in stage 111 melanoma is on-going. A new scheme of Temozolomide (low dose for 21/28 days) is tested in patients with stage IV ocular and mucosal melanoma. Wolkers MC, Toebes M, Okabe M, Haanen jbag, SchumacherTNM. 5 basic rules for optimized epitope-directed DNA vaccination. ] Immunol2002; 168: Renal cell carcinoma (ReC) A phase 11 study with pegylated Interferon-a (PEG-Intron R ) in progressive metastatic RCC is nearly finished. It showed no additional value compared to standard IFN-a (1 CR, 1 PR in 20 patients). A phase 11 study oftemozolomide in IFN-a resistant patients is on-going. A phase 11 study has been initiated in close collaboration with the Amsterdam Medical Center and the NIH, hematology branch in the USA. Metastatic RCC patients with disease progression after first line immunotherapy-based treatrnent who have an HLA-identical sibling may be enrolled in a non-myeloablative allogeneic peripheral blood stem cell transplantation protocol. Response rates obtained so far in other centers vary between 20 and 50%. Depending on the experience with allogeneic transplantation, the transplantation-related mortality varies between 10-20%. Peri-operative immunotherapy A phase I study of escalating doses ofthree cytokines has been completed showing a maximum tolerated dose (MTD) of 2.5 Ilg for GM-CSF, 1.0 MIU /m 2 for IL-2 and 3 MIU for IFN-a which is administered for 8 days from day-3 before until 5 days after nephrectomy for RCe. Higher doses led to collection of proteinacious fluid in the contra-iateral pleura and abdominal cavity. The immunologie data showed that post-operative immune depression oft celis, NK cells and monocyte DR expres sion in peripheral blood was completely prevented with the MTD. Immune histology of the nephrectomy specimen showed a significant increase of CD8+ TeelIs and dendritic cells in the tumor area but not in normal renal tissue. A tendency for a delay of relapse or progression was found clinicaliy, but has to be confirmed in a phase 111 study. Tumor specific immunity in melanoma patients With the aid ofmhc tetramers, peripheral blood samples from stage 111 and stage IV melanoma patients are being analyzed for the presence of naturally occurring melanoma-specific T cell immunity. In the analysis, T cell immunity specific for the melanocyte differentiation antigens

109 Selected publications (continued) Autologous hematopoietie progenitor eell transplantation program Bosma AJ, Weigeit B, Lambrechts AC, Verhagen OJ, Pruntel R, Hart AA, Rodenhuis S, Van 't Veer LJ. Deteetion of eireulating breast tumor eells by differential expression of marker genes. Clin Caneer Res 2002; 8: De Jonge ME, Mathöt RAA, Dalesio 0, Huitema AD, Rodenhuis S, Beijnen JH. Relationship between irreversible alopeeia and exposure to cyclophosphamide, thiotepa and earboplatin (CTC) in high-dose ehemotherapy. Bone Marrow Transplant 2002; 30: Huitema ADR, Spaander M, Mathöt R, Tibben MM, Holtkamp MJ, Beijnen JH, Rodenhuis S. Relationship between exposure and toxieity in high-dose ehemotherapy with cyclophosphamide, thiotepa and carboplatin. Ann Oneol 2002; 13J Schrama JG, Faneyte IF, Schornagel JH, Baars JW, Peterse J L, Van de Vijver MJ, Dalesio 0, Van Tinteren H, Rutgers EJ, Richel DJ, Rodenhuis S. Randomized trial ofhigh-dose ehemotherapy and hematopoietie progenitor-eell support in operabie breast caneer with extensive lymph node involvement: final analysis with 7 years offollow-up. Ann Oneo12002; 1]: MART-I, tyrosinase and gproo and antigens belonging to the CaneerjTestis group are being examined. In a cohort of 38 HLA-A2+ metastatic melanoma patients, 50% had MART-I, tyrosinase or gploo-speeifie TeelIs in the peripheral blood. Interestingly, the presenee of melanoma-speeifie TeelIs in these patients was assoeiated with the memory T eell phenotype, indieating that these eells have been activated in vivo. Also, several patients with melanoma-specific T cell immunity had tumors which had lost HLA-A expression, suggesting immunological escape. AUTOLOGOUS HEMATOPOIETIC PROGENITOR CELL TRANSPLA TATION PROGRAM Sjoerd Rodenh uis, Joke Baars, Jos Beijnen, Jan Paul De Boer, Milly De Jonge, Marjo Holtkamp, Martijn Kerst, Jan Schellens, Jan Schornagel The main objective of this program is to offer potentially curative therapy to patients with high-risk primary breast cancer and to patients with so-called oligometastatic disease. Oligometastatic disease is usually defined as stage IV disease, in which all tumor localizations could, in principle, be managed by local therapy such as surgery or radiation therapy. As in adjuvant chemotherapy, the high-dose therapy serves to eradicate microscopic disease. Advanced breast cancer The program in which patients with oligometastatic breast cancer receive three subsequent courses of cyclophosphamide, thiotepa and carboplatin (CTC) was continued. With this approach, ab out one-third of the patients responsive to conventional chemotherapy achieved long-term disease-free survival. In an effort to increase this fraction of surviving patients, the weekly administration of oral paclitaxel for three months was explored and found to be tolerabie. Both the high-dose chemotherapy and the oral paclitaxel have been shown to lead to widely varying blood levels of the compounds and of their metabolites. Therapeutic drug monitoring has now become standard to reduce this variability, both for the high-dose therapy and for paelitaxel (Figure X.6). It is hoped that this approach will be able to prevent the occasionally severe organ toxicity associated with high-dose therapy and to ensure adequate exposure to paclitaxel. H igh risk primary breast ca ncer In 2002, the first complete analysis of the Dutch national trial ofhigh-dose chemotherapy in the adjuvant treatment of high-risk breast cancer was performed. This trial, which was conducted in ten Dutch cancer centers Figure X. 6: Blood samplingfor therapeutie drug monitoring in high-dose ehemotherapy.

110 and which has been designed and analyzed in The Netherlands Cancer Institute, suggests that tumors that do not express the HER-2jneu receptor particularly benefit from high-dose alkylating agents. Conversely, HER-2jneu positive tumors appear to be quite resistant, confirming the findings of eight smaller uncontrolled studies, most of which have been published in the last two years. This and other findings of the analysis suggest that candidates for high-dose therapy should be selected based on age (below 50), HER-2jneu receptor negativity and possibly lower tumor grade. Based on this we have adapted the selection criteria for our trial in oligometastatic breast cancer. This alone could improve the long-term survival fraction in future cohorts of patients. (Figure X.7) Selected publications (continued) Thoracic oncology Baas P. Chemotherapy for malignant mesothelioma: from doxorubicin to vinorelbin. 5em Oncol 2002; 29:62-9. Baas P. Inductive and adjuvant treatment strategies for localized non-smalt celtlung cancer. Current Opinion Oncol 2002; Van Schooten FJ, Nia AB, De Flora S, D'Agostini F, Izotti A, Camairano A, Balm AJM, Dallinga JW, Bast A, Haenen GRRM, Van 't Veer Lj, Baas P, Sakai H, Van Zandwijk N. Effects oforal administration of N-Acetylcysteine: a multi-biomarker study in smokers. Cancer Epidemiol Biomarkers Prev 2002; 11: Van Zandwijk N. New methodsfor early diagnosis oflung cancer. Lung Cancer 2002: 38:59. Figure X.T Nurse practitioner Marjo Holtkamp, making rounds in the high-dose chemotherapy unit. THORACIC ONCOLOGY Nico Van Zandwijk, Paul Baas, Jan Schellens Non-Small Cell Lung Cancer (NSCLC) The dose intensification studies with the gemcitabinejcisplatin (GemjCis) combination in patients with advanced and metastatic NSCLC were completed. Doubling of cisplatin dose intensity was feasible when the drugs we re combined on day I (Gem) and day 2 (Cis) in a cyele of two weeks. Unfortunately, no indication could be found for a translation of increased drug exposure to better efficacy. Phase I studies with the Cyclin inhibitor Ro given in combination with Gemcitabine were terminated. Drug metabolism showed unpredictable inter-individual differences and some patients with high exposures had excessive toxicity. Over IlO NSCLC patients failing after chemotherapy have received ZD1839 in a daily dose of 250 mg p.o. day as a part of an expanded access program. A small group of patients showed impressive responses oflong duration while the side effects were very mild in the majority of patients. In a fair number of patients significant periods of disease stabilization were also recorded. The international randomized phase 11 study evaluating trastuzumab (Herceptin R ) in patients with advanced and metastatic NSCLC underwent its final analysis. The proportion ofnsclc patients with significant EG FR expression turned out to be quite low «10%). Although in the rare patients with significant (+++) EGFR expression activity of trastuzumab was suspected, the phase 11 comparison did not reveal any difference between the two study arms. Sm all Cell Lung Cancer (SCLC) A phase 11 study in patients with limited disease SCLC with carboplatin, etoposide, paclitaxel and concurrent radiotherapy was completed. A total of 38 patients was accrued. An impressive objective response rate

111 Selected publications (continued) Gastroenterology Boot H, De Jong D. Diagnosis, treatment decisions, and follow-up in primary gastric lymphoma. Gut 2002; 51: Van den Bongard HJ, Boot H, Baas P, Taal BG. The role ofparallel stent insertion in patients with esophagorespiratory fistulas. Gastraintest Endosc 2002; 55: Van der Gaag LC, Renooij 5, Witteman CL, Aleman BM, Taal BG. Probabilities for a probabilistic network: a case study in oesophageal cancer. Artif Intell Med 2002; 25: Zuetenhorst JM, Bonfrer JM, Korse CM, Bakker H, Van Tinteren H, Taal BG. Carcinaid heart disease: the role of urinary 5-H1AA excretion and plasma levels of ANP, TGF-b and FG. Cancer (in press). of 95% and a median survival time of 22 months could be documented and the planning of a national study combining platinum containing chemotherapy with early radiotherapy is ongoing. Chemoprevention The placebo controlled phase II chemoprevention study with inhalational fluticasone in 'healthy' smokers accrued more than 20 volunteers. Spiral CT-scans and bronchoscopies we re performed before and at the end of a 6-months intervention period. In this study, the P53 and htert expressions in bronchial biopsies serve as markers for the effect of fluticasone on carcinogenesis. Preparatory studies for an Optical Coherence Tomography project to visualize the central airways in semihistological fashion we re started. Malignant Mesothelioma Tomudex was tested within the framework of the EORTC (08992). An encouraging objective response rate of over 20% was observed. STI 571 (Clivec), chosen for its potential interference with the PDCF receptors has been used as single agent in 15 patients and Thalidomide was employed in first and second line in more than 40 patients. Thalidomide was rather weil tolerated and induced a prolonged stabilization of disease in about 25% of patients tested. Surgical treatment of mesothelioma has focussed on the few patients with early stage disease. Since previous experience with intraoperative therapy (photodynamicjchemotherapeutic) has been accompanied by significant toxicity, pleuropneumonectomy followed by radiotherapy remains our standard for patients selected for radical treatment. GASTROEN EROLOGY Annemieke Cats, Henk Boot, Babs Taal Castrointestinal Iymphomas The pathogenesis of gastric MALT lymphomas is strongly associated with a chronic Helicobacter pylori infection. In low-grade MALT lymphomas, translocation t(n;i8) is the most frequent single chromosomal abnormality. Translocation leads to a fusion-product of the API2-gene (Apoptosis Inhibitor 2) on chromosome n and the MALTI-gene on chromosome 18 (Figure X.8). This fusion-product leads to a strong survival advantage for the lymphoma cells. In early stages of the disease, H. pylori eradication leads to a histological remission in 70-80% of patients, although it often takes months to achieve this. Factors that may prediet histological response indude stage as assessed by endoscopie ultrasound, subgrading of gastric low-grade MALT lymphoma and the presence of the translocation t(n;i8). This translocation is associated with more advanced disease stages. In a large multicentre european study in In gastric MALT lymphoma patients, we recently demonstrated the dinically relevant finding that it prediets non-responsiveness to H. pylori eradication, as weil. Histological remis sion should still be based on conventional histology, and despite earlier reports, the role of endoscopie ultrasound seems to be limited. Future research will focus on therapeutic interventions to re duce the time required to obtain histological remis sion. Carcinoid tumours In 56 patients with metastatic neuroendocrine tumors, it was studied wh ether histological grading could accurately prediet prognosis. I BIR2 I IIBIR3 I 2% 93% 1% 4% t ~ 1 t API2 I BIR1 CARD _ I RING 11 MALT1 11 DO [ 19 1 t 19 I Caspase like t t tt 9% 42% 32% 17% Figure X.8: Percentages ofbreakpoints in API2-gene (chromosome 11) and MAL Tl-gene (chromosome 18) in 111 patients with gastric MALT-lymphoma (Gastroenterology 2002: 122; ).

112 1 ~ ~;~I~~(.!W' ~~(~~q.~[a ~ ~ ~ Immunostaining was only oflimited value. Low-grade and high-grade tumors could be recognized by the Capella and the WHO classification. Mitotic activity was of additional value in low-grade tumors. Bone metastases in metastatic carcinoid patients are assumed to be rare, but in our series occurred in 10% of patients, with a median interval of 37 months following diagnosis. Nuclear imaging with IIIIn-pentetreotide and 13 I I_MIBG is not able to detect these metastases with SO to 80% false-negatives, respectively. Standard bone scintigraphy, however, is a sensitive technique. In some patients, skin metastases are extremely painful. It has been postulated that this may be due to neuroinvasion. Histology, however, did not show a difference in neuroinvasion between asymptomatic metastases and painful nodules in four patients. Heart failure is an important cause of death in carcinoid patients. Increased urinary S-hydroxyindoleacetic acid excretion is associated with the presence of right-sided he art valve fibrosis. Transforming Growth Factor-B (TGF-B) and Fibroblast Growth Factor (FGF) are both associated with pulmonary and renal fibrosis development. In 37 carcinoid patients, however, these factors we re not associated with carcinoid heart disease. In conclusion, plasma levels may not adequately reflect the local process. Endoscopie stents in gastrointestinal malignancies The endoscopic insertion of expandable stents in oesophageal and biliary malignant stenoses has enabled better palliative treatment of these patients. When a narrowing of the central airways occurs, parallel stenting ofboth tracheo-bronchial tree and oesophagus results in immediately relief in most patients. Our experience has now expanded to over 20 patients during the last years. Further improvement in stents and delivery systems also enables stenting of stenoses in the stomach, duodenum and colon. Thus, such patients can be rapidly treated with endoscopic intervention without the need of surgery. The fitst 40 patients who underwent this treatment for gastroduodenal obstruction were recently analysed. Fracture of stent material (nitinol wire) in four patients was an unexpected problem and led to serious adverse events in three patients.

113 XI DIVISION OF SURGICAL ONCOLOGY Division head, group leader Bin Kroon Board Bin BR Kroon MD PhD FRCS (Head) Fons JM Balm MD PhD FRCS FACS Omgo E Nieweg MD PhD General Surgical Oncology Bin BR Kroon MD PhD FRCS Academic staff, Head Division Houke M Klomp MD Academic staff Omgo E Nieweg MD PhD Academic staff Hester SA Oldenburg MD PhD Academic staff Emiel JTh Rutgers MD PhD FRCS Academic staff Frits Van Coevorden MD PhD Academic staff Frans AN Zoetmulder MD PhD Academic staff Susanne H Estourgie MD Academic staff Geertje Govaert MD Academic staff Reinie Kaas MD Academic staff Eduard LAR Mutsaerts MD Academic staff Eva M Noorda MD Academic staff Marjan Piek-den Hartog MD Academic staff Pieter J Tanis MD PhD Academic staff Peter Van Duijvendijk MD Academic staff Richard Van Hillegersberg MD PhD Fellow Johan C Van Mourik MD Academic staff Serge Van Ruth MD Academic staff Johanna Van Sandick MD PhD Academic staff Vic J Verwaai MD Academic staff Bart C Vrouenraets MD PhD Academic staff Jan Wijsman MD PhD Fellow Arjen J Witkamp MD Academic staff Yaïr Acherman Undergraduate student Lysette Broekhuizen Undergraduate student Claudia Dekker Undergraduate student Jaap DH De Vries Undergraduate student R Houtkamp Undergraduate student Bart Takkenberg Undergraduate student Head and Neck Oncology Frans JM Hilgers MD PhD Academic staff, Head Fons JM Balm MD PhD FRCS FACS Academic staff Michiel MW Van den Brekel MD PhD Academic staff I Bing Tan MD PhD Academic staff Annemieke H AckerstaffPhD Academic staff Joeri Buwalda MD Academic staff Lucille Dorresteyn MD Academic staff Tom Geurts MD Academic staff Steven Gonggrijp DOS Academic staff Luuk M Janssen MD Academic staff Frans HM Kroon DOS PhD Academic staff Peter JFM Lohuis MD PhD Fellow RolfPostema MD Academic staff lym PHATIC MAPPI NG Omgo E Nieweg, Simon Horenbias, Bin BR Kroon, Emiel JTh Rutgers, Susanne H Estourgie, Pieter J Tanis Lymphatic mapping with sentinel node biopsy is a staging technique devised to detect non-palpable tumor involvement of the regionallymph node fields. This approach allows for selection of patients for early regional treatment and for adjuvant systernic therapy. The lymphatic mapping studies are logical constituents of the research program of the Division of Surgical Oncology, which involves pathophysiology, early diagnosis, staging and treatment of regional dissemination of solid cancers. The tumor types that are subject to investigation within the lymphatic mapping research program are melanoma, breast cancer, carcinoma of the penis, testicular cancer and carcinoma of the larynx. The general aims of this branch of the surgical research program are to improve the technique oflymphoscintigraphy and surgery, and to determine the clinical benefits: rates of survival and regional control following implementation oflymphatic mapping. An essential element in the program is the multidisciplinary approach. The team involves surgeons, urologists, head and neck surgeons, nuclear medicine physicians, pathologists, radiologists and a physicist. Studies are done in cooperation with the Medisch Spectrum Twente, the WHO Melanoma Programme and The John Wayne Cancer Center. A selection of the work is presented here. Breast cancer The Netherlands Cancer Institute pioneered lymphatic mapping in breast cancer. The technique that was developed involves administration of the tracers into the primary breast cancer. The success of this procedure led to the desire to also use it in non-palpable lesions. A technique was devised to enable administration of the tracers into non-palpable breast cancers. This approach not only allows the removal of the sentinel node, but also enables excision of the primary tumor guided by the gamma ray detection probe instead of a guide-wire. Sixty patients were studied in this fashion. In S8 of them, a sentinel node was identified. The node was tumor-positive in ten. An interesting observation was that a whopping 24 patients had their sentinel node outside the axilla. Comparison of the results of the segmental excisions of the primary tumors showed that better margins we re obtained than in an earl ier group of patients who underwent wire-guided excision. This new technique is a relevant step forward not only because of the benefits for the patients concemed but also because the screening programs identify growing numbers of patients with early, non-palpable breast cancers. Other institutions are now following our approach. Melanoma Lately, disappointing results oflymphatic mapping in melanoma have been published. Reputable institutions have presented false negative rates ranging from r6% to 40%. These results were not appreciated by the surgical community and we have initiated efforts to increase awareness. One of the presumed causes of the poor results is an insufficient leaming process of the surgeons concerned. A study was performed to determine the statistical soundness of the learning phase. It was found that the currently recommended learning period of ten to 80 operations lacks statistical significanee. Unfortunately, it was also found that a statistically sound learning phase was clinically unattainable. Another disturbing finding is the r8% incidence of in-transit metastases in a large series of sentinel node-positive patients at MD Anderson Cancer Center. Given these recent unfavourable developments elsewhere, efforts are now underway at The Netherlands Cancer Institute to analyse our false negative rate after a median follow up of six years and to establish the incidence of in-transit metastases. Urologie tumors The Netherlands Cancer Institute continues to be the leading institution in lymphatic mapping for penile cancer. Testicular cancer is another type

114 Figure XI.l: Lymphatic drainage of a testicular carcinoma (T) via an afferent lymphatic vessel to a sentinel node (arrow) in the para-aortic region. of disease in which the role of elective lymph node dissection is being debated. Efforts were initiated to examine the feasibility oflymphatic mapping in patients with this disease. Funicular administration of the radiopharmaceutical resulted in inguinallymph node visualisation. Intratesticular administration resulted in retroperitoneal sentinel node depiction in three of four patients (Figure XLI). This is the technique that was adopted for further study. ISOLATED LlMB PERFUSION Bin BR Kroon, Omgo E Nieweg, Gooike W Van Slooten, Eva M Noorda, Bart C Vrouenraets Isolated limb perfusion (I LP) in elderly patients Older patients are assumed to have a higher risk of complications from ILP. A study was performed evaluating the safety and efficacy of ILP in patients older than 75 years. Two hundred and eighteen therapeutic ILPS with Melphalan ± tumor necrosis factor-alpha (TNFa) were performed in 202 patients with recurrent measurable melanoma and analysed retrospectively. Fifty-three patients (28%) were 75 years or older. Complete response rates we re 56% for these ol der patients and 58% for the younger age group (P=0.79) with locoregional relapse in 56% and 51% of these patients respectively (P=0.6I). In the older patients, 19% had a grade lil/iv toxicity (28% in the younger group, P=0.I8). Systemic toxicity, local complications and long-term morbidity were similar in both age groups. Older patients stayed in hospital for a median of 23 days (younger patients 19 days, p<o.oi). ILP results in similar response rates in the elderly with recurrent melanoma without increased toxicity, complications and long-term morbidity compared to younger patients. Older age in itself is not a contraindication for ILP. Early mortality after 1 LP for melanoma Risk factors for mortality within one year after ILP were determined to identify patients who might not live long enough to experience the possible benefits of ILP but would risk its harm. Data from 439 patients who underwent a therapeutic ILP for extremity melanoma were analyzed. Ninety percent of these patients had MD Anderson stage I1b/III disease. Various ILP regimes were used over the years. Median follow-up was 38 months (25-75% interval months) with a minimum of one year. A multivariable logistic regression analysis was performed to determine risk factors for death within one year from ILP. Tested varia bles were: gender, age, Breslow thickness and ulceration of the primary tumor, interval between primary and ILP, number of previous recurrences, number and size of the lesions at the time of ILP, stage of disease and ILP regimen. Adriaan Timmers DOS Academic staff Corina J Van As PhD Academic staff Guido Van den Broek MD Academic staff Juliëtte F Van Hagen MD Academic staff Vincent lm Vander Poorten MD PhD Academic staff Jan Wedman MD Fellow Urological Oncology Simon Horenbias MD PhD Academic staff, Head Axel Bex MD PhD Academic staff Wim Meinhardt MD PhD Academic staff Henk G Van der Poel MD PhD Academic staff Anne P lont MD Academic staff Jacco A Nieuwenhuijzen MD Fellow Gynecological Oncology Matthé PM Burger MD PhD Academic staff, Head Marc Van Beurden MD PhD Academic staff Arnold CM Van Lindert MD PhD Academic staff Nine Van der Vange MD PhD Academic staff (till ) lottie AC lubsen-brandsma MD Academic staff Marianne AC Slot MD PhD Academic staff Arjanneke C Van Hof MD Academic staff Dana S Acherman MD Academic staff Roelien Olivier undergraduate student Plastic and Reconstructive Surgery J Joris Hage MD PhD Academic staff, Head leonie AE Woerdeman MD Academic staff louis De Weerd MD Fellow Brigit Drost MD Academic staff Petra Koster MD PhD Fellow Paris Melis MD Fellow Jan Van loon MD Fellow Anne Van Schijndel Undergraduate student Anesthesiology Peter FE Schutte MD Academic staff, Head Dick R Buitelaar MD Academic staff Myrtille C De Vries MD Academic staff Hans Huitink MD PhD Academic staff May Ronday MD Academic staff Hans Visscher MD Academic staff Secretary Noëlle Tameris Marion Van Zuilen

115 Selected publications Full papers Ackerstaff AH, Tan IB, Rasch CRN, Balm AJM, Keus RB, Schornagel JH, Hilgers FJM. Quality-oflife assessment after supradose selective intra-artenal cisplatin and concomitant radiation (RADPLAT) for inoperable stage IV head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2002; 128: Buskens Cl. Van Coevorden F, Obertop H, Van Lanschot J). Disturbed anastomotic healing after esophagectomy: a novel treatment of a benign tracheo-neoesophageal fistula. Dig Surg 2002; 19: De Bree E, Van Coevorden F, Peterse J L, RusselI NS, Rutgers EJ. Bilateral angiosarcoma of the breast after conservative treatment ofbilateral invasive carcinoma: genetic predisposition? EurJ Surg Onco12002; 28:] Hilgers FJM, Jansen HA, Van As CJ, Polak MF, Muller Ml. Van Dam FSAM. Long-term results of olfaction rehabilitation in laryngectomized individuals using the nasal airfiow-inducing 'polite yawning' maneuver. Arch Otolaryngol Head Neck Surg 2002, 128: Karim RB, Hage Jl. Ahmed AKl. De Wit FS, Van de Sandt MM, Daemen A. Digital photography as a means to enhance inter-consultant communication in oncologic cutaneous surgery. Ann Plast Surg 2002; 48: Kroon BBR, Noorda EM, Vrouenraets BC, Nieweg OE. Isolated limb peifusionfor melanoma. Review article. ] Surg Oncol 2002; 79: Nieweg OE, Tanis Pl. De Vries JDH, Kroon BBR. Sensitivity of sentinel node biopsy in melanoma. ] Surg Oncol2001; 78: O'Higgins N, Kroon BBR. Observations on surgical oncology and the Livre blanco Bull Cancer 2002; 89:S Rutgers EJTh, Nieweg OE. Finding lymph node metastases in invasive breast cancer: sampling or sentinel node procedure? EurJ Surg Oncol2002; 28:569. Sixty-nine patients died within one year af ter ILP, 64 due to metastatic melanoma. Patients with regionallymph node metastases combined with alocal recurrence (stage I1lb) or in-transit metastases (stage Illab) had a factor 4.6 (95% ci ) and 3.6 (95% ci ) increase in risk of dying within one year respectively (p<o.ooi). Five patients with distant metastases disease died (71.4%) within one year, increasing this risk 22 times (95% ei , P=O.OOI) for stage IV. The indication for ILP in patients with regionallymph node involvement or distant metastases should be carefully considered and if inevitabie, the use oftnfa is recommended since higher response rates are attained within a shorter time after ILP. ILP and recurrence free interval In 2-3% ofpatients with extremity melanoma, intransit metastases recur repeatedly without development of distant metastases. We studied whether ILP prolongs the limb recurrence-free interval (LRFI) after repeated excisions in such patients. Forty-three patients were selected from our computer database because they had their first ILP for a third or further limb recurrence. Eighteen patients had resectable lesions and 25 had irresectable lesions at ILP. All had a total of 269 intervals between treatrnent of their primary melanoma and last recurrence or last follow-up (in the case of a final complete remission af ter ILP). Median follow-up was 35 months. Overall, the median LRFI decrease over time from primary melanoma to the third or subsequent recurrence for which ILP was performed (P<O.OOI). The median LRFI is 4-7 (95% ci: , P<O.OOI) times longer af ter ILP compared to the last interval before ILP. For patients with resectable lesions the median LRFI is 5.9 (95% ei: , p<o.ooi) times longer after ILP. ILP lengthens the LRFI significantly compared to these intervals before ILP in patients with repeatedly recurrent limb melanoma and could be a valuable adjunct to exeision oflesions in these patients whose LRFI tends to shorten over time. I LP for irresectable soft tissue sarcoma of the extremities Since 1992, ILP with TNFa and Melphalan has been applied for irresectable soft tissue sarcomas of the extremities. From 1992 to 2000, 49 patients (29 men (58%) underwent 50 ILP'S for irresectable soft tissue sarcomas of the extremities in our institution. All patients received Melphalan and TNFa, four also had IFNy. Residual tumors were resected if rendered operabie and post-operative radiation therapy was applied for tumorinvolved resection margins. Mean follow-up was 72 months (range 9-II5 months). One complete response was attained (2%) and in 31 patients resection of the tumor remnant could be performed (63%). Final response was complete in four (8%) and partial in 27 patients (54%). A limb salvage rate of74% (36 patients) was attained. Seven of these patients had persistent local disease but were not amputated because of progressive distant metastases. Four patients (13%) who had been rendered tumorfree developed alocal recurrence. Acute limb toxieity af ter ILP was mild with 74% grade Ijll reactions, 24% grade 111 reactions (blistering) and one 2% grade IV reaction (compartrnental syndrome). There was little systemic toxicity. Seven patients (23%) had wound problems after resection of the tumor remnant; four patients had irreversible nerve damage. Limb salvage surgery in this patient group could be performed in the majority of patients with acceptable morbidity and a low local recurrence rate. H A AND CK 0 "OlOey Fons JM Balm, Michiel WM van den Brekel, Marcel P Copper, Frans JM Hilgers, Bing Tan, Peter J FM Lohuis The head and neck oncology group continues its efforts in research on rehabilitation af ter totallaryngectomy in close collaboration with Division XII and on organ sparing treatrnent in collaboration with Divisions IX and X. Translational research is focused on prognostic relevance ofhypoxic markers (see also Division VIII) and development of a conditional knock-out mouse model using the Cre-Iox system (see also Division VII). Rehabilitation In the ongoing postlaryngectomy rehabilitation program, the project started in 2000 to develop an improved handsfree device for postlaryngectomy

