THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2001 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2001

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1 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2001 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2001 THE NETHERLANDS CANCER INSTITUTE SCIENTIFIC ANNUAL REPORT 2001 The Netherlands Cancer Institute Plesmanlaan CX Amsterdam

2 SCIENTIFIC ANNUAL REPORT

3 4 Patron HM The Queen Beatrix

4 6 COPYRIGHT Scientific Annual Report 2001 Illustrations and unpublished data in these reports should not be used without permission of the author. Copyright : The Netherlands Cancer Institute Antoni van Leeuwenhoek Huis Plesmanlaan CX Amsterdam The Netherlands Phone Fax ISSN

5 7 CONTENTS CONTENTS Board Members 8 Research Divisions 9 Introduction 11 Education in Oncology 14 I Division of Cell Biology 15 II Division of Molecular Carcinogenesis 23 III Division of Cellular Biochemistry 29 IV Division of Immunology 39 V Division of Molecular Biology 48 VI Division of Tumor Biology 54 VII Division of Molecular Genetics 63 VIII Division of Experimental Therapy 69 IX Division of Radiotherapy 80 X Division of Medical Oncology 92 XI Division of Surgical Oncology 101 XII Division of Psychosocial Research and Epidemiology 109 XIII Division of Diagnostic Oncology 115 Biometrics Department 122 Research Facilities 126 Ongoing Trials 135 Invited Speakers 146 Projects 148 Personnel-Project Index 160

6 8BOARD MEMBERS President of Board of Governors WF Duisenberg BOARD MEMBERS International Scientific Advisory Board JR Bertino, American Cancer Society Professor of Medicine and Pharmacology, Yale University School of Medicine, New Haven, USA RA Flavell, Professor of Immunobiology, Yale University, New Haven, USA S Hellman, AN Pritzker Distinguished Service Professor, The University of Chicago, Chicago, USA WGJ 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 of Texas, 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 of Technology, Whitehead Institute, Cambridge, USA C Weissmann, Professor of Molecular Biology University of Zürich, Institut für Molekularbiologie Zürich, Switzerland Board of Directors AJM Berns, chairman and director of research L Neeleman, director organization and management (until ) S Rodenhuis, director clinical research and development W Van Harten, director organization and management (from ) Board of Governors WF Duisenberg, president HCJ Van der Wielen, vice-president R Hazelhoff, treasurer (until ) PJ Kalff, treasurer (from ) D Sinninghe-Damsté (from ) JHM Temmink GNJ Tytgat PF Van der Heyden S Van der Kooij (until ) J Van der Meer GP Vooys (from ) MWM Vos-Van Gortel Scientific Advisory Council AJM Berns, president AM Kruisbeek, secretary S Rodenhuis A Sonnenberg H Te Riele FE Van Leeuwen National Scientific Advisory Board LA Aarden, Professor of Molecular Immunology, Amsterdam SWJ Lamberts, Professor of Internal Medicine, Rotterdam B Löwenberg, Professor of Hematology, Rotterdam CJLM Meijer, Professor of Pathological Anatomy, Amsterdam CJM Melief, Professor of Immunohematology, Leiden HM Pinedo, Professor of Clinical Oncology, Amsterdam FH Schröder, Professor of Urology, Rotterdam GNJ Tytgat, Professor of Gastroenterology, Amsterdam AJ Van der Eb, Professor of Fundamental Tumor Virology, Leiden PC Van der Vliet, Professor of Physiological Chemistry, Utrecht

7 9 RESEARCH DIVISIONS RESEARCH DIVISIONS I Cell Biology E Roos (head) J Calafat JG Collard K Jalink A Sonnenberg II Molecular Carcinogenesis R Bernards (head) R Beijersbergen (from ) A Perrakis TK Sixma M Van Lohuizen (until ) III Cellular Biochemistry WH Moolenaar (head) J Borst N Divecha P Ten Dijke WJ Van Blitterswijk M Verheij IV Immunology H Spits (head) JBAG Haanen MJ Kersten AM Kruisbeek TNM Schumacher FA Vyth-Dreese K Weijer V Molecular Biology H Te Riele (head) P Borst (honorary staff member) R Medema HGAM Van Luenen J Wijnholds (honorary staff member) VI Tumor Biology J Neefjes (head) R Agami (from ) M Fornerod J Hilkens RJAM Michalides P Peters AA Verstraeten (stationed at the Free University) VII Molecular Genetics P Demant (head) R Beijersbergen (until ) AJM Berns P Krimpenfort M Snoek MA Van der Valk M Van Lohuizen (from ) VIII Experimental Therapy AC Begg (head) KMG Haustermans JHM Schellens AH Schinkel FA Stewart MJ Van de Vijver LJ Van 't Veer IX Radiotherapy H Bartelink (head) BMP Aleman JSA Belderbos LJ Boersma J Borger EMF Damen RW De Boer LGH Dewit RLM Haas AAM Hart JV Lebesque EAH Masselink BJ Mijnheer AWH Minken LMF Moonen CRN Rasch NS Russell JG Salverda C Schneider BNFM Van Bunningen M Van Herk M Verheij FW Wittkämper X Medical Oncology S Rodenhuis (head) JW Baars P Baas EM Bais JH Beijnen W Boogerd H Boot A Cats JP De Boer GC De Gast JBAG Haanen MJ Kersten H Neering JHM Schellens JH Schornagel BG Taal WW Ten Bokkel Huinink JJ Van der Sande M Van der Weide N Van Zandwijk APE Vielvoye-Kerkmeer

8 10 RESEARCH DIVISIONS Research Nurses D Batchelor AC Dubbelman MMJ Holtkamp H Mallo ME Schot M Swart XI Surgical Oncology BBR Kroon (board; head) AJM Balm (board) OE Nieweg (board) A Bex DR Buitelaar MPM Burger MC De Vries S Gonggrijp JJ Hage FJM Hilgers S Horenblas H Huitink H Klomp FHM Kroon W Meinhardt HSA Oldenburg JM Ronday EJTh Rutgers PFE Schutte IB Tan AP Timmers M Van Beurden F Van Coevorden MWM Van den Brekel HG Van der Poel N Van der Vange ACM Van Lindert J Visscher LAE Woerdeman FAN Zoetmulder XII Psychosocial Research and Epidemiology NK Aaronson (head) MA Rookus FSAM Van Dam FE Van Leeuwen XIII Diagnostic Oncology MJ Van de Vijver (head) APE Besnard JMG Bonfrer D De Jong MPW Gallee K Gilhuijs CA Hoefnagel FBL Hogervorst E Joekes W Koops R Kröger WJ Mooi SH Muller PM Nederlof WJ Nooijen FA Pameijer JL Peterse L Schultze Kool F Sivro M Smid-Geirnaerd LMTh Sterk RA Valdes Olmos O Van Tellingen LJ Van 't Veer MLF Van Velthuysen S Verhoef Biometrics Department OB Dalesio (head) H Van Tinteren HEADS OF GENERAL RESEARCH SERVICES Audiovisual Service JM Lomecky Central Cancer Library S Bakker Financial Administration B Simmelink General Facilities R Clement Laboratory Animal Department RGM Ten Berg Research Coordination AM Kruisbeek, laboratory research coordinator H Van Luenen, laboratory research manager EJ Vos, clinical research manager

9 11 Director of Research Anton Berns The Scientific Annual Report 2001 is presented in a new style. We hope that this format increases the accessibility for the reader. You will find less detailed information about the various projects; additional information can be found on the websites of the various divisions. In line with this new concept this introduction has also been trimmed. The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital (NKI/AvL) is an integrated cancer institute, 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 and active research groups in epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on mouse tumor models, mouse (reverse) genetics, cell 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 NKI/AvL. Information on patient care can be found in our General Report. In 2001, the actual construction of our new hospital building and the expansion of our Radiotherapy unit were started. Seeing this building rise from its foundation is a special experience. It is one of the embodiments of a thriving organization. In this important year we also reached agreement with the Ministry of Health about new financing rules for the hospital - which will now receive funding similar to that of academic hospitals. This averted a major deficit that was expected over the year In spite of these positive perspectives we also faced unexpected complications. The future of our neighbouring public hospital, Slotervaart Hospital, on which we rely for a number of general hospital and support functions, has become uncertain. This will have consequences for the NKI/AvL as well. Due to the tight job market we are more than ever before encountering the difficulty in attracting qualified health care workers at all levels. In the hospital this has compromised our productivity and we have had to take drastic measures to prevent unacceptable delays between diagnosis and treatment. We are energetically trying to attract personnel but this remains a difficult task especially since hospitals in large cities do not receive financing to compensate their personnel for more expensive housing or longer commutes. This has also affected research, in which it has proven to be more difficult to fill the positions of graduate students, post-docs and support staff. We have encountered a steep increase in labor costs during The absence of a matched increase in our basic funding puts pressure on our research budget. We are trying to tackle this problem along several routes. Our primary attempts are focused on renegotiating the financing rules of the Ministry of Health so that the basic funding follows the increasing costs of labor and materials in biomedical research. Secondly, we are attempting to better exploit our intellectual property. We have signed a contract with Cancer Research Ventures in Britain who now serves as our technology transfer agent. In 2001 a number of new patent applications have been filed and new licensing agreements have been negotiated. We will have to wait and see to what extent this will contribute to our research budget. Thirdly, we also stressed the importance that grant agencies cover overhead costs. This is particularly relevant for us since a large percentage of our research is funded by competitive grants. This wish has been partly fulfilled by the introduction of the Veni, Vidi, Vici grant system of the Netherlands Organization for Scientific Research which includes overhead funding and even offers funding for salaries of talented tenured investigators. Budgets (mln hfl) NKI research budgets *) Year * estimate Total budget Project grants Department of health Dutch Cancer Society (KWF) Other revenues Highlights The new research initiatives taken during the last few years have begun to bear fruit. Most appealing are the results in the area of micro-array analysis. In collaboration with Rosetta Impharmatics in Seattle, an in house small task force was set up to take advantage of the large series of tumor tissues stored at the NKI/AvL for over twenty years. This project in which now nearly 400 mammary tumors have been analyzed has been propelled by Laura Van t Veer, Marc Van de Vijver and René Bernards, on this side of the ocean. It has yielded highly significant results in identifying patients with either a very good or a poor prognosis more accurately than was possible thus far. With this knowledge, treatments can be better individualized. In a time frame of years this should enable us to reduce the burden of adjuvant chemotherapy for a substantial proportion of the patients with early stages of breast cancer, as the profiles can predict whether patients have either a very low or high probability of recurrent disease. The poor prognosis patients could possibly benefit from more extensive adjuvant therapy. Subsequently, we hope to extract information from these analyses that will allow us to predict which therapeutic intervention is most suited for the individual patient.

10 12 The assessment of several large-scale European clinical trials evaluating the effects of radiotherapy and adjuvant chemotherapy and hormone treatment on disease-free survival of patients with localized breast cancer has shown that optimal treatment can reduce recurrent disease by 50%. Especially younger women profit from the combined treatments. Harry Bartelink and coworkers showed that in the group of patients under 40 years additional radiotherapy of the tumor bed leads to a substantial further reduction of recurrent disease. Other projects have started to yield returns for the investment made. René Bernards and colleagues successfully continued their in vitro screens to identify new genes that contribute to escape of cells from senescence. Titia Sixma and coworkers received much acclaim for the elucidation of the 3D crystal structure of the acetylcholine binding protein. The structure of this transmembrane protein reveals unique features of this important group of pentameric ligand gated ion-channels. This is also extremely relevant for the design of drugs against these receptors. Peter Demant and coworkers identified a mouse colon tumor susceptibility gene. The gene encodes a receptor-like tyrosine phosphatase. Subsequently, they showed that loss of heterozygosity is frequently observed in human colon cancers. In my own group we designed a number of mouse tumor models closely mimicking the cognate cancers in man. Mice carrying a point mutation in the Ink4a tumor suppressor gene exhibited an enhanced predisposition for malignant melanoma. The study resolved some of the disputes on the relative contribution of the Ink4a and p19arf genes in tumorigenesis. Utilizing conditional knockout approaches, models for lung and breast cancer were developed as well. We hope that with these tools we can begin to better understand the correlation between distinct genetic lesions and phenotypic characteristics of tumors. These systems will prove valuable in screening new drugs that target defined cellular pathways. Recent literature shows that this specific targeting of pathways is a very promising treatment strategy. The astonishing success of Glivec, a drug targeting the Abelson tyrosine kinase and the kit receptor kinase hopefully serves as the forerunner of a whole battalion of new drugs. 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 NKI staff) has shown a steady increase since the beginning of the eighties. In the last years the number of citations and impact are leveling off (Table I) with yearly fluctuations indicating that we have reached a steady state. Our competitiveness in obtaining grants is another measure of quality. We have been quite successful during the last decade in obtaining grant support, scoring on average two- to three-fold better than our competitors. This translates in funding of the vast majority of our grant applications. In 2001 our success rate was exceptionally high. There is an inevitable fluctuation; however, Table 1 Short-term citations and impact of scientific articles published by the NKI research staff Publication year Citations Impact in spite of this, our score remains very favorable compared to most of our competitors in the field of biomedical research. The third measure of quality is based upon external site visits, in which international leaders in a particular field of research review the work of a division or research groups with a similar theme on a quinquennial basis. It requires the group leaders to prepare a report and to reflect on their research activities. This by itself appears very beneficial as it helps to bring focus in the research. A next series of site visits will be held in New appointments Our Director of Management and Organization, Leo Neeleman, left after having served the institute for seven years, to become a member of the Board of Directors of the Academic Medical Center (St Radboud) in Nijmegen. We wish him success in this new environment. Fortunately, we have found an excellent successor, Wim Van Harten, bringing the Board of Directors back to full strength. Ada Kruisbeek, Laboratory Research Coordinator and staff member in the Division of Immunology, has left to join Crucell, a Dutch biotech firm, as senior vice president and head of Oncology and Inflammatory diseases. We wish her much success in this new position. The NKI/AvL cannot award university degrees. However, many of our staff members hold special parttime chairs at Dutch universities. This facilitates the supervision and awarding of degrees to graduate students receiving their training at The Netherlands Cancer

11 13 Institute. Three staff members were appointed as professor: Maarten Van Lohuizen at the University of Utrecht, Simon Horenblas at the Free University Amsterdam and Adrian Begg at the University of Nijmegen. Titia Sixma and Ton Schumacher, AvL fellows in the Divison of Molecular Carcinogenesis and Immunology, respectively, were promoted to permanent staff member after rigorous evaluation by outside experts in their field of research. René Medema, coming from the University of Utrecht, joined the Division of Molecular Biology as permanent staff member, continuing his work on cell cycle regulation at the G2/M boundary. Reuven Agami from Israel was appointed as AvL-fellow in the Division of Tumor Biology, his research focusing on cell cycle regulation, a theme he worked on before during his postdoctoral period in the group of René Bernards. Bas Van Steensel, a KNAW fellow at the Swammerdam Institute for Life Sciences at the University of Amsterdam, will be joining us as AvL-fellow shortly. He will continue his work on chromatin profiling, establishing databases with specific protein-dna interactions. Table 2 total IF00 journal Cell Nat Genet N Engl J Med Nat Med Nature Science Curr Opin Cell Biol Immunity Genes Dev Trends Cell Biol Mol Cell Immunol Today J Exp Med EMBO J J Cell Biol J Natl Cancer Inst Curr Opin Genet Dev Adv Immunol Trends Bioch Sci Trends Genet Clin Microbiol Rev J Clin Invest Nat Cell Biol Proc Natl Acad Sci USA Ann Intern Med Mol Cell Biol Am J Hum Genet Lancet Development Outlook Last year in these paragraphs I expressed my concerns about biomedical research in the Netherlands. A year later, my concerns are not markedly diminished although there are a few positive points to be mentioned as well. Currently, both the level and the manner in which research is funded in the Netherlands remain of great concern and this inevitably will jeopardize the future of biomedical research. One concern is the increasing tendency of dedicating funds to special programs, an invention particularly appealing to politicians. Usually these special programs are less effective than steady support for research, selected on the basis of quality rather than the hype of the day. We should attempt to avoid that the poor practices of academic research funding we know so well from the EC are also going to dominate the national funding scene. The positive side is obviously the political willingness to increase funding for genomic research, although the efforts remain very modest in comparison with initiatives in other western countries. Undoubtedly, research will profit from this extra injection. In addition, the new Veni, Vidi, Vici program of NWO is a major step forward and a significant incentive to young talented investigators. The negative effects of unnecessary restrictive governmental regulations is of increasing concern. Some of the regulations, e.g. on the use of genetically modified cells and animals, are becoming more and more prohibitive. They slow down research efficiency while substantially increasing costs, as it almost invariably requires extra technical provisions, administration, reporting, etc. We have not been very successful in convincing our politicians that money is better spent on actual research than on red tape. In the meantime we will do our utmost to shield research in the NKI/AvL from these inhibitory effects. Focus of the Board of Directors is now on negotiating with various ministries for more appropriate funding parameters for our basic research. These continue to stay far behind the actual increase in the cost of research that almost doubles every ten years. The budget we receive from the Ministry of Health has increased less than 40% over 20 years. With the leveling off of funding we receive from the KWF (Dutch Cancer Society) there is also an urgent need to increase our basic funding. The steep rise in salaries during this last year is hardly compensated and has created a deficit for the coming year. The need for a dedicated internationally renowned cancer institute in the Netherlands is recognized by both the government and the public, and we are confident that a funding system will be put in place that meets the requirements of an institute striving for excellence. We are grateful to those who have supported us. For all our research activities we depend on the 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 will 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

12 14 EDUCATION IN ONCOLOGY EDUCATION IN ONCOLOGY The Oncology Graduate School Amsterdam (Onderzoeksschool Oncologie Amsterdam, OOA) is a collaboration between the NKI/AvL, the medical faculty of the Free University Amsterdam and the medical faculty of the University of Amsterdam. The main objective of the OOA is the organization of a tutorial program focusing on various aspects of cancer research for graduate students. Since the OOA harbors many top labs involved in various aspects of cancer research, a diverse and high quality tutorial program is provided for graduate students. This year six different courses were orga- nized on topics like mouse anatomy and histology (February), microscopic techniques (May), pharmacological innovation in cancer treatment (June), viral oncogenesis (June) and protein structure and function (October). Each course was taken by about 20 graduate students. The OOA also organizes another type of course aimed at teaching graduate students how to critically evaluate scientific publications and write short reviews. These longitudinal programs are headed by two staff members. They correct and discuss the reviews and the papers. In addition, a so-called special seminar series is organized for graduate students within the OOA. A highly renowned scientist is invited to give a general lecture followed by a discussion with the graduate students about the topic presented. This is preceded by an introduction by the host on the particular topic. The graduate student committee is now planning to host such special seminars as well. Apart from the tutorial program, the OOA also organizes an annual evaluation on the scientific progress made by each individual graduate student. This evaluation is carried out during a discussion with the group leader, head of the division and at least two other staff members. One of the highlights of the OOA is the annual oio -retreat aimed at allowing graduate students the experience of public presentations and the opportunity to initiate collaborations. This year the retreat was held on the island of Texel. In a sort of mini-symposium, more than 90 graduate students from the OOA presented their work in 15 minute presentations during two simultaneous symposia. This year a highly successful poster session was organized for graduate students within 6 months of the start of their project. For more information: Prof Dr J Neefjes (jneefjes@nki.nl), Marieke van der Velde (marieke@nki.nl). COURSES AT THE OOA GRADUATE SCHOOL 2001 May Microscopy symposium In the footsteps of Antoni van Leeuwenhoek P Peters, G Meijer, R Van Noorden June Pharmacological Innovations in Cancer Treatment WJF Van der Vijgh, J Lankelma, GJ Peters June Viral Oncogenesis J Middeldorp, PJF Snijders October Protein Structure and Function T Sixma, J Neefjes October OOA retreat J Neefjes, J Van Denderen November The anatomy and histology of the house mouse (3) WH Lamers, CJF Van Noorden

13 I DIVISION OF CELL BIOLOGY CELL BIOLOGY ADHESION MECHANISMS IN METASTASIS Our aim is to elucidate mechanisms of metastasis. We focus mainly on two highly metastatic lymphoma cell lines: TAM2D2 T-cell hybridoma and ESb T-lymphoma. In vitro models, invasion into rat embryo fibroblasts and into BMS2 bone marrow stromal cells for TAM2D2 and ESb cells, respectively, accurately predict in vivo behavior of these cells. Remarkably, although the mode of invasion in e.g. the liver is quite similar, the two cells use quite different mechanisms. TAM2D2 cells are dependent on Gi proteins and the integrin adhesion molecule LFA-1, whereas ESb cells do not require Gi and use the integrin α4b1. Chemokines and lymphoma metastasis Metastasis of TAM2D2 cells depends on Gi protein signaling and thus on a factor that acts on a Gi protein-coupled receptor. We established that this factor is SDF-1 (CXCL12), a chemokine that is constitutively expressed in many tissues. To demonstrate this, we transfected SDF-1 fused to a KDEL sequence. The SDF-KDEL fusion protein is retained in the ER by the KDELreceptor (see figure I.1). It binds to its receptor CXCR4 which is therefore also retained and never reaches the surface. This lack of surface CXCR4 has completely prevented metastasis to many different tissues. Recently, we also transfected another KDEL-conjugated chemokine, TARC (CCL17) which binds to CCR4, also expressed by TAM2D2 cells. This blocked TARC-induced migration but had no effect on metastasis, showing that SDF-1-KDEL specifically affects CXCR4. However, CXCR4 is not sufficient for metastasis, since the non-invasive BW5147 cells from which the T-cell hybridoma was made, do express CXCR4 but do not metastasize. Relevant signaling pathways must be blocked since the cells do not migrate towards SDF-1. It is thus important to further study these pathways. Chemokine receptors in tumor progression of carcinomas Chemokine receptors are also expressed by carcinoma cells, although often at low levels. We found, however, that on CT26 mouse colon carcinoma cells, freshly derived from a tumor, CXCR4 levels are high, and rapidly decline upon in vitro culture. CXCR4 levels are modulated by factors such as TGF-β, produced by stromal cells in vivo. Antibodies against CXCR4 were recently shown to reduce breast carcinoma metastasis, and this was ascribed to a block of invasion into the tissues (Müller et al., Nature 2001; 410:50). To test this for CT26 cells, we transfected pertussis toxin, which blocks chemokine-induced migration. Strikingly, this led to increased instead of diminished metastasis. However, we also transfected the SDF-1-KDEL construct (see above) to reduce CXCR4 surface levels and this did reduce the formation of metastases in both lungs and liver dramatically. Thus, CXCR4 is apparently not required for invasion, but it is essential for growth of the metastasized cells. Since SDF-1 is likely produced by stromal cells and CXCR4 is probably upregulated by factors from stromal cells, the stimulation of the SDF-1/CXCR4 pathway may be an important contribution of the stroma to tumor growth. The verification of this working hypothesis is being actively pursued. G-protein and integrin signaling during lymphoma metastasis Invasion by TAM2D2 cells is initiated by CXCR4 signals triggered by SDF-1, that cause polarization and movement of the cells but also activation of the integrin adhesion molecule LFA-1. This can then bind to its ligand, ICAM-1 or -2, and this interaction elicits signals from LFA-1 that are also required for invasion. This process is now being studied using a simplified model, i.e chemotaxis towards low SDF-1 levels (1 ng/ml) through filters coated with ICAM-1. As illustrated in figure I.2, the relevant signaling pathways are the same for invasion in vitro and, as far as tested, for metastasis in vivo. In contrast, chemotaxis towards 100 ng/ml SDF-1 does not require LFA-1-triggered signals. Division head, Group leader E Roos E Roos PhD Group leader AB Alvarez Palomo PhD Post-doc FJM Opdam PhD Post-doc PJM Stroeken MSc Graduate student IS Zeelenberg MSc Graduate student MK Kamp Technical staff EMF Ruuls-Van Stalle Technical staff HWM Van Deutekom Technical staff YM Wijnands Technical staff NS Borsje-Maeyer Secretary Golgi SDF-1 CXCR4 SDF-1 KDEL- Receptor KDEL Endoplasmic reticulum CXCR4 KDEL Figure I.1

14 16 CELL BIOLOGY Director of Research Anton Berns Key Publications Soede RDM, Zeelenberg IS, Wijnands YM, Kamp M, Roos E. SDF-1-induced LFA-1 activation during T-lymphoma dissemination requires Gq/11, RhoA and myosin, as well as Gi and Cdc42. J Immunol 2001; 166: Zeelenberg IS, Ruuls-Van Stalle L, Roos E. Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T-cell hybridoma. J Clin Invest 2001; 108: Role of Gq/11, PLC and G-protein bg subunits The role of heterotrimeric Gi proteins in chemokine-induced migration was established long ago, but we have found that Gq/11 proteins are essential as well. Our initial results suggested that Gq/11 was only required for SDF-1-induced LFA-1 activation, but we recently found that migration towards high SDF-1 levels, which is independent of LFA-1, depends on Gq/11 as well. Phospholipase C (PLC) is required for migration and can be activated by the Gαq subunit, but also by the βγ dimer. To study this, we expressed the C-terminal tail of the kinase GRK2 which binds the βγ subunit. Surprisingly, this led to increased chemotaxis. It had no effect on initial invasion but, whereas invasion of control cells normally stops after minutes, GRK2-Ct led to continued invasion. The βγ subunit binds kinases such as GRK2 and thus translocates them to the plasma membrane, where they phosphorylate G- protein-coupled receptors. This ultimately leads to desensitization. We conclude that the βγ subunit mediates the downregulation of the CXCR4 signal and is not required for PLC activation, which is thus likely triggered by Gαq. Jak kinase We previously demonstrated involvement of the ZAP-70 tyrosine kinase downstream of LFA-1, which required both the ZAP-70 SH2 domains, suggesting that binding to an ITAM sequence was essential. However, the T-cell receptor complex that contains ZAP-70-binding ITAMs is not expressed in TAM2D2 cells and Src-like kinases, which phosphorylate these ITAMs, are not involved in invasion by TAM2D2 cells. We are thus searching for an ITAM-containing protein and a kinase that phosphorylates its ITAM(s). A possible candidate for the latter is Jak2, since the Jak2 inhibitor AG490 strongly inhibits invasion. Although others have suggested that Jak2 acts downstream of the chemokine receptor and even upstream of G- proteins, our results indicate that it is only involved in the LFA-1 signal. Chemotaxis towards high SDF-1 levels, independent of LFA-1, is hardly affected by AG490. ICAP-1 Metastasis of ESb lymphoma cells, as well as invasion into monolayers of BMS2 bone marrow stromal cells, is completely dependent on the integrin α4b1, as we have shown, amongst others, by targeted disruption of both β1 alleles. Metastasis and invasion was restored upon transfection of β1 cdna. Several b1 mutants that did not restore metastasis had in common that they did not interact with ICAP-1, a protein that binds specifically to the β1 cytoplasmic domain. To study the potential role of ICAP-1, we have performed a yeast two hybrid analysis and identified the Rhoassociated kinase ROCK as an ICAP-1-binding protein. This was confirmed by coimmune precipitation from transfected COS cells. The proteins colocalize in ruffles. The binding site is being mapped with the aim of generating mutants that can block the binding and can be transfected to study the relevance of this interaction for invasion. Migration: High SDF-1 Migration: Low SDF-1 + ICAM-1 Invasion into fibroblast layers Metastasis of i.v. injected cells to liver a.o. ICAM-1 Gi Gq Cdc42 RhoA ZAP-70 myosin PLC calpain + _ + SDF n.d. n.d. n.d. Figure I.2

15 17 13 CELL BIOLOGY GENETIC CONTROL OF INVASION AND METASTASIS Rho family proteins control dynamic cytoskeletal changes Rho-like GTPases, which include Cdc42, Rac and RhoA, have been implicated in the control of a wide range of biological processes. In particular, Rho-like proteins act as key control 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 cells. Our recent studies implicate Rho-like GTPases in invasion and metastasis of tumor cells. The invasion-inducing Tiam1 gene encodes an activator of Rac We have identified the invasion-inducing Tiam1 gene, which encodes an activator (GEF) of the Rho-like GTPase Rac. Overexpression of Tiam1 in NIH3T3 fibroblasts causes extensive membrane ruffling, similar to V12Rac1-expressing cells. We have developed pull down assays with GST-Pak- and GST-Rhotekin fusion proteins, to determine the activation state of Rho-like GTPases in vivo in cells. The use of these assays confirmed that Tiam1 predominantly regulates the activity of Rac. PI3-kinase activity is required for Tiam1-mediated Rac activation, indicating that PI3-kinase acts upstream of Tiam1. Products of PI3-kinases, such as PIP3, bind to the N-terminal Pleckstrin Homology (PH) domain of Tiam1, which is required for the membrane localization of Tiam1. Only membrane-localized Tiam1 is threonine phosphorylated and is able to activate Rac, suggesting that phosphorylation events play a role in activation and/or localization of Tiam1. Group leader JG Collard JG Collard PhD Group leader M Driessens PhD Post-doc A Malliri PhD Post-doc C Olivo PhD Post-doc L Price PhD Post-doc RC Roovers PhD Post-doc S Van Es PhD Post-doc A Hajdo-Milasinovic MSc Graduate student E Damen Undergraduate student J Kind Undergraduate student K Clark Technical staff RA Van der Kammen Technical staff Rho-like GTPases and invasion of T-lymphoma cells Similar to Tiam1, constitutively active V12Rac induces an invasive phenotype in T-lymphoma cells consistent with the findings that Tiam1 activates Rac. Activated V12Cdc42 also induces invasion of T-lymphoma cells, which is not caused by Cdc42-mediated activation of Rac. Most likely, one of the many common downstream signaling pathways of Cdc42 and Rac are instrumental in the induction of invasion. Activated V14RhoA potentiates invasion but fails by itself to mimic Rac and Cdc42. Expression of Tiam1 and V12Rac1 promotes integrin-mediated adhesion to various substrates and correlates with invasive capacity, suggesting that enhancing cell-substrate adhesion of lymphoid tumor cells promotes their invasion. Tiam1-Rac signaling and invasion of epithelial cells Invasion and metastasis of epithelial carcinomas are often associated with reduced E-cadherin-mediated cell-cell adhesion. Tiam1 localizes to adherens junctions and ectopic expression of Tiam1 in epithelial cells inhibits HGF-induced cell scattering by increasing E-cadherinmediated adhesion. In addition, increased Tiam1-Rac signaling inhibits invasion and migration of fibroblastoid V12Ras-transformed MDCK cells by restoring E-cadherinmediated adhesions and an epithelial phenotype. Interestingly, Tiam1/Rac-induced cellular responses with respect to cell-cell adhesion and cell migration are dependent on integrin-mediated cell substrate interactions. On fibronectin and laminin-1, Tiam1/Rac signaling inhibits migration of MDCKf3 cells by restoring E-cadherinmediated cell-cell adhesion, whereas on collagen Rac activation promotes motile behavior. Invasion of epithelial cells appears to be determined by a balance between invasion-inhibitory cell-cell interactions and invasion-promoting cell-substrate interactions, which are both regulated by Tiam1-Rac signaling. The proto-oncogene Ras is frequently mutated in epithelial tumors resulting in uncontrolled growth and transition towards an invasive, mesenchymal phenotype. In epithelial cells, we found that sustained 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 of Tiam1. Reconstitution of Rac activity by exogenous expression of Tiam1 decreases Rho activity and restores the epithelial phenotype in mesenchymal V12Ras- or RafCAAX-transformed cells. Similarly, E1a reverts a mesenchymal phenotype into an epithelial phenotype by increasing Tiam1 expression levels, which is accompanied by increased Rac and decreased Rho activity. All data together indicate that a balance between Rac and Rho activity determines epithelial- Ctrl Arf6 epithelial cells Figure I.3 Activated Arf6 induces cell scattering in epithelial cells

16 18 CELL BIOLOGY Director of Research Anton Berns Key Publications Engers R, Springer E, Michiels F, Collard JG, Gabbert HE. Rac Affects Invasion of Human Renal Cell Carcinomas by Upregulating Tissue Inhibitor of Metalloproteinases (TIMP)-1 and TIMP-2 Expression. J Biol Chem 2001; 276: Malliri A, Ten Klooster JP, Olivo C, Collard JG. Determination of the activity of Rho-like GTPases in cells. Methods in Mol Biol (in press). Negre-Aminou P, Van Erck M, Van Leeuwen RE, Collard JG, Cohen LH. Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells. Biochem Pharmacol 2001; 61: Palacios F, Price L, Collard JG, D Souza- Schorey C. Arf6 regulates adherens junction assembly in epithelial cells. EMBO J 2001; 20: Price L, Collard JG. Regulation of the cytoskeleton by Rho-family GTPases: Implications for tumour cell invasion. Seminars in Cancer Biology 2001; 11: Average number of tumours per mouse Tiam1 +/+ Tiam1 +/- Tiam1 -/ Weeks post initiation Tumor formation in wild-type and Tiam1-mutant mice induced by DMBA/TPA treatment Tiam1 +/+ Tiam1 -/- Tiam1 expression in the skin of Tiam1+/+ and Tiam1-/- mice mesenchymal transition. High Rac and low Rho activity promotes E-cadherin-based adhesions and thereby an epithelial phenotype, whereas low Rac and high Rho activity leads to loss of cadherin-mediated adhesion and a mesenchymal highly migratory phenotype. Arf6 regulates adherens junctions Increased Arf6 activity results in a disruption of adherens junctions and cell scattering, whereas dominant negative Arf6 inhibits HGFinduced cell migration. Arf6, a regulator of vesicular trafficking, co-localizes with cadherin and induces its internalization into the cytoplasm. Accompanying increased Arf6 activity is an increase in Rho and a decrease in Rac activity, which is again consistent with the acquisition of a mesenchymal phenotype. These findings suggest that the regulated trafficking of cadherin by Arf6 coordinates with Rac- and Rho-dependent changes in the cytoskeleton in the control of epithelial-mesenchymal transition. How Arf6 influences Rac and Rho activities is currently under investigation. Tiam1 interacts with the Arp2/3 complex The Arp2/3 protein complex initiates nucleation of actin filaments at sites of submembraneous actin polymerization and consists of the actin-related proteins (Arp) 2 and 3, and five additional arc (Arp complex) proteins. Rho-family proteins regulate actin cytoskeleton dynamics but how in particular Rac connects to the actin-nucleating complex is still not clearly understood. Using yeast two-hybrid screening we found that Tiam1 interacts with the p21-arc subunit of the Arp2/3 complex through the N-terminal Pleckstrin homology domain and adjacent coiled-coil region. Tiam1 co-localizes with the Arp2/3 complex at sites of extensive actin polymerization such as in membrane ruffles and in epithelial cell-cell contacts. The binding of Tiam1 to p21-arc may thus provide a mechanism for linking receptor-mediated activation of Rac to Arp2/3-controlled actin dynamic changes. Tiam1 mutant mice In vitro studies have implicated Rho-like GTPases in processes such as cell migration, cell-cycle progression and Ras-induced focus formation, suggesting that these GTPases play a role in the formation and progression of tumors in vivo. We have generated Tiam1-deficient mice by gene targeting to investigate the consequences of changes in Tiam1/Rac signaling in tumorigenesis in vivo. Genotyping revealed the successful generation of Tiam1+/- and Tiam1-/- mice. Western blot analysis confirmed the absence of Tiam1 protein in different tissues of Tiam1-/- mice, whereas Tiam1+/- mice showed an intermediate expression level of Tiam1. Tiam1-/-mice develop, grow and reproduce normally. Analysis of the mice failed to reveal aberrations in organs, tissues or haematopoiesis. Most likely, other GEFs capable of activating Rac compensate for the loss of Tiam1. Tiam1 is widely expressed, with the highest levels detected in brain and testis. In mouse skin, Tiam1 was present in basal and suprabasal keratinocytes of the interfollicular epidermis and in hair follicles where it is predominantly expressed in the infundibular portion. Because of this specific expression pattern, skin tumors were initiated by application of a two-stage chemical carcinogenesis protocol. This protocol entails tumor initiation in epidermal keratinocytes by a single treatment with the carcinogen DMBA, which invariably induces oncogenic activation of the c-ha-ras gene. Subsequent repeated treatments with the tumor promoter TPA result in the outgrowth and progression of initiated cells. Benign tumors (largely papillomas) appeared in wild-type mice within 7-10 weeks from DMBA initiation. Significantly, Tiam1-/- mice displayed a dramatic resistance to tumor formation. Papillomas developed only after a period of latancy of ten weeks and their number was on average reduced ten-fold during the promotion period. Immunohistochemical staining revealed high levels of Tiam1 protein expression (comparable to normal epidermis) in papillomas of wild-type mice. Tiam1 deficiency affected not only tumor burden but also tumor growth. The few tumors that developed in Tiam1-/- mice were significantly smaller than the many tumors appearing in Tiam1+/+ mice. Interestingly, the frequency and growth of tumours in heterozygous Tiam1+/- mice was intermediate to that of Tiam1+/+ and Tiam1-/- mice. From these data we conclude that the Tiam1 gene dosage 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. The mechanisms involved and the role of Tiam1 in the progression of these Ras-induced skin tumors is currently being investigated.

17 19 13 CELL BIOLOGY RECEPTORS FOR MATRIX ADHESION Role of a6b4 in the formation of hemidesmosomes The integrin α6β4 is a major component of hemidesmosomes. We have shown that inactivation of β4 in mice results in severe blistering of the skin, due to the absence of hemidesmosomes. The atypical and exceptionally long cytoplasmic domain of β4 plays an essential role in the formation of hemidesmosomes by providing binding sites for various hemidesmosomal components, one of which is plectin. Recently, we have shown that two missense mutations in β4, discovered in patients with the non-lethal form of junctional epidermolysis bullosa, render β4 unable to interact with plectin and prevent the localization of plectin in hemidesmosomes. This finding further attests for a specific role of α6β4 as a scaffolding molecule for the formation and function of hemidesmosomes. Dynamics of hemidesmosome assembly We have performed time lapse fluorescence microscopy of living PA-JEB cells that stably express (through retroviral infection) GFP chimeras of the β4 subunit. One chimera consisted of the full-length β4 subunit fused to GFP at its carboxy terminus. This chimera (β4-egfp) can bind to α6 and thus interact with laminin-5. In a second chimera, EGFP-β4, the extracellular domain of β4 was replaced by EGFP, rendering it unable to bind to laminin-5. This, however, did not prevent the chimera from inducing hemidesmosomes. Both β4-egfp and EGFP-β4 hemidesmosomes are dynamic structures displaying characteristic movements and rearrangements during migration and division of cells. They dynamically appear at the cell margins, including the leading edge of migrating cells, where they induce the formation of hemidesmosome-like structures containing plectin. Many of the b4-egfp hemidesmosomes are converted into retraction fibers during cell migration and division, whereas those containing EGFP-β4 are not. The β4-egfp chimera is retained in retraction fibers, probably because it cannot be easily dissociated from laminin-5. It is not connected to the intermediate filament system at these sites, since plectin is not present in retraction fibers, and neither is BP180 or BP230. The presence of the β4-egfp chimera in retraction fibers suggests that it retards cell migration. Indeed, PA-JEB cells expressing β4-egfp migrate considerably more slowly than those expressing EGFP-β4. Group leader A Sonnenberg A Sonnenberg PhD Group leader S Bessone PhD Post-doc E Danen PhD Post-doc CAW Geuijen PhD Post-doc F Malergue PhD Post-doc GJ Smits PhD Post-doc LMTh Sterk MD Post-doc S Van Wilpe PhD Post-doc J Koster MSc Graduate student SHM Litjens MSc Graduate student A Van der Flier MSc Graduate student S Van Leeuwen MSc Graduate student D Kramer Technical staff M Kreft Technical staff I Kuikman Technical staff P Sonneveld Technical staff A link between a6b4 and growth factor receptors In collaboration with L Borradori in Geneva, we found that the integrin α6β4 binds to ERBIN, a cytoplasmic component that belongs to the LAP family of proteins. This family of proteins is characterized by the presence of 16 leucine rich repeats at their amino terminus and a single PDZ domain at their carboxy-terminus. ERBIN has previously been identified as an ErbB-2 interacting protein in yeast-two hybrid studies and was found to play an important role in the localization of the ErbB-2 receptor at the basolateral surface of a variety of cells. Binding of ERBIN to ErbB-2 is mediated via its PDZ domain, while the interaction with β4 involves the central region of ERBIN. We have suggested that interaction of α6β4 with ErbB-2 mediated by ERBIN enhances the signaling function of the ErbB-2 receptor. Generation of mouse strains with a conditional mutation in the genes for a3 or b4 Knockout mice for the α3 or β4 subunit die perinatally due to kidney defects or extensive blistering of the skin, respectively. In order to study the loss of function of the α3β1 and α6β4 integrins in adult mice, we have generated mice in which one or more exons of the integrin subunit genes have been flanked with loxp sites. Mice carrying the conditional mutation on both alleles are viable and do not show obvious signs of tissue defects. They are currently being crossed with transgenic mice, expressing a regulated Cre recombinase that can be activated with tamoxifen. Control of cell cycle progression by integrin mediated adhesion Attachment of cells to extracellular matrix components via cell surface receptors of the integrin family regulates progression through the cell cycle. We have found that α5β1- mediated attachment to fibronectin supports S-phase entry in a Rho-dependent fashion. We observed that reduction of the transcription of the p21cip/waf cyclin-

18 20 CELL BIOLOGY Director of Research Anton Berns Key Publications Danen EHJ, Sonneveld P, Sonnenberg A, Yamada KM. Dual stimulation of Ras/Mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression. J Cell Biol 2000; 151: Favre B, Fontao L, Koster J, Jaunin F, Shafaatian R, Saurat J-H, Sonnenberg A, Borradori L. The hemidesmosomal proteins bullous pemphigoid antigen 1 and the integrin b4 subunit bind to ERBIN. Molecular cloning of multiple alternative splice variants of ERBIN and analysis of their tissue distribution. J Biol Chem 2001; 276: Fontao L, Geerts D, Kuikman I, Koster J, Kramer D, Sonnenberg A. The interaction of plectin with actin: evidence for crosslinking of actin filaments by dimerization of the actin binding domain of plectin. J Cell Sci 2001; 114: Klinowska TCM, Alexander CM, Georges- Labouesse E, Van der Neut R, Kreidberg JA, Jones CJP, Sonnenberg A, Streuli CH. Epithelial development and differentiation in the mammary gland is not dependent on a3 or a6 integrin subunits. Develop Biol 2001; 233: Koster J, Kuikman I, Kreft M, Sonnenberg A. Two different mutations in the cytoplasmic domain of the integrin b4 subunit in non-lethal forms of epidermolysis bullosa prevents interaction of b4 with plectin. J Invest Dermatol 2001; 117: Van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell Tissue Res 2001; 305: Van der Flier A, Sonnenberg A. Structural and functional aspects of filamins. Biochem Biophys Acta 2001; 1538: Van Leusden MR, Pas HH, Gedde-Dahl Jr T, Sonnenberg A, Jonkman MF. Truncated type XVII collagen expression in a patient with non-herlitz junctional epidermolysis bullosa caused by a homozygous splice-site mutation. Lab Invest 2001; 81: dependent kinase (cdk) inhibitor (cki) and enhancement of cyclin D1 protein levels in fibronectin-adhered cells both depended on the higher levels of GTP-bound Rho in these cells. Currently, the molecular cascade underlying this control mechanism is being investigated in more detail. Thus far, we have found that Rho kinase-mediated organization of actin is involved in the PI3K and mtor dependent stimulation of cyclin D1 accumulation by fibronectin. The cyclin D-cdk4/6 complex plays a dual role in the G1 phase of the cell cycle: it provides initial phosphorylation of the family of Rb pocket proteins, and it sequesters cki s away from the cyclin E-cdk2 complex. We use embryonic fibroblasts lacking p21cip/waf, p27kip, or both cdk inhibitors, to determine the relevance of fibronectin-mediated cyclin D1 control in the absence of these inhibitors. Our initial observations show that p21-deficient cells can enter S-phase in the absence of fibronectin-mediated stimulation of cyclin D1 while cells lacking p27 cannot. Regulation of cadherin-mediated cell-cell adhesion by integrin signaling We have shown that the expression of β1 in a β1-deficient epithelial cell line, GE11, induces an epithelial-mesenchymal-like transition. The morphological change induced by the expression of β1 is associated with the downregulation of cadherin function. The induction of loss of cadherin mediated cell-cell adhesion does not require the effect of adhesion of β1 integrins to ligand, since the expression of an IL2-R chimera, containing the extracellular domain of the IL2 receptor and the cytoplasmic domain of β1 induces the same the effect. Overexpression of either α- catenin or vinculin, two molecules that reinforce cadherin-mediated cell-cell junctions, prevents the disruption of intercellular adhesions by β1 expression. These results suggest that the balance between cell-cell and cell-matrix adhesions largely determines the phenotype of a given cell. Expression of αvβ3 in GE11 cells induces a similar EMT-like transition. In contrast to the GE11-β1 cells, the β3-expressing cells do not assemble a fibronectin matrix, excluding this condition as being instrumental for EMT. Interaction of b1 with splice variants of filamin-a and -B In a yeast-two hybrid screen for proteins interacting with the cytoplasmic domain of β1d we isolated a novel splice variant of filamin-b, termed filamin-b var-1. In this variant a stretch of 41 amino acids that is located between repeats 19 and 20 is deleted. We have shown that a similar variant exists of filamin-a and that both filamin-a var-1 and filamin-b var-1 bind more strongly than their wild-type isoforms to different integrin β subunits. In all instances, the binding to β1a proved to be stronger than to β1d. Both filamin- A var-1 and filamin-b var-1 are expressed in various tissues but are relatively minor components. A filamin-b variant in which the deletion of the var-1 region is combined with the deletion of the variable hinge-1 region is specifically localized in focal contacts. These results show that specific combinations of splicing events of filamin-b mrna lead to alterations in the cellular localization and binding affinities for integrins. The role of plakoglobin in desmosome assembly Plakoglobin is a member of the armadillo family of proteins, expressed in both desmosomes and adherens junctions. It has been suggested that plakoglobin plays a key role in the sorting of desmosomal and adherens junction components. We have generated plakoglobin double knockout ES cells to better explore the role of plakoglobin in cardiac and skeletal muscle cell differentiation in vitro. Preliminary studies suggest that the in vitro differentiation of the double knockout ES cells into cardiac muscle cells is delayed. Furthermore, epithelial cells that are also formed as a result of the applied differentiation protocol contain only few rudimentary desmosomes. Currently, the double knockout ES cells are being infected with retroviruses containing mutants of plakoglobin that cannot bind one or more junctional proteins, to assess the importance of these interactions with plakoglobin for the formation of desmosomes.

19 21 13 CELL BIOLOGY BIOPHYSICS IN CELL SIGNALLING Detailed understanding of signals that are short-lived or exist very locally within the cell requires techniques with high spatiotemporal resolution applied to single living cells. To provide this resolution, we employ a range of biophysical approaches, including electrophysiological and spectroscopical techniques. Membrane phosphatidylinositol bisphosphate (PIP 2 ) levels in vivo in single cells To study regulation of Gq-mediated phospholipase C (PLC) activation by G protein-coupled receptors, we have developed an assay to monitor PIP 2 in vivo that is based on fluorescence resonance energy transfer (FRET) between GFP-tagged, PIP 2 -binding pleckstrin homology (GFP-PH) domains. FRET allows subsecond monitoring of cellular PIP 2 kinetics and reveals that significant PIP 2 hydrolysis occurs within seconds after stimulation. FRET gives an accurate reflection of agonistinduced PLC activation and reveals receptor-specific differences in the dynamics of PIP 2 hydrolysis that are not uncovered by other assays. We are using this method to study desensitization of G protein-coupled receptors by monitoring the kinetics of inactivation in C-terminal receptor mutants. Furthermore, in collaboration with N Divecha (Division of Cellular Biochemistry), the effects of expressed PIP kinases are being investigated. Distribution of PIP 2 along the plasma membrane of single cells It has been suggested that PIP 2 in the plasma membrane serves a role as a localized signal to relay extracellular stimuli into local cytoskeletal responses. However, direct evidence for this notion is still lacking. We undertook an in-depth analysis of the spatial distribution of this lipid along the plasma membrane in several cell lines. Membrane PIP 2 was labeled in vivo with GFP-PH and visualized at high resolution using numerical deconvolution of confocal images. While at first glance these images seemed to support the notion of spatially differentiated membrane PIP 2 content, a detailed analysis of the colocalization of GFP-PH fluorescence with various membrane markers indicated that PIP 2 distributes completely homogeneously along the plasma membrane (see figure I.5). The apparent concentrations of PIP 2 were found to be due to membrane folds, at which sites the presence of multiple membrane layers cause increased GFP-PH labeling. Role of membrane potential in cellular signaling In N1E-115 neuroblastoma cells and many other cell types, agonists such as lysophosphatidate, thrombin and sphingosine-1-phosphate induce prominent, prolonged depolarization of the plasma membrane. A main outstanding question concerns the function of depolarization in non-excitable cells. We are addressing a putative role for agonist-induced changes in membrane potential as a modulator of signaling cascades by performing whole-cell patch clamp studies combined with continuous monitoring of the effects on known signaling cascades using fluorescent sensors for intracellular Ca 2+ levels, membrane PIP 2 content and the cyclic nucleotides cgmp and camp. Group leader K Jalink K Jalink PhD Group leader M Langeslag MSc Graduate student J Van Rheenen MSc Graduate student B Ponsioen Undergraduate student S Heerkens Technical staff W Van Donselaar Trainee technician Key Publications Miyawaki A, Mizuno H, Llopis J, Tsien RY, Jalink K. Cameleons as cytosolic and intraorganellar calcium probes. In: Calcium Signalling (in press). Postma FR, Jalink K, Hengeveld T, Offermanns S, Moolenaar WH. Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells. Curr Biol 2001; 11: Van der Wal J, Habets R, Varnai P, Balla T, Jalink K. Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J Biol Chem 2001; 276: Figure I.5 Intensity distribution of GFP-PH and the red membrane dye DiI in the basal membrane of a N1E-115 neuroblastoma cell. The tight colocalization demonstrates that local enrichment of PIP 2 does not occur. Scale bar, 5 micrometer.