116 Selected publications (continued) Tanis PJ, Deurloo EE, Valdés Olmos RA, I Automatic speaking valve Rutgers EJTh, Nieweg OE, Kroon BBR. Single intralesional tracer dose for radioguided excision of clinically occult breast cancer and sentinel node. Ann Surg Onco12001; 8: Tanis PJ, Lont AP, Bex A, Meinhardt W, Valdés Olmos RA, Nieweg OE, Horenbias S. Dynamic sentinel node biopy for penile cancer: reliabilitty of a staging technique. J Urol 2002; 186: Figure XI.2: Provox FreeHands HME, consisting Of a disposable heat and moisture exchanger (HME) as its indispensable 'core' and areusabie, multi-magnet, automatic speaking valve, which has a front opening cough-relief valve on top and a side-ways closing membrane, to be placed in an adhesive attached to the peristomal skin. From top to bottom; the speaking valve, the HME, and the adhesive. Tanis PJ, Nieweg OE, Hart AAM, Kroon BBR. The iuusion ofthe learning phase for lymphatic mapping. Ann Surg Onco12002; 9: prosthetic voice restoration could be finalized. The objectives were to develop and test the prototypes of a novel postlaryngectomy rehabilitation tooi, incorporating an obligatory, disposable Heat-and-Moisture Exchanger (HME) and areusabie, multimagnet automatic speaking valve (ASV) (Figure XI.2). Twenty laryngectomized individuals (15 males and 5 females) participated in this prospective clinical study, 5 of whom already were using an ASV and 15 not. Three successive prototypes were tested. Data were collected with structured questionnaires (e.g. patient compliance, skin adhesion, voicing, coughing aspects), and voice and speech quality assessment (e.g. maximum phonation time, dynamic loudness range). The results were as follows: of the 15 non-users, 5 were non-compliant in this study due to peristomal skin adhesion problems. Of the remaining 15 patients, all 5 ASV users and 6 of the 10 non-users were fuuy compliant with the new device. The cough-relief valve of the new device functions properly, as does the valve position adjustment for physical exertion. With this new device the maximum phonation time was longer than with the regular ASV (15.2 versus 11.6 seconds; P=.006) and the dynamic range was larger (33-0 versus 24-8 db; P<.OOI). From this study it could be concluded that this new device with its advanced features (obligatory HME and multi-magnet valve systems) offers additional benefits for further improving vocal and pulmonary rehabilitation after totallaryngectomy. A multi-center prospective long-term clinical trial (in collaboration with the ENT departments of the Universities of Rotterdam, Nijmegen, and Maastricht) has been conducted in 2002, and results can be expected in Evaluation of organ sparing treatment strategies The effectiveness of 53 primary therapeutic selective neck dissections (SND) (levels II-V) in patients with laryngeal and hypopharyngeal squamous cell carcinoma has been investigated in a group of 48 patients. Of the primarily treated necks, 45/53 (85%) were irradiated postoperatively. Regional recurrences in level I occurred in 1.8% (1/53) and in level II-V 9-4% (5/53) The actuarial overall survival at 4 years was 36.5%. In selected cases therapeutic SND (level II-V) in node positive (NI,2) patients with laryngeal or hypopharyngeal carcinoma does not lead to increased risk for recurrence in level I or other levels of the neck and is therefore a safe procedure. Function preserving treatment ofti,2 N2,3 upper aero digestive tract carcinoma by neck dissection followed by radiotherapy ofboth primary tumor and neck was evaluated in 37 patients. All patients received radiotherapy to the primary tumor (66-7oGy) and adjuvant radiotherapy to the neck (46-66 Gy). With a minimal follow-up of 2 years, the 5-year overall survival as estimated by the Kaplan-Meier method was 47% ( % confidence interval). Local failure occurred in 4 patients (10.8%). Regional failure was detected in 5 cases (13.5%), of which 2 (5 4%) concemed outgrowth of occult metastases in the contralateral, non-irradiated and non-dissected neck. In this selective group of patients, neck dissection followed by radical radiotherapy to the primary tumor and neck represents a valid treatment Tanis PJ, Nieweg OE, Valdés Olmos RA, Peterse J L, Rutgers EJTh, Hoefnagel CA, Kroon BBR. Impact ofnon-axiuary sentinel node biopsy on staging and treatment of breast cancer patients. Br J Cancer 2002; 8T Tanis PJ, Valdés O lmos RA, Hoefnagel CA, Meinhardt W, Nieweg OE, Horenbias S. Feasibility ofsentinel node lymphoscintigraphy in stage I testicular cancer. Eur J Nucl Med 2002; 29: Tanis PJ, Van Sandick JW, Nieweg OE, Valdés Olmos RA, Rutgers EJTh, Kroon BBR. The hidden sentinel node in breast cancer. Eur J Nucl Med 2002; 29: Van Ruth S, Bronkhorst MW, Van Coevorden F, Zoetmulder FA. Peritoneal benign cystic mesothelioma: a case report and review of the literature. Eur J Surg Oncol2002; 28: Van Ruth S, Van Coevorden F, Peterse JL, Kroon BBR. Alveolar soft part sarcoma. A report of 15 cases. Eur J Cancer 2002; 38: Van Sandick JW, Van Coevorden F. Plexiform neurofibroma with intraspinal extension. J Am Coll Surg 2002; 195:572. Vos SCB, Hage Jj, Woerdeman LAE, Noordanus RP. Acute renal failure during dextran-40 antithrombotic prophylaxis: Report of two microsurgical cases. Ann Plast Surg 2002; 48:

117 Selected publications (continued) Zuur CL, Van Velthuysen MLF, Schornagel JH, Hilgers FJM, Balm AJM. Diagnasis and treatment of isolated neck metastases of adenocarcinomas. EurJ Surg Oncol2002; 28: In press Hilgers FJM, Ackerstaff AH, van As CJ, Balm AJM, Van den Brekel MWM, Tan IB. Development and clinical assessment of a heat and moisture exchanger with a multi-magnet automatic tracheostoma valve (Provox FreeHands HME) for vocal and pulmonary rehabilitation after total laryngectomy. Acta Otolaryngol (in press). Jol JAD, Van Velthuysen MLF, Hilgers FJM, Keus RB, Neering H, Balm AJM. Treatment results ofregïonal metastasis from cutaneous head and neck squamous ceu carcinoma. Eur J Surg Oncol (in press). Noorda EM, Vrouenraets BC, Nieweg OE, Van Geel AN, Eggermont AMM, Kroon BBR. Safety and efficacy ofisolated limb perfusion in elderly melanoma patients. Ann Surg Oncol (in press). option, providing maximal control of the neck and minimal disturbance at the primary site. To evaluate the long-term outcome of Foscan mediated Photodynamic therapy, 25 patients with 29 TI-2 No tumors of the aral cavity and/or oropharynx were retrospectively analyzed. The mean follow-up of the patients af ter treatrnent was 37 months (20-69 months). In 25 of 29 tumors (86%) a complete remis sion of the primary tumor was obtained. The four residual tumors could all be salvaged by conventional therapy. In none of the patients was any long-term functional deficit detected. Regional recurrences occurred in 5 patients. One patient died because of a second primary tumor. This study confirms that photodynamic therapy is a powerful treatrnent modality, which could be considered as an alternative to surgery or radiotherapy in specific cases ofhead and neck cancer with full preservation of functions. Research on prognostic markers Tissue array blocks we re made from 184 he ad and neck ca neer specimens. Three co re tissue biopsies (0.6mm x 3-4mm) were taken from individual 'donor' paraffin-embedded tumor blocks and arrayed into a new 'recipient' paraffin block. Seventy-four percent (475/64 ) of samples placed into tissue arrays we re confirmed to represent tumor tissue. The remaining samples we re lost during processing or contained too few tumor cells _ Only 6% of cases were completely lost while 55%, 28% and n% of cases we re judged on 3, 2 or I disks respectively. The Cohens Kappa coefficient was 0.66 for Cyclin-DI (Figure XI-3), 0-40 for EGFR and -41 for Rb. Tissue array technology is a rapid and efficient method for retrospective analysis of protein expres sion in large series ofhead and neck squamous cell carcinoma. The agreement in scoring of the full section and the tissue arrays is reasonable. Part of the discrepancies might be caused by heterogeneity within tumors as weil as intra-individual variation and la ck of robustness of the scoring. 5 4 >- I!.( ~3 VI VI 1= Ci Ë 2 ü?i o o Cycllne Di Full SectIon Figure XI.]: Diagram showing the number of cases (sizes of the dots) assigned an intensity score for cyclin Dl for the whole section (horizontal bar) versus the tissue array (vertical bar). Note that for cyclin Dl most ofthe dots are on, or close to the square line (Cohen-Kappa: 0,66). T J Joris Hage, leonie AE Woerdeman TRUCTIVE SURGERY Since April 2000, the clinical activities of the Department of Plastic and Reconstructive Surgery are restricted to reconstructive surgery following oncological resections. Historically, the main topics of interest of the department are reconstruction of the breast and the head and neck region, but the plastic surgeons are nowadays increasingly involved in surgery of the trunk, limbs, and genitalia.

118 Immediate breast reconstruction Breast conserving therapy (BeT) is widely accepted as an appropriate method of primary treatment of TI and T2 breast cancers that measure up to 5 cm. Although large tumor size alone is not considered a contraindication for BeT in terms oflocal tumor control, size plays the main role in determining the feasibility of BeT in terms of cosmetic results. The volume reduction associated with oncologically safe margins is too large in some cases oft2, and in almost all cases oft3 breast cancers. For safe and cosmetically acceptable BeT in these cases, the tumor volume must be reduced preoperatively and lost tissue volume ought to be replaced af ter the resection (Figure XI.4A). Such immediate autologous volume replacement is controversial. To evaluate the long-term oncologic safety and cosmetic outcome of a combination of preoperative radiotherapy and immediate tissue replacement by myo-(sub)cutaneous latissimus dors i flap transplantation, we studied the results obtained in 20 patients with a minimum follow-up of 5 years (Figure XI.4A and B). Local recurrence was observed in the one patient who refused adjuvant therapy and both the observed 5-years overall survival and the expected ro-years overall survival in our series equaled that of more radical surgical therapy. The cosmetic outcome was similar to that obtained by conventional BeT modalities for TI and relatively small T2 breast cancers. However, combining radiotherapy, ablative surgery, and reconstructive surgery in the proposed tight time schedule proved difficult. We conclude that the proposed therapy is an oncologically safe and cosmetically rewarding, but logistically straining modality of treatment of relatively large T2 and T3 breast cancers. Oelayed breast reconstruction In 1986, the combined use of the lateral thoracodorsal (LTD) flap and an implant was introduced as an alternative method of delayed reconstruction of small- to medium-size breasts for postmastectomy patients. From our experience with the fitst 60 LTD flaps we conclude that this technique is a weil reproducible technique for breast reconstruction with few complications leading to failure. The LTD flap allows for reconstruction ofbreasts oflarger than medium size by using it in combination with tissue expanders. Moreover, we successfully applied fully de-epithelialized LTD flaps for additional indications. Statistical significance of post-mastectomy radiotherapy as a risk-factor could not be confirmed (P=0.I5) but we proved age (P=0.04), body mass index (P=0.03) and smoking (P=0.04) to be significant patient-related risk factors. Of five surgery-related characteristics, only increased flap length (P=O.OI) was proven to negatively influence the outcome of surgery. Figure XI+ Preoperative (A) and postoperative ( B) view of a young patient who underwent preoperative radiotherapy to decrease the load of a breast tumor in her right breast and immediate volume replacement by transplantation of the latissimus dorsi myocutaneous flap after oncologie resection. Tissue engineering in uro-genital reconstructions To study the differences between a muscle flap and a musculoperitoneal flap used for urinary bladder wall substitute, a prospective study was performed in 24 Wistar albino rats. In twelve rats a rectus abdominis muscle flap including the overlying peritoneum (RAMP flap) was sutured in a surgical defect in the bladder wall, whereas the flap was interpositioned without peritoneum (RAM flap) in twelve other rats. The animals were sacrificed on postoperative day 6 (6 RAMP flaps and 6 RAM flaps), and day 90 (5 RAMP flaps and 5 RAM flaps) to obtain early and late histologic results. The two remaining rats were lost before 90 days. Partial resurfacing of the flaps' muscle surf ace with uroepitheleum was observed on the 6th postoperative day in both the RAMP, and the RAM group. No significant histologic difference of this uroepithelium was observed between the RAMP and the RAM flaps at early examination. Af ter 90 days, resurfacing with uroepithelium was complete in both groups. Histologically, the earlier presence of peritoneum in the RAMP flaps seemed to have had a positive effect on the quantity and quality of transitional uroepithelium. HYPERTHERMIC INTRAPERITONEAL CHEMOTHERAPY (HIPEC) Frans AN Zoetmulder, Vic J Verwaai, Van Ruth S, Witkamp AJ The life expectancy of patients with peritoneal carcinomatosis of colorectal cancer is short when treated with palliative surgery and systemic chemotherapy. Aggressive cytoreduction in combination with hyperthermic intraperitoneal chemotherapy has shown promising results in several non-randomized studies, with a 5-year survival of

119 over 20%. In a phase 111 study, 105 patients were randomized to undergo either standard treatment consisting of systemic chemotherapy (5-FU jleucovorin) and palliative surgery or experimental therapy consisting of cytoreduction with Hyperthermic Intra Peritoneal Chemotherapy (HIPEC), followed by the same systemic chemotherapy. Af ter a median follow-up period of 21.6 months, the median survival was 12.6 months in the standard therapy arm and 22.3 months in the experimental therapy arm (logrank test: P=0.032) (Figure XI.S). l.0 overall survival by treatment _, '": ol, t. -.. L _-;, p= 0.032, 10grank: test, two-sided tol_ol L----_ l t-- _ HIPEC ~ ~ ~ ~ months from randomization 5 11 con trol 4 HIPEC Figure XI.S: Statistical significant difference (P=o.oJ2 ) in overall survival between patients who were randomized to undergo either standard treatment consisting of systemic chemotherapy (s -F U / Leucovorin) (continuous line) and palliative surgery or experimental therapy consisting of cytoreduction with Hyperthermie Intra Peritoneal Chemotherapy (HIPEC) (interrupted line). A subanalysis of the HIPEC group showed that patients with 0 to 5 of the 7 regions of the abdominal cavity affected at the time of the HIPEC procedure had a significantly better survival than patients with 6 or 7 affected regions (logrank test: P<O.OOOl). If the cytoreduction was macroscopically complete (R-l), the median survival was also significantly better than in patients with limited (R-2a), or extensive residual disease (R-2b) (logrank: P<O.OOOl). Patients in the experimental arm experienced a significant drop in quality oflife 6 weeks to 3 months af ter HIPEC Their quality oflife normalized 6 months after HIPEC Cytoreduction followed by intraperitoneal chemotherapy improves survival in patients with peritoneal carcinomatosis of colorectal origin. However, in patients with involvement of six or more regions of the abdominal cavity or grossly incomplete cytoreduction, the benefit is limited. It is probably better to exclude these patients from HIPEC therapy. The quality oflife is impaired by the treatment during the first six months after HIPEC HIPEC therapy costs 24,576.- in direct medical expenses. The total cost per patient over their complete live span was 26,620.- higher in the HIPEC arm, compared to the standard therapy arm. The ratio is 17,286.- per life year gained.

120 XII DIVISION OF PSYCHOSOCIAL RESEARCH AND EPIDEMILOGY PSYCHOSOCIAL ONCOLOCY The subdivision of psychosocial oncology is pursuing three major research lines: (I) quality oflife assessment in clinical research and clinical practice; (2) symptom perception and mangement; and (3) psychosocial issues in cancer clinical genetics. The role of health-related quality of life (H RWL) considerations in palliative chemotherapy treatment decisions This observational study investigated the frequency with which HRQL considerations lead to modification or discontinuation of pal1iative chemotherapy. Four consecutive medical consultations of 203 patients were audiotaped and content analyzed to determine the frequency of and reasons for treatment alterations. Physicians rated their patients' HRQL using the COOP /WONCA Charts. Treatment was modified in 54 (26%) and discontinued in 40 (20%) cases. The primary reasons for modifying treatment were toxicity (n=22), HRQL considerations (n=18) and tumor progression (n=14). The primary reasons for discontinuation of treatment were tumor progression (n=23), HRQL considerations (n=6) and toxicity (n=3). For eight patients, a combination of tumor progression and HRQL issues resulted in discontinuation of treatment. Treatment decisions were associated significantly with physicians' global ratings of patients' HRQL, but not with more specific HRQL domains. In the presence of tumor progression or serious toxicity, HRQL considerations played little or no role in treatment decisions. Approximately 70% of patients without evidence of tumor progression or toxicity, but with seriously impaired HRQL, continued to receive their treatment as planned. Contrary to previous findings based on physicians' self-report data, HRQL considerations appear to play a relatively minor role in decisions regarding modification or discontinuation of palliative chemotherapy. The impact of epilepsy on the cognitive functioning and H RQL of low-grade glioma patients This cross-sectional study investigated the influence of epilepsy and antiepileptic drug (AED) treatment on the cognitive functioning and HRQL of patients with low-grade glioma. Included were 156 patients who, at the time of assessment, had had no clinical or radiological signs of tumor recurrence for at least I year fol1owing diagnosis. Patients' cognitive test performance and self-reported HRQL were compared with age-, sex- and education-matched healthy controls. Eighty- six percent of the patients had epilepsy, and only 50% of those using AEDs were seizure-free. The patients had significantly poorer cognitive functioning and HRQL in the majority of domains assessed than the controls. Epilepsy burden was strongly associated with decrease in working memory capacity and executive functioning, and poorer self-reported physical and mental health. Division Head, Group leader Neil Aaronson Neil Aaronson PhD Group leader Joanna Madalinska MA, MSc Academic staff Fred Menko MD, PhD Academic staff Jan Schornagel MD, PhD Academic staff Babs Taal MD, PhD Academic staff Heiddis Valdimarsdottir PhD Academic staff Marc Van Beurden MD, PhD Academic staff Sen no VerhoefMD Academic staff Eveline Bleiker PhD Post-doc Kommer Sneeuw PhD Post-doc Doranne Hilarius MSc Graduate student Rianne Hoopman MSc Graduate student Ruud Knols MSc Graduate student Martin Muller MSc Statistica I analyst Fatima Bouali Research assistant Sahsenem Celik Research assistant Fadoua EI Haddouchi Research assistant Miranda Gerritsma Research assistant Masjda Idrissi Research assistant Esther Janssen Research assistant Amina Rourou Research assistant Ed Caffin Undergraduate student Marian Chin-A-Kwie Secretary Quality of life af ter curative radiotherapy in stage I non-smal! cel! lung (NCSLC) cancer This longitudinal study investigated changes in pulmonary function and HRQL among 46 medically inoperable, stage 1 NSCLC patients treated with curative radiotherapy. Twenty-seven patients were treated only at the primary site; 19 received irradiation of the regionallymph nodes. A gradual but significant increase over time was observed in dyspnea, fatigue, and appetite loss, together with deterioration in role functioning. Significantly higher levels of dysphagia, which persisted for up to 12 months, we re observed in those patients who received irradiation of the lymph nodes. Radiation-induced pulmonary changes, assessed with chest radiography, were more pronounced in the group treated with loco regional radiotherapy. Considering the low incidence of regional recurrence after local irradiation, the higher incidence and severity of radiation-induced changes, and the higher levels of dysphagia, local irradiation of the primary tumor without elective irradiation of the regionallymph nodes may be the most appropriate treatment for patients with small, peripherally located tumors.

121 Selected publications Aaronson NK. Cross-cultural use ofhealthrelated quality oflife assessments in clinical oncology. In: Lipscomb j, Gotay Cc, Snyder CF (editors), Outcomes Assessment in Cancer. Cambridge: Cambridge University Press 2002 (in press). Aaronson NK, Fayers P. Quality of lift In RL Souhami RL, Tannock I, Hohenberger P, Horiot j-c (editors). Oxford Textbook of Oncology, 2nd Edition. Oxford: Oxford University Press 2002; Detmar SB, Muller MJ, Wever LDV, Schornagel JH, Aaronson NK. The role of health-related quality oflife in palliative chemotherapy treatment decisions. j Clin Onco12002; 20: Detmar SB, Muller MJ, Schornagel JH, Wever LDV, Aaronson NK. Health-Related Quality-ofLife Assessments and Patient Physician Communication. A Randomized Controlled Trial. JAMA 2002; 288(23):] Hagedoorn M, Sneeuw KCA, Aaronson NK. Changes in physical functioning and quality oflife in patients with cancer: Response shift and relative evaluation of one's condition. j Clin Epidemiol2002; 5P Klein M, Heimans Jl, Aaronson NK, Van der Ploeg HM, Grit J, Muller M, Mooij JJ, Boerman RH, Beute GN, Ossenkopele GH, Van ImhoffGW, Dekker AW, Jolles J, Slotman BJ, Struikmans H, Taphoorn MJB. Mid- to long-term cognitive sequelae in low-grade gliomas: The impact of radiotherapy and other treatment-related factors. Lancet 2002; 360: Scientifk Advisory Committee of the Medical Outcomes Trust (Aaronson NK, Alonso J, Burnam A, Lohr KN, Patrick D, Perrin E, Stein R). Assessing health status and quality-oflift instruments: Attributes and review criteria. Qual Life Res 2002; 11: Sneeuw KC, Sprangers MA, Aaronson N K. The role ofhealth care providers and significant others in evaluating the quality oflift of patients with chronic disease. j Clin Epidemiol 2002; 55(11): H RQL assessment among ethnic minority cancer patients In 2002, data collection continued for a study whose primary aims are: (I) to translate, adapt and validate two widely used generic QL questionnaires (the SF-36 and the COOP jwonca charts) and two cancer-specific questionnaires (the EORTC QLQ-C30 and the Rotterdam Symptom Checklist) for use among Turkish and Moroccan cancer patients in the Netherlands; and (2) to investigate the accuracy of proxy (family member) ratings of these patients HRQL. To date,los Turkish and 85 Moroccan patients and 40 Turkish and 25 Moroccan proxies have been recruited into the study. Data collection and analysis are on-going. The psychosocial and behavioral impact of genetic counseling for colorectal cancer (erc): a prospective, multicenter study This multicenter (AvL, AZVU, AMC, RUG, and LUMC) prospective study is assessing: I) the effect of genetic counselingjtesting on risk perception, distress, family relationships, work, and family and financial planning; 2) risk factors for poor psychological adjustrnent to and early withdrawal from the genetic counseling process; and 3) rates of short-term compliance with recommended screening practices. Preliminary analyses indicate that the most important reasons for undergoing genetic counselingjtesting are to determine personal cancer risk (64%), to determine the cancer risk of off-spring (59%), and to take preventive actions (58%). Less than 10% of counselees exhibit heightened levels of distress immediately following intake. Collection of follow-up data and data analyses are on-going. The physical and psychosocial impact of gynecological screening or prophylactic oophorectomy among women from hereditary breast/ovarian (HBOC) cancer families This multicenter, cross-sectional and longitudinal study (AvL, AZVU, AMC, LUMC, AZN, AZG, AZM, UMCU) is investigating: I) the decision-making process surrounding the choice of preventive health options among women at increased risk of developing ovarian cancer; 2) the impact of screening versus prophylactic surgery on psychosocial well-being; 3) compliance with screening advice; and 4) the prevalence and severity of menopausal symptoms among women who opt for surgery, and the use and perceived benefit ofhormone-replacement therapy. To date, 337 women have been recruited into the cross-sectional component of the study. Preliminary results indicate that those women who underwent preventive oophorectomy (n=123) have significantly lower levels of cancer worries and perceive their cancer risk as lower than those women who opted for screening (n=214). Levels of generalized distress are low in both groups. Recruitrnent for the longitudinal component of the study began in June, To date, 72 women have been recruited into the prospective study. 'No shows' in a family cancer clinic Self-report questionnaires were sent to 73 women who had requested genetic counseling for breastjovarian cancer in the period January 1999 to March 2000, but who ultimately chose not to attend andjor complete the counseling process. Completed questionnaires were received from 48 women (66%). The best predictors of non-attendance were: I) having no or only few worries ab out cancer; and 2) being the first and only member in the family requesting genetic counseling. Twenty percent of the women indicated that their decision to discontinue counseling was related to concerns about not being able to cope with an unfavorable test re sult. None reported complaints about the services provided as a reason for discontinuing counseling. The assessment of the risk/benefit ratio of phase 11 cancer clinical trials by institutional review board (I RB) members Semi-structured interviews were conducted with 53 members of IRBs from six research hospitals and specialized cancer centers in the Netherlands. While the toxicity and side-effects of treatrnent were most often identified as risks associated with participating in a phase 11 trial, approximately two-thirds of IRB members also cited psychosocial andjor quality-oflife risks. Conversely, 68% of the respondents identified psychosocial benefits of trial participation, while 25% cited treatrnent effectiveness as a possible benefit. Between one-quarter and two-thirds of respondents indicated that trial protocols provide insufficient information about the likelihood, magnitude and duration ofboth risks

122 -r ~.~p~.~.r~.i:~~~~~~~ :~~1~~~~:~ ~ and benefits. Between I5% and 34% of IRB members reported feeling less than fully competent to evaluate various aspects of phase 11 protocols (e.g., originality and feasibility of the study, adequacy of the methods and analysis procedures, etc.). This was particularly the case for non-physician IRB members. Few IRB members reported weighing risks and benefits in a systematic manner, but rather relied on global impressions or preferred to leave such matters to the IRB as a whole or to their patients.

123 SYMPTOM PERCEPTION AND MANAGEMENT IN CANCER PATIENTS The principal research line of the symptom perception and management group is the investigation of the impact of cancer chemotherapy on cognitive functioning of ca neer patients. Group leader Frits van Dam Frits Van Dam PhD Group leader Willem Boogerd MD, PhD Academic staff Frans Hilgers MD, PhD Academic staff Sjoerd Rodenhuis MD, PhD Academic staff Corinne Van As PhD Academic staff Aafke Donker Registered nurse Hilda Hauer Registered nurse Suzy Oppeneer Registered nurse Karin Van Rooijen Registered nurse Sanne Schagen PhD Post-doc Baudewijntje Kreukels MSc Graduate student Martin Muller MSc Statistica I analyst Angelique Biervliet Research assistant Hesje Andersson Undergraduate student Kirsten Douma Undergraduate student Heleen Faasen Undergraduate student Marthe Ford Undergraduate student Janna Fortuin Undergraduate student Sanne Koemans Undergraduate student Tjerk Maas Undergraduate student Nina Rozenschoon Undergraduate student Sietske Sikkes Undergraduate student Elena Van der Puij Undergraduate student Judith Versloot Undergraduate student Miriam Zaagsma Undergraduate student Neuropsychological and neurophysiological evaluation of cognitive deficits af ter chemotherapy The impact of cytostatic drugs on the patients cognitive functioning is evaluated with neuropsychological and neurophysiological techniques. We are investigating cognitive functioning fol1owing chemotherapy in (high-risk) breast cancer patients, lymphoma patients, patients with testicular cancer and patients who have been treated during childhood for osteosarcoma and Ewing sarcoma. In all of these studies the same standard battery of neuropsychological tests is used, making possible a comparison between tumor types and chemotherapy regimens. In examining cognitive functioning of cancer patients, the persistency of deficits potential1y compromised by organic impairment attributable to the cytotoxic treatrnent, is of great relevance. A study was designed to obtain insight into the long-term sequelae of the neuropsychological deficits and their course in time. The cognitive functioning of all patients who participated in our first

124 - ~------~~~~. ~~.~I::~~~~~I~.4~1~~~~:1~ ~ neuropsychological studies and who were still free of disease, was tested for a second time with an additional two years test-retest interval, i.e. 4 years after completion of chemotherapy. 22 of the original34 CTC patients, 23 of the 36 FEC patients, 31 of the 39 CMF patients and 27 of the 34 control patients were reexamined with a neuropsychological battery of tests, identical to the previous investigations. For all chemotherapy groups improvement in test performance was observed, while in the control group a slight deterioration of test results was seen. None of the previously observed differences in cognitive functioning between patients treated with high-dose chemotherapy, patients treated with various regimes of conventional dose chemotherapy and patients who received no systemic therapy for breast cancer, could be detected. Also, no significant differences were seen in self-reported complaints on cognitive functioning for the patients treated with CTC or FEC chemotherapy in comparison to the con trol patients, while the patients treated with CMF chemotherapy expressed significantly more cognitive problems in daily life than the patients in the control group. Akin to the findings of the fust examinations, no clear relation was found between test performance on the one hand and anxiety, depression, fatigue and self-reported complaints of cognitive functioning on the other hand. A differential attrition was observed among the groups, with a relatively high percentage of initially cognitively impaired patients from the CTC group dropping out due to factors related to disease progression. Based on the results of our prior neurophysiological work, a new, four-year study has been initiated to gain a better understanding of specific stages of information processing using event-related potential measurements in addition to qeeg in various groups of patients. In a pilot study in healthy controls we tested the feasibility and validity of a task based on the Additive Factors Method (AFM) while at the same time registering the electrocortical activity. With the AFM task we will be able to obtain information on which specific stages of information processes in the brain have been affected by chemotherapy. To assess the feasibility of this AFM task, a pilot study has been performed in healthy controls (n=16). In order to check if the experimental manipulations in the task succeeded, the individual ma in effects and interaction effects between the three factors have been examined on behavioral as weil as psychophysiological measures. The analysis of the reaction times (RT) showed main effects for the factors stimulus-discriminability and responsecomplexity. No effect of the central factor, stimulus-response compatibility on the RTs was found, so the manipulation of this factor on the behavioral measures failed. The ERP data are presently being analyzed. Selected publications De Wit R, Van Dam F. From hospital to home care: a randomized controlled trial of a Pain Education Programme for cancer patients with chronic pain. J Adv Nurs 2001; Hilgers Fj, jansen HA, Van As CJ, Polak MF, Muller Mj, van Dam FSAM. Long-term results of ejjèctive rehabilitation in laryngectomized individuals using the nasal airjlow-inducing 'poute yawning' maneuver. Archives of Otolaryngology Head ei( Neck Surgery 128, Schagen SB, Muller Mj, Boogerd W, Rosenbrand RM, Van Rhijn D, Rodenhuis S et al. Late effects of adjuvant chemotherapy on cognitive function: a follow- up study in breast cancer patients. Ann Oncol2002; 1]: Schagen SB, Muller MJ, Boogerd W, Van Dam FS. Cognitive dysfunction and chemotherapy: neuropsychological findings in perspective. Clin Breast Cancer 2002; 3 SUppl]:SlOO-8. The need for care by palliative patients being treated at the outpatient department The received care and the unmet care needs of patients undergoing palliative treatrnent at the outpatient clinic of a cancer hospital were investigated. There were 155 patients (87%) who agreed to participate. The results show that patients have, on average, three severe complaints, with fatigue and pain being the most prevalent. Of all patients with 'severe' complaints, 44% has one or more complaints of which (according to the patient) no caretaker is aware. According to the patients, ab out 64% of all severe problems, were known to the medical specialist, 39 to the general practitioner and 27% to the oncology nurse. All patients have contact with three care professionals and 25% have special cure/care facilities at home. The women in this study had, on average, more unmet care needs than the men. A quarter of the patients reported that they were not weil informed about their disease and treatrnent.

125 EPIDEMIOLOGY The cancer epidemiology group is currently concentrating on two principal research lines: (I) the etiology ofhormone-related cancers; (2) the long-term health consequences of cancer treatment, particularly in terms of the risk of developing a second cancer. Group leader Floor Van Leeuwen Group leader Matti Rookus Floor Van Leeuwen PhD Group leader Matti Rookus PhD Group leader Berthe Aleman MD Academic staff Petra Nederlof Academic staff Nicola RusselI PhD Academic staff Emiel Rutgers MD, PhD Academic staff Laura Van 't Veer PhD Academic staff Senno VerhoefPhD Academic staff Lisette Hoogendoorn PhD Post-doc Helen Klip PhD Post-doc Richard Brohet MSc Graduate student Evelien De Boer MSc Graduate student Maart je Hooning MD Graduate student Agnes Van Rosmalen MSc Graduate student janneke Verloop MSc Graduate student Willem Klokman MD, MSc Statistical analyst Thea Mooij MSc Statistical analyst Vivianne Hartog Research assistant Nandy Hofland Research assistant Esther janssen Research assistant Marianne Kuenen Research assistant Monica Legdeur Research assistant Bart Maertzdorf Research assistant Manita Van Baaien Research assistant Sandra Van den Belt-Dusebout Research assistant Annewil Van der Meij Research assistant Gabey Owens Research assistant judith Versloot Research assistant Mireille Wolfers Research assistant Ralph Bergsma Undergraduate student Co ra Busstra Undergraduate student Merel Linthorst Undergraduate student Maaike Van Veen Undergraduate student Risk factors for hormone-related cancer Concerns have been raised that ovarian stimulation for in vitro fertilization (IVF) may lead to an increased risk of hormonerelated cancers. Therefore, we are examining cancer risk in a nationwide historical cohort of 25,I52 women treated for subfertility in the Netherlands between I980 and The first linkage with the Netherlands Cancer Registry, af ter 5.7 years of followup, revealed no increased risks ofhormone-related cancers associated with IVF. Recently, we also reported that there was no increased risk of childhood cancer in the 9484 children conceived by IVF in this cohort. Sixteen children with cancer were observed till I997, against 16 expected. However, in the nearly complete Dutch registry of retinoblastoma we recently ( ) observed five retinoblastoma cases born after IVF_ Two patients had the sporadic bilateral form of retinoblastoma and three patients the sporadic unilateral ferm. We estimated the relative risk assuming that 1% of all recently bom children in the N etherlands is conceived by IVF. This resulted in a significant 7.2-fold increased risk compared to the population. Further research is needed to confirm the association and to explore a possible causal mechanism. In the same cohort we are examining determinants of age at menopause, such as cause of subfertility, number of retrieved oocytes at IVF and IVF treatment itself. We already reported that women with a low number of retrieved oocytes at first IVF treatment more often have an earlier menopause. Women with a poor response (0-3 oocytes) at their first IVF attempt had a 3-fold increased risk of entering the menopausal transition in the first six years following IVF treatment as compared with a normal response of more than three oocytes. In 2002 we continued our cohort study on hormone-related cancer risk in women who were exposed to diethylstilbestrol (DES) in utero. In total, we have received 7,779 risk factor questionnaires from DES daughters registered at the Netherlands DES Center. In addition, we identified 574 DES daughters from historical archives in seven hospitals and also started to identify controls not exposed to DES in utero from the same archives. Following case reports, we reported an increased risk ofhypospadias among DES grandsons in the IVF cohort. Therefore, we have investigated in this new cohort of DES daughters whether the association between hypospadias and in utero DES exposure of the mother could be confirrned. In a recent screening program among Rotterdam newborns new data became available on the prevalenee ofhypospadias in the general population. As compared to this prevalenee estimate, we found a sevenfold increased risk of severe hypospadias among DES grandsons, confirming our earlier findings. Mutation specific breast cancer risk: delex p = delex22 other BRCA1 mutations.2 o. -.2 Io;--..,--..,--..,----r----r----r----r- 10 o age

126 On the basis of one of our population-based case-control studies we collaborated in a meta-analysis on breastfeeding and risk ofbreast cancer. The relative risk of breast cancer decreased by 4.3% for every 12 months ofbreastfeeding in addition to a decrease of7.0% for each birth. It was estimated that the cumulative incidence ofbreast cancer in developed countries would be reduced by more than half, from 6.3 to 2.7 per 100 women by age 70, if women had the average number ofbirths (6 5 instead of 2.5) and duration ofbreastfeeding (24 months per child instead of 3.5 months per child) that had been prevalent in developing countries until recently. We have continued the accrual of participants for our national cohort study in breast andjor ovarian cancer families (GEO-HEBON study), aimed I) to examine whether hormonaljlife-style factors modify cancer risk in BRCAlj2 and non-brcai/2 families, 2) to assess the age-specific cumulative risks ofbreast, ovarian and other cancers based on full pedigree inforrnation. So far, for 664 BRCAlj2 and 725 non BRCAlj2 families, clinical, pedigree and DNA information has been collected comprising 78, 38 family members. We started risk assessment analyses and the figure shows one of the mutation-specific curves. 593 families have been approached to complete a mailed questionnaire on horrnonaljlife-style factors. Up to now, 4,257 questionnaires have been received with an overall response rate of75%. We have also contributed data to an international cohort study among 971 BRCAlj2 mutation carriers, coordinated by IARC, Lyon. Preliminary analyses of this cohort study show no clear association between oral contraceptive use and risk of breast cancer. Related to the GEO-HEBON study we wrote a methodological paper on the analyses of efficacy studies evaluating prophylactic surgery among BRCAlj2 carriers. We are also closely involved in an intervention study focusing on the insulin-like growth factor system in relation to breast cancer and colorectal carcinogenesis (see Division of Experimental Therapy j Division of Diagnostic Oncology). We continued data collection for a case-control study ofyoung sister-pairs (n=i30, non-brcalj2) with pre-menopausal breast cancer and no affected first- or seconddegree relative. Eligible control sister-pairs consist of a sister-in-law with her sister. The purpose of this study is to examine prenatal and early life-style factors, as weil as environmental factors, that rnight play a role in the etiology ofbreast cancer. Late effects of ca neer treatment Now that curative treatment is available for a substantial group of cancer patients, it is increasingly important to evaluate how the occurrence oflate complications of treatrnent affects their long-term survival. Our first aim is to evaluate the risk of second cancers in 5-year survivors of Hodgkin's disease (HD) (n=2,800), testicular cancer (n=2,250) and breast cancer (n=8,000) over a period of up to 30 years after primary treatrnent. The second aim is to evaluate the risk of vascular disease. In 2002, we completed collection of treatrnent and follow-up data on 8,000 survivors ofbreast cancer treated between 1970 and 1985 in The Netherlands Cancer Institute or the Dr. Daniel den Hoed Cancer CenterjEUR. In a first analysis, we studied second cancer risk in a group of 3,750 patients who were treated for early stage breast cancer between 1970 and For 91% of the patients medical status was complete up to at least J anuary Median follow-up time was 12 years; for 25% of the patients follow-up time was longer than 20 years. The diagnosis of contralateral breast cancer (CLBC) was only accepted when no distant metastases were manifest up to six months af ter CLBC diagnosis (n=243). In al1, 535 patients developed a second cancer as compared to 270 cases expected on the basis of cancer incidence rates in the general population (standardized incidence ratio (SIR): 2.0; 95% Cl: ). Significantly increased SIRs were observed for CLBC (SIR=2.9), lung cancer (n=42; SIR=3.8), oesophageal carcinoma (n=9; SIR=5.2), melanoma (n=n; SIR=2.2) and ovary cancer (n=32; SIR=2.2). The risk ofleukernia was raised (n=8; SIR=I.7) but not significantly so. After 20 years of follow-up, the SIR oflung cancer was 9-fo1d increased, which meant a 4-fold excess as compared to patients with a shorter follow-up, suggestive of an effect of radiation. Indeed, the SIR oflung cancer was significantly greater for irradiated patients than for patients treated with mastectomyalone (4-6 vs 2.0). Among patients treated for breast cancer before age 45, the risk oflung cancer was much higher (SIR=5.3) than in patients of 55 years and older (SIR=2.8). Patients treated with RT had a sirnilar risk of CLBC as patients Selected publications Collaborative Group on Hormonal Factors in Breast Cancer. Breast cancer and breasifeeding: collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries, including women with breast cancer and women without the disease. Lancet 2002; 360: Klaren H M, Van 't Veer Lj, Van Leeuwen FE, Rookus MA. Potential for bias when evaluating reduction of cancer risk ajter prophylactic surgery in BRCAIand BRCA2-carriers. jnci (in press). Klip WHA, Burger CW, De Kraker j, Van Leeuwen FE. Risk of cancer in the olfspring of women who underwent ovarian stimulation for IVF. Human Reproduction 2001; 16: Klip H, Verloop j, Van Gooi JO, Koster META, Burger CW, Van Leeuwen FE. Hypospadias in sons of women exposed to Diethylstilboestrol in utero: a cohort study. Lancet 2002; 359:11021 Klip H. Long-term health elfects of subfertility treatment. Thesis 2002, Amsterdam. Moll AC, ImhofSM, Cruysberg JRM, Schouten-van Meeteren AYN, Boers M, Van Leeuwen FE. Higher incidence of retinoblastoma in children bom ajter in vitro fertilization. Lancet (in press). Ronckers CM, Van Leeuwen FE, Hayes RB, Verduijn PG, Stovall M, Land CE. Cancer incidence ajter nasopharyngeal radium irradiation. Epidemiology 2002; 13: Travis LB, Gospodarowicz M, Curtis RE, Cl arke EA, Andersson M, Glimelius B, Joensuu T, Lynch CF. Van Leeuwen FE et al. Lung cancer following chemotherapy and radiotherapy for Hodgkin's disease. jnci 2002; 94:

127 treated with mastectomy only (SIR=3.3 resp. SIR=2.6). However, the SIR of CLBC in patients treated with RT only as compared to patients treated with RT and chemotherapy (CT) was 2.4, indicating a protective effect of CT which is likely due to CT-related premature menopause. In a previous study on tamoxifen's late effects, we showed that the risk of poorprognosis endometrial cancers increased with duration of tamoxifen treatment for breast ca neer. Recently, we started a new nationwide study to confirm our previous findings and to also obtain insight into the mechanism of tamoxifen-induced carcinogenesis. In 600 cases of endometrial cancer af ter breast cancer we will investigate whether the clinicopathologic characteristics and prognosis of endometrial cancer differ between women with and without long-term tamoxifen treatment for breast ca neer. We will also search for tamoxifen-specific genomic aberrations (using comparative genomic hybridization) and differentially expressed genes (using micro-array techniques). In 2002, we also assessed long-term cause-specific mortality of patients treated for Hodgkin's disease (HD) before the age of 40 years. The study population consisted of 1261 patients treated for HD at 40 years or younger between 1965 and Followup regarding vital status was complete tilloetober 2000 and the exact cause of death could be determined for 95% of the deceased patients. The long-term cause-specific mortality was compared with general population rates and the relative and absolute excess risks of death were assessed. Af ter a median follow-up time of 18 years, 534 patients had died. The 25-year actuarial risk of death was 44% for all causes, 24% for HD, 14% for second cancer (sq, and 7% for cardiovascular disease (CVD). The relative risk (RR) of death from all causes other than HD was 7-fold increased as compared to the general population. Even after a follow up of more than 30 years, the risk of death from all causes other than Hodgkin's disease was still elevated as compared to the general population (RR=5.1). The overall absolute excess risk (AER) of death from eaus es other than HD was 97 per 10,000 patients per year and this AER increased throughout follow-up. The RRs for dying of solid tumors and CVD were increased overall, but especially for patients treated at the age of 20 years or less (RR 14.8 and 15.1 respectively). However, when these patients attained older ages we observed trends of decrease of the excess mortality from solid tumors and CVD. Hopefully, this indicates that the excess second cancer and cardiovascular disease burden mayabate when patients grow older. Patients who received salvage CT had a much greater mortality from causes other than HD as compared to patients whose treatment was restricted to initial RT or initial combined modality treatment (RR of 8.7 versus RRs of 5.4 and +5, respectively). In collaboration with Division VIII (Annegien Broeks, Laura Van 't Veer) we have started a new project to determine the contribution of cancer susceptibility genes to the development of radiation-induced breast cancer. To examine the separate and joint effects of radiation exposures and ATM germline mutations on breast cancer risk, we will perform a case-control study of contralateral breast cancer.!l Long-term excess mortality in 1,261 survivors of Hodgkin's disease ; 350 ;; ft! ~ g 300 Cl ei :: ~ ~ o ; 150 lil GI u = 100 ii :::I C ~ 50 All causes HO deaths o Other deaths! 0-5 yrs 5 10 yrs yrs yrs yrs yrs > 30 yrs Time since first treatment

128 XIII DIVISION OF DIAGNOSTIC ONCOLOGY DEPART ENT OF CLiN CAL CHFMISTRY HA Bardelmeijer, T Buckle, CG) Cleypool, EM Kemper, CM Korse, TC Linders, M Ouwehand, H R van der Woude, J MG Bonfrer, WJ Nooijen, 0 van Tellingen Pharmacological studies in mice This year, Heleen Bardelmeijer defended her thesis entitled 'Oral administration of taxanes: A preclinical mechanistic study'. In this project we have used mouse models to elucidate the factors that determine the usefulness of the oral route for delivery of taxane drugs (paclitaxel/docetaxel) to cancer patients. Major findings described in this thesis are: (r) P-glycoprotein (Pgp) is very effective in preventing the oral up take of paclitaxel, however, for docetaxel metabolism by cytochrome P 450 enzymes is the most important limiting factor. (2) Next to their Pgp inhibitory potencies, the effects of Pgp inhibitors on stomach emptying and passage rate through the gastro-intestinal tract determine uptake of paclitaxel from the gut. (3) Micellar encapsulation in Cremophor EL, which is part of the current formulation, limits the oral uptake at higher doses explaining the less than proportional increase of AUC with dose observed in patients. Our data are important for designing better oral formulations and drug combinations for use in clinical studies with oral taxanes. We have also studied the metabolism of taxanes using murine bile and feces samples. Interestingly, the profile of metabolic products in rnice appears to be very similar to that in humans. We have identified several novel docetaxel metabolites. The previously identified metabolite M4 found in the faeces of man and mice appears to be a degradation product from an initially formed but unstable carboxylic acid metabolite excreted in the bile (Figure XII!.r). We have studied the role of Pgp and mrpr in the protection of the bone marrow against chemotherapy induced toxicity. Lethally irradiated wildtype mice received bone marrow of mdnab, mrpl, mdnabjmrpl knockout mice or wildtype mi ce and were subsequently challenged with vincristine, a substrate ofboth drug transporters. The results show that mrpr protects the bone marrow from vincristine more than Pgp does, but the absence ofboth transporters caused the severest toxicity. This o Time (min) Figure XIII.1: UV-chromatograms ofbile obtained from a mouse receiving docetaxel (A) and ofblank mouse bile (B). Besides oxidative metabolites that are also found in humans (peak 10 and 11) and unchanged docetaxel (peak 15), va rio us other products were present. Peak 3 represents an unstable carboxylic acid metabolite, which subsequently degrades to a product that was previously identified in frees ofhumans Division Head, Group leader Marc Van de Vijver Marc Van de Vijver MD PhD Head Divis ion XIII DEPARTMENT OF CLINICAL CHEMISTRY Willem Nooijen PhD Academic staff Hans Bonfrer PhD Academic staff Olafvan Tellingen PhD Academic staff Heleen Bardelmeijer Graduate student Marleen Kemper Graduate student Jan Hendrik Beumer Graduate student Arjan Roelofs Undergraduate student Miranda Verheij Undergraduate student Mariët Ouwehand Technical Staff Wanda Douwenga Techn ical Staff Tessa Buckle Technical Staff Tiny Korse Technical Staff Dorothé Linders Technical Staff DEPARTMENT OF NUCLEAR MEDICINE Cornelis A. Hoefnagel MD PhD Academic Staff Ferida Sivro-Prndelj MD Academic Staff Renato A Valdés Olmos MD PhD Academic Staff Saar H Muller PhD Academic Staff Sas ki a Baank Techn ical Staff Martine C Bakker Technical Staff Carolien M Bauhuis Technical Staff Petra A Doodeman Technical Staff Ineke C. GrolIe Technical Staff Iris J van den Heuvel Technical Staff Bert A Pool Technical Staff Gerrie van Steeg Technical Staff DEPARTMENT OF PATHOLOGY Hester van Boven MD PhD Academic Staff Frans Hogervorst PhD Academic Staff Daphne De Jong MD PhD Academic Staff Wolter Mooi MD phd Academie Staff Petra NederlofPhD Academic Staff Hans Peterse MD Academic Staff Marc Van de Vijver MD PhD Academic Staff, Head Division XIII Loes Van Velthuysen MD PhD Academ ic Staff Laura Van 't Veer PhD Academic Staff Annuska Glas PhD Post-doc Douwe Atsma Technical Staff Lucy Boerrigter-Barendsen Technical Staff Leonie Delahaye Technical Staff Jeroen Poodt Technical Staff Carla Schippers-Gillissen Technical Staff Anke Witteveen Techn ical Staff Christine Arkes Secretary Guido Brink Quality Staff

129 THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC laura van 't Veer PhD Academic Staff Frans Hogervorst PhD Academic Staff Senno VerhoefMD PhD Academic Staff Marjanka Schmidt PhD Post doc Marielle Ruijs MD Clinical Fellow Marieke Bronk Genetic Consultant Anja Van Rens Genetic Consultant Gea Wigbout Genetic Consultant Rob Plug Technical Staff Cecile Ottenheim Technical Staff RoelofPruntel Technical Staff leonie Ran Technical Staff Paul van der Voort Technical Staff Rein Regnerus Technical Staff Marjella Boutmy-de lange Technical Staff Bart Maertzdorf Research Assistant Anouk Pijpe Research Assistant Rob van der Spruit Research Assistant Carla van Tiggelen Secretary Guido Brink Quality Staff DEPARTMENT OF RADIOLOGY Peter Besnard MD Academic Staff Kenneth Gilhuijs PhD Academic Staff Elisabeth Joekes MD Academic Staff Wim Koops MD Academic Staff Robert Kröger MD Academic Staff Saar Muller PhD Academic Staff Frank Pameijer MD PhD Academic Staff leo Schuitze Kool MD PhD Academic Staff Claudette loo MD Clinical Fellow Eline Deurloo MD Graduate student William Klein Zeggelink Graduate student Angelique SchliefTechnical Staff Marja van Vliet Technical Staff suggests that the bone marrow is well protected by various transporters and that inhibition of only one of these may have only limited consequences. Ongoing is a project to enhance the delivery of paclitaxel and docetaxel to tumors growing in the brain by inhibition of Pgp in the blood-brain barrier. Pharmacokinetic studies have shown that only potent and selective inhibitors can significantly increase drug penetration in the brain with minimal effects on systemic drug clearance. We are currently testing the efficacy of taxa ne chemotherapy combined with an inhibitor ofpgp against brain tumors growing in wildtype or mdnab knockout mice. We are using luciferase transfected tumor celllines for our brain tumor modeis, which allows convenient non-invasive longitudinal follow up with the IVIS camera (Xenogen). Tumor markers: prognostic significance of pretreatment levels of tumor markers in gastric carcinoma We have assessed the possible contribution of pretreatrnent tumor marker measurements to evaluate patients with gastric cancer. The determination of tumor markers in gastric cancer management is not common; the main reason for this is the low sensitivity for most markers tested to date. Because the assay of Tissue Polypeptide Antigen (TPA) was renewed on an automated analyzer we correlated levels oftpa with CA72-4 and CEA in 140 gastric cancer patients (98 male to 42 female). Histologie classification of the tumors was as follows: 25 signet cell carcinomas; all others were of intestinal type (including seven of mucinous type and one of neuro-endocrine type). The location was cardiac in 56, the pylorus in 29 and other locations in 55 cases. Increased levels we re found in 68, 44 and 46% of the cases for TP A, CEA and CA72.4 respectively. For cases with signet cell histology these figures were 48,25 and 44%. This was significantly different for all markers from results with adenocarcinomas. Interestingly, male patients had more often increased CA72.4levels than female patients. In the univariate survival analysis (Cox proportional hazard) we found that only TPA had significant prognostic value (p=o.0001). In the multiple stepwise analysis stage (P=O.001), age (P=0.03) and TPA (p=0.0003) were all significant factors. DEPART T OF NUCLEAR MEDICINE APE Besnard, E Deurloo, C.A. Hoefnagel, SH Muller, F. Sivro, R.A. Valdés Olmos The departrnent of nuclear medicine is actively involved in a number of clinical research projects, which, in part, are presented elsewhere in this report. The emphasis of these studies are on tumor characterization (using radiopharmaceuticals highlighting, for instanee, hypoxia, apoptosis, specific metabolic functions), lymphatic mapping prior to surgery (sentinel node procedures) and radio nuclide therapy. In 2002, positron emission tomography (PET) was introduced in the institute: the departrnent was the first in the Netherlands to operate a mobile PET-camera system for specific oncological indications (Figure XIII.2). Tracer administration guided by ultrasound, stereotaxis or endoscopy for sentinel node visualization and gamma probe guided tumor excision Intratumoral administration of 99 m Tc-nanocolloid guided by ultrasound or stereotaxis was performed in 146 patients with clinically occult breast tumor in order Figure XIII.2: PET introduced in NKIjAvL (left to right): mobile unit, camera system, PET scan showing metastases in the liver.