20 22 CELL BIOLOGY Director of Research Anton Berns IMMUNO-EM FACILITY Group leader J Calafat J Calafat PhD Group leader JWRM Janssen Technical staff Key Publications (continued) De Melker AA, Van Der Horst G, Calafat J, Jansen H, Borst J. c-cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor associated throughout the endocytic route. J Cell Sci 2001; 114: Egesten A, Calafat J, Janssen H, Knol EF, Malm J, Persson T. Granules of human eosinophilic leucocytes and their mobilization. Clin Exp Allergy 2001; 31: Giepmans BN, Verlaan I, Hengeveld T, Janssen H, Calafat J, Falk MM, Moolenaar WH. Gap junction protein connexin-43 interacts directly with microtubules. Curr Biol 2001; 11: McEuen AR, Calafat J, Compton SJ, Easom NJ, Buckley MG, Knol EF, Walls AF. Mass, charge, and subcellular localization of a unique secretory product identified by the basophil-specific antibody BB1. J Allergy Clin Immunol 2001; 107: Human cathelicidin, hcap-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3 Cathelicidins are a family of antimicrobial proteins found in the peroxidase-negative granules of neutrophils. The known biologic functions reside in the C-terminus, which must be cleaved from the holoprotein to become active. Bovine and porcine cathelicidins are cleaved by elastase from the azurophil granules to yield the active antimicrobial peptides. The aim of this study was to identify the physiological setting for cleavage of the only human cathelicidin, hcap-18, to liberate the antibacterial and cytotoxic peptide LL-37 and to identify the protease responsible for this cleavage. Immunoelectron microscopy demonstrated that both hcap-18 and azurophil granule proteins were present in the phagolysosome. Immunoblotting revealed no detectable cleavage of hcap-18 in cells after phagocytosis. In contrast, hcap-18 was cleaved to generate LL-37 in exocytosed material. Of the three known serine proteases from azurophil granules, proteinase 3 was solely responsible for cleavage of hcap-18 after exocytosis. This is the first detailed study describing the generation of a human antimicrobial peptide from a promicrobicidal protein, and it demonstrates that the generation of active antimicrobial peptides from common proproteins occurs differently in related species. This study was performed in collaboration with OE Sorensen of the National University Hospital, Copenhagen, Denmark. Collaboration with other groups from NKI/AvL In collaboration with B Giepmans (Division of Cellular Biochemistry), we have shown that microtubules are in contact with the gap junction protein connexin-43. With A De Melker (Division of Cellular Biochemistry), we demonstrated that c-cbl associates with the EGF receptor at the plasma membrane prior to receptor recruitment into clathrin-coated pits and remains associated throughout the clathrin-mediated endocytic pathway. c-cbl and the EGF receptor also colocalize in internal vesicles of multivesicular endosomes. With J Neefjes (Division of Tumor Biology), the intracellular localization of DM/DO and class II in Mel JuSo cells and transfectants was analyzed. Intracellularly both DM(/DO) and class II primarily localize to multivesicular bodies (MVB) (MIIC). Strikingly, the distribution between the limiting perimeter membrane and the internal vesicles of both DM(/DO) and class II within the multivesicular MIICs is altered in the wtdo transfectant compared with the DOCD8 transfectant or Mel JuSo itself. Quantification of the distribution demonstrates that in Mel JuSo and the DOCD8 transfectant DR is equally distributed over perimeter membrane and internal membranes, whereas DR is enriched on the perimeter membrane in the wtdo transfectant. The redistribution to the perimeter membrane is also seen for DM(/DO) going from 30% in Mel JuSo and the DOCD8 transfectant to 50% in the wtdo transfectant. The redistribution seems to be specific for DR and DM/DO complexes, since the distribution of the lysosomal marker CD63 residing in the same MIICs is not altered. Further, we have shown that, in cells transfected with virus encoding the Rab7 effector protein RILP, sligthly swollen MVB that were positive for RILP were densely clustered around the microtubules organiser center (MTOC). Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS, Hiemstra PS, Borregaard N. Human cathelicidin, hcap- 18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 2001; 97: Van Lith M, Van Ham M, Griekspoor A, Tjin E, Verwoerd D, Calafat J, Janssen H, Reits E, Pastoors L, Neefjes J. Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body. J Immunol 2001; 167:

21 II DIVISION OF MOLECULAR CARCINOGENESIS MOLECULAR CARCINOGENESIS REGULATION OF THE MAMMALIAN CELL CYCLE Following the completion of the human genome sequence, a major challenge is to functionally annotate the tens of thousands of genes for which this information is currently lacking. A main goal of the group has been the development of high throughput functional genetic screens to identify novel components of cancer-relevant pathways. By using this approach we hope to obtain detailed insights in the function for a number of hitherto anonymous genes. We have concentrated on two major tumor suppressor pathways: the p19 ARF -MDM2-p53 pathway and the p16 INK4A - cyclin D-pRb pathway. Both pathways are disrupted in virtually all forms of human cancer. Therefore, novel components of these pathways have a high probability of being deregulated in cancer as well. The p19 ARF -MDM2-p53 pathway p53 functions as a transcription factor that can activate expression 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 cells undergo senescence during in vitro propagation. Upregulation of p19 ARF (p14 ARF in man), leading to activation of p53, is a critical early event in the senescence response as rodent cells lacking p19 ARF escape from senescence and are immortal. As a first approach in identifying novel regulators of the p19 ARF -MDM2-p53 pathway, we have generated a mouse fibroblast cell line conditionally immortalized by a temperature sensitive allele of SV40 Large T antigen. Upon shift to the non-permissive temperature, these cells undergo rapid senescence. Introduction of a retroviral cdna expression library prior to temperature shift led to the identification of several genes that allow escape senescence. The first gene that we identified in this screen was BCL6, an oncogene originally identified as the target of an oncogenic translocation in non-hodgkin s lymphoma. BCL6 efficiently immortalizes primary mouse embryonic fibroblasts and co-operates with RAS in oncogenic transformation. Our data indicate that BCL6 overrides the senescence response downstream of p53 through a process that requires induction of cyclin D1 expression, as cyclin D1 knockout fibroblasts are specifically resistant to BCL6-mediated immortalization. Using additional retroviral cdna expression libraries, we 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 potently represses expression of both mouse p19 ARF and human p14 ARF. Interestingly, point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome patients, have lost the ability to inhibit senescence and fail to repress mouse p19 ARF and human p14 ARF expression. These data raise the possibility that the hypo-proliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14 ARF. More recently, we have identified a G protein coupled receptor, a guanine nucleotide exchange factor, an immediate early response gene and a homeobox transcription factor as senescence inhibitory genes, using the same approach. The interaction of these factors with the p19 ARF -MDM2-p53 pathway is currently under investigation. Primary fibroblasts respond to an activated RAS oncogene by prematurely undergoing cell cycle arrest, which resembles normal replicative senescence. This irreversible fail-safe mechanism against oncogenic transformation requires p19 ARF and p53 as their disruption causes RAS-expressing cells to escape from senescence. To identify novel genes that allow escape from RAS-induced senescence, we again used an unbiased, retroviral cdna library screen. Using this approach we identified DRIL1, the human orthologue of the mouse Bright and Drosophila Dead Ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS-induced, anti-proliferative signaling by p19 ARF /p53. Thereby, DRIL1 not only rescues RASinduced senescence, but also causes these fibroblasts to become highly oncogenic. Immortalization by DRIL1, which binds the prb-controlled transcription factor E2F1, correlates with induction of E2F activity. As a result, DRIL1 expression leads to induction of the E2F target gene CYCLIN E1, over-expression of which is sufficient to trig- Division head, Group leader R Bernards R Bernards PhD Group leader R Agami PhD Post-doc K Berns PhD Post-doc M Edel PhD Post-doc P Eichhorn PhD Post-doc D Peeper PhD Post-doc B Scheijen PhD Post-doc A Shvarts PhD Post-doc T Van der Meer PhD Post-doc L Wang MD PhD Post-doc R Oosterkamp MD Clinical fellow T Brummelkamp MSc Graduate student M Creyghton MSc Graduate student M Epping MSc Graduate student H Masselink MSc Graduate student S Nijman MSc Graduate student B Rowland MSc Graduate student M Bronk MSc Technical staff S Douma MSc Technical staff M Hijmans MSc Technical staff R Kortlever Technical staff H Winterwerp Technical staff E Bogaards Undergraduate student D Markusic Undergraduate student L Vredeveld Undergraduate student M Sonne Secretary

22 24 MOLECULAR CARCINOGENESIS Director of Research Anton Berns Key Publications Masselink H. Transcriptional repression and oncogenesis studies on adenovirus E1A interacting repressors. Thesis 2001, Utrecht. Masselink H, Vastenhouw N, Bernards R. B-myb rescues ras-induced premature senescence, which requires its transactivation domain. Cancer Letters 2001; 171: Oosterkamp HM, Bernards R. Androgen receptor and estrogen receptors. In: La Thangue NB, Bandara LR, editors. Targets for cancer chemotherapy: transcription factors and other nuclear proteins. Totowa NJ Humana Press (in press). Peeper DS, Dannenberg J-H, Douma S, Te Riele H, Bernards R. Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nature Cell Biology 2001; 3: Peeper DS, Shvarts A, Brummelkamp T, Douma S, Koh EY, Daley GQ, Bernards R. A functional screen identifies Hdril1 as an oncogene that rescues RAS-induced senescence. Nature Cell Biology (in press). Van t Veer L, 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 clinical outcome of breast cancer. Nature (in press). Figure II. 1 Model for p19 ARF /p53-induced cell cycle arrest. Both spontaneous and RAS V12 - induced senescence cause induction of a variety of anti-proliferative proteins, including p16 INK4a and p19 ARF. Our results suggest that both p19 ARF and its target p53, require active prb- E2F repressor complexes to induce senescence. Anti-proliferative signaling by p16 INK4a has been reported by others to require the prb-e2f repressor. ger escape from senescence. Thus, DRIL1 disrupts cellular protection against RASinduced proliferation downstream of the p19 ARF /p53 pathway. The p16ink4a-cyclin D-pRb pathway The p16-cyclin D-pRb pathway is deregulated in virtually every form of human 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 G1 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 such genes, we have developed a stable reporter cell line in which the activity of the E2F transcription factors can be monitored through measurement of luciferase activity. Our collaborator on this project, Galapagos Genomics in Leiden, has developed a collection of over 100,000 recombinant adenoviruses, each expressing a single human cdna. At this point, we have screened the E2F-luciferase reporter cell line with 11,000 of these recombinant adenoviral vectors using a highly automated robotic system. We identified three cdnas that can significantly modulate E2F activity. Two of these were known repressors of the E2F transcription factor and one unknown gene (designated H1-9) activated E2F. To study the function of E2F transcription factors in vivo, we have generated a panel of (conditional) E2F transgenic mice. Our data indicate that the five known E2Fs differ widely in their ability to induce cancer in vivo. Transgenic expression of E2F1 did not predispose to tumorigenesis. However, these animals were small due to a specific defect in endochondral bone formation. No defects in differentiation or apoptosis were seen in E2F1 transgenic mice. In contrast, expression of E2F2 predisposes to the development of thymoma in multiple independent strains of E2F2 transgenic animals. Elevated expression of E2F4 and 5 is well tolerated in transgenic animals and does not lead to any detectable pathology. Interestingly, the presence of the E2F4 transgene in a p107 null genetic background was an early embryonic lethal combination due to massive apoptosis in the peripheral nervous system, whereas the E2F4 transgene and p130 null combination was tolerated during development. To compare the oncogenic activity of the three prb interacting E2Fs (E2F1, 2 and 3) directly, we made three strains of conditional transgenic mice. The three E2Fs were inserted by site-specific recombination in the ubiquitously expressed ROSA26 locus. In these mice, expression of transgenic E2F occurs only after a CRE-mediated recombination event. At this point, we have crossed these conditional E2F transgenics with mouse strains expressing the CRE recombinase off a keratin promoter, an actin promoter and the POMC promoter. The effects of conditional E2F expression on development and oncogenesis are under investigation. Connections between the p53 and prb pathways Using primary fibroblasts deficient for the retinoblastoma tumor suppressor family members Rb, p107, or both, we found that only simultaneous ablation of both Rb and p107 disrupted RASinduced replicative arrest. This occurred despite activation by RAS of the p19 ARF /p53 pathway, identifying prb and p107 as essential downstream mediators of antiproliferative p19 ARF /p53 signaling. Unexpectedly, in contrast to p19 ARF or p53 deficiency, loss of prb/p107 function did not result in oncogenic transformation by RAS. The observed escape from RAS-induced replicative arrest without concomitant oncogenic transformation demonstrates that these processes can be uncoupled. In a related study, we found that p19arf-induced cell cycle arrest is accompanied by transcriptional repression by E2F. Inhibition of E2F-mediated repression caused a sharp increase in p19arf expression, but at the same time prevented growth arrest by both p19 ARF and p53 and rendered cells resistant to both spontaneous and oncogenic RAS-induced senescence. Together, these data indicate that the prb-e2f transcriptional repressor complex is a critical downstream mediator of anti-proliferative p19 ARF /p53 signaling. Thus, the prb and p53 pathways converge at the level of prb/e2f.

23 25 13 MOLECULAR CARCINOGENESIS CONTROL OF TELOMERASE ACTIVATION DURING IMMORTALIZATION AND TUMORIGENIC TRANSFORMATION Research in our group is primarily focused on the activation of telomerase in human cells. Normal human somatic cells, which generally lack telomerase activity, exhibit progressively shortened telomeres with repeated cell divisions and enter senescence after a limited number of cell divisions. When the life span of normal cells is extended beyond senescence, life span terminates at crisis, a point at which telomere loss results in chromosome instability and cell death. However telomere shortening is stopped by the expression of htert, the catalytic subunit of the telomerase enzyme, in those cells that have become immortal. htert mrna can not be detected in most normal cell lineages, but is present in a variety of tumor cell lines and is detectable in 90% of human tumors. The ectopic expression of htert is sufficient to restore telomerase activity and maintain or extend telomeres. The introduction of htert in several primary human cells prevented senescence, allowed them to bypass crisis and become immortal. In contrast, inactivation of telomerase in immortal cells results in telomere shortening and cell death. Together these results indicate that htert expression represents the rate limiting determinant that regulates the levels of telomerase enzyme activity in tumor cells and that the process of cell immortalization is closely or completely linked to the expression of htert. We are studying the molecular mechanisms responsible for derepression of the htert gene that occurs upon immortalization. We have isolated, sequenced and analyzed genomic fragments containing the promoter and potentially upstream regulatory elements of the human htert gene. We have set up an htert-reporter system that allows for the identification of proteins or pathways that can either repress or activate htert expression through introduction cdna expression libraries. A 1.2 Kb promoter fragment of the htert gene directs the expression of GFP in human embryonic kidney cells transformed with SV40 Large T and immortalized by the introduction of an htert cdna. This integrated reporter, as well as the endogenous htert gene, can be activated upon introduction of c-myc, to a level comparable to spontaneous immortalized cells of the same origin. We are currently isolating cdnas that, after retroviral transduction, result in the upregulation of this integrated htert reporter. We will confirm the effect of the cdnas on the endogenous htert gene in cells of the same origin. In addition, we have identified a DNA element in the htert promoter that upon mutation causes upregulation of the htert reporter in telomerase negative cells to a level identical to telomerase positive cells. This DNA binding element is currently under investigation. By deletion analysis and electromobility bandshift analysis, we are in the process of identifying factors that interact with this DNA element. This isolated element will also be used in reporter assay-based screens with retroviral cdna libraries to identify interacting proteins. We expect that the isolation of these factors will contribute to our understanding of the repressive mechanisms that act on the htert gene. The result of these studies may allow us to elucidate the molecular mechanism that enables cancer cells to overcome two barriers, senescence and crisis, before they become immortal. The identification of the proteins or pathways that are involved in regulation of telomerase expression may have implications for interference with the process of immortalization and provide us with a molecular event, specific in the generation of tumor cells. Group leader R Beijersbergen R Beijersbergen PhD Group leader LM Carlee Graduate student WABE Nijkamp Technical staff M Roseboom Undergraduate student M Van Lun Undergraduate student

24 26 MOLECULAR CARCINOGENESIS Director of Research Anton Berns PROTEIN STRUCTURE AND FUNCTION Group leader TK Sixma TK Sixma PhD Group leader K Brejc PhD Post-doc P Celie PhD Post-doc V Notenboom PhD Post-doc P Knipscheer Graduate student M Lamers Graduate student G Natrajan Graduate student S Watkins Graduate student WJ Van Dijk Technical staff S Van Rossum Technical staff HW Winterwerp Technical staff Development of cancer is generally caused by errors occurring in cellular pathways. Structural biology can help to understand these errors at the atomic level, by studying the proteins and the DNA involved. We use X-ray crystallography as a tool to provide three-dimensional structures. These provide more insight into the molecular processes and they can also provide targets for drug design studies. In our group we focus mainly on proteins involved in DNA stability (transposition, mismatch repair), transmembrane signaling and cell cycle control. DNA mismatch repair Mutations in DNA mismatch repair genes predispose for the most prevalent type of familial cancers, hereditary non-polyposis colon carcinoma (HNPCC). This type of DNA repair is specific for a single mismatch or a small stretch of unpaired bases in DNA. It involves a cascade of proteins that is highly conserved from bacteria to humans. The initial step of the repair is the recognition and binding of mismatched DNA by the MutS protein (E. coli) or its heterodimeric MSH2/MSH6 or MSH2/MSH3 homologs (eukaryotes). This complex is then recognized by the MutL protein or its homologs. Last year we solved the crystal structure of E. coli MutS in complex with a G:T mismatched DNA oligomer. In this structure we found that the symmetric MutS molecules bind to the DNA in an asymmetric manner. Only one monomer contacts the mismatch directly. In our structure this mismatch also binds ADP while the other ATPase domain is empty. To understand whether the observed asymmetry of the ATPase domains is functional, and what the role of the ATPase domain in the protein is, we have soaked ATP into our crystals and shown that it is possible to bind nucleotide to both monomers. However, our crystal contacts seem to prevent the hydrolysis and the conformational changes that are usual upon ATP binding. Mapping of the mutations that are found in HNPCC families to the E. coli protein is sometimes useful in deciding if a mutation is likely to be the cause of the disease. To study such mutations in more depth we have created a series of these mutants, in collaboration with N De Wind (Leiden). Currently we are studying these using functional studies and crystallography. Homolog of the nicotinic acetylcholine receptor Acetylcholine binding protein (AChBP) is a water-soluble protein greatly resembling the extracellular domains of ligand-gated ion channels. This superfamily includes the nicotinic acetylcholine receptor, as well as the GABA A and GABA C, the glycine and the 5HT 3 serotonin receptors. Most of these are important drug targets. AChBP forms homopentamers with pharmacology that resemble the α 7 nicotinic receptors. The protein is secreted from glial cells in the fresh-water snail, Lymnaea stagnalis, where it modulates neuronal transmission. Since there were no highresolution structures of any member of the pentameric ligand-gated ion-channel family, we solved the crystal structure of AChBP at 2.7 Å resolution. Figure II.2. Ribbon diagram of AChBP, close homolog of the Nicotinic Acetylcholine Receptor extracellular domain (Brejc et al, 2001). Each monomer in a different colour. Ligand binding residues are shown in ball-and-stick model, showing the positions of the acetylcholine binding sites. In the crystal structure each protomer has a modified immunoglobulin fold. Almost all residues shown to be involved in ligand binding in the nicotinic 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 nicotinic acetylcholine receptors. Surprisingly, the interface between protomers is relatively weakly conserved between family members in the superfamily of pentameric ligand-gated ion channels. The lack of conservation has implications for the mechanism of gating of the ion-channel. By comparing AChBP with a bungarotoxin peptide complex we could build a good model of the interaction of this toxin with the nicotinic receptor, showing how it inserts a loop into the ligand-binding site. Our crystal structure can serve as a basis for structure-based drug design targeting Alzheimer s, certain forms of schizophrenia and smoking. Ubiquitin dependent conjugation Ubiquitin conjugation processes are emerging as a general addressing system essential for cell stability, by controling degradation of

25 27 13 MOLECULAR CARCINOGENESIS short-lived proteins, DNA repair and targeting to specific areas in the cell through endocytosis. Because of its importance for regulating cell cycle (e.g. p53, MDM2, E2F s, p27, cyclin D), apoptosis (e.g. ccbl, IAPs) and DNA repair (e.g. Rad6, Rad18, Brca1) 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 E1/E2/E3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, the atomic details have been lacking and the specific determining factors are unclear. We are studying several E2/E3 complexes. We have created E. coli and baculovirus expression systems for the human SCF components Skp1, Cullin and several F-box proteins (e.g. Skp2, Fbx4, Fbl7). We have purified the Skp1-Fbx4 complex to homogeneity and are currently studying target interaction, in collaboration with W Boelens (Nijmegen). The E2/E3 complex of mammalian Rad6/Rad18 is involved in error-free postreplicative DNA repair. In collaboration with J Hoeijmakers (Rotterdam) we have purified the complex from E. coli and purified it to homogeneity. The complex is stable through a number of purification steps. On gel-filtration it behaves as a dimer of heterodimers. This agrees with the size estimated in mass-spectrometry studies of cross-linked complex that we obtained in collaboration with the group of A Muysers (UvA, Amsterdam). Human Rad6 has also been purified separately from E. coli, crystallized, yielding good diffracting crystals (2.0 Å), and the structure has been solved. Key Publications Brejc K, Van Dijk WJ,. Klaassen RV, Schuurmans M, Van der Oost J, Smit AB, Sixma TK. Crystal structure of an AChbinding protein reveals the ligand-binding domain of nicotinic receptors. Nature 2001; 411: Harel M, Kasher R, Nicolas A, Guss JM, Balass M, Fridkin M, Smit AB, Brejc K, Sixma TK, Katchalski-Katzir E, Sussman JL, Fuchs S. The binding site of acetylcholine receptor as visualized in the X-ray structure of a complex between a-bungarotoxin and a mimotope peptide. Neuron 2001; 32: Sixma TK. DNA mismatch repair: MutS structures bound to mismatches, Current Opinion in Structural Biology 2001; 11: Smit AB, Syed NI, Schaap D, Van Minnen J, Klumperman J, Kits KS, Lodder H, Van der Schors RC, Van Elk R, Sorgedrager B, Brejc K, Sixma TK, Geraerts WPM. A gliaderived acetylcholine-binding protein that modulates synaptic transmission. Nature 2001; 411: Figure II.3. Human Rad6 (Ubc2) crystals and electron density with model superimposed. This ubiquitin conjugating enzyme (E2) is involved in postreplicative DNA repair. Currently the structure is being refined. It resembles previously solved E2 enzymes, including the yeast Rad6 structure, but it will be useful for determining the Rad6- complex structures, and for analyzing the effects of complex formation on Rad6 itself. Earlier data on yeast Rad6/Rad18 indicated that these proteins bound to DNA. In a preliminary experiment we could confirm this for the human proteins. The E2 structures solved by others and us, have made it clear that understanding their mechanism will require structures of complexes with the proteins involved.

26 28 MOLECULAR CARCINOGENESIS Director of Research Anton Berns STRUCTURAL BIOLOGY Group leader A Perrakis A Perrakis PhD Group leader K Verschueren PhD Post-doc O Weichenrieder PhD Post-doc K Repanas MSc Graduate student N Rocha MSc Graduate student M Kakaris MSc Software engineer Key Publications Lamzin VS, Perrakis A. Current state of automated crystallographic data analysis. Nat Struct Biol 2000; 7 suppl: Perrakis A, Harkiolaki M, Wilson KS, Lamzin VS. ARP/wARP and molecular replacement. Acta Crystallogr D Biol Crystallogr 2001; 57: Our newly established group aims to combine biological interests that adapt and complement the NKI scientific research objectives, culture and experience with technical innovation and expertise in structural biology. Our research plan is two-fold: to study biological systems from a structural viewpoint by using X-ray crystallography and single particle reconstruction transmission electron microscopy and to develop computational methods for high throughput structure determination for X-ray crystallography. During the past year, we proceeded with a major refurbishment of the crystallographic computing facilities, which included the installation of a four-processor Compaq Alpha-server (in large part generously loaned by Compaq Europe). The first steps for starting up single particle transmission electron microscopy work were also made by establishing a collaboration with the University of Leiden Electron Microscopy unit, giving us access to a state of the art Tecnai 200 kv Field Emission Gun (FEG) microscope, and by making our first pictures of protein particles in Leiden and using the in-house microscope. Most importantly however we have set the foundation for two projects in collaboration with in-house groups: With J Neefjes, building on the recent discovery of RILP in his group, we will study components of the late lysosomal trafficking machinery, which among other roles is responsible for antigen presentation and an attractive target for immunity intervention. Soluble RILP protein is being produced and we are proceeding with the formation of the complex between RILP/RAB7 and crystallization trials. With the group of A Berns, we have started studying the structural characterization of the LINEs retroposition machinery, responsible for creating about one quarter of human DNA, most of these commonly known as junk DNA. We are currently constructing various expression constructs of an active L1 retroposase. We have also developed the conceptual design for two projects that will commence early next year. The study of regulators of the CTLA-4 receptor (with A Kruisbeek) and of various nucleoporins and its complexes with cargo carrier proteins, in collaboration with M Fornerod. Interest of this group in the development of high throughput methods for macromolecular X-ray crystallography focuses on the ARP/wARP software package for automated model building and refinement in macromolecular X-ray crystallography, which is actively being pursued with V Lamzin at the EMBL. ARP/wARP has eliminated, to a large extent, the need for the time consuming step of manually building crystallographic models in graphics workstations. During the last years, its user community has increased to over 500 academic laboratories and 34 commercial users, while publications citing the software have exceeded 200 within The commencement of the AUTOSTRUCT EU network early this year, for a Europe-wide collaboration on crystallographic methods development, and the high ranking of our NIH application for further development of the ARP/wARP package will allow the building of a strong research team focussing on that subject. We expect to further develop the ARP/wARP package to meet the increasing needs of the high-throughput structural genomics efforts worldwide and widen its applicability towards complicated structural biology projects. Fig II.4. An electron density map and the corresponding final crystallographic model after conventional crystallographic techniques (top) and after ARP/wARP refinement and automated model building (bottom). ARP/wARP not only drammatically improves the quality of the electron density map, but also automatically builds the molecular model - instead of the crystallographer spending laborius hours in a graphics workstation.

27 III DIVISION OF CELLULAR BIOCHEMISTRY CELLULAR BIOCHEMISTRY LYSOPHOSPHOLIPID SIGNALING: SMALL GTPASES AND CYTOSKELETAL REGULATION Lysophosphatidic acid (LPA) is serum-borne lysophospholipid that evokes a host of cellular responses, ranging from rapid cytoskeletal reorganization to stimulation of cell proliferation and survival. LPA is produced by activated platelets and has been implicated in wound healing and tissue remodeling. LPA levels are found elevated in ascitic fluid from ovarian cancer patients. Precisely how and where LPA is produced in vivo remains an outstanding question. LPA signals through three distinct G protein-coupled receptors (GPCRs), whose individual properties remain to be characterized. Since LPA receptors drive tumor cell migration, invasion and proliferation, whilst LPA levels are elevated in cancer patients and the receptors are found overexpressed in certain tumors, LPA receptors qualify as potential targets for therapy. Our studies explore LPA receptor signaling pathways mediated by small GTPases of the Ras/Rho family, with emphasis on the control of cytoskeletal remodeling and cell migration. This should lead to novel ways of interfering with unscheduled LPA receptor signaling in cancer cells and with LPA production in their microenvironment. The ins and outs of LPA receptor signaling A major unresolved question is how and where LPA production occurs in vivo. Increasing evidence suggests that LPA can be generated by an as-yet-unidentified extracellular lysophospholipase D (LPLD) acting on lysophosphatidylcholine (LPC). Since the putative LPLD gene has not been cloned yet, bacterial (lyso)pld may serve as a useful model. We find that bacterial PLD (from S. chromofuscus), a virulence factor, reproduces the multiple actions of LPA in many cell types, but only if these cells express functional LPA receptors and extracellular LPC is not limiting. Exogenous PLD mimics LPA in provoking rapid internalization of LPA receptors Thus, mammalian LPA receptors can mediate the pathogenic effects of bacterial PLDs. Recently, we have identified a human cdna that may encode a secreted (L)PLD. Studies to examine the putative PLD-like properties of this gene product are under way. We have analyzed the effects of LPA receptor signaling on cell behavior by expression of LPA 1,2 (Edg2,4) in receptor-negative cells. After ligand addition, LPA receptors are rapidly internalized as determined by live-cell imaging (Figure III.1). LPA 1,2 activation promotes activation of the Rac1 GTPase, which is a Gi-mediated and PI3-kinase-dependent response. Rac activation is accompanied by myosin heavy chain phosphorylation and leads to lamellipodia formation, cell spreading and migration. In collaboration with J Collard and co-workers (Division I), we established that the invasion-inducing Tiam1 gene product mediates LPA-induced Rac activation. Thus, LPA 1,2 can act as chemoattractant receptors by activation of a Gi-PI3kinase-Tiam1-Rac signaling pathway. Regulation of c-ras by cholesterol Cholesterol-rich and caveolin-containing microdomains of the plasma membrane, termed caveolae, have been implicated in signal transduction. However, the role of caveolae in regulating the Ras-MAP kinase cascade is incompletely understood. The mammalian Ras isoforms (H, N and K) use different membrane anchors to attach to the plasma membrane and thereby may localize to functionally distinct microdomains, which might explain isoform-specific signaling. We have established that in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both caveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We have found that both LPA and epidermal growth factor (EGF) activate N-Ras, but fail to activate K-Ras in these cells. Extraction of cholesterol with methyl-b-cyclodextrin disrupts complex formation between caveolin and K- and N-Ras and, strikingly, enables EGF to activate both K-Ras and N-Ras. While cholesterol depletion enhances GTP-loading on total c-ras, activation of the downstream MEK-MAP kinase cascade by EGF and LPA is inhibited. It thus appears that membrane cholesterol is crucial for two distinct steps in the receptor-ras-map kinase pathway: (1) it negatively regulates c-ras activation, presumably by promoting the formation of caveolin-ras complexes, and (2) it is required for downstream signaling from active c-ras to MEK and MAP kinase. Division head, group leader WH Moolenaar WH Moolenaar PhD Group leader BNG Giepmans PhD Post-doc L Sayas PhD Post-doc F Van Leeuwen PhD Post-doc S Grivell MSc Graduate student J Mulder MSc Graduate student FPG Van Horck MSc Graduate student L Van Meeteren MSc Graduate student A Van Rossum MSc Graduate student L Van Zeijl MSc Graduate student R Bernad Undergraduate student GM Hengeveld Technical staff P Ruurs Technical staff I Verlaan Technical staff A Van Leeuwen Technical staff H Wieldraaijer Secretary Figure III.1. LPA induces rapid internalization of LPA1/Edg2 receptors (fused to green fluorescent protein, GFP) in HEK293 cells. Figure III.2. Microtubule localization at Cx43 cellcell contacts. Note that microtubule distal ends terminate at Cx43-based gap junctions (Rat-1 cells). Lower right panel: Immunoelectron microscopic localization of tubulin at Cx43 gap junctions (GJ). Arrows point to tubulin staining (10 nm gold). Cx43 staining: 15 nm gold (Giepmans et al., 2001).

28 30 CELLULAR BIOCHEMISTRY Director of Research Anton Berns Key Publications Andreev J, Galisteo M, Kranenburg O, Logan S, Chiu E, Okigaki M, Cary L., Moolenaar W, Schlessinger J. Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor but are not required for coupling to the mitogen-activated protein kinase signaling cascade. J Biol Chem 2001; 276: Giepmans BNG, Hengeveld T, Postma FR, Moolenaar WH. Interaction of c-src with gap junction protein connexin-43: role in the regulation of cell-cell communication. J Biol Chem 2001; 276: Giepmans BNG, Verlaan I, Hengeveld T, Janssen H, Calafat J, Falk MM, Moolenaar WH. Gap junction protein connexin-43 interacts directly with microtubules. Curr Biol 2001; 11: Kranenburg O, Moolenaar WH. Ras-MAP kinase signaling by lysophosphatidic acid and other G protein-coupled receptor agonists. Oncogene 2001; 20: Kranenburg O, Verlaan I, Moolenaar WH: Regulating c-ras function: cholesterol depletion affects caveolin association, GTP loading and signaling. Curr Biol 2001; 11: Moolenaar WH. The ins and outs of lysophosphatidic acid signaling. BioEssays (in press). Postma F, Jalink K, Hengeveld T, Offermanns S, Moolenaar WH. G(alpha)13 mediates activation of a depolarizing chloride current that accompanies RhoA activation. Curr Biol 2001; 11: Van Horck FP, Ahmadian MR, Haeusler LC, Moolenaar WH, Kranenburg O. Characterization of p190rhogef: a RhoAspecific guanine nucleotide exchange factor that interacts with microtubules. J Biol Chem 2001; 267: Cytoskeletal regulation by p190rhogef and p116rip Rho family GTPases, which control cytoskeletal remodeling, are activated by guanine nucleotide exchange factors (GEFs). We previously isolated p190rhogef, which specifically acts on RhoA. When overexpressed in neuronal cells, p190rhogef induces cell rounding and inhibits neurite outgrowth. Immunofluorescence studies show that endogenous p190rhogef localizes to distinct RhoA-containing regions at the plasma membrane and along microtubules. In vitro binding experiments show that p190rhogef directly interacts with microtubules via its C-terminal region. Microtubule disruption promotes RhoA activation, similar to what is observed with p190rhogef overexpression. Thus, p190rhogef may provide a link between microtubule dynamics and RhoA signaling. We are also characterizing p116rip, a putative scaffold protein containing two PH domains and a coiled-coil region. p116rip localizes to the actin cytoskeleton and is also present in the nucleus. In vitro binding studies show that the N-terminal region of p116rip binds directly to f-actin. Overexpression of the actin-binding domain leads to disruption of the actin cytoskeleton, suggesting that p116rip is essential for maintaining cytoskeletal integrity. We are currently performing two-hybrid studies to identify the binding partners of this novel actin-binding protein. Cytoskeletal regulation by a Trp-related cation channel / MHC kinase There is still little known about how agonists such as LPA induce cytoskeletal relaxation and cell spreading. LPA and other GPCR ligands induce a Ca 2+ -dependent phosphorylation of the myosin II heavy chain (MHC), accompanied by Rac activation and disassembly of the cortical 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 bifunctional protein is now known as TRP-PLIK, LTRPC7 or Channel Kinase (ChaK), but its functions are unknown. We find that ectopic expression of ChaK promotes cell spreading and matrix adhesion, accompanied by striking actomyosin remodeling. Furthermore, overexpression of ChaK enhances Ca 2+ -influx in response to LPA, but not when internal calcium stores are depleted by thapsigargin pretreatment. We find that ChaK associates with actomyosin and can phosphorylate myosin II heavy chain, which correlates with an increase in basal Ca 2+ levels. These results identify ChaK as a potential MHC kinase, and they provide a link between store-operated Ca 2+ influx, regulation of the cortical actomyosin network and cell spreading. Cell-cell communication: connexin-43 and microtubules Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by LPA and other mitogens. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. We found that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35 amino-acid juxtamembrane region in the Cx43 tail mediates microtubule binding. Immunofluorescence and electron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells (Figure III.2). However, intact microtubules are dispensable for the regulation of gap junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.

29 31 13 CELLULAR BIOCHEMISTRY LYMPHOCYTE ACTIVATION AND SURVIVAL Regulation of lymphocyte activation Lymphocyte activation and ensuing proliferation is initiated by antigen receptors, which signal according to the protein tyrosine kinase (PTK) principle. The c-cbl proto-oncogene product associates with PTK-coupled receptor systems and negatively regulates their function, at least in part by promoting their ubiquitination. This function impinges on a Ring finger domain, which interacts with E2-type ubiquitin conjugating enzymes, according to our findings and those of other groups. The collective data classify c-cbl as an E3-type ubiquitin ligase. Using the EGF receptor as a model system, we investigate the importance of ubiquitination in receptor endocytosis and trafficking and examine whether c-cbl has functions in addition to being an E3. We have found that ubiquitination occurs at the cell surface prior to receptor endocytosis via clathrin coated pits. To examine whether ubiquitination regulates receptor internalization, as has been shown for specific membrane receptors in yeast, we have generated a variety of EGF receptor mutants lacking potential ubiquitination sites. The sites employed are being mapped by mass spectrometry (collaboration with L De Jong, Chemistry Department, University of Amsterdam). c-cbl chimeras composed of a receptor binding domain and a functional E2 restore ubiquitination function and are used to determine whether c-cbl harbours additional activities. Group leader J Borst J Borst PhD Group leader A De Melker PhD Post-doc S Tait PhD Post-doc Y Xiao PhD Post-doc J Hendriks MSc Graduate student A Werner MSc Graduate student I Bontjer Technical staff E De Vries Technical staff G Van der Horst Technical staff Regulation of lymphocyte survival The size of the antigen-specific lymphocyte pool depends not only on cell proliferation, but also on survival. We are studying cross-talk between antigen receptors and members of the Tumor Necrosis Factor (TNF) receptor family in the regulation of lymphocyte survival both in vitro and in vivo. Jurkat leukemia T cells are being employed as a model, since they reflect the process of activation-induced cell death, which is instrumental in down-regulation of the immune response and selection of memory T cells. We are studying the mechanism of apoptosis resistance displayed by cells, selected for resistance to agonists of the CD95 death receptor. These cells display cross-resistance to DNA damaging anti-cancer drugs and ionizing radiation. Using exogenous mitochondria and recombinant proteins made by in vitro transcription/translation, we have established that a factor newly expressed in the cytosol of these resistant cells impedes mitochondrial cytochrome c release in the presence of truncated Bid (tbid). Such a mechanism of inhibition has not been described previously. A Bid-interacting protein, Bfl-1/A1 was isolated, which performs such a function. Bfl-1 interacts with full length Bid, but does not impede Caspase-8-mediated Bid processing or association of tbid with mitochondria. It remains selectively and strongly associated with tbid in the mitochondrial membrane and impedes cytochrome c release induced by tbid and its cooperation with Bax or Bak. Bfl-1 is, however, not the inhibitor operating in our variant cells. We have set up assays to monitor relocation of proteins to the mitochondria and cytochrome c release in fixed cells and in real time, which will be used to define the mechanism of action of inhibitors of death receptor-induced apoptosis. Such inhibitors will be identified by protein interaction screens as well as functional screens, for which the conditions have been created. Regulation of lymphocyte survival is being studied in vivo in mouse model systems. We are focusing on the role of CD27 and its close relatives OX-40 and 4-1BB. These are members of the TNF receptor family associating with Traf adaptor molecules, which have been implicated in anti-apoptotic signaling. We are examining lymphocyte proliferation, differentiation and migration in response to influenza virus infection or challenge with tumors engineered to express an influenza virus nucleoprotein fragment. This allows us to follow the antigen-specific T-cell response with MHC tetramers. CD27 makes an important contribution to the memory T-cell response to virus as well as tumor cells. The primary T-cell response is also diminished in absence of CD27 but to a lesser extent. Adoptive transfer experiments using TCR transgenic T cells, as well as in vitro experiments show that CD27 influences the size of the responding T-cell pool by promoting cell survival rather than proliferation. Moreover, CD27 may influence T cell migration to the effector site. Analysis of mice deficient for CD27, CD28 or both show that CD27 and CD28 have complementary roles in determining the size of the effector T-cell pool. CD27 function is controlled by CD28, presumably through regulation of its ligand CD70.

30 32 CELLULAR BIOCHEMISTRY Director of Research Anton Berns Key Publications Arens R, Tesselaar K, Van Schijndel GMW, Baars PA, Pals ST, Krimpenfort P, Borst J, Van Oers MHJ, Van Lier RAW. Constitutive CD27/CD70 interaction induces expansion of effector-type T cells and results in IFNmediated B cell depletion. Immunity (in press). Recently, we have established that CD28-driven germinal center formation and ensuing antibody production to influenza virus depends on the presence of CD27 on B cells. Future experiments are directed at linking receptor directed gene expression profiles with cellular responsiveness in vivo. Borst P, Borst J, Smets L. Does tumor cell resistance to apoptosis affect the outcome of chemotherapy? Drug Res Updates 2001; 4: De Melker AA, Van der Horst G, Calafat J, Jansen H, Borst J. c-cbl ubiquitinates the EGF receptor at the plasma membrane and remains receptor-associated throughout the endocytic route. J Cell Sci 2001; 114: Haks MC, Cordaro TA, Van den Brakel J, Haanen JB, De Vries EF, Borst J, Krimpenfort P, Kruisbeek AM. A redundant role of the CD3 immunoreceptor tyrosine-based activation motif in mature T cell function. J Immunol 2001; 166: Tepper AD, Ruurs P, Borst J, Van Blitterswijk WJ. Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis. Biochem Biophys Res Comm 2001; 280: Van Blitterswijk WJ, Van der Luit AH, Caan W, Verheij M, Borst J. Sphingolipids related to apoptosis from the point of view of membrane structure and topology. Biochem Soc Trans (in press).

31 33 13 CELLULAR BIOCHEMISTRY LIPID METABOLISM IN SIGNAL TRANSDUCTION Membrane lipids undergo continuous breakdown and resynthesis, inherent to their active participation in signal transduction. During this process, second messengers are transiently generated to recruit and activate specific enzymes. Such lipid turnover is also found in the nucleus, where its function is not understood. Signals from the cell surface are internalized via tightly regulated endocytic and vesicular trafficking processes. New in this respect is the role of membrane microdomains, called rafts and caveolae, which are enriched in sphingolipids. We are investigating how certain unnatural lysophospholipids interfere with signaling and trafficking processes and can thereby act as apoptotic and anti-tumor agents. Diacylglycerol kinases (collaboration with N Divecha) Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to yield phosphatidic acid (PA). Since DAG is an activator of protein kinase C (PKC), DGK may serve to attenuate PKC activity. The DGKθisotype, cloned in our laboratory, may play a role in vascular smooth muscle physiology. Adrenergic stimulation of arterial smooth muscle and endothelial cells causes rapid translocation of DGKθfrom the nucleus to membranes. There, the enzyme becomes transiently activated in a phosphoinositide 3-kinase-dependent manner and is found associated with Akt/PKB (collaboration with J Ohanian, University of Manchester, UK). Purinergic receptor stimulation evoked translocation of DGKθfrom the cytosol towards the plasma membrane in A431carcinoma cells. Such translocation is also observed with phorbol ester, suggesting that PKC may counteract its own activation by recruiting and activating DGKq. By using DGKqmutants, we are currently investigating which domains are responsible for translocation and in which signaling pathway DGKqis operating. Another DGK isotype, termed DGKζ,localizes to the nucleus in a PKC-dependent manner. We have found that DGKζ can bind to the retinoblastoma protein, prb, a key regulator of E2F-mediated gene transcription and cell cycle progression. We have found that DGKζ promotes Rb-mediated E2F inactivation. Since prb can also bind to other lipid (PIP) kinases in the nucleus (group N Divecha), these findings suggest a link between lipid metabolism in the nucleus and the cell cycle machinery. Group leader WJ Van Blitterswijk WJ Van Blitterswijk PhD Group leader M Verheij MD PhD Academic staff J Van Baal PhD Post-doc A Van der Luit PhD Post-doc RJ Veldman PhD Post-doc A Los MSc Graduate student GA Ruiter MSc Graduate student M Budde Technical staff JJM De Widt Technical staff SF Zerp Technical staff Sphingolipids in rafts/caveolae Sphingolipids and their metabolites have emerged as important regulators of cellular function. Some of these lipids (e.g. sphingosine 1-phosphate) can act extracellularly, by binding to specific G proteincoupled receptors. However, the majority of sphingolipids determine membrane fine-structure in a dynamic way and, as such, are specific co-regulators of receptor signaling and vesicular trafficking. Sphingolipids, together with cholesterol, are physically assembled in microdomains called lipid rafts and caveolae. The latter structures, containing caveolin, are particularly enriched in endothelial cells (Figure III.3). Breakdown of sphingomyelin, the major phospholipid in these domains, to ceramide is an almost universal phenomenon during apoptosis. We have challenged the common belief that ceramide may signal apoptosis. Instead, we propose that the Figure III.3. Caveolin staining in normal human colon tissue (left) and colon carcinoma tissue (right) from the same patient. Note the staining of the endothelial lining in microvessels, which is lost in the malignant tissue. (Materials obtained from D De Jong, pathologist). We are investigating possible colocalization of caveolin with bioactive sphingolipid metabolites.

32 34 CELLULAR BIOCHEMISTRY Director of Research Anton Berns Key Publications Ruiter GA, Verheij M, Zerp SF, Van Blitterswijk WJ. Alkyl-lysophospholipids as anti-cancer agents and enhancers of radiation-induced apoptosis. Int J Radiat Oncol Biol Phys 2001; 49: Tepper AD, Ruurs P, Borst J, van Blitterswijk WJ. Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis. Biochem Biophys Res Commun 2001; 280: Van Blitterswijk WJ, Van der Luit AH, Caan W, Verheij M, Borst J. Sphingolipids related to apoptosis from the point of view of membrane structure and topology. Biochem Soc Trans 2001; 29: Walker AJ, Draeger A, Houssa B, Van Blitterswijk WJ, Ohanian V, Ohanian J. Diacylglycerol kinase-theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries. Biochem J 2001; 353: changes in raft composition following sphingomyelin hydrolysis facilitate domain clustering and membrane vesicle formation. Since rafts contain many signaling proteins, sphingolipid metabolism may thus modulate signaling pathways and vesicular traffic that emanate from rafts. To test this, we are isolating caveolae and are using a novel HPLC system, with sensitivity in the femtomolar range, to analyze their sphingolipid composition. In addition to more complex sphingolipids, we have also found ceramide and sphingosines preferentially localized in caveolae. We suggest that these microdomains are sensors for (oxidative) stress and that stress signals emanating from these domains are propagated via endocytosis. How the various sphingolipids participate in internalizing these signals is now under investigation Apoptosis induced by alkyl-lysophospholipids (ALPs) Synthetic alkyllysophospholipids (ALPs) such as 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Edelfosine), hexadecylphosphocholine (HePC; Miltofosine) and its piperidine analog D (Perifosine) induce apoptosis in many tumor cells and are used clinically as anti-cancer agents. ALPs act on cell membranes and interfere with lipid signaling pathways that feed into major protein kinase cascades. In this way, ALPs inhibit the ERK/MAP kinase proliferative pathway and the Akt/PKB survival pathway. On the other hand, the JNK/SAP kinase stress pathway is stimulated by ALPs. We have investigated how ALP (Edelfosine) is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC) and how this relates to the induction of apoptosis. To this end, we have compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49 AR, which shows cross-resistance to HePC and D-21266, and, intriguingly, also to other forms of stress, such as H 2 O 2 and ionizing radiation. ALP inhibited PC synthesis by CTP:phosphocholine cytidylyltransferase (CT). Exogenous lysophosphatidylcholine (LPC), leading to PC generation probably in endosomes, can rescue cells from ALP-induced apoptosis. This indicates that continuous rapid PC synthesis is essential for cell survival. Apoptosis induced by other apoptotic stimuli such as ionizing radiation, that does not target PC synthesis, remained unaffected by LPC. We have found that S49 cells internalize ALP by endocytosis, which is not observed in S49 AR cells. This may involve a previously unrecognized rapid and constitutive form of endocytosis, operating via rafts. S49 AR cells show a reduced biosynthesis of sphingomyelin (SM) (Figure III.4) and other complex sphingolipids. Our data point to a defect in the translocation of precursor sphingolipids across the Golgi membrane, eventually resulting in anomalous rafts, the likely cause of defective endocytic vesicular traffic of stress signals. ALP-induced cellular stress leads to formation of reactive oxygen species. S49 AR cells may escape the toxic effects of continuous ALP treatment by shutting off the endocytosis of ALP via inhibition of their sphingolipid synthesis and functional raft formation. Other aspects of anti-cancer activities of ALPs are reported under IX Division of Radiotherapy (M Verheij). Figure III.4. Phospholipid biosynthesis in S49 cells and S49AR cells in time. A thin-layer chromatograph of 14C-choline-labeled phospholipids is shown. SM, spingomyelin; PC, phosphatidylcholine; LPC, lysopc; ori (origin), site of application.

33 35 13 CELLULAR BIOCHEMISTRY SIGNALING THROUGH INOSITOL PHOSPHOLIPIDS Oncogenic mutations of nuclear DNA leads to the up-regulation of intracellular signaling pathways which stimulate proliferation, block apoptosis and increase motility of cells. Normal cells use phosphoinositide signaling pathways to interpret extracellular environmental signals, such as growth factors, to regulate many of the above processes. Our group is interested in how phosphoinositide pathways can be deregulated in order to support tumor progression. Our research concentrates on the action and regulation of the phosphatidylinositol phosphate (PIP) kinase enzymes, which synthesize the phosphoinositide, PtdIns(4,5)P2, a central lipid in cellular signaling. In vitro, PtdIns(4,5)P2 is generated by Type I PIPkinases, which phosphorylate PtdIns(4)P on the 5-OH position of the inositol ring, or by Type II PIPkinases, which phosphorylate PtdIns(5)P on the 4-OH position. While Type I PIPkinases are found in all eukaryotic cells and are essential for viability, no functional role has been found for Type II PIPkinases. Type I PIPkinases and the regulation of extracellular matrix adhesion In vivo many cellular processes require the ability to modulate the interaction between the cell surface and the extracellular matrix. This is essential for cell migration and motility in normal cells and is drastically deregulated in cancer cells. Malignant cells characteristically lose appropriate contacts with the extracellular matrix and show anchorage independent growth. Modulation of extracellular contacts is also fundamental to the development of the nervous system and is controlled in part by the small molecular weight G protein Rho, an upstream activator of Type I PIPkinases. In collaboration with F Van Horck and W Moolenaar, we have uncovered an essential role for Type I PIPkinases in the regulation of neurite remodelling in response to both growth factors and endogenous neuronal guidance cues such as Semaphorin-3A. We suggest that activation of Type I PIPkinases is required downstream of Rho for the negative regulation of integrin/extracellular matrix interactions. Group leader N Divecha N Divecha PhD Group leader CS D Santos PhD Post-doc JR Halstead PhD Post-doc D Jones PhD Post-doc J Van Baal PhD Post-doc D Weinkove PhD Post-doc AP Los MSc Graduate student M Roefs Technical staff Redox regulation of Type I PIPkinases in tumors Oxidative stress is an important factor in both the generation of somatic cell mutations that lead to cancer, and the pathophysiology of cancer itself. Furthermore, neurodegenerative and inflammatory diseases exhibit elevated levels of reactive oxygen species (ROS). We have recently discovered that the phosphoinositide, PtdIns(3,4,5)P 3 produced in response to oxidative stress, is generated by Type Iα PIPkinase, which 5- phosphorylates PtdIns(3,4)P 2, another lipid that accumulates in response to oxidative stress. The accumulation of 3-phosphorylated phosphoinositides upregulates cell survival pathways and may delay cell death to allow cellular damage to be repaired. Cells unable to repair damage wili ultimately undergo cell death. Intriguingly, prolonged exposure to oxidative stress negatively regulates Type Ia PIPkinase and leads to a dramatic decrease in the levels of both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 which may be a mechanism to initiate cell death. How tumor cells, which are often exposed to prolonged oxidative stress, overcome this problem is not known. As a first step toward better understanding of how this may occur, we are studying how oxidative stress regulates Type I PIPkinases catalytic activity. Further analysis of Type I Iα PIPkinase during oxidative stress induced tumorigenesis (for which there is a number of models) will define how phosphoinositide pathways are deregulated in tumor cells in order to prevent their elimination. Type II PIPkinase regulates oxidative stress signaling in vivo In mammals, there are three Type I and three Type II PIPkinase isoforms, whereas C. elegans contains only one isoform of each. We have isolated a deletion mutant for the C. elegans Type II PIPkinase (in collaboration with R Plasterk), which does not show any obvious phenotype. However, we found that Type II PIPkinase mutant larvae are more sensitive to developmental arrest, induced by oxidative stress (H 2 O 2 ) as compared to wild type larvae. Genes in the insulin receptor/pi3kinase/forkhead pathway regulate aging and stress responses in C. elegans and also appear to be important in the developmental response to H 2 O 2. Furthermore, these genes interact genetically with Type II PIPkinase in this response, suggesting that Type II

34 36 CELLULAR BIOCHEMISTRY Director of Research Anton Berns Key Publications Clarke JH, Letcher AJ, D Santos CS, Halstead JR, Irvine RF, Divecha N. Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem J 2001; 357: Halstead JR, Roefs M, Ellson CD, D Andrea S, Chen C, D Santos CS, Divecha N. A novel pathway of cellular phosphatidylinositol(3,4,5)-trisphosphate synthesis is regulated by oxidative stress. Curr Biol 2001; 11: Itoh F, Divecha N, Brocks 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 localisation of SARA in early endosomes and regulates TGFb/SMAD signalling. Genes Cells (in press). Jones DR, D Santos CS, Merida I, Divecha N. T lymphocyte nuclear diacylglycerol is derived from both de novo synthesis and phosphoinositide hydrolysis. Int J Biochem Cell Biol (in press). PIPkinase may modulate the insulin pathway. To define the level at which this occurs, we have developed methods to measure phosphoinositides in larvae and have shown that exposure to H 2 O 2 leads to the generation of PtdIns(3,4,5)P 3. The goal of this project is to link genetic and biochemical analyses in both C. elegans and mammalian cells to understand how metazoans, from worms to humans, use phosphoinositides to cope with oxidative stress. Phosphoinositides in the nucleus Phosphoinositides are produced in the nucleus where they may regulate a number of cellular functions including mrna splicing and export, cell cycle progression, non-homologous end joining of double stranded DNA breaks and the association of chromatin remodelling complexes with DNA. Using sensitive mass assays, we have shown that mass levels of nuclear phosphoinositides change as cells progress from G1 to S-phase of the cell cycle. We have discovered that the retinoblastoma protein (Rb), which regulates G1 to S-phase transition and is inactivated in the majority of human tumors, interacts with and regulates the activity of both the Type I and Type II PIPkinases. Furthermore, using large t-antigen (a tumor virus protein) to inactivate RB, we have demonstrated a role for Rb-mediated activation of PIPkinases in the regulation of nuclear phosphoinositides in vivo. Our data suggest that the regulation of PIPkinases by RB is not critical for cell proliferation but may represent a nuclear signaling function in response to cellular stress such as DNA damage. The regulation of multiple nuclear processes by phosphoinositides requires the maintenance and regulation of several pools of phosphoinositides. We have identified at least two PI 4-kinases in the nucleus, one of which regulates the synthesis of a rapidly turned over pool of PtdIns(4,5)P 2 while the other may be involved in the regulation of a larger, more inert pool of PtdIns(4,5)P 2. Meijer HJG, Berrie CP, Iurisci, C, Divecha N, Musgrave A, Munnik T. Identification of a new polyphosphoinositide in plants; phosphatidylinositol 5-phosphate and its accumulation upon osmotic stress. Biochem J 2001; 360: Van Horck FPG, Lavazais E, Eickholt BJ, Moolenaar WH, Divecha N. Essential role of Type Ia phosphatidylinositol-4-phosphate 5-kinase in neurite remodelling. Curr Biol (in press).