130 to enable both sentinel node biopsy and gamma probe guided tumor-excision the day after scintigraphy. Sentinel nodes were visualized in 134 patients (92%). Extra-axillary sentinel nodes were seen in 63 patients (43%). Radical tumor-excision was achieved in lol out of II4 patients. Intratumoral tracer administration guided by endoscopy was performed in five patients with laryngeal carcinoma. In two patients sentinel nodes were visualized. In three patients with recurrences after previous radiotherapy no lymphatic drainage was observed. Endoscopy-guided tracer administration is also being performed in a feasibility study conceming bladder carcinoma. I maging of tumor hypoxia SPECT using a nove12-nitro-imidazole hypoxia marker e 9m Tc-BRU 59-21) was found to be feasible for imaging of tumor hypoxia in head and neck cancer. Tracer up take was found in seven of ten primary tumors with tumor/non-tumor (T/NT) ratios increasing from 1.6 (range ) at 30 min to 2.1 (range ) at three hours. Late SPECT showed better tumor delineation than early images. T /NT ratios for primary tumors correlated with exvivo pimonidazole staining for both early and late SPECT whereas the inclusion oft /NT ratios for involved lymph nodes resulted in correlation only with early SPECT. Whole body images showed activity in liver, gall bladder, intestine and urinary bladder. On SPECT, activity was seen in salivary glands, anterior oral cavity, soft palate and thyroid. Unexpected cardiac uptake probably associated to subclinical cardiomyopathy was seen in four patients. Selected publications Bardelmeijer HA, Ouwehand M, Malingre MM, Schellens JH, Beijnen JH, and Van Tellingen O. Entrapment by Cremophor EL decreases the absorption of paclitaxel from the gut. Cancer Chemother Pharmacol2002; 49(2): Bardelmeijer HA, Ouwehand M, Buckle T, Huisman MT, Schellens JHM, Beijnen JH, Van Tellingen O. Low systemic exposure of oral docetaxel in mice resultingfrom extensive first pass metabolism is boosted by ritonavir. Cancer Res 2002; 62: Bijker N, Peterse JL, Fentiman IS, Julien JP, Hart AA, Avril A et al. Effects of patient selection on the applicability of results from a randomised clinical trial (EORTC 10853) investigating breast-conserving therapy for DCIS. BrJ Cancer 2002; 87(6): Imaging of apoptosis Planar and SPECT images with the tracers 99 m Tc Phentiotate-rh-Annexin V and 99 m Tc Hynic-rh-Annexin V led to the visualization of apoptosis in five of seven patients with low-grade non-hodgkin lymphoma investigated before and after localized low-dose radiotherapy. In comparison to 99 m Tc Phentiotate-rh-Annexin, 99 m Tc Hynic-rh-Annexin V scintigraphy V, shows a more favorable biodistribution which improves the documentation of pathology in thorax and abdomen. In a multicenter study this radiopharmaceutical will be used to assess the role of apoptosis in predicting therapy response in patients with non-small celilung cancer. Anthracycline-related cardiotoxicity The evaluation ofiiiln-antimyosin cardiac scintigraphy in the assessment of myocyte damage associated to anthracyclines was completed. In 24 patients receiving standard doses of mg/m 2 doxorubicin or 90-II2.5 mg/m 2 epirubicin IIIln-antimyosin heart-to-lung ratios became abnormal in 18 patients (75%) after two anthracycline cycles, in 22 (92%) after four cycles, and in 24 (100%) after six cycles whereas left ventricle ejection fraction remained within normallimits in all patients. This early myocyte damage preceding left ventricle systolic and diastolic dysfunction may be of importance to predict long-term risk of cardiomyopathy. Parathyroid imaging with 99 m Tc_M IBI In 48 patients clinically suspected ofhaving hyperparathyroidism planar 99 m Tc-MIBI images were compared with SPECT performed at 120 min after administration of the radiopharmaceutical. The conclusiveness of planar scintigraphy and SPECT was respectively 63% (30/48) and 94% (45/48). SPECT also improved the localization ofthe parathyroid abnormalities in relation to the thyroid gland. CP RT E T OF PA-- OLOG I Douwe Atsma, Lucy Boerrigter-Barendsen, Leonie Delahaye, Annuska Glas, Frans Hogervorst, Wolter Mooi, Hans Peterse, Jeroen Poodt, Carla Schippers-Gillissen, Loes Van Velthuysen, Anke Witteveen, Daphne De Jong, Petra Nederlof. Laura Van 't Veer, Marc Van de Vijver Gilhuijs KGA, Deurloo EE, Muller SH, Peterse, JL Schultze Kool LJ. MR breast imaging of wamen at increased lifètime risk ofbreast cancer: Clinical system for computerized assessment ofbreast lesions - Initial results, Radiology 2002, (In press). Gille JJ, Hogervorst FB, Pais G, Wijnen J, Van Schooten Rl. Dommering CJ, Meijer GA, Craanen ME, Nederlof PM, De Jong D, McElgunn CJ, Schouten JP, Menko FH. Genomic deletions ofmsh2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br ] Cancer 2002 Oct 7; 87: Hoebers FJP, Janssen HLK, Valdes Olmos RA, Sprong D, Nunn AD, Balm AJM, Hoefnagel CA, Begg AC, Haustermans KMG. Phase 1 study to identify tumor hypoxia in patients with head and neck cancer using TC-99m BRU EurJ Nucl Med Mol Imag 2002; 29: Klaren HM, Van 't Veer LJ, Van Leeuwen FE, Rookus MA. Potential for bias when evaluating reduction of cancer risk after prophylactic surgery in BRCA1 and BRCA2 carriers. ] Natl Cancer Inst 2002, (in press). Genetic alterations in tumors The Department ofpathology, which includes the Family Cancer Clinic, has a limited number of research projects which are carried out within the department itself. In addition, several research projects are carried out in the Division of Experimental Therapy. The main focus within the departrnent in the past year has been setting up the logistics needed to perform gene expres sion

131 Selected publications (continued) Lakhani SR, Van de Vijver MJ, Jacquemier l. Anderson Tl. Osin PP, McGuffog L et al. The pathology ojjamilial breast cancer: predictive value oj immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and P53 in patients with mutations in BRCA1 and BRCA2.] Clin Onco12002; 20: Mooi WJ. Spitz nevus and its histologic simulators. Adv Anat Pathol 2002; 9: Rebbeck TR, Lynch HT, Neuhausen SL, Narod SA, Van 't Veer Ll. Garber JE, Evans G, Isaacs C, Daly MB, Matloff E, Olopade Ol, Weber B, PROSE. Prophylactic oophorectomy in carriers oj BRCA1 and BRCA2 mutations. New Eng] Med 2002; 346: Ruivenkamp CA, Van Wezel T, Zanon C, Stassen AP, Vlcek C, Csikos T et al. Ptprj is a candidate Jor the mouse colon-cancer susceptibility locus SCC1 and is frequently deleted in human cancers. Nat Genet 2002; 31: Taal BG, Van Tinteren H, Van 't Veer LJ. Adjuvant treatment in colorectal cancer: microsatellite instability as a prognostic marker. Br. J. Cancer 2002; 86: Tanis PJ, Horenbias SJ, Valdes Olmos RA, Hoefnagel CA, Nieweg OE. Feasibility oj sentinel node lymphoscintigraphy in stage I testicular cancer. Eur] Nuc! Med 2002; 29:67-3. Tanis PJ, Nieweg OE, Valdes Olmos RA, Peterse J L, Rutgers EJTh, Hoefnagel CA, Kroon BBR. Impact oj non-axillary sentinel node biopsy on staging and treatment oj breast cancer patients. Br ] Cancer 2002; 8T Van de Vijver MJ, He YD, Van 't Veer LJ, Dai H, Hart AAM,Voskuii DW, Schreiber Gl. Peterse J L, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, Van der Velde T,Bartelink H, Rodenhuis S, Rutgers Eth, Friend SH, Bernards R. A gene expression signature as a predictor oj survival in breast cancer. New Engl] Med, 2002; 347: prohling using micro array analysis on tumor samples from our tissue bank. The first large study using micro array analysis ofbreast cancer is described in the next paragraph. A number of projects involving expression prohling of various tumor types in collaboration with the NKI micro-array facility have recently started and first results of a study on malignant lymphoma are presented. Gene expression profiling of breast ca neer In breast cancer, assessment of prognostic and predictive factors are important to tailor optimal treatment. The strongest predictors for metastases, e.g., lymph node status, and histological grade, fail to accurately elassify breast tumors according to their clinical behaviour. In a collaboration with Rosetta Inpharmatics (Kirkland, Washington, USA), we have previously used DNA micro arrays on primary breast tumours of II7 young patients and applied supervised elassification to identify a gene expres sion signature strongly predictive of a short interval to distant metastases in lymph node negative patients. This prognosis signature consists of 70 genes and ineludes genes regulating cell cyele, invasion, metastasis, and angiogenesis. In addition, a signature was established that identifies tumours of BRCAI carriers. To validate this prognosis signature, we have elassified a consecutive series of 295 primary breast carcinomas as having either a 'poor prognosis' or 'good prognosis' signature. All patients had stage I or 11 breast cancer before age 53 and had lymph node negative (n=151, ineluding 61 tumors from our training series to avoid selection bias in the consecutive series) or positive disease (n=144). A ~0.8! ~0. 6. i -= 0.4rr-::---=---;-~"'--=,..., ;: ~02 P<O.OO) n ~0.8 i 10.6 i Time ey.ars) S Tine ey..rs) -= O.4 t r-.--::good,...-:-; si-gnallr...,.-s-:c(5=5)" ;: n pq()( SI!JI8Iure (89) ~02 P<O.OO) S Tine(yNrs) B Good signallrs (115) n p Q()( SlgnalLrS (180) O~~2--74~6~~8...,.-1~0~12 TIme ey.ars) 0.8 i 0.6! 0.4 tr-::--==-=,=:::::-;~ I!. ~Slsi=8(~~) I 0.2 P<O.OO) U so Time ey..rs) F, 0.8 i 0.6! ~ 0.4 I Good SlgnaILre (55) I. n PQ()( SlgnalLrS (89) 02 P<O.OO) 1 55 n S TIme ey.ars) All patients Lympb node negative patients Lympb node positive patients Figure XII!.3: Kaplan-Meier plotsjor 295 breast cancer patients. (A) Metastasis-free probability oj all 295 patients according to 'good prognosis' (YI=115, I) and 'poor prognosis' (YI=180, II) signature. (B) Overall survival oj all 295 patients according to 'good prognosis' and 'poor prognosis' signature. Metastasis-free probability and overall survival similarly Jor lymph node negative (C and D) and lymph node positive (E and F) patients. Patients at risk at each time point (years) are indicated in Jor 'good signature' (I) or 'poor signature' (II) patients. P-value is calculated by the log-rank test.

132 -, J ~~~~~~~~,.(~~.~"~~".~~.~.. ~----~ 180 tumors had a 'poor prognosis' signature and II5 tumors a 'good prognosis' signature. Overall Io-year survival for the 'poor' and 'good prognosis' signature groups was 54.6% (SE 4-4%) and 94.5% (SE 2.6%). Distant metastasis-free probabilities for the 'poor' and 'good prognosis' signature groups were 50.6% (SE 4.5%) and 85.2% (SE 4.3%) at ten years. The hazard ratio for distant metastases of'poor' versus 'good prognosis' signature is estimated to be 5.1 (95% Cl: , P<O.OOI) and retains its significance in lymph node negative and positive subgroups separately (Figure XIII.3), also when the 61 tumors from the training series were excluded. Multivariable Cox regression analysis reveals that the prognosis profile is a strong independent parameter in predicting disease outcome. We have validated the 'pro gnosis profile' in lymph node negative and positive breast cancer. This profile is by far the strongest predictor of prognosis available for breast cancer to date and can be used to optirnize adjuvant therapy selection in premenopausal breast cancer patients. Gene expression profiling in malignant Iymphoma Malignant lymphomas are a collection of about 30 different disease entities. Each entity is characterized by a specific morphological spectrum, a characteristic immunofenotype, often characteristic genomic alterations and a typical clinical presentation and clinical course. Follicular lymphoma (FL) is one of the commonest types oflymphoma in adults. Typically, the patients present with advanced disease, that responds mostly very wen to relatively mild chemotherapy. The disease is characterized by frequent relapses, however, and ultimately becomes therapy resistant or transforms into a more aggressive phase called diffuse large B-celllymphoma (DLBCL). Using morphological and immunohistochemical methods, it is very difficult to predict the clinical course in an individual patient. Currently, a morphological grading system is used to stratify patients into indolent and transformed types of FL, indicted as grades land 2 versus 3 respectively. This method, however, is highly subjective and poorly reproducible, precluding meaningful implementation in patient management. DLBCL can be recognized more reliably. Using gene-expres sion profiling, we have analyzed the genes and pathways that are involved in transformation of FL in order to develop a predictor to better stratify patients with FL and to be used as a guideline in the choice of therapy. Paired samples of FL and transformed FL within 21 single patients we re collected from the collaborating Departrnents ofpathology (NKljAvL, AZ Groningen, AZN ijmegen, L VFriesland). The use of paired samples was chosen to minimize the influence of inter-individual variation and tissue localization. In all cases, detailed clinical information on presentation, clinical course, treatment protocols and outcome was collected into a central Access database, that was specifically developed for this purpose (A Van de Velde, Departrnent of Biometrics and M Kersten, Division of Medical Oncology NKljAvL in collaboration with R Kibbelaar, LVFriesland, data collection by M Kersten, P Joosten and G Van Imhoff). All specimens we re reclassified and graded according to the WH 0 classification for hematological malignancies by at least two hematopathologists. RNA from tumor samples and pool of normal tonsillar lymphoid tissue was isolated and amplified and co-hybridized to 18K micro-arrays produced by the NKljAvL Micro-Array Facility. All hybridizations were performed in duplo (color-reverse). Mter normalization, 7 00 potentially relevant genes were selected using the Rosetta error model. From this set, genes were selected on the basis of signal-to-noise ratios per sample pair to collect pair-independent data as demonstrated by Monte Carlo randomization and the most significant gene set (P<O.OI) was collected and further optimized by a leave-out approach. U sing this 80-gene-set, cases clustered into three major clusterbranches. A very strict, mutually exclusive distinction could be made between indolent FL, grade land 2 versus transformed lymphoma. The latter cluster contained both FL grade 3 and DLBCL cases, indicating that a follicular growth pattem is of relatively minor importance as compared to the expres sion of transformation defining genes. The third cluster showed more similarity to the 'transformed' cluster than to the 'indolent' cluster except for three distinct groups of related genes. The third cluster contained a mixture of DLBCL cases and FL grades I, 2 and 3. Clinical evaluation, however, revealed that the morphologically indolent cases that showed a clinically Selected publications (continued) Van 't Veer LJ, Dai H, Van de Vijver MJ, He YD, Hart AA, Mao M et al. Gene expression profiling predicts clinical outcome ofbreast cancer. Nature 2002; 415(6871}:s30-6. Van 't Veer LJ, de Jong D. The microarray way to tailored cancer treatment (NB[V) Nature Medicine 2002; 8:1314. Valdes Olmos RA, Carrio I, Hoefnagel CA, Estorch M, Ten Bokkei Huinink WW, Lopez-Pousa J, Dalesio O. High sensitivity of radiolabelled antimyosin scintigraphy in assessing anthracycline related early myocyte damage preceding cardiac dyifunction. Nucl Med Commun 2002: 23: Wessels LFA, Hart AAM, van 't Veer LJ, Reinders MJT, Nederlof PM. Molecular classification ofbreast carcinomas by CGH: aspecific somatic genetic profile for BRCA1 tumors. Cancer Res. 2002;

133 divergent behaviour of aggressive disease (no or insufficient response to CVP of Chlorambucil or very rapid transformation at another site) ail clustered in this third cluster branch in contrast to the morphological indolent cases that behaved as expected. These data are highly suggestive, that this approach wiil result in a predictive profile for FL. Further validation on a independent, unselected set of morphologically indolent FL, FL grade 3 and DLBCL as transformation offl is currently being performed. M LPA as a new tooi in molecular pathology A new technique called Multiplex Ligation-dependent Probe Amplification (MLPA) was developed in close collaboration with G PaIs of the Free University Medical Center (VUMC) and a biotech company (MRC-Holland) both located in Amsterdam. This method allows the relative quantitation of specific sequences (e.g. the deletion of regions in genomic DNA) in a multiplex PCR. The method was developed the detection of exon deletions and gains in the BRCAl gene, which is involved in hereditary breast and ovarian cancer, and a test for the MLHl and MSH2 genes, involved in hereditary colorectal cancer. Methods currently used to detect such mutations are very laborious and lack sensitivity and are therefore only used in a minority of families. Finally, we applied MLPA to detect HERz copy number changes (amplification) in breast tumors, which is an important diagnostic test for optimal treatrnent planning. Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair (MMR) genes, in particular MLH1, MSH2 and MSH6. Mutation screening ofmmr genes was performed in li6 colorectal cancer families selected on the basis of clinical criteria and, in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumors. Thirty-six germline mutations were detected in 35 (30%) of these kindreds, 30 of which have a predicted pathogenic effect. Among families with MSI-high tumors 66% harbored gerrnline MMR gene defects. Genomic deletions in ML Hl or MSH2 accounted for 53% of the pathogenic mutations. A complete deletion of the MLHl gene not described previously was detected in two families (Figure XIII-4). Mutation analysis of the BRCAl and BRCA2 genes is usually restricted to the analysis ofpcr fragments amplified from genomic DNA. However, a wide variety oflarge genomic exon deletions and duplications in the BRCAl gene have been described which in some populations constitute a substantial part of all BRCAl mutations. We have tested 66r families in which routine DNA-diagnostic analysis of BRCAl or BRCA2 was inconclusive. Five different mutations were identified, four of which 4 ~ , ~ MLH1 MSH2 3 -(I).c ~ 2 Z ~ C. o 1 o c o >< w 0 del MLH1 ~ 8 '" ~ g g 3 ~ ~ ~ del exon 12 MLH1 del exon 3 del exon 1-6..,... e» 8 E '" '" E ~ CD 0 '"... ~ ~ ~ ~ ~ ~ w ~ )( N ~ ~ g N N g N w 3 3 g 3 3 :I: :I: :I: N N ",- UI i3i UI UI :I: :I: :I: ~ ~ ~ ~ ~ ~ UI UI UI ~ ~ ~ ~ ~ ~ ~ ~ MLH1 and MSH2 exons Figure XIII+ Graphical representation ofmlpa results ofhnpcc patients shows exon deletions in MLH1 and MSH2. Each line connects the exon copy numbersfor a particular patient. In case of a deletion, the copy number for the deleted exons dropsfrom 2 to 1. One patient shows a deletion ofthe complete MLH1 gene, the others show deletion of one single or multiple consecutive exons.

134 have not been reported before: deletions of exon 8 or exons 20-22, duplications of exon 13 or exons 21-23, and a triplication of exons Subsequent Southem blotting and PCR confirrned all mutations. Application of the MLPA test clearly improves the yield of mutation detection in hereditary breast and/or ovarian cancer families. The presence of HER2 gene amplification occurs in approximately ls-20% ofbreast cancer patients. HER2 positive tumors may be reponsive to antibody based therapy (Herceptin@); and there is strong evidence that HER2 positive tumors benefit more from optimal anthracylin dosing. In practice, assessment of HER2 status is mainly do ne using immunohistochemistry and, with much lower frequency, with fluorescent in situ hybridization (FISH). We have tested the MLPA on DNA isolated from snap-frozen tumor samples and formalin-fixed paraffin embedded tissue from the pathology archive. All fresh samples were also analyzed by Southem blotting, and the paraffin material was also subjected to FISH. Southem blotting and MLPA are mghly concordant, and re sult in similar gene copy number estimations. FISH and MPLA are also concordant, however, the copy number estimation by FISH is in general higher than in the MLPA assay. THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC Prophylactic surgery in carriers of BRCAl and BRCA2 mutations The aim of this project is to evaluate the efficacy of prophylactic mastectomy in high-risk breast cancer women. Since the number of women that opt for a preventive bilateral mastectomy or oopherectomy in one single hospital is rather low (80 and 180 women at the NKI with relatively short follow-up to date respectively) the data of several institutions are incorporated in an international NIH-study coordinated by Timothy Rebbeck, University of Pennsylvania USA, that will thus obtain sufficient power. The efficacy of prophylactic oophorectomy was recently published and showed a hazard rate reduction for coelomic epithelial cancer of 0.04 (9S%CI ) and a breast cancer hazard rate reduction of 0-47 (9S%CI ). At present the data for mastectomy are collected and within the next four years new follow-up data will be added to this retrospectively identified, though prospectivily followed cohort. Clinical outcome of breast cancer in BRCAl and BRCA2 carriers In a retrospective cohort study, stratified by BRCAl/2 carriership, we will evaluate clinical outcome in a large and unselected, non-family based, sample ofbreast cancer patients diagnosed under age SO (e.g. overall, breast-cancer-specific and disease-free survival, as weil as the occurrence of ovarian and contralateral breast cancer). The prevalence of BRCA1/2 mutation carriership in this group is expected to be around 6%. The cohort to be evaluated will include approx. 6soo patients, treated in eight hospitals from S. Af ter anonymization, forty-one BRCA1/2 founder and recurrent mutations, representing 82% of the Dutch BRCA1/2 mutations, will be determined by (eighteen) mutation specific tests in DNA isolated from paraffin embedded blocks. Before linkage with the BRCA1/2 mutation data, the patient database will be anonymized to guarantee optimal privacy. This procedure will obviate the need for specific informed consent. In a multivariate We anticipate to have 93% power to detect at least a 10% breast-cancer-specific survival difference by Cox-model analysis at 13 years of follow-up. Substantiation of a worse outcome of survival will guide more extensive genetic screening in young breast cancer patients and will result in tailored therapeutic strategies for BRCA1 and BRCA2 carriers. For presymptomatic BRCA1 and BRCA2 carriers, important information will be generated that can be used in decision making between surveillance by mammography or bilateral prophylactic mastectomy. In the first year of this project ethical approval was obtained that will allow us to conduct the study in a 'samplecoded' fasmon. The key to this code will be kept at a notary and will allow us to update follow-up of the patients at later time points, keeping the actual status of the patients up-to-date. The ethical protocol recently drew a lot of attention from national ethical committees and law makers.

135 ~. ~. ~~.'-- ~ ~ :.. ~ DEPARTM E T OF RADIOLOGY E.J.Th. Rutgers, H. Bartelink, S. Muller, E.E. Deurloo, M. van Vliet, W. Klein Zeggelink, J.L. Peterse, M. van de Vijver, K.G.A. Gilhuijs Figure XI I IT Example of a grade 2 infiltrating ductal carcinoma automatically segmented in 3-D using all time frames in contrast-enhanced M RI. Tumor volume and largest diameter are automatically computed after segmentation. Computer-aided Diagnosis of Lesions in Contrast-enhanced M RI of the breast The purpose of this ongoing study is to reduce the number ofbiopsies on benign lesions without compromising the sensitivity to trap malignant lesions. In a previous study, a system was developed for automated characterization oflesions in contrast-enhanced MR images of the breast. Currently, training of the system has been updated to 100 lesions: 50 malignant and 50 benign lesions. After training, the system was tested prospectively on the MR images obtained at our clinic. Application of the system was done parallel to the reading by our radiologists without consequences for the patient. So far, 136 lesions have been analyzed. The prospective performance of the system was found to be comparable to the performance during training (area under the ROC curve 0.93 and 0.91, respectively). In addition, the prospective performance of the computerized analysis was found to be comparable to the performance of a team of well-trained radiologists. First results indicate that combination of the radiologists and the computerized analysis leads to improvement of accuracy of clinical reading. M ulti-modality analysis and radiological guidance in breast conserving therapy This year, our MARGINS database, which is aimed at providing preoperative estimation of tumor margins and guidance to surgery, has increased to include the data of 441 patients with breast cancer. As of yet, the database includes mammograms (426), ultrasound images (410), MR images (166), and x-ray specimen photographs (259). A pilot study was launched to quantify intern al shifts ofbreast tissue during ultrasound-guided fine needle aspiration. This study is a continuation of prior studies that quantified dis placement of compressed breast tissue during stereotactic core biopsies and uncompressed breast tissue during repeat supine setup at MR examination. These studies will eventually lead to better feed-back of diagnostic imaging information to surgery. Table XII 1.1 Systematic and random variations in the assessment of tumor extent (largest diameter) by a team of experienced breast MR radiologists, by automated assessment of tumor extent in 3-D, and by pathology examination (n=40). Team of Radiologists Systematic Deviation (mm) -0.2 Random Variation (mm) 4 3 Validation of semi-automated lesion segmentation in contrast-enhanced M RI of the breast The aim of this study was to validate a previously developed system for semi-automated quantification of tumor extent in three dimensions (3-D) from contrast-enhanced MR images of the breast (Figure XIII.5). In a test set of 40 benign and 40 malignant lesions, two operators independently used the system to quantify tumor extent. The mean difference in extent found by the two operators was small (o.n mm for benign lesions and 0.22 mm for malignant lesions), and did not prove to be statistically significant (P=0.29). In a subsequent study, the accuracy of the automated system was determined with respect to examination of tumor extent by pathology in 40 malignant lesions. The mean deviation was found to be small (0.9 mm). An analysis of variance technique was employed to assess the random variations in tumor extent determined by the automated system, the radiologists, and the pathologists (TabIe XII!.I). The results indicate that the automated system is highly reproducible across the clinical population of tumors and between operators. It also indicates that the reproducibility of the system is superior to the reproducibility of the gold standard. Automated Assessment 2.8 Pathology Assessment (*) The systematic deviation in tumor extent from pathology assessment is 0 mm by definition (gold standard).