35 37 13 CELLULAR BIOCHEMISTRY TGF-B/SMAD SIGNAL TRANSDUCTION Transforming growth factor-b (TGF-b) family members, which include TGF-bs, activins and bone morphogenetic proteins (BMPs), regulate a broad spectrum of biological responses in a large variety of cell types. TGF-b family members play critical roles during embryogenesis and in maintaining tissue homeostasis during adult life. Subversion of TGF-b family signaling has been implicated in multiple developmental disorders and in various human diseases, including cancer, fibrosis and autoimmune diseases (Figure III.5). Our goals are to elucidate the molecular mechanisms by which TGF-b family members elicit their effects on cell proliferation, differentiation, migration and apoptosis and to generate animal models for human diseases that are caused by perturbed TGF-b family signaling. TGF-b family members transduce their signals across the plasma membrane by inducing the formation of specific heteromeric complexes of type I and type II serine/threonine kinase receptors. The type I receptor, also known as activin receptorlike 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 members of the Smad family, receptor regulated (R)-Smads, at their extreme C-terminal serine residues. Whereas Smad2 and Smad3 act downstream of TGF-b and activin type I receptors, Smad1, Smad5 and Smad8 are phosphorylated by BMP type I receptors. Phosphorylated R-Smads form complexes with the common partner (Co)-Smad, i.e. Smad4, which accumulate in the nucleus, where they control gene expression in a cell-type specific manner through interaction with other transcription factors, co-activators and corepressors. Inhibitory (I-) Smads, i.e. Smad6 and Smad7, form a distinct subclass among Smads by acting opposite from the signal transducing R- and Co-Smads. Group leader P Ten Dijke P Ten Dijke PhD Group leader S Dennler PhD Post-doc M-J Goumans PhD Post-doc S Itoh PhD Post-doc O Korchynskyi PhD Post-doc F Lebrin PhD Post-doc M Nishita PhD Post-doc F Itoh MSc Graduate student G Valdimarsdottir MSc Graduate student M Brugman Undergraduate student M Thorikay MSc Technical staff Smad function and regulation The anti-mitogenic and pro-apoptotic effects of TGF-β family members are mediated via Smad proteins. Certain Smads have been found to be mutated in specific types of cancer and gene ablation of particular Smads in mice has revealed an increased rate of tumorigenesis. To advance our understanding of Smad function and regulation, we have characterized interaction partners of Smad proteins. Smad anchor for receptor activation (SARA) regulates the subcellular localization of R-Smads, and presents R-Smads to the TGF-β receptor complex. We have found that SARA is localized in early endosomes and that its FYVE domain is sufficient for this localization. Moreover, we have demonstrated that the FYVE domain preferentially binds to PtdIns(3)P in vitro. Consistent with this finding, wortmannin, an inhibitor of PI3 kinase, induces a mislocalization of SARA and inhibits TGFβ/Smad signaling. Furthermore, ectopic expression of only the FYVE domain from SARA, induced a dissociation of wild-type SARA from early endosomes and inhibited TGF-β/Smad signaling. Our results raise the possibility that the activated TGF-β receptor complex has to undergo internalization to induce the phosphorylation of SARA-bound R-Smads (Figure III.6). In collaboration with C Ibanez (Karolinska Institute, Sweden), we identified GATA3, a key regulator of T helper cell development, as a tissue-restricted partner of Smad3. We found that TGF-β induces a specific and direct interaction between Smad3 and GATA-3 in T-cells, thereby recruiting Smad3 to GATA DNA binding sites and allowing TGF-β regulation of GATA-3 target promoters. I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β family members by interacting with activated TGF-β family type I receptors and thereby preventing the activation of R-Smads, or by competing with activated R-Smads for heteromeric complex formation with Co-Smads. Using the yeast two-hybrid interaction assay we have identified a cytoplasmic protein previously termed Associated Molecule with SH3 domain of STAM (AMSH), as a direct binding partner for I-Smads. AMSH was found to interact with Smad6 and Smad7, but not R- and Co-Smads upon BMP (and TGF-β) stimulation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of I-Smad with AMSH. Ectopic expression of AMSH prolonged BMP-induced Smad1 phosphorylation and potentiated BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. Furthermore, our obtained data strongly suggest that the molecular mechanism by which AMSH promotes BMP-induced responses is formed by inhibiting the binding of I-Smads to activated type I receptors or activated R-Smads. Figure III.5. TGFb family members are multifunctional cell-to-cell signaling proteins. TGFb regulates cell proliferation, differentiation, migration and apoptosis of in cell types. Deregulated TGFb signaling has been implicated in diverse human diseases, including cancer, fibrosis, vascular and bone disorders and auto-immune diseases.

36 38 CELLULAR BIOCHEMISTRY Director of Research Anton Berns Key Publications Dennler S, Goumans, M-J, Ten Dijke P. Transforming growth factor b signal transduction. J Leukocyte Biol (in press). Fan X, Valdimarsdottir G, Larsson J, Brun A, Magnusson M, Jacobsen SE, Ten Dijke P, Karlsson S. Transient disruption of autocrine transforming growth factor-b signaling leads to enhanced survival and proliferation potential in single primitive human hematopoietic progenitor cells. J Immunol (in press). Itoh F, Asao H, Sugamura K, Heldin C-H, Ten Dijke P, Itoh S. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. EMBO J 2001; 20: Itoh F, Divecha N, Brocks 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-b/Smad signaling. Genes Cells (in press). Korchynskyi O, Ten Dijke P. Identification and functional characterization of distinct critically important BMP-specific response elements in the Id1 promoter. J Biol Chem (in press). Larsson J, Goumans MJ, Sjostrand LJ, Van Rooijen MA, Ward D, Leveen P, Xu X, Ten Dijke P, Mummery CL, Karlsson S. Abnormal angiogenesis but intact hematopoietic potential in TGF-b type I receptor-deficient mice. EMBO J 2001; 20: Tgf-b and angiogenesis TGF-β family members play a pivotal role in vascular morphogenesis. Under most culture conditions TGF-β inhibits the proliferation and migration of endothelial cells, stimulates extracellular matrix accumulation and stimulates the differentiation of mesenchymal cells into pericytes and smooth muscle cells. However, stimulatory effects of TGF-β on angiogenesis have also been reported in vivo and endothelial cells in vitro. Genetic studies in humans and mice have revealed the importance of TGF-β family members in vascular morphogenesis. Mutations in TGF-β and BMP type II receptors have been linked to the human vascular disorders heriditary hemorrhagic telangiectasia and primary pulmonary hypertension. In a similar manner, as shown previously for mice lacking TGF-β1 or TGF-β type II receptor, we (in collaboration with S Karlsson, Lund University, Sweden and C Mummery, Netherlands Institute for Developmental Biology, Utrecht) have found that mice lacking TGF-β type I receptor (ALK-5) die at midgestation due to defects in angiogenesis. We have observed that TGF-β in endothelial cells, in contrast to other cell types, signals via two pathways, i.e. TGF-β can activate the broadly expressed TGF-β type I receptor (also termed ALK-5), which induces the phosphorylation of Smad2 and Smad3 and TGF-β can activate the endothelial specific ALK-1 that mediates the Smad1 and Smad5 phosphorylation. Interestingly, whereas activation of ALK-5 by TGF-β inhibits endothelial cell migration and proliferation, TGF-β-induced activation of ALK-1 induces migration, proliferation and promotes tube formation. The activation state of the endothelium may thus be dependent on the balance of ALK-1 versus ALK-5 activation that is induced by TGF-β. Our results provide a framework to understand previously conflicting reports in which pro- and anti-angiogenic properties were ascribed to TGF-β. BMP signal transduction BMPs have important roles in directing cell fate choices of mesenchymal cells. The mechanisms that govern the ability of BMPs to stimulate osteoblast differentiation and inhibit myogenic differentiation are incompletely understood. We have found that Id1, a dominant negative inhibitor of basic helixloop-helix proteins, is a direct target gene for BMP (but not TGF-b) in mesenchymal cells. We have identified two BMP responsive regions in the Id1 promoter, which contain distinct critically important sequence elements. Smads are part of nuclear transcription factor complexes that specifically bind to these sequence elements. Multimerization of these sequence motifs generates a powerful BMP/Smaddependent specific enhancer. Furthermore, Id1 was found to cooperate with another BMP-induced target gene, Cbfa1/RUNX2, in promoting osteoblast differentiation and inhibiting myoblast differentiation. Ten Dijke P, Goumans M-J, Itoh F, Itoh S. Regulation of cell proliferation by Smad proteins. J Cell Phys (in press). Figure III.6. A model for TGF-β/Smad signaling in early endosomes. The activated TGF-β receptor complex internalizes to early endosomes. SARA located in early endosomes through its interaction with PtdIns(3)P, presents R-Smads to the activated TGF-β receptor complex. After phosphorylation by TGF-β type I receptor, activated R-Smads dissociate from the TGF-β receptor and SARA, and initiate downstream signaling responses. Wortmannin (an inhibitor of PI3 kinase) and ectopic expression of the isolated FYVE domain of SARA induce a dissociation of wild type SARA from early endosomes, and inhibit TGF-β/Smad signaling.

37 IV DIVISION OF IMMUNOLOGY IMMUNOLOGY DEVELOPMENT AND FUNCTION OF HUMAN T CELLS Regulation of lymphoid development by transcription factors and cytokines Pluripotent hematopoietic stem cells develop into mature lymphoid cells (T, B, NK and lymphoid dendritic cells) via the common lymphoid progenitor cell (CLP). The mechanisms of cell fate determination of CLP are as yet incompletely understood. Obviously, transcription factors ultimately control cell-lineage diversification. Of considerable interest are master genes that are essential for development of certain cell lineages. In recent years it became clear that master genes direct differentiation in part by shutting off programs for other, competing, lineages. One example is the helix loop helix (HLH) factor Id2, which is required for NK-cell development and inhibits T, B and lymphoid dendritic cell (DC) development (Spits et al. J Exp Med 2000; 192:1795). Mice lacking the negative transcription regulator Id2, which inhibits transcriptional activities of basic HLH transcription factors, have an NK deficiency due to an absence of committed NK-cell precursors. Since IL-15-deficient mice also lack NK cells, we have investigated whether there is a link between the IL-15 receptor and Id2 activity. Addition of IL-15 to an in vitro fetal thymic organ culture (FTOC) stimulated NK and concomitantly inhibited TCRαβ cell development. TCRγδ development was affected only at high concentrations of IL-15. These effects of IL-15 were completely neutralized by overexpression of the bhlh factor HEB in thymic precursors but not by an HEB mutant lacking the transcriptional domain. We furthermore found that expression of a constitutive active mutant of STAT5a inhibited TCRαβ and stimulated NKcell development. As IL-15 activates STAT5, our data indicate that IL-15 and STAT5 are linked to Id2 and HEB in controlling NK-cell development. Id2 promotes NK and inhibits development not only of T cells but also of type 2 dendritic cell (DC) precursors, also called plasmacytoid DC (pdc). These cells are able to produce high levels of type I IFNs (α and β) and are frequently referred to as natural interferon producing cells. Besides in the periphery, these cells are present in the medulla of the thymus. Their function within that organ is poorly understood. Recently we presented evidence that these cells are of lymphoid origin and probably share a common precursor with T and NK cells. However direct evidence that pdc2 develop within the thymus from an intrathymic precursor was lacking. We now clarified this issue, in a collaborative study with C Uittenbogaart (UCLA School of Medicine, Los Angeles, CA, USA) and FA Vyth-Dreese (this division), by showing that CD34 + cells labeled with the fluorescent dye CSFE and injected into a human thymus transplanted in immunodeficient RAG-2 -/- IL-2Rγ -/- mice develop within that organ into pdc. In an effort to identify genes that control pdc development we collaborated with the group of F Brière at Schering Plough France in Dardilly to search for genes restricted to human pdc using a cdna subtraction technique with activated monocyte-derived DC (Mo-DC) as competitor. The transcription factor Spi-B was found to be expressed in pdc2 but not in Mo-DC. Spi-B expression in pdc2 was maintained upon in vitro maturation of pdc2. Overexpression of Spi-B in hematopoietic progenitor cells resulted in promotion of pdc2 development and inhibition of T- and NK-cell development. Our results indicate that Spi-B is involved in the control of pdc2 development and may be a master gene for development of pdc. Recently we have demonstrated that IL-7 activates PI-3K and its downstream effector PKB/Akt. PI-3K couples to the IL-7R through tyrosine residue 449 in its cytoplasmic domain. IL-7 promotes survival and cell cycle progression of T cells undergoing TCR rearrangements. The lipid phosphatase PTEN is a negative regulator of the PI-3K pathway. PTEN is a tumor suppressor frequently mutated in a remarkable variety of tumors. By dephosphorylating phosphoinositol 3,4,5 triphosphate (PIP3) it counteracts PI-3K, which catalyzes the reverse reaction. PTEN is highly expressed in the thymus and may be a negative regulator of the IL-7R signal transduction. To investigate this we made use of conditional PTEN deficient mice generated by P Krimpenfort (Division of Molecular Genetics) in which PTEN was specifically deleted exclusively Division head, group leader H Spits H Spits PhD Group leader K Weijer DVM PhD Academic staff S Alexeeva PhD Post-doc B Blom PhD Post-doc R Luiten PhD Post-doc S Müller PhD Post-doc M Naspetti PhD Post-doc S Ligthart MSc Graduate student F Scheeren MSc Graduate student R Schotte MSc Graduate student T Hagenbeek Undergraduate student C Van Schieveen Undergraduate student F Couwenberg Technical staff E Kueter Technical staff M Nagasawa Technical staff A Voordouw Technical staff P Weder Technical staff MA Van Halem Secretary

38 40 IMMUNOLOGY Director of Research Anton Berns Key Publications Reche PA., Soumelis V, Gorman DM, Clifford T, Liu MR, Travis M, Zurawski SM, Johnston J, Liu J, Spits H, De Waal Malefyt R, Kastelein RA, Bazan JF. Human thymic stromal lymphopoietin preferentially stimulates myeloid cells. J Immunol 2001; 167: Spits H. Dendritic cells in the thymus. In: Lotze MT, Thompson AW, editors. Dendritic Cells: Biology and Clinical Applications (2nd Ed). Academic Press 2001: Spits H, Couwenberg F, Bakker AQ, Weijer K, Uittenbogaart CH. Id2 and Id3 inhibit development of CD34 + stem cells into pre-dendritic cell (pre-dc)2 but not into pre-dc1. Evidence for a lymphoid origin of pre-dc2. J Exp Med 2001; 192: 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 cells in vivo. Blood (in press). in the T-cell lineage through the CRE lox system. Strikingly, these mice develop lymphomas within the thymus at months of age. All lymphomas expressed high levels of the TCR/CD3 complex and were mostly CD4 and CD8 double positive. Analysis of PTEN-deficient embryos and mice less than 1 month old revealed several abnormalities in T-cell development, suggesting enhanced survival and proliferation of late pre-t cells resulting in an accelerated T-cell development. By generating compound mice with deficiencies in both PTEN and elements of the IL-7R or the pre- TCR complex we aim to obtain more insight in the molecular mechanism of abnormal T-cell differentiation and malignant transformation caused by PTEN-deficiency. T-cell-mediated immunity against melanoma; immortalizing tumor-specific CTL for application in therapies Studies in various mouse models, for example prostate cancer, liver tumors and brain tumors, have demonstrated that adoptive therapies are superior to vaccination in the induction of anti-tumor immunity. Therefore, several years ago we started to optimize techniques for establishment and culture of large numbers of tumor-specific T-cell clones for use in therapeutic strategies involving adoptive transfer of tumor-specific CTL. One major roadblock in achieving this goal is that human T lymphocytes have a limited replicative life span preventing large-scale expansion of cultured CTL. Recently we demonstrated that the limited life span of the T cells is due to progressive shortening of telomere ends. We have shown that ectopic expression of htert immortalizes both CD4 + and CD8 + T cells without loss of function, antigen-specificity and growth characteristics. The immortalized T cells remained dependent on activation and cytokines for continued growth, indicating that they were not transformed. It is known that human T cells can express endogenous htert upon activation through the TCR, which raises the question why this is not sufficient to cause immortality. In theory, the telomeric loss caused by the proliferative response of T cells upon activation might be repaired by the simultaneous increase in telomerase activity. Indeed, our finding that forced expression of a dominant-negative mutant of the htert gene in human T cells reduces their replicative life span indicates that endogenous htert regulates this process. Our subsequent analysis of the upregulation of endogenous htert expression in activated T-cell populations with different replicative history revealed that the capacity of T cells to upregulate htert decreases with progressing replicative age. Thus T cells eventually lose their capacity to reverse telomere end loss resulting in replicative senescence and death. Having solved the problem of restricted cell expansion posed by limitations of the replicative life span, we are now working on streamlining isolation and expansion of T-cell clones using direct sorting of CD8 + T cells that react with HLA tetramers loaded with melanocyte-specific peptides (tyrosinase, Mart and gp100). We have furthermore demonstrated in a mouse model, in collaboration with N Verra (this division), that htert immortalized melanoma-specific CTL clones recognize melanoma cells in vivo. Further research will be focused on strategies to eliminate htert-transduced CTL after they have performed their task in vivo.

39 41 13 IMMUNOLOGY T-CELL DEVELOPMENT AND ANTI-TUMOR IMMUNITY The work in this laboratory focusses on two major lines of investigation: (1) identification of pathways that regulate the development and function of T cells; and (2) manipulation of mature T- cell responsiveness in the context of anti-tumor immunity and auto-immunity. Checkpoints in T-cell development and activation T-cell development and survival is regulated by the mature T-cell antigen receptor (TCR) and its developmental forerunner, the pre-tcr, but the molecular events controlled by these receptors remain ill-defined. Understanding the biology of signaling through these receptors requires identification of the contribution of the different structural and signaling components of the pre-tcr and the TCR complex. We have therefore generated a number of mutant mouse strains whose dysfunctional pre-tcrs and TCRs provide model systems to address these issues. We have found that early T-cell development is severely impaired in mice lacking the CD3γ chain of the pre-tcr as a consequence of a profound cell survival defect, and have identified p53 as a downstream target of the pre-tcr. Furthermore, we have created mutants in which either the immunotyrosine activation motifs (ITAM) or the di-leucine-based (dil) internalization motifs of CD3 chains are disrupted, and these are currently being studied for their role in TCR driven expansion and differentiation. An additional goal of this program is to identify and characterize novel genes that act in pre-tcr and TCR-driven survival and differentiation, and to place these in pathways that control key checkpoints in development, be they lymphocyte-specific or of broader scope. Organization and control of genetic pathways can be studied very efficiently in the T-cell lineage: the activity of genes of interest can be followed in primary cells, and the different developmental stages can be easily distinguished on the basis of expression of a wide range of markers. We have now created a framework in which complementary searches for candidate genes in established T-cell lines representative of the early and late stages of T-cell development (i.e., micro-array and survival/growth rescue screens with cdna libraries) are combined with functional screens in primary cells in fast in vitro systems. In addition, the available mutant mouse strains with dysfunctional pre-tcrs and TCRs provide excellent model systems of primary cells for gene complementation approaches. Finally, we are studying how T-cell responses are down-regulated, and have concentrated our studies on the dominant negative regulator, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor. This is an activation-induced integral membrane receptor which inhibits the T-cell response by extinguishing T-cell expansion. Given its central role in immune modulation, the CTLA-4 signaling pathway is a primary therapeutic target for preventing autoimmune disease, graft-versus-host disease and graft rejection, and promoting tumor immunity. Therefore, there is great interest in identification of the mechanisms of action of CTLA-4, which will permit the design of molecules that can modulate its function. We are using biochemical, unbiased screening methods that can distinguish between interactions with phosphorylated and non-phosphorylated CTLA-4. This is important since the phosphorylation status of CTLA-4 determines its intracellular location. We have identified various CTLA-4 interacting proteins that are now the subject of further analysis. An interaction with cofilin is among the most interesting of those that we chose to follow-up: cofilin is a protein essential for actin rearrangement, and polymerization of T-cell actin in the APC contact area is impaired when CTLA-4 signaling occurs. These observations imply a direct intervention of CTLA-4 with focal reorganization of the T-cell actin cytoskeleton. Further Biacore and crystallization studies will supply more detail on the character of this interaction, and the success of the unbiased protein-protein interaction approach has triggered its application to three other regulatory receptors on T cells. Together, leads from these studies will generate a broader arsenal of options for manipulating the T-cell response. Group leader AM Kruisbeek AM Kruisbeek PhD Group leader W Marissen PhD Post-doc W Overwijk PhD Post-doc Q Valent PhD Post-doc Y Zevering PhD Post-doc T Cordaro MSc Graduate student K De Visser MSc Graduate student A Keller MSc Graduate student S Smeele MSc Graduate student A Nooijen Undergraduate student R Van De Ven Undergraduate student E De Jong Technical staff F Tirion Technical staff J Van Den Brakel Technical staff E Wijnands Technical staff T-cell tolerance and anti-tumor immunity Questions such as how self-reactive T cells can survive, expand and differentiate into effector and memory populations are central to our understanding of both autoimmunity and anti-tumor immunity. We are interested in answering these questions, since insights into how self-specific

40 42 IMMUNOLOGY Director of Research Anton Berns Key Publications Bischof F, Wienhold W, Wirblich C, Malcherek G, Zevering O, Kruisbeek AM, Melms A. Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by selfepitopes. Proc Natl Acad Sci USA 2001; 98: Carleton M, Haks M, Smeele SAA, Jones A, Belkowski SM, Berger MA, Linsley P, Kruisbeek AM, Wiest DL. Early growth response (Egr) transcription factors are essential for pre-tcr driven thymocyte differentiation. J Immunol (in press). Cordaro TA, De Visser KE, Tirion FH, Schumacher TNM, Kruisbeek AM. Exploiting the low avidity self-specific T cell repertoire for tumor rejection. J Immunol (in press). De Visser KE, Cordaro TA, Kessels HWHG, Tirion FH, Schumacher TNM, Kruisbeek AM. Low avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands. J Immunol 2001; 167: Haks MC, Cordaro TA, Van Den Brakel JHN, Haanen JBAG, De Vries EFR, Borst J, Krimpenfort P, Kruisbeek AM. A redundant role of the CD3g-immunoreceptor tyrosine-based activation motif in mature T cell function. J Immunol 2001; 166: Kessels HWHG, De Visser K, Kruisbeek AM, Schumacher TNM. Circumventing T cell tolerance to tumor antigens. Biologicals (in press). T cells can differentiate into effector and memory populations will allow us to apply these cells for tumor rejection. At the same time, designing effective means to preclude undesirable autoimmunity also requires insights into how the responsiveness of self-specific T cells is regulated. Thymic expression of self antigens results in the deletion of T cells with high affinity self-specific TCRs from the T-cell repertoire, leaving behind a residual population of T cells with low affinity TCRs for MHC-self-peptide ligands. We have examined the functional competence of CD8 + T cells with such low affinity self-specific TCRs, and found that low affinity interactions between TCRs and antigenic peptides are associated with selective loss of critical T-cell functions. Triggering of IFNγ production and cytolytic activity through low affinity TCRs readily occurs, provided high ligand doses are used, but induction of IL-2 production and clonal expansion are abrogated. Intriguingly, variant peptides that bind with high affinity and slow off-rates to selfspecific TCRs can easily be identified (collaboration with T Schumacher, this division), and these can rescue the expansion defect. Our results therefore define a mechanism for the weak T-cell responses against tumor self antigens, and a means to improve their function. To examine the in vivo functional capabilities of CD8 + T cells with low affinity TCRs in more detail, we are now defining the conditions under which growth of self-antigen expressing tumors can be delayed or prevented. A growth delay can be induced under optimal inflammatory conditions for tumors growing in the lung. For subcutaneously growing tumors, only memory T-cell responses were found capable of quenching tumor growth. This most likely reflects the combination of both quantitative and qualitative features that renders memory responses more robust. The reduction of tumor growth during the memory CD8 + T-cell response against tumor selfantigens is associated with a dramatic increase in the number of self-specific CD8 + cells infiltrating the tumor, and we are currently evaluating whether the observed difference between primary and memory responses is purely quantitative, or whether the memory response distinguishes itself in other aspects (i.e., migratory behavior, differential ligand sensitivity or effector function differences). Finally, we are interested in studying how the immune system can be made aware of small tumor burdens as they arise, through vaccinations and/or induction of inflammatory responses. Transplantable tumor models, specially selected for rapid growth, will not provide answers to this crucial question. We are therefore developing tumor models (in collaboration with Berns/Krimpenfort) in which tumor growth is induced in a tissue-specific and time-controlled fashion, and expression of tumor antigens with T-cell epitopes will be induced concomitant with cellular transformation. We are generating both B-cell lymphoma and melanoma models, and will first study the ability to contain tumor-progression by adoptively transferred CD8 + T cells expressing TCRs specific for deliberately introduced foreign tumor antigens or tumor-self-antigens. In addition, we plan to define how the hosts endogenous T-cell repertoire can be exploited to control tumorigenesis, both for model tumor antigen that represents a foreign antigen, for model tumor antigens that are also self-antigens and for non-mutated tissue antigens that are expressed by tumors. Within this framework, we expect to identify the parameters that trigger a T-cell mediated immune response against tumors whose growth pattern more faithfully resembles tumor growth in man.

41 43 13 IMMUNOLOGY TUMOR-SPECIFIC T-CELL IMMUNITY The aim of our research is straightforward 1) to design novel technologies that can be used to examine and modify protein interactions that control T-cell immunity; 2) to use these tools to unravel and manipulate the molecular processes underlying immune recognition by T lymphocytes. Tumor-specific T-cell responses in mouse model systems In a subset of human tumors such as melanoma, significant tumor-specific T-cell responses can be observed in patients (see Haanen, this division). There has been a long-standing debate on the mechanism through which the immune system is alerted to such solid tumors that at least initially grow outside of the lymphoid organs. It has been postulated that induction of cytotoxic T cells against tumor antigens occurs through direct interaction of CD8 + T cells and tumor cells that have migrated to the draining lymph nodes. Alternatively, evidence has been obtained for cross priming of tumor antigens as the relevant mechanism, in which host antigen presenting cells take up antigens and present these antigens to tumor-specific T cells in the lymphoid organs. To address the relative merits of these two processes, we have set up a murine tumor model in which tumor-specific T-cell responses can be directly visualized by MHC tetramer technology (see Figure IV.1). We have used this model to demonstrate that both direct priming and cross priming can be very efficient mechanisms for the induction of tumor-specific T-cell immunity. Ongoing experiments suggest that antigen-specific parameters such as epitope location may affect the relative efficiency of the two processes. The observation that MHC class-i negative tumors can induce very prominent tumor-specific cytotoxic T-cell responses through antigen cross priming suggests that the development of tumor-specific T-cell immunity is not necessarily a positive sign, an observation that is corroborated by our recent findings in melanoma patients (see Haanen, this division). Over the past years it has become increasingly clear that the role of CD4 + T cells in the generation of tumor-specific T-cell immunity may be greater than previously expected. However, the tools with which to analyze tumor-specific CD4 + T-cell immunity have in large part been lacking. We have therefore embarked on the development of reliable protein expression strategies for the generation of MHC class II tetramers, to allow a direct analysis of CD4 + T-cell immunity. In the past years we have developed a successful strategy for the production of murine and more recently also human MHC class II tetramers. In collaboration with F Ossendorp (LUMC, Leiden) and FA Vyth-Dreese (this division), we have started the use of these reagents to analyze the contribution of CD4 + T cells to tumor control and to determine the relationship between the development of CD4 + and CD8 + T-cell immunity in a retrovirus-induced sarcoma model. In this model, sarcoma regression is CD4 + T-cell dependent and antigen-specific CD4 + T cells can be visualized directly ex vivo by MHC tetramer technology. Analysis of the kinetics and distribution of the antigenspecific CD4 + T-cell response reveals the presence of antigen-specific helper T cells both within the lymphoid organs and at the effector site. Depletion studies suggest a dual role for CD4 + T-cell immunity, both in the promotion of the development of CD8 + T-cell immunity and in the generation of an inflammatory environment at the effector site. Group leader TNM Schumacher TNM Schumacher PhD Group leader N Brouwenstijn PhD Post-doc M Coccoris MSc Graduate student HWHG Kessels MSc Graduate student K Schepers MSc Graduate student MC Wolkers MSc Graduate student M Ameziane Undergraduate student Z Rivero Undergraduate student M Toebes Technical staff G Sotthewes Technical staff J Urbanus Technical staff P Van den Berk Technical staff M Van Den Boom Technical staff Vaccination-induced T-cell immunity In related experiments we have set out to utilize MHC tetramer technology to assess and improve the efficiency of various vaccination strategies that are aimed at the induction of CD8 + T-cell immunity. In these experiments we have analyzed the factors that are essential for the induction of high magnitude T-cell responses by DNA vaccination in a mouse model system. These experiments have led to a set of five simple rules for efficient epitope-directed DNA vaccination indicating that carboxy-terminal fusion of the epitope to a carrier protein of foreign origin is the most favorable strategy. DNA vaccines that are based on these rules induce high magnitude CD8 + T-cell responses in >95% of vaccinated animals. Figure IV.1: MHC class I tetramers.

42 44 IMMUNOLOGY Director of Research Anton Berns Key Publications Cordaro TA, De Visser KE, Tirion FH, Schumacher TN, Kruisbeek AM. Can the low avidity self-specific T cell repertoire be exploited for tumor rejection. J Immunol (in press). De Visser KE, Cordaro TA, Kessels HW, Tirion FH, Schumacher TN, Kruisbeek AM. Low-avidity self-specific T cells display a pronounced expansion defect that can be overcome by altered peptide ligands. J Immunol 2001; 167: Gamadia LE, Rentenaar RJ, Baars PA, Remmerswaal EB, Surachno S, Weel JF, Toebes M, Schumacher TNM, Ten Berge IJM, Van Lier RAW. Differentiation of cytomegalovirus-specific CD8 + T cells in healthy and immunosuppressed virus carriers. Blood 2001; 98: Kessels HW, Wolkers MC, Van Den Boom MD, Van Der Valk MA, Schumacher TN. Immunotherapy through TCR gene transfer. Nature Immunol 2001; 2: Schumacher TNM, Haanen JBAG. Ex vivo and in situ detection of tumor-specific T cell immunity. In: s: Stauss HJ, Kawakami Y, Parmiani G, editors. Tumor Antigens Recognized by T cells and Antibodies with MHC tetramers. Tumor Immunology Series, Vol. III. Harwood Academic Publishers (in press). Stromnes IM, Schumacher TN, Schepers K, Messer RJ, Evans L, Peterson KE, Race B, Hasenkrug KJ. Temporal effects of IFNgamma deficiency on the course of Friend retrovirus infection in mice. J Virol (in press). Sutmuller RP, Van Duivenvoorde LM, Van Elsas A, Schumacher TN, Wildenberg ME, Allison JP, Toes REM, Offringa R, Melief CJM. Synergism of cytotoxic T lymphocyteassociated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 2001; 194: Wolkers MC, Stoetter G, Vyth-Dreese FA, Schumacher TN. Redundancy of direct priming and cross-priming in tumor-specific CD8+ T cell responses. J Immunol 2001; 167: Figure IV.2: T-cell immunity through TCR gene therapy. Kinetics of T-cell responses of redirected T cells in mice receiving TCR-transduced T cells (closed circles) or mock-transduced T cells (open circles) and subsequently infected with influenza A virus. TCR gene therapy The main limitation in the use of vaccination-based strategies for curative cancer treatment is the scarcity of proper T-cell antigens. The majority of tumor-specific antigens that are currently targeted are self antigens that are overexpressed within the tumor tissue, or antigens with a restricted tissue distribution. Because of self tolerance, the T-cell repertoire specific for these antigens is often of low affinity and the effect of vaccination may therefore be limited. As an alternative to vaccination-based strategies we have begun to explore the feasibility of TCR gene transfer as a strategy for the induction of tumor-specific T-cell immunity. The antigen-specificity of T lymphocytes is solely dictated by the T-cell receptor (TCR) α and β chains. Consequently, genetic transfer of TCR chains suffices to impose a desirable viral- or tumor-antigen specificity onto cytotoxic or helper T-cell populations. We have recently described the genetic introduction of a virus-specific T-cell receptor into peripheral T cells in a mouse model system. These experiments reveal that T cells redirected by TCR gene transfer expand dramatically upon viral infection of mice (see Figure IV.2) 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. These data suggest the redirection of T cells by TCR gene therapy as a new strategy for the rapid induction of virus- or tumor-specific immunity. In subsequent experiments we have begun to address the feasibility of TCR transfer for human T-cell receptors in collaboration with the groups of J Haanen and MJ Kersten. In these experiments we will test the effect of TCR transfer in murine preclinical models for melanoma and EBV lymphoma. Treatment of EBV lymphoma through TCR gene transfer is particularly attractive because the effectiveness of EBV specific T-cell infusions has previously been demonstrated in transplant recipients. In parallel, we aim to create high affinity T-cell receptors specific for self antigens such as CD20 through the use of a TCR optimization strategy that we have previously developed. In addition, a number of technical issues relating to TCR gene therapy is currently being addressed. For instance, in TCR gene therapy a single T-cell receptor may be used in patients that share the relevant MHC allele with the MHC haplotype of the TCR-donor but that are mismatched for other MHC alleles. 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. To address this issue, the merits and risks of allogeneic TCR transfer are currently being examined in mice and preliminary data are encouraging. Finally, in T cells that are redirected by TCR gene transfer a total of four different T-cell receptors can be present at the cell surface and two of these receptors (those that are formed by pairing of endogenous and exogenous chains) have an unpredictable specificity. Efforts to remodel to interface of the TCR α and β chain in order to circumvent the assembly of these mixed dimers are in progress.

43 45 13 IMMUNOLOGY MELANOMA-SPECIFIC T-CELL IMMUNITY AND IMMUNOTHERAPY In our research we aim to understand and to exploit melanoma-specific T-cell immunity in melanoma patients. Melanoma is considered an immunogenic tumor and tumor-specific CD8 + and CD4 + T cells can be found in many advanced-stage melanoma patients. This T-cell immunity is directed against an array of tumor antigens. These tumor antigens are either melanocyte differentiation antigens, which are shared with normal melanocytes, such as MART-1/melanA, tyrosinase and gp100, or gene products of so-called Cancer/Testis genes that are normally expressed in HLAnegative sperm cells and aberrantly expressed in tumor cells. Although it is assumed that the presence of these cells in blood and tumor samples from melanoma patients reflects in vivo T-cell activation and expansion, evidence for a beneficial effect of this melanoma-specific T-cell immunity on the clinical outcome of melanoma patients is lacking. Group leader JBAG Haanen JBAG Haanen MDPhD Group leader M Van Oijen PhD Post-doc A Bins MD Graduate student P Weder Technical staff Melanoma-specific T-cell immunity in stage IV melanoma patients is not correlated with better clinical outcome In order to study the role of naturally occurring melanoma-specific T-cell immunity in the course of disease, we have systematically analyzed peripheral blood samples from untreated metastatic melanoma (stage IV) patients using flow cytometry to test for the presence of melanoma-specific CD8 + T cells. All patients were enrolled in a phase II clinical trial (collaboration with the group of G De Gast) studying the effect of a combined chemoimmunotherapy treatment modality. In collaboration with the groups of T Schumacher and H Spits, HLA-A2.1/peptide tetramers were generated. These tetramers containing peptides that correspond to the CD8+ T-cell epitopes from melanocyte differentiation antigens MART-1, tyrosinase and gp100 were used for the detection of melanoma-specific T cells in peripheral blood samples from these patients. Results were correlated with course of the disease and response to treatment. In total, 38 out of 39 HLA-A2 + metastatic melanoma patients could be evaluated. In 50% of the blood samples MART-1, tyrosinase or gp100-specific T cells were detectable, in some patients amounting to 20% of peripheral CD8 + T-cell pool. Strikingly, the presence of melanoma-specific T cells was not correlated with better clinical outcome, since the presence of these cells was preferentially found in nonresponders. The difference in number of melanoma-specific T cells between responder and non-responder patients was statistically highly significant (p=0.09, Fisher Exact Test). Furthermore, melanoma-specific T cells in these patients have a memory phenotype, indicating that these cells have been activated and have expanded in vivo. Immunohistochemical analysis of tumor biopsies revealed that complete loss of HLA-A expression appeared to be a frequent event in these patients. Interestingly, HLA-A loss was more common in patients with circulating melanoma-specific T cells, suggesting that immunological escape mechanism has played a role. This rather unexpected inverse correlation between naturally occurring melanomaspecific T-cell immunity and clinical outcome has yet to be explained. Preliminary results indicate that expansion of the melanoma-specific T-cell pool is a late event in the course of the disease, as detection of these cells in patients with earlier disease stage is rather rare. Therefore, the expansions that were found in rapidly progressing patients may reflect massive tumor growth and spreading. Based on these results we propose that patients with stage IV disease probably do not benefit from immunotherapy strategies directed at expansion of melanoma-specific cytotoxic T cells. These strategies are more likely to be effective in patients with earlier stage disease. Accordingly, we have now designed vaccination-based immunotherapy studies for patients with stage III disease. Key Publications Haks MC, Cordaro TA, Van Den Brakel JH, Haanen JB, De Vries EF, Borst J, Krimpenfort P, Kruisbeek AM. A redundant role of the CD3 gammaimmunoreceptor tyrosine-based activation motif in mature T cell function. J Immunol 2001; 166: Schumacher TNM, Haanen JBAG. Ex vivo and in situ detection of tumor-specific T cell immunity. In: Stauss HJ, Kawakami Y, Parmiani G, editors. Tumor Antigens Recognized by T Cells and Antibodies with MHC Tetramers. Tumor Immunology Series, Vol. III. Harwood Academic Publishers (in press).

44 46 IMMUNOLOGY Director of Research Anton Berns IMMUNOTHERAPY Group leader GC De Gast GC De Gast MD PhD Group leader FA Vyth-Dreese PhD Academic staff N Verra MSc Graduate student TAM Dellemijn Technical staff J Sein Technical staff WF Van de Kasteele Technical staff Adoptive transfer of htert transduced human CTL in mice bearing autologous human melanoma lung tumors To test the anti-tumor effect of human CTLs against their autologous tumor, in a close collaborative study in our laboratory with K Weijer, H Spits and R Luiten (this division) a model has been developed using Rag-2/common γ chain double knockout mice, lacking T, B and NK cells, to facilitate transplantation of human cells. In this model a human melanoma cell line is used to generate lung metastases and the anti-tumor activity of two autologous CTL clones, both recognizing the same Mart-1 peptide in HLA-A2, and a control clone recognizing a Flu peptide in HLA-A2 was tested. All clones had been immortalized by transduction with htert to enable large-scale expansion of the cells. Upon iv injection of MelAKR melanoma cells, anti-tumor effects of CTL, their number found in the lungs as well as the number and size of the metastases, were determined using immunohistochemical analysis. Single injection of Mart-1 CTL reduced the number of lung metastases by approximately 30%. Repeated injections resulted in further reduction in size. When the control Flu clone was used in either single or repeated dosages no anti-tumor effect was observed. In conclusion, human CTL recognizing their autologous tumor are able to exert in vivo anti-tumor activity in this model. However, total tumor eradication is not achieved. Higher expression of the antigen on the tumor might enhance recognition of the tumor cells by CTL and may therefore lead to higher anti-tumor activity. To test this, a melakr variant overexpressing the Mart-1 epitope has been generated. These tools will allow us to further optimize the protocol to ultimately achieve complete tumor eradication. Effect of peri-operative immunotherapy in peripheral blood and at the tumor site in renal cell cancer and head and neck cancer patients Renal cell carcinoma (RCC) and potentially head and neck squamous cell carcinoma (HNSCC) are being recognized as immunogenic tumors. The current treatment for metastatic RCC is immunotherapy with the cytokines interleukin-2 (IL-2) and interferon α (IFNα). IL-2 stimulates T cells whereas IFNα enhances MHC class I expression. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) may be a potentially enhancing additive in immunotherapy by generating and differentiating dendritic cells (DC) which are necessary to initiate specific immune responses. To test this combination of cytokines, peri-operative immunotherapy from day 3 till day +5 after the operation has been given to RCC and HNSCC patients (in collaboration with S Horenblas and A Balm, Division XI and D De Jong, Division XIII) in a phase I dose-escalation study, where radical surgery can remove all locally visible tumors. 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. Secondly, in a situation of minimal residual disease, the immune system, if activated, should be able to attack this minimal amount of tumor and so might prevent the outgrowth of micro-metastases. To date, eleven RCC and six HNSCC patients have been treated. In peripheral blood samples, it was shown that the post-operative immune depression was prevented. More activated monocytes were observed, and the fraction of activated T cells had increased five-fold. Furthermore, using immunohistochemistry, tissue analysis of the tumors showed increased numbers of DC and more activated CD25+ cells in comparison to controls (see Figure IV.3). Detection of dendritic cells of various subtypes and maturation stages in developing thymus, inflammatory colon and tumor tissues In situ analysis using 3-color confocal laser scanning microscopy (CLSM) was performed to discriminate between subtypes of dendritic cells and other accessory cells. In three different settings, the value of this type of in-depth analysis was confirmed. First, in an ongoing collaborative study with the group of H Spits, this division, the in vivo development and maturation of precursor populations of dendritic cells, injected into rag 2 common γ chain double knockout mice, was followed in situ. Analyses included the activation and maturation status of subsets of both lymphoid and myeloid lineages (see group Spits, this division). Second, inflammatory lesions of patients

45 47 13 IMMUNOLOGY Key Publications De Gast CG, Vyth-Dreese FA, Nooijen W, Van Den Boogaard CJC, Sein J, Holtkamp M, Linthorst G, Baars JW, Schornagel JH, Rodenhuis S. Re-infusion of autologous lymphocytes with GM-CSF induces rapid recovery of CD4+ and CD8+ T cells after high dose chemotherapy for metastatic breast cancer. J Clin Oncol (in press). Figure IV.3: Dendritic cells infiltrating into head and neck tumor before (left) and after (right) peri-operative immunotherapy. suffering from inflammatory bowel disease were studied for expression of antigen presenting cells (in collaboration with the group of S Van Deventer and A Te Velde, Academic Medical Center, University of Amsterdam, Amsterdam). The CLSM technique proved to be very useful in discriminating dendritic subsets from macrophages in these lesions as well as in non-affected colon tissues from healthy donors or unrelated patients. Furthermore, using multicolor analysis, insight was gained in the state of activation and maturation of accessory and lymphoid cells and their cytokine potentials. These studies may have direct implications for future immunotherapy approaches. Third, experiments are ongoing to further examine the in situ localization and cytokine expression of precursor lymphoid and myeloid dendritic cells in infiltrates of tumor tissues of patients with HNSCC and RCC upon triple cytokine immunotherapy. 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 cells in vivo. Blood (in press). Wolkers MC, Stötter G, Vyth-Dreese FA, Oosterwegel MA, Schumacher TNM. Redundancy of direct priming and crosspriming in tumor specific CD8+ T cell responses. J Immunol 2001; 167: Detection of antigen specific T lymphocytes in experimental and human tissues Immunohistological techniques have been developed to visualize tetrameric peptide/mhc complexes binding antigen specific and virus specific CD8 + as well as CD4 + T lymphocytes (Haanen et al. Nature Med 2000; 6: ) (see also groups Schumacher and Kruisbeek, this division). The technique has been successfully applied for the detection of minor antigen specific T cells in a skin explant model of graft versus host reactivity in a collaborative study with the group of E Goulmy (Leiden University Medical Center, Leiden). Studies are ongoing to further refine this technique to not only be able to detect antigen specific T cells in fresh, but also cryopreserved tissues and to visualize the functional status of cells in situ. This novel immunohistochemical approach will provide a tool for further detailed in situ studies of human T-cell / tumor-cell interactions, the unraveling of the graft versus host reaction in human skin and the monitoring of immunotherapy trials.

46 48 MOLECULAR BIOLOGY Director of Research Anton Berns V DIVISION OF MOLECULAR BIOLOGY MOUSE MODELS FOR HEREDITARY CANCER Division head, Group leader H Te Riele H Te Riele PhD Group leader J-H Dannenberg MSc Post-doc J Hansen PhD Post-doc N Claij MSc Graduate student F Foijer MSc Graduate student M Sonneveld MSc Graduate student V Doodeman Undergraduate student E Van Riet Undergraduate student O Van den Broek Undergraduate student C Brouwers Technical staff M Dekker Technical staff S De Vries MSc Technical staff L Schuijff Technical staff A Van der Wal Technical staff Genetic instability and deregulated cell cycle control are hallmarks of human cancer. Research in our group involves both aspects focusing on (1) 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 stem (ES) cells and analyses of the phenotypic consequences in homozygous, heterozygous and chimeric knockout mice and in cell lines derived thereof. DNA mismatch repair The hereditary form of non-polyposis colorectal cancer (HNPCC) is caused by defects in genes that encode essential components of the DNA mismatch repair (MMR) system. Mismatched nucleotides in DNA can arise by erroneous replication or occur in heteroduplex regions formed at initial stages of recombination between homologous but non-identical DNA molecules. Mismatches in DNA are recognized by two heterodimeric protein complexes: MutSα, a dimer of MSH2 and MSH6, recognizes base-base mismatches and single unpaired nucleotides; MutSβ, a dimer of MSH2 and MSH3, preferentially targets small loops of two to five nucleotides. To study the role of mismatch recognition proteins in mutation avoidance, antirecombination and suppression of cancer, we have generated ES cells and mice carrying disruptions in the central MMR genes Msh2, Msh6 and Msh3. We found that some tumor types rapidly developed upon homozygous disruption of Msh6, while another, intestinal cancer, required concomitant inactivation of Msh6 and Msh3. These results prompted us to address the specific mutation avoidance functions of MutSα and MutSβ. As readout, we use frameshift and recombination reporter constructs that allow precise assessment of the role of different MMR functions in the processing of mismatched nucleotides within different contexts. Corroborating previous results, we observed that correction of dinucleotide slippage errors can be mediated by either Msh2/Msh6 or Msh2/Msh3, while mononucleotide slippage errors are mainly targeted by Msh2/Msh6. Thus, development of intestinal tumors in mice appears to require frameshift mutations involving two or more nucleotides. To study MMR-dependent anti-recombination, we have constructed a recombination reporter consisting of a neo gene that was rendered 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 -/- cells was 10- to 50-fold higher than in wild-type cells. Thus, a single base-pair difference is already sufficient to activate MMR-dependent anti-recombination. Further experiments are aimed at identifying the role of the different mismatch-recognition complexes in mismatch-directed anti-recombination. In addition to cell lines with complete ablation of mismatch recognition proteins, we have constructed a cell line carrying a hypomorphic Msh2 allele leading to a tenfold reduced MSH2 protein level. Remarkably, cells with low MSH2 protein level became resistant to the toxic, but highly sensitive to the mutagenic effects of methylating agents. We have now observed that deficient or reduced MMR capacity also rendered cells highly sensitive to mutation induction by another alkylating agent, ENU, although the toxicity of this compound was hardly affected. These observations suggest that MMR activity not only counteracts spontaneous mutagenesis, but also DNA damage induced mutagenesis, suggesting an explanation for the strong carcinogenicity of low doses of ENU in MSH2-deficient mice. The retinoblastoma gene family Loss of function of the retinoblastoma suppressor gene, RB, is a common event in the development of human cancer. prb acts as a key regulator of the G 1 -S transition of the cell cycle by virtue of its capacity to modulate the activity of E2F transcription factors. prb can exist in a hyper- and hypophosphorylated form, the latter binding to E2Fs and suppressing the

47 49 13 MOLECULAR BIOLOGY transcription of E2F target genes which inhibits progression of the cell cycle. Phosphorylation of prb through cyclin D1/CDK4 kinase activity releases the transcription activity of E2Fs and promotes G 1 -S transition. prb shares its E2Fregulating activity with two homologues, p107 and p130. Overexpression of each of these proteins (also called pocket proteins ) in vitro can cause cell cycle arrest, an activity that can be relieved through phosphorylation. Thus, the three pocket proteins may to some extent be redundant in blocking cell cycle progression. We have obtained further evidence for this notion by analyzing the growth characteristics of primary mouse embryonic fibroblasts (MEFs) carrying single and compound loss-of-function mutations in the retinoblastoma gene family including a triple knockout (TKO) line that is completely devoid of all three pocket proteins. The limited lifespan of wild-type MEFs in culture has been attributed to the gradual accumulation of the cell cycle inhibitor p19 ARF causing stabilization of the tumor suppressor protein p53. Also premature senescence induced by expression of oncogenic RAS (RAS V12 ) has been attributed to activation of the p19 ARF /p53 pathway. P19 ARF or p53 deficiency alleviates both spontaneous and RASV12-induced senescence and leads to oncogenic transformation by RAS V12. We have shown that MEFs deficient for all three pocket proteins were also immortal and could not be arrested by ectopic p19 ARF expression. Also prb/p107- or prb/p130-deficient MEFs appeared to be immortal: after having adapted to in vitro culturing, they sustained a moderate but constant proliferation rate upon prolonged passaging. A picture thus emerges in which either prb alone or p107 plus p130 are required for the senescence response revealing an intimate interaction between p19 ARF /p53- and prb-mediated growth control. Loss of pocket proteins also alleviated the senescence reponse following expression of oncogenic RAS, however, unlike abrogation of the p19 ARF /p53 pathway, this did not lead to oncogenic transformation by RAS V12. Furthermore, we observed that triple knockout MEFs cultured under certain growthrestricting conditions, sustained continuous cell turnover without net increase in cell number. We envisage that loss of pocket protein function in vivo may also cause increased cell turnover, which could strongly facilitate the acquisition of additional oncogenic events leading to development of cancer. Although evidence for a role of p107 or p130 defects in human cancer is scarce, in mice we observed that p107 and p130 activity can modulate the tumor suppressor function of prb. While Rb -/- chimeras only developed pituitary gland tumors, Rb -/- p107 -/- and Rb -/- p130 -/- chimeras developed retinoblastomas originating from mutant cells. Interestingly, in developing retinas, ES cell-derived lesions could be detected that showed elevated incorporation of BrdU but also extensive apoptosis (Figure V.1). This suggests that simultaneous loss of prb and either p107 or p130 sustained prolonged cell turnover which may prove to be essential for retinoblastoma development later in life. In addition to retinoblastoma, Rb -/- p130 -/- chimeras developed other ES cell-derived tumors, including adrenal tumors (pheochromocytomas), neurological tumors located to the hypothalamus, ovarian tumors and many polyp-like lesions at the bronchial epithelium which are likely precursors of small cell lung cancer (Figure V.1). Finally, we generated a cohort of 28 Rb +/- p107 -/- chimeras. All animals succumbed within one year to tumors located to the pituitary gland, coecum, bone, ovary, thyroid gland and thymus, all of ES cell origin and in most cases showing loss of the Rb wild type allele. In conclusion, loss of pocket proteins has severe consequences for the growth characteristics of cells both in vitro and in vivo. Our current work is focused on the identification of additional genetic events that collaborate with loss of the Rb gene family in oncogenic transformation. Key Publications Ciarmatori S, Scott PH, Sutcliffe JE, McLees A, Alzuherri HM, Dannenberg J-H, Te Riele H, Grummt I, Voit R, White RJ. Overlapping functions of the prb family in the regulation of rrna synthesis. Mol Cell Biol 2001; 21: Peeper DS, Dannenberg J-H, Douma S, Te Riele H, Bernards R. Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nature Cell Biol 2001; 3: Te Riele H, Brouwers C, Dekker M. Generation of double-knockout embryonic stem cells. Methods Mol Biol 2001; 158: Van den Broek W, Nelen MR, Wansink DG, Coerwinkel M, Te Riele H, Groenen PJTA, Wieringa B. Somatic expansion behavior of the (CTG) n -repeat in Myotonic Dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatchrepair proteins. Human Mol Genet (in press). Figure V.1: ES cell derived hyperplasia (arrows) in Rb -/- p130 -/- chimeras.

48 50 MOLECULAR BIOLOGY Director of Research Anton Berns CELL CYCLE CHECKPOINTS Group leader R Medema R Medema PhD Group leader J Laoukili PhD Post-doc S Lens PhD Post-doc M Schmidt PhD Post-doc M Stahl MSc Graduate student B Van de Weerdt MSc Graduate student M Van Vugt MSc Graduate student D Elouarrat Undergraduate student J Kauw Technical staff R Klompmaker Technical staff Cell division of eukaryotic cells is a highly regulated process. Cells can respond to a variety of extracellular signals, which together dictate cellular behavior, including the decision to proliferate, differentiate or undergo apoptosis. Cell proliferation is controlled by multiple growth-regulatory pathways that act together to ensure proper cell division. At the late G1 restriction point (R point) the cell weighs the activity of positive and negative regulatory signals. Part of our research is focused on the mechanisms by which growth factors, through their cognate receptors, modulate the activity of cell cycle regulatory proteins that control passage through G 1. 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 signals. Instead, cells come to rely upon the intrinsic regulators of the cell cycle machinery for orderly progression through the remainder of the cell cycle. For example, re-replication normally cannot take place before cells have passed through mitosis. This coupling of cell cycle events is ensured by cell cycle checkpoints, which regulate the dependency of one cell cycle event on the other. In addition, checkpoint controls can modulate cell cycle progression in response to adverse conditions, such as those following damaged DNA. Progression through the cell cycle is mediated by the activation of a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). Activation of a CDK requires binding to a specific regulatory subunit, termed a cyclin. Together, these cyclin/cdk complexes are the universal cell cycle regulators, with each complex controlling a specific transition between the subsequent phases in the cell cycle. Cell cycle control by checkpoints functions through interference with activation of these complexes. For example, for the onset of mitosis the activation of Cyclin B/Cdc2 complexes is required, whereas activation of the G 2 DNA damage checkpoint results in inhibition of these complexes, leading to an arrest in G 2 progression. Recently, new insights into the targets of the DNA damage checkpoint are helping to unravel more of the complex mechanisms of cell cycle checkpoints. In the past years our group has studied the function and regulation of CDKinhibitors and cyclins, both in the G 1 phase, as well as in the G 2 phase of the cell cycle. Two major themes are worked on in this group. Forkhead transcription factors and cell cycle regulation In close collaboration with a number of groups at the University Medical Center in Utrecht, we have been studying the function of a variety of Forkhead transcription factors in regulating cell cycle progression. We have shown that expression of the CDK-inhibitor p27 kip1 is directly regulated through the PI3-kinase/PKB signaling pathway, via modulation of AFX-like Forkhead transcription factors, resulting in the description of a full signaling cascade connecting receptors to cell cycle regulatory proteins. In addition, we have shown that the same cascade exists in lymphocytes, regulating p27 kip1 expression and subsequent apoptosis. In parallel, we have been studying the role of another forkhead transcription factor, Trident, that does not fall within the AFX-like subfamily. We have shown that Trident is involved in checkpoint control that prevents DNA re-replication during G 2. This work is being performed in collaboration with: B Burgering (Laboratory for Physiological Chemistry, UMC Utrecht), P Coffer (Department of Pulmonary Diseases, UMC Utrecht) and H Clevers (Department of Immunology, UMC Utrecht). DNA damage checkpoints in G2 and mitosis During the last three years we have been concentrating on checkpoint functions in G 2 and mitosis, in particular the role of p21 and Polo-like kinase-1 (Plk1) in DNA damage responses during these phases of the cell cycle. As part of these efforts, we have shown that p21 blocks the activating phosphorylation of Cdc2 on Thr161 and shown that Plk1 is inhibited by DNA damage in G2 and in mitosis, which constitutes a novel mechanism by which DNA damage can prevent activation of Cdc25C. Indeed, we have demonstrated that DNA damage inhibits Plk1-mediated phosphorylation of Cdc25C, and most importantly, have shown that expression of mutants of Plk1 that are not inhibited by DNA damage causes an override of the G2 arrest that is normally seen in response to DNA damage. This clearly demonstrates that inhibition of Plk1 is an important event in the

49 51 13 MOLECULAR BIOLOGY Key Publications Fajas L, Paul C, Vie A, Estrach S, Medema RH, Blanchard, JM, Sardet C, Vignais ML. Cyclin A is a mediator of p120e4f-dependent cell cycle arrest in G1. Mol Cell Biol 2001; 21: Smits VAJ, Medema RH. Checking out the G2/M transition. Biochim Biophys Acta 2001; 1519:1-12. Figure V.2: G 2 /M DNA damage checkpoint. Different pathways that influence Cdc2 phosphorylation in response to DNA damage. Upon checkpoint activation, negative regulatory phosphorylation of Cdc2 is rapidly stabilized due to inhibition of Cdc25C. This can occur via direct phosphorylation of Cdc25C by Chk1/Chk2 resulting in inhibition of Cdc25C phosphatase activity. Chk1/Chk2 can also phosphorylate Cdc25C on Ser216 which creates a binding site for proteins, leading to tethering of Cdc25C in the cytoplasm. Although initially invoked as an important mechanism, recent data show that this unlikely to play a major role in the DNA damage response. In addition, DNA damage causes inhibition of Plk1, which in turn is involved in the activation of Cdc25C. Plk1 inhibition is mediated by ATM/ATR kinases, yet the involvement of Chk1/Chk2 in this pathway is not known at present. Also, Plk1 was recently shown to play a role in the nuclear translocation of Cyclin B and Cdc25C, and its inhibition could in this way contribute to the arrest in G 2. In addition to these rapid response, a slow response involving activation of p53 and induction of p21 expression, is required for the maintenance of the G 2 arrest. This pathway appears to target positive regulatory phosphorylation of Cdc2 by inhibition of CAK-mediated phosphorylation of the Thr161 residue on Cdc2. Van Oirschot BA, Stahl M, Lens SM, Medema RH. Protein kinase A regulates expression of p27(kip1) and cyclin D3 to suppress proliferation of leukemic T cell lines. J Biol Chem 2001; 276: Van Vugt MATM, Smits VAJ, Klompmaker R, Medema RH. Inhibition of Polo-like Kinase-1 by DNA Damage Occurs in an ATM- or ATR-dependent Fashion. J Biol Chem 2001; 276: DNA damage-induced inhibition of mitotic entry. Thus, our data provide important extensions to the existing models on DNA damage checkpoints (Figure V.2). Our data with p21 demonstrate the existence of multiple DNA damage activated pathways that inhibit Cdc2 activity. In addition, our experiments with Plk1 have identified a target of the DNA damage pathway in mammalian cells that is involved in mitotic entry, as well as mitotic exit. Interestingly, we have shown that Plk1 is also inhibited in cells that have passed the G2/M 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. In this respect, it is interesting to note that we have been able to show that degradation of Cyclin B and mitotic exit, which both require Plk1-activity, are also inhibited by DNA damage in mammalian cells. Taken together, our data point to Plk1 as an important target of the DNA damage checkpoint, involved in cell cycle arrests in G2, as well as during mitosis. We are currently investigating the relative importance of the different DNA damage-induced effects in the overall response of a cell to genotoxic stress, and studying the fate of a cell that escapes checkpoint responses during interphase.

50 52 MOLECULAR GENETICS Director of Research Anton Berns ANTIGENIC VARIATION IN TRYPANOSOMES Group leader P Borst P Borst MD PhD Group leader H Van Luenen PhD Academic staff J Wijnholds PhD Academic staff (honorary) R Mussmann PhD Post-doc G Reid PhD Post-doc P Wielinga PhD Post-doc PA Genest MSc Graduate student C Toaldo MSc Graduate student S Ulbert MSc Graduate student Z Yu MSc Graduate student N Zelcer MSc Graduate student M Franken Undergraduate student J Koppes Undergraduate student M De Haas Technical staff R Kieft Technical staff A Kuil Technical staff L Van Deemter Technical staff I Van der Heijden Technical staff B Van Houten Secretary The African 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 surface 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 mice and in culture. Our recent work has focussed on two issues: the antigenic variation of the transferrin receptor of T. brucei and the biosynthesis and function of the unusual DNA base J. Base J (β-glucosyl-hydroxymethyluracil), which we discovered in trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993; 75: ) 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 the telomeric repeats. We have considerable evidence that base J is made from thymine in DNA in two steps as illustrated in Figure V.3, but we have been unable to identify the enzymes involved thus far. This remains a major goal in the group, however. We have isolated and characterized a protein that binds with high specificity to J, but only in duplex DNA (Chaves et al., EMBO J 1999; 18: ). Targeted disruption of this gene for JBP in T. brucei had no effect on growth, on repression of inactive genes, or on the stability of repeat sequences, but resulted in a global decrease in the level of J to only 5% of the wild type level. We have shown that this decrease is not due to an increased turnover of J and we now think that JBP acts as a local J maintenance and propagation factor, which helps the enzymatic machinery that introduces J to increase local J levels, once a basic J level has been introduced in the DNA. Little JBP is required for this task, as there appears to be only enough JBP in the cell to bind to less than 1% of the J residues. To cover its need for iron the trypanosome takes up transferrin (Tf) from its mammalian host. Uptake takes place in an invagination of the surface membrane, the flagellar pocket, and is mediated by a heterodimeric transferrin receptor (Tf-R) attached to the membrane via a GPI anchor. We have shown that T. brucei can make about 20 different transferrin receptors differing slightly in amino acid sequence and we have presented evidence that 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: ). Both the variant surface glycoprotein (VSG), the major surface glycoprotein of T. brucei and the Tf-R are GPI-anchored and both enter the surface in the flagellar pocket, but the VSG spreads all over the surface, whereas the Tf-R is retained in the pocket. We have shown that a modest overproduction (three-fold) of receptor results in the spreading of the Tf-R all over the surface. This occurs not only when we force overproduction of receptor by transfecting in extra copies of the Tf-R genes, but also when we starve trypanosomes for Tf, which leads to a rapid increase in the levels of receptor RNA and protein. Obviously, the flagellar pocket retention mechanism for the receptor is easily saturated. The nature of the retention mechanism is under investigation. MULTIDRUG RESISTANCE OF CANCER CELLS We are interested in mechanisms of drug resistance in cancer cells and focus on resistance caused by increased transport of drugs out of the cell. We have isolated the genes for these transporters and are characterizing their substrate specificity and sen- Figure V.3: Scheme for the biosynthesis of base J in Trypanosoma brucei.

51 53 13 MOLECULAR GENETICS sitivity to inhibitors in transfected cells. We make extensive use of transfected polarized kidney cell monolayers in which the transporters either route to the apical or the basolateral membrane, allowing the study of vectorial transport through the cell layer. We also use the baculovirus system for producing insect cell membrane vesicles containing high amounts of transporter protein and suitable for vesicular transport studies. Drug transporters causing chemotherapy resistance in tumors are present in (some) normal tissues and we are also interested in their physiological function in metabolism and defence of the body against drugs and xenotoxins. We have therefore inactivated several genes for drug transporters by targeted gene disruption in mice. Initially we looked at P-glycoproteins; more recently we have studied the family of Multidrug Resistance Proteins (MRPs), and we are now concentrating on MRP3, 4 and 5. MRP3 We identified MRP3 as one of the members of the MRP family, cloned the gene, expressed it in a variety of cell systems and raised monoclonal antibodies against fusion proteins containing segments of MRP3 (Kool et al., Proc Natl Acad Sci USA 1999; 96:6914-9). We have shown that MRP3 is a typical drug-conjugate (GS-X) pump, but with a preference for glucuronosyl-conjugates. MRP3 is located in the basolateral membrane of intrahepatic bile ducts and adrenal glands, but also present in gut, kidney, pancreas and lung. MRP3 is highly upregulated in the liver under cholestatic conditions and, as MRP3 can also transport some bile salts, it may act to remove toxic anions, including bile salts, from the liver when bile flow is disturbed. In view of its high affinity for glucuronosyl conjugates, MRP3 may also play a role in removing toxic compounds, detoxified by glucuronidation, from cells. We have studied the resistance against anti-cancer drugs caused by MRP3 in transfected fibroblasts from mice lacking MDR1a/1b P-glycoprotein and MRP1, cells with low background drug resistance. We have found only resistance to etoposide/teniposide and not to other MDR drugs. Etoposide resistance is due to increased extrusion of etoposide by itself, not to extrusion of glucuronosyl-etoposide or co-transport of etoposide and glutathione (as found for MRP1). We are making a more detailed study of the substrate specificity of MRP3 in membrane vesicles of insect Sf9 cells highly overproducing MRP3. We have also generated a Mrp3 (-/-) mouse. This is healthy and fertile and under study. MRP4 and MRP5 We have shown that MRP5 is an organic anion pump, able to extrude nucleotide analogs (Wijnholds et al., Proc Natl Acad Sci USA 2000; 97: ) and similar results have been obtained by J Schuetz (St Jude s, Memphis, Figure V.4: Simplified scheme showing the intracellular metabolism of USA) for MRP4. We have 6-mercaptopurine and thioguanine, and the transport of thiopurine made a detailed study of nucleoside monophosphates by MRP4 and MRP5. the resistance against 6-mercaptopurine (6MP) and thioguanine (TG) in HEK293 cells overproducing either MRP4 or MRP5 in collaboration with J Beijnen (Division X) and J Schuetz. We find that these cells excrete the timp, txmp and tgmp made from 6MP and the tgmp made from TG, but not the thio-nucleoside di- and triphosphates (Figure V.4). Following reports from other labs that MRP4 and 5 can also transport cyclic nucleotides at low rate, we have set up systems to study cyclic nucleotide transport. No effect has yet been found in the Mrp5 (-/-) mice generated, but in the HEK293 cells we find that the presence of MRP4 or MRP5 decreases the induced accumulation of intracellular cgmp and that this decrease is associated with increased cgmp export. It is possible that the apparent lack of a phenotype in the Mrp5 (-/-) mice that we have generated is due to the presence of several transporters with similar substrate specificity, MRP4, MRP5 (or the recently identified MRP8 and 9?), and we are collaborating with other laboratories to study this problem. Key Publications Borst P, Borst J, Smets LA. Does resistance to apoptosis affect clinical response to antitumor drugs? Drug Resistance Updates 2001; 4: Borst P, Oude Elferink R. Mammalian ABC-transporters in health and disease. Chapter Vol 71 Annual Review of Biochemistry 2001 (in press). Borst P, Ulbert S. Control of VSG gene expression sites. Mol Biochem Parasitol 2001; 114: Gerrits H, Mussmann R, Bitter W, Kieft R, Borst P. The physiological significance of transferrin receptor variations in T. brucei. Mol Biochem Parasitol 2001 (in press). Sabatini R, Meeuwenoord N, Van Boom J H, Borst P. Recognition of base J in duplex DNA by J-binding protein. J Biol Chem (in press). Scheffer GL, Kool M, De Haas M, De Vree JML, Pijnenborg ACLM, Bosman DK, Oude Elferink RPJ, Van der Valk P, Borst P, Scheper RJ. Tissue distribution and induction of human MRP3. Lab Invest (in press). Schultz MJ, Wijnholds J, Peppelenbosch MP, Vervoordeldonk MJBM, Speelman P, Van Deventer HJ, Borst P, Van der Poll T. Mice lacking the multidrug resistance protein 1 are resistant to Streptococcus pneumoniae. J Immunol 2001; 166: Zelcer N, Saeki T, Reid G, Beijnen JH, Borst P. Characterization of drug transport by the human multidrug resistance protein 3(ABCC3). J Biol Chem (in press).

52 54 TUMOR BIOLOGY Director of Research Anton Berns VI DIVISION OF TUMOR BIOLOGY ANTIGEN PRESENTATION BY MHC CLASS I AND II MOLECULES Division head, Group leader J Neefjes J Neefjes PhD Group leader M Fernandez-Borja PhD Post-doc C Herberts PhD Post-doc E Reits PhD Post-doc A Griekspoor MSc Graduate student I Jordens MSc Graduate student M Marsman MSc Graduate student M Van Lith MSc Graduate student C La Sage Undergraduate student J Neijssen Undergraduate student L Janssen Technical staff J Overwater Secretary M Van der Velde Secretary We have continued our studies aimed at understanding the molecular basis of the specific cellular immune response. This response determines the specificity of antivirus and anti-tumor responses by CTL and the initiation of a specific antibody response. Understanding these principles should result in predictions of the specificity of immune responses based on the primary sequence of an antigen. Antigen presentation by MHC class I molecules MHC class I molecules are critical in mounting a specific immune response. They present fragments (also called peptides) of endogenous proteins to circulating cytotoxic T cells (CTL). CTL are selected such that they recognize all peptides except those derived from self proteins. When CTL recognize a non-self peptide, for example derived from a mutated cancer protein or a virus, presented by MHC class I molecules, they initiate a program resulting in the elimination of the cell. MHC class I molecules may thus present specific tumor peptides. If so, CTL can kill these tumors. In order to predict the outcome of such a process, the components involved have to be characterized. Especially the specificity of these processes in conjunction with their dynamics are of interest because they allow further predictions of presented peptides and analysis of variations in the process of antigen presentation. We have established techniques to visualize in vivo the rate of peptide formation by visualizing the mobility of the peptide transporter TAP in living cells. TAP transports peptides from the cytoplasm into the ER lumen for consideration by MHC class I. TAP moved slowly when pumping peptides and quickly when in the inactive state. These techniques have now been used to visualize never realized effects of γ irradiation. γ irradiation on cells rapidly increased the amount of intracellular peptides and also peptide-loaded class I molecules, in a dose dependent manner. We isolated these class I molecules from cells, eluted the peptides and analyzed these by HPLC-mass spectrometry. A number of peptides were found exclusively in irradiated cells. Five peptides were sequenced and were derived either from proteins involved in DNA repair or protein degradation. One unknown protein was identified. We are currently trying to raise CTL against these peptides in a process that may ultimately combine immunotherapy with radiotherapy. Other experiments concentrate on the role of the proteasome and peptidases in the final outcome of an MHC class I-restricted CTL response. It is unclear how peptides find the peptide transporter TAP. We have followed the fate of fluorescent peptides in living cells. These peptides rapidly diffuse in the cytoplasm and nucleus. In addition, they associate to chromatin components including histon 2B and histon 4(identified by mass spectrometry). Interestingly, the nucleus and cytoplasm are different from the peptide s perspective. For example, two major cytosolic peptidases are excluded from the nucleus. Since we have shown that TAP is not located in the inner membrane of the nuclear envelope, nuclear peptides still have to diffuse into the cytosol for binding to TAP. These studies visualized the step between the moment of peptide generation by the proteasome and binding to TAP. To determine the half-life of peptides in vivo, we have established technologies to introduce peptides containing fluorophores and quenchers in cells and measure their degradation. Specific sequence and size dependent differences in half-life of individual peptides were detected. These experiments will add to the growing knowledge of the specificity of the individual components in the cascade of events resulting in antigen presentation by MHC class I molecules. Finally, some - more physical - characteristics of the peptide pump TAP in this cascade are being studied, including the rotation and mobility in the membrane of the endoplasmic reticulum. These studies are aimed at improving our understanding of the alterations occurring in ABC transporters during substrate binding and transport. Antigen presentation by MHC class II molecules MHC class II molecules basically perform a function similar to MHC class I molecules. A major difference is

53 55 13 TUMOR BIOLOGY Key Publications De Leeuw HP, Fernandez-Borja M, Reits EA, Romani de Wit T, Wijers-Koster PM, Hordijk PL, Neefjes J, Van Mourik JA, Voorberg J. Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells. Arterioscler Thromb Vasc Biol 2001; 21: Jordens I, Fernandez-Borja M, Marsman M, Dusseljee S, Janssen L, Calafat J, Janssen H, Wubbolts R, Neefjes J. The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors. Curr Biol 2001; 11: Fig. VI.1: A schematic representation of the cell biological mechanism of peptide generation, transport and loading onto MHC class I molecules. the source of peptides. MHC class II molecules mainly present peptides fragments from proteins degraded in the endo/lysosomal pathway. They are transported transiently through these compartments, called MIIC, where exchange of peptides occurs in a process mediated by a specific chaperon called HLA-DM. In some cells and tumors, another molecule is expressed that controls HLA-DM activity. The function of this molecule (called HLA-DO) is being studied. HLA-DO associates to HLA-DM, inhibits its function and is mainly expressed in non-activated B cells. Its expression rapidly drops after activation of a B cell. Clear differences in HLA-DO expression are seen between different B cell tumor types. This item is being studied further. We have studied the cell biology of HLA-DO in detail. The B chain of HLA- DO contains a classical internalization signal. Using electronmicroscopy, we showed that the tail of HLA-DO determined the location within a multivesicular body and thus controlled the contacts made between MHC class II molecules and HLA-DO. We are currently studying a novel interaction of HLA-DO with the free class I H-chain. These are expressed at the plasma membrane but the function of this interaction is unclear. Furthermore, the exact nature of the MIIC s and the mechanism that controls their transport to the plasma membrane are being studied. Electronmicroscopy has shown that the MIIC can be a multivesicular structure (MVB) containing many small internal vesicles. To test whether these vesicles are free or interconnected allowing lateral interactions between proteins located on different vesicles, we analyzed these MVB by EM and subcellular fractionation. This showed that the internal vesicles were interconnected by protein-like structures. Although interconnected, the nature of these interconnections does not allow lateral diffusion of molecules from one vesicle to the next. MIIC s are structures on the crossroad of incoming and departing material. To understand these processes better, we have studied the small GTPase RhoB and Rab7. RhoB is recruited to early endosomes and affects the local actin cytoskeleton. Rab7 is a small GTPase located on MIIC s that is controling fusion with later compartments. Using yeast two hybrid, we identified a Rab7-effector protein called RILP that induces the local recruitment of the minus-end directed motor protein complex dynein/dynactin. As a result, the MIIC s are transported to the extreme minus end of microtubules, which is around the centrioles. We thus identified one segment of a pathway that controls the transport of MIIC s. The molecular details of this pathway, and the possibility that it also affects the intracellular survival of intracellular mycobacteria, are currently being studied. Knuehl C, Spee P, Ruppert T, Kuckelkorn U, Henklein P, Neefjes J, Kloetzel PM. The murine cytomegalovirus pp89 immunodominant H-2L(d) epitope is generated and translocated into the endoplasmic reticulum as an 11-mer precursor peptide. J Immunol 2001; 167: Koppers-Lalic D, Rijsewijk FA, Verschuren SB, Van Gaans-Van Den Brink JA, Neisig A, Ressing ME, Neefjes J, Wiertz EJ. The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1. J Gen Virol 2001; 82: Neisig A, Neefjes J. Application of onedimensional isoelectric focusing to separate different major histocompatibility complex class I alleles and determine their allelic interactions with transporter associated with antigen processing (TAP). Methods Mol Biol 2001; 156: Reits EA, Neefjes JJ. From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 2001; 3:E Schüller S, Neefjes J, Thole J, Young D. Separation and characterisation of mycobacterial compartments in human macrophages: Involvement of coronin in bacterial uptake but not in phagosome maintenance. Infect Immunol (in press). Van Lith M, Van Ham M, Griekspoor A, Tjin E, Verwoerd D, Calafat J, Janssen H, Reits E, Pastoors L, Neefjes J. Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body. J Immunol 2001; 167:

54 56 TUMOR BIOLOGY Director of Research Anton Berns ULTRASTRUCTURAL STUDIES ON ENDOCYTOSIS Group leader PJ Peters PJ Peters PhD Group leader A Mironov MD PhD Post-doc N Van der Wel PhD Post-doc T Groothuis MSc Graduate student G Schreibelt Undergraduate student E Bos MSc Technical staff Membrane traffic We are continuing the investigation into membrane trafficking 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 cell. Our work concentrates on the vesicular transport mechanism within the endocytic pathway and its link to pathogenesis. Cryoimmunogold-electron microscopical methods are the primary techniques used, which allow the subcellular detection of gene products at high resolution. The research is done in three projects. Differential intracellular pathways of antigen presentation by MHC class II and CD1 molecules in developing dendritic cells CD1 isoforms have been identified in humans as membrane proteins structurally related to MHC class I and II molecules. This novel class of antigen presenting molecules presents mycobacterial lipid antigens from intracellular bacteria to the immune system. We previously found that the subcellular localization between the CD1 isoforms differs and is dependent on a cytoplasmic targeting signal. CD1b localizes to lysosomal compartments known to contain MHC class II (MIIC s). CD1a lacks an endosomal-targeting signal and was exclusively detected in endocytic recycling vesicles. Such differences are likely to be of critical functional importance since they determine the compartments in which antigen loading may occur. Infection with pathogens such as M. tuberculosis results in the activation or maturation of dendritic cells, as surface expression of costimulatory molecules as well as MHC class II is elevated and inflammatory cytokines are released. This year, in collaboration with M Brenner of the Harvard Medical School Boston US, the pathway taken by CD1b was determined upon addition of the bacterial component lipopolysaccharide (LPS) using cryo-immunogold labeled transmission electron microscopy. LPS is known to mature dendritic cells and to trigger redistribution of MHC class II molecules from MIIC s to the plasma membrane. We determined the redistribution of different CD1 molecules in LPS treated dendritic cells and compared this to MHC class II molecules. During LPS induced maturation the localization of the two antigen presenting molecules segregate. CD1b molecules remain intracellular and are present in small-noncoated vesicles or tubules and in lysosomal compartments. The highly arranged laminar membrane organization of the lysosomal MIIC s is rapidly modified after LPS stimulation and long electron dense structures are formed. Thus, in dendritic cells, lysosomes are highly dynamic structures from which molecules can be variously sorted disproving the current dogma that lysosomal compartments are the terminal end stations. A novel caveolae-mediated endosomal/lysosomal pathway used by cellular prion protein Prion diseases are fatal neurodegenerative disorders. Biochemical purification of the infectious material and transmission studies employing transgenic and PrP-ablated (Prnp0/0) mice have shown that transmission and neurodegeneration are mediated by the abnormally folded prion protein (PrP Sc ). PrP Sc is mostly an insoluble, partially protease-resistant glycoprotein, which accumulates in affected brains. PrP Sc is formed 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 PrP C molecules are converted into beta-sheet structures. Conversion is a late event in the lifecycle of PrP C and occurs after PrP C has passed the Golgi complex. After conversion, PrP Sc accumulates in a late endocytic lysosomal compartment of the infected cell. The mechanism involved in the conversion of PrP C into PrP Sc and the exact subcellular site of PrP Sc formation have not yet been determined. Here we have, in collaboration with S Prusiner, University California San Francisco, studied the trafficking of PrP C in CHO cells stably transfected with either Syrian hamster (SHa) or chimeric mouse-hamster (MH2M) PrP C using cryo-immunogold electron microscopy. PrP C was found to be highly enriched in caveolae of these cell lines. We also found that PrP C gained access to a non-classical endocytic pathway, not enriched for clathrin-mediated transferrin receptor, but by internalization through caveolin-coated caveolae. Thus, caveolae mediate uptake of a GPI-anchored protein into caveolin-positive endocytic structures. This novel pathway is the primary

55 57 13 TUMOR BIOLOGY internalization route of PrP C and is therefore likely to determine the subcellular location in which PrP C :PrP Sc interaction leads to the generation of nascent infectious prions. We are now in the process of establishing the ultrastructural localization of PrP C and PrP Sc in hippocampus, cerebellum and neocortex. Regulation of recycling of endosomal membrane 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, cell spreading, cell migration and perhaps invasion. Thus, we have focused our efforts on understanding the cellular basis of ARF6-regulated endosomal recycling. In collaboration with V Hsu (Harvard Medical School), we are characterizing the nature of the ARF6 endosomal compartment using both biochemical and morphological investigations. While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding transport carriers that mediate this pathway. We overexpressed a point mutant of ADP- Ribosylation Factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. These vesicles were found mostly aggregated with larger non-coated membranes, and could be released with high salt treatment. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-snare cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling. In collaboration with the group of J Neefjes we have found that endocytic recycling vesicles migrate in Density Gradient Electrophoresis (DGE) fractions that are distinct from conventional recycling compartments. One of the hallmarks of these structures is that they are enriched in the tetra spanner called SCAMP. We are now trying to characterize these vesicles by whole mount immunogold electron microscopy to identify protein markers and other characteristics of these vesicles in order to understand the molecular composition of this novel endocytic recycling compartment. PJ Peters has also been active as dean of postdoctoral affairs. See Key Publications Ameen NA, Van Donselaar E, Posthuma G, De Jonge H, McLaughlin G, Geuze HJ, Marino C, Peters PJ. Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic. Histochem Cell Biol 2000; 114: Lennon-Dumenil AM, Roberts RA, Valentijn K, Driessen C, Overkleeft HS, Erickson A, Peters PJ, Bikoff E, Ploegh HL, Wolf Bryant P. The p41 isoform of invariant chain is a chaperone for cathepsin L. EMBO J 2001; 20: Peters PJ, Gao M, Gaschet J, Ambach A, Van Donselaar E, Traverse JF, Bos E, Wolffe EJ, and Hsu VW. Characterization of coated vesicles that participate in endocytic recycling. Traffic (in press). Peters PJ, Hunziker W. Subcellular localization of Rab17 by cryo-immunogold electron microscopy in epithelial cells grown on polycarbonate filters. Methods Enzymol 2001: 329: Sugita M, Peters PJ, Brenner MB. Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide. Traffic 2000; 1: Teaching at the Medical School, Free University PJ Peters has been teaching Cell Biology at the Medical School of the Free University in Amsterdam. In addition, as a semester chair for first year students he coordinated the semester program, From molecule to cell with the various faculty members.

56 58 TUMOR BIOLOGY Director of Research Anton Berns GENES INVOLVED IN BREAST CANCER METASTASIS Group leader J Hilkens J Hilkens PhD Group leader I Gaemers PhD Post-doc M Kimm MSc Graduate student V Theodorou Graduate student Z Ali Alas Undergraduate student M Boer Technical staff Formation of metastases is the main cause of death of most cancer patients. However, the biological processes which lead to the metastatic phenotype are poorly understood. We aim to identify novel genes involved in breast cancer metastasis and to understand the biological function of cell surface associated mucins, notably episialin/muc1, which have been implicated in breast cancer progression. Identification of novel genes involved in mammary tumor progression and metastasis by MMTV tagging We have developed a mouse model system that can be used to identify novel metastasis genes in a BALB/c substrain that acquired mouse mammary tumor virus (MMTV) by foster-nursing (denoted BALB/c+ strain). Almost all of the breeders of this strain develop mammary tumors, which often give rise to micrometastases in the lung, which are, however, too small for genetic analysis. We found that subcutaneously (s.c.) transplantation of lung tissue from tumor bearing animals develops into a tumor if metastases of the mammary carcinoma were present. These s.c. tumors have provided us with a source of mammary tumor metastases derived tissue for genetic analysis, which was previously not available. A series of independent sets of primary tumors and s.c. grown lung metastases of these tumors has been collected and was examined for novel MMTV tagged genes by using a splinkerette PCR. We have identified the Wnt-3A as a novel target for MMTV. Interestingly, Wnt-3A was frequently found activated only in the subcutaneously growing metastases. Membrane-associated mucins in breast cancer Membrane associated mucins are frequently upregulated in various types of cancer cells. These glycoproteins are characterized by a heavily O-glycosylated mucin domain protruding more than 200 nm above the plasma membrane. Several lines 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), encoded by the MUC1 gene, is a paradigm of this class of mucins to which also belong epiglycanin, MUC4 and several others. We have previously shown in vitro and in vivo that this elongated and relatively rigid molecule, when overexpressed, can mask other cell surface molecules such as adhesion receptors, thus impeding cell-cell and cell-matrix interactions and contributing to the invasive and metastatic potential of tumor cells. It has recently been shown that episialin can associate with the EGF receptor and Erb-B2 upon which it becomes tyrosine phosphorylated. We have now shown that episialin can be present in cholesterol rich membrane rafts. Raft localization appears to be dependent on EGF receptor activation. These results suggest that episialin may be involved in cell signaling. Regulation of episialin gene expression In order to understand the overexpression of episialin in cancer cells we have embarked on an analysis of its promotor. The episialin promoter contains a cluster of regulatory elements around 500 nt before the transcription start site, one of which is a STAT3 response element. We have shown that this element is involved in regulating episialin expression. Since STAT3 is constitutively active in breast and several other types of carcinomas, it may thus constitutively upregulate episialin expression. However, other factors are also needed since addition of IL-6 only (which stimulates STAT3) has only a minor effect on episialin expression in most carcinoma cell lines. We found that hydrocortisone (HC) in addition to IL-6 synergistically stimulates episialin expression and that the glucocorticoid receptor (GR) and STAT3 signaling pathways interact directly and indirectly. A direct interaction of GR with STAT3 was shown by co-immunoprecipitation, while the indirect interaction became apparent by HC dependent serine 727 phosphorylation of STAT3, which is known to increase the transactivation potential of STAT3. Interestingly, GR binds to STAT3 even without being activated by glucocorticoids. Analysis of the sequence adjacent to the STAT3 binding site also revealed three GR binding sites. Mutation of all three sites reduced both the HC and IL-6 induced promoter activity and also the synergistic activity to approximately 25%. Conversily, reporter assays with constructs in which the STAT3 response element was mutated

57 59 13 TUMOR BIOLOGY revealed that IL-6 still stimulated the HC induced promoter activity. Based on these results we propose the following model: GR binds to STAT3 and probably acts as a coactivator of STAT3 upon binding of glucocorticoids, however the activity can be increased even further upon stabilization of the complex by binding of the GR to its DNA binding element. Conversely, STAT3 can act as a coactivator of GR. We have generated several mutated forms of STAT3 and GR to further substantiate this model. Key Publications Gaemers IC, Vos HL, Volders HH, Van der Valk SW, Hilkens J. A STAT-responsive element in the promoter of the episialin/ MUC1 gene is involved in its overexpression in carcinoma cells. J Biol Chem 2001; 276: Steelant WF, Goeman JL, Philippe J, Oomen LC, Hilkens J, Krzewinski-Recchi MA, Huet G, Van der Eycken J, Delannoy P, Bruyneel EA, Mareel MM. Alkyllysophospholipid 1-O-octadecyl-2-O-methylglycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human. Int J Cancer 2001; 92:

58 60 TUMOR BIOLOGY Director of Research Anton Berns CELL CYCLE CONTROL GENES AND BREAST CANCER Group leader R Michalides R Michalides PhD Group leader E Bindels PhD Post-doc A Balkenende Technical staff D Verwoerd Technical staff Key Publications Germano D, Pacillio D, Cancemi M, Cicatiello L, Altucci L, Petrizzi VB, Sperandio C, Salzano S, Michalides R, Taya Y, Bresciani F, Weisz A. Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1. Breast Cancer Res Treatment 2001; 67: Michalides R, Van Tinteren H, Balkenende A, Vermorken JB, Benraadt J, Huldij J, Van Diest P. Cyclin A is a prognostic indicator in early stage breast cancer with and without tamoxifen treatment. Br J Cancer (in press). Mommers E, Leonhart A, Falix F, Michalides R, Meijer C, Baak J, Van Diest P. Similarity in expression of cell cycle proteins between in situ and invasive ductal breast lesions of the same differentiation grade. J Pathology 2001; 194: We are studying aberrations in cell cycle regulators in tumor progression and have previously found that cyclin D1 itself can activate the estrogen receptor, ER, in the absence of estrogen, and, moreover, that this activation is not impeded by anti-estrogens (Zwijsen et al, Cell 1997; 88:405). We have now investigated the clinical impact and the consequences on estrogen dependent growth of that finding. We, therefore, transiently overexpressed various cell cycle regulators in estrogen-dependent T47D breast cancer cells. Excessive overexpression of cyclin D1 alone led to hormone-independent growth, which was not inhibited by anti-estrogens. Combinations of cyclin D1 with a constitutive active cdk2, cdk2-af, or an activator of cdk2 activity, cdc25a, had no additional effect. The cyclin D1-KE mutant, that does not bind to cdk4 but still exerts an estrogen-independent activation of ER, did not, however, yield a hormone independent and anti-estrogen insensitive growth. In stable transfected estrogendependent MCF7cells we found that overexpression of cyclin D1 protein up to six-fold did not result in estrogen-independent growth and only delayed the effects of antiestrogens. In stable transfected cells the effect of anti-estrogens is due to the downregulation of the cyclin D1 and A proteins, resulting in a redistribution of cyclin kinase inhibitors from cyclin D1 complexes to cyclin E-cdk2-containing complexes, thereby causing a G1 arrest. The clinical impact of the hormone-independent activation of ER by cyclin D1 was investigated in a retrospective study on the association between deregulation of a variety of cell cycle regulators and recurrence of breast cancer in 394 patients with early stage breast cancer. By univariate analysis, only expression of cyclin A, Neu, Ki- 67 index and lack of ER expression were significantly associated with poor prognosis, both in tamoxifen treated as well as in non-treated patients. By multivariate analysis, only cyclin A and Neu were significantly associated with poor prognosis (HR 2.024, p= ). Cyclin A is, therefore, 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. In conclusion, cyclin D1 when present in excessive protein levels achieved by transient transfection, may activate estrogen independent growth in an anti-estrogen insensitive manner. This independent growth does not occur by activation of the estrogen receptor, but, most likely, by influencing the balance of cyclin-dependent kinase inhibitors between the cdk complexes. These excessive levels of cyclin D1 are apparently not reached in a stable transfected MCF7 cell line with a six-fold overexpression of cyclin D1 nor in breast cancer samples of a clinical study, in which cyclin D1 overexpression was not found to be associated with insensitivity to tamoxifen treatment. Overexpression of cyclin A seemed much more promising in that respect. Cyclin D1 overexpression has an effect on other treatment modalities. Constitutive overexpression of cyclin D1 protein in levels comparable to those found in breast tumors with overexpression of cyclin D1, delays the effect of mevalonate/protein prenylation inhibitor Simvastatin (Germano et al, 2001). On the other hand, it enhances a G2 arrest upon gamma radiation and upon treatment of cells with paclitaxel by an elevated induction of p21 affecting the G2/M transition (Michalides et al, submitted 2001; Coco-Martin et al, Cancer Research 1999; 59:1134). The manner in which overexpression of cyclin D1, which is frequently observed in breast cancer, affects treatment of breast cancer depends on the extent of overexpression and the mechanism behind the treatment modality. Van Diest P, Michalides R. Cell cycle regulators: interactions and their role in diagnosis, prognosis and treatment of cancer. In: Cell cycle inhibitors in cancer therapy. Cancer drug discovery and development; (in press).

59 61 13 TUMOR BIOLOGY NUCLEOCYTOPLASMIC TRANSPORT AND THE ROLE OF THE NUCLEAR PORE COMPLEX In our recently established research group we are studying macromolecular transport between the nucleus and the cytoplasm, and investigating how this fundamental process is used in the regulatory circuits of the cell. Eukaryotes are characterized by a separation of (most of) their transcription and translation machineries in different subcellular compartments, 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 are 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 level of nuclear transport. Group leader M Fornerod M Fornerod PhD Group leader H Pickersgill PhD Post-doctoral fellow R Bernad-Fernandez MSc Graduate student J Hendriksen MSc Graduate student D Engelsma Undergraduate student H Van der Velde BSc Technical staff Nuclear 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, Fig. VI.2), the channel forming units between the nucleus and the cytoplasm. These 125 MDa complexes restrict free diffusion of molecules or particles with a diameter larger than 9 nm, corresponding to the size of a globular protein of 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. Nucleocytoplasmic transport is accomplished by distinct classes of soluble transport receptors that interact with transport signals and nuclear pore complex. One such class consists of importins and exportins, a superfamily of transport receptors that is responsible for the majority of nuclear transport pathways identified to date. Importin and exportin mediated transport is dependent on a signal within the transport substrate. Examples of transport signals are the basic nuclear localization signal (NLS) that is found in many nuclear proteins, and the nuclear export signal (NES) that mediates rapid nuclear export. Leucine-rich-type NESs have recently been identified in a diverse range of cellular proteins that include cell cycle regulators, stress response signal transducers and mediators of apoptosis. A second mechanism to maintain nuclear and cytoplasmic identities is the existence of the nuclear marker protein RanGTP. Nuclear RanGTP binds to and dissociates importin-cargo complexes and thereby provides direction to nuclear import. The import substrate is released into the nucleoplasm, and the RanGTP-bound importin recycles back to the cytoplasm (Fig. VI.3). RanGTP imposes direction to nuclear export as well, since RanGTP/exportin/cargo heterotrimeric complexes are several orders of magnitude more stable than exportin/cargo heterodimers. After translocation through the nuclear pore complex, trimeric export complexes are destabilized by RanBP1 or RanBP1-like domains in RanBP2/Nup358. The export reaction is made irreversible by RanGAP1-stimulated RanGTPase activity (Fig. VI.3). With the identification of transport signals, transport receptors and Ran the specificity of cargo recognition is relatively well understood. However, specificity within the nuclear pore complex remains largely unknown. 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 different proteins that are collectively named nucleoporins, and occupy distinct positions within the NPC. Nuclei and pronuclei form efficiently in vitro from crude mitotic or meiotic cytosol upon 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 to study their function in transport and ultrastructural morphology. In this way we aim to create a functional map of the nuclear pore complex. Fig. VI.2: Nuclear pore complexes in Xenopus laevis oocyte nuclear envelopes visualized by scanning electron microscopy from the cytoplasmic (upper) or nuclear (lower) side. Arrows point to cargoes caught in transit (Images courtesy of Terry Allen, Patterson Institute, Manchester, UK).

60 62 TUMOR BIOLOGY Director of Research Anton Berns Key Publications Englmeier L, Fornerod M, Bischoff FR, Petosa C, Mattaj IW, Kutay U. RanBP3 facilitates nuclear protein export by stabilising the interaction between certain export substrates and CRM1. EMBO Rep 2001; 2: Trotman LC, Mosberger N, Fornerod M, Stidwill RP, Greber UF. Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1. Nat Cell Biol 2001; 3: Walther TC, Fornerod M, Pickersgill H, Goldberg M, Allen TD, Mattaj IW. The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins. EMBO J 2001; 20: Fig. VI.3: Nuclear RanGTP regulates the direction of importin and exportin mediated nucleocytoplasmic transport (adapted from Fornerod et al., Cell 1997; 90:1051). Role of the nuclear pore complex in b type catenin nuclear signaling β type catenins constitute a family of structurally related vertebrate proteins that have dual roles in cell adhesion and signal transduction. β catenin is an essential mediator in the Wnt signaling pathway, which regulates cell proliferation during embryogenesis and is a major oncogenic target in humans. Mutations in β catenin are strongly connected with colon cancers and melanomas, while γ catenin is also implicated as an oncoprotein. Necessary for β catenin s oncogenic activity is its translocation to the nucleus, where it coactivates a cell proliferative genetic program in conjunction with the transcription factor TCF. γ Catenin has a transient presence in the nucleus, and can transactivate certain promotors in a TCF dependent way, suggesting that it may have a similar function. The mechanism of nuclear entry of these proteins is largely unknown, but is independent of conventional transport pathways and dependent on the nuclear pore complex. By investigating the role of the nuclear pore complex in their nucleocytoplasmic transport we aim to define a new level of regulation in this signal transduction pathway. For more information, please visit

61 VII DIVISION OF MOLECULAR GENETICS MOLECULAR GENETICS CANCER GENETICS The influence of host genetic factors on the development of cancer is obvious in familial cancer syndromes that are caused by germ-line mutations in tumor suppressor or mismatch repair genes like APC, Rb, NF2, MSH2. These familial cancer syndromes comprise only a small fraction of all cancers and the largest proportion of cancers is of a non-familial, sporadic, type. However, also in these apparently noninherited cancers the host genetic factors play an important causative role (e.g. Lichtenstein et al. New Engl J Med 2000; 343:78). As the family and population studies do not possess sufficient resolving power to identify the low-penetrance genes involved in the development of non-familial cancer (Ponder, Nature 2001; 411:336), experimental animals, with the advantage of defined genetic constitution, the possibility of producing informative crosses, and standardized tumor-induction procedures, are being used to resolve the genetics of cancer susceptibility. We have developed a genetic tool, the recombinant congenial strains (RCS), specifically designed to dissect genetics of traits controlled by multiple genes (Demant and Hart 1986; Stassen et al. 1996). In a series of 20 homozygous RCS each strain carries a different random portion of 12.5% of the genes of one inbred strain (the donor strain) on the genetic background of the second inbred strain (the background strain). Thus the 20 RCS represent 20 different random combinations of genes of the donor strain and the background strain. Using the RCS, susceptibility to colon and lung tumors and leukemias are being studied. In addition, the RCS are being used in a number of collaborative projects on susceptibility to skin and mammary tumors and other common diseases: infections (leishmaniasis, tuberculosis, pertussis), asthma, T-cell responsiveness, atherosclerosis, lipid metabolism, and others. The increasing importance of genetic studies has led to the need for more powerful techniques of genotyping. A part of the capacity of the group has been dedicated to the development of a high trough-put SNP (single nucleotide polymorphism) genotyping system. Division head, Group leader P Demant P Demant MD PhD Group leader M Snoek PhD Academic staff T Csikos PhD Post-doc M Van Kooy PhD Post-doc C Ruivenkamp MSc Graduate student C Zanon MSc Graduate student T De Boer Undergraduate student H Horlings Undergraduate student K De Groot Technical staff E Delzenne-Goette Technical staff J De Moes Technical staff A Klous Technical staff V Theodorou Technical staff M Treur-Mulder Technical staff H Van Vugt Technical staff T Philippo Secretary K Van den Berg Secretary Colon cancer susceptibility Using the CcS/Dem RCS each of which contain a different set of 12.5% genes from the colon-tumors susceptible STS strain on the genetic background of the resistant BALB/c strain, we mapped nine Scc (Susceptibility to colon cancer) loci, five of them (Scc1 through Scc5) in the (CcS-19 x BALB/c) F2 hybrids after combined treatment with the carcinogens DMH (di-methyl hydrazine) and ENU (N-ethyl-N-nitrosourea). A new group of (CcS-19 x BALB/c) F2 hybrids was produced and treated with a different carcinogen, AOM (azoxymethane). The linkage analysis indicated susceptibility genes in the same regions as in the previous experiment and three novel Scc loci that were suggested by previous experimentation but of which the evidence was not statistically significant. One of the previously mapped loci, Scc1, has been limited by recombinant mapping to a region of less than 200 Kb. Sequencing of the BAC-PAC contig bridging this region revealed a single gene, the receptor-type protein tyrosine phosphatase eta (Ptprj). The Ptprj products of the susceptible and resistant strain differ at several amino-acids in their finbronectin type 3 domains. Homologous part of the human chromosome (11p) has been tested for LOH and has been found to be frequently deleted in human colon, breast and lung cancers. Homologous recombination in ES cells is being used to produce Ptprj knock-out mice for further study of the role of this gene in tumor suppression. Figure VII.1

62 64 MOLECULAR GENETICS Director of Research Anton Berns Key Publications Havelkova H, Badalova J, Demant P, Lipoldova M. A new type of genetic regulation of allogeneic response. A novel locus on mouse chromosome 4, Alan2 controls MLC reactivity to three different alloantigens: C57BL/10, BALB/c and CBA. Genes Immun 2000; 1: Pajukanta P, Bodnar JS, Sallinen R, Chu M, Airaksinen T, Xiao Q, Castellani LW, Sheth SS, Wessman M, Palotie A, Sinsheimer JS, Demant P, Lusis AJ, Peltonen L. Fine mapping of Hyplip1 and the human homolog, a potential locus for FCHL. Mamm Genome 2001; 12: Tripodis N, Demant P. Genetic linkage of nuclear morphology of mouse lung tumors to the Kras-2 locus. Exp Lung Res 2001; 27: Tripodis N, Demant P. Three-dimensional patterns of lung tumor growth: association with tumor heterogeneity. Exp Lung Res 2001; 27: Tripodis N, Hart AA, Fijneman RJ, Demant P. Complexity of lung cancer modifiers: mapping of thirty genes and twenty-five interactions in half of the mouse genome. J Natl Cancer Inst 2001; 93: Van Kooij M, De Groot K, Van Vugt H, Snoek M. Genotype versus phenotype: conflicting results in mapping a lung tumor susceptibility locus to the G7c recombination interval in the mouse MHC class III region. Immunogenetics (in press). Van Wezel T, Lipoldova M, Demant P. Identification of disease susceptibility genes (modifiers) in mouse models: cancer and infectious diseases. In: Malcolm S, Goodship J, editors. Genotype to Phenotype. Oxford: BIOS Scientific Publishers Ltd, 2001: Wandersee NJ, Roesch AN, Hamblen NR, De Moes J, Van der Valk MA, Bronson RT, Gimm JA, Mohandas N, Demant P, Barker JE. Defective spectrin integrity and neonatal thrombosis in the first mouse model for severe hereditary elliptocytosis. Blood 2001; 97: Lung cancer susceptibility Lung cancer, the leading cause of cancer deaths worldwide also exhibits familial clustering both in smokers and non-smokers, indicating the involvement of presently unknown genes. 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. However, little is known about the total number of genes predisposing to lung cancer in humans or in animals. Therefore we have used the mapping efficiency of the RCS to screen systematically for Sluc (Susceptibility to lung cancer) loci in the mouse. The strain O20 develops, after ENU (N-ethyl-N-nitrosourea) treatment, significantly more and larger tumors than the strain B10.O20. OcB/Dem RCS have each only a random 12.5% subset of genes derived from the resistant strain B10.O20, and the rest from the strain O20. The strains OcB-3, OcB-6, OcB-9 and OcB-16 are more resistant than O20; OcB-4 is intermediate. Linkage analysis of susceptibility to lung cancer was performed F 2 crosses between each of these OcB strains and O20. Collectively, these tests screened approximately half of the mouse genome. Using statistical SAS-GLM models to analyze the genetics of tumor size, we extended the number of mapped Sluc loci to 30. Most loci were detected in pair-wise interactions, in which the effect of each locus depends on the genotype of the interacting locus (Figure VII.1). The microscopic appearance of cancer has been the major predictor of its clinical behavior. More recently, with the advent of the DNA array technology, complex molecular profiles, based on expression patterns of hundreds or thousands of genes, are being built that correlate with previously known tumor types/classes and suggest the existence of new types. In order to be able to study the genes affecting lung tumor progression and correlate them to gene expression patterns, 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. Analysis of lung tumors derived from various inbred and RC strains has shown that the new 3-D shape variable, called Rratio, is correlated to tumor heterogeneity and tumor asymmetry. Eight new lung tumor shape determining (Ltsd) loci loci have been detected that influence the overall three dimensional (3-D) shape of a tumor. These studies lay the basis for a more detailed study of the mechanisms of genetic control of tumor progression. Mouse genome profiling Genetic background may play 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 function and modify the pathways in which they are involved. In designing mouse models for cancer controlling the genetic background is of importance. 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 results. Using marker assisted selection protocols speed-congenic mouse strains can be generated in order to obtain the transgene or mutant on a desired genetic background. For genotyping we will be using SNPs (single nucleotide polymorphisms). The first-generation SNP map of the mouse is freely available on 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/Ola 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. Micro-arrays are generic and primers complementary to the unique sequence tags spotted 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 in making 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 genetic control of the colony.