136 BIOMETRICS DEPARTMENT CLINICAL STUDIES AND OVERVIEWS Overview of randomized trials in prostate cancer Since 1989, we coordinate, in collaboration with R Peto and rus group at the University of Oxford, an overview of all randomized trials of the treatment of pro state cancer. The Prostate Cancer Trialists' Collaborative Group (PCTCG) was created to bring together as many of the randomized trials as possible and, where different trials have addressed similar questions, to produce a formaloverview of their findings. To identify all relevant randomized trials, a wide variety of sources is used, including literature databases and data nets, the Cochrane Controlled Trials Register, abstract books from relevant meetings and contact with individuals, trial groups and pharmaceutical companies. For each trial identified, data on each of the patients randomized is sought from the different trialists, institutes and cooperative groups. These data are analyzed centrally and the results of the different trials are combined appropriately to pro duce an overall estimate of the differences between the treatments assessed. The initial cycles of this collaboration assessed the use of maximum androgen blockade (MAB). In 2002, we have obtained funding for two projects presented to the EC Quality of Life program, to support the continuation of our central tasks of the Overview and for the organization of the PCTCG Second Main Meeting that took place in Oxford, in September. The most important question addressed by this cycle is the effect of immediate endocrine therapy as compared to deferred endocrine treatment in patients with no recorded metastases (M-). Nineteen trials were identified and Individual patient data we re obtained from 16 of them on 13,000 patients out of a total of 14,000. Results of the effects on prostate cancer mortality and non-prostate cancer mortality were discussed. In addition, the question of the effect of the addition of chemotherapy on mortality in comparison to the standard treatment was revisited. Thirty trials were identified: 26 first line (three of these in patients with localized disease) and six af ter failure to hormonal treatment. For 16 trials data were not available and in a majority of cases not recoverable (the studies we re too old and data we re not kept.) Individual patient data were obtained for 3015 patients out of the 4492 entered in all trials. In general, trials we re small typically including a few dozens of patients and the drugs used in the chemotherapy were diverse, in some studies given as mono-therapy and in others in combinations. We are preparing the report of the results of these two questions for publication in 2 3 Collaboration with national and international trial groups We have developed collaborations with several cooperative groups; industry and clinical research organizations, and we function as partner for clinical trials, being involved from the generation of the idea, protocol setting, the planning, and providing randomization services, data management and statistical expertise. The collaboration with the Dutch Chest Physician Association (NV ALT) already resulted in three randomized studies. In one study, treatment with docetaxel + carboplatin in a three weekly schedule is compared to docetaxel in a weekly schedule in patients with stage IIIb and IV NSCLC. In October, after accruing 297 patients, the study was stopped following advice of the Independent Data Monitoring Committee based on the results of an interim analysis including the first IS0 patients. A full report is in preparation. The second study, also performed in collaboration with the British Medical Research Council, is aimed at evaluating the benefit of neo-adjuvant chemotherapy in operabie NSCLC. The third study is being planned in elderly patients. Collaboration with the Dutch Colorectal Cancer Group (DCCG) has resulted in the CAIRO study for patients with previously untreated advanced colorectal carcinoma. This is a randomized trial that aims to ac crue 640 patients to establish whether it is preferabie to use two drugs in a sequential way or in combination. A study in the adjuvant setting is currently being planned. Group leader Otilia Dalesio Otilia Dalesio MSc Head Harm van Tinteren MSc Academic staff Ninja Antonini MSc Academic staff Danny Baars Technical staff Vanessa Bouwman Technical staff Astrid Diemeer Technical staff Wilma Deurloo Technical staff Marjolijn de Waal MSc Technical staff Antje Fierens Technical staff Annelies Hiemstra Technical staff Lea Kooij Technical staff Jan Lieverst MSc Technical staff Jolanda Maaskant Technical staff Marianne Mahn-Schaefers MSc Technical staff Ingrid Mandjes MSc Technical staff Carla Modder Technical staff Anja Muusers Technical staff Jacolien Nicolai Technical staff Ellen Peter PhD Academic staff Anneke Reinders-Som Technical staff Suzanne Reitsma Technical staff Jolanda Remmeizwaai Technical staff Danny Roberts Technical staff Dea Storm Technical staff Heleen Vaessen MSc Technical staff Renato Valdes Olmos Technical staff Ludy Valkenet MD Technical staff Emile van der Donk Technical staff Ivy van der Schilde Technical staff Tony van der Velde Technical staff Els van Oosterwijk Technical staff Wil van Waarden berg Technical staff Anneke Wals Technical staff Lidwina Wever Technical staff Els Willemse Technical staff Lonny Ziblat Technical staff Hans Oosting PhD Guest Annamarie van der Kwast Student Joke van Gulik Secretary

137 Selected publications De jonge ME, Mathot RA, Dalesio 0, Huitema AD, Rodenhuis S, Beijnen jh. Relationship between irreversible alopecia and exposure to cydophosphamide, thiotepa and carboplatin (CTC) in high-dose chemotherapy. Bone Marrow Transplant. 2002; 30: Michalides R, Van Tinteren H, Balkenende A, Vermorken j B, Benraadtl. Huldij l. Van Diest P. Cydin A is a prognostic indicator in early stage breast cancer with and without tamoxifen treatment. BrJ Cancer ; 86: Schoemaker NE, Van Kesteren C, Rosing H, jansen S, Swart M, Lieverstl. Fraier D, Breda M, Pellizzoni C, Spinelli R, Grazia Porro M, Beijnen jh, Schellens jh, Ten Bokkei Huinink Ww. A phase I and pharmacokinetic study ofmag-cpt, a water-soluble polymer conjugate of camptothecin. Br ] Cancer ; 8T The study of the Dutch Breast Cancer Study group on breast cancer patients with at least four tumor-positive axillary lymph nodes but no distant metastases, in which 885 patients we re randomized by ten Dutch centers was extensively analyzed for first publication of the results. Conventional treatment of five courses of FEC (fluorouracil50o mgjm 2, epirubicin 90 mgjm 2 and cyclophosphamide 500 mgjm 2 ; q 3 weeks) followed by radiation therapy and tamoxifen was compared to high-dose treatment, where the fifth FEC course was replaced by high-dose chemotherapy (CTC, cyclophosphamide 6 gjm 2, thiotepa 480 mgjm 2 and carboplatin 1600 mgjm 2 ) with peripheral blood progenitor cell transplantation. In July 2002,319 events (36%) we re reported and the median follow-up was 57 months. The totality of data included, among others, information on almost 4000 cycles of FEC and 8000 follow-up records. 842 (95%) of the surgical specimens were centrally reviewed at the NKI. Also, the HIPEC trial was analyzed in depth for publication. This is a study in patients with peritoneal carcinomatosis of colorectal cancer that were randomized between standard adjuvant chemotherapy (5FU jleucovorin) on outpatient basis and experimental therapy consisting of cytoreduction with Hyperthermic Intra PEritoneal Chemotherapy (HIPEC), followed by the same systemic chemotherapy. Between January 1998 and August 2001,105 patients from our institute were randomized in the study and at the time of analysis 55 patients had died. Definite publications for both these studies can be expected early 2003 and the results ofboth trials will have a major impact on clinical practice and for the design of new studies in these types of cancer. IeT PROJ EeTS Schrama jg, Faneyte IF, Schornagel jh, Baars jw, Peterse j L, Van de Vijver Mj, Dalesio 0, Van Tinteren H, Rutgers El. Richelt Dj, Rodenhuis S. Randomized trial ofhigh-dose chemotherapy and hematopoietic progenitor-ceu support in operable breast cancer with extensive lymph node involvement: final analysis with 7 years of follow-up. Ann Oncol 2002; 1]: Taal BG, Van Tinteren H, Van 't Veer L. Adjuvant treatment in colorectal cancer. BrJ Cancer 2002; 86: Valdes Olmos RA, Carrio I, Hoefnagel CA, Estorch M, Ten Bokkei Huinink WW, Lopez-Pousal. Dalesio 0. High sensitivity of radiolabeued antimyosin scintigraphy in assessing anthracydine related early myocyte damage preceding cardiac dysfunction. Nud Med Commun 2002; 23: For more than a decade we have been involved in research and development in the area oftelematics applied to healthcare. We have developed information and communication technology (ICT) projects and we have obtained funding under the various framework programs of the EC. In 2002, we were a partner in two project funded within the Information Society Program of the EC: RUBIS and BEPRO with a focus on standardization in Clinical Data Interchange and Information System for Clinical Guidelines, respectively. BEPRO: Information System for Clinical Guidelines BEPRO, Best Practice in Oncology has been funded in the EC fifth framework programme Best Practice call for proposals. The project was successfully completed in June The consortium comprised both technology solution providers and end users. Our role in BEPRO was to assess the added value of new technology in the delivery of online information to nurses in the hospital. The current nursing guidelines at the NKI in the 'Vraagbaak' were converted into an XML format and the added value of new technology was assessed in three fields: improved access to the information, simplified and better management of the information, and better quality of the information. A simple example of one of the advantages was alerting_ Clinical guidelines need to be reviewed periodically. In the Vraagbaak the date oflast review was stored in the HTML code and therefore not machine-readable. In order to see which guidelines needed revision, all guidelines had to be 'scanned' manually for outdated guidelines. With the XML based system, guidelines include data such as date last modified and author. This allows us to automatically send messages to authors on guidelines that need revision. In addition to the XML system, collaboration with the UK based organization InferMED was established to assess what is referred to as 'Active Guidelines'. With active guidelines, all specific steps in a guideline are entered into a Decision Support system based on Arrezzo technology. For each patient, a guideline is 'instantiated', meaning that the system knows about the patient and the steps in the guideline (in our pilot a chemotherapy treatment), which the patient has already undergone. Although the technology is very promising and useful, the ma in obstacle for use in clinical practice is the huge amount of work required to enter all guidelines (including nursing guidelines and instructions) into such a system and the fact that nurses will need to modify their way of working. Instead of checking a step performed on paper, the nurses will have to enter this into the system. This has high

138 requirements on end-user equipment. Nurses should be equipped with PDA (Personal Digital Assistants) and the wards and outpatient clinic should be equipped with Wireless LAN. A pilot with PDA and WLAN was included in BEPRO and was successful: a PDA in the hospital could access resources stored in an isolated server at the department. However, the clinical take-up is not to be expected in the coming two years. In order to pilot the active guidelines in the clinical setting, we wrote a project in 2002, aiming to develop and implement an 'Active Guidelines' system. This system should accept guidelines from different sources, and make them accessible to different us er groups in the clinical setting. Funding will be sought in 2003 within a larger Integrated Project in the EC 6 th Framework (Figure I). Selected publications (continued) Van Tinteren H, Hoekstra OS, Smit EF, Van den Bergh JH, Schreurs Aj, Stallaert RA, Van Velthoven PCM, Comans EF, Diepenhorst FW, Verboom P, Van Mourik JC, Postmus PE, Boers M, Teu Ie G J. Effectiveness of positron emission tomography in the preoperative assessment of patients with suspected non-small-eell lung cancer: the PLUS multicentre randomised trial. Lancet ; 359: Vielvoy-Kerkmeer A, Van Tinteren H, Mattern C, Schüller J. Sustained-release morphine sulphate in cancer pain. Eur J Pall Care 2002; 9: Standards in Care Point of Care RUBIS3: standardisation in Clinical Data Interchange In order to make two software packages 'interoperable' beyond the level of shallow integration, an interface must be created to map (meta) data from one application to the other application and vice versa. For three packages, two interfaces would be required for each package, four packages would require three interfaces per package, etcetera. This scales negatively for environments such as RUBIS, a 'Reseau UBiquitaire a Integration de Services' which typically gives home to a large number of applications: DPWEB, RISA, MACRO, ALEA, PDB2000 and eforms. For such an environment, a 'pivot' structure should be implemented that will require only a single interface per software package. Such a pivot should dictate a uniform and complete format for the representation of data and metadata. Obviously specific pivots will be developed for different application domains: e.g. industrial production environments, financial services sector, and health care. Within the scope, several pivots wiil describe representation of data for a specific scope. The development of such a pivot format is typically a collaborative effort of entities involved in the design, implementation, operation and use of software systems. Since overlaps may exist between scopes, the representation system should provide mechanisms to mix definitions from different pivots. XML is an ideallanguage for the development of such pivots, and many research groups are developing de facto standards for representation of data in different domains and for different scopes within the application domain. In health care, a dozen groups are partly working together, semi-independently, on such pivots. RUBIS3 started with a detailed study of the state-of-the-art of the different standardization bodies, which yielded three applicable pivots: ODM by CDISC, prenvi3607 by CEN TC251 and RIM by HL7. Since each of these standards is still in development, it was expected that an implementation effort would highlight a number of shortcomings in the existing models. Therefore, in RUBIS3 the concept of nested DTD's was chosen to ailow for adoption of the pivot, yet give the technical option to complement the pivot with proprietary extensions required to operate the pivot. The proprietary extensions were divided in

139 two groups: extensions required to overcome shortcomings of the pivot, and extensions required to overcome shortcomings of our own software packages. Shortcomings of the pivot were bundled for each implementation and forwarded to CDISC as proposed Operational Model extensions. In order to allow the consolidated forwarding of extensions to (pre-) standards, RUBIS3 adopted a model for collaborative operation, development and contribution to International Standardization efforts. In this model the forces of the various consortium partners was bundled in order to create a cri tic al mass of industrial backing for proposed extensions to the standard (Figure 2). Nesting DTD to extend the Data Models A model tor collaborative operation, development and cont ribution to Internatio nal standardizatio n AC:TION OTO OTO Common to all interoperabie applications prenv13606 COISC OOM RIM CENTC251 COISC HL7 PILOT EXTRANET ONCONNECT.NET Sec:urlty, Single Sign On, otstrtbuted Searchlng, Vldeoc:onr.renclng, forum Specific results achieved in the third phase ofrubis3 in the context of contribution to standardization of (meta) data representation and interoperability between different ACTION products are: 1. MACRO software provides Remote Data Entry for clinical trials, while ALEA is software for the online randomization of patients in clinical trials. The data representation level integration of ALEA and MACRO developed in RUBIS3 enabled two 'interoperabilities'. Data interoperability enables the completion of an ALEA randomization form with a MACRO RDE front end, be sent by the MACRO RDE back end in an ODM format to an ALEA randomization service which sends back a patient number and treatrnent allocation in an ODM format to the MACRO RDE back end which in turn is displayed with the MACRO RDE front end. Metadata interoperability enables the exchange of study definitions from MACRO to ALEA and vice versa. 2. The ALEA Web Service for randomization of patients in clinical trials can be invoked from DPWeb. A study definition in ODM format is retrieved from the ALEA Study Definition Web Service. Next, the Randomization form is retrieved from the study defmition and presented in DPWeb. Next, the DPWeb user completes the form and DPWeb sends it on to the Randomization Service. The DPWeb server receives a treatrnent back and displays this onto the user DPWeb screen. This mechanism also works with generic eforms, such as ADOBE eforms. The extensions required for the CDISC Operational Data Model in order to support the link between MACRO and ALEA were bundled according to the model described above. The extensions for CDISC were submitted to the CDISC consortium and will be evaluated by the consortium for the next version of ODM.

140 RESEARCH FACI LITI ES The research divisions within the NKIjAvL are supported by a number of indispensable research service facilities. Each facility comprises a group of experts who provide information, instruction and service assistance and houses state of the art equipment. Besides the facilities described below, the institute has a central cryogenic storage facility, a glassware kitchen, a(n) (electro-) technical workshop and an audiovisual department. B 0 CT C Otilia Dalesio MSc, Ninja Antonini MSc, Danny Baars, Vanessa Bouwman, Wilma Deurloo, Marjolijn de Waal MSc, Astrid Diemeer, Antje Fierens, Annelies Hiemstra, Lea Kooij, jan Lieverst MSc, jolanda Maaskant, Marianne Mahn-Schaefers MSc, Ingrid Mandjes MSc, (aria Modder, Anja Muusers, Hans Oosting PhD, Ellen Peter PhD, Anneke Reinders-Som, Suzanne Reitsma, jolanda Remmeizwaai, Danny Roberts, Dea Storm, Heleen Vaessen MSc, Renato Valdes Olmos, Ludy Valkenet MD, Emile van der Donk, Annemarie van der Kwast, Ivy van der Schilde, Tony van der Velde, joke van Gulik, Els van Oosterwijk, Harm van Tinteren MSc, wil van Waardenberg, Anneke Wals, Lidwina Wever, Els Willemse, Lonny Ziblat In addition to its own research activities (described separately), the Biometrics Department is the data center of the institute and provides the infrastructure for clinical and fundamental research concerning bio-statistical support, centralized patient data collection and documentation, data processing and coordinated administration and monitoring of clinical trials. The statisticians and data managers collaborate in several clinical and research projects both within the institute and for national and international multicenter studies. The department maintains a Tumor Register database, with information on patients with benign tumors, pre-malignant and malignant tumors seen in the institute since This database is a valuable resource for research currently containing more than 100,000 registrations. Between June 3,2001 and July I, 2002 a total of 5,596 new tumors were seen in the hospita!, including 4,652

141 malignancies, 50 pre-malignancies and 894 non-malignancies. Breast, lung and pleura, genitourinary tract and gastro-intestinal are the most frequently seen sites. Of the malignancies 1,266 were seen only for a second opinion, while 3,386 malignancies we re actually treated. In excess of 200 questions from investigators within the institute have been processed and data was extracted from the database which will be used in specific projects. Separate databases of several of these projects have also been developed. Support of the medical divisions for central administration, patient registration and data collection of clinical trials is provided through the departrnent's Trial Office. Between July 2001 and June 2002, 783 patients we re entered in one of the trials open to accrual. There we re 135 such studies. Forty-five percent of them we re included in studies sponsored by the institute, thirty percent in studies supported by the NKB or by the Health Insurance, and twenty-five percent in studies sponsored by the industry. Half of the patients were entered in early clinical trials (pilot, phase I and II). Two of the department data managers visited 31 institutes afmiated to the Dutch Chest Physician Organization (NVALT), across the Netherlands, to collect data on the 240 patients entered in the first randomized trial of the group. Furthermore, central registration and randomization services for ten other multi-center studies were provided. TRION, the information system on current clinical trials developed and maintained by the departrnent and made available over the Intranet, has been further improved by the standardization and inclusion of nursing protocols. The course on Medical Statistics for researchers and physicians, which has been so successful previous years, was organized for the sixth time. COMPUTERS AND NETWORK SUPPORT Tjeerd Canrinus, Evert-Jan de Kruijf, Fred Kasiander, Ton Luts, Albert Pauw, Jan Stoltz, Iija van de Pavert, Gert-Jan van der Stroom The central ICT department provides general IT support for all research divisions as weil as specific 'site wide' services (such as facilities) for all personnel. Tasks include: management of routers, Ethernet switches, me servers, job-and-database servers, configuration of network software on PCs and Apple Macintosh computers of end us ers and, if necessary, the development and maintenance of custom server andjor client side software. We are providing IT services in line with the latest technology standards. Two examples are the introduction of Gigabit Internet technology and a storage infrastructure for images. We will also intro duce Virtual Private Networks to exchange data between protected zones of the NKI-infrastructure and people outside the organization in a secure way. OGITAL MlrROSCOPY Lauran Oomen PhD, Lenny Broeks PhD The Digital Microscopy Facility provides four imaging systems: two confocallaser scanning microscopes and two microscope setups with cooled CCD-cameras. Each of these systems has its own special functionality and together they form a complementary set of instruments. A large group of researchers from all research divisions make use of one or more of these central microscope systems. This year the study of processes in living cells has become more and more important. The possibilities offered by the confocals make it the system of choice in performing high-resolution microscopie imaging ofliving cells. Most important are the study of protein synthesis, assembly, transport and dynamics in living cells, making use of the green fluorescent protein (GFP) and its analogs (YFP, CFP). These processes are followed for minutes to up to more than twelve hours. The black and white microscopejccd-camera system is equipped with a highly sensitive cooled CCD-camera and can operate in a fully automated image acquisition mode without the need of user intervention. Therefore, it is very weil suited to study very low fluorescent signals, also in time in living cells, which are difficult to be recorded on a (less sensitive) confocal system. Apart from this, the system is also

142 used to: (r) analyze genetic alterations in human tumors (comparative genomic hybridization [CGH] and fluorescent in situ hybridization [FISH] studies) and (2) the effects of radiotherapy on human tumors and normal tissue. The color microscopejccd-camera system is primarily suited to capture digital color images directly from (immuno-)histochemicaliy stained tissue sections. In the past year, the system has also been extensively used to capture bright field (phase-contrast or DIC-Nomarski) time-lapse image series from living celis (time periods of up to 20 hours). The main two subjects studied we re factors influencing celi migration and cell division. ELEC RON Jero Cal afat PhD, Hans Janssen C OSCOPY The facility consists of an FEl CMro and an FEl Tecnai r 2 G2 transmission electron microscope and Leica (cryo)-ultramicrotomes. In addition to its own research activities (see Division of Celi Biology), the facility assists researchers in their ultrastructural morphological analysis of macromolecules, viruses, cells and tissues. lmmunoelectron microscopy on ultrathin cryosections reveals a great wealth of structural details in the 2 to 100 nm range providing information that can be obtained in no other way. This technique is used for the following studies: the exact localization of antigensjmolecules in the celi organelies; localization of two different antigens simultaneously using colloidal gold of different sizes; trafficking of molecules in the cell; localization of molecules in the celi during signal transduction events and celi-celi and celi-matrix interactions. FLOW CYTO T Anita Pfauth, Frank van Diepen The Flow Cytometry facility provides instrumentation and technical assistance for flow cytometric analysis and sorting. Current instrumentation includes a multiple colour Moflo high speed sorter, a FACStar sorter for more fragile celis and sorting on DNA-content. Both sorters are equipped with three lasers and a device to perform sortings in 96-well plates. The sorting experiments are carried out by the operators.

143 The experiments on the analyzers can be carried out by the researchers themselves, after they had a training on one of the machines. The facility has four analyzers: two double-laser FACSCalibur, one single-laser FACScan and one FACScan suited to measure GFP and YFP together. Measurements on cell surface markers, cell cycle progression and apoptosis are examples of experiments possible on these analyzers. Different software packages to analyze the data are available via the research computer network. LABORATORY AN I MAL DEPARTM ENT A large part of the oncology research in the institute is carried out using rodent models (mice and rats). The Laboratory Animal Facility is located in a separate wing in order to maintain a good microbiological barrier. The departrnent provides the infrastructure for animal experiments: production and maintenance of mice and rats, rendering ofbiotechnical assistance, sanitation of strains and production of gnotobiotic animals. The facility possesses a large isolator unit for gnotobiology, sanitation and quarantine, plus a separate microbiological diagnostic laboratory for monitoring animais, with special emphasis on sick animal screening. LABORATO Y ANIMAL PATHOLOGY AND HISTOLOGY jurjen Bulthuis, Cees de Goeij, ji Ying Song, Marlon Tjin-A Koeng, Martin van der Valk PhD Due to the increasing use of animal models in tumor biology and clinical cancer research, it is expected that transgenic and knock-out mouse pathology will gain further interest, and in collaboration with clinical pathology, will play an important role in present day cancer research. Experienced technicians perform a wide range of technical procedures, including all standard methods such as tissue processing, sectioning and staining, cryosectioning and immune histology. The pathologists perform histopathology on request, either on aregular, project-related basis, or incidentally, for most research groups in the institute. The staff also gives advice on subjects involving post-mortem examination, tissue sampling, handling and processing and different staining methods. LI BRARY Suzanne Bakker MSc, Irene Benne, Ina Goede, Meena Kanhai, Truud Kroeskamp The Central Cancer Library, serving the research, clinical, nursing and paramedical departrnents of the institute, is incorporating an ever increasing number of electronic publications and services. In recent years, the journal collection expanded with electronic access to full-text over the Internet under site-license agreements. Current awareness bulletins are available as hypertext links to PubMed (including the expert search strategy) and are to be distributed by . The new library catalogue and databases are available through a web-browser interface on the Intranet. The bibliography ofnkijavl publications will be fully integrated with the library catalog, including hypertext linking to the electronic fulltext. Digital formats of pictures and other archivalia will be cataloged for easy access in the near future and further support ofthe activities ofthe 'NKIjAvL Historisch Genootschap' (The NKIjAvL Oncology History Interest and Study Group). Electronic books and applications on CD-ROM are now a substantial part of the collection; some will be made available on the Intranet, many more will be available for loan to NKIjAvL staff. Electronic discussion lists are in use for comrnunication with library users (e.g. CKB-AANVRAGEN@nic.surfnet.nl is available for requests ofliterature retrieval or interlibrary loans). The library is responsible for the annual scientometric analyses (based on impact factors and citations). The library offers training opportunities and research projects for students in library and information science.

144 C 0 A,. L T I Ron Kerkhoven PhO, Arno Velds Msc, Mike Heimerikx, Marcel Oaemen, Arenda Schuurman The production of significant numbers of high complexity human and mouse cdna micro-arrays was the main aim this past year. Production of micro-arrays increased significantly with the installation of two new and improved printing robots. We have focussed on the production of the human 18K and the mouse 15K cdna microarrays, of which 4632 have been delivered to users. The spectrum of produced arrays was extended to SNP, BAC, and oligo-arrays. This year, the Micro-array Facility fulfilled its role as a national resource of DNA micro-array technology with ongoing support from the Dutch Cancer Society. As a re sult, the facility produced and delivered 1399 cdna arrays for Dutch Cancer Society-sponsored research projects nationwide. Major study was conducted this year at our institute with the support of the Micro-array facility (Van 't Veer LJ, Dai H, Van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, Van der Kooy K, Marton MJ, Witteveen AT, Schreiber CJ, Kerkhoven RM, Roberts C, Linsley PS, Bemards R, Friend SH. Gene expression profiling predicts clinical outcome ofbreast cancer. Nature 2002; 415:484-5.). Protocols for RNA-amplification and labeling have been further developed and introduced successfully. Better post-processing protocols resulted in the production of top quality micro-arrays. Our ongoing collaboration with the National Cancer Institute in Bethesda, USA has led to the full implementation of the madb-software for micro-array data analysis and data storage (see The installation of computer hardware and software was made possible thanks to a generous grant from the Josephine Nefkens Foundation. An automated backup system was tested and subsequently implemented, which connects the madb-software to the SARA computer center, and which safeguards the precious data sets. New software tools for data normalization, data combining and extraction of significant outliers (the R-routines) have been developed in-house and made available for all users through a web based interface (see website a demonstration data set is available after login 'KWF' and password 'KWF'). For data Microarray Ana/ysis Lunginduced P o += c CL + o ~ V) Calveolin-l Cesl, surfaetant adipocy1e P27 Pecam ~1e gly Tlmp-3 ~:.r-:,i!l-.... ~:tj~~~-,--.,...,o ~ 3x ~r-.-~n. ;----,.c'r-~-i 2X IGF-II 1;1 fa 2X 3x o 19 Embryonie zeta-g bin o 16 o1~ Cl2~6 ~~ e6 010 o Stathmin Y1-globin MARCKS- like MuscIe specifie Nete1 o I Embryo induced L0g2 (average Intenslty) 16 Spot Intensity Example of a micro-array hybridization analysis. Highlighted are some significant outliers where two tissues (embryo and lung) vary in their gene expression.