63 65 13 MOLECULAR GENETICS MOUSE MODELS FOR CANCER The mouse is used as a model organism for establishing the role of oncogenes and tumor suppressor genes in tumor development. Using Cre/Lox approaches 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 are established in this fashion. These models are also suited for testing 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. Functional analysis of tumor suppressor genes We have generated mouse strains carrying conditional tumor suppressor gene alleles. These include Rb, Nf2, Atr, Pten, Brca1, Brca2, p53, Dcc, E-cadherin and Ink4a/p19Arf. A number of these conditional alleles have been combined by crossbreeding. As part of this project (inducible) Cre transgenic lines and switchable oncogenes have also been produced. In refining the methodology, we found that Cre recombinase introduces double strand DNA breaks in chromosomal DNA. Therefore, we have begun to modify the way in which we control Cre expression. These compound conditional knockout mice are being used to study the involvement of various tumor suppressor genes in the development of mammary tumors, lung tumors, mesotheliomas and melanomas. We are focusing on combinations of lesions that are found in the corresponding tumors in man. Mammary tumors Inheritance of one defective BRCA1 or BRCA2 allele predisposes humans to breast cancer. To establish a mouse model for Brca1 and Brca2-associated breast cancer, we generated conditional mouse mutants with Brca1 or Brca2 in various epithelial tissues, including mammary gland epithelium. Mammary tumors developed with high incidence in females carrying conditional Brca and p53 alleles. The presence of one wild-type Brca1 or Brca2 allele resulted in a markedly delayed mammary tumor formation. Moreover, loss of the wild-type Brca1 or Brca2 allele was found in a subset of the tumors. Our results demonstrate that BRCA and p53 loss-offunction collaborate in mammary tumorigenesis and imply a pivotal role of p53 in BRCA-associated breast cancer. In follow-up studies we will search specifically for genes and pathways that contribute to tumor progression. Group leader A Berns A BernsPhD Group leader P Krimpenfort PhD Academic staff J Jongsma PhD Post-doc J Jonkers PhD Post-doc S Lyons PhD Post-doc R Meuwissen PhD Post-doc M Nawijn PhD Post-doc K Quon PhD Post-doc S Linn MD, PhD Clinical research fellow A Loonstra MSc Graduate student C Martins MSc Graduate student H Mikkers MSc Graduate student R Van Amerongen MSc Graduate student E Van Montfort MSc Graduate student M Vooijs MSc Graduate student Y Bronkhorst Undergraduate student A Groen Undergraduate student K Van Vliet Undergraduate student F Matthesius Technical staff L Rijswijk Technical staff F Van de Ahé Technical staff H Van der Gulden Technical staff K Van t Wout Technical staff K Van Veen Technical staff N Wilson Technical staff J Zevenhoven Technical staff Lung tumors Mutations in K-Ras play a pronounced role in human small cell lung cancer, whereas loss of Rb and p53 are more predominant in small cell lung cancer. We have developed a mouse lung tumor model in which K-Ras is sporadically activated through Cre-lox mediated somatic recombination. Adenoviral mediated delivery of Cre recombinase in lung epithelial cells gives rise to rapid onset of tumorigenesis, yielding pulmonary adenocarcinomas with 100 % incidence after a short latency. The lung tumor lesions share many features with human non-small cell lung cancer. When Rb and p53 were inactivated by AdenoCre injection, primarily small cell lung cancer was found. Both the loss of Rb and p53 were required for the development of SCLC. The tumors metastasize to the same tissues as observed in man. This nicely illustrates the cell type specific oncogenicity of these different cancer associated genes. This model will be utilized both to identify genes involved in tumor progression and to test intervention strategies using existing and new drugs. Mesotheliomas Very little is known about the genetic lesions contributing to the development of mesotheliomas. In a subset of tumors in man, inactivation of 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 Rb, Ink4a/Arf, or p53 by Adeno-Cre infection of mesothelial cells of the thoracic and intraperitoneal cavity. Mesotheliomas ensued upon loss of Nf2. Concomitant loss of Ink4a/Arf or p53 strongly accelerated tumorigenesis. We are in the process of analyzing the tumors by array CGH and expression arrays to gain access to progression events. In addition, we are exploring whether retroviral insertional mutagenesis can be utilized to accelerate tumorigenesis and identify genes involved in mesothelioma progression. Figure VII.2: Increase in abnormal karyotypes in embryo fibroblasts after temporal expression of Cre recombinase.

64 66 MOLECULAR GENETICS Director of Research Anton Berns Key Publications Berns A. Cancer. Improved mouse models. Nature 2001; 410: Jonkers J, Meuwissen R, Van der Gulden H, Peterse H, Van der Valk M, Berns A. Induction of mammary tumors by somatic mutation of Brca2 and p53. Nature Genetics (in press). Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A. Loss of p16ink4a confers susceptibility to metastatic melanoma in mice. Nature 2001; 413:83-6. Loonstra A, Vooijs M, Berna Beverloo H, Al Allak B, Van Drunen E, Kanaar R, Berns A, Jonkers J. Growth inhibition and DNA Damage Induced by Bacteriophage P1 Cre- Recombinase in mammalian cells. Proc Natl Acad Sci USA 2001; 98: Meuwissen R, Jonkers J, Berns A. Mouse models for sporadic cancer. Exp Cell Res 2001; 264: Meuwissen R, Linn S, Van der Valk M, Mooi W, Berns A. Mouse model for lung tumorigenesis through Cre/Lox controlled sporadic activation of the K-Ras oncogene. Oncogene 2001; 20: Quon K, Berns A. Haplo-insufficiency? Let me count the ways. Genes & Dev (in press). Vooijs M, Jonkers J, Berns A. A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent. EMBO Rep 2001; 2: Figure VII.3: Brca2 loss causes accelerated mammary tumorigenesis in conjunction with p53 deficiency. Melanomas CDKN2A (INK4a/ARF) is frequently disrupted in various types of human cancer, and germline mutations of this locus can confer susceptibility to melanoma and other tumors. However, because CDKN2A encodes two distinct cell cycle inhibitory proteins, p16 INK4a and p14 ARF (p19 ARF in mice) the mechanism of CDKN2A tumor suppression has remained controversial. Genetic disruption of Arf predisposes mice to tumorigenesis, demonstrating that Arf is a tumor suppressor gene in mice. Here we describe mice specifically mutated in Cdkn2a (p16 Iink4a ). These mice do not show a significant predisposition to spontaneous tumor formation within 17 months, and embryo fibroblasts derived from them proliferate normally, are mortal and are not transformed by oncogenic HRAS. However, mice that are Ink4a deficient and heterozygous for Arf spontaneously develop a wide spectrum of tumors, including melanoma. Carcinogen treatment of these mice results in an increased incidence of melanoma, with frequent metastases. Our results demonstrate that in the mouse, Ink4a is a tumor suppressor gene whose loss can recapitulate the tumor predisposition seen in man. In vivo imaging of tumors In order to follow tumor development in vivo, we are exploring, in collaboration with Xenogen Corp, the use of tissue-specific and Cre/Lox-switchable Luciferase reporter constructs that permit the detection of small numbers of tumor cells in mice by in vivo imaging. This allows us to follow their growth and regression in time. In this way chemoprevention and therapy protocols can be efficiently tested in the fore-mentioned spontaneous tumor models. Identification and characterization of new oncogenic pathways Proviral insertional mutagenesis in transgenic and compound mutant mice 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 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 were earlier found as one of the most potent oncogenes collaborating with Myc in lymphomagenesis. We have generated compound mice carrying the Eµ-myc transgene on a Pim1/Pim2 double knock-out background. In these mice lymphomas have been induced by proviral infection. In a high throughput screen we identified a number of genes that can compensate for PIM deficiency. We are currently characterizing these genes. We expect that this will give insight into the oncogenic pathway in which PIM oncoproteins act. Frat1-3 is the second gene family being studied. Frat1 has been cloned as a tumor progression gene using retroviral transposon tagging. The FRAT1 protein binds to GSK3 and Dishevelled and is intimately connected with the beta-catenin pathway. The three members of this protein show substantial overlap in expression. Inactivation of the Frat1 gene does not lead to any obvious developmental aberrations. We are now generating and analyzing compound mutant mice with inactivated Frat1, Frat2, and Frat3 alleles for phenotypic alterations. Currently, compound Frat1/Frat2 knockout mice are being analyzed in detail. Utilizing proviral insertional mutagenesis we have also searched for oncogenes that collaborate with p21 and/or p27 deficiency. An intriguing and unexpected synergy was found between p27 loss and Myc overexpression. This is surprising since MYC has been reported to counteract p27 directly and therefore loss of p27 would be expected to reduce the requirement for MYC activation. Interestingly, both overexpression of MYC and loss of p27 appear to contribute to higher CDK2 activity, suggesting that some of the action of these mutations converge on the same pathway.

65 67 13 MOLECULAR GENETICS ROLE OF MOUSE POLYCOMB-GROUP GENES IN TRANSCRIP- TIONAL REPRESSION AND TUMORIGENESIS We are studying the mechanism of stably inherited transcriptional repression of individual target genes by Polycomb-group (Pc-G) protein complexes in the mouse, and the effects of deregulation of Pc-G genes on Homeobox (Hox) gene expression, development, cell cycle control and tumorigenesis. In addition, we are performing 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 expression patterns 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 biochemically distinct evolutionary highly conserved Pc-G protein complexes can be distinguished. The first contains Enx1/Enx2, Eed and histone deacetylases and is required during early development when Pc-G repression is initiated. The second large complex encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2 and Ring1b/ Ring1a 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 Bmi1 and Mel18). To study Pc-G function we are focusing on representative members of these groups: Bmi1, Mel18, M33, Ring1b and Eed, respectively in gain- and loss-offunction studies in mice. In collaboration with H Koseki, Chiba University, Japan, we have recently shown that Mel18 and Bmi1 are required in synergy and in a dosedependent and cell type specific manner to keep Hox cluster genes repressed and mediate cell survival during embryonic development. In addition, we demonstrated that Mel18 and Bmi1 are required for maintenance but not initial establishment of Hox gene expression. Further insights have come from our recent analysis of Ring1bdeficient mice. Unlike other single Pc-G gene knockout mice (which display various defects but are viable, see below), null mutant mice die shortly after gastrulation. This may relate to the central positioning of the Ring1b protein in the Pc-G complex, and in addition suggests that the closely related Ring1a gene cannot compensate for loss of Ring1b. This severe phenotype resembles that of the Eed null mutant mice (the only Pc-G homolog known so far to be represented by a single gene), and suggests that loss of Ring1b results in complete loss of Pc-G maintenance function. This phenotype has been studied in detail by in situ hybridization with early developmental markers (collaboration with J Deschamps, Hubrecht Lab, Utrecht). In addition, we have generated Ring1b-conditional knockout ES cell lines and mice, which are valuable tools for studying Pc-G function in relation to differentiation and development in a timed and cell type specific manner. Regulation of PC-G complexes in association with chromatin We discovered a marked cell cycle regulation of Pc-G-chromatin interaction: nuclear Pc-G staining dissipates in late S-phase and is rapidly re-established in early G1 phase. Chromatin association of Bmi1 and Mph1 inversely correlates with their phosphorylation status: at G1/S hypophosphorylated Bmi1 and Mph1 is present in chromatin-associated nuclear protein fractions, whereas during G2/M, hyperphosphorylated Bmi1 and Mph1 complexes dissociate from chromatin, although a small but significant fraction of Bmi1 remains associated with specific sites on mitotic chromosomes. These results provide new insights into the dynamic regulation of Pc-G complex-chromatin association, and suggest that extra-cellular cues may influence this process through kinase/phosphatase pathways (collaboration with D Schweizer, University of Vienna). To study the molecular mechanism of Pc-G repression we are characterizing local changes in chromatin structure as a function of changes in the expression levels of Pc-G proteins, using Chromatin Immunoprecipitation (CHIP) and DNAse1 accessibility assays. Main focus of these studies is on the currently best-characterized in vivo relevant cell cycle target gene: Cdkn2A (p16ink4a/p19arf), see below. Group leader M Van Lohuizen M Van Lohuizen PhD Group leader M Hernandez PhD Post-doc J Jacobs PhD Post-doc P Keblusek PhD Post-doc A Lund PhD Post-doc M Prudenziati PhD Post-doc E Boutsma MSc Graduate student M Lingbeek MSc Graduate student P Taghavi MSc Graduate student P Van der Stoop MSc Graduate student K Kieboom Technical staff J-P Lambooij Technical staff E Tanger Technical staff E Verhoeven Technical staff E Wientjens Technical staff Figure VII.4: In vitro senescence-bypass high-copy suppressor screen. Bmi1-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 Bmi1-/- MEFs with human tumorderived retroviral cdna libraries at passage 2, followed 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.

66 68 MOLECULAR GENETICS Director of Research Anton Berns Key Publications Akasaka T, Van Lohuizen M, Van der Lugt N, Mizutani-Koseki Y, Kanno M, Taniguchi M, Vidal M, Alkema M, Berns A, Koseki H. Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression. Development 2001; 128: Bea S, Tort F, Pinyol M, Puig X, Hernandez L, Hernandez S, Fernandez PL, Van Lohuizen M, Colomer D, Campo E. BMI-1 gene amplification and overexpression in hematological malignancies occur mainly in mantle cell lymphomas. Cancer Res 2001; 61: Brock HW, Van Lohuizen M. The Polycomb group no longer an exclusive club? Curr Opin Genet Dev 2001; 11: Vonlanthen S, Heighway J, Altermatt HJ, Gugger M, Kappeler A, Borner MM, Van Lohuizen M, Betticher DC. The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression. Br J Cancer 2001; 84: Figure VII.5: Northern blots illustrating that Bmi1-deficient MEFs show depression of p19arf and p16ink4a (top). Bmi1-containing Pc-G protein complexes act as repressors of the INK4a/ARF fail-safe mechanism, which is activated by oncogenes such as Myc and RAS in primary cells (Bottom). Connections between PC-G gene repression, cell cycle regulation and tumorigenesis We originally identified Bmi1 as an oncogene that cooperates with the cmyc oncogene in the induction of B and T-cell leukemia in mice, underscoring the connection between deregulation of Pc-G repression and cancer. In line with this, Bmi1- overexpressing transgenic mice have a high incidence of B and T-cell lymphomas. In contrast, Bmi1 knockout mice show severe proliferation defects and increased apoptosis of lymphoid and myeloid cells, resulting in severe lymphopenia. In addition, primary embryo fibroblasts (MEFs) and neurons in the cerebellum of Bmi1 knockouts also show such defects. We recently found that these defects are due to increased levels of the INK4a/ARF-encoded tumor suppressors p16ink4a and p19arf, that in turn are critical regulators of the RB and the p53 tumor suppressor pathways. Conversely, overexpression of Bmi1 facilitates immortalization and neoplastic transformation by hyper-repressing p16ink4a and p19arf. This connects Pc-G repression to cell cycle control and control of the senescence fail-safe governed by these tumor suppressors (Figure VII.4). This connection explains the strong cooperation between Bmi1 and cmyc: Bmi1 represses the p19arf fail-safe which is induced by cmyc. The relevance of these findings for human cancer is underscored by the amplification of Bmi1 in Mantle cell lymphomas and the overexpression of BMI1 in various tumor types including non-small cell lung cancer. In vivo and in vitro genetic screens to identify new groups of collaborating oncogenes As loss of the INK4a/ARF fail-safe is common to all cancers, we have initiated a genetic screen in INK4a/ARF-deficient mice, using MuLV-insertion mutagenesis on a genome-wide scale. Sequences flanking the proviral insertions are obtained in a high-throughput manner using efficient PCR methods, and are mapped using the mouse genome databases. Aim is ultimately to get a full complement of groups of oncogenes that cooperate in vivo with INK4a/ARF loss. We also started two types of in vitro high-copy suppressor screens. The first is to bypass premature senescence of Bmi1 knockout MEFs using tumor-derived retroviral cdna library transduction (Figure VII.5). Since the INK4a/ARF locus is intact in these MEFs, this screen has the potential to uncover both new upstream regulators and downstream effectors of the INK4a/ARF fail-safe pathways. This is underscored by our recent identification of the T-box member TBX2 as a potent immortalizing gene that acts by repressing p19arf transcription. 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 prior to retroviral library transduction. Transformed clones 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 cells is the topic of intensive study.

67 VIII DIVISION OF EXPERIMENTAL THERAPY EXPERIMENTAL THERAPY PREDICTION AND MODIFICATION OF TREATMENT OUTCOME Role and prediction of tumor hypoxia Tumor hypoxia measured by oxygen electrodes is a negative prognostic factor for patients undergoing radiotherapy, chemotherapy or surgery. We previously reported studies on 14 head and neck patients treated with primary surgery in which pimonidazole (a bioreductive hypoxic marker drug) and IUdR (iododeoxyuridine; S-phase marker) were administered the night before surgery. We have accrued a further 15 patients and have set up a colloboration with Nijmegen for participation in this study (J Kaanders, A van der Kogel). We have assessed chronic hypoxia using image analysis of vasculature (Diffusion Limited Fraction, DLF), the fraction of tumor area positive for pimonidazole staining, and the fraction of CD31/34 blood vessels with surrounding IUdR-negative but Ki67- positive tumor cells (a measure of perfusion deficit). Marked inter-patient variation was seen for all parameters. We have also looked at HIF-1α (Hypoxia Inducible Factor) staining as an endogenous marker for hypoxia. Co-localization of HIF-1α with pimonidazole was done on matched, reconstructed whole-tumor-section images (mosaics from consecutive smaller images) stained for the two markers and for blood vessels. Using a distance transform approach, area and intensity profiles with distance from blood vessels were generated, showing a peak at a distance for pimonidazole, but a flatter, more homogeneous distribution for HIF-1α (see figure VIII.1a and 1b) In ten head and neck tumors studied to date, these different profiles and a lack of correlation for mean values for fractional stained area suggest that HIF-1α is not a good marker for chronic hypoxia. We will also test whether it is an independent prognostic factor for outcome after surgery with or without radiotherapy. A disadvantage of the immunohistochemical approach is the relatively small fraction of tumor area assessed. We have therefore also looked at an external scanning approach using a novel technetium-labeled 2-nitroimadazole (similar to pimonidazole) employing SPECT scanning (collaboration with R Valdes Olmos and C Hoefnagel, Nuclear Medicine, NKI; and Bracco Diagnostics, USA). Of ten head and neck primary tumors, uptake was seen in eight. These patients also received pimonidazole within two weeks. Significant correlations were found between scan data 0.5 and 3h after administration, with the fraction of tumor area stained by pimonidazole in tissue sections. Division head, Group Leader AC Begg AC Begg PhD Group leader KMG Haustermans MD PhD Principal investigator G Blommestijn PhD Academic staff F Hoebers MD Radiation Oncologist C Vens PhD Post-doc H Janssen MSc Graduate student B Ramakers Undergraduate Student E Dahmen Technical staff I Hofland Technicical staff D Sprong Technical staff M Verwijs Technical staff TEA Eggenhuizen Secretary Mechanisms and modulation of radiosensitivity We have previously shown that expression of the DNA binding domain of DNA polymerase β, an enzyme involved in base excision repair (BER), radiosensitized two human cell lines, presumably by dominant negative action resulting in successful competition for binding sites with the wildtype protein. We have now shown that this radiosensitization occurs primarily in cells in S and G2, with little radiosensitization in G1. In contrast, confluent cells primarily in G0 do show radiosensitization. We hypothesize that much of the damage incurred in G1 can be primarily repaired during the next S-phase where the Area at distance (pixels) Tumor ( 10) HIF1α Uncorrected Pimo Percent area at distance HIF1α Pimo Area corrected Distance from blood vessels (pixels) Figure VIII.1: Expression profiles of HIF-1α and pimonidazole as function of distance from blood vessel in head and neck tumor; raw data (left), corrected for tumor area at each distance (right).

68 70 EXPERIMENTAL THERAPY Director of Research Anton Berns Key Publications Begg AC, Vens C. Genetic manipulation of radiosensitivity. Int J Radiat Oncol Biol Phys 2001; 49: Begg AC, West CML. Individualization of therapy. In: Steel GG, editor. Basic Clinical Radiobiology, 3rd Edition, Pub. London, Arnold (in press) Begg AC, Janssen H, Sprong D, Hofland I, Blommestijn G, Raleigh JA, Varia M, Balm AJ, Van Velthyzen L, Delaere P, Sciot R, Haustermans KMG. Hypoxia and perfusion measurements in human tumors: initial experience with pimonidazole and IUdR. Acta Oncologica (in press) McMillan TJ, Begg AC. Genetic control of the cellular response to ionising radiation. In: Steel GG, editor. Basic Clinical Radiobiology, 3rd Edition, Pub. London, Arnold (in press) replication apparatus or translesional synthesis pathways remove the lesions. In G0, cells do not reach S in time. This is presently being tested. We have also included MEFs from DNA polymerase knock-out mice in this study. We found greater radiosensitization in these cells than in wildtype MEFs. This is consistent with the known two BER pathways, short and long patch, in which polymerase β operates primarily on the short patch pathway. Radiation-induced base damage is repaired primarily via the long patch pathway. The dominant negative protein has been shown to bind base lesions and we hypothesize that this will inhibit both pathways. Greater sensitization in knock-out cells would result from the lack of a back-up repair pathway (short patch). This is under investigation using plasmid repair assays using cell extracts from wildtype and knock out clones. Our studies on putative dominant negative peptides against ku80, a critical component in DNA double strand break repair by non-homologous end-joining, showed modest radiosensitization of both human and murine cell lines by expressing the ku80 DNA-PKCS activation domain. In similar studies using the ku70 binding domain as a putative dominant negative, only a small degree of radiosensitization was observed in two human tumor cell lines. The ku70 binding fragment was shown to be expressed, and to localize to the nucleus. However, levels were considerably less than the wildtype protein, despite attempts to select the most highly expressing cells, probably explaining the lack of effect. These data illustrate the difficulty of using ku80 as a target for any gene therapy approach. As a spin-off from these studies, we developed a rapid assay to study radiosensitization after introducing modifying genes. We have employed a retroviral vector coexpressing green fluorescence protein (EGFP) with the gene of interest. After infection, the ratio of infected to uninfected cells can be measured rapidly by flow cytometry analysis of green fluorescence. For cells with equal radiosensitivities, this ratio will remain constant with time in culture after radiation. If the genetically manipulated cells are more radiosensitive, the ratio will decrease with time. This was validated by comparing ratio assay results with those from colony assays for the DNA polymerase β and ku80 dominant negatives. The assay is in principle applicable to any type of treatment and can be adapted to any fluorochrome(s).

69 71 13 EXPERIMENTAL THERAPY VASCULAR MEDIATED TISSUE DAMAGE AND THERAPY Radiation induced renal injury The aim of this project is to identify molecular mechanisms involved in development of radiation nephropathy, with a view to designing strategies for pharmacological intervention for patients treated with abdominal radiotherapy. In mice, pronounced changes in micro-vascular structure (telangiectasia) and function (leakage and reduced glomerular perfusion) were seen from 20 weeks after irradiation of the kidneys. To investigate whether this was due to changes in VEGF levels we analyzed mrna levels in total kidney extracts or from isolated glomeruli. There was no significant upregulation of VEGF, and no change in the ratio of the three isoforms, from one day to 30 weeks after irradiation. Protein levels have not yet been examined and it is still possible that post-transcriptional regulation may occur after irradiation in the absence of increased VEGF mrna. The expression of thrombotic and inflammatory markers of endothelial cell (EC) damage was examined in relation to the subsequent development of nephropathy in mice. Significantly decreased levels of glomerular ADPase and increased glomerular Vwf protein were seen from four and 20 weeks respectively after irradiation. These pro-thrombotic changes were associated with increased fibrin/fibrinogen deposits in the glomeruli, indicative of micro-thrombus formation. These events were, however, not sensitive to changes in total radiation dose or dose per fraction, whereas the subsequent deterioration in renal function (from about 25 weeks after irradiation) was strongly related to both total dose and dose per fraction. A progressive increase in leukocyte invasion of the renal cortex was seen from ten weeks after irradiation; this inflammatory marker was quantitatively dependent on both total dose and dose per fraction. Linear-quadratic analysis of the leukocyte dose response curves at 40 weeks after irradiation yielded an α/β ratio of 7.7 Gy, which is significantly greater than the α/β ratio of 2.7 Gy determined for nephropathy, indicating less fractionation sensitivity for the inflammatory response. We conclude that inflammatory changes contribute to, but do not entirely explain, radiation nephropathy. The role of thrombotic changes is less clear. We have investigated the potential of anti-platelet therapy (continuous administration of the COX inhibitor ASA or the ADP receptor blocker Clopidogrel) and oxygen radical scavengers (short term administration of catalase and SOD or continuous administration of NAC) for ameliorating late radiation nephropathy in mice. Modest reductions in kidney damage were obtained for daily oral administration of ASA but none of the other drugs tested provided effective protection against radiation nephropathy at tolerable dose levels. Group leader FA Stewart FA Stewart PhD Group leader P Baas MD PhD Academic staff L Dewit MD PhD Academic staff N Van der Vange MD PhD Academic staff P Cramers MSc Graduate student J Krüse MSc Graduate student M Triesschijn MSc Graduate student S Zeamari MSc Graduate student G Rumpin Undergraduate student I Wijdenes Undergraduate student BGJ Floot Technicial staff H Oppelaar Technicial staff MC Ruevekamp Technicial staff JAM Te Poele Technicial staff A Van der Wal Technicial staff Photodynamic Therapy (PDT) Clinical protocols for PDT are based on the assumption that optimum intervals between photosensitizer administration and illumination are times at which there is a 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. To this end we have investigated the relationship between uptake, distribution and clearance of the sensitizer meta-tetrahydroxyphenylchlorin (mthpc) and efficacy of PDT in: 1) human endothelial or fibroblast cells and tumor cell lines in vitro; 2) human tumor xenografts in nude mice and normal mouse skin; 3) patients with multiple basal cell carcinomas (BCC). In vitro results showed that cellular uptake of mthpc increased linearly with increasing drug concentration in human tumor cell lines (HNXOE, HMES01) and human endothelial cells (HUVECs). The rate of drug uptake was faster in HUVECs and maximum cellular drug levels were achieved within 4h, compared with only 20-40% of maximum in the tumor lines. In vivo studies demonstrated a marked dissociation between times of maximum drug uptake in tissue and illumination times for maximum PDT effect. Maximum drug uptake in tumor (HMES01) and skin was not until 72h after administration of mthpc. The PDT response was, however, maximal for illumination at 3-6h and minimal for illumination at 48-72h (Figure VIII.2).

70 72 EXPERIMENTAL THERAPY Director of Research Anton Berns Key publications Baas P, Saarnak AE, Oppelaar H, Neering H, Stewart FA. Photodynamic therapy with meta-tetrahydroxyphenylchlorin for basal cell carcinoma: a phase I/II study. Br J Dermatol 2001; 145:75-8. Kuin A, Citarella F, Oussoren YG, Van der Wal AF, Dewit LG, Stewart FA. Increased glomerular vwf after kidney irradiation is not due to increased biosynthesis or endothelial cell proliferation. Radiat Res 2001; 156:20-7. Schouwink H, Oppelaar H, Ruevekamp M, Van der Valk M, Hart G, Rijken P, Baas P, Stewart FA. Oxygen depletion during and after mthpc mediated photodynamic therapy in RIF1 and H-MESO 1 tumors. Radiat Res (in press). Schouwink H, Ruevekamp M, Oppelaar H, Van Veen R, Baas P, Stewart FA. Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols. Photochem Photobiol 2001; 73: Schouwink H, Rutgers ET, Van der Sijp JR, Oppelaar H, Van Zandwijk N, Van Veen R, Burgers S, Stewart FA, Zoetmulder F, Baas P. Intraoperative photodynamic therapy after pleuropneumonectomy in patients with malignant pleural mesothelioma: dose finding and toxicity results. Chest 2001; 120: 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 functional damage and vascular mediated inflammatory and thrombotic changes. Acta Oncologica (in press) Te Poele JA, Van Kleef EM, Van der Wal AF, Dewit LG, Stewart FA. Radiationinduced glomerular thrombus formation and nephropathy are not prevented by the ADP receptor antagonist clopidogrel. Int J Radiat Oncol Biol Phys 2001; 50: Van Veen RLP, Schouwink JH, Star WM, Sterenborg HJ, Van der Sijp JR, Stewart FA, Baas P. Wedge-shaped applicator for additional light delivery and dosimetry in the diaphragmal sinus during photodynamic therapy for malignant pleural mesothelioma. Phys Med Biol 2001; 46: The PDT response correlated much better with plasma drug levels (excluding the first 20 minutes distribution time) at the time of illumination than with tissue drug levels. This is consistent with our hypothesis that vascular mediated tissue damage predominates over direct cellular phototoxicity. Plasma pharmacokinetic data were also analyzed from six courses of mthpc given to four patients with multiple BCC. High plasma drug levels were maintained during the first 12h, with exponential clearance thereafter. A total of 132 BCC tumors in these four patients were treated with illumination at intervals of 12-96h and tumor response is now being correlated with plasma pharmacokinetic profiles and with tumor drug levels at the time of illumination. Inhibition of tumor cell seeding in surgical wound sites During surgery for abdominal cancer the normally non-adhesive and non-thrombotic peritoneum is disturbed and tumor cell seeding in surgical scar tissue may occur. The purpose of this study is to determine the influence of growth factors and adhesion molecules released at wound sites on the growth of residual peritoneal tumor and on tumor re-seeding. At a later stage, this information will be used to design and test intervention strategies. Initial studies demonstrated preferential seeding and growth of HT29 or CC531 cells (human or rat colon carcinoma cells) in nude mice or Wag Rij rats which had received a small peritoneal wound. A model for peritoneal granulation tissue was then set up to investigate the invasion of tumor cells into a wound in relation to the expression of specific growth factors. Small pieces of sterile gelatin sponge were implanted in the mesenteric fan of nude mice and the sponges were harvested for analysis at 1-28d after implantation. Inflammatory cell invasion (granulocytes, macrophages, mast cells and lymphocytes) was evident at 1-3d. This was followed by infiltration of fibroblasts and the formation of extracellular matrix at 7-14d. By 28 days blood vessels were clearly visible in the mature granulation tissue. In separate experiments, mice were inoculated with HT29 cells eight hours to ten days after sponge implantation and the excised sponges were examined for tumor invasion at 28 days. Inoculation of cells during the first three days induced tumor seeding and rapid growth in 100% of the cases, whereas inoculation after >10d resulted in only limited growth in 17% of the sponges. These results suggest that factors are released at early stages in the development of granulation tissue which stimulate tumor cell infiltration and growth. Preliminary analyses of mrna showed increased VEGF expression at 14 days after initiation of granulation tissue, with return to control levels at later times. Edg2, one of the receptors for lysophosphatidic acid (which is produced by activated fibroblasts and stimulates cell migration, invasion and proliferation) was also highly expressed at 28 days. These studies will be expanded to identify stimulatory factors for tumor cell invasion of wound tissue, with a view to blocking their expression in the post-surgical period. Tumor regrowth time (days) co ntr ol 5' 20' 1 h 3 h 6 h 24 h Drug-light interval Figure VIII.2: PDT response (tumor growth delay) was much greater for short drug-light intervals of 1-6h than at times when tumor drug levels were highest (48-72h). HMES01 xenografts treated with 0.3 mg/kg mthpc and 20 J/cm2. 48 h 72 h

71 73 13 EXPERIMENTAL THERAPY GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG RESISTANCE AND PHARMACOKINETICS Our research focuses on genes and proteins that can cause (multi-)drug resistance in tumors, and/or influence the pharmacological behavior of anticancer and anti- HIV/AIDS drugs. Insight into these systems may improve current and novel chemotherapy strategies used to treat cancer and HIV/AIDS, as well as pharmacotherapy in a broader sense. To study the physiological and pharmacological roles of the genes involved, we generate and analyze knock-out mice lacking the encoded proteins. Furthermore, cell lines obtained from such knock-out mice are used as tools to search for new drug resistance genes. Use of mice lacking Mdr1-type P-glycoproteins Mdr1-type P-glycoproteins (P-gps) are plasma membrane proteins of the ATP binding cassette (ABC) family of proteins that actively export a wide range of anticancer, anti-hiv/aids and many other drugs from the cell. The ATP-dependent drug extrusion can cause multidrug resistance (MDR) in tumor cells. P-gps localize to the apical membrane of polarized epithelial cells, resulting in vectorial transport of drug substrates. Previous pharmacological experiments in Mdr1a/1b knock-out mice have taught us that the drug-transporting P-glycoproteins (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 orally administered substrates from the intestinal lumen and by mediating active excretion of substrates via liver and intestine. We have shown previously with Mdr1a/1b (-/-) mice that P-gp is an important factor in limiting brain and fetal (i.e., pharmacological sanctuary site) penetration of the HIV protease inhibitor (HPI) saquinavir, and that it also limits saquinavir oral bioavailability to some extent. The low penetration of HPIs in pharmacological sanctuary sites may allow continued replication of HIV in these tissues, thus lowering the chance of complete viral eradication. The low oral bioavailability of HPIs results in a very high pill burden for patients, making strict therapy adherence much more difficult. Using the Mdr1a/1b (-/-) mouse model we tested the feasibility and safety of in vivo P-glycoprotein (P-gp) inhibition to enhance the oral uptake and penetration into pharmacological sanctuary sites of saquinavir, when orally co-administered with the HPI ritonavir. Ritonavir is a strong inhibitor of Cytochrome P450 3A4 (CYP3A4) and therefore frequently co-administered with saquinavir to increase the bioavailability of saquinavir, which is otherwise rapidly metabolized by CYP3A4. Although ritonavir has moderate P-gp-inhibiting activity in vitro, we found that even high-dose ritonavir does not abrogate P-gp function, which therefore still limited the oral uptake of saquinavir and its penetration into brain and fetus. Next, we orally co-administered ritonavir and saquinavir to mice, with or without a potent P-gp inhibitor. Genetic and chemical P-gp deficient animals became more sensitive to ritonavir and suffered from impaired gastric emptying, even when exposed to a decreased ritonavir dose. Using a potent P-gp inhibitor and a low ritonavir dose, we showed that P-gp function can be inhibited efficiently as demonstrated by increased saquinavir concentrations in brain and fetus. Including the P-gp inhibitor in a multiple dosing scheme, we demonstrated that steady-state HPI concentrations are not yet reached within several days, as evidenced by continuous saquinavir accumulation in brain and testis, and transient ritonavir-related toxicity after the final drug dosing. Clinical attempts to inhibit P-gp function for improved HPI disposition may therefore be useful, but they should be performed without ritonavir and monitored carefully for unexpected side effects. Group leader AH Schinkel AH Schinkel PhD Group leader JD Allen PhD Post-doc E Rocchi PhD Post-doc JW Smit PhD Post-doc MT Huisman MSc Graduate student JW Jonker MSc Graduate student A Otten Undergraduate student M Buitelaar Technical staff SC Jackson Technical staff CMM Van der Kruijssen Technical staff E Wagenaar Technical staff Analysis of the BCRP/Bcrp1 multidrug transporter: BCRP inhibitors Inhibitors of BCRP are of interest as chemosensitizers for clinical drug resistance, for improving the pharmacokinetics of substrate chemotherapeutic drugs and in functional assays of BCRP activity for tailoring chemotherapy. The fungal toxin fumitremorgin C (FTC) is a potent and specific inhibitor of BCRP but its neurotoxic effects preclude use in vivo. In collaboration with A Van Loevezijn and GJ Koomen (University of Amsterdam), we screened a panel of 42 analogs of fumitremorgin C, a known inhibitor of BCRP, as potential inhibitors of Bcrp1 and BCRP. From this panel,

72 74 EXPERIMENTAL THERAPY Director of Research Anton Berns Key publications Huisman MT, Smit JW, Wiltshire HR, Hoetelmans RM, Beijnen JH, Schinkel AH. P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir. Mol Pharmacol 2001; 59: Jonker JW, Wagenaar E, Mol CA, Buitelaar M, Koepsell H, Smit JW, Schinkel AH. Reduced hepatic uptake and intestinal excretion of organic cations in mice with a targeted disruption of the organic cation transporter 1 (Oct1 [Slc22a1]) gene. Mol Cell Biol 2001; 21: Schellens JH, Maliepaard M, Scheper RJ, Scheffer GL, Jonker JW, Smit JW, Beijnen JH, Schinkel AH. Transport of topoisomerase I inhibitors by the breast cancer resistance protein. Potential clinical implications. Ann N Y Acad Sci 2000; 922: Schellens JH, Malingré MM, Kruijtzer CM, Bardelmeijer HA, Van Tellingen O, Schinkel AH, Beijnen JH. Modulation of oral bioavailability of anticancer drugs: from mouse to man. Eur J Pharmaceut Sci 2000; 12: Schinkel AH. The roles of P-glycoprotein and MRP1 in the blood-brain and bloodcerebrospinal fluid barriers. In: Snyder et al, editors. Biological Reactive Intermediates VI: Chemical and biological mechanisms in susceptibility to and prevention of environmental disease. Kluwer Academic/Plenum Publishers (in press). Van Loevezijn A, Allen JD, Schinkel AH, Koomen GJ. Inhibition of BCRP-mediated drug efflux by fumitremorgin-type indolyl diketopiperazines. Bioorg Med Chem Lett 2001; 11: promising candidates were selected for further analysis, and one compound, Ko134, was further modified by adding a methoxy group also present in native FTC, yielding the new compound Ko143. We have now extensively evaluated three tetracyclic analogs of FTC as practical inhibitors of BCRP, including Ko143. All three compounds are effective inhibitors of both mouse Bcrp1 and human BCRP, proving highly active for increasing the intracellular drug accumulation and reversing Bcrp1/BCRP-mediated multidrug resistance. Indeed, Ko143 appears to be the most potent BCRP inhibitor known. In contrast, the compounds have only low activity against P-glycoprotein or the Multidrug Resistance-associated Protein (MRP1) or other tested drug transporters. They are non-toxic in vitro at useful concentrations and preliminary tests evinced no signs of toxicity in mice at high oral or intraperitoneal doses. Administered orally to inhibit intestinal Bcrp1, Ko143 markedly increases the oral availability of topotecan in mice. FTC analogs of this type are thus effective, potent and specific inhibitors of Bcrp1/BCRP, and potential candidates for development as clinical BCRP inhibitors. Mice lacking the Oct1 organic cation transporter Organic cations, including many clinically applied drugs, are usually taken up in cells via carrier proteins. The polyspecific Organic Cation Transporter 1 (OCT1/SLC22A1) mediates facilitated transport of small (hydrophilic) organic cations, including clinically applied drugs. OCT1 is localized at the basolateral membrane of epithelial cells in liver, kidney and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. Moreover, several OCT1 substrates are shared with the apically localized P-glycoprotein, so there might be a complementary functional role between these transporters. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout mice. Oct1 (-/-) mice were viable, healthy and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1 (-/-) mice. In Oct1 (-/-) mice, accumulation of TEA in liver was four to six fold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about two fold. Excretion of TEA into urine over one hour was 53% of the dose in wild-type compared to 80% in knockout mice, probably because in Oct1 (-/-) mice less TEA distributes to liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to a decreased liver accumulation of the anticancer drug metaiodobenzylguanidine (MIBG) and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine. The possible complementary roles of P-gp and Oct1 are currently being studied in combined P-gp/Oct1 knock-out mice. We are working further on the generation and analysis of Oct2 and Oct1/2 knock-out mice, as there are several indications there may be redundancy between Oct1 and Oct2 function. Zwart R, Sleutels F, Wutz A, Schinkel AH, Barlow DP. Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes. Genes Dev 2001; 15: Figure VIII.4: Left: Subcellular localization of P-glycoprotein and Oct1 in the hepatocyte. Right: Uptake of the organic cation TEA into the liver is substantially reduced in Oct1 knockout mice.

73 75 13 EXPERIMENTAL THERAPY CLINICAL AND PRECLINICAL PHARMACODYNAMICS OF ANTICANCER DRUGS Regulation of breast cancer resistance protein (bcrp/mxr/abcg2) and mrp2 In order to establish whether variability in the clinical pharmacokinetics of camptothecin analogs, such as topotecan, can be explained by interindividual variation in activity of BCRP or MRP2 in excretory organs, we tested the inducibility of bcrp1 and mrp2 in kidneys, small intestine and the liver. Groups of FVB mice each were given a pre-treatment of days of valproic acid, phenobarbital, phenytoin, carbamazepine or dexamethasone at pharmacological daily (oral) doses. Preliminary results revealed that phenobarbital, valproic acid, phenytoin and carbamazepine increased the tissue expression of bcrp1 in mouse kidney 1.5 to 2-fold, the latter three being statistically significant (p<0.05). Phenytoin and carbamazepine also induced bcrp1 levels significantly in the liver. No significant upregulation was observed of bcrp1 by dexamethasone in any of the tissues tested, and even a slight reduction of hepatic bcrp1 levels was observed (mechanism under investigation). Mrp2 levels appeared to be much less sensitive for induction, although carbamazepine increased mrp2 levels significantly in the liver. Cyp3a11 levels increased as expected. These experiments will be expanded with measurement of tissue staining with a monoclonal antibody against bcrp1 (collaboration with AH Schinkel s group). The effect of inducers of BCRP on the clinical pharmacokinetics of BCRP substrate drugs in cancer patients is also planned. Immunohistochemical studies using the monoclonal antibody BXP34 against human BCRP were performed on xenografts of human colon cancer exposed to CPT11 (model established by G Vassal, Institut Gustave Roussy, Villejuif). CPT11 and its active metabolite SN38 are camptothecin derived BCRP substrates. Results revealed intensive plasma membrane staining with BXP34, indicating that BCRP expression probably mediates resistance to CPT11 in this model. For confirmation, tissue mrna expression of BCRP is currently being measured. This model may be very useful for exploring effects of BCRP reversing agents on tumor cell uptake and antitumor activity. In a proof of principle study in patients with advanced cancer, we showed that co-administration of oral topotecan and the BCRP reversing agent GF resulted in a 2.5-fold increase in the oral bioavailability of topotecan. The oral bioavailability increased from 40.0% (range 32-47%) to 97.1% (range %), indicating complete bioavailability of oral topotecan in combination with GF After a single administration the combination was well tolerated by patients. This is the basis for a series of clinical studies to establish: 1) the minimal effective dose of GF needed to achieve a maximal effect on the oral bioavailability of topotecan; 2) the maximum tolerable dose and safety of topotecan in combination with the reversal agent and 3) the antitumor activity of this combination. Group leader JHM Schellens JHM Schellens MD PhD Group leader JH Beijnen MD PhD Academic staff J Brouwer PhD Academic staff K Mohrmann PhD Post-doc RCAM Van Waardenburg PhD Post-doc N Appels MSc Graduate Student P Breedveld MSc Graduate student S Bocxe Undergraduate student M Brons Undergraduate student LA De Jong Technical staff D Pluim Technical staff M Van Eijndhoven Technical staff Mechanism of synergistic cytotoxicity of scheduled incubation of cisplatin and camptothecin We previously reported a schedule dependent synergistic cytotoxicity of cisplatin followed by topoisomerase I (topi) inhibitors in various human cancer cell lines. The most active schedule was cisplatin followed by the topi inhibitor. We established that topi is attracted to the platinum DNA adduct, binds to it and is trapped by the adduct onto the DNA, hindering the religation step. This results in an accumulation of topi cleavable complexes. The involvement of DNA repair pathways in the mechanism of synergy was further investigated since both agents induce DNA damage. We used Saccharomyces cerevisiae, Schizosaccharomyces pombe and Chinese hamster cells deficient Nucleotide or Base Excision Repair, Mismatch Repair, Homologous Recombination Repair or Non-Homologous End Joining. All cell systems showed loss of synergy in homologous recombination mutants, whereas synergy remained in mutants of other DNA repair pathways. Analysis of rad52, rad51 (yeast) and xrcc2, xrcc3 (Chinese hamster cells) revealed that gene conversion was the major pathway involved in synergy, while mutations in rad52 or ercc1 established that single strand annealing and break-induced replication played a minor role. This shows for the first time that homologous recombination is essential for the outcome of combination treatment with the clinically important platinum agents and topi inhibitors.

74 76 EXPERIMENTAL THERAPY Director of Research Anton Berns Key publications Crul M, Van den Bongard HJ, Tibben MM, Van Tellingen O, Sava G, Schellens JH, Beijnen JH. Validated method for the determination of the novel organo-ruthenium anticancer drug NAMI-A in human biological fluids by Zeeman atomic absorption spectrometry. Fresenius J Anal Chem 2001; 369: Maliepaard M, Scheffer GL, Faneyte IF, Van Gastelen MA, Pijnenborg AC, Schinkel AH, Van De Vijver MJ, Scheper RJ, Schellens JH. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 2001; 15: Maliepaard M, Van Gastelen MA, Togho A, Hausheer FH, Van Waardenburg RC, De Jong LA, Pluim D, Beijnen JH, Schellens JH. Circumvention of Breast Cancer Resistance Protein (BCRP)-mediated Resistance to Camptothecins invitro Using Non-Substrate Drugs or the BCRP Inhibitor GF Clin Cancer Res 2001; 7: Malingré MM, Beijnen JH, Rosing H, Koopman FJ, Van Tellingen O, Duchin K, Ten Bokkel Huinink WW, Swart M, Lieverst J, Schellens JH. A phase I and pharmocokinetic study of bi-dialy dosing of oral paclitaxel combination with cyclosporin A. Cancer Chemother Pharmacol 2001; 47: Malingré MM, Beijnen JH, Rosing H, Koopman FJ, Van Tellingen O, Duchin K, Ten Bokkel Huinink WW, Swart M, Lieverst J, Schellens JH. The effect of different doses of cyclosporin A on the systemicexposure orally administered paclitaxel. Anti Cancer Drugs 2001;12: Malingré MM, Beijnen JH, Schellens JHM. Oral delivery of taxanes. Invest New Drugs 2001; 19: Malingé MM, Richel DJ, Beijnen JH, Rosing H, Koopman FJ, Ten Bokkel Huinink WW, Schot ME, Schellens JH. Coadministration of cyclosporine strongly enhances the oral bioavailability of docetaxel. J Clin Oncol 2001;19: Pharmacodynamic endpoints in clinical studies and anticancer drug biotransformation One of our main fields of interest continues to be modulation of oral bioavailability of anticancer drugs by applying combinations of substrate drugs and inhibitors of the drug transporters P-glycoprotein and BCRP and/or metabolic enzymes, especially cytochrome P450 (CYP) 3A. (collaboration with AH Schinkel s group). Phase II studies revealed high activity and good tolerance of the oral combination of paclitaxel and cyclosporin A in patients with anthracycline pretreated advanced breast cancer (overall response rate 44%), with platinum pre-treated advanced non-small cell lung cancer (overall response rate 23%) and chemo-naive gastric cancer (overall response rate 41%). Oral docetaxel combined with cyclosporin A revealed an overall response rate in anthracycline pretreated breast cancer of 45%. (Clinical studies in collaboration with the Pharmacy Department, head JH Beijnen) The randomized phase I study in patients with advanced non-small cell lung cancer to explore the toxicities and maximum tolerated dose (MTD) of cisplatin plus gemcitabine given weekly or every two weeks is still ongoing. The two-weekly schedule was selected for further clinical and pharmacological studies because of its higher dose intensity. The dose of cisplatin at the maximum tolerated dose in combination with gemcitabine was approximately 50% higher than in standard treatment with this combination. In the 69 patients entered, there was a highly significant interaction, such that gemcitabine caused a significant reduction in the level of platinum DNA-adducts in white blood cells (WBC) of both GG and AG adducts without affecting the unbound clearance of platinum. The mechanism of the interaction and possible clinical implications are subjects of current investigations. A phase I and pharmacological study to explore the maximum tolerated dose, doselimiting toxicities, pharmacokinetics and pharmacodynamics of the combination of carboplatin and topotecan supports preclinical mechanistic studies with this combination. Platinum DNA adducts could be measured in 15/17 patients of whom tumor biopsies were taken. Average DNA adduct levels in tumor cells were three-fold higher than in WBC collected at the same time as the tumor biopsy. The MTD has been reached in the schedule carboplatin followed by topotecan. The trial is proceeding with the reverse schedule. The phase I and pharmacologic study with the ruthenium organic compound NAMI-A showed linear pharmacokinetics over a wide dose-range of 12 to 1500 mg/m2/course. DNA-adducts in WBC, measured by the postlabeling assay, were more than ten-fold lower compared to levels usually obtained in patients treated with comparable doses of cisplatin. In experiments using calf thymus DNA, the reactivity of NAMI-A for DNA was equal to that of cisplatin. Intracellular pharmacokinetics and dynamics of NAMI-A in vivo therefore seem to be substantially different from cisplatin. Drug targeting is being explored in a clinical and pharmacologic study using a platinum polymer (AP5280). The pharmacokinetics of platinum were linear between 90 and 3300 mg/m2. DNA-adduct levels of platinum could be measured in WBC but were more than ten-fold lower than after standard cisplatin or carboplatin at equal doses. The ongoing mass balance study with the novel disulphonamide anticancer agent E7070, using an HPLC assay with online radioisotope detection, revealed that there are at least ten new peaks that probably represent previously unknown metabolites. The major metabolic pathway elucidated thus far is glucuronidation of the parent drug (study in collaboration with the Pharmacy Department.). Schellens JHM, Maliepaard M, Scheper RJ, Scheffer GL, Jonker JW, Smit JW, Beijnen JH, Schinkel AH. Transport of Topoisomerase I Inhibitors by the Breast Cancer Resistance Protein. Potential Clinical Implication. In: JC Liehr, Giovanella BC, Verschraegen CF, editors. The Campotothecins Unfolding Their Anticancer Potential. Annals of the New York Academy of Sciences Volume 2001; 922:

75 77 13 EXPERIMENTAL THERAPY MOLECULAR PATHOLOGY ATM heterozygous germline mutations contribute to breast cancer susceptibility Approximately 0.5-1% of the general population has been estimated to be heterozygous for a germline mutation in the ataxia-telangiectasia mutated gene (ATM) and epidemiological studies have indicated that female heterozygous carriers 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 cancer. The frequency of ATM germline mutations is being examined in three casecontrol studies of women who developed breast cancer subsequent to exposure to various levels of ionizing radiation (1. breast cancer (n=250), 2. Hodgkin s disease (n=200) and 3. mammography (n=300)). The results of our first case-control study indicate that ATM heterozygotes have an increased risk of developing a type of breast cancer characterized by frequent bilateral occurrence, early age at onset and long-term survival (approximate RR 3.1). Moreover, ATM heterozygous breast cancer patients have a 3.5-fold increased risk to develop a contralateral breast cancer. The higher frequency of ATM heterozygotes in the bilateral breast cancer group (4/60 bilateral vs. 4/200 unilateral breast cancer patients, p=0.05), suggests that radiation might indeed be an induction trigger. In a new study we will assess whether ATM carriership predisposes to breast cancer in general, or whether ATM-radiation interactions contribute to this high frequency of contralateral breast cancers. In addition, the role of other breast cancer susceptibility genes in radiation induced breast cancers is being examined. Long-term survivors of Hodgkin s disease who received mantle-field irradiation at a young age have a strong increased risk of developing breast cancer, in large part attributable to the radiation exposure. In the second case-control study we concluded that disease causing ATM germline mutations, however, are not a major component underlying this increased risk (35 cases tested). Comparative genomic hybridization, however, showed that these breast cancers have a specific pattern of genomic alterations. The pattern is being studied in more detail to reveal the underlying etiology of these cancers. Group leader LJ Van t Veer LJ Van t Veer PhD Group leader FE Van Leeuwen PhD Academic staff S Rodenhuis MD PhD Academic staff NS Russell MD PhD Academic staff A Broeks PhD Post-doc D Voskuil PhD Post-doc B Weigelt MSc Graduate student H Halfwerk Undergraduate student AJ Bosma Technical Staff AN Floore Technical Staff S Klaver Technical Staff B Nota Technical Staff JHM Urbanus Technical Staff Molecular methods for minimal disease detection in breast cancer Evaluation of the presence of breast cancer cells in blood and bone marrow has become increasingly important for the staging of breast cancer at diagnosis and in predicting relapse. We undertook a systematic approach to identify additional marker genes for the detection of minimal residual disease (MRD) with molecular assays. Using serial analysis of gene expression (SAGE), we identified a range of genes that were expressed in breast cancer but were absent in the profiles of blood or bone marrow cells. Increased marker gene expression of p1b, ps2, CK19 and EGP2 was seen in 33 out of 103 peripheral blood mononuclear cells (PBMC) of patients with metastatic breast cancer compared to PBMCs of healthy volunteers (real-time quantitative PCR). Quadratic discriminant analysis including the four marker genes provided us with a discriminant value with 30% positivity in the breast cancer patient group and no false positives among the healthy volunteers. Preliminary data revealed that positivity is seen in a subgroup of patients known to have bone marrow metastases at the time of blood sampling and was absent in patients with metastatic disease in other organs. Moreover, the MRD detection in peripheral blood seems indicative of 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 increased in individuals with relatively high serum concentrations of IGF-1. Experimental evidence shows that at the tissue level, IGF-1 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.

76 78 EXPERIMENTAL THERAPY Director of Research Anton Berns In a series of 40 female BRCA1/BRCA2 gene carriers who had undergone a prophylactic mastectomy, we performed preliminary analyses of levels of IGF-system components in serum and tissue. IGF-1 and IGF-binding protein 3 (IGFBP-3) serum concentrations were assessed by radioimmunoassays, while IGF-1, IGF-2, IGF type 1 receptor, and IGF type 2 receptor mrna levels were quantitatively determined by real-time PCR. In the small series of normal breast tissue and serum collected within one year of mastectomy, expression levels of tissue IGF-1 and IGF type 1 receptor mrnas were positively correlated with serum levels of IGF-1. Serum and tissue IGFsystems either have common determinants, or high levels of serum IGF-1 induce a high expression of IGF type 1 receptor in normal breast tissue.

77 79 13 EXPERIMENTAL THERAPY MOLECULAR ANALYSIS OF BREAST CANCER Multidrug resistance-related proteins in breast cancer The mechanism of chemotherapy resistance in breast cancer is unresolved. We have previously shown that it is unlikely that MDR1/P-glycoprotein overexpression plays a role in conferring multidrug resistance in clinical breast cancer. We have now investigated whether the breast cancer resistance protein (BCRP) and the multidrug resistance (MDR) associated proteins MRP1, MRP2 and MRP3 are involved in multidrug resistance in breast cancer. BCRP and MRP1, MRP2 and MRP3 mrna expression were determined with real-time RT-PCR in nine breast cancer cell lines and in samples of 25 primary breast carcinomas and 27 patients who received pre-operative anthracycline-based therapy. In the cell lines, only MCF7 and BT20 had BCRP mrna levels above 0.15 compared with Igrov/T8(p75)-40 cell line, consistent with membrane bound immunostaining. In clinical samples, BCRP expression ranged from 0.01 to 0.86 (mean 0.18). With immunohistochemistry, BCRP was detected in vessels and normal breast epithelium but not in tumor cells. There was no difference in BCRP expression between primary and anthracycline treated tumour samples. BCRP expression was not associated with decreased response or survival. The MRP1-3 encoding mrna s were detected in all breast cancer cell lines and samples. No increase of expression was detected in samples taken after anthracycline treatment. IHC was not suitable for detection of the proteins at these expression levels. MRP expression was not associated with tumor response to treatment. We conclude that there is no indication that elevated BCRP, MRP1, MRP2 or MRP3 expression in breast carcinomas confers resistance to anthracyclines. Group leader MJ Van de Vijver MJ Van de Vijver MD PhD Group leader H Bartelink MD PhD Academic staff JL Peterse MD Academic staff EC Robanus Maandag PhD Post-doc I Faneyte MD Graduate student CAJ Bosch Technical staff P Kristel Technical staff Key publications Faneyte IF, Kristel PM, Van de Vijver MJ. Determining MDR1/P-glycoprotein expression in breast cancer. Int J Cancer 2001; 93: 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, particularly those 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 twelve 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. In some tumors, we observed a few distinct differences between otherwise identical genome profiles of both 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 high-level amplification of C-MYC in the invasive component only in one tumor (figure VIII.3). To validate this correlation, from a panel of 188 invasive carcinomas we identified 18 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. The minimal amplified region was defined at 8q24.1-8qter using BAC clones. C-MYC amplification was correlated with overexpression of C-MYC and two target genes, TERT and 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 of breast carcinomas.

78 80 RADIOTHERAPY Director of Research Anton Berns IX DIVISION OF RADIOTHERAPY IMAGE ACQUISITION AND PROCESSING R De Boer, BJ Mijnheer, C Rasch, P Remeijer, H Bartelink, JV Lebesque, M Van Herk Division head, H Bartelink H Bartelink MD, PhD Head BMP Aleman MD Academic staff JSA Belderbos MD Academic staff N Bijker MD PhD Academic staff LJ Boersma MD PhD Academic staff JH Borger MD PhD Academic staff EMF Damen PhD Academic staff RW De Boer PhD Academic staff P De Haan MD Academic staff K De Jaeger MD Academic staff LGH Dewit MD PhD Academic staff RLM Haas MD Academic staff AAM Hart MSc Academic staff FJP Hoebers MD Academic staff B Kreike MD Academic staff JV Lebesque MD PhD Academic staff J Maas MD Academic staff EAH Masselink MD Academic staff BJ Mijnheer PhD Academic staff AWH Minken PhD Academic staff LMF Moonen MD PhD Academic staff D Nuyten MD Academic staff CRN Rasch MD Academic staff NS Russell MD PhD Academic staff JG Salverda MD Academic staff C Schneider PhD Academic staff CJA Tissing MD Academic staff JF Ubbels MD Academic staff BNFM Van Bunningen MD Academic staff M Van Herk PhD Academic staff M Verheij MD PhD Academic staff C Vrieling MD Academic staff FW Wittkämper Academic staff N Dekker MSc Graduate student M Engelsman MSc Graduate student W Heemsbergen MSc Graduate student CW Hurkmans MSc Graduate student LAJ Ploeger MSc Graduate student Y Seppenwoolde MSc Graduate student R Steenakkers Graduate student L Bos PhD Post-doc K Deurloo PhD Post-doc M Hoogeman PhD Post-doc R Louwe PhD Post-doc A Saarnak PhD Post-doc JJ Sonke Post-doc P Remeijer PhD Post-doc A Betgen Technical staff B Brand Technical staff JA De Bois Technical staff J De Goede Technical staff J Duppen Technical staff Feasibility of using patient contours for setup verification The aim of this study is to establish the efficacy of body contours to verify patient setup in 3D. Body contours are acquired on a simulator at the same moment that simulator images were taken as a reference. To determine the patient setup, both modalities are matched in 3D to the planning CT scan: the contours on the skin, the images on the bone. Data of a breast phantom, 24 lung patients and 9 head & neck patients were collected. The phantom study showed an agreement of better than one mm. Movement of the head in the applied mask (the body contour method registers the mask), movement of the skin and the small magnitude of the setup errors cause a poor correlation between contour data and simulator images. When the patient setup is corrected using contour data, the magnitude of the setup deviations decreases only slightly compared with marker-based setup. In conclusion, body contours can improve the patient setup for these two patient groups slightly, but cannot replace portal imaging. Portal and reference image matching using gray value cost functions Anatomy matches between a portal and a reference image for patient setup verification often have to be performed manually. The purpose of this study is to test gray value based matching method on a large number of images of most common treatment sites. A set of 96 images was selected from rectum, salivary gland, brain, prostate and lung treatments. The following gray value cost functions were tested: mutual information (MI), the mean and RMS pixel wise difference and image correlation. Cost function values calculated the range of expected setup errors in clinical practice: translations up to +/- 1 cm and rotations up to +/- 10 degrees. Our clinical database was used as gold standard. The differences between the tested cost functions are small. MI performs poorest with an overall success rate of about 80%. For the other cost functions it is about 90%. In addition, the number of local minima with MI is much larger, complicating automatic matching. The SD of the difference with manual registration was about 1 mm and 1 degree, which is close to the accuracy of the clinical matches. In conclusion, gray value matching of portal images works well for many treatment sites and is a useful alternative for segmentation-based methods. Implementation of a novel flat panel electronic portal-imaging device (EPID) We have implemented our first flat panel imager based on an amorphous silicon area image detector coupled to a fluorescent plate in clinical practice. The detector is mounted on a retractable support and has been fully integrated with our in-house developed software for clinical use (Figure IX.1). We developed software for auto- Figure IX.1: (left) Novel amorphous silicon detector as implemented in our clinic. (right) Example of one of the first clinical images obtain with the new detector. It is a processed lateral head and neck image that exquisitely displays the internal anatomy.

79 81 13 RADIOTHERAPY matic image acquisition such that an optimal image quality is obtained for any exposure. At a dose of 5 MU or less, the accelerator has no time to reach a constant output, i.e., all frames show an uneven exposure (with variations in intensity of up to 50% over the image area) due to image scanning. A circular buffer is used to store the images. The trigger algorithm starts the image recording when the maximum intensity in the downsized image rises above a threshold. The buffer is a few frames larger than needed so that some frames from before the trigger event and after beamoff are recorded. The trigger algorithm works correctly for fields sizes down to 2x2 cm2 at exposures as low as 1 MU. When all frames are combined, the effect of the uneven exposure is reduced to less than 2% for 5 MU. Next, we investigated the impact of focal spot motion of the linear accelerator on portal image acquisition. A slightly tilted narrow slit phantom was placed at the isocenter of the accelerator and three image frames per second were acquired with the flat panel imager. The motion of the focal spot was determined by using these images on all our linear accelerators. The older linacs show focal spot motion between 0.4 mm and 1 mm during beam start-up, which stabilizes after 2-3 seconds. The motion of the focal spot has the following effect on the portal images: for a dose of 1 MU, anatomy shifts by 0.2 mm-0.4 mm and field edges shift by 1 mm-2.5 mm in the isocenter plane. In conclusion, deviations due to focal spot motion are relatively small compared to other uncertainties in radiotherapy and are mainly found during the start-up phase of the irradiation. This effect, however, limits the accuracy of portal images taken at low exposure. Hence, a more accurate adjustment of the linacs would be beneficial for portal image registration. High precision image-guided radiotherapy of the prostate The basis of the image-guided radiotherapy project forms a cone beam CT scanner on the treatment machine that consists of a kilovolt (diagnostic) radiation source and a flat panel detector, which is being developed by D Jaffray and co-workers at the William Beaumont Hospital in Royal Oak, USA. Preliminary tests of such a system show that volumetric CT data with diagnostic image quality can be obtained with a single gantry rotation. We are co-operating in this NCI project in the fields of image analysis and statistics. An important step in on-line image guided radiotherapy of the prostate is fast localization of the prostate. We investigated in how far the prostate can be considered a rigid organ, i.e., we quantified shape variation of prostate and seminal vesicles. Repeat CT scans were made during the course of conformal radiotherapy for eleven patients with prostate cancer. Three scans were obtained in the first week of treatment, followed by eight weekly scans. One observer contoured the GTV in all scans and volume variations were measured. The GTVs of the repeat CT scans were matched onto the planning CT by rotation and translation, and visualized in movie loops. For each patient the average GTV was computed. The perpendicular distance between the average GTV and the individual GTVs was measured for each point of the average GTV. Their variation was expressed in terms of local SD, displayed in P Filius Technical staff M Frenay Technical staff C Goedbloed Technical staff TJ Minderhoud Technical staff P Muller Technical staff E Roosjen Technical staff MHP Smitsmans Technical staff B Smulders Technical staff R Tielenburg Technical staff A Van Assen Technical staff PJH Van de Ven MSc Technical staff L Zijp Technical Staff X Artignan MD Guest W Bär MSc Guest J Cho MD Guest L McDermott Ba/Bs (Hons) Guest C McGibney MD Guest A Olszewska MSc Guest M Schwarz MSc Guest C Van Vliet-Vroegindeweij PhD Guest D Brandwijk Secretary P Fewer Secretary E Hofmann Secretary Figure IX.2: Results of shape variation analysis of the prostate and seminal vesicles. Prostates defined in CT scans of 12 patients were registered, and the residual shape variation was quantified for each position of the prostate (left), with an average SD of 1.7 mm. However, intra-observer variation plays an important role, with an average SD of 1.2 mm. After correcting for this uncertainty, the average SD of the prostate shape variation is small, about 1 mm SD (right).

80 82 RADIOTHERAPY Director of Research Anton Berns Key publications Bartelink H. Commentary on the paper A preliminary report of intraoperative radiotherapy (IORT) in limited-stage breast cancers that are conservatively treated. A critical review of an innovative approach. Eur J Cancer 2001; 37: Bartelink H. From translational research to improved local control and survival: the Gilbert Fletcher award lecture, Lugano, March Int J Radiat Oncol Biol Phys 2001; 49: Bartelink H, Horiot JC, Poortmans P, Struikmans H, Van den Bogaert W, Barillot I, Fourquet A, Borger J, Jager J, Hoogenraad W, Collette L, Pierart M, for the European Organization for Research and Treatment of Cancer Radiotherapy (EORTC) and Breast Cancer Groups. Recurrence rates after treatment of breast cancer with standard radiotherapy plus supplementary radiation. N Eng J Med 2001; 345: Bartelink H, Schellens JHM, Verheij M. The combined use of radiotherapy and chemotherapy in the treatment of solid tumors. Eur J Cancer (in press). 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 head and neck cancer. Eur J Cancer (in press). color wash, and averaged over the whole GTV. It turns out that the motion of the GTV is well described by a translation and rotation. The remaining variations were due to volume and delineation differences, in combination with variations in shape of the real prostate. No significant variations in GTV volume were observed. The overall SD of shape variations (averaged over the whole GTV and all patients) is 1.7 mm. Most of this variation can be attributed to delineation variability, of which the magnitude is 1.2 mm SD. The color wash and movie loop displays revealed that the largest variations occurred at interface between bladder and prostate (1.7 mm SD) and at the tip of the seminal vesicles (1.9 mm SD). In conclusion, shape variations of prostate and seminal vesicles during external beam radiotherapy are small compared to delineation variation. Delineation variability The aim of this project is to evaluate the current level of reproducibility of target volume delineation in different institutions, and to improve the quality of target volume delineation. We are currently implementing the infrastructure to collect sample cases of lung, head & neck and prostate cancer from the associated institutions. A simple standard PC-based 3D delineation tool has been developed and is now ready for distribution to the associated institutions. This system will be used to delineate Gross Tumor Volume (GTV), Clinical Target Volume (CTV) and Planning Target Volume (PTV) according to protocols. For each tumor site, three to four institutions (specialized in the selected tumor site) will participate in submitting the full treatment planning data (including 3D dose distributions) of five cases per site. Software was developed to analyze shape variations in three dimensions, which was tested to investigate shape variability of the prostate and seminal vesicles (see above). Inclusion of geometrical uncertainties in treatment plan evaluation The purpose of this study is to correctly evaluate realistic treatment plans in terms of absorbed dose to the Clinical Target Volume (CTV) and tumor control probability (TCP) in the presence of execution (random) and preparation (systematic) geometrical errors. The dose matrix is blurred with all execution errors to estimate the total dose distribution of all fractions. To include preparation errors, the CTV is randomly displaced (and optionally rotated) many times with respect to its planned position, while computing the dose and TCP for the CTV using the blurred dose matrix. Probability distributions of these parameters are computed by combining the results with the probability of each particular preparation error. Probability levels for the Bijker N, Peterse JL, Duchateau L, Julien JP, Fentiman IS, Duval C, Di Palma S, Simony-Lafontaine J, De Mascarel I, Van de Vijver MJ. Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma-in-situ: analysis of European Organization for Research and Treatment of Cancer Trial J Clin Oncol 2001; 19: Bijker N, Peterse JL, Duchateau L, Robanus-Maandag EC, Bosch CA, Duval C, Pilotti S, Van de Vijver MJ. Histological type and marker expression of the primary tumor compared with its local recurrence after breast-conserving therapy for ductal carcinoma in situ. Br J Cancer 2001; 84: Figure IX.3: (Left) Relation between execution errors (S), preparation errors (s), margin and TCP loss due to geometrical misses. The contours represent the situation where there is 1% tumor control probability (TCP) loss due to geometrical misses. For instance, if a margin is used of 10 mm, the situation with 4 mm s and 4 mm S is adequately covered. (Right) Population-averaged tumor control probability (<TCP>pop) as function of the dose level for a prostate case (including seminal vesicles) treated with three multi-leaf collimator shaped fields with varying margins treated to a dose of 80 Gy. The curves are almost the same for margins of 10, 12 and 14 mm. The <TCP>pop levels start to decrease when smaller margins are used, indicating the presence of geometrical misses. Increasing the dose can partly counteract the loss in <TCP>pop. For instance, to maintain the <TCP>pop level for a margin of 10 mm at 80 Gy (see the horizontal line) at a margin of 6 mm a dose increase of 7 Gy is required.

81 83 13 RADIOTHERAPY minimum dose, computed with the new method, are within 1% of an analytical solution. The impact of rotations depends strongly on the CTV shape. A margin of 10 mm between CTV and PTV is adequate for three-field prostate treatments given the accuracy level of our department, i.e., the TCP in a population of patients, <TCP>pop, is reduced less than 1% due to geometrical errors. When reducing the margin to 6 mm, the dose must be increased from 80 to 87 Gy to maintain the same <TCP>pop (Figure IX.3). Only in regions with a high dose gradient will such a margin reduction lead to a decrease in normal tissue dose for the same <TCP>pop. Based on a rough correspondence of minimum dose with EUD, a margin recipe was defined. For less than 1% TCP loss, the PTV margin must approximately be 2.5 Σ σ 3 mm, where σ and Σ are the combined standard deviation of the preparation and execution errors. In conclusion, the new method computes the influence of geometrical errors on the statistics of target dose and <TCP>pop in clinical treatment plans in a few minutes. Margins which are too small lead to significant loss of <TCP>pop that is difficult to compensate by dose escalation. Key publications (continued) Bijker N, Rutgers EJ, Duchateau L, Peterse JL, Julien JP, Cataliotti L. Breastconserving therapy for Paget disease of the nipple: a prospective European Organization for Research and Treatment of Cancer study of 61 patients. Cancer 2001; 91: Bos LJ, Damen EMF, De Boer RW, Mijnheer BJ, Lebesque JL, McShan DL, Fraas BA, Kessler ML. Reduction of rectal dose by integration of the boost in the largefield treatment plan for prostate irradiation. Int J Radiat Oncol Biol Phy 2002; 52: Cho BJC, Hurkmans CW, Damen EMF, Zijp LJ, Mijnheer BJ. Intensity modulated versus non-intensity modulated radiotherapy in the treatment of the left breast and upper internal mammary lymph node chain: a comparative planning study. Radiother Oncol (in press). Damen EM, Brugmans MJ, Van der Horst A, Bos L, Lebesque JV, Mijnheer BJ, McShan DL, Fraas BA, Kessler ML. Planning, computer optimization, and dosimetric verification of a segmented irradiation technique for prostate cancer. Int J Radiat Oncol Biol Phys 2001; 49: Figure IX.4: Off-axis factor as a function of off-axis distance, perpendicular to the wedge direction for an 18-MV photon beam. The lines represent the average of the values for different field sizes. DOSIMETRY AWH Minken, M Van Herk, FW Wittkämper, BJ Mijnheer Dose calculation Algorithms for calculating monitor units (MUs) in wedged asymmetric high-energy photon beams as implemented in treatment planning systems have their limitations. Therefore an independent method for MU calculation has been developed to determine MUs for points at the center of wedged fields, asymmetrical in two directions. The method is based on the determination of an off-axis factor (OAF) that corrects for the difference in dose between wedged asymmetric and wedged symmetric beams with the same field size. Measurements were performed in a water phantom irradiated with 6 and 18 MV photon beams produced by Elekta accelerators, which are fitted with an internal motorized wedge that has a complex shape. The OAF perpendicular to the wedge direction changed significantly in depth for the 18 MV beam (Figure IX.4). Dose values measured for a set of 18 test cases were compared with those calculated with our new method. The maximum difference found was 6.5 % and in 15 cases this figure was less than 2.0 %. Published methods were also tested and showed errors up to 12.8 %. It can be concluded that our simple formalism makes it possible to calculate MUs in wedged asymmetric fields with an acceptable accuracy in most clinical situations. Transmission dosimetry After the development of transmission dosimetry using an electronic portal imaging device (EPID) as a research tool, the main goal of our current activities is the introduction of EPID dosimetry in the clinic. To test the accuracy of transmission dosimetry during the treatment of breast cancer, first two anthropo- Danciu C, Proimos BS, Rosenwald JC, Mijnheer B. Variation of sensitometric curves of radiographic films in high-energy photon beams. Med Phys 2001; 28: Engelsman M, Damen EM, De Jaeger K, Van Ingen KM, Mijnheer BJ. The effect of breathing and set-up errors on the cumulative dose to a lung tumor. Radiother Oncol 2001; 60: Engelsman M, Damen EM, Koken PW, t Veld AA, Van Ingen KM, Mijnheer BJ. Impact of simple tissue inhomogeneity correction algorithms on conformal radiotherapy of lung tumors. Radiother Oncol 2001; 60: Engelsman M, Remeijer P, Van Herk M, Lebesque J V, Mijnheer B J, Damen EMF. Field size reduction enables ISO-NTCP escalation of tumor control probability for irradiation of lung tumors. Int J Radiat Oncol Biol Phys 2001; 51:

82 84 RADIOTHERAPY Director of Research Anton Berns Key publications (continued) Hurkmans CW, Cho BJC, Damen EMF, Mijnheer BJ. Reduction of cardiac and long complication probabilities after breast irradiation using conformal radiotherapy with or without intensity modulation. Radiother Oncol (in press). Hurkmans CW, Borger JH, Pieters BR, Russell NS, Jansen EP, Mijnheer BJ. Variability in target volume delineation on CT scans of the breast. Int J Radiat Oncol Biol Phys 2001; 50: Hurkmans CW, Borger JH, Van Giersbergen A, Cho J, Mijnheer BJ. Implementation of a forearm support to reduce the amount of irradiated lung and heart in radiation therapy of the breast. Radiother Oncol 2001; 61: Hurkmans CW, Remeijer P, Lebesque JV, Mijnheer BJ. Set-up verification using portal imaging; review of current clinical practice. Radiother Oncol 2001; 58: Olszewska AM, Saarnak AE, De Boer RW, Van Bunningen BN, Steggerda MJ. Comparison of dose-volume histograms of the rectum of patients treated with intracavitary brachytherapy. Radiother Oncol 2001; 16: Ong F, Moonen LM, Gallee MP, Ten Bosch C, Zerp SF, Hart AA, Bartelink H, Verheij M. Prognostic factors in transitional cell cancer of the bladder: an emerging role for Bcl-2 and p53. Radiother Oncol 2001; 61: Ploeger LS, Smitsmans MH, Gilhuijs KG, Van Herk M. Accurate measurement of the dynamic response of a scanning electronic portal imaging device. Med Phys 2001; 28: Ruiter GA, Verheij M, Zerp SF, Van Blitterswijk WJ. Alkyl-lysophospholipids as anticancer agents and enhancers of radiation-induced apoptosis. Int J Radiat Oncol Biol Phys 2001; 49: Seppenwoolde Y, Lebesque JV. Partial irradiation of the lung. Semin Radiat Oncol 2001; 11: Van Blitterswijk WJ, Van der Luit AH, Caan W, Verheij M, Borst J. Sphongolipids related to apoptosis from the point of view of membrance structure and topology. Biochem Soc Transactions 2001; 29: morphic phantoms were irradiated. EPID, film and ionization chamber dosimetry were applied during the irradiation, and midplane dose values obtained with these techniques were compared with results of 3D dose calculations. The differences in the central part of the midplane were generally small; less than 1.5% (after normalization of the film measurements) but may increase up to 10% in the lung region. From these results we concluded that the EPID is a reliable tool for the verification of the dose delivered during the treatment of breast cancer. Next, we evaluated the occurrence and magnitude of the breast dose inhomogeneities and heart dose for left-sided breast cancer patients. For 14 patients the midplane dose distribution was reconstructed using EPID measurements. Small systematic patient positioning errors were observed, probably due to the single-sided arm support. After correction for these positioning errors, the average dose delivered to the isocenter was 1.2% lower than the prescribed dose. Dose variations in the midplane, relative to the prescribed dose, of up to ± 10% observed for large breasts. The NTCP values for late excess cardiac mortality, based on observed treatment parameters, are systematically lower than those based on pre-treatment parameters. We also started with in vivo dosimetry of prostate treatments using the EPID and compared the results with corresponding data obtained using diodes. For a group of 30 patients both the EPID and diodes yielded a systematic difference of 0.5% compared to the prescribed dose. The standard deviation of the diode measurements was about 1% but the EPID data showed a standard deviation of about 1.7 %. A further investigation of the various factors influencing the EPID response, including its temperature dependence, has been started. Film dosimetry Verification of irradiation techniques is often performed by means of film dosimetry. Existing data concerning the influence of irradiation geometry on the sensitometric curve of radiographic films are, however, conflicting. We therefore investigated the variation of optical density, OD, with field size and depth in a phantom, as well as the effect of beam energy and film plane orientation on OD, for two types of film, Kodak X-Omat V and Agfa Structurix D2. Films were positioned in a solid phantom, either perpendicular or (almost) parallel to the beam axis, and irradiated to different dose levels using various photon beams. It was found that the sensitometric curves of the Kodak films derived at different depths are almost identical for the various photon beams, within 2%, except for the Co-60 beam, where the difference is about 4% at 10cm depth. The slope of the sensitometric curve of the Agfa film is somewhat more dependent on photon beam energy, depth and field size. The sensitometric curves of both types of film are almost independent of the film plane orientation, except for shallow depths. Good dosimetric results can be obtained if films from the same batch are irradiated with small to moderate field sizes, at moderate depths, using a single calibration curve, e.g., for a 10 cm x 10 cm field. Limited information is available on the magnitude of the variations in sensitometric curves applied in clinical practice in different institutions. In a recent study we assessed the effect of the various parameters influencing the shape of the sensitometric curve: batch composition, irradiation conditions, film processing and film scanning, in a systematic way. Two types of film, Kodak X-Omat V and CEA TVS, were irradiated, processed, and analyzed in three different institutions. The observed interinstitutional variation of the sensitometric curves was rather large; variations in absolute OD at 50 cgy may be as high as 32%. This difference is caused mainly by a variation in film batch composition and differences in processing conditions. By using relative OD values rather than absolute OD values, this difference can be reduced to 2%. Consequently, one sensitometric curve is sufficient to derive relative dose values. If processing conditions are well controlled, it may therefore be advantageous to determine the absolute OD only at one or two dose values, in combination with a universal relative sensitometric curve. Boron Neutron Capture Therapy (BNCT) The aim of this work was to provide quantitative information about the effect of bone, lung tissue and air cavities on the dose distributions encountered in BNCT beams. For this purpose various cubical phantoms were irradiated using the Petten epithermal neutron BNCT beam. The

83 85 13 RADIOTHERAPY first phantom is a water-filled cube inside of which a piece of bone could be positioned or an air gap could be created. The second phantom was built to simulate the thorax region while inside a third water-filled phantom a hollow PMMA tube could be positioned to study the effect of an air cavity on the dose distribution. Activation foils and a diode detector were used for the determination of the thermal neutron fluence rate. The gamma-ray dose rate and the fast neutron dose rate were determined using paired ionization chambers. The effect of the piece of bone in a small air gap on the fluence and dose components is small. For instance, the thermal neutron fluence immediately behind the bone is reduced by about 5% while at larger distances behind, as well as in front of the bony structure, the difference is less than 3%. At depths larger than approximately 10 cm in the lung-equivalent material, the thermal neutron fluence as well as the gamma-ray dose is higher than in the homogeneous phantom. TREATMENT PLANNING JSA Belderbos, JH Borger, L Bos, JV Lebesque, NS Russell, BJ Mijnheer, EMF Damen General ICRU Report 62 suggests drawing margins around Organs at Risk (ORs) to produce Planning Organ at Risk Volumes (PRVs) to account for geometric uncertainty in the radiotherapy treatment process. The benefit of the PRV defined in this way is that, despite the geometric uncertainties, the dose calculated within the PRV by the treatment planning system can be used to represent the dose in the OR to a given level of accuracy. A suitable level is where, in the majority of cases (90%), the dose-volume histogram (DVH) of the PRV does not under-represent the high-dose components in the OR. In order to provide guidelines on how to do this in clinical practice, an algorithm has been suggested for producing such margins where the OR PRV margin width is given by 1.3 Σ (+ 0.5 σ in the case of large and parallel organs). Here, Σ is the standard deviation of the combined systematic uncertainties and σ is the standard deviation of the random set-up uncertainties during treatment execution. Examples show that the application of this algorithm leads to margin widths around ORs similar to those used clinically. Lung cancer Using a phantom study, we showed that the probability of tumor control of lung tumors, expressed as the equivalent uniform dose (EUD) of the clinical target volume (CTV), can be increased by dose escalation in combination with fieldsize reduction. In this simulation, movement of the CTV due to patient breathing and set-up errors was included and the maximum attainable EUD was calculated as a function of field size under the constraint of a constant mean lung dose (MLD), which is an estimator of the complication probability for lung. The applicability of this approach for patient treatment was tested in a non-small cell lung cancer (NSCLC) patient using the beam directions of the clinically applied treatment plan. For this patient, the EUD could be increased from an initial 62.2 Gy for the conventional plan, to 83.2 Gy at maximum. In this maximum, the prescribed dose is 88.1 Gy, and the minimum dose in the CTV is 67.4 Gy. In this case, the 95 % isodose surface is conformed closely to the static CTV during treatment planning. The phantom simulation was extended to investigate the influence of the number and directions of beams and the effect of beam-fringe (50%-90%) sharpening using intensity-modulated radiotherapy (IMRT). The results, obtained both for coplanar and non-coplanar treatment plans, show that when dose homogeneity in the CTV is pursued, use of intensity modulation for sharpening the beam fringe allows a large increase in EUD up to a maximum of about 100 Gy. Without the dose homogeneity constraint the ratio between EUD and NTCP of the lungs can be further increased, but the benefit of sharpening the beam fringe decreases. From these studies we concluded that iso-ntcp escalation of the probability of tumor control is possible for lung tumors by reducing field sizes and allowing a larger dose inhomogeneity in the CTV. Optimum field sizes can be derived, having the highest EUD and highest minimum dose in the CTV under condition of a constant NTCP of the lungs. If dose homogeneity in the CTV is pursued, beam-fringe sharpening using IMRT might be beneficial to increase the EUD even further.

84 86 RADIOTHERAPY Director of Research Anton Berns Prostate cancer To investigate the benefit of the simultaneous integrated boost (SIB) compared to the serial boost for prostate treatment, we performed a treatment planning study on five different patients having a varying degree of overlap between planning target volume (PTV) and rectal wall. For all patients the SIB resulted in a lower dose to the rectal wall compared to the serial boost (Figure IX.5). The NTCP for severe proctitis/necrosis decreased on average by a factor of almost two. Thanks to this study, the SIB is now used for all our IMRT treatments of prostate cancer. To investigate the benefit of inverse planning versus forward planning, two methods for creating beam segments for step-and-shoot IMRT were compared. In forward planning, segments are created manually using a recipe based on experience. In inverse planning, first an optimal fluence map is automatically calculated for each beam, using computer optimization minimizing a score function. Subsequently, the fluence map is converted automatically into a number of static segments that approximate the original fluence map as good as possible. The first results show that the inverse planning process does not result in better dose distributions with respect to target coverage and the dose to the rectal wall. Figure IX.5: DVHs of the rectal wall comparing the results of the serial boost and the SIB technique. Breast Cancer Methods have been developed to reduce cardiac and lung complication probabilities after breast irradiation. One of the simplest ways to do this is by changing the patient position in such a way that less heart or lung is in the treatment beam. We therefore compared simulator images of tangential fields taken in two positions: (1) with the ipsilateral arm abducted, holding an L-bar armrest and (2) with both arms extended above the head in a forearm support. The average maximum heart distance as well as the central lung distance decreased significantly, by 3.4 ± 0.9 mm and 4.7 ± 1.1 mm, respectively, when the new forearm support was used. The estimated normal tissue complication probability for excess cardiac mortality decreased with on average 3.1%. For some patients a greater amount of the axilla was included in the field. We recommend the use of the forearm support during breast cancer treatment with tangential fields to decrease the amount of heart and lung inside the field. Another approach to reduce the cardiac and lung dose might be the application of conformal tangential beam irradiation of the intact left breast, with or without intensity modulation. For 17 left-sided breast cancer patients, three different tangential beam techniques were compared: 1) rectangular fields with optimized wedges, 2) blocked conformal fields with optimized wedges and 3) intensity modulated fields. Plans were evaluated using DVHs for the PTV, the heart and the lungs. NTCPs for radiation pneumonitis and late cardiac mortality were calculated using the DVH data. The results showed that the use of conformal tangential fields decreases the NTCP for late cardiac toxicity on average by 30% compared to using rectangular fields, while the tangential IMRT technique can further reduce this value by an additional 50%. To investigate whether intensity modulated (IMRT) radiotherapy techniques would

85 87 13 RADIOTHERAPY also be advantageous for the treatment of the left breast and upper internal mammary lymph node chain (IMC), these organs as well as the heart and lungs were delineated on a CT-scan for 12 patients. Three different treatment plans were created: 1) tangential photon fields with oblique IMC electron-photon fields with manually optimized beam weights and wedges, 2) wide split tangential photon fields with a heart block and computer optimized wedge angles and 3) IMRT tangential photon fields. For the IMRT technique, an inverse planning program (KonRad) generated the intensity profiles and a clinical 3D treatment planning system (UM-Plan) optimized the segment weights. UM-Plan calculated the dose distribution for all three techniques. The resulting average mean dose for the breast PTV was the same for the three techniques. The average mean dose to the IMC was 97.2, and 99.6% for the oblique electron, wide split tangent and IMRT techniques, respectively. The average NTCPs for the ORs (i.e. heart and lungs) were comparable between the oblique electron and IMRT techniques. The wide split tangent technique resulted in higher NTCP values for these ORs (Figure IX.6). It can be concluded that the lowest NTCP values were found with the oblique electron and the IMRT techniques. The IMRT technique had the best breast and IMC target coverage. Figure IX.6: NTCP for excess late cardiac mortality by patient and technique. wide = wide split tangent, obl = oblique electrons, imrt = IMRT tangent. Head and neck cancer An ethmoid sinus cancer case was sent as an IMRT planning task to all participants of the ESTRO teaching course on IMRT and Other Conformal Techniques in Practice, held in Amsterdam in June. A comparison was made of the planning strategies and dose statistics of nine IMRT plans generated for this case. For the same prescribed dose and dose constraints for organs at risk, IMRT strategies for this complex head and neck case differed in many aspects. The most striking differences concern beam setup, total number of segments, PTV coverage and dose statistics for organs at risk. It can be concluded that the planning of IMRT is a complicated issue, which needs close co-operation between the various disciplines involved in the preparation of a radiotherapeutical treatment. STUDIES OF RADIATION RESPONSE IN NORMAL TISSUE JSA Belderbos, LJ Boersma, JV Lebesque Pulmonary function after radiotherapy for NSCLC Changes in pulmonary function were studied in 74 patients receiving high dose 3D conformal RT for inoperable NSCLC. CT scans of the chest, Single Photon Emission CT (SPECT) lung perfusion

86 88 RADIOTHERAPY Director of Research Anton Berns scans and pulmonary function tests (PFTs) including the forced expiratory volume in one second, diffusion capacity for carbon monoxide, alveolar volume and vital capacity were evaluated before RT and at 3 months follow up. The 3D-dose distribution in lung was converted to the mean lung dose (MLD) and weighted with the 3D pretreatment perfusion, resulting in the mean perfusion-weighted lung dose (MpLD). The relative decline of all PFTs was significantly associated with the MpLD but the correlation coefficients were small. Tumor regression, the MLD, measured perfusion reduction and reperfusion were not correlated with changes in PFTs. Reperfusion was associated with the pre-treatment perfusion deficiency. For central tumors, reperfusion was related to tumor regression but did not translate to an improvement of pulmonary function tests. Perfusion-related variables as the mean perfusionweighted lung dose and the predicted perfusion reduction, revealed the best available parameters to estimate the post-rt pulmonary function. Tumor motion Information about tumor motion in lung tissue is required to model tumor movement that can be used for accurate (gated or breath-hold) radiotherapy or CT scanning. Therefore, 3D motion of lung tumors during radiotherapy was studied in real-time in 20 lung cancer patients. Before treatment a gold marker was implanted in the tumor. A real-time tumor tracking system used fluoroscopy image processor units to determine the 3D position of the marker at a high sample rate. Breathing-induced tumor motion was largest (12 mm) in the CC direction for tumors that were situated in the lower lobes and were not attached to the bony anatomy. In other directions the tumor motion was small (2 mm), both for upper and lower lobe tumors. Tumor motion could be modeled with a sinusoid with varying asymmetry because the tumor spends more time in exhalation than in inhalation phase. Shifts in tumor position were observed between and within fractions, due to gravity, patient relaxation or movement. The 3D trajectory of the tumor showed 1-5 mm hysteresis for ten of the 21 tumors. Frequency analysis revealed that for seven of the 21 tumors, a measurable tumor motion was caused by the cardiac beat. CONFORMAL RADIOTHEARPY JSA Belderbos, LJ Boersma, BJ Mijnheer, C Rasch, M Van Herk, JV Lebesque There was an increase in the number of patients treated with conformal radiation therapy (CRT): 310 in 2001 against 265 in 2000 and 187 in 1999, which is an increase of 66% over the past two years. A major contribution to this increase in treated patients is the head & neck group treated with IMRT techniques; this patient group increased from 10 patients in 1999 to 60 in Prostate cancer Another 35 prostate patients were recruited at our institution for the dose escalation trial. Closure of patient recruitment is planned for April 2002, when we expect to have included 600 patients, balanced over the 68 Gy and the 78 Gy arms. To study changes in the rectal shape and its effect on the actual administered dose, we have developed a method to unfold the delineated rectal wall and project the received dose or the co-ordinates on a 2D angular map. The advantage of this geometrical reconstruction is that, to a first order, the same rectal tissue is projected on the same map position irrespective of rectal volume differences. This means that dose or co-ordinate rectal maps from repeat CT scans of the same patient can be added. We investigated rectal maps of 19 patients who each received eleven repeat CT scans. Rectal deformations predominantly occurred at the anterior upper part of the rectum. Furthermore, we found that large rectal volumes (> 100 cm 3 ) in the planning CT scan, led to an overestimate of up to 15 % of the actual administered dose to the anterior upper side of the rectum. Lung cancer The use of 3D-CRT with smaller volumes and higher doses might improve clinical outcome in patients with inoperable NSCLC. In October 1998 a phase I/II dose escalation trial using 3D-CRT was initiated in our institution. The primary objective of this trial is to establish the maximum tolerable dose (MTD). Five risk groups were defined according to the relative mean lung dose (rmld). The starting dose levels in the five defined groups were 81 Gy (I), 74.3 Gy (II-IV) and 60.8 Gy

87 89 13 RADIOTHERAPY (V). The MTD has been reached if two out of six patients experience dose-limiting toxicity. Dose limiting toxicity is defined as pneumonitis grade 3 (SWOG), grade 3 early and grade 2 late esophagus toxicity or any other grade 3 / 4 complications (RTOG). Until November 2000, 64 patients had been enrolled in this study. An interim analysis of the first group of 56 patients was performed. The majority of this patient group received a dose of 74.3 Gy (n=16) or 81.0 Gy (n=21). Radiation pneumonitis occurred in six patients: three grade II; two grade III and one grade IV. Acute esophagitis grade II was recorded in ten patients and late esophagitis grade I in eight patients. The current dose levels for the risk groups are 94.5 (I), 87.7 (II, III), 81 (IV), and 60.8 (V) Gy. The maximum tolerable dose has not been reached yet in any group in this ongoing study. BREAST CANCER E Damen, J Borger, N Russell, H Bartelink Breast cancer research programs This year we were able to evaluate the effect of additional radiation dose to the tumor bed on the rates of local recurrence among patients who received radiotherapy after breast-conserving surgery for early breast cancer in patients treated within a large phase III clinical trial. After lumpectomy and axillary dissection, patients with stage I or II breast cancer received 50 Gy of radiation to the whole breast in 2-Gy fractions over a five-week period. Patients with a microscopically complete excision were randomly assigned to receive either no further local treatment (2657 patients) or an additional localized dose of 16 Gy, usually given in eight fractions by means of an external electron beam (2661 patients). During a median follow-up period of 5.1 years, local recurrences were observed in 182 of the 2657 patients in the standard-treatment group and 109 of the 2661 patients in the additional-radiation group. The five-year actuarial rates of local recurrence were 7.3 percent (95 percent confidence interval, 6.8 to 7.6 percent) and 4.3 percent (95 percent confidence interval, 3.8 to 4.7 percent), respectively (P<0.001), yielding a hazard ratio for local recurrence of 0.59 (99 percent confidence interval, 0.43 to 0.81) associated with an additional dose. Patients 40 years old or younger benefited the most. At five years, their rate of local recurrence was 19.5 percent with standard treatment and 10.2 percent with additional radiation (hazard ratio, 0.46 (99 percent confidence interval, 0.23 to 0.89; P=0.002)). In patients 41 to 50 years old, the rates were 9.5 percent and 5.8 percent, respectively; P=0.02. Between both treatment arms, no differences were found in distant metastasis-free rate or overall survival (which were 87 and 91 percent, respectively). We concluded that in patients with early breast cancer who undergo breast-conserving surgery and receive 50 Gy of radiation to the whole breast, an additional dose of 16 Gy of radiation to the tumor bed reduces the risk of local recurrence, especially in patients younger than 50 years of age. Other activities towards improvement of the treatment technique for patients with breast cancer to prevent late normal tissues damage are reported elsewhere in this section, while the possible reasons of the higher local recurrence rate are investigated in close cooperation with the molecular pathologists of the institute. MODULATION OF RADIATION-INDUCED APOPTOSIS R Haas, SF Zerp, H Bartelink, M Verheij Apoptosis is now recognized as an important component of radiation-induced cell death and co-determinant of radiosensitivity. Our line of research focuses on developing pharmacological strategies to increase the apoptotic response after irradiation. Alkyl-lysophospholipids (ALPs) represent a group of synthetic antitumor agents known to accumulate in plasma membranes and to interfere with lipid-mediated signaling. We studied the effect of ALPs on radiation-induced cell death in a wide variety of tumor cell lines and several types of normal cells. Three of these compounds, Et- 18-OCH3 (Edelfosine), HePC (Miltefosine) and D (Perifosine), were found to enhance radiation-induced apoptosis by inhibiting mitogenic/survival signaling and activating pro-apoptotic pathways. These effects were preferentially observed in malignant cells, leaving normal cells relatively unaffected. The apoptotic effect of ALPs on

88 90 RADIOTHERAPY Director of Research Anton Berns vascular endothelial cells, however, depended on the proliferative status of these cells and their ALP uptake capacity. Whereas confluent, resting endothelial cells incorporated small amounts of ALPs and failed to undergo apoptosis, proliferating endothelial cells showed high levels of ALP uptake and significant apoptosis. Based on these observations, we tested the anti-angiogenic properties of ALPs in two different in vitro angiogenesis models. ALPs interfered with bfgf/tnfa- and VEGF/TNFa-mediated endothelial tube formation in a dose-dependent manner. More recently, we found that ALPs reduce clonogenic survival after radiation. This radiosensitizing effect is most likely due to an interference with radiation-induced DNA damage repair. Whereas the above-described cytotoxic actions of ALPs were observed in the micro molar range of these drugs, in A431 cells sub apoptotic, Nan molar concentrations ALPs induce activation of the MAPK/ERK pathway. The exact mechanism by which this occurs remains unclear, but experimental evidence strongly suggests that subtle changes in the plasma membrane microenvironment causes ligand-independent clustering and subsequent internalization of the EGF receptor. These studies confirm that the plasma membrane is the primary site of action of ALPs. In two other projects (in collaboration with W Van Blitterswijk, Division III) we are investigating mechanisms of cellular ALP uptake using radio labeled compounds and an ALPresistant cell line (S49 AR). These S49 AR cells show cross-resistance to DNA-damaging agents including ionizing radiation and etoposide, and are characterized by an sphingomyelin deficiency. The underlying mechanism and physiological significance of this latter observation remains to be established. Follicular lymphomas, frequently characterized by (14; 18) translocation and overexpression of the anti-apoptotic bcl-2 gene product, are highly radioresponsive even to very low doses of ionizing radiation. The N97HOR study has shown a response rate of 88% in 90 patients with recurrent follicular lymphoma. In October, a phase III study (HOVON 47) was initiated, randomizing untreated patients with follicular lymphoma between Chlorambucil and low dose (2x2 Gy) involved field radiotherapy. We are currently investigating the mechanisms of this antitumor effect, focusing on apoptosis as the predominant mode of cell death. One of the earliest events during the apoptotic process is the exposure of phosphatidylserine (PS) at the outer leaflet of the plasma membrane lipid bilayer. The human endogenous protein annexin V, which has a high affinity for PS, is often used as a marker for apoptosis in vitro. Recently, 99mTc labeled annexin V became available for in vivo imaging of apoptosis. In five patients with lowgrade lymphoma a baseline and 24 hours post-radiotherapy 99mTc-annexine V scintigraphy were performed. It was possible to image radiation-induced apoptosis in four out of five patients (Figure IX.7). Immediately following the post-treatment scan the irradiated lymph node was biopsied for (immuno-) histochemical analysis. The increase in uptake of the radiopharmaceutical marker correlated with histolopathological evidence of apoptosis. Future investigations will focus on the appropriate timing between the lymph node biopsy and 99mTc-annexin V scintigraphy. Figure IX.7: 9mTc-annexine V scintigraphy of the pelvic region. Left: before radiotherapy, right: 24 hours post-treatment (arrow: apoptotic lymph node, bladder activity was shielded). COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY JSA Belderbos, H Bartelink, C Rasch A total number of 100 patients have been entered in the Phase III radplat trial (M99RAD), comparing 70 Gy radiation + intra-arterial cisplatin or intravenous cisplatin for inoperable head and neck carcinoma. The trial is ongoing; a total num-

89 91 13 RADIOTHERAPY ber of 240 patients are planned. An interim analysis to detect large difference between the two treatment arms is scheduled for early The first results of the Phase II radplat trial confirm the first impressions. 79 patients were evaluable, median age was 51 years and median follow-up was 2.1 years. The complete response rate was 91% and 96% for the primary tumor and lymph nodes, respectively. At two years the local control was 79%, lymph node control was 91%, DM free rate was 89%, overall survival 40% and disease free survival 58%. A study measuring the DNA- (cisplatin)-adduct formation in primary tumor and lymph nodes was initiated in September. The EORTC trial is a randomized phase III trial comparing induction chemotherapy to daily low dose cisplatin both combined with high dose radiotherapy in patients with inoperable NSCLC stage I, II and low volume stage III. This trial, that was designed in the AvL and AMC opened in 1999 and has now reached 25% of the accrual. Most participating centers are in the Netherlands. The rate of accrual is steady.

90 92 MEDICAL ONCOLOGY Director of Research Anton Berns X DIVISION OF MEDICAL ONCOLOGY Division head, S Rodenhuis S Rodenhuis MD PhD Academic staff, Head Division X JW Baars MD PhD Academic staff P Baas MD PhD Academic staff EM Bais MD Academic staff JH Beijnen PhD Academic staff W Boogerd MD PhD Academic staff H Boot MD PhD Academic staff M Bülbül MD Academic staff A Cats MD Ph Academic staff JP De Boer MD PhD Academic staff GC De Gast MD Ph Academic staff JBAG Haanen MD PhD Academic staff ADR Huitema PhD Academic staff ALT Imholz MD Academic staff JM Kerst MD PhD Academic staff MJ Kersten MD PhD Academic staff CMF Kruijtzer MD Academic staff PMM Kuijer MD Academic staff RAA Mathôt MD Academic staff H Neering MD Ph Academic staff CH Rikers MD Academic staff JHM Schellens MD PhD Academic staff JH Schornagel MD PhD Academic staff JG Schrama MD Academic staff M Spaander Academic staff BG Taal MD PhD Academic staff WW Ten Bokkel Huinink MD Ph Academic staff JJ Van der Sande MD PhD Academic staff M Van der Weide MD PhD Academic staff N Van Zandwijk MD PhD Academic staff APE Vielvoye-Kerkmeer MD PhD Academic staff J Wanders MD Academic staff JM Zuetenhorst MD Academic staff N Appels Graduate student H Bardelmeijer Graduate student J-H Beumer Graduate student M Bouma Graduate student KML Crommentuyn Graduate student M Crul Graduate student ME De Jonge Graduate student MM De Maat Graduate student MWJ Den Brok Graduate student B Kappelhof Graduate student M Kemper Graduate student M Klous Graduate student MM Malingré Graduate student K Morhmann Graduate student JM Rademaker-Lakhai Graduate student L Rook Graduate student NE Schoemaker Graduate student A Schrijvers Graduate student CLINICAL PHARMACOLOGY JHM Schellens, JH Beijnen, WW Ten Bokkel Huinink, P Baas, EM Bais, S Rodenhuis, JH Schornagel, N Van Zandwijk Major research topics include: 1) modulation of oral bioavailability through inhibition of drug transport proteins, especially P-glycoprotein and BCRP (MXR/ABCG2) and drug metabolizing cytochrome P450; 2) modulation of drug resistance in solid tumors through inhibition of P-glycoprotein; 3) phase I and pharmacological studies; 4) clinical investigation of novel polymer drug carriers for drug-targeting; 5) modeling of (sparse) pharmacokinetic and pharmacodynamic data by the population approach using NONMEM and 6) studies to unravel the metabolism of anticancer drugs. Phase I studies and drug scheduling The number of active studies is comparable to last year. At present, 18 phase I/II and pharmacologic studies are being conducted, of which eleven studies are open for accrual. One of the major translational research activities continues to be the development of an oral treatment schedule for paclitaxel and docetaxel in combination with oral cyclosporin A (CsA). We have modified the oral formulation of paclitaxel. Results reveal that when paclitaxel is dissolved in polysorbate 80, the area under the concentration-time curve (AUC) of paclitaxel is 50% higher and much less variable than when paclitaxel is dissolved in Cremophor EL. We have shown that gut epithelial BCRP plays an important role in the absorption of oral topotecan. Follow-up studies are starting up to determine the minimal effective dose of the BCRP blocker GF that results in a maximal enhancement of the oral bioavailability of topotecan. LY is a strong inhibitor of P-glycoprotein, currently being developed in combination with iv paclitaxel. LY is given orally in two doses of 450 mg one hour prior to paclitaxel (225 mg/m2 given, q three weeks) and 12 hours later. Dose-limiting toxicities (DLT) are reversible ataxia and visual disturbances. In another study we are investigating the combination of iv docetaxel ( mg/m2 per three weeks) and LY In future studies, this combination will be tested in advanced breast cancer. In the phase I study with the farnesyltransferase inhibitor R in combination with cisplatin and gemcitabine, the dose-level of 75 mg/m2 cisplatin day one, 1000 mg/m2 gemcitabine day one and eight and R mg day 1-7, q three weeks is being tested. The main toxicities are myelosuppression, nausea and vomiting. The randomized phase I study with either weekly or two-weekly gemcitabine and cisplatin in patients with NSCLC is ongoing. The aim is to develop a dose-dense schedule for NSCLC. To date, 69 patients have been recruited. The dose-intensity is highest in the two-weekly schedule and its maximum was reached at 105 mg/m2 of cisplatin plus 1500 mg/m2 of gemcitabine per week. The dose-intensity of cisplatin is 50% higher than in the standard schedule of this combination. Interestingly, gemcitabine significantly reduces platinum DNA-adduct formation in leukocytes, without affecting the plasma pharmacokinetics of platinum. The mechanism of this interaction is being explored in in vitro systems. Novel metabolites of E7070 have been identified in the mass balance study with 14 C-labeled E7070. One of the major pathways is glucuronidation. The excretion of unchanged drug is negligible. A new phase I study with Kahalalide F (KF), a novel dehydroaminobutyric acid-containing peptide isolated from the Hawaiian herbivorous marine species of mollusk Elysia rufescens, was initiated in androgen resistant prostate cancer. Twelve patients have been recruited thus far. Doses could be increased rapidly from 20 µg/m2/day on a daily times 5 schedule q three weeks to the currently tested 560 µg/m2/day. A decrease in PSA was seen in three of the twelve patients, coinciding with meaningful clinical improvement in one patient during six months. The combination study of carboplatin and topotecan is still ongoing. Tumor biopsies have been collected in all 18 recruited patients to measure platinum-dna adducts and catalytic activity and expression of topoisomerase I. DLT is myelosuppression.