145 quantification we have acquired six licenses for the Imagene software and the one for the Spotfire analyses package. The combination of improvement of micro-array production and micro-array analysis has facilitated the generation ofhigh-quality data sets for many users. Both the human and the mouse micro-arrays have proven their value in experimental research. To provide training and education, eight 2-day micro-array courses were given for 64 new us ers from a number of different research laboratories in the Netherlands. In total, we have now introduced 120 people to the micro-array research field. We have provided training to two trainee technicians and one bioinformatics student. Our website ( underwent significant revision and is now weli-adapted for the exchange of technical information and for ordering micro-arrays. A successful user group meeting was held this year and an discussion list was started at surfnet ('NKI-CMF NKI-Central Micro-array Facility Informatie voor CMF gebruikers'). The Celera database, which is hosted by the Micro-array Facility, has been updated to the latest version and is used by the NKI community to search information on the human and mouse annotated genomes and SNP coliections. ONOCL NAL ANTIBODY UNIT Els Groeneveld The Monoclonal Antibody Unit provides support and advice conceming antibody production, development and characterization. Mice and rats are used for immunization. Immunochemical techniques, such as different immunoassays, celi staining, immunohistochernistry and Western blotting, are used to characterize the monoclonal antibodies and the antibody-antigen interaction. PEPT D Henk Hilkmann SVNTH ESIS There is an ever increasing demand for peptides in the institute. In the last eleven months 223 peptides were synthesized. A new Pioneer synthesizer was installed to replace the 9050 synthesizer. This synthesizer is capable of monitor two different synthesis simultanously. This in contrast to the Syro IJ robot synthesizer which can synthesize, without monitoring, 60 different peptides. Peptides were mainly

146 produced for members of our institute but also for other research institutions and universities collaborating within the framework of the Oncology Graduate School Amsterdam. As part of routine quality control each peptide is checked by HPLC. For elucidating the cause of a less successful synthesis and for a fiuther control of the peptides, mass spectroscopy is performed in collaboration with the Free University of Amsterdam and the Departrnent of Pharmacy and Pharmacology of the Slotervaartziekenhuis of Amsterdam. Most of the peptides synthesized are used for biological studies in tissue culture or for raising antiserum, however a shift is noted for peptides used for in vitro binding experiments. Peptides used for the production of antibodies are now routinely synthesized onto a branching lysine core. The result of the multiple antigenic peptides (MAP's) is satisfactory. There is always a need for peptides with special features. Examples include the synthesis ofbiotinylated or acylated peptides, peptides containing phosphorylated amino acids and peptides existing of two chains of amino acids. It is also possible now to synthesize cyclic peptides. In collaboration with Division IV, small amounts of different peptides are synthesized on the Syro multi peptide synthesizer. RADION UCLI DE LABORATORY Henny van Rooij, Theo Lamers In addition to departrnentallaboratories licensed for radioactive work, which are present on nearly each fioor of the research building, there is a central radiochemistry facility (class C and B) available for specific and general experimental use. The staff of the Radionuclide Laboratory offers help and advice on all aspects of radioactive work. The departrnent is equipped with up to date gamma and scintillation counters, gamma analyzers, a tissue oxidizer and HPLC apparatus for online radiodetection. There are also separate facilities for animal experiments with radioactive tracers for cancer research (e.g. multidrug-resistance and metabolic studies of rádiopharmaca). The departrnent provides regular courses on Radiation Protection, level5b, for all new personnel (including students and guests) whose work entails the use of radioactive material. There are also practical courses level 4B for therapeutic laborants of the Inholland College. SEQUENCE FACILITY Frans Hogervorst PhD, Rob Plug, Roelof Pruntel The Sequence Facility offers a service for DNA sequence and fragment analyses to the users in the research departrnents and the DNA-diagnostics laboratory of the Departrnent of Pathology. The Sequence Facility has an important role in the diagnostic analyses of patient samples and therefore its procedures and protocols are qualified by Sterlab. The facility is equipped with an ABI 3700 capillary sequence machine, which can handle up to 96 samples simultaneously and a 377XL slab-gel automated sequencer. Also this year, we noticed an increasing number of samples to be analyzed, at the moment 3500 samples per month. To ensure the continuity of the facility and to assist researchers with the implementation of novel applications, a second technician has recently joined the team. Furthermore, we have introduced a purification step of the cycle-sequenced samples, which has resulted in better quality and long er reads of the samples. In total more than 130 researchers representing all divisions make use of the service provided by the facility. TRANSGENIC MOUSE SERVICE Paul Krimpenfort PhD, Rahmen Bin Ali, Fina van de Ahe The Transgenic Mouse Service and Animal Departrnent are currently in the process of reorganizing their activities involved in the production of genetically modified mouse strains. The aim of this reorganization is to allow the Transgenic Mouse

147 Service to invest more time in the development of new technologies and to share responsibilities with the Animal Department. The consequence of this change is that the Transgenic Mouse Facility will now carry out the actual embryo manipulation, while the Animal Department will provide as si stance during the routine procedures such as embryo isolation and transfer. With respect to technology development, the Transgenic Mouse Facility will focus on a) in vitro fertilization using cryo-preserved sperm, b) the testing of new ES celllines for the generation of modified strains with a more suitable gene tic background and c) the development of alternative methods for transgenesis. Recently, we have started to use recombinant Lenti viruses for the transfer of exogenous DNA constructs into pre-implantation embryos. STAFF GENERAL RESEARCH FACILITIES: Cryogenic storage: Minze Dijkstra, Erwin Kambey Glassware kitchen: Trees Holman-Van Doorn, Moustapha Aboutalib, Esther Holman, Erwin Kambey, Harry Kempff (Electro)-Technical workshop: Rob Van der Weiie, Kees Sier, Sanny Kraan, Rob Gans, Dennis Roedoe Audiovisual department Martin Lomecky, Simone Bakker, SM Drent je, ARM Jagt, ER Tragter

148 CLINICAL TRIALS IN THE NETHERLANDS CANCER INSTITUTE

149 liiector. esearc1lantanjl~ms type of study coordinator ph ase activated no pts. cancer study in AvL (in italics: inter- (closed) in AvL national coordinator) per 30. ALL SITES MooAPC A phase I study to determine the safety of AP 5280 as i.v. Schellens, j.h.m infusion once every 3 weeks in patients with a solid tumor ( ) MooROG Phase Ijll study of Ro , a novel cell cycle inhibitor, Zandwijk van, N. Ijll administered in escalating oral doses in combination ( ) with i.v. gemcitabine in adults with solid tumors, and at MTD in first line NSCLC M010CC Dose-finding phase I clinical and pharmacokinetic study Schellens, j.h.m of orally administered irinotecan (CPT-ll) once daily for 14 days as single agent or in combination with capecitabine twice daily for 14 days every three weeks in patients with advanced solid tumors M02GFT A Phase I, Randomized, Open-Label, Parallel-Cohort, Schellens, j.h.m Dose-Finding Study of Elacridar (GF120918) in Combination with 2.0 mg Oral Topotecan in Cancer Patients M02LGC A phase I clinical and pharmacokinetic evaluation Schellens, j.h.m of oral LY in combination with Gemcitabine and Cisplatin in patients with advanced cancer M02TTA A clinical trial on TopoTect (dexrazoxane) in the Schornagel, j.h. lil treatment of accidental extravasation of antracycline ant-cancer agents M97TIF A prospective, open-label, non-comparative Schellens, j.h.m two-center ph ase I trial to evaluate the dosage and ( ) safety ot topotecan administered as daily x 5, 30 minutes intravenous infusions every 3 weeks in combination with ifosfamide in patients with advanced malignancies M99DIL Safety and tolerability of long-term administration Vielvoye-Kerkmeer, A.P.E. lil of Dilaudid SR (Hydromorphone HCI) in cancer pain NooCEP The effect of the co-solvent Cremophor EL on the oral Schellens, j.h.m bioavailability of paclitaxel in combination with CsA ( ) NooRGC Phase I maximum tolerated dose (MTD) trial to Schellens, j.h.m determine the safety and pharmacokinetics of 7 days oral administration offarnesyl transferase inhibitor Rl15777 N01DLY A ph ase I dose ranging study of LY Schellens, j.h.m administered in combination with docetaxel ( ) N01DXR Oral bioavailability of docetaxel in combination with Schellens, j.h.m OCl ( ) N010RH Evaluation of pharmacokinetics and safety after oral Schellens, j.h.m dose R115777, in at least 21 cancer patients with normal, ( ) mild and moderate impaired, hepatic function NOlPER A clinical ph ase I study of combined treatment with Verheij. M the alkyl-iysophospholipid Perifosine and ionizing radiation in patients with locally advanced solid tumors

150 type of study coordinator phase activated no pts. ca neer study in AvL (in italics: inter- (closed) in AvL national coordinator) per N02MET Mass balance study with ET-743 as an 3 or 24-hous Schel lens, j.h.m intravenous infusion to patients with advanced cancer N97PIF Inter- and intrapatient variation in disposition of Schellens, j.h.m ifosfamide and its metabolites in various treatment ( ) schedules N97PPA Pharmacokinetic study of a comparison of blood Schellens, j.h.m sampling of paclitaxel in different sampling ( ) N98CTO A phase I study to determine the maximum tolerated Bokkei Huinink, w.w. ten doses of Carboplatin and Topotecan administered intravenously every 28 days to patients with malignant tumors. N98NAM Phase land pharmacologic study with NAM I-A, a novel Schel lens, j.h.m ruthenium anti- cancer agent BRAIN I CNS NooGL! Temozolomide and combined immunotherapy in Boogerd, W. 1/ malignant glioma BREAST E10951 Randomized phase II study in first line hormon al Schornagel, j.h. II treatment for meta- static breast cancer with Exemestane ( ) or Tamoxifen in postmenopausal patients E A double-blind phase III clinical trial to compare the Oldenburg, H III effects of a pre-operatively administered single dose of ( ) Faslodex (Iong-acting ICI ) with placebo on tumor recurrence in pre- and postmenopausal women treated for operabie first primary breast cancer E10981 After Mapping of the Axilla: Radiotherapy Or Surgery? Rutgers, E.j.Th. III E22922 Phase III randomized trial investigating the role of Bartelink, G.M.M. III internal mammary and medial supraclavicular Iymph node chain irradiation in stage breast cancer MooHDO Open label, comparative, randomized, multicenter, Schornagel, j.h. II multinational study of Herceptin (trastuzumab) given ( ) with docetaxel (Taxotere) versus docetaxel as a single-agent in first-line metastatic breast cancer patients with H ER2-neu overexpression MooPCM Phase II study of weekly Paxoral (Oral Paclitaxel) Schellens, J.H.M. II with Cyclosporin A for advanced breast cancer M01VIC Phase I study of oral Vinorelbine in combination with Schellens, j.h.m oral Cyclophosphamide in patients with metastatic breast cancer M02TEA An open label, randomized multicenter comparative Oldenburg, H III trial of 5 years adjuvant Exemestane treatment versus 5 years adjuvant Tamoxifen treatment in postmenopausal women with early breast cancer

151 Director of Research Anton Berns type of study coordinator phase activated no pts. cancer study in AvL (in italics: inter- (closed) in AvL national coordinator) per M96ATLAS Reliable assessment of the efficay and safety of Rutgers, E.j.Th. III prolonging the use of adjuvant tamoxifen; a large simple randomized study M99BLU Lymphatic drainage analysis through preoperative Nieweg, O.E. Pilot intradermal injection of blue dye in patients undergoing a modified radical mastectomy M99XEL Treatment guideline ofxeloda (capecitebine) in patients Schornagel, j.h who have failed or are resistant to previous treatment ( ) with paclitaxel (Taxol) or docetaxel (Taxotere) for locally advanced andjor metastatic breast cancer. NooDCD Single-institution randomized study of doxorubicin- Rodenhuis, S. III cyclophosphamide versus doxorubicin-docetaxel in locally advanced breast cancer NooRLY Reproducibility oflymphoscintigraphy for Iymphatic Nieweg,O.E. Pilot mapping in patients with breast carcinoma N01NIG Nutrients en insulin-like growth factor (IGF) system Vosku il, D.W. Pilot N02SNB Validation of the sentinel node procedure in patients Nieweg,O.E with a breast les ion after prior excisional biopsy N99FCO Feasibility and Phase 11 Study offec-tctc-otax Rodenhuis, S in Hormone-Refractory Stage IV Breast Cancer PooCOG Psycho physiological study of long-term cognitive deficits Boogerd, W as a consequence of high- dose chemotherapy: study in high-risk breast cancer patients PooMRI MRI-scan bij patienten met borstkanker ter bepaling Gilhuijs, K.GA van de afmetingen van de afwijking P99SHB Impact van regelmatige controle (screening) bij vrouwen Rutgers, E.j.Th met een verhoogd risico op borstkanker vanwege een familiaire predispositie GASTRO INTESTI NAL E22921 Four arms phase III clinical trial for T3-T 4 resectable Aleman, B.M.P rectal cancer comparing operative pelvic irradiation ( ) to pre-operative irradiation combined with Fluorouracil and leucovorin with or without post-operative adjuvant chemotherapy M01BAX POCASTER trial (J-POuch- Colo-Anale anastomose Zoetmulder, FAN versus Side-To-End colo-rectale anastomose na preoperatieve radiotherapie en Totale Mesorectale Excisie (TME) bij Rectumcarcinoom; een multicenter, gerandomiseerd onderzoek M01EPC An open-label ph ase lia trial evaluating the safety and Bokkei Huinink, W.w. ten II efficacy of EP0906 as therapy in patients with advanced ( ) colorectal cancer

152 type of study coordinator phase activated no pts. cancer study in AvL (in italics: inter- (c1osed) in AvL national coordinator). per M99CCC CPT-11 in combination with Capecitabine as first line Bokkei Huinink, w.w. ten chemotherapy for metastatic colorectal cancer M99 ESB Palliatie bij patienten met passageklachten t.g.v. een Aleman, B.M.P inoperabel slokdarmcarcinoom: plaatsing van een ( ) zelf-ontplooi bare stent of brachytherapie in de slokdarm NooSNC Lymphatic mapping and Sentinel Node Biopsy in colonic Coevorden, F. van Pilot cancer NOlDNR Onderzoek naar een dag/nacht ritme en de invloed van Taal, B.G dagel ijkse bezigheden op de uitscheiding van s-hiaa ( ) in de urine bij patienten met carcinoid N02ECC A single institution phase 11 study ofecc (Epirubicin, Boot, H Cisplatin, Capecitabin) in locally advanced or metastatic gastric cancer and adenocarcinoma of the oesophago-gastric junction N98ECF A phase 11 feasibility study of ECF (Ep irubicin, Cisplatin Taal, B.G and continuous 5-FU) in locally advanced metastatic ( ) gastric cancer and adenocarcinoma at the oesophagogastric junction N99SAN Conversion of short-acting octretide to the long-acting Taal, B.G compound octreotide (SandostatinLAR) : a phase 11 dose finding study in metastatic carcinoid tumors GYNAECOLOGICAL MOlEPO An open-label phase Ila trial evaluating the safety and Bokkei Huinink, w.w. ten efficacy of EP0906 in patients with advanced ovarian, ( ) primary fallopian, or primary peritoneal cancer M02MCA Measurement of cisplatinum adduct in patients with Verheij. M. Pilot cervical cancer treated with chemo radiotherapy N99 FMO Study of fluorescein for the detection of metastases of Vange, N. van der Pilot ovarian tumor in the abdominal cavity ( ) HEAO ANO NECK MooCET Randomized phase 111 trial to compare radiation therapy Verheij. M alone with radiation therapy and concomitant anti-egfr ( ) anti body (Cetuximab) for locally advanced squamous cell carcinomas of the head and neck MooQPI Hypoxia in head and neck tumors: quantification using Balm, A.J.M pimonidazole and IdUrd M99 RAD Phase 111 multi-institutional trial of targeted supradose Rasch, C cisplatin chemoradiation versus systemic chemo radiation in locally advanced squamous cell carcinoma of the head and neck NooVOX Development and clinical assessment of the Hilgers, F.J.M Provox FreeHands H M E tracheostoma valve

153 ectm:4 :search nton..b.ems type of study coordinator phase activated no pts. ca neer study in AvL (in italics: inter- (closed) in AvL national coordinator) per N01TSI Effecten van behandeling op longfunctie van patienten Zandwijk van, N. Pilot met een tracheoatoma met inhalatie medicatie ( ) (salbutamol-ipratropiumbromide dosisareosol) met en zonder voorzetkamer N02SNL Early detection of Iymph node metastasis of squamous Lohuis, P.j.F.M. Pilot cell carcinoma of the larynx with Iymphatic mapping and sentinel node biopsy N96-126S NKB S ; Prediction of radiotherapy response using Balm, A.j.M. Pilot FISH to assess chromosome damage in tumors and ( ) normal tissue N98RTB Carotid arterial acclusive disease following radiation Boogerd, W. 1/ therapy; A prospective study in patients with a parotid t umor N99BRU A clinical evaluation to assess the safety and Balm, A.j.M biodistribution oftc-99mbru S9-21 in identifying areas ( ) of tumor hypoxia in patients with head and neck cancer LEUKAEMIA I MOS M01H43 Randomized induction and post induction therapy in Baars, j.w. III older patients (>= 61 yrs of age) with acute myelocytic leukemia (AM L) and refractory anemia with excess of blasts (RAEB, RAEB-t) M01Hso A randomized phase III study on the effect of thalidomide Baars, j.w. III combined with adriamycin, dexamethason (AD) and high dose melphalan in patients with multiple myeloma M99H37 Early intensifkation by (un)related allogeneic autologous Baars, j.w. II stem cell transplantation in adult acute Iymphoblastic leukemia. HOVON 37 - CKVO MHOVON20 A randomized ph ase III study comparing low dose versus Baars, j.w. III high dose interferon alpha-2b (Intron A) both combined ( ) with adapted doses ofhydroxy-urea in chronic myeloid leukemia N98LPB De Lymfomen Plasma Bank Haas, R.L.M LUNG E08941 Randomized trial of surgery versus radiotherapy in Zandwijk van, N. III patients with stage lila NSCLC after a response to induction chemotherapy E08972 A randomized phase III study comparing induction Beiderbos, j.sa III chemotherapy to daily low dose cisplatin both combined with high dose radiotherapy in patients with inoperable NSCLC stage I, II and low volume E08984 Taxotere and cisplatin as induction chemotherapy in Zandwijk van, N. II patients with stage lila, N2 non small celilung cancer (NSCLC)

154 type of study coordinator phase activated no pts. ca neer study in AvL (in italics: inter- (closed) in AvL national coordinator) per 30. ~ -. ',-'.. '... J..' - '.~ ,T... I j MOlALl A randomized phase 3 trial comparing All MTA plus best Baas, P supportive care versus best supportive care alone in previously treated patients with locally advanced or metastatic pleural mesothelioma M01FLU The influence of f1uticasone inhalation on intermediate Zandwijk van, N markers of carcinogenesis in the bronchial epithelium of a high risk population: A double blind placebo-controlled randomized phase 11 study M01NLU Pre-operative chemotherapy in resectable NSCLC Baas, P M01TCY The international Tirazone Triple Trial (i3t). A ph ase 111, Zandwijk van, N randomized efficacy and safety study of the combination ( ) chemotherapy with Tirapazamine+Cisplatin+Yinorelbine ' versus Cisplatin+Yinorelbine in subjects with inoperable, previously untreated, Non-Small Cell Lung Cancer M98CCR Combined chemotherapy (Carboplatin, Paclitaxel, Baas, P Etoposide) and concurrent radiotherapy as first line ( ) treatment in Smal I Cell Lung Cancer Limited Disease M98TDR A phase Ijll dose escalation study using three Beiderbos, j.s.a. Ijl I dimensional conformal radiation therapy in patients with inoperable non-small celilung cancer M99GMA International multicenter randomized phase 11 study Zandwijk van, N evaluating the feasibility of concomitant chemotherapy ( ) and radiotherapy or radiotherapy alone, following an induction chemotherapy regimen in patients with locally advanced, unresectable NSCLC (st. lila, multiple N2jIIIB) M99HER An open-label randomized controlled phase 2 study of Zandwijk van, N Herceptin (trastuzumab) in combination with ( ) chemotherapy in patients with H ER-2 over- expressionj amplification in advanced andjor metastatic Non-Small-Cell Lung Cancer (NSCLC) - Randomized part M99PET Positron emission tomography in the assessment of Zandwijk van, N neoadjuvant chemotherapy for locally advanced NSCLC ( ) M99POQ Randomized study in NSCLC comparing conventional Zandwijk van, N and FDG PET staging, the POORT study ( ) NooALF Endoscopic detection op pre-neoplastic lesions and Baas, P. Pilot carcinoma in the bronchial tree with delta-aminolevunic acid f1uorescence NooTHM Phase 11 trial of the antiangiogenic agent Thalidomidei in Baas, P patients with malignant pleural mesothelioma NOl PAL Feasibility study of targeting the plasma paclitaxel Schellens, j.h.m concentration above 0.1 umoljl during a defined exposure-time in patients with Non-Small Cell Lung Cancer N02GLM Phase 11 study of Glivec in malignant mesothelioma Zandwijk van, N

155 irector..dj: esearch Anton.Bems type of study coordinator phase activated no pts. cancer study in AvL (in italics: inter- (closed) in AvL national coordinator) per N98CIG Phase I study of dose-intensive Cisplatin + Gemcitabine Schellens, j.h.m in NSCLC N98MDM A phase II feasibility study of high dose Methotrexate Baas, P. II with or without Doxorubicin in malignant mesothelioma ( ) N99HIM Cytoreduction and Hyperthermic Intra-Thoracic Zoetmulder, FAN. Pilot Chemotherapy (H ITHOC) in patients with pleu ral metastases of thymoma or limited mesothelioma Lymphoma - Hodgkin's Disease E20011 A randomized trial of BEAM plus PBSCT versus single Baars, j.w III agent high dose therapy followed by BEAM plus PBSCT in patients with relapsed Hodgkin's disease E20982 Prospective controlled trial in clinical stages 1-11 Baars, j.w III supradiaphragma- tic Hodgkin's Disease. Evaluation oftreatment efficacy, (long term) toxicity and quality of life in two different prognostics groups. Trial H9 NooMEG Ex vivo expanded megakaryocytes and their precursor Baars, j.w cells to flil the platelet gap after high-dose chemotherapy N98LPB De Lymfomen Plasma Bank Haas, R.L.M LYMPHOMA - NON-HODGKIN'S E20981 Chimeric anti-cd20 monoclonal anti body (Mabthera) Baars, j.w III in remission induction and maintenance treatment of relapsed follicular non-hodgkin's Iymphoma: a ph ase III randomized clinical trial MOlH46 A randomized phase III study of chimeric anti-cd20 Baars, j.w III monoclonal antibody (Rituximab) with 2-weekly CHOP chemotherapy (CHOP14) in elderly patients with intermediate- or high-risk non-hodgkin's Iymphoma M01H47 Chlorambucil versus 2X2 Gy involved field radiotherapy in Haas, R.L.M. III patients Stage lil/iv previously untreated follicular Iymphoma M02H44 A randomized phase III study on the effect of chimeric Boer, de, j.p anti-cd20 monoclonal anti body (MabThera) during sequential chemotherapy followed by autologous stem cell transplantation in patients with relapsed B-cell non-hodgkin's Iymphoma M96FNL Treatment with BEAM and total nodal irradiation, both Baars, j.w Pilot followed by PSCT support as consol idation after remis sion induction for patients with high risk relapsed or refractory low grade (including certain subtypes intermediate grade) non-hodgkin's Iymphoma. A feasibility study M99H26 Intensifled CHOP of 12-weeks duration plus G-CSF as Baars, j.w III compared with standard CHOP of 24-weeks duration for patients with intermediate prognosis non-hodgkin's Iymphoma (HOVON26)

156 type of study coordinator phase activated no pts. cancer study in AvL (in italics: inter- (closed) in AvL national coordinator) per M99H40 Intensified CHOP followed by triple high-dose Baars, j.w chemotherapy with autologous stem cell support as ( ) first line treatment for high-risk Non-Hodgkin's Iymphoma: A Ph ase II study NooMEG Ex vivo expanded megakaryocytes and their Baars, j.w precursor cells to fill the platelet gap after high-dose chemotherapy N97HOR Low dose radiotherapy (2X 2 Gy) in low grade Haas, R.L.M. II Non-Hodgkin Iymphomas N98LPB De Lymfomen Plasma Bank Haas, R.L.M MELANOMA I SKIN MooCIS Chemo-immunotherapy as neo-adjuvant and adjuvant Gast, G.C de therapy in combination with surgery in in-transit or clinically involved Iymph node metastases of malignant melanoma MooTIM Temozolomide adjusted dose with or without Gast, G.C de III combined immunotherapy with GM-CSF, IFN-alpha and low dose IL-2 in patients with metastatic melanoma. M94MSL A multicenter clinical study of wide excision plus Nieweg,O.E. III intra operative Iymphatic mapping with selective ( ) Iymphadenectomy versus wide excision ofthe primary melanoma in the treatment of patients with melanoma NooDER Specific immunotherapy bij intradermal vaccination with Haanen, j.bag tumor-(associated) peptides and GM-CSF in melanoma. A Phase Ijll study NooPEM Aanvullende waarde van positron-emissie tomografie Nieweg,O.E. Pilot met FDG bij de opsporing van melanoom metastasen N01TMM Protracted daily temozolomide patients with metastatic Gast, G.C de melanoma and poor prognosis N99TPC mthpc mediated PDT and basal cell carcinoma: a Baas, P. Ijll phase Ijll study to determine optimal treatment parameters and its related pharmacological profile M ISCELLAN EOUS M02BMC Bruikbaarheid van MR-angiografie (MRA) en Hage, j.j color-flow-doppler voor de beoordeling van cruropedale arterieën voorafgaand aan de microchirurgische transplantatie van een fibula lap M02RFA None Coevorden, F. van Pilot N01RIT Identifications of molecular mechanisms involved in RusselI, N.S. Pilot radiation-induced telangiectasia

157 irecto. of. search-anton.bern type of study coordinator phase activated no pts. cancer study in AvL (in italics: inter- (closed) in AvL national coordinator) per N99EOL Endoscopisch echo-onderzoek van de centrale Baas, P. Pilot luchtwegen onder narcose: feasibility study NIET WMO STUDIES NWMOO1 DELTA study: Eloxatin, 5FU and folinic acid in patients Boot, H. IV not pretreated for metastatic colorectal cancer SOFT TISSUE I OSTEOSARCOMA E62005 Ph ase III randomized, intergroup, international trial Rodenhuis, S. III assessing the cl inical activity ofsti-571 at 2 dose levels ( ) in patients with unresectable or metastatic gastrointestinal stromal tumors (GIST) expressing the KIT receptor tyrosine kinase (CD117) E62011 Efficacy and safety of Brostallicin in patients with locally Rodenhuis, S. II advanced or metastatic soft tissue sarcoma failing one prior chemotherapy regimen E62931 Adjuvant high dose Doxorubicin, Ifosfamide and Rodenhuis, S. III Filgrastim in patients with high grade soft tissue sarcoma E62971 Randomized Phase III trial oftwo investigational schedules Rodenhu is, S. III of Ifosfamide vs standard dose Doxorubicin in patients ( ) with advanced or metastatic soft tissue sarcoma M01EUR EURO-E.W.I.N.G. 99. EUROpean Ewing tumor Working Rodenhuis, S. III Initiative of National Groups M01ROS Ph ase II study of rosiglitazone in advanced liposarcoma Rodenhuis, S. II SOFT TISSUE I OSTEOSARCOMA X01STI Open-label trial of Glivec in patients with unresectable Rodenhu is, S. IV or metastatic malignant gastrointestinal stromal tumors ( ) expressing c-kit URO GENITAL E30983 Randomized phase lijlil study oftaxol-bep versus BEP Schornagel, j.h. Iijll in patients with intermediate prognosis germ cell cancer E30986 Randomized phase lijlil study assessing Gemcitabinej Schornagel, j.h. Iijll Carboplatin and MethotrexatejCarboplatinjVinblastine in previously untreated patients with advanced urothelial cancer ineligible for Cisplatinum based chemotherapy E30987 Randomized phase III study comparing paclitaxelj Schornagel, j.h. III cisplatinjgemcitabine and cisplatinjgemcitabine in patients with metastatic or Ically advanced urothelial cancer without systemic therapy MooLMT Identification of occult Iymph node metastases in Horenbias, S. Pilot testicular cancer to select patients for adjuvant treatment. Feasibility of a laparoscopie selective retro peritoneal Iymphadenectomy