91 93 13 MEDICAL ONCOLOGY The phase I study with the organic ruthenium compound NAMI-A is still actively recruiting patients. The schedule is daily times 5 q 3 weeks. Doses could be rapidly escalated from the starting level of 12 mg/m2/course to 1500 mg/m2/course in only ten steps. The pharmacokinetics of total and unbound ruthenium are linear over the wide dose range. One DLT at the highest level was observed being hypotension and renal dysfunction. The two-center phase I study with the polymer platinum compound AP5280 is actively recruiting patients at dose-level 6. Doses were rapidly increased from the starting dose-level of 90 mg/m2 to 3300 mg/m2. The main toxicity is gastrointestinal. The two-center phase I study with the combination of the cell cycle inhibitor Ro and gemcitabine was put on hold because of unexpected liver dysfunction. Ro is given orally on an intermittent schedule and gemcitabine iv on day 1 and 8 of a three-weekly schedule. The interim safety analysis is being finalized and the results will guide further development of this combination. The phase I study with topotecan and ifosfamide has reached DLT at a dose of topotecan of 1.4 mg/m2 and ifosfamide of 1.2 g/m2, both given at a daily times 3, q 3 weeks schedule. A new phase I study with paclitaxel and carboplatin in NSCLC was initiated. The aim is to individualize the dose and infusion duration of paclitaxel in order to reach the target level of 0.1 µm during a time-period of at least 16 hours. Carboplatin is dosed to achieve an AUC of 5 mg.ml -1.hr. Phase II and pharmacologic studies The patient recruitment in four phase II studies exploring the activity of oral paclitaxel plus CsA in advanced breast cancer, gastric cancer and NSCLC and docetaxel plus CsA in breast cancer is nearly finished. Paclitaxel (90 mg/m2) and CsA (10 mg/kg) are given orally bidaily on a weekly schedule, resulting in reproducible concentration time-curves (Figure X.1). The overall response rate of 23% in stage IIIb/IV NSCLC is promising. The phase II study in second line of breast cancer with oral docetaxel plus CsA revealed a high response rate of 45% (three complete and eleven partial remissions were noted in 31 patients). The safety of this oral weekly schedule is comparable or better than that of the standard three weekly iv schedule. Docetaxel is given at a fixed dose of 100 mg. The dose of CsA is 15 mg/kg. Preliminary data in first line of advanced gastric cancer reveal that seven out of 17 patients developed a partial remission. Population pharmacokinetics and dynamics Population analysis of sparse data with NONMEM is extensively applied to support the Biomed2 project and phase II studies with ET743 and E7070. Population analysis of E7070 showed non-linear metabolism and non-linear distribution at the higher dose-levels (Figure X.2). The DHJG Van den Bongard Graduate student C Van Kesteren Graduate student RCAM Van Waardenburg Graduate student P Quaedvlieg Undergraduate student I Bedeker Technical staff E Boerhorst Technical staff CGJ Cleypool Technical staff MJX Hillebrand Technical staff SE Jansen Technical staff FJ Koopman-Kroon Technical staff L Nan-Offeringa Technical staff B Nuyen Technical staff M Ouwehand Technical staff H Rosing Technical staff RW Sparidans Technical staff MM Tibben Technical staff R Van Gijn Technical staff D Batchelor Research nurse AC Dubbelman Research nurse M Holtkamp Research nurse M Keessen Research nurse H Mallo Research nurse M Schot Research nurse M Swart Research nurse Y Arts-Blom Secretary MEG De Kwant Secretary Figure X.1: Plasma concentration-time curves of three different patients who received oral paclitaxel bid at a dose of 90 mg/ma plus cyclosporin A.

92 94 MEDICAL ONCOLOGY Director of Research Anton Berns Key publications Crul M, De Klerk GJ, Beijnen JH, Schellens JHM. Ras biochemistry and farnesyl transferase inhibitors: a literature survey. Anticancer Drugs 2001; 12: Huitema ADR, Mathôt RAA, Tibben MM, Rodenhuis S, Beijnen JH. A mechanismbased pharmacokinetic model for the cytochrome P450 drug-drug interaction between cyclophosphamide and thiotepa and the autoinduction of cyclophosphamide. J Pharmacokinet Pharmacodyn 2001; 28: Huitema ADR, Mathôt RAA, Tibben MM, Schellens JHM, Rodenhuis S, Beijnen JH. Population pharmacokinetics of thiotepa and its active metabolite TEPA in patients undergoing high-dose chemotherapy. Br J Clin Pharmacol 2001; 51: Kerbusch Th, De Kraker J, Keizer JH, Van Putten WG, Groen HJM, Jansen RLH, Schellens JHM, Beijnen JH. Clinical pharmacokinetics and pharmacodynamics of ifosfamide and its metabolites. Clin Pharmacokinet 2001; 40: Maliepaard M, Scheffer GL, Faneyte IF, Van Gastelen MA, Pijnenborg AC, Schinkel AH, Van de Vijver MJ, Scheper RJ, Schellens JHM. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissue. Cancer Res 2001; 61: Maliepaard M, Van Gastelen MA, Tohgo A, Hausheer FH, Van Waardenburg RC, De Jong LA, Pluim D, Beijnen JH, Schellens JHM. Circumvention of breast cancer resistance protein (BCRP)-mediated resistance to camptothecins in vitro using non-substrate drugs or the BCRP inhibitor GF Clin Cancer Res 2001; 7: Figure X.2: Population pharmacokinetic model of E7070. The model comprises 3 compartments, a linear as well as a non-linear elimination pathway and a non-linear tissue distribution pathway. AUC of E7070 was shown to be significantly correlated with neutropenia and thrombocytopenia (Figure X.3). PHARMACY JH Beijnen, JHM Schellens, WW ten Bokkel Huinink The research programs of the Department of Pharmacy and Pharmacology deal with pharmaceutical formulation and drug level monitoring of (investigational) anticancer drugs to support preclinical (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. Maintenance of our GMP (Good Manufacturing Practice) and GLP (Good Laboratory Practice) status of the formulation unit and laboratory requires our continuous attention. This year the department acquired an ISO Certification for Purchasing, storage and distribution of drugs for world-wide clinical investigations (GDP, Good Distribution Practice). Formulation Our formulation research is mainly focused on cytotoxic agents originating from marine organisms (ecteinascidin-743 or ET-743, kahalalide F, aplidine, spisulosine), heavy metals (platinum, ruthenium complexes), the aziridinylquinone EO9 and peptides for vaccination. A systematic stability study of the ruthenium-containing antitumor agent encoded NAMI-A was completed. Light and ph were the main determinants for instability. The drug is being tested in a phase I trial in our institute. A pharmaceutical formulation for an HPMA(hydroxypropyl methacrylamide)-polymer (with spacer) bound platinum compound (AP-5280) was designed Malingré MM, Beijnen JH, Rosing H, Koopman FJ, Jewell RC, Paul EM, Ten Bokkel Huinink WW, Schellens JHM. Co-administration of GF significantly increases the systemic exposure to oral paclitaxel in cancer patients. Br J Cancer 2001; 84:42-7. Figure X.3: Relationship between the AUC of E7070 and the thrombocyte counts during the first course.

93 95 13 MEDICAL ONCOLOGY using the freeze-drying technique. We have manufactured many batches to support clinical testing in our hospital and in Lyon. Kahalalide F (from the mollusc Elysia rufescens) is a novel marine derived investigational anticancer drug. We have been responsible for the entire pharmaceutical development. A stable lyophilized formulation with reconstitution with a 5/5/90 % v/v/v Cremophor EL, ethanol, water mixture has been designed. Last year we manufactured several Kahalalide F batches (600 vials per run) for clinical studies in both our hospital and a center in Spain. The development of a formulation of the extremely poorly water-soluble cytotoxic spisulosine (originating from Spisula polynoma) based on complexation and inclusion into hydroxypropyl-ß-cyclodextrins is still ongoing. Toxicology tests in mice are in progress and it is expected that the first phase I trial can start in the beginning of We designed a new, stable formulation for EO9 (freeze dryed from 40% t-butanol) which can be reconstituted with a propyleneglycol/water/sodium edetate/sodium bicarbonate (60/40/0.02/2; v/v/w/w) mixture. A clinical trial with intravesical instillation for superficial bladder cancer will be starting soon in the UK. Three peptides, intended for melanoma vaccination investigations, are the subject of current formulation research. Poor water solubility and instability are the main problems to be dealt with for these compounds D - V a l L - V a l L - T h r O H O O D - P ro N H O N N H O NH O R NH L- Orn N H 2 O N H O D- allo- Thr L- Val D- allolle NH O NH N H O O O H N O O NH Z - Dh b NH O D - all- Thr D - Val Key publications(continued) Malingré MM, Beijnen JH, Rosing H, Koopman FJ, Van Tellingen O, Duchin K, Ten Bokkel Huinink WW, Swart M, Lieverst J, Schellens JHM. A phase I and pharmacokinetic study of bi-daily dosing of oral paclitaxel combination with cyclosporin A. Cancer Chemother Pharmacol 2001; 47: Malingré MM, Richel DJ, Beijnen JH, Rosing H, Koopman FJ, Ten Bokkel Huinink WW, Schot ME, Schellens JHM. Coadministration of cyclosporine strongly enhances the oral bioavailability of docetaxel. J Clin Oncol 2001; 19: Meerum Terwogt JM, Ten Bokkel Huinink WW, Schellens JHM, Schot M, Mandjes IA, Zurlo MG, Rocchetti M, Rosing H, Koopman FJ, Beijnen JH. Phase I clinical and pharmacokinetic study of PNU166945, a novel water soluble polymer-conjugated prodrug of paclitaxel. Anticancer Drugs 2001; 12: Quaedvlieg PFHJ, Visser O, Lamers CBH, Jansen-Heijen MLG, Taal BG. Epidemiology and survival in patients with carcinoid disease in the Netherlands. Ann Oncol 2001; 12: D - Val R ~NH O KF ~NH O I S L- Phe Rodenhuis S. The role of high-dose chemotherapy in the treatment of breast cancer. In: Bonadonna G, Hortobagyi G, Gianni AM, editors. Textbook of Breast Cancer. A clinical guide to therapy. Second edition. London: Martin Dunitz Ltd., 2001: Figure X.4: Structures of Kahalalide F and butyrated internal standard. Fragmentation pattern of Kahalalide F is presented. Bioanalysis and clinical pharmacology Bioanalytic and clinical pharmacologic research comprise the following compounds (and metabolites): topoisomerase-i inhibitors (irinotecan/sn-38, topotecan/n-desmethyltopotecan, lipotecan, DRF- 1644), gemcitabine, mitomycin C, melphalan, taxoids (paclitaxel, docetaxel), platinum/ruthenium compounds, the drugs in the CTC high dose regimen (carboplatin-thiotepa-cyclophosphamide) and novel marine cytotoxics (ET-743, aplidine, thiocoraline, kahalalide F, spisulosine). The latter group of compounds is typically given in very low dosages (µg/m2) yielding blood levels in the ng/ml and pg/ml range. Sensitivity is therefore a major requirement for the bioanalytical methodologies developed in our laboratory. Due 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 pretreatment. New HPLC assays with fluorescence detection have been developed for lipotecan, a liposomal formulation of topotecan and the orally active topoisomerase-i inhibitor DRF The assays were used for preclinical pharmacokinetic studies as a reference for the planned clinical phase I studies. Gemcitabine and its deaminated Schellens JHM, Maliepaard M, Scheper RJ, Scheffer GL, Jonker JW, Smit JW, Beijnen JH, Schinkel AH. Transport of topoisomerase I inhibitors by the breast cancer resistance protein. Potential clinical implications. Ann N Y Acad Sci 2000; 922: Schornagel JH, Verheij M, Bartelink H. Concurrent chemotherapy with radiation for locally advanced squamous cell cancer: what is the schedule of choice? Curr Oncol Rep 2001; 3:1-2. Schouwink JH, Ruevekamp M, Oppelaar H, Van Veen R, Baas P, Stewart FA. Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols. Photochem Photobiol 2001; 73:

94 96 MEDICAL ONCOLOGY Director of Research Anton Berns Key publications (continued) Schouwink JH, Rutgers EJTh, Van der Sijp J, Oppelaar H, Van Zandwijk N, Van Veen R, Burgers S, Stewart FA, Zoetmulder F, Baas P. Intraoperative photodynamic therapy after pleuropneumonectomy in patients with malignant pleural mesothelioma: dose finding and toxicity results. Chest 2001; 120: Schrama JG, Baars JW, Holtkamp MJ, Schornagel JH, Beijnen JH, Rodenhuis S. Phase II study of a multi-course high-dose chemotherapy regimen incorporating cyclophosphamide, thiotepa, and carboplatin in stage IV breast cancer. Bone Marrow Transplant 2001; 28: metabolite are measured in plasma and urine with an HPLC-UV assay; gemcitabinetriphosphate is quantified in leukocytes. Ex-vivo instability of the triphosphate is a complicating factor. The stability is now being investigated systematically to find the most optimal conditions for sample handling; temperature and acidity appear crucial. Taxoids (paclitaxel, docetaxel) are being measured with validated HPLC assays for the oral program with these agents. We are measuring mitomycin C levels in plasma and peritonal fluid for the HIPEC (Hyperthermic Intraperitoneal Chemotherapy) studies (Division XI Surgical Oncology). Melphalan is being measured in biological samples from liver perfusion studies executed by investigators in Leiden (LUMC). Kahalalide F levels are being measured with HPLC coupled with positive turbo-ion spray tandem mass spectrometry (Figures X.4 and X.5). Interestingly, this tridecadepsipeptide could be measured very selectively and sensitively in a positive ion mode employing a basic mobile phase. The lower limit of quantitation is 1 ng/ml which was sufficient to monitor the drug even at the first dose level (20 µg/m2 iv) in the phase I trial. From in vitro experiments (microsomes-, plasma-incubations) it appeared that the compound is metabolically fairly stable. Vasen HFA, Stormorken A, Menko FH, Nagengast FM, Kleibeuker JH, Griffioen G, Taal BG, Moller P, Wijnen JT. MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers: a study of hereditary nonpolyposis colorectal cancer families. J Clin Oncol 2001; 19: Voermans C, Kooi ML, Rodenhuis S, Van der Lelie H, Van der Schoot CE, Gerritsen WR. In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation. Blood 2001; 97: In press De Gast GC, Vyth-Dreese FA, Nooijen W, Van den Boogaard CJC, Sein J, Holtkamp M, Linthorst G, Schornagel JH, Rodenhuis S. Re-infusion of autologous lymphocytes with GM-CSF induces rapid recovery of CD4+ and CD8+ T-cells after high dose chemotherapy for metastatic breast cancer. J Clin Oncol (in press). Meernum Terwogt JM, Groenewegen G, Maliepaard M, Tibben M, Pluim D, Ten Bokkel Huinink WW, Schot M, Welbank H, Blijham G, Schellens JHM, Beijnen JH. Phase I and pharmacokinetic study of SPI- 77, a liposomal encapsulated dosage form of cisplatin. Cancer Chemother Pharmacol (in press). Taal BG, Van Tinteren H, Zoetmulder FAN. Adjuvant 5FU plus levamisole in colonic or rectal cancer: improved survival in stage II and III. Br J Cancer (in press). Figure X.5: Tandem mass spectrum of Kahalalide F (selected ion m/z 740; double protonated parent molecule Mw 1478). ET-743 is now being evaluated in phase II trials with pharmacokinetic monitoring. The use of a stable isotope of ET-743 as internal standard has greatly improved the accuracy of our LC/MS/MS assay. An LC/MS/MS assay is now being developed for spisulosine (ES-285). It is expected that the drug will enter phase I in 2002 with pharmacokinetic monitoring. We have designed an LC/MS/MS assay for the matrix metalloproteinase inhibitor ABT-518 with 6 putative metabolites. The sample pretreatment was a solid phase extraction. The lower limit of quantitation was 10 ng/ml. Six patients have been treated with the drug. The assay appeared suitable to follow the parent drug and 5 metabolites could be detected and quantified in plasma. Preliminary evaluations of pharmacokinetic-pharmacodynamic relationships in CTC have revealed a correlation between the occurrence of alopecia and thiotepa + tepa and carboplatin exposures. Pharmacokinetic target values have been set and treatment individualization has become the daily practice in the CTC-scheme. These studies will continue within the setting of a new NKB project. E7070, a cell cycle inhibitor, is being measured by LC/MS/MS in different phase II studies. The results will be used to explore any pharmacokinetic-pharmacodynamic relationships. The metabolic disposition of 14C-E7070, administered to patients, has been investigated. The study showed that E7070 is metabolized to a great extent with the formation of several metabolites. These compounds have been isolated and subjected to mass spectrometry for structural elucidation. Several metabolites have been identified. Glucuronidation appears to be a major metabolic pathway in humans. Platinum and ruthenium analyses are being performed by atomic absorption spectroscopy (AAS) and support ongoing phase I studies with AP-5280 and NAMI-A, respectively. Linear kinetics and extensive renal excretion of the compounds are evident. Our LC/MS/MS equipment is also used for quality control purposes of peptides synthesized in the institute. To support cellular transport and metabolic studies (group P Borst) in MRP4 and MRP5 transfected cells, we measure several purines and pyrimidines with an accurate gradient-hplc assay with photo-diode array detection.

95 97 13 MEDICAL ONCOLOGY IMMUNOTHERAPY GC De Gast, JBAG Haanen, W Boogerd, MJ Kersten Metastatic melanoma The results of a phase II study of Temozolomide followed by combined immunotherapy showed a response rate of 31% (23 of 74 patients), with a therapeutic effect on CNS metastases in two of 13 patients and the prevention of CNS metastasis in responders and SD patients. These findings have led to a randomized phase III study of Temozolomide with dose adjustment in which stage IV melanoma patients are randomized to additional combined immunotherapy or no further treatment. Until the time of this writing only patients from the NKI/AvL have been included, but several other are going to participate. A phase II study neoadjuvant and adjuvant chemo-immunotherapy study in sequence with surgery is on-going in stage III melanoma. Key publications (continued) Van de Kolk LE, Baars JW, Van Oers MJH. Rituximab treatment results in impaired secondary humoral immune responsiveness. Blood (in press). Van de Kolk LE, Grillo-Lopez AJ, Baars JW, Hack CE, Van Oers MJH. Complement activation plays a key role in the side-effects of rituximab treatment. Br J Haematol (in press). Renal cell carcinoma (RCC) The phase II multi-center study of combined immunotherapy (GM-CSF, low dose IL-2 and IFNα) showed an overall response rate of 19% (eleven of 64 patients) and a relationship between T cell levels but not NK cell levels after immunotherapy and long survival (10+ months), suggesting that T cell activation may be more important than NK cell activation in this disease. A study evaluating the value of PEG-Intron as first line therapy and Temozolomide in IFNα resistant patients was started. Peri-operatieve immunotherapy In a phase I study of escalating doses of GM-CSF, IL-2 and IFNα in (metastatic) renal cell carcinoma or relapsed head and neck carcinoma patients, the maximum tolerated dose has not yet been reached. Apart from prevention of post-operative immune depression, an increase of dendritic cells and CD8+ T-cells between the tumor cells was observed in the surgical specimens. Tumor specific immunity in melanoma patients With the aid of MHC tetramers, peripheral blood samples from stage III 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 MART-1, tyrosinase and gp100 and antigens belonging to the Cancer/Testis group are being examined. In a cohort of 38 HLA-A2+ metastatic melanoma patients, 50% had MART-1, tyrosinase or gp100-specific T cells in the peripheral blood. Interestingly, the presence of melanoma-specific T cells in these patients was associated with the memory T cell phenotype, indicating that these cells 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. Peptide vaccination in metastatic melanoma patients A phase I study is being initiated to test both toxicity and immunity of a peptide-based vaccine regimen in stage IV HLA-A2+ melanoma patients. The peptide vaccine consists of three melanocyte-differentiation antigen (MDA) derived peptides that will be injected together with tetanus toxoid GM-CSF. AUTOLOGOUS HEMATOPOIETIC PROGENITOR CELL TRANSPLANTATION PROGRAM S Rodenhuis, JW Baars, JH Beijnen, JP De Boer, MJ Kersten, JHM Schellens, JH Schornagel High-risk primary breast cancer In 2001, high-dose chemotherapy with peripheral blood progenitor cell transplantation was no longer employed in high-risk breast cancer because of the continuing uncertainty regarding its efficacy. A preliminary analysis of the Dutch randomized study in 885 patients previously showed a trend towards a disease-free survival advantage for patients who had received high-dose therapy. The final analysis of the study is planned for In 2001, essential preparations such as an extensive review of the pathology specimens (JH Peterse, Pathology Department) was done. In addition, an internal audit which included source document verification was performed in the ten collaborating centers.

96 98 MEDICAL ONCOLOGY Director of Research Anton Berns Further maturation of the randomized high-dose studies must be awaited. The current treatment approach in high-risk early breast cancer is a randomized study in which two anthracyline-based pre-operative chemotherapy regimens are being compared (doxorubicin with either cyclophosphamide or docetaxel). Importantly, prechemotherapy biopsies from the primary tumor are taken in order to analyze the RNA-expression profiles. It is hoped that profiles can be identified that predict sensitivity or resistance to specific (groups of) chemotherapeutic agents. Advanced breast cancer A phase II/feasibility study is in progress, that evaluates the effect of weekly treatment with orally administered paclitaxel and cyclosporine A following triple high-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin. The high-dose part of this treatment strategy has been shown to achieve approximately 30% disease-free survival beyond three years in a suitably selected patient group. Based on the first five patients, the weekly paclitaxel is tolerable but leads to marked variations in plasma kinetics between patients. Pharmacokinetics/pharmacodynamics of high-dose therapy The combination of cyclophosphamide, thiotepa and carboplatin (CTC) ranks among the most frequently used high-dose regimens in solid tumors. When peripheral blood progenitor cell transplantation is employed, its myelosuppressive properties are no longer doselimiting and end-organ toxicity such as hemorrhagic cystitis, veno-occlusive disease of the liver and a hemolytic uremic syndrome may occur in some patients, particularly when the courses are repeated. Previous work from our pharmacology group has led to a comprehensive set of analytical methods to obtain detailed pharmacokinetic information of the three compounds and their metabolites. We have now shown that a population pharmacokinetic approach does not accurately predict plasma levels in this group of patients. In an attempt to prevent severe end-organ toxicity by high-dose chemotherapy, therapeutic drug monitoring is now routinely performed in patients who receive CTC. Plasma levels during the first day of the 4-day course are used to adapt the doses on days 3 and 4 to arrive at pre-determined target values. This unique service has also recently been offered to other centers in the Netherlands who employ CTC. THORACIC ONCOLOGY N Van Zandwijk, P Baas, JHM Schellens Non-Small Cell Lung Cancer (NSCLC) Based on the favorable results of our gemcitabine/cisplatin (GC) combination in the pre-operative setting, a following study was designed with GC combined with ZD 1834 (IressaR), an EGFR tyrosine kinase blocking agent. Remarkably swift responses have been observed with this agent in pretreated metastatic NSCLC patients (compassionate use program). Activity in second and third line and the favorable toxicity profile of ZD 1834 prompted us to plan a study in stage IIIA patients within the framework of the EORTC Lung Cancer Group. Dose intensity studies revealed that bi-weekly dosing of GC is better tolerated than a weekly schedule. The bi-weekly regimen also allows the delivery of a higher dose intensity. Attention is now focussing on the effects of drug dose and on the sequence of administration of gemcitabine and cisplatin and their relationships with platinum- DNA adducts in white blood cells (surrogate marker) and tumor response. The international randomized phase II study evaluating the effects of trastuzumab (HerceptinR) added to standard chemotherapy for patients with advanced NSCLC and a positive Her-2 receptor completed accrual. A preliminary analysis did not exclude a favorable effect of trastuzumab administration in individual patients but could also not reveal survival differences between the two arms of the study. The phase II study with oral paclitaxel in pretreated NSCLC patients has been completed successfully. Objective responses were seen in 26% of patients and a comparative study with this well- tolerated regimen for old or unfit patients is being planned. Small Cell Lung Cancer (SCLC) The Phase II (CKVO) study with the carboplatin, etoposide and paclitaxel given concurrently with radiotherapy has accrued 37 patients with limited disease. This combined modality approach is very active (objective

97 99 13 MEDICAL ONCOLOGY response rate: 100%) and a formal comparison with standard therapy is warranted. Unfortunately, previous studies with the CDE regimen, that is still regarded as the standard in the Netherlands, have shown that this regimen cannot be combined with concurrent radiotherapy. Chemoprevention and early detection The analysis of the N-acetylcysteine (NAC) study in healthy smoking volunteers was completed. In this primary preventive setting, NAC was found effective in modulating smoking-associated biomarkers within specific cells (compartments). The study also revealed that the positive effect of NAC on carcinogen-dna adduct formation was influenced by polymorphisms of the detoxification enzyme glutathione-s-transferase, which suggests an NAC-gene interaction. This result and the inability of NAC to prevent second primary tumors in the Euroscan study underline the complexity of the chemopreventive approach. The next (placebo-controlled) phase II study focuses on a similar group of volunteers. A six-months intervention with inhalational fluticasone, active in preclincal models, is being tested. The expressions of p53 and telomerase in bronchial biopsies are being used as intermediate markers and fluorescence bronchoscopy is included to enhance the detection of preneoplastic lesions. Malignant Mesothelioma The combined-modality studies, employing intra-operative photodynamic and hyperthermic chemotherapy in early stage patients advanced significantly in Complete responses and long-term survival have been observed in a subgroup of patients. But it has also become increasingly clear that combined treatment is associated with significant morbidity. Analyses are now being performed to determine whether we are able to predict the risks and benefits on an individual basis. The poor results of chemotherapy and the marked neo-vascularization of mesothelioma formed the basis for a phase II study with the anti-angiogenic agent thalidomide. Over 31 patients have now been included. Side effects of thalidomide are minimal and some patients have reported positive symptomatic effects. It is anticipated that this agent may have a modest effect on the natural course of the disease. The current experience has led to the planning of additional studies with targeted therapy. GASTROENTEROLOGY BG Taal, H Boot, A Cats Esophageal and gastric cancer Narrowing of both the esophagus and the central airways with or without fistulas in advanced esophageal or lung cancer has an enormous impact on the quality of life in patients. Insertion of expandable stents in both trachea and esophagus (interval days) was shown to be feasible and leads to amelioration of dyspnea and improved swallowing in such patients (n=13). The insertion of expandable stents for esophageal carcinoma is now a routine procedure and can be done on an outpatient basis. With technical improvement of delivery systems, expandable stents can also be inserted in the stomach and the duodenum, obviating surgical bypass procedures in the upper GI-tract. Colonic stenting reduces the need for colostomies and stabilizes patients presenting with large bowel obstruction, enabling a more definitive surgical procedure. Chemotherapy with the ECF regimen (epiadriamycin, cisplatin and continuous 5FU) in patients with cancer of the stomach or of the gastro-esophageal junction has been used in approximately 80 patients, with either locally advanced or metastatic disease. An interim analysis of the first 57 patients in this prospective study confirms the response rate of 45% reported in literature. In several patients, surgical resection became possible after chemotherapy and in metastatic disease several long-term remissions were obtained. Breast cancer metastases in the GI-tract In patients with the lobular type of breast cancer, gastric metastases are found more often than large bowel metastases. Symptoms, endoscopic findings and therapeutic results were studied in 51 patients. Metastatic disease at other sites, especially in the skeleton, was found in the vast majority of patients.

98 100 MEDICAL ONCOLOGY Director of Research Anton Berns In a separate study, the toxicity of intra-arterial chemotherapy with mitomycin C for liver metastases was analyzed in 61 patients, in whom a total of 119 infusions were given. In general, toxicity was mild. Abdominal pain grade 3 occurred in only four patients. Endoscopically confirmed gastroduodenal ulceration occurred in four patients. One patient had a fatal duodenal ulcer bleeding. Gastric lymphoma Early gastric low-grade MALT lymphomas regress in about 70% of cases after H. pylori eradication. The stage is the dominant predictive factor. However, for stage I patients additional predictive factors are needed to determine the optimal management. Histological subgrading of low-grade lymphoma and immunological parameters in 23 patients showed that these characteristics can be employed to recognize the transition into an antigen-independent phase. Characteristic chromosomal abnormalities have been described in low-grade gastric MALT lymphoma, especially the t(11;18) translocation. In a large collaborative international study of 111 patients, the presence of this translocation was shown to be the hallmark of stage I low-grade gastric MALT-lymphoma that is resistant to H. pylori eradication. Colorectal cancer Adjuvant chemotherapy is currently the standard in stage III, but not in stage II colonic cancer. An analysis of adjuvant chemotherapy with 5FU/levamisole in the NACCP study, which accrued patients between 1990 and 1995, showed benefit in disease free and overall survival for adjuvant chemotherapy in both stage II and III colonic cancer. In rectal cancer there was no benefit. Hereditary non-polyposis colorectal cancer is caused by different mutations in DNAmismatch-repair genes. In a collaborative study on 79 families of the Netherlands Foundation of the Detection of Hereditary Tumors, it was shown that MSH2 mutation carriers are at higher risk of cancer development than MLH1 mutation carriers. Carcinoid tumors An epidemiologic study showed a female predominance in younger patients with carcinoid disease, suggesting a possible role for hormonal factors. Moreover, the improved survival after 1992 might be related to the clinical introduction of octreotide, enabling better symptomatic treatment and often tumor stabilization.

99 XI DIVISION OF SURGICAL ONCOLOGY SURGICAL ONCOLOGY LYMPHATIC MAPPING BBR Kroon, S Horenblas, EJTh Rutgers, OE Nieweg Lymphatic mapping with sentinel node biopsy can be used to detect tumor involvement of the regional lymph node fields and to select patients for therapeutic regional lymph node dissection and adjuvant systemic and regional therapy. The lymphatic mapping work is a logical constituent 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 subject to investigation within the lymphatic mapping research program are breast cancer, melanoma, carcinoma of the penis and testicular cancer. The general aims of the lymphatic mapping research are improving the technique of lymphoscintigraphy and surgery and determining the clinical benefits: rates of survival and regional control following implementation of lymphatic mapping. An essential element in the program is the multidisciplinary approach. The team involves surgeons, nuclear medicine physicians, pathologists, radiologists and a physicist. Studies are done in cooperation with the cancer collaboration program of the regional hospitals, with The IJsselland Hospital in Capelle aan de IJssel, The Red Cross Hospital in The Hague, Medisch SpectrumTwente, The WHO Melanoma Programme and The John Wayne Cancer Center. A selection of the work is presented here. Breast cancer Controversy over the best technique to perform lymphatic mapping prompted a study of the anatomy and physiology of lymph drainage of the breast. Despite studies spanning two centuries, the intricate lymphatic system of the breast is not completely understood, causing differences of opinion concerning one of the most interesting topics of lymphatic mapping in breast cancer, namely the site of tracer administration. Knowledge of physiological mechanisms underlying tracer clearance from the injection site, lymphatic flow and lymph node uptake is important because of its implications for tracer characteristics and timing of lymphoscintigraphy and surgery. Another study evaluated adjustment in the colloid particle concentration and tracer dosage to optimise mammary lymphoscintigraphy. Scintigraphy was performed in 151 breast cancer patients. For the first 75 patients (group A), a standard labelling of 0.5 mg nanocolloid with 99mTc was performed. For the subsequent 76 patients (group B), the labelling dilution volume was reduced from 4 to 2 ml. The volume of injection was 0.2 ml in both groups. Lymph node uptake was evaluated by a four-step visual score (from 0=absent to 3+=very intense) and by count quantification at four hours in the first draining lymph node. The sentinel node visualisation rate increased from 93% in group A to 99% in group B. The percentage of patients with uptake 3+ was significantly higher in group B (51% vs. 35% in group A, P=0.001). Sentinel node counts were significantly higher in group B in comparison with group A (P=0.0001). More lymph channels were visualised in group B (53% vs. 35% in group A, P=0.033) and for a longer time (26% vs. 4% at four hours). This study shows that enhancement of colloid particle concentration and adjustment of tracer dosage led to improved sentinel node identification by substantial increase in lymph node uptake and lymph vessel depiction. Penile cancer A total number of 90 clinically node-negative patients with squamous cell carcinoma of the penis were prospectively studied. Preoperative lymphoscintigraphy was performed, followed by intraoperative lymphatic mapping with the aid of intradermally administered patent blue dye and a gamma-ray detection probe. Regional lymph node dissection was performed only when metastasis was found in a sentinel node. Lymphoscintigraphy visualised 217 sentinel nodes in 159 inguinal regions of 88 patients. A total of 208 sentinel nodes were intraoperatively identified in 149 inguinal regions of 88 patients. Sentinel node metastasis was found in 19 inguinal regions of 18 patients. Four of eight patients with unilateral clinically N1 Division head, BBR Kroon BBR Kroon MD PhD FRCS Academic staff, Head Board AJM Balm MD PhD FRCS FACS Academic staff, Board member OE Nieweg MD PhD Academic staff, Board member AH Ackerstaff PhD Academic staff A Bex MD PhD Academic staff DR Buitelaar MD Academic staff MPM Burger MD PhD Academic staff MC De Vries, MD Academic staff S Estourgie MD Academic staff IF Faneyte MD Academic staff T Geurts MD Academic staff S Gonggrijp DDS Academic staff JJ Hage MD PhD Academic staff FJM Hilgers MD PhD Academic staff S Horenblas MD PhD Academic staff JM Huitink MD PhD Academic staff LM Janssen MD Academic staff R Kaas MD Academic staff H Klomp MD Academic staff FHM Kroon DDS PhD Academic staff AP Lont MD Academic staff W Meinhardt MD PhD Academic staff EM Noorda MD Academic staff HSA Oldenburg MD PhD Academic staff R Postema MD Academic staff JM Ronday MD Academic staff EJTh Rutgers MD PhD FRCS Academic staff PFE Schutte MD Academic staff IB Tan MD PhD Academic staff PJ Tanis MD Academic staff AP Timmers DDS Academic staff CJ Van As PhD Academic staff M Van Beurden MD PhD Academic staff F Van Coevorden MD PhD Academic staff MWM Van den Brekel MD PhD Academic staff HG Van der Poel MD PhD Academic staff VLM Vander Poorten MD Academic staff N Van der Vange MD PhD Academic staff JF Van Hagen MD Academic staff ACM Van Lindert MD PhD Academic staff S Van Ruth MD Academic staff J Van Sandick MD PhD Academic staff J Visscher MD Academic staff AJ Witkamp MD Academic staff LAE Woerdeman MD Academic staff FAN Zoetmulder MD PhD Academic staff C Bei MD Fellow PJFM Lohuis MD PhD Fellow Th Van Dalen MD PhD Fellow

100 102 SURGICAL ONCOLOGY Director of Research Anton Berns V Verwaal MD Fellow J Wijsman MD PhD Fellow YIZ Acherman MSc Undergraduate student JDH De Vries MSc Undergraduate student C Dekker MSc Undergraduate student R Houtkamp MSc Undergraduate student S Klein MSc Undergraduate student ELAR Mutsaerts MSc Undergraduate student GK Roozendaal MSc Undergraduate student D Van Poll MSc Undergraduate student S Veerman MSc Undergraduate student GMM Van Zuilen Secretary stage had a tumor-positive sentinel node on the opposite site. During a median follow-up of 32 months (range 3-91), regional recurrence after excision of a tumor-negative sentinel node was seen in two patients. This resulted in a false-negative rate of 10% (2/20). Three-year disease specific survival was 98% and 71% for patients with a tumor-negative or tumor-positive sentinel node respectively (P=0.0018). This study shows that occult lymph node metastases in penile cancer can be detected with a sensitivity of 90% by lymphatic mapping and sentinel node biopsy. This approach allows for performing early lymphadenectomy in most lymph node-positive patients and provides important prognostic information. Lymph node-negative penile cancer patients can be spared substantial morbidity associated with elective lymphadenectomy. BREAST CANCER HSA Oldenburg, EJTh Rutgers Detection method and staging of breast cancer in BRCA-1 and BRCA-2 mutation carriers Breast cancer in BRCA-1 and BRCA-2 mutation carriers develops at a relatively young age, tends to be high grade, quickly growing and difficult to discern on mammography because of dense breast tissue. We examined the hypothesis that mammography may be an insufficient screening technique for such tumors. A retrospective study was carried out to determine the method that identified the cancer and to determine its stage. All carriers were followed according to a protocol that consisted of monthly breast self-examination, clinical breast examination every six months and annual mammography. A total of 29 breast cancers were detected in 28 mutation carriers (one bilateral). Of these lesions, 15 were first breast cancers and 14 were contralateral cancers that developed during follow-up for an earlier cancer. Selfexamination or physical examination at the clinic revealed 72% of the cancers, 21% were found with mammography and the remaining 7% by MRI. In half of the patients, the tumor was not visible on mammography. The average tumor size was 1.8 cm (+/- 0.9 cm). The smaller lesions were encountered in mammographydetected cancers. One lesion was an in situ carcinoma. Lymph node metastases were absent in 72% of the patients. Stage distribution does not appear to differ from the population screening experience. It is concluded that breast self-examination and clinical examination improve early detection in this population, as only 21% of the cancers were detected by mammography. Stage in women screened because of a positive family history does not differ from stage in population screening Stage distribution of breast cancers detected during screening in women at increased risk for developing breast cancer was analysed. Two groups at high risk were retrieved from the breast screening clinic database, group I (BRCA-1/2 mutation carriers, n=29) and group II (women with a life-time risk of at least 15%, n=61). A total of 91 cancers was present in these 90 patients, 51 first cancers and 40 contralateral additional breast cancers. These patients were compared to the recently published results of the Dutch Population Screening Program. The mean age in group I was 43 years and the mean age in group II was 54 years. In the Dutch Screening Program, the mean age is more than 60 years. In spite of the younger age, stage distribution in the high-risk groups was found to be comparable to that of the population screening: stage ptis and pt1 was 66% in group I, 79% in group II and 78% in the population screening group. The percentages for pn0 were 72, 69 and 70 respectively. The BRCA-1/2 cancers had a substantially higher grade and mitotic index as compared to group II: grade III 79% versus 31% and mean mitotic index 22.7 (±13.2) versus 5.9 (±7.8). Three of the 90 patients have died, two of breast cancer and one of oral cancer. Screening for breast cancer in high-risk women appears to be as effective as screening of the general population. A retrospective search of all breast cancer patients treated at The Netherlands Cancer Institute between 1989 and 1998 revealed 95 patients with multifocal disease. Nodal metastases were found in 61 of 93 axillary clearances (66%). Even in patients in whom both tumors were smaller than 2 cm, the node positive rate was 56% (22/39). The risk of nodal involvement compared unfavorably to patients with unifocal cancer: 21% of T1

101 SURGICAL ONCOLOGY cancers and 40% of T2 tumors were node-positive in a database of 25,000 patients (SEER database). A higher risk of nodal involvement in multifocal breast cancer means that less patients will benefit from sentinel lymph node biopsy. All patients who had node-positive disease received adjuvant systemic treatment. Of the 32 node-negative patients, 20 did not receive adjuvant systemic treatment and they all remained diseasefree after a median follow-up of 50 months. The twelve remaining patients did receive systemic treatment and three relapsed. We conclude that multifocal cancer does not require a separate approach with regard to adjuvant systemic treatment. HEAD AND NECK ONCOLOGY AJM Balm, FJM Hilgers, IB Tan, MWM Van den Brekel Rehabilitation In the ongoing research projects on rehabilitation after total laryngectomy, voice quality assessment in tracheoesophageal speech was further evaluated. Forty patients were studied using a sustained /a/. Four acoustic signal types, based on a narrow-band spectrogram, were developed for better visual representation of voice quality. Three pitch dependent acoustic parameters (fundamental frequency, standard deviation of fundamental frequency and jitter) could be determined in 77% of the voice samples, whereas four pitch independent measures (percentage of voiced, harmonics-to-noise ratio, glottal-to-noise excitation ratio, band energy difference) could be calculated for the entire patient group (Figure XI.1). The four signal types were significantly related to the overall judgment of voice quality as good, rea- Key publications Full papers Horenblas S. Lymphadenectomy for squamous cell carcinoma of the penis. Part 1: diagnosis of lymph node metastasis. Br J Urol Int 2001; 88: Horenblas S. Lymphadenectomy for squamous cell carcinoma of the penis. Part 2 diagnosis of lymph node metastasis. Br J Urol Int 2001; 88: Horenblas S, Meinhardt W, IJzerman W, Moonen LMF. Sexuality preserving cystectomy and neobladder: initial results. J Urol 2001; 166; Karim RB, Hage JJ, Ahmed AKJ, Westerga J. Pedicled scrotal island skin flap in treatment of anal basal cell carcinoma: Report of an extraordinary case. Br J Plast Surg 2001; 54: Koppe M, Zoetmulder FAN, Van Zandwijk N, Hart AAM, Baas P, Rutgers EJTh. The prognostic significance of a previous malignancy in operable non-small cell lung cancer. Lung Cancer 2001; 32: Mutsaerts EL, Zoetmulder FAN, Meijer S, Baas P, Hart AA, Rutgers EJ. Outcome of thoracoscopic pulmonary metastasectomy evaluated by confirmatory thoracotomy. Ann Thorac Surg 2001; 72: Nieweg OE, Tanis PJ, Kroon BBR. The definition of a sentinel node. Ann Surg Oncol 2001; 8: Schouwink H, Rutgers ET, Van der Sijp J, Oppelaar H, Van Zandwijk N, Van Veen R, Burgers S, Stewart FA, Zoetmulder FAN, Baas P. Intraoperative photodynamic therapy after pleuropneumonectomy in patients with malignant pleural mesothelioma: dose finding and toxicity results. Chest 2001; 120: Figure XI.1: Acoustic signal types I II III IV: in the top bar the oscillogram of the signal is shown, below that a 100 ms selection of the oscillogram, followed by the result of pitch extraction, the narrow-band spectrogram showing the harmonics, and, in the lower bar, the long-term average spectrum giving an indication of the high-frequency noise in the signal. Signal type I is stable and harmonic (stable signal for longer than 2 seconds, and clear harmonics up to at least 1000 Hz); Signal type II is stable and at least one harmonic (stable signal for longer than 2 seconds, and at least one stable harmonic at the fundamental frequency for longer than 2 seconds); Signal type III is unstable or partly harmonic (no stable signal for longer than 2 seconds, or harmonics in only part of the sample (for longer than one second)); Signal type IV is barely harmonic (no or only short-term detectable harmonics (shorter than 1 second)). Also, the poorer the signal type, the more energy is present in the high frequency noise range (as can be seen in the lower bar, showing the long-term average spectrum). Smit M, Balm AJM, Hilgers FJM, Tan IB. Pain as a sign of recurrrent disease in head and neck squamous cell carcinoma. Head Neck 2001; 23: Strackee SD, Kroon FHM, Bos KE. Fixation methods in mandibular reconstruction using fibula grafts: a comparative study into the relative strength of three different types of osteosynthesis. Head Neck 2001; 23:1-7.

102 104 SURGICAL ONCOLOGY Director of Research Anton Berns Key publications Strobbe LJA, Jonk A, Hart AAM, Peterse JL, Wobbes Th, Nieweg OE, Kroon BBR. The value of Cloquet s node in predicting melanoma nodal metastases in the pelvic lymph node basin. Ann Surg Oncol 2001; 8: Tanis PJ, Nieweg OE, Valdés Olmos RA, Kroon BBR. Anatomy and physiology of lymphatic dranaige of the breast in perspective of sentinel node biopsy. J Am Coll Surg 2001; 192: Tanis PJ, Nieweg OE, Valdés Olmos RA, Rutgers EJTh, Kroon BBR. History of sentinel node and validation of the technique. Breast Cancer Res 2001; 3: Valdés Olmos RA, Tanis PJ, Hoefnagel CA, Jansen L, Nieweg OE, Meinhardt W, Horenblas S. Penile lymphoscintigraphy for sentinel node identification. Eur J Nucl Med 2001; 28: Valdés Olmos RA, Tanis PJ, Hoefnagel CA, Nieweg OE, Muller SA, Rutgers EJTh, Kooi MLK, Kroon BBR. Improved sentinel node visualization in breast cancer by optimizing the colloid particle concentration and tracer dosage. Nucl Med Commun 2001; 22: Van As CJ, Op de Coul BMR, Van den Hoogen FJA, Koopmans-Van Beinum FJ, Hilgers FJM. Quantitative videofluoroscopy: a new evaluation tool for tracheoesophageal voice production. Arch Otolaryngol Head Neck Surg 2001; 127: Van Veen SJAM, Balm AJM, Valdés Olmos, Hoefnagel CA, Hilgers FJM, Tan IB, Pameijer FA. Occult head and neck primaries: accuracy of 201Thallium SPECT and CT/MRI. Arch Otolaryngol Head Neck Surg 2001; 127: Witkamp AJ, De Bree E, Kaag MM, Van Slooten GW, Van Coevorden F, Zoetmulder FAN. Extensive surgical cytoreduction and intraoperative hyperthermic intraperitoneal chemotherapy in patients with pseudomyxoma peritonei. Br J Surg 2001; 88: sonable or poor (p<.001). Significant correlations were found between the acoustic parameters and the perceptual judgments ( pitch and fundamental frequency; voice quality and percentage of voiced, harmonics-to-noise ratio and band energy difference; tonicity and glottal-to-noise excitation ratio). These results show that both these acoustic signal types and the acoustic measures are related to perceptual evaluation of voice quality and thus can be used for objective analyses of tracheoesophageal voice quality in future studies. Results of olfaction rehabilitation after total laryngectomy were further evaluated. A new odor test (Zürcher Geruchstest) appeared to be useful for this type of research in laryngectomized individuals. Furthermore, consistent positive long-term results were found in almost half of the patients, at the same time underlining the necessity of a more vigorous training program to ensure consequent use of the recently developed nasal airflow inducing method, in order to achieve compensation for the loss of passive smelling. Under the N00VOX protocol, a new pulmonary and vocal rehabilitation tool was developed, enabling handsfree speech after total laryngectomy, while at the same time ensuring optimal pulmonary protection and rehabilitation. A prospective clinical trial with 20 patients showed a high compliance rate with significant improvement in maximum phonation time and dynamic range. A multicenter trial is planned for Evaluation of treatment strategies Operation reports and postoperative wound drainages of 389 neck dissections ( ) were analyzed. In 22 patients a chylus leakage was peroperatively closed (including nine local Tissucol applications) by using 5x0 atraumatic Prolene (n = 20) and Vicryl (n=2). Six patients developed a postoperative leakage. Remarkably, three out of 23 patients with a preventively tied thoracic duct showed postoperative chylus leakage as well. In six cases postoperative chylus leakage occurred without identification of a peroperative leakage. Parenteral feeding was needed in two cases and one patient underwent renewed operative closure. No serious (>1l/24hr) protracted wound drainages (> 15 days) were observed. Atraumatic closure of thoracic duct endings by using 5x0 Prolene in case of macroscopic peroperative chylus leakage seems effective in 2/3 of the cases. Local application of Tissucol has no added effect. A cyto-histologic correlation study was performed in 1023 patients with a salivary gland lesion, including 373 cases with histological follow up. The positive predictive values for malignant and benign cytologies were 95% and 99% respectively. In case of the cytological diagnosis cyst four out of 19 cases proved to be malignant. These results demonstrate an improvement of cytological accuracy in our institute.1 Twenty-two patients with a cytologically confirmed neck node metastasis of unknown primary origin were examined with Short Tau Inversion Recovery (STIR) MRI (coronal and/or axial STIR, axial and/or coronal T1WI with fat suppression before and after intravenously administered contrast and axial T2WI). Two radiologists interpreted MRI scans (+/-STIR) independently from each other. Ten out of 22 patients had biopsy-proven carcinoma. The sensitivity of STIR MRI was 90% and 80% for the two observers versus 20% and 50% without STIR (Figure XI.2a,b). STIR MRI seems a helpful tool for the detection of unknown primary head and neck tumors. Epidemiology To re-evaluate earlier described associations between salivary gland tumors and breast cancer 2,3 a database of 439 salivary gland tumor patients ( ) was analyzed with a median follow-up duration of 5.5 years. Fifteen patients (pleomorphic adenoma n=8; adenoid cystic carcinoma n=1; adenocarcinoma n=1; basal cell adenoma n=1; Warthin s tumor n=1; muco-epidermoid carcinoma n=2; acinic cell carcinoma n=1) developed a breast cancer. On the basis of incidence rates in the general population, only 6.07 were to be expected. The relative risk is 2.47 (95% confidence interval, ; P=0.003). Patients with a salivary gland tumor have a more than two-fold increased risk of developing breast cancer. Intensified screening of these patients is recommended. REFERENCES 1 Van Heerde P, Peterse JL. Fine needle aspiration cytology of salivary glands. Verh. Dtsch. Ges. Zyt. 1993; 18: Moertel CG, Elveback LR. The association between salivary gland cancer and breast cancer. JAMA 1969; 210:306-8.

103 SURGICAL ONCOLOGY Key publications Full papers in press Dorresteijn LDA, Kappelle AC, Boogerd W, Klokman WJ, Balm AJM, Keus RB, Van Leeuwen FE, Bartelink H. Increased risk of ischemic stroke after radiotherapy on the neck. J Clin Oncol (in press). Figure XI.2a: Nasopharyngeal carcinoma (long arrow) and neck node metastasis (short arrow) as visible on MRI STIR coronal view 3 Dunn JE, Bragg KU, Sautter C, Gardipee C. Breast cancer risk following a major salivary gland carcinoma. Cancer 1972; 29: INTRACAVITARY THERAPY FAN Zoetmulder Figure XI.2b: Small superficially growing nasopharyngeal carcinoma (arrow) detected by guidance of MRI STIR (Figure XI.2a) Hyperthermic Intra Peritoneal Chemotherapy (HIPEC) Phase III study in peritoneal carcinomatosis of colorectal origin) During 2001 the entry of patients in the randomized phase III study comparing cytoreduction and HIPEC plus systemic chemotherapy with systemic chemotherapy alone in patients with peritoneal carcinomatosis of colorectal origin was completed, with a total of 105 patients being randomized. The main goal of this study was survival. Quality of life and cost-efficacy were secondary goals in this study. A first report was prepared based on 94 patients with a minimal follow-up of three months. Forty-six patients were treated in the control arm and 48 in the HIPEC arm. Kaplan-Meier survival curves are shown in Figure XI.3. The results demonstrate an increase of the median survival from one year in the control arm to two years in the HIPEC arm (P=0.0181) and an estimated long-term survival of 25%. The final report on this study including cost efficacy and quality of life, will be prepared by early The results of this study show that peritoneal carcinomatosis is potentialy curable similar to liver metastases and lung metastases. Another study related to HIPEC concerns pharmacokinetics of intra-peritoneal Mitomycine C in relation to local and systemic toxicity, with the aim to develop a dosing schedule that limits some of the variations observed in the present schedule based on body surface. A third study concerns penetration depth of intra-abdominal hyperthermia. Hilgers FJM, Jansen HA, Van As CJ, Polak MF, Muller MJ, Van Dam FSAM. Longterm results of olfaction rehabilitation in laryngectomized individuals using the nasal airflow-inducing polite yawning maneuver. Arch Otolaryngol Head Neck Surg (in press). Karim RB, Hage JJ, Ahmed AKJ, De Wit FS, Van de Sandt MM, Daemen A. Digital photography as a means to enhance interconsultant communication in oncologic cutaneous surgery. Ann Plast Surg (in press) Kummer EE, Rasch CRN, Keus RB, Tan IB, Balm AJM. T-stage as prognostic factor in irradiated localized squamous cell carcinoma of the nasal vestibule. Head Neck (in press). Spermon JR, Roeleveld TA, Van der Poel HG, Hulsbergen-Van de Kaa CA, Ten Bokkel Huinink WW, Van de Vijver M, Witjes JA, Horenblas S. Comparison of surveillance and retroperitoneal lymph node dissection in stage I non-seminomatous germ cell tumors. Urol (in press). Strackee SD, Kroon FHM, Jaspers JEN, Bos KE. Modeling a fibula transplant in mandibular reconstructions: evaluation of the effects of a minimal number of osteostomies on the contour of the jaw. J Plast Surg (in press). Hyperthermic Intra Thoracic Chemotherapy (HIThoC) A phase I dose finding study using Cisplatin (80 mg/m2) and Doxyrubicin (15-35 mg/m2) was carried out in stage I malignant mesothelioma patients. After cytoreductive surgery, the thoracic cavity was perfused for 90 minutes at a temperature of 42 C. The approach proved feasible. The area under the curve (AUC) of Doxorubicin in the perfusate was 200 to 250 times the AUC in plasma. No systemic toxicity of either Cisplatin or Doxorubicin was observed. After 20 patients the study had to be stopped at a dose level of 35 mg/m2 of Doxorubicin due to lack of funding. UROLOGICAL ONCOLOGY A Bex, S Horenblas, W Meinhardt, HG Van der Poel Minimally invasive surgery (Laparoscopic surgery) Laparoscopic surgery is becoming increasingly important in urological oncology. Nowadays, daily practice consists of laparoscopic lymph node dissection for staging bladder and prostate can-

104 106 SURGICAL ONCOLOGY Figure XI.3: Kaplan-Meier curves of patients with peritoneal carcinomatosis demonstrating the survival probability after HIPEC ( ) compared with control patients ( ) cer and laparoscopic removal of kidneys with or without the ureter. Three clinical trials are ongoing. In order to reduce post radiation symptoms in prostate carcinoma patients, a trial was initiated in collaboration with the radiotherapy department. Patients at risk of involvement of seminal vesicles, assessed on the basis of PSA, T- stage and Gleason score, undergo a laparoscopic excision of the seminal vesicles as an extension of the laparoscopic lymphadenectomy. This allows the radiation oncologist to reduce the radiation field with 30% on average. This procedure appeared to be feasible in the first ten patients. The second clinical trial concerns the value of laparoscopic mobilization of the seminal vesicles together with a perineal prostatectomy to decrease erectile dysfunction after total prostatectomy by means of better preservation of the autonomic nerves. The third trial is part of the sentinel node testis pilot study. After identification of the sentinel node in the retoperitoneum the node is removed via laparoscopic route. Feasibility was proven in one patient. The rationale of the trial is better guidance of management in clinical stage I testicular tumors, irrespective of histological diagnosis. These patients can be spared unnecessary treatment, surgery or radiation therapy leading to overtreatment in 70%-80% of them, if the nodes are free of metastasis. GYNECOLOGICAL ONCOLOGY MPM Burger, M Van Beurden, ACM Van Lindert, N Van der Vange Imiquimod in the treatment of multifocal Vulvar Intra-epithelial Neoplasia (VIN) The efficacy of topical treatment with Imiquimod 5% cream, an immune response modifier, was tested in patients with VIN II/III. Fifteen women (age 35 to 51) with histologically proven multifocal VIN II/III, without invasiveness, were entered into a prospective observational pilot study. The patient herself applied Imiquimod 5% cream on the vulvar lesions, (1 3x/week). Clinical response was analyzed by macroscopic examination. A complete response was found in four patients (27%) and a partial response in nine patients (60%) after six to 34 weeks of treatment. Two patients discontinued treatment. This pilot study shows a potential beneficial effect of Imiquimod 5% cream in multifocal VIN II/III, preserving the anatomy and function of the vulva. A prospective, randomized, double-blind, placebo-controlled clinical trial has been started. Psychosocial consequences of preventive health options among women at increased risk of developing ovarian cancer Women at increased familial or

105 SURGICAL ONCOLOGY genetic risk of (breast and) ovarian cancer may choose to undergo a prophylactic oophorectomy or annual gynecological screening. We investigated the psychosocial consequences of these two preventive health strategies and the sociodemographic, clinical and psychosocial factors related to the choice between these strategies. The study sample included 36 women who attended the Department of Gynecology between 1995 and 1999 because of familial or hereditary (breast-) ovarian cancer. The sample was stratified according to preventive health strategy and personal history of breast cancer. In total 29 women (81%) completed a self-administered questionnaire by mail. Sixteen of them had undergone a prophylactic oophorectomy and 13 had opted for periodic screening. Women who had undergone an oophorectomy reported, on average, significantly less cancer-related worries, less generalized distress, fewer intrusive thoughts about cancer and perceived their risk of developing (extra-) ovarian cancer as lower than did women who opted for screening. Women who had undergone preventive surgery were also significantly more satisfied with their choice of preventive health action as compared to those who opted for screening. In a logistic regression model, only two variables were found to be significantly associated with the choice of prophylactic oophorectomy: mutation status (DNA carriership) and an active coping style characterized by information-seeking behavior. This pilot investigation is currently being replicated in a larger, multicenter study. PLASTIC AND RECONSTRUCTIVE SURGERY JJ Hage, LAE Woerdeman Breast reconstruction Since April 2000, the clinical research program focuses prospectively on the use of the lateral thoraco-dorsal flap for primary or secondary breast reconstruction. So far, no such series have been reported outside of Sweden, where the technique was developed. The purpose of study goes beyond confirmation of the Swedish results as it seeks to establish the span of indications for and versatility of this flap and its reliability in irradiated and non-irradiated patients. The study is being continued within the scope of a Ph-Doctorate. Likewise, all patients undergoing direct reconstruction after skin-sparing mastectomy (prophylactic or curative) are prospectively studied. The series is unique as all patients are operated on in accordance to a surgical protocol, thereby reducing the variables drastically. Skin-sparing mastectomy is a frequently used procedure in The Netherlands Cancer Institute allowing a rapid build-up of a large series. As is often the case in studies performed in the Netherlands, stringent long-term follow-up is performed. The purpose of this study is to establish risk factors for post-operative complications, in particular the loss of implant or tissue expander, and the long-term cosmetic outcome. The data on a similar but retrospective study on reconstruction after skin-sparing mastectomy are being processed. Head and neck reconstructions The research program of reconstructive head and neck surgery focused on the use of the fibula free flap for mandibular reconstruction. Bone staples made of a nickel titanium alloy exert dynamic compression, require little dissection, and may provide an alternative to conventional fixation in mandibular reconstruction with a free vascularized fibula graft. To evaluate its stability relative to conventional methods of fixation with interosseous wires or miniplates, an in vitro model using beech dowels was developed. Torsional stiffness and strength, and compression stiffness and strength were examined. The compression test results showed that maximum strength of interosseous wires is significantly less than memory staples, which in turn are significantly weaker than titanium miniplates. Miniplates are the most rigid form of fixation. Torsional testing showed no significant difference in strength between staples and miniplates and only a marginal difference in elasticity. Interosseous wires show a rapid decrease in strength and rigidity during torsional stress. It was concluded that memory staples can provide enough stability to ensure consolidation, provided that interosseous wires are considered the least, and miniplates the most stable form of fixation by which bone healing can occur. Peripheral arterial occlusive disease (PAOD) or congenital anomalies of the major crural arteries may limit the use of the fibula free flap and should be detected preoperatively. Conventional selective angiography is the gold standard for this, but it has

106 108 SURGICAL ONCOLOGY Director of Research Anton Berns its drawbacks. A safer, cheaper, accurate and non-invasive alternative is desirable. In 2001, research was done to test the hypothesis that the ankle-arm index (AAI) of each of the three crural arteries combined with pencil-doppler examination of the peroneal skin perforators provides adequate information to restrict angiography to cases where either or both are insufficient. Data on the AAI of each of the three crural arteries and pencil-doppler examination of the peroneal skin perforators of both legs of nine prospectively included patients and of the non-operated legs of 13 retrospectively included patients were compared statistically with the findings of preoperative angiography in four different ways. It was concluded that a combined AAI and pencil-doppler examination is not accurate enough to detect legs or arteries with subclinical PAOD or vascular variation and, hence, is not sufficient to base the surgical plan of a fibula free flap on. Thirdly, the outcome of reconstructive surgery after RadPlat therapy was evaluated in To improve treatment outcome for the patients with stage IV head and neck cancers and to prevent mutilating surgery, recent clinical trials have focused on nonsurgical treatment combining supradose intra-arterial targeted cisplatin therapy with concomitant radiotherapy (RadPlat). The outcome of 25 reconstructive procedures as performed in 21 patients treated with intensive chemoradiation was investigated. Six patients underwent seven flap reconstructions after additional neck dissections for persistent or recurrent lymph node involvement, whereas 15 patients had 18 flap reconstructions following salvage surgery for recurrent local disease or osteoradionecrosis. Twenty-one of the 25 flaps remained fully viable but the postoperative course was uneventful after only nine of the reconstructive procedures. We feel that the complications were directly related to poor tissue conditions of the receptor area treated with RadPlat in the other twelve cases. Surgeons should be aware that the incidence of wound complications is increased after RadPlat therapy, and surgical procedures should be planned with caution and tissue handled accordingly. Figure XI.4: Normal vascular anatomy of the crural arteries and congenital vascular anomalies that may limit the use of the fibula free flap (left to right): normal vascular anatomy; class IIIA vascular anomaly; class IIIB anomaly; class IIIC anomaly; class IV anomaly. In class IIIC legs use of the peroneal artery (pr) for fibula free flap transplantation would lead to an ischaemic foot; in class IV, the fiblua free flap lacks a vascular pedicle.

107 XII DIVISION OF PSYCHOSOCIAL RESEARCH AND EPIDEMIOLOGY PSYCHOSOCIAL RESEARCH PSYCHOSOCIAL ONCOLOGY Health-related quality of life assessment (HRQL) in clinical practice An observational study was conducted to investigate oncologist-patient communication regarding four HRQL issues (daily activities, emotional functioning, pain and fatigue.) Ten oncologists and 240 of their patients receiving outpatient, palliative chemotherapy were included in the study. Data were collected via questionnaires and audiotapes of medical consultations. The results indicated that physicians devoted 64% of their conversation to medical/technical issues and 23% to HRQL issues. Patients communication behavior was divided more equally between these two broad topics. Self-reported HRQL was the strongest predictor of discussing HRQL issues. In 20% to 54% of the consultations no time was devoted to discussing patients (serious) HRQL problems. Discussion of HRQL issues was not found to be related significantly to evaluation of tumor response. In a prospective, randomized study we evaluated the efficacy of incorporating standardized HRQL assessments as a routine part of outpatient palliative chemotherapy treatment in terms of two primary outcomes: 1) facilitating doctor-patient communication, and 2) increasing physicians awareness of patients physical and psychosocial health problems. The study included ten medical oncologists and 214 of their patients treated with palliative chemotherapy. For each patient, four follow-up consultations were audiotaped. Physicians and patients rated the patients HRQL on the COOP/WONCA charts. Patients physical and psychosocial functioning and symptoms were discussed significantly more frequently in the intervention group than in the control group. Physicians in the intervention condition identified a significantly greater percentage of patients with moderate to severe problems in several HRQL domains than did those in the control condition. Incorporating standardized HRQL assessments in daily clinical oncology practice facilitates the discussion of HRQL issues and heightens physicians awareness of their patients problems. Division head, Group leader N Aaronson N Aaronson PhD Group leader I Kluijt MD Academic staff F Menko MD, PhD Academic staff J Schornagel MD, PhD Academic staff B Taal MD, PhD Academic staff H Valdimarsdottir PhD Academic staff M Van Beurden MD, PhD Academic staff S Verhoef MD Academic staff E Bleiker PhD Post-doc M Klein PhD Post-doc S Detmar MSc Graduate student J Hollenstein MSc Graduate student R Hoopman MSc Graduate student R Knols MSc Graduate student J Madalinska MSc Graduate student M Muller MSc Statistical analyst S Celik Research assistant M Gerritsma Research assistant F El Haddouchi Research assistant M Idrissi Research assistant M Chin-A-Kwie Secretary Figure XII.1

108 110 PSYCHOSOCIAL RESEARCH Director of Research Anton Berns Key publications Aaronson NK, Fayers P. Quality of life. In Souhami RL, Tannock I, Hohenberger P, Horiot JC, editor. Oxford Textbook of Oncology, 2nd Edition. Oxford: Oxford University Press 2001; Detmar SB, Muller MJ, Wever LD, Schornagel JH, Aaronson NK. The patientphysician relationship. Patient-physician communication during outpatient palliative treatment visits: an observational study. JAMA 2001; 285: Detmar SB, Muller MJ, Wever LDV, Schornagel JH, Aaronson NK. The role of health-related quality of life in palliative chemotherapy treatment decisions. J Clin Oncol (in press). Fossa SD, Slee PH, Brausi M, Horenblas S, Hall RR, Hetherington JW, et al. Flutamide versus prednisone in patients with prostate cancer symptomatically progressing after androgen-ablative therapy: a phase iii study of the European organization for research and treatment of cancer genitourinary group. J Clin Oncol 2001; 19: Klein M, Taphoorn MJ, Heimans JJ, Der Ploeg HM, Vandertop WP, Smit EF, et al. Neurobehavioral status and health-related quality of life in newly diagnosed high-grade glioma patients. J Clin Oncol 2001; 19: Langendijk JA, Aaronson NK, De Jong JM, Ten Velde GP, Muller MJ, Lamers RJ, et al. Prospective study on quality of life before and after radical radiotherapy in non-smallcell lung cancer. J Clin Oncol 2001; 19: Langendijk JA, De Jong J, Tjwa M, Muller M, Ten Velde G, Aaronson NK, et al. External irradiation versus external radiation in inoperable non-small cell lung cancer: A prospective randomised study. Radiother Oncol 2001; 58: Sneeuw KC, Albertsen PC, Aaronson NK. Comparison of patient and spouse assessments of health related quality of life in men with metastatic prostate cancer. J Urol 2001; 165: The HRQL and cognitive functioning of patients with high-grade glioma In 2001, we completed the analysis of baseline (post-surgical) data from a longitudinal study of the HRQL and neuropsychological status of patients with high-grade glioma (HGG). The study sample included 68 newly diagnosed HGG patients, 50 patients with non-small cell lung cancer (NSCLC), and 68 age- and gender-matched healthy controls. The HRQL of the two patient groups, as assessed by the SF-36, was similar, but significantly lower than that of the healthy controls. HGG patients reported significantly more neurological symptoms, and poorer objective and subjective neuropsychological functioning than the NSCLC patients. Cognitive impairment assessed at the individual patient level was observed in all glioma patients and in 52% of the NSCLC patients. Cognitive functioning was related significantly to tumor lateralization, but not to the extent of resection. Corticosteroid use was associated with better recognition memory, while antiepilectic drug use was correlated negatively with working memory. Neuropsychological functioning of mid-to long-term survivors of low-grade A cross-sectional study of the neuropsychological functioning of patients treated previously for low-grade glioma (LGG) was completed. A total of 195 LGG patients was compared with 100 low-grade hematological patients and 195 age- and gendermatched healthy controls. LGG patients had lower performance levels on all cognitive domains than low-grade hematological patients, and performed even worse when compared to healthy controls. Radiotherapy (RT) was associated with poorer cognitive functioning, in general. However, cognitive disability was found only in patients who had received RT fraction doses exceeding 2 Gy. Antiepileptic drug use was associated strongly with disability in attentional and executive function. These results suggest that the tumor itself has the most deleterious effect on cognitive function and that RT does not lead to additional long-term cognitive disability, unless elevated fraction doses are used. The impact of antiepileptic drugs on cognitive functioning merits further study. Changes in physical functioning and quality of life in patients with cancer: response shift and relative evaluation of one s condition This study examined the hypothesis that patients persistence in comparing themselves with others who are perceived as being worse off might induce a response shift in self-reported quality of life (QL). Specifically, we tested whether patients ratings of how they are doing as compared to others (i.e., relative evaluation) moderates the link between physical deterioration and decline in self-reported QL. A heterogenous sample of patients undergoing palliative chemotherapy (N=240) rated their physical functioning and QL (i.e., emotional functioning and global quality of life) twice, with an interval of three months. Consistent with the buffering model of response shift, patients who felt that they were better off than others appeared to be able to sustain their quality of life in the face of a worsening physical condition. In contrast, those who felt the same or worse off than others could not. The HRQL of non-small cell lung cancer (NSCLC) patients receiving radical radiotherapy This study investigated changes in respiratory symptoms and HRQL among 164 NSCLC patients receiving radical radiotherapy (60 Gy). HRQL assessments were made pretreatment, and at 2, 6, 26 and 52 weeks post-treatment. The HRQL response rates were relatively high for hemoptysis (83%), chest pain (68%), arm/shoulder pain (60%), but low for dyspnea (37%), cough (31%) and fatigue (28%). The HRQL response for functional scales of the QLQ-C30 varied between 35% for physical and role functioning to 55% for social and cognitive functioning. A significant, albeit modest association was observed between tumor response and HRQL response. HRQL assessment among ethnic minority cancer patients In 2001, data collection continued for a study whose primary aims are: (1) to translate, adapt and validate two widely used generic QL questionnaires (the SF-36 and the COOP/WONCA 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 mem-

109 PSYCHOSOCIAL RESEARCH ber) ratings of these patients HRQL. To date, 65 Turkish and 36 Moroccan patients have been recruited into the study from seven hospitals. This project will facilitate inclusion of ethnic minority cancer patients in HRQL investigations, and will provide a wealth of descriptive information on the impact of cancer and its treatment on the functional health and well being of these populations. Key publications (continued from page 112) Hilgers FJ, Jansen HA, Van As CJ, Polak MF, Muller MJ, Van Dam FSAM. Longterm results of effective rehabilitation in laryngectomized individuals using the nasal airflow-inducing polite yawning maneuver. Archives of Otolaryngology Head & Neck Surgery (in press). Schagen SB, Hamburger HL, Muller MJ, Boogerd W, Van Dam FS. Neurophysiological evaluation of late effects of adjuvant high-dose chemotherapy on cognitive function. J Neurooncol 2001; 51: Schagen SB, Boogerd W, Muller MJ, Rodenhuis S, Van Dam FSAM. Central neurotoxicity following adjuvant chemotherapy in breast cancer patients. Anemia & Quality of Life in Oncology 2001; 4:7-10. The psychosocial and behavioral impact of genetic counseling for colorectal cancer (CRC): a prospective, multicenter study This multicenter (AvL, AZVU, AMC, RUG and LUMC) study is: 1) assessing the effect of genetic counseling/testing on risk perception, distress, family relationships, work and family and financial planning; 2) identifying risk factors for poor psychological adjustment to and early withdrawal from the genetic counseling process; and 3) establishing rates of short-term compliance with recommended screening practices. Questionnaires are administered at three points in time: after the first counseling session, three weeks and sixmonths after the final genetic counseling session. To date, 443 individuals have been recruited into the study. Data collection and analysis are on-going. The physical and psychosocial impact of gynecological screening or prophylactic oophorectomy among women from hereditary breast/ovarian (HBOC) cancer families This cross-sectional, multicenter (AvL, AZVU and AMC) study is investigating: 1) the decision-making process surrounding the choice of preventive health actions 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 of hormone-replacement therapy. In 2001, 443 questionnaires have been returned (75% response rate). Data collection and analysis are on-going. De Wit R, Van Dam F, Loonstra S, Zandbelt L, Van Buuren A, Van der Heijden K, Leenhouts G, Kuijer Abu-Saad H. The Amsterdam Pain Management Index compared to eight frequently used outcome measures to evaluate the adequacy of pain treatment in cancer patients with chronic pain. Pain 2001; 91: Van der Zouwe N, Van Dam FSAM. De betekenis van alternatieve behandelwijzen voor patiënten met kanker. In: De Haes JCMJ, Gualthérie van Weezel LM, Sanderman R, Van der Wiel HBM, (redactie). Psychologische patiëntenzorg in de oncologie. Assen: Koninklijke van Gorcum 2001: Client satisfaction with family cancer clinics This study is evaluating client satisfaction with the care provided at three family cancer clinics in Amsterdam (AvL, AZVU and AMC). All individuals who complete the genetic counseling process are asked to complete a questionnaire immediately following completion of the counseling process and six months later. The first questionnaire has been completed by 271 individuals; the second by 220. Data collection and analysis are on-going.

110 112 PSYCHOSOCIAL RESEARCH Director of Research Anton Berns SYMPTOM PERCEPTION AND MANAGEMENT IN CANCER PATIENTS Group leader F Van Dam F Van Dam PhD Group leader W Boogerd MD, PhD Academic staff F Hilgers MD, PhD Academic staff S Rodenhuis MD, PhD Academic staff J Van As MSc Academic staff A Vielvoye-Kerkmeer MD, PhD Academic staff M De Rond MSc Graduate student B Kreukels MSc Graduate student S Schagen MSc Graduate student H Andersson Undergraduate student K Douma Undergraduate student H Faasen Undergraduate student J Fortuin Undergraduate student B Heemskerk Undergraduate student B Huinink Undergraduate student J Jansen Undergraduate student S Koemans Undergraduate student T Overbeek Undergraduate student M Puts Undergraduate student N Rozenschoon Undergraduate student J Versloot Undergraduate student M Zantvoord Undergraduate student A Donker Registered nurse H Houer Registered nurse S Oppeneer Registered nurse K Van Rooijen Registered nurse M Muller MSc Statistical analyst A Biervliet Research assistant R Kuiper Research assistant KEY PUBLICATIONS (continued on page 111) De Rond M, De Wit R, Van Dam F. The implementation of a Pain Monitoring Programme for nurses in daily clinical practice: results of a follow-up study in five hospitals. J Adv Nurs 2001; 35: De Wit R, Van Dam F, Loonstra S, Zandbelt L, Van Buuren A, Van der Heijden K et al. Improving the quality of pain treatment by a tailored pain education program for cancer patients in chronic pain. Eur J Pain 2001; 5: De Wit R, Van Dam F, Litjens MJ, Van Buuren A, Van der Heijden K, Zandbelt L et al. Assessment of pain cognitions in cancer patients with chronic pain. J Pain Symp Man 2001; 22: Neuropsychological and neurophysiological evaluation of cognitive deficits after chemotherapy The impact of cytostatic drugs on the patients cognitive functioning is evaluated with neuropsychological and neurophysiological techniques. In the main neuropsychological study we are investigating cognitive functioning prior to and following (adjuvant) chemotherapy in high-risk breast cancer patients and highgrade lymphoma patients. Neuropsychological status is assessed at three points in time with a standard battery of tests. Patient accrual started in April 1998 and has finished in December In total 750 patients have been entered in this study. Recently, a cross-sectional study was finished in a group of patients with non- Hodgkin lymphoma treated with CHOP-chemotherapy using a standard neuropsychological test battery and a quality of life questionnaire. The results of this patient group (n = 54) were compared with a control group of patients of a matched group of healthy controls matched in age and gender. The patients were tested on average four years after completion of treatment. Patients reported significantly more complaints on the EORTC QLQ-C30 regarding cognitive, physical, emotional functioning and fatigue than controls. There were no differences with regard to anxiety and depression between the two groups. There were no significant differences in the number of cognitively impaired patients and controls as measured with the neuropsychological test battery. It was concluded that cognitive complaints of NHL patients treated with CHOP-chemotherapy were not reflected in cognitive functioning tests. The objective of this four-year study is to gain better understanding of these effects by studying specific stages of information processing using event-related potential measurements 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. Olfactory rehabilitation after total laryngectomy In cooperation with head and neck surgeons and speech therapists, a method to rehabilitate olfaction in laryngectomized patients has been developed and evaluated. This Nasal Airflow Inducing Maneuver (NAIM), can be learned, in a short period of time, usually 30 minutes of training are sufficient. Training programs have been set up and carried out in the Netherlands and abroad to teach speech therapists in this technique. The need for care by palliative patients being treated at the outpatient department Most cancer patients are being treated in the outpatient department and day-care. Compared to the hospital situation, nurses and doctors of the outpatient department and day-care, have only limited time to help patients cope with the adversities of their disease and treatment. We developed a simple method to be used by nurses of the outpatient department to trace those patients in need of extra help. An implementation study has been started in which we will develop care-protocols attuned to the needs of patients in the home situation. The emphasis will be on educating patients and their next of kin on how to cope with their symptom. Why do patients with localized prostate cancer choose for radiotherapy or surgery? Eighty-six patients treated for a localized prostate cancer, stage T1 or T2 with radiotherapy or surgery have been interviewed with regard to their motivation in choosing either radiotherapy or surgery. Patients came from the Netherlands Cancer Institute as well as two regional hospitals. Patients who chose for surgery assumed (wrongly) that their survival would be better than with radiotherapy, whereas patients who chose for radiotherapy were motivated to do so by their fear of the operation itself and the side-effects of a prostatectomy.

111 11313 PSYCHOSOCIAL RESEARCH EPIDEMIOLOGY 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. We examined cancer risk in a nation-wide historical cohort of 25,152 women treated for subfertility in the Netherlands between 1980 and In total, 19,136 women received one or more cycles of IVF and 6,016 did not undergo IVF. Treatment data were collected from the medical records of the twelve participating clinics and all women were asked to complete a mailed risk factor questionnaire. Cancer incidence through 1997 was ascertained through linkage with the populationbased Netherlands Cancer Registry. After a median follow-up of 5.7 years, the incidence rates of endometrial cancer and melanoma were greater than expected on the basis of population rates, both in the IVF group among unexposed controls (overall relative risks of 3.0 and 1.5, respectively). However, direct comparisons with our control group revealed no increased risks for breast, endometrial and ovarian cancer and melanoma associated with IVF. 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. Our first objective was to study whether women with a low number of retrieved oocytes at first IVF treatment more often have an early age at menopause. Women with a poor response (0-3 oocytes) at their first IVF attempt had an 11.6-fold elevated risk of having an early menopause as compared to women with a normal response (> 3 oocytes). Alerted by two case reports, we also examined in this cohort whether the risk of hypospadias was increased in male offspring from women exposed to diethylstilbestrol (DES) in utero. There were 205 boys whose mother reported DES exposure in utero. Four of these children were reported to have hypospadias. Among all other 8,729 children, only eight cases of hypospadias were reported, resulting in a significant 21-fold increased risk of hypospadias among DES grandsons. This first observation of a transgenerational effect of DES in humans warrants additional studies. In 2001, we continued our cohort study on hormone-related cancer risk in women who were exposed to diethylstilbestrol (DES) in utero. In total, 13,373 DES daughters registered at The Netherlands DES Center were sent a risk factor questionnaire. Up to now, 7697 questionnaires have already been received; non-responders are now being approached by telephone. In a preliminary analysis we investigated the prevalence of hypospadias among the 3,020 sons of the first 5,459 responding DES daughters. In accordance with the prevalence found in the IVF study (19.5/1000) we found a prevalence of 16.6/1000 sons, supporting an increased risk of hypospadias among DES grandsons. On the basis of one of our population-based case-control studies we collaborated in a meta-analysis on familial breast cancer. It was found that during their lifetime women with one or two affected first-degree relatives had a 5.5% or 13.3% higher risk of breast cancer, respectively. We have continued the accrual of participants for our national cohort study in breast and/or ovarian cancer families, aimed at: 1) examining whether hormonal/life-style factors modify cancer risk in BRCA1/2 and non-brca1/2 families; 2) assessing the age-specific cumulative risks of breast, ovarian and other cancers based on full pedigree information. So far, for 507 BRCA1/2 and 573 non-brca1/2 families, clinical, pedigree and DNA information has been collected comprising 59,738 family members. 440 families have been approached to complete a mailed questionnaire on hormonal/life-style factors. Up to now, 2539 questionnaires have been received with an overall response rate of 81.3%. We also have contributed data to an international cohort study among 971 BRCA1/2 mutation carriers, co-ordinated by IARC, Lyon. We just started data collection for a case-control study of young sister-pairs (n=130, non-brca1/2) 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 well as environmental factors, that might play a role in the etiology of breast cancer. Group leader F Van Leeuwen Group leader M Rookus M Rookus PhD Group leader F Van Leeuwen PhD Group leader B Aleman MD Academic staff N Russell PhD Academic staff E Rutgers MD, PhD Academic staff L Van t Veer PhD Academic staff S Verhoef PhD Academic staff R Brohet MSc Graduate student E De Boer MSc Graduate student M Hooning MD Graduate student H Klip MPH Graduate student C Ronckers MSc Graduate student A Van Rosmalen MSc Graduate student J Verloop MSc Graduate student R Bergsma Undergraduate student M Busstra Undergraduate student K Janssen Undergraduate student M Van de Kerkhof Undergraduate student H Van Onzenvoort Undergraduate student W Klokman MD, MSc Statistical analyst V Hartog Research assistant N Hofland Research assistant E Janssen Research assistant M Kuenen Research assistant M Legdeur Research assistant B Maertzdorf Research assistant M Van Baalen Research assistant A Van den Belt-Dusebout Research assistant A Van der Meij Research assistant M Wolfers Research assistant Late effects of cancer treatment Now that curative treatment is available for a substantial group of cancer patients, it is increasingly important to evaluate how the occurrence of late complications of treatment affects their long-term survival. Our

112 114 PSYCHOSOCIAL RESEARCH Director of Research Anton Berns Key publications Dorresteijn LD, Kappelle AC, Boogerd W, Klokman WJ, Balm AJ, Keus RB, et al. Increased risk of ischemic stroke after radiotherapy on the neck. J Clin Oncol 2002; 20: Klip WH, Burger CW, De Kraker J, Van Leeuwen FE. Risk of cancer in the offspring of women who underwent ovarian stimulation for IVF. Human Reproduction. 2001; 16: Noorda EM, Somers R, Van Leeuwen FE, Vulsma T, Behrendt H. The Dutch Late Effects Study Group. Adult height and age at menarche in childhood cancer survivors. Eur J Cancer 2001; 37: Ronckers CM. Long-term health effects of nasopharyngeal radium irradiation. A retrospective cohort study in the Netherlands. Thesis 2001, Amsterdam. Ronckers CM, Land CE, Verduijn PG, Hayes RB, Stovall M, Van Leeuwen FE. Cancer mortality after nasopharyngeal radium irradiation in the Netherlands; a cohort study. J Nat Cancer Inst 2001; 93: Treffers PE, Hanselaar AG, Helmerhorst ThJ, Koster ME, Van Leeuwen FE. Gevolgen van diëthylstilbestrol in de zwangerschap: na 50 jaar nog steeds een actueel probleem. Ned Tijdschr Geneeskd 2001; 145: Van Leeuwen FE, Schornagel JH. Bewaren of vernietigen? Het belang van het medisch dossier voor de patiënt van gisteren en morgen. Ned Tijdschr Geneeskd 2001; 145: first aim is to evaluate the risk of second cancers in 5-year survivors of Hodgkin s disease (HD) (n=2650), testicular cancer (n=2250) and breast cancer (n=8000) over a period of up to 30 years after primary treatment. The second aim is to evaluate the risk of vascular disease. In 2001, we collected treatment and follow-up data on 3300 breast cancer patients treated between 1976 and 1985 in The Netherlands Cancer Institute or the Dr Daniel den Hoed Cancer Center/EUR. A preliminary analysis in 1500 breast cancer patients showed no significant effect of radiotherapy on cerebrovascular mortality as a late complication of breast cancer treatment. We have also studied cerebrovascular mortality after treatment for HD in a cohort of 1300 patients treated before age 40 between 1966 and With a median followup of 28 years, cerebrovascular mortality was increased 3.6-fold compared to general population rates. Our previous studies showed that female survivors of HD have a highly increased risk of breast cancer. We conducted a case-control study in a cohort of 2500 patients treated for HD between 1966 and Treatment information was collected for 48 cases of breast cancer and 175 matched controls, in whom breast cancer had not developed. For each case-control set, the radiation dose to the area of the breast where the case s tumor had developed was estimated on the basis of radiotherapy charts, simulation films of all radiation treatments and mammograms. The risk of breast cancer increased significantly with increasing radiation dose (p trend= 0.01), with a relative risk of 4.5 for patients who received 38.5 Gy or more, as compared to those who received less than 4 Gy. Patients who received chemotherapy and radiotherapy had significantly decreased risk as compared to those treated with radiation alone. The risk reduction from CT could be attributed to an effect of CT on ovarian function. Apparently, ovarian hormones are necessary to propel tumorigenesis once radiation has produced an initiating event. In close collaboration with the Reinaert Clinic in Maastricht and the US National Cancer Institute we conducted a nationwide cohort study to assess long-term cancer risk among 5358 patients who have undergone nasopharyngeal radium irradiation (NR) and a control group of 5265 patients treated without radiation for ear-nosethroat conditions. The average radiation doses were 275, 11, 1.8 and 1.5 cgy for nasopharynx, pituitary, brain and thyroid, respectively. Cancer incidence was ascertained through a linkage with The Netherlands Cancer Registry. Among NR-exposed subjects, 168 invasive tumors were observed (O) after a median follow-up time of 32 years, compared to expected (E) based on sex-, age- and calendar specific reference data (O/E=1.2). In accordance with our mortality analyses, statistically significantly elevated risks were observed for breast cancer (O/E=1.5) and non- Hodgkin s lymphoma (O/E=2.3), but not for head-and-neck cancers (O/E=1.4). The risk for thyroid cancer was not significantly increased (O/E = 2.8). Strong doseresponse trends could not be demonstrated for any cancer although relative risks were significantly elevated in the highest dose category for head-and-neck cancer and breast cancer. Since the median attained age of the cohort was 41 years, prolonged follow-up for cancer incidence in this cohort is warranted. Van Leeuwen FE, Travis LB. Second cancers. In: DeVita Jr VT, Hellman S, Rosenberg SA, editors. Cancer Principles and Practice of Oncology. Philadelphia, PA: Lippincott Williams & Wilkins 2001; 6: Figure XII.3

113 XIII DIVISION OF DIAGNOSTIC ONCOLOGY DIAGNOSTIC ONCOLOGY DEPARTMENT OF CLINICAL CHEMISTRY HA Bardelmeijer, T Buckle, CGJ Cleypool, EM Kemper, CM Korse, TC Linders, M Ouwehand, HR Van der Woude, JMG Bonfrer, WJ Nooijen, O Van Tellingen Pharmacological studies in mice We have unravelled the mechanisms underlying the poor oral bioavailability of docetaxel using wild-type (WT) and P-glycoprotein knockout (KO) mice. The drug transporter P-glycoprotein (Pgp) is a less important transporter for docetaxel than for paclitaxel, as a substantial fraction of a 10 mg/kg dose (about 60%) is absorbed from the intestines. However, very efficient first-pass metabolism by cytochrome P450 (CYP) enzymes and hepato-biliary excretion results in very low systemic levels after oral docetaxel. In vitro transport studies using Pgp transfected pig renal epithelial cells and studies in Pgp deficient fibroblasts have confirmed that docetaxel is a weaker substrate than paclitaxel. It has been postulated that the concerted action of Pgp and CYP builds the barrier for uptake of substances from the intestines with Pgp limiting the amount of drug reaching CYP, thus preventing its saturation. The important contribution of first-pass metabolism in limiting the oral bioavailability is highlighted by the effect of ritonavir. The concomitant use of this very potent CYP inhibitor increases the oral bioavailability by more than 50-fold (Figure XIII.1). We have further explored the brain penetration of paclitaxel by the concomitant use of so called third generation P-gp blockers, including LY335979, R and XR9576. Using these potent blockers it remained impossible to achieve brain levels of paclitaxel similar to those reached in KO mice. Building on the results from our studies with oral docetaxel, which suggest that this is a much weaker substrate than paclitaxel, we are currently testing whether this taxane derivative can be delivered more easily to the brain. In addition, we are investigating the metabolites, formed by CYP isoenzymes of paclitaxel and docetaxel. Tumor markers In our institute 63 patients with pseudomyxoma peritonei were treated with cytoreductive surgery combined with HIPEC. The serum for the mea- Figure XIII.1: Plasma concentration of docetaxel in wild type (closed symbols) and Pgp knockout mice (open symbols) receiving 10 mg/kg orally with (squares) or without (circles) 12.5 mg/kg of ritonavir. The apparent oral bioavailability of docetaxel increases by more than 50-fold when using this potent cytochrome P450 inhibitor. Division head, MJ Van de Vijver MJ Van de Vijver MD PhD Academic Staff, Head APE Besnard MD Academic Staff JMG Bonfrer PhD Academic staff D De Jong MD PhD Academic Staff KGA Gilhuijs PhD Academic Staff CA Hoefnagel MD PhD Academic Staff FBL Hogervorst PhD Academic Staff E Joekes MD Academic Staff W Koops MD Academic Staff R Kröger MD Academic Staff WJ Mooi MD PhD Academic Staff S Muller PhD Academic Staff PM Nederlof PhD Academic Staff WJ Nooijen PhD Academic staff FA Pameijer MD PhD Academic Staff JL Peterse MD Academic Staff LJ Schultze Kool MD PhD Academic Staff F Sivro MD Academic Staff MJA Smid-Geirnaerdt MD PhD Academic Staff LMTh Sterk MD Academic Staff RA Valdés Olmos MD PhD Academic Staff O Van Tellingen PhD Academic staff L Van t Veer PhD Academic Staff MLF Van Velthuysen MD PhD Academic Staff S Verhoef MD PhD Academic Staff A Glas PhD Post-doc K Van der Kooy PhD Post-doc HA Bardelmeijer Graduate student EE Deurloo MD Graduate student EM Kemper Graduate student HE Klaren MD Graduate Student BGH Roelofs Undergraduate student R Tolboom Undergraduate student MBM Verheij Undergraduate student D Atsma Technicical Staff LH Boerrigter-Barendsen Technical Staff T Buckle Technical Staff CGJ Cleypool Technical Staff L Delahaye Technical Staff W Klein Zeggelink Technical Staff CM Korse Technical Staff TC Linders Technical Staff M Ouwehand Technical Staff CPE Ottenheim Technical Staff J Poodt Technical Staff R Pruntel Technical Staff L Ran Technical Staff R Regnerus Technical Staff

114 116 DIAGNOSTIC ONCOLOGY Director of Research Anton Berns C Schippers-Gillissen Technical Staff A Witteveen Technical Staff A Schlief Technical Staff HR Van der Woude Technical Staff M Van Vliet Technical Staff G Brink Quality Staff M Bronk Genetic Consultant A Van Rens Genetic Consultant G Wigbout Genetic Consultant C Arkes Secretary surement of CEA and CA19.9 was collected before therapy and at 3-monthly intervals during follow-up. Pre-operative CEA and CA19.9 levels were elevated in respectively 75% and 58% of the patients. At two years the recurrence-free percentage is 94% (SE 10%) for the 25 patients with normal baseline CA19.9, 55% (SE 17%) for the 20 patients with CA19.9 between 37 and 200 and 37% (SE 17%) for the 15 patients with CA19.9 over 300 (p=0.012). During follow-up, elevated CA19.9 levels (p=0.0005) or further increase of the marker are predictive of imminent recurrence (p<0.0001). The median lead-time of elevated CA19.9 to recurrence is nine months. CA19.9 appears to be a more useful tumor marker than CEA for follow-up. Serum samples of 79 patients with non-hodgkin s lymphoma (NHL) obtained during their first visit at our institute were used to compare Thymidin Kinase (TK) and scd27 with standard used LDH and β2m. The median serum TK level in stage I/II was 8 U/L and in stage III/IV 12 U/L (p=0.011). For scd27 the median serum level in stage I/II and III/IV were, respectively, 160 ku/l and 514 ku/l (p<0.001). For LDH and β2m the difference in serum levels with regard to stage was only significant for β2m (p=0.011). ROC curves were made for TK, scd27, β2m and LDH to demonstrate their ability to distinguish between low (I and II) and high stage (III and IV). Areas under the curves (AUC) were (p<0.001) for scd27 and (p=0.007) for β2m in the indolent group. For TK (0.634) and LDH (0.502) the AUC were not significant. The ROC curves for scd27 and TK showed that both could discriminate between low and high stage aggressive tumors (AUC=0.71; p=0.03). For patients with low serum scd27 levels 91% was free of progression after three years. For patients with intermediate elevated levels 64% and for the high scd27 level patients the three year survival was only 8% (p<0.001). DEPARTMENT OF PATHOLOGY D Atsma, LH Boerrigter-Barendsen, L Delahaye, A Glas, FBL Hogervorst, K Van der Kooy, WJ Mooi, JL Peterse, J Poodt, C Schippers-Gillissen, MLF Van Velthuysen, A Witteveen, D De Jong, PM Nederlof, L Van t Veer, MJ Van de Vijver Genetic alterations in tumors The Department of Pathology, 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 and these are described in that chapter. The main focus within the department in the past year has been on setting up the logistics needed to perform gene expression profiling using micro array analysis on tumor samples from our tissue bank. This is an extension of the Markerstudy, our effort to make DNA and RNA from various tumor types available for research purposes. The first large study using micro array analysis of breast cancer is described in the next paragraph. A number of projects involving expression profiling of various tumor types in collaboration with the NKI micro-array facility are presently being started. Gene expression profiling of breast cancer In a collaboration with Rosetta Inpharmatics (Kirkland, Washington, USA) we have used gene expression profiling with DNA microarrays on primary breast tumors of young patients. We selected tumors from 98 patients with primary breast cancer: 34 from sporadic lymph node negative patients who had developed distant metastases within five years, and 44 from sporadic patients who continued to be disease-free after a period of at least five years. In addition, 18 tumors from patients with BRCA1 germline mutations, and two from BRCA2 mutation carriers were selected. RNA from each tumor was amplified and crna 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 regulated across the group of samples, i.e., there was at least a two-fold difference with a significant p-value of less than 0.01 in more than five tumors. The overall gene expression patterns were examined by two-dimensional clustering analysis. Two distinct groups of tumors are the dominant feature in this two-dimensional display, which differ in ER status and lymphocytic infiltration. The 78 sporadic patients were all younger than 55 years and specifically selected to search for a

115 DIAGNOSTIC ONCOLOGY prognostic signature in their gene expression profiles. This group consisted of 44 patients who had remained disease-free after their initial diagnosis for an interval of at least five years ( good prognosis group, mean follow-up 8.7 years) and 34 patients who had developed distant metastases within five years ( poor prognosis group, mean time to metastases 2.5 years). To unambiguously discriminate between good and poor prognostic tumors, we employed a supervised classification method. Using this method, 70 genes were identified which optimally predict which tumors will metastasize. The expression pattern of the 70 genes in the 78 samples is shown in the plot of Figure XIII.2 (left panel), where tumors were rank ordered according to their correlation coefficient with the average good prognosis profile (Figure XIII.2, middle panel). Thus, the classifier correctly predicted the actual outcome of disease Key publications Bardelmeijer HA, Ouwehand M, Beijnen JH, Schellens JHM, Van Tellingen O. Determination of cyclosporin A in human and mouse plasma by reversed-phase highperformance liquid chromatography. J Chromatogr B Biomed Sci Appl 2001; 763: Bijker N, Peterse JL, Duchateau L, Julien JP, Fentiman IS, Duval C et al. Risk factors for recurrence and metastasis after breastconserving therapy for ductal carcinoma-insitu: analysis of European Organization for Research and Treatment of Cancer Trial J Clin Oncol 2001; 19: Bijker N, Peterse JL, Duchateau L, Robanus-Maandag EC, Bosch CA, Duval C et al. Histological type and marker expression of the primary tumour compared with its local recurrence after breast-conserving therapy for ductal carcinoma in situ. Br J Cancer 2001; 84: Deurloo EE, Gilhuijs KGA, Schultze Kool LJ, Muller SH. Displacement of breast tissue and needle deviations during stereotactic procedures. Invest Radiol 2001; 35: Hoefnagel CA, Rutgers M, Buitenhuis CKM, Smets LA, De Kraker J, Meli M, Carrel F, Amstutz H, Schubiger PA, Noval-Hofer I. A comparison of targetting neuroblastoma with mibg and anti L1- CAM antibody mab chce7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients. Eur J Nucl Med 2001; 28: Figure XIII.3: Supervised classification for the identification of prognosis signatures in breast cancer. (a) The use of prognostic reporter genes to optimally identify two types of disease outcome of 78 sporadic breast tumours into a poor prognosis group and a good prognosis (b) Expression data matrix for tumours of 78 breast cancer patients over the 70 optimal prognostic marker genes (left panel). Each row represents a tumor and each column a gene. Genes were ordered according to their correlation coefficient with the two prognostic groups. The prognostic classifier was defined at two threshold settings: optimal for accuracy. Above the dashed line, patients have a good prognosis signature and below the dashed line a poor prognosis signature. The metastasis status for each patient is shown in the right panel with white for patients who presented with distant metastases within 5 years after the primary diagnosis and black for patients who continued to be disease-free for at least 5 years. (c) Similar to b, but for the expression data matrix of tumors of 19 additional breast cancer patients using the same 70 optimal prognostic marker genes. Thresholds in the correlation coefficients (solid and dashed line) are identical to b. Kemper EM, Jansen B, Brouwer KR, Schellens JH, Beijnen JH, Van Tellingen O. Bioanalysis and preliminary pharmacokinetics of the acridonecarboxamide derivative GF in plasma of mice and humans by ion-pairing reversed-phase highperformance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Appl 2001; 759: Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A. Loss of p16ink4a confers susceptibility to metastatic melanoma in mice. Nature 2001; 413:83-6. Mooi WJ. Histopathology of Spitz naevi and Spitzoid melanomas. Curr Top Pathol 2001; 94:65-77.

116 118 DIAGNOSTIC ONCOLOGY Director of Research Anton Berns Key publications Van Tellingen O. The importance of drugtransporting P-glycoproteins in toxicology. Toxicol Lett 2001; 120: Valdés Olmos RA, Nieweg OE. Reproducibility of cutaneous lymphoscintigraphy: same or different lymphatic routes after reinjection? J Nucl Med 2001; 42: Valdés Olmos RA, Tanis PJ, Hoefnagel CA, Jansen L, Nieweg OE, Meinhardt W, Horenblas S. Penile lymphoscintigraphy for sentinel node identification. Eur J Nucl Med 2001; 28: Valdés Olmos RA, Tanis PJ, Hoefnagel CA, Nieweg OE, Jansen L, Muller SH, Rutgers EJTh, Kooi MLK, Kroon BBR. Improved sentinel node visualization in breast cancer by optimizing the colloid particle concentration and tracer dose. Nucl Med Commun 2001; 22: Valdés Olmos RA, Hoefnagel CA, Nieweg OE. Optimized mammary lymphoscintigraphy using larger colloid particles. J Nucl Med 2001; 42:826. Van t Veer LJ, Dai H, Van de Vijver MJ, He YD, Hart AAM, Mao M, Peterse HL,Van der Kooy, Marton MJ, Witteveen AJ, Schreiber GJ, Kerkhoven R, Roberts C, Linsley PS, Bernards R, Friend SH. Gene expression profiling of breast cancer accurately predicts clinical outcome of disease. Nature (in press). Van Veen SAJM, Balm AJM, Valdés Olmos RA, Hoefnagel CA, Hilgers FJM, Tan IB, Pameijer FA. Occult primary tumors of the head and neck: accuracy of Thallium-201 single-photon emission computed tomography and computed tomography and/or magnetic resonance imaging. Arch Otolaryngol Head Neck Surg 2001; 127: Voogd AC, Nielsen M, Peterse JL, Blichert-Toft M, Bartelink H, Overgaard M et al. Differences in risk factors for local and distant recurrence after breast-conserving therapy or mastectomy for stage I and II breast cancer: pooled results of two large European randomized trials. J Clin Oncol 2001; 19: for 65 of the 78 patients (83%), with respectively five poor prognosis and eight good prognosis patients assigned to the opposite category. An additional independent set of primary invasive tumours of 19 young breast cancer patients was selected to further validate the prognosis marker genes and the classifier. This group consisted of seven patients that remained metastasis-free for at least five years and the twelve patients that developed distant metastases within five years. The disease outcome was predicted for each of these 19 patients by the classifier as established above. Using the optimal accuracy threshold 3.2 patients would be expected to be misclassified and using the optimized sensitivity threshold, 3.7 patients would be expected to be misclassified in this set of 19 patients. The actual number of misclassified patients was two in both cases. Thus, the classifier showed a comparable performance on the validation set of 19 independent sporadic tumors and thereby confirmed the predictive power and robustness of prognosis classification using the 70 optimal marker genes. Using multivariate analysis, it was shown that the prognosis classification was independent of clinical and pathological factors. Using supervised cluster analysis, 550 genes that optimally report the dominant pattern associated with ER status were identified. BRCA1 is associated with low expression of the oestrogen receptor, but within our ER-negative group, nearly half of the tumors are from patients without known BRCA1 germline mutations. Within the group of ER-negative tumors, as defined by the ER classifier, we used supervised classification and identified a BRCA1 classifier consisting of an optimal set of 100 genes. Malignant lymphoma In an international collaborative study, 111 patients with stage I gastric MALT-lymphoma who were treated with H.pylori eradication only were studied for the presence of the t(11;18) translocation. The presence of t(11;18) was highly predictive for treatment failure after H.pylori eradication with 67% translocation-positive non-responsive cases (42/63), while only 2/48 responders carried the translocation. Moreover, translocation and non-responsiveness was paralleled by nuclear expression of bcl-10. In view of the recently identified immunoglobulin and T-cell receptor signalling pathway, linking the MALT-1 and bcl-10 proteins in activating NFkB, these findings may give a basis for therapy selection of patients as well as for the development of novel therapy approaches. DEPARTMENT OF NUCLEAR MEDICINE APE Besnard, E Deurloo, CA Hoefnagel, SH Muller, F Sivro, RA Valdés Olmos Three protocols integrating 131I-MIBG therapy in the management of neuroblastoma Within the Department of Nuclear Medicine, there is extensive experience with 131I-MIBG therapy in the treatment of neuroblastoma