158 type of study coordinator phase activated no pts. ca neer study in AvL (in italics: inter- (closed) in AvL national coordinator) per 30. Q -.',.~ " J - ', M01CHI Chimerism-inducing immunotherapy in patients with Haanen, ),BAG, advanced renal cell carcinoma M01IGR I mage-guided radiotherapy of the prostate, A study to Lebesque, )V determine the intra-fraction motion of the prostate and normal tissues in patients receiving external beam radiation therapy for adenocarinoma ofthe prostate M01MMI Improvement of Delineation quality in conformal Rasch, C Pilot radiotherapy of the prostate through multi-modality imaging M94SAL Salvage regimen incorporating repeated ablative Rodenhuis, S chemotherapy with autologous PSCT, a phase 11 study M97RAD Ph ase 111 study for prostate cancer, randomizing between Lebesque, )V two radiation dose levels (68 Gy vs 78 Gy) and utilizing three dimensional conformal radiotherapy (CKVO 96-10) NooKAH Phase I clinical and pharmacokinetic study to determine Schellens, ),H,M, the safety of Kahalalide F administered as a daily x 5 over 1 hour infusion every 21 days in patients with advanced or metastatic prostate cancer NooPRO Laparoscopically assisted peri neal prostatectemy Meinhardt, W. Pilot NooVES Laparoscopische bekkenklierdissectie met vesiculectomie Meinhardt, W. Pilot N01PEG PEG-Intron in metastatic renal cell carcinoma: Gast, G,c' de A phase 11 study of the Netherlands Cancer Institute N01TMR Temozolomide in metastatic renal cell carcinoma Gast, G,c, de N02SBC Feasibility study of sentinel node Iymph scintigraphy Meinhardt, W. Pilot and radio guided surgery for the Iymph node dissection prior to cystectomy for bladder carcinoma N02VBB Volume dependence of bladder shape Moonen, LM,F, Pilot N97PCI Pre-operative combined immunotherapy in renal cell Gast, G,c, de Pilot carcinoma

159 INVITED SPEAKERS Alber M, Germany Can PET-hypoxia imaging be used for biologically conformal boosts in Head 'n Neck tumours? Albert M, USA Antigen cross-presentation and tumor immunity. Benitah S, Spain Regulation of Stat3, Statsa and NFkB transcription factors by Rho GTPases. Implications in Rho-induced neoplastic transformation. Blasco M, Spain Telomeres and chromosomal stability: cancer and aging. Bradley A, UK Cancer predisposition by elevated recombination in Bloom mice. Buchwald G, Germany How the Salmonella toxin SopE activates Rho-GTPases. Bussemaker Hl. USA Modeling micro array data in terms of transcription factor activities. Cella D, USA From quality-of-life measurement science to clinical application. Cl arke R, UK Human mammary stem cells. Clevers H, The Netherlands APC, signal transduction and genetic instability in colorectal cancer. Daemen M, The Netherlands Unique gene expres sion of unstable atherosclerotic plaques. Daemen T, The Netherlands The virosome concept. Dai H, USA Microarray data acquisition and data interpretation. Damalas A, Israel Deregulated beta catenin activates the PS3 pathway De Marco V, Germany Heterodimerization of Xklp3A/B: insights into a novel mechanism for coiled-coil formation. Dikomey E, Germany Individual radiosensitivity: clinical implications and biological mechanisms. Ellenberg 1. Germany Nuclear envelope and chromosome dynamics in living cells. Engstler M, Gerrnany Secretion of G PI -anchored proteins: visualization and analysis by RNA interference. Fayers P, Scotland - Analysis of quality-of-life data in clinical trials; - Quality-of-life research designs; sample size issues. Fischer A, France SCID: Block in T cell differentiation, from mechanisms to therapy. Fishel R, USA Examining the mechanism of mis match repair. Flavell RA, USA Regulation of the immune, autoimmune and antitumor immune response by TG F beta. Fortes P, Spain Inhibiting expres sion of specific genes in mammalian cells using modified UI snrnps. Galjart N, The Netherlands CLIP-IIS: linking microtubules to behaviour? Hahn S, USA Farnesyltransferase inhibitors in (pre)clinical studies. Hoeijmakers 1. The Netherlands Regulation of nucleotide excision rep air in mammais. Hoogenboom H, Belgium Introduction to a mature antibody display technology platform and its applications for the isolation of MHC-peptide specific antibodies. Howell S, USA DepoCyte for the treatment oflymphomatous and solid tumor neoplastic meningitis. Ishikawa T, Japan Human ABC transporters. From functional genomics to drug molecular design. J ackson S, UK Mechanisms for detecting DNA damage. Jacobs H, The Netherlands Secondary diversification of Ig genes: transcription DNA double-strand breaks and error prone repair. Jallepalli P, USA Chromosomal instability in cancer: cutting through the mystery. Jones Y, UK Structural insights into ligand binding sites: the strong, the weak and the very, very specific. Jonkers 1. UK Mammary tumorigenesis in conditional tumor suppressor gene knockout mice: disease progression, gene discovery and cellular pathways.

160 Kind P, UK - Health utilities: implicit values and their effects on health-related quality-of-life measures; - Health utilities: expliciet values and their measurement. Korswagen R, The N etherlands Wnt signaling and celi migration in C. elegans. Kurts C, Germany Investigation of antigen presentation and T cell tolerance in transgenic mice. Leone GW, USA Rb function in the placenta is essential for embryonic development and viability. Lukas J, Denmark Cell cycle checkpoint response to DNA strand breaks: molecular mechanisms and spatio-temporal dynamics in mammalian cells. Mäkelä T, Finland Mouse model ofpeutz-jegher Syndrome. McNiven MA, USA The dynamin family of GTPases: key players in membrane and cytoskeletal dynamics. Melchior F, Germany Smali Ubiquitin-related Modifier SUMO-I. 'Big' functions for a small protein. Mons B, The Netherlands Don't read us, we will read you: computer-assisted conceptual meta-analysis oflarge numbers ofbiological publications. Nakatani Y, USA A 600 kda microtubule-associated factor that contributes to genomic stability. Niessen C, Germany Regulation of celi-cell adhesion and celi sorting mediated by cadherins. O'Garra A, UK Development and function of IL-IO-producing regulatory Teelis. Oosterhuis JW, The Netherlands Pluripotency in germ cell tumours. Osoba D, Canada - How to design clinical trials with quality-of-life outcomes; - How to assess the clinical significanee of quality-oflife outcomes. Ovaa H, USA The ubiquitin-proteasome system: chemistry-based functional proteornies to target deubiquitinating enzymes. Plasterk R, The N etherlands A genome wide RNAi screen for genes that proteet the genome against mutations. Princiotta MF, USA Kinetics of peptide-mhc complex production from model protein substrates. Rapp U, Germany MALDI TOF in Proteomics: principals and new developments. Reiner S, USA How celis decide, and remember, their fate. Rucker, V, USA Control of gene expression and fluorescent detection of dsdna by smal!, sequence-specific dsdna binding molecules. Sause WT, USA Phase 111 lung cancer efforts in the USA. Schoenberger S, USA Programming of CD8+ T cell responses. Scully R, USA Recombination functions ofbrcar and BRCA2. Shenoy A, India - N ear totallaryngectomy; - Quality oflife in HN cancer patients, a disabilityrelated issue? Taya Y, Japan Regulation of the functions of P53 and the RB protein by phosphoryla tion. Taylor S, UK Quality control mechanisms which ensure accurate chromosome segregation. Tsien R, USA Genetically encoded reporters of signal transduction and protein sociology. Van den Heuvel S, USA Developmental control of cell division. Van Haastert PJM, The Netherlands Signal transduction in four dimensions: chemotaxis in Dicteostelium. Vidal M, USA From genomes to systems biology. Weppler SA, The Netherlands Role of the Pl3-kinase pathway and translation initiation factors in HIF-ra expression: impact on tumour angiogenesis? Wilmanns M, Germany Differential activities of transcription factors of the Oct family by DNA induced interface swapping. Xu Y, USA Bioactive lysophospholipids, receptors and cancer. Yan D, USA Image-guided radiotherapy of non-small celllung cancer: new technical developments.

161 PROJECTS SUPPORTED BY THE DUTCH CANCER SOCIETY Number of Title Div. Principal Starting Ended project I nvestigator Date NK The role of the sphingomyelin signal transduction pathway in radiation-induced apoptosis. IX M Verheij H Bartelink June'97 May '02 11 I W Van Blitterswijk NKI Implementation of a Pain Education Program (PEP) for cancer patients with chronic pain by nurses. XII X F Van Dam J Passchier A Vielvoye-Kerkmeer Dec '98 NKI Chemokine receptor homologs: role in dissemination of hematopoietic malignancies. XIII E Roos D De Jong Jan '98 JUly'02 NK Patient setup and treatment verification for conformal t herapy using dynamic beam intensity modulation. IX M Van Herk K Gilhuijs Jan '98 Apr '02 NKI Quality oflife assessment in ethnic minorities cancer patients in the Netherlands: A study of the SF-36, the COOP jwonca charts, XII N Aaronson Nov '98 the EORTC QLQ-C30 and the Rotterdam Symptom Checklist. NK Long-term cognitive deficits as a consequence of high-dose chemotherapy: a role in high-risk breast cancer patients and high-grade Iymphoma patients. XII X F Van Dam W Boogerd Febr '98 NK Rho signaling: role in differentiation and transformation. lil W Mooienaar Od '98 NK Genetic alterations involved in local recurrence and progression of invasive and non-invasive breast cancer after breast conserving therapy. XIII IX M Van de Vijver J Peterse H Bartelink Oct '98 Oct '02 NK Pharmacology of orally administered taxanes: a preclinical mechanism study and clinical evaluation. X J Beijnen J Schellens Sept '98 Sept '02 NKI Dose-volume-effect relationships for local control and normal tissue complications for prostate patients treated with three-dimensional conformal radiotherapy. IX J Lebesque L Boersma P Koper Ju1Y '98 NKI Clinical implementation of conformal radiotherapy of lung cancer using dynamic beam intensity modulation. IX B Mijnheer E Damen Nov '98 Nov '02 H Bartelink NK The SCC-1 (Susceptibility to colon cancer - 1) gene: isolation and functional analysis. VII P Demant MaY'98 Dec '02 NK Long-term risk of second cancer following treatment of Hodgkin's disease, testicular cancer and breast cancer. XII IX F Van Leeuwen BAleman June '98 Different response of RER+ and RER- tumors to chemotherapy. V X H Te Riele S Rodenhuis Jan '98 Dec '02 XIII D De Jong NK The role oftiam1 in E-cadherin-mediated homophilic adhesion. J Collard MaY'98

162 Numberof Title Div. Principal Starting Ended project Investigator Date NK Risk assessment and gene-environmental interactions in breast XII F Van Leeuwen Jan '98 andjor ovarian cancer families. XIII L Van 't Veer NK The psychological and behavioral impact of genetic counseling for XII N Aaronson May'98 colorectal cancer: a prospective multicenter study. X F Menko B Taal KUN Trinucleotide repeat expansion and human cancer: the role of MSH3 V H Te Riele May'99 and FEN-1 in unpaired DNA processing. B Wieringa J Hoeijmakers NK Transport of taxoids across the blood-brain barrier and CNS-tumor X J Beijnen Sept '99 penetration: a preclinical pharmacologic study and clinical XIII o Van Tellingen 'proof of concept' testing in glioma patients. W Boogerd NK Quantitative assessment of treatment margins and implementation IXjXll1 K Gilhuijs Sept '99 of multimodality imaging in breast conserving therapy. XI E Rutgers IX H Bartelink NK Molecular analysis of the cytotoxic T cell response. IV T Schumacher Nov '99 A Kruisbeek NK The role of the integrin alpha6jbeta4 in signal transduction A Sonnenberg Aug '99 controlling cell proliferation. NK G protein control of ce 11 proliferation and transformation. III W Mooienaar Dec '99 NK Dose escalation study for non-small celilung cancer (NSCLC) IX J Lebesque April '99 using three-dimensional conformal radiotherapy with tight treatment J Beiderbos margins and functionally optimized radiation treatment plans. L Boersma NKI Induction of apoptosis by alkyl-iysophospholipids and related III W Van Blitterswijk Dec '99 anticancer agents. IX M Verheij NK Characterization of antigen specific and biological responses of IV H Spits Jan '99 CD4+ melanoma specific T cells. X G De Gast NK Modulation of normal human T cell development by ectopic IV H Spits April '99 expression oftal-1, -2 and LYL in T cell progenitor ceiis. NK Structure-function studies of cyclin Dl in complex with nuclear II T Sixma Jan '99 hormone receptors andjor coactivators. R Bernards NK Dephosphorylation of retinoblastoma family proteins by protein II R Bernards Sept '99 phosphatase 2A. NK The role of interieukin-lo in immunosuppression of tumor responses. VI J Neefjes Febr '99 IV H Spits NK The role of the nuclear inositide pathway in cell cycle progression. III N Divecha Dec '99 NK Mechanism of cell cycle regulation and control of cellular lifespan VII M Van Lohuizen May'99 by the Polycomb-group and oncogene BmÏ1. NK Development of a mouse model to study the genetic basis of VII A Berns Aug '99 mesothelioma. NK Mechanisms of reduced cellular accumulation of topoisomerase I X J Schellens June '99 inhibitors. VIII A Schinkel

163 Numberof Title Div. Principal Starting Ended project Investigator Date NKI Control of cell cycle progression by integrin mediated adhesion. A Sonnenberg April '99 NK Modulation of cytochrome P450 (CYP), P-glycoprotein (P-gp), and X J Schellens Dec '00 BCRP activity in gut wall and liver as major determinants of poor VIII A Schinkel oral bioavailability of anticancer drugs: preclinical and clinical studies. X J Beijnen NK Regulation of the G2jM transition by extracellular factors. V R Medema March '00 NK The role of the Forkhead transcription factor Trident in cell cycle V R Medema Oct '00 regulation. H Clevers NK Hypoxia and perfusion in hu man tumors: quantification and VIII A Begg July'oo implications for treatment outcome. XI K Haustermans A Balm NK The regulation of the expression ofhtert, the catalytic subunit of 11 R Beijersbergen May'oo telomerase during immortalization and tumorigenesis. NK The function of E2F transcription factors studied in vivo. 11 R Bernards Jan '00 NKI Role of diacylglycerol kinases in cell cycle progression and 111 W Van Blitterswijk Sept '00 cytoskeletal organization. N Divecha NK O Ras and Rho-signaling networks and epithelial-mesenchymal J Collard Nov '00 transition. NK Design and clinical implementation of optimized irradiation IX E Damen Dec '00 techniques using static and dynamic intensity modulation. B Mijnheer J Lebesque NK Communication to the mitochondria in apoptosis signaling by 111 J Borst July'oo death receptors and DNA damaging regimens. NK Identification and functional characterization ofintracellular signaling 111 P Ten Dijke Jan '00 proteins controlling transforming growth factor-beta-induced growth arrest. NK Specific and redundant functions of the retinoblastoma suppressor V H Te Riele Aug '00 gene family in growth control and differentiation. NK Genetic and environmental modifiers of cancer predisposition in V H Te Riele Dec '00 mismatch-repair-deficient mice. NK Improvement of delineation following conformal radiotherapy IX M Van Herk June '00 through multimodality imaging. C Rasch P Nowak NK Dissecting the role of CD4+ T cells in anti-tumor immunity. IV A Kruisbeek Sept '00 Sept '02 T Schumacher NKI Self-nonself discrimination by tumor specific CTL. IV A Kruisbeek Dec '00 T Schumacher NK Genetic screens to identify new components of the VII M Van Lohuizen Jan '00 senescencejimmortalization pathway. NK Functional analysis of BMI1-RING finger interacting proteins and VII M Van Lohuizen March '00 application of microarray technology to identify BMl1-responsive genes.

164 Number of Title Div. Principal Starting Ended project Investigator Date NK Dose verification and optimization of radiotherapy using portal IX B Mijnheer Oct '00 imaging. M Van Herk NK Cell-cell communication and connexin signalling: suppression of III W Mooienaar Sept '00 cell transformation. NK The role and mechanism of escape from Ras-induced senescence VII D Peeper Jan '00 in oncogenesis. II R Bernards NK Signal pathways controlling Iymphoma dissemination. E Roos April '00 NK Physiological and pharmacological analysis of the novel multidrug VIII A Schinkel Sept '00 resistance gene Bcrp (breast cancer resistance protein) using Bcrp knockout mice. NK Control of survival, replication and differentiation of hu man IV H Spits July 'oo T cell precursors. NKI mth PC-mediated photodynamic therapy (PDT) : how do drug uptake VIII F Stewart Sept '00 and distribution influence tumor and normal tissue response? X P Baas J Schellens NK The impact of prophylactic oophorectormy in women from hereditary XII N Aaronson Dec '01 breast/ovarian cancer families on psychosocial health and symptom XI M Van Beurden experience. NK The role ofhla-do and HLA-DM in effective MHC class II-mediated VI J Neefjes Oct '01 immune activation. M Van Ham NK Y irradiation and the generation of novel peptides for M HC VI J Neefjes Febr '01 class I molecules. NK Analysis of vaccine-induced melanoma specific T cell immunity: IV/X J Haanen Sept '01 a combined human and mouse study. IV T Schumacher X G de Gast NK In vitro isolation ofhigh affinity melanoma-specific T cell receptors. IV T Schumacher April '01 H Spits NK Bayesian adaptive dosing in cyclophosphamide-thiotepa-carboplatin X J Beijen Febr '01 (CTC) and mitoxantrone-thiotepa (MT) high-dose chemotherapy J Schellens regimens. S Rodenhuis NK Clinical outcome of breast cancer in BRCA1 and BRCA2 carriers. XIII/VIII L Van 't Veer Jan '01 R Tollenaar NK Genetic profiling of tumors from patients with a genetic susceptibility XIII P Nederlof Jan '01 for breast cancer. XIII/VIII S Verhoef L Van 't Veer NK Cancer susceptibility genes that predispose for radiation-induced VIII A Broeks Nov '01 breast cancer. XIII/VIII L Van 't Veer XII F Van Leeuwen NK Long-term effects of exposure to DES in utero on the risk of XII M Rookus Nov '01 hormone-related cancers. F van Leeuwen T Helmerhorst UU Control of cellular proliferation in norm al and tumor cells by the V R Medema Sept '01 PI3K/PKB/Forkhead pathway. B Burgering

165 Numberof Title Div. Principal Starting Ended project I nvestigator Date NK MRP5 and resistance to purine analogs. V P Borst March'02 J Wijnholds NK Identification ofthe PIM regulatory network by microarray analysis VII A Berns JUly'01 and high throughput retroviral tagging in compound mutant mice. NK Structure/function analysis of Muts homologs in HNPCC. II T Sixma Jan 'Ol NK Subversion oftgf-beta/smad signaling in cancer: Analysis of III P Ten Dijke Dec 'Ol TGF-beta receptor initiated intracellular responses distinct from Smad activation. NK Initiation and progression of Iymphomas and skin ca rcinomas in J Collard Dec 'Ol Tiam1 mutant mice. A Berns NK Integrin-associated protein regulation, migration and invasion. E Roos Jan 'Ol NK Regulation of cadherin-mediated adhesion by integrin signaling. A Sonnenberg May'01 NK Cloning of novel mammary tumor progression and metastasis genes. VI J Hilkens JUly '01 VII A Berns NK T cell tolerance and immunity during spontaneous tumor IV A Kruisbeek Sept 'Ol development. P Krimpenfort NK Antigen processing events in cross-presentation of exogenously IV N Brouwenstijn May'01 acquired antigens. NK Improvement of tumor response by combined modality treatment: IX M Verheij Oct 'Ol a translational approach. NK The Insulin-like Growth Factor (IGF) system in breast and colorectal VIII D Voskuil Oct 'Ol carcinogenesis: dietary intervention and molecular studies. XIII/VIII L Van 't Veer E Kampman NK Function-based screening for genes involved in invasion of J Collard Dec '02 epithelial tumor cells. NK Microarray analysis ofbreast cancer as a diagnostic tooi to guide M Van de Vijver Jan '02 optimal treatment. H Bartelink L Van 't Veer NK Pathological and Molecular Characteristics of tamoxifen-induced XII F Van Leeuwen Jun '02 endometrial Tumors. XIII H Hollema P Nederlof NK Regulation of cellular life span of antigen-specifk T cells and IV H Spits JUly'02 application of immortalized CTL for therapy of melanoma. II R Beijersbergen NK Determinants of radiosensitivity in mammalian cells: elucidating VIII A Begg JUly '02 mechanisms using a putative dominant negative to DNA polymerase beta. NK A functional genomics approach to identify genes which Influence II R Bernards Jan '02 tumor cell behavior in vivo. NK Regulation oftumor cell motility and invasion by myosin III F van Leeuwen Aug '02 heavy-chain kinase. W Mooienaar NK Functional genomics of gene regulation by heterochromatin proteins. VIII B Van Steensel May'02

166 Numberof Title Div. Principal Starting Ended project Investigator Date NK The identification of oncogen ic events collaborating with loss of V H Te Riele Nov '02 function ofthe retinoblastoma gene family in tumor development and progression. NK Characterisation of a redox regulated PI P3 synthesis pathway. III N Divecha Aug '02 NK Inelusion of geometrie uncertainties in treatment planning for IX M Van Herk JUIY '02 intensity modulated radiotherapy. J Lebesque C Schneider NK The role of survivin in cell cyele regulation and Iymphomagenesis. V R Medema March '02 S Lens NKI02-277' The incidence, nature and etiology of cognitive problems following XII S Schagen Nov '02 chemotherapy for cancer. F Van Dam

167 MAJOR PROJECTS SUPPORTED SY OTHER ORGANIZATIONS Granting agency Title Div. principal project number I nvestigator Dutch Kidney Foundation The role of the integrin a3~1 in the development and pathophysiology of the A Sonnenberg C glomerus. J Weening EEC-Bio-CT Tiaml/Rac signaling in neuronal differentiation. J Collard Dystrophic Epidermolysis Assembly of hemidesmosomes and identification of novel hemidesmosomal A Sonnenberg Bullosa Research components. Association (U K) NWO Localized phosphatidylinositol signals in single cells. K Jalink Fondation pour la Development of a mouse model to study the role of the integrin alpha-3/beta-l A Sonnenberg Recherche Médicale in skin carcinogenesis. IARC Analysis ofthe role of alpha-6/beta-4 in tumorigenesis. A Sonnenberg ZON-MW Molecular dissection of the negative regulation oft-cell signalling by CTLA4. A Sonnenberg IV Q Valent EU HPMF-CT Ras- and Rac signalling in tumour formation. J Collard STW VBI-4568 Program for discovery of novel drugs, based on the utilization of a naturally I1 T Sixma occurring soluble protein analog of the nicotinic acetylcholine receptors. NWO MW Crystal structure determination of the components of an E2/E3 ligase ubiquitination 11 T Sixma system involved in cell-cycle control. NWO CW The Process of transposition: three-dimensional structure analysis oftcl and 11 T Sixma TC3 transposase from Caenorhabditis elegans. NWO CW JC Structural studies of DNA mismatch repair. 11 T Sixma HFSPRG0249/1999-M 103 Upstream and downstream regulatory controls in the Rb pathway. I1 K Helin R Bernards NWO MW Genetic screens to identify novel cancer-relevant genes. II R Bernards Leukemia & Lymphoma Career development program: personal grant V Notenboom. 11 T Sixma Society EC Netwerk Autostruct: Determination of macromolecular crystal structures: integrated, 11 A Perrakis QLRI-CT automated and user-friendly approaches. EC Netwerk MAX-INF: European macromolecular crystallography infrastructure network. 11 A Perrakis H RPI NIH Structural Genomics JCSG: European macromolecular crystallography infrastructure network. 11 A Perrakis EMBO YIP 0333 Structure of macromolecular multicomponent complexes and computational 11 A Perrakis Macromolecular crystallography.

168 Granting agency Title Div. Principal project number Investigator EC IHP-HPMF- Structural characterisation ofthe human L1 retrotransposition machinery. II o Weichenrieder CT A Perrakis EC QLRT Structural proteomics in Europe. II T Sixma A Perrakis NWO-CW Structural analysis of the mechanism of ubiquitin conjugation, a generalized cellular II T Sixma PI /01690 addressing system that controls protein and DNA stability. NWO-CW Structural and functional analysis ofthe human L1/Alu retrotansposition machinery. II A Perrakis /03653 NIH Automatic model building and reflnement in crystallography. II A Perrakis NWO Role of the CD27/CD70 receptor-ligand pair in control of the immune response. 111 J Borst R Van Lier BTS/Senter project Sphingolipiden. III W Van Blitterswijk CBG Lysophospholipid receptor signaling. III W Mooienaar EEC Trans Mobility Signaling and neural induction. 111 P Ten Dijke Research network ERBFMRXCT Ludwig Institute for TGF-beta signal transduction. 111 P Ten Dijke Cancer Research NWO ALW BMP target genes in the differentiation of extraembryonic endoderm and bone 111 P Ten Dijke Netherlands Heart Functional analysis of endothelial cell defects associated with endogl;n and ALK-1 III P Ten Dijke Foundation NHS mutations in hereditary hemorrhagic telangiectasia. EU QLRT Targeting of angiogenic TGF-beta signalling in cancer and cardiovascular diseases. III P Ten Dijke ZON MW TGF-beta signaling in vasculogenesis and angiogenesis. 111 P Ten Dijke EEG HPMF-CT Stress mediated regulation of phosphositides. 111 N Divecha (European TMR) EU (MCFI ) Communication to the mitochondria in apoptosis signaling by death receptors 111 J Borst and DNA damaging regimens. S Tait NWO-MW The role of the I L-7 receptor in development of human T cells. IV H Spits NWO-MW Identiflcation of small molecule ligands for cellular proteins. IV T Schumacher NWO-ALW Identiflcation and characterization of genes involved in T-cell commitment. IV H Spits National Multiple Sclerosis How antigen processi ng pathways affect negative selection of M SP reactive T cells. IV A Kruisbeek Society (USA) RG 2940-A-1 Human Frontier Science Pre-TCR expression and function in developping T cells. IV A Kruisbeek Program Organization (HFSPO) RG 0335/1998-M NWO-MW (Program) Cytoplasmic and nuclear signaling pathways involved in negative selection IV A Kruisbeek of MBP reactive T ceiis.

169 Granting agency Title Div. Principal project number I nvestigator NWO-MW Dissecting virus specific cytotoxic T cell immunity. IV T Schumacher NWO-MW (Program) Functional and molecular characterization of the role of cytokines in development of hu man Iymphocytes. IV H Spits NWO-MW (Pionier) Analysis and manipulation of tumor specific T cells and T-cell receptors. IV T Schumacher ZON-MW Antigen processing in cross-presentation and cross-priming. IV N Brouwenstijn ZON-MW Functional characterization of a novel I L-12-like cytokine; role in IV B Blom dendritic-t cell interaction. Senter BTS Detection and isolation of antigen-specific cellular immunity for diagnostic and IV T Schumacher therapeutic purposes. EEC HPMF-CT Interleukin-7 mediated signalling events in early human T cell development. IV H Spits M Naspetti ZON-MW Pioneer Analysis and manipulation of tumor-specific T cells and T cell receptors. IV T Schumacher NWO Investigation of membrane transport proteins involved in multidrug resistance V P Borst of cancer. NWO-CW Function and biosynthesis of a new modified base discovered in trypanosome DNA, V P Borst B-D-glucosyl-hydroxymethyluracil or J. European Community Role of genomic instability in environmental carcinogenesis. V H Te Riele ENV4-CT NOW-MW The role oftrident in cell cycle contro!. V R Medema NWO-MW Checkpoint function in mitosis. V R Medema NOW-MW (Program) Phosphoinositide 3-kinase signaling: Regulation and function. V R Medema B Burgering P Coffer AFM 8292 (160438) CTG repeat instability in Myotonic Dystrophy: deciphering the instability V G Gourdon mechanism (s) towards therapeutic apparoaches. D Monckton H Te Riele B Wieringa NWOjPGS Processes involved in antigen presentation by MHC molecules and the release VI J Neefjes of cytosolic granules. NIH R01-CA Molecular studies on MUC ljepisialin Promoter. VI J Hllkens UCSF Prion protein localization. VI P Peters ILEP Cell biology of CDl and mycobacteria. VI P Peters Dutch Leprosy Relief NWO Role of the nuclear pore complex in b type catenin nuclear signalling. VI M Fornerod NWO-ALW Identification of specific transport pathways through the nuclear pore complex VI M Fornerod using in vitro nuclear reconstitution. NWO Characterization of SNARE's. VI P Peters

170 Granting agency Title Div. Principal project number I nvestigator NWO-CW Function and structure characterisation of the RI LP /RAB7 complex VI J Neefjes /02696 controlling Iysosomal transport. II A Perrakis NWO Genes and mechanisms in the multigenic control of cancer susceptibility: the VII POemant mouse model and application to human cancer. NWO Pionier Mechanism of gene repression by mammalian Polycomb-group proteins: VII M Van Lohuizen connections to cell cycle regulation and other silencing processes. EEC QLGl Inducible Melanoma Model. VII A Berns EC 5th framework Molecular mechanisma of senescence and ageing. VII M Van Lohuizen QLK6-CT NWO (genomics program) Genome-wide mapping of oncogenic pathways by high-throughput insertional VII A Berns 2002, mutagenesis. M Van Lohuizen NWO/Vidi Novel functional genomic screens to identify pathways that protect mouse and VII o Pee per human cells against oncogenic transformation by mutant RAS. AIDS Fonds project 4011 The role of P-glycoprotein in the oral bioavailability and penetration of VIII A Schinkel HIV protease inhibitors into HIV sanctuary sites. NWO-Genomics Chromatin proflling ofhuman gene silencing complexes. VIII B Van Steensel (Program ) EMBO-Young Functional genomics of chromatin and gene regulation. VIII B Van Steensel I nvestigator Program EEC (DGXII) A code of practice for dosimetry of boron neutron capture therapy (BNCT) in IX B Mijnheer SMT 4-CT Europe. STW Application of an electronic portal imaging device for dosimetry in radiotherapy. IX B Mijnheer BGN M Van Herk STW (BGN ) Portal image analysis. IX M Van Herk EC Biomed-2 Population analysis of anticancer drug treatment. X J Schellens PL Schumacher-Kramer Feasibility and phase 11 study offec-tctc-otax in hormone-refractory X S Rodenhuis Stichting stage IV breast cancer. NCI POl CA Multicenter selective Iymphadenectomy trial. XI o Nieweg Health Insurance Council Hl PEC in peritonitis carcinomatosa of colorectal origin. XI F Zoetmulder Fund for I nvestigative Medicine Health Insurance Council Breast cancer screening in cases of familial pre-disposition. XI E Rutgers Fund for Investigative J Klijn Medicine 98-3 Atos Medical Development and evaluation of a third generation Provox voice prosthesis. XI F Hilgers I Tan A Ackerstaff Dutch Cancer Society Olfactory rehabilitation after laryngectomy. XI F Hilgers Patient Education Program C Van As XII F Van Dam

171 Granting agency Title Div. Principal project number Investigator International Health IVF treatment, unexplained subfertility and number of retrieved oocytes in XII F Van Leeuwen Foundation relation to age at menopause. C Burger US Army BC Early life exposure and risk of breast cancer: a case-control study of young affected XII F Van Leeuwen sister-pairs without a family history. IARC International BRCA1/2 Carrier Cohort Study. XII F Van Leeuwen ZAO Between two stools an intervention study into the needs of palliative patients XII F Van Dam undergoing treatment at the outpatient department. Nefkens Foundation Effects of chemotherapy on cognitive skilis and evoked potentials in women XII F Van Dam receiving adjuvant treatment for breast cancer. X W Boogerd EORTC An international field study ofthe reliabil ity and validity of the EORTC QLQ-C30 XII N Aaronson and a disease-specific questionnaire module (QLQ-PR25) for assessing the quality of life of patients with prostate cancer. COPZ Palliative care: the development of an educational program for nurses. XII F Van Dam XI A Donker Bayer Pharmaceutical A phase 111, multi-center randomized, trial to compare survival and to evaluate the XII N Aaronson BAY efficacy, safety and tolerance of BAY-8862 versus best supportive care in patients with non-small cell lung cancer (NSCLC) with brain metastases. Lance Armstrong Long term risk of second cancers and cardiovascular disease following treatment XII F Van Leeuwen Foundation of testicular cancer. IX BAleman Schering Plough Development and testing of a patient self-report measure of peripheral neuropathy. XII N Aaronson EC Telematica Reseau Ubiquitaire a Integration de Services (RUBIS). Biom o Dalesio 4th framework E Van der Donk EC Telematica Retransplant. Biom o Dalesio 4th framework E Van der Donk EC Telematica Enabling BEst PRactices in Oncology (BEPRO). Biom o Dalesio E Van der Donk EC QLG1-CT PCTCG overview. Biom o Dalesio EC QLG1-CT PCTCG meeting. Biom o Dalesio

172 FUNDING AIDS Fonds Atos Medical Bayer Pharrnaceutical Biomed 2 CBG COPZ Dutch Cancer Society Dutch Kidney Foundation Dutch Leprosy Relief Dystrophic Epiderrnolysis Bullosa Research Association (UK) EEC: Bio-CT; Biomed; DGXII; HPMF; Trans Mobility Research Netwerk EC 5 tb framework EC Telematica 4 tb Framework EC QLGr-CT EMBO Fondation pour la Recherche Médicale Health Insurance Council Fund for Investigative Medicine (Ziekenfondsraad) Human Frontier Science Program Organization (HFSPO) IARC Lance Armstrong Foundation Leukemia and Lymphoma Society Ludwig Institute for Cancer Research National Multiple Sclerosis Society (USA) Nefkens Foundation Netherlands Heart Foundation Netherlands Organization for Scientific Research: NWO-ALW; NWO-CW; Pionier; Program; ZON-MW NIH Structural Genomics Ross Products Schering Plough Schumacher-Kramer Foundation Senter BTS UCSF US Army

173 PERSONNEL INDEX Aalders M 80 Aaronson N 121 Aboutalib M 148 Acherman D S II S Acherman YIZ II4 Ackerstaff AH II4 Agami R 62 Aleman BMP 92, 126 Alendar A 72 Alvarez Palomo B 19 Andersson H 124 Antonini N 137, 141 Appels N 84, 104 Ariaens A 36 Arkes C 129 Artignan X 93 Arts-Blom Y los Atsma D 129 Baank S 129 Baars D 137, 141 Baars JW 104 Baas P 80, 104 Bais EM 104 Bakker A 48 Bakker MC 129 Bakker S S6 Bakker S 144 Bakker S 148 Balkenende A 69 Balm AJM II4 BärW 93 Bardelmeier H 104 Bardelmeijer HA 129 Bartelink H 89, 92 Batchelor D los Bauer M 23 Bauhuis CM 129 Begg AC 78 Beijersbergen R 31 Beijnen JH 84, 104 BeIderbos JSA 92 Benne I 144 Bergsma R 126 Bernad-Fernandez R 64 Bernardini D 66 Bernards R 28 Berns K 28 Berns T 72 Besnard P 130 Bessone S 23 Betgen A 92 Beumer JH 104, 129 Bex A IIS Biervliet A 124 Bijker N 92 Bin Ali R 72,147 Bins A 52 Bleiker E 121 Blom B 46 Blommestijn G 78 Bocxe S 84 Boer M 66 Boerhorst E los Boerrigter-Barendsen LH 129 Boersma LJ 80, 9 2 Bolscher E 82 Bonfrer JMG 129 Boogerd W 104, 124 Boot H 104 Borlado L 28 Borsje-Maeyer NS 19 Borst J 38 Borst P S6 Bos E 67 Bos L 92 Bosch CAJ 89 Bosch T 104 Bosma AJ 86 Bouali F 121 Bouma M 104 Boutmy-de Lange M 130 Boutsma E 70 Bouwman V 137, 141 Brand B 93 Bras A S8 Breedveld P 84 Brink G 129, 130 Brocks L 142 Broekhuizen L II4 Broeks A 86 Brohet R 126 Bronk M 130 Brouwenstijn N 48 Brouwers C S4 Brummelkamp T 28 Buchwald G 32 Buckle T 129 Budde M 40 Buitelaar DR IIS Buitelaar M 82 Bülbül M 104 Bulthuis J 144 Bultsma Y 42 Burger MPM IIS Busstra C 126 Buwalda J II4 Caffin E 121 Calafat J 27, 143 Calogero A 48 Canrinus T 142 Carlee LM 31 Cats A 104 Celie P 32 Celik S 121 Chessa T 38 Chin-A-Kwie M 121 Cho J 93 Christodoulou E 34 Claij N S4

174 Clark K 21 Coccoris M 48 Cohen S 34 Collard J G 21 Creyghton M 28 Crommentuyn KML I04 Crul M I04 Csikos T 76 Daemen M 145 Dalesio 0 137, 141 Damen EMF 92 Danen E 23 Dannenberg J -H 54 De Boer E 126 De Boer JP I04 De Boer R 92 De Bois JA 93 De Bruijn R 19 De Gast GC 50, 104 De Goeij CCJ 144 De Groot K 76 De Grouw E 86 De Haan P 92 De Haan S 66 De Haas M 56 De Jaeger K 92 De Jong D 129 De Jong LA 52 De Jonge ME I04 De Kruijf E-J 142 De Kwant MEG 105 De Leeuw H 66 De Maat MM I04 De Melker A 38 De Moes J 72, 76 De Visser K 48 De Vries E 38 De Vries H 56 De Vries JDH li4 De Vries MC li5 De Vries S 54 De Waal M 137,141 De Weerd L li5 De Widt JJM 40 Dekker C li4 Dekker M 54 Dekker N 92 Delahaye L 129 Dellemijn TAM 50 Delzenne-Goette E 76 Demant P 76 Den Brok MWJ 104 Dennler S 44 Deurloo E 130 Deurloo K 92 Deurloo W 137,141 Dewit LGH 86,92 Diemeer A 137, 141 Dijkstra M 148 Dirac A 28 Divecha N 42 Donker A 124 Dontje W 46 Doodeman PA 129 Doodeman V 54 Dorrestein L li4 Douma K 124 Douma S 74 Douwenga W 129 Drentje S 148 Drost B li5 Dubbelman AC I05 Duppen J 93 DuursmaA 62 Edel M 28 Eggenhuizen TEA 78 Eichhorn P 28 El Haddouchi F 121 Engelsma D 64 Engelsman M 92 Epping M 28 Estourgie S li4 Faasen H 124 Feenstra M 90 Feldmann N 72 Ferring D 34 Fierens A 137, 141 Floore AN 86 Floot BGJ 80 Foijer F 54 Ford M 124 Fornerod M 64 Fortuin J 124 Franken M 56 Frederiks F 36,44 Frenay M 93 Frische E 32 FuJ 44 Gangaram-panday S 36 Gans R 148 Genest P-A 56 Gerritsma M 121 Geuijen CAW 23 Geurts T li4 Giepmans BNG 36 Gilhuijs KGA 130 Gimeno R 46 Glas A 129 Goedbloed C 93 Goede IN 144 Göker E 104 Gomez R 52 Gonggrijp S li4 Goumans M -J 44 Govaert G li4 Graversen L 78 Greil F 90 Griekspoor A 60 Grivell S 36 Groeneveld E 146 Grolle IC 129 Groothuis T 67

175 ':J; ~~. Haanen JBAC 52, 104 Haas RLM 92 Hage JJ II5 Hajdo-Milasinovic A 21 Halfwerk H 89 Halstead JR 42 Hannemann J 89 Hansen J 54 HartAAM 92 Hartog V 126 Hauer H 124 Haye-Fewer P 93 Heemsbergen W 92 Heimeriks M 145 Helgason H 104 Hendriks J 38 Hendriksen J 64 Hengeveld CM 36 Herberts C 60 Hernandez M 70 Hiemstra A 137, 141 Hijmans M 28 Hilarius D 121 Hilgers FJM II4, 124 Hilkens J 66 Hilkmann H 146 Hillebrand MJX 105 Hoebers FJP 78, 92 Hoefnagel CA 129 Hofland I 78 Hofland N 126 Hogervorst F 129, 13, 147 Holman E 148 Holman-Van Doorn T 148 Holtkamp M 105 Hoogeman M 92 Hoogendoorn L 126 Hooning M 126 Hoopman R 121 HorenbIas S II5 Houtkamp R II4 Huisman MT 82 Huitema ADR 104 Huitink JM II5 Hulsman D 70 Huseinovic A 86 Huveneers S 21 Idrissi M 121 Itoh F 44 Itoh S 44 Jagt A 148 Jalink K 25 Jansen E 92 Jansen SE 105 Janssen E 121, 126 Janssen H 78 Janssen JWRM 27, 143 Janssen L 60 Janssen LM II4 Joekes E 130 Joling J 48 Jones D 42 Jongsma J 72 Jonker JW 82 Jonkers J 72 Jordens I 60 J orritsma A 52 Kaas R II4 Kakaris M 34 Kambey E 148 Kamp MK 19 Kanhai M 144 Kappelhof B 104 KasIander F 142 Kauw J 58 Kedde M 38 Keessen M 105 KeIler A 38 Kemper M 104, 129 KempffH 148 Kerkhoven R 145 Kerst JM 104 Kersten M-J 5, 1 4 Kessels HWH C 48 Kieft R 56 Kimm M 66 Klaver S 86 Klein Zeggelink W 130 Klip H 126 Klokman W 126 Klomp H II4 Klompmaker R 58 Klous A 76 Klous M 104 Knipscheer P 32 Knols R 121 Koemans S 124 Kooij L 137 KoolJ 72 Koopman-Kroon C 105 Koops W 130 Koppes J 56 Korchynskyi 0 44 Korse CM 129 Kortlever R 28 Koster J 23 Koster P II5 Kraan S 148 Kreft M 23 Kreike B 89, 92 Kreukels B 124 Krimpenfort P 72, 147 Kristel P 89 Kroeskamp T 144 Kröger R 130 Kroon BB R II4 Kroon FHM II4 Kruijtzer CMF 104 Kruse J 80 Kuenen M 126 Kuijer PMM 104 Kuiken J 31

176 Kuikman I 23 Kuil A 56 Kuppens I I04 Kuyl C 60 Lakhai W I04 Lambooij J -P 70 Lamers M 32 Lamers T 145 Langeslag M 25 Laoukili J 58 Lascaris R 90 Le Sage C 62 Lebbink J 32 Lebesque JV 9 2 Lebrin F 44 Legdeur M 126 Lens S 58 Lieverst J 137, 141 Ligthart S 46 Linders TC 129 Lingbeek M 70 Linn S 104 Linthorst M 126 Litjens SHM 23 LiuX 72 Lohuis PJFM II4 Lomecky M 148 LontAP IIS Loo C 130 Loonstra A 72 Los AP 4,42 Louwe R 92 Lubsen-Brandsma LAC IIS Luiten R 46 LundA 70 Luts T 142 Lyons S 72 Maas T 124 Maaskant J 137, 141 Madalinska J 121 Madiredjo M 28 Maertzdorf B 126, 130 Mahn-Schaefers M 137,141 Malingré MM 104 Malliri A 21 Mallo H IOS Mandjes lam 137, 141 Markusic D 28 MarsmanM 60 Martinez M unoz C 74 Martins C 72 Masselink EAH 92 Matentzoglu K 70 McDermott L 92 McGibney C 93 Medema R 58 Meinhardt W IIS Melis P IIS Menko F 121 Mertens S 21 Meuwissen R 72 Michalides R 69 Michaloglou C 74 Mijnheer BJ 92 Mikkers H 72 Minderhoud TJ 93 Mironov A 67 Modder C 137, 141 Mohrmann K 84, I04 Mooi WJ 129 Mooij T 126 Mooienaar WH 36 Moonen LMF 92 Mulder J 36 Muller M 121, 124 Müller M 34 Muller P 93 Muller S 129,13 Mussmann R 56 Mutsaerts ELAR II4 Muusers A 137,141 Muyrers-Chen I 70 Nagasawa M 46 Nan-Offeringa L IOS Naspetti M 46 Natrajan G 32 Nawijn M 72 NederlofPM 86,126,129 Nee~es J 60 Neering H 104 Neijssen J 60 Nicolai J 137,141 Nieuwenhuijzen JA IIS N ieweg 0 E II4 Nijkamp WABE 31 Nijman S 28 Nishita M 44 Noback S 48 Nooijen WJ 129 Noorda EM II4 Nota B 86 Notenboom V 32 Nuyen B IOS Nuyten D 92 Oldenburg HSA II4 Olivier R IIS Olivo C 21 Olszewska A 92 Ono N 56 Oomen L 142 Oosterkamp R I04 Oosting H 137, 141 Opdam FJM 19 Oppeneer S 124 Ottenheim CPE 130 Ouwehand M l0s, 129 Overwater J 60 Overwijk W 52 Owens G 126 Pameijer FA 130 Pauw A 142 Peeper D 74

177 Pengel K 93 Perrakis A 34 Persson R 34 Peter E 137, 141 Peters PJ 67 Peterse JL 89, 129 pfauth A 143 Philippo T 70 Pickersgill H 64 Piek E 44 Piek-Den Hartog M!I4 Pijpe A 130 Ploeger LAJ 9 2 Plug R 13,147 Pluim D 84 Ponsioen B 25 Poodt J 129 Pool BA 129 Pos F 92 Post L 31 Posterna R 114 Price L 21 Prudenziati M 70 Pruntel R 13,147 Radernaker-Lakhai JM!O5 Ran L 130 Rasch C 92 Raymond K 23 Regnerus R 130 Reid G 56 Reinders-Sorn A 137,141 Reits E 60 Reitsma S 137, 141 Rerneijer P 92 Remmelzwaal J 137, 141 Repanas K 34 Rijswijk L 72 Rikers CH 104 Robanus Maandag EC 89 Roberts D 137,141 Rocha N 34 Rodenhuis S 86,!O4, 124 Roedoe D 148 RoeIen B 44 Roelofs BG H 129 Rorneijn M 67 Rörner L 56 Ronday JM!I5 Rook L 105 Rookus MA 126 Roos E 19 Roovers RC 21 Rosing H!O5 Rossi M 93 RourouA 121 Rowland B 74 Rozendaal M 58 Rozenschoon N 124 Ruevekarnp MC 80 Ruijs M 130 Ruiter GA 40 Ruivenkamp C 76 Rumping G 78 Russell NS 80, 86, 92, 126 Rutgers EJTh!I4, 126 Ruuls-Van Stalle EMF 19 Ruurs P 36 SaamakA 92 Salverda JG 9 2 Sayas L 36 Schagen S 124 Scheeren F 46 Schellens JHM 84, 104 Schepers K 48 Schinkel AH 82, 84 Schippers-Gillissen C 129 Schlief A 130 Schrnidt M 58 Schmidt M 130 Schneider C 92 Schoernaker NE!O5 Scholte M 54 Schornagel JH 104, 121 Schot M!O5 Schotte R 46 Schrarna JG!O4 Schreibelt G 67 Schultze Kool L 130 Schurnacher TNM 48 Schutte PFE!I5 Schuurman A 145 Schwarz M 92 Sein J 50 Seppenwoolde Y 92 Sier K 148 Sikkes S 124 Singh S 42 Sivro-Pmdelj F 129 Sixma TK 32 Slot MAC!I5 Srnits GJ 23 Srnitsrnans MHP 92 Smulders B 93 Sneeuw K 121 Snoek M 76 Song J-Y 144 Sonke TT 9 2 Sönrnezer Ö 84 Sonne M 28 Sonnenberg A 23 Sonneveld M 54 Sonneveld P 23 Sotthewes G 48 Spits H 46 Sprong D 78 Stahl M 58 Steenbakkers R 92 Stewart FA 80 Stokvis E!O 5 Stoltz J 142 Storm D 137,141 Stroeken PJM 19

178 Stroom J 92 Swart E 48 Swart M los Taal BG lo4, 121 TaborV 72 Taghavi P 70 Tait S 38 Takkenberg B II4 Tameris N IIS Tan IE II4 Tanger E 70 Tanis PJ II4 Te Poele JAM 80 Te Riele H 54 Ten Bokkel Huinink WW lo4 Ten Dijke P 44 Theodorou V 66 Thijssen BIOS Thorikay M 44 Tibben MM los Tielenburg R 93 Timmers AP II 5 Tirion F 52 Tissing-Tan C 92 Tjin-A-Koeng M 144 Toaldo CB 56 Toebes M 48 Tragter E 148 Treur-Mulder M 72,76 Triesscheijn M 80 Ubbels JF 92 Ulbert S 56 Urbanus J 48 Vader G 58 Vaessen H 137,141 Valdés Olmos RA 129,137,141 Valdimarsdottir G 44 Valdimarsdottir H 121 Vale nt Q 23 Valken et L 137, 141 Van 't Veer LJ 86, I26, 129, 130 Van Amerongen R 72 Van As CJ IIS, 124 Van Baal J 40, 42 Van BaaIen M I26 Van Beers E 86 Van Beurden M IIS, 121 Van Blitterswijk WJ 40 Van Boven H 129 Van Bunningen BNFM 92 Van Coevorden F II4 Van Dam F 124 Van Dam S l0s Van de Ahé F 72,147 Van de Kasteele WF 50 Van de Pavert I 142 Van de Ven PJH 93 Van de Vijver MJ 89, 129 Van de Weerdt B 58 Van Deemter L 56 Van den Belt-Dusebout A 126 Van den Berg K 70 Van den Berk P 50 Van den Bongard DHJG los Van den Boom M 48 Van den Boomen D 36 Van den Bout I 23 Van den Brekel MWM II4 Van den Broek G IIS Van den Broek 0 54 Van den Heuvel IJ 129 Van der Donk E 137, 141 Van der Gulden H 72 Van der Heijden I 56 Van der Horst G 38 Van der Kammen RA 21 Van der Kruijssen CMM 82 Van der Kwast A 137, 141 Van der Luit A 40 Van der Meer T 28 Van der Meij A 126 Van der Poel HG IIS Van der Puij E 124 Van der Sande JJ lo4 Van der Schilde I 137, 141 Van der Schoot S l0s Van der Spruit R 130 Van der Stoop P 70 Van der Stroom G-J 142 Van der Torre J 54 Van der Valk M 144 Van der Van ge N IIS Van der Velde A 137,141 Van der Velde H 64 Van der Velde M 60 Van der Velde M 60 Van der Voort P 130 Van der Wal A 54 Van der Weide M lo4 Van der Wel N 67 Van der Welle R 148 Van Diepen F 143 Van Dijk WJ 32 Van Dinther M 44 Van Duijvendijk P II4 Van Eijndhoven M 84 Van Es S 21 Van Gijn R los Van Gulik J 137, 141 Van Hagen JF IIS Van Halem MA 46, 70 Van Herk M 92 Van Herwaarden A 82 Van Hillegersberg R II4 Van Hof AC IIS Van Horck FPG 36 Van Houten B 56 Van Kesteren C los Van KooyM 76 Van Laar T 74 Van Leeuwen B 62 Van Leeuwen FE 86, I26

179 Van rindert ACM II5 Van Lith M 60 Van Lohuizen M 70 Van Loon J II5 Van Luenen H 56 Van Maanen R I05 Van Meeteren L 36 Van Montfort E 72 Van Mourik JC 114 Van Oosterwijk E 137,141 Van Rens A 130 Van Rheenen J 25 Van Riet E 54 Van Rooij H 147 Van Rooijen K 124 Van Rosmalen A 126 Van Rossum A 36 Van Rossum-Fikkert S 32 Van Ruth S II4 Van Sandick J II4 Van Schijndel A II5 Van Steeg G 129 Van Steensel B 90 Van Tellingen Van Tienen F 70 Van Tiggelen C 130 Van Tinteren H 137,141 Van Veen M 126 Van Velthuysen MLF 129 Van Vliet M 130 Van Vliet-Vroegindeweij C 92 Van Vugt H 76 Van Vugt M 58 Van Waardenburg W 137,141 Van Warmerdam L 104 Van Welsem T 86 Van Wilpe S 23 Van Zandwijk N I04 Van Zeijl L 36 Van Zuilen M II5 Vander Poorten VLM II5 Veldman RJ 40 Velds A 145 Vens C 78 Verheij M 4,92 Verheij MBM 129 Verhoef S 86, 121, 126, 130 Verhoeven E 70 Verloop J 126 Vermeulen C 78 Verra N 50 Verschueren K 34 Versloot J 124, 126 VerwaaI V II4 Verwijs M 78 Verwoerd D 69 Vielvoye-Kerkmeer APE 104 Visscher J II5 Vogel M 90 Voordouw A 46 Voorhoeve M 62 VormerT 54 Voskuil D 86 Vredeveld L 74 Vrieling A 86 Vrieling C 92 Vrouwenraets BC II4 Vyth-Dreese FA 50 Wagenaar E 82 Wals A 137,141 Wanders J I04 Wang L 28 Watkins S 32 Weder P 46 Wedman J II5 Weichenrieder 0 34 Weigelt B 86 Weijer K 46 Weinkove D 42 Wemer A 38 Wever L 137,141 Wiebenga 0 54 Wielders C 54 Wielinga P 56 Wigbout G 130 Wijnands E 23 Wijnands YM 19 Wijnholds J 56 Wijsman J II4 Willemse E 137,141 Winterwerp HW 28, 32 Witkamp AJ 114 Witteveen A 129 Wittkämper FW 92 Woerdeman LAE II 5 Wolfers M 126 Wolkers MC 48 Wolthuis R 58 Xiao Y 38 YuZ 56 Zaagsma M 124 Zanon C 76 Zeamari S 80 Zeelenberg IS 19 Zeker N 56 Zerp SF 40 Zevenhoven J 72 Ziblat L 137, 141 Zijp L 93 Zoetmulder FAN II4 Zuetenhorst JM I04 ZwartW 60

180 COLOFON Editors F Balm IS Benne M Fomerod RJAM Michalides T Philippo WJ Van Blitterswijk MA Van Halem (coordinator) H Van Luenen EJ Vos FA Vyth-Dreese Photograph HM The Queen Beatrix Enquiry' s / permission: Rijksvoorlichtingsdienst, Afd. Pers en Publiciteit/FOTO Postbus EA 's-gravenhage Photo: Ruud Taal/Capital Photos Copyright RVD Photograph AJM Berns Loek Zuijderduin Other Photographs and Illustrations Audiovisual Services The N etherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan I2I 1066 CX Amsterdam The N etherlands Designed by B@seline vormgeving, Utrecht Printed by De Bussy Ellerman Harms

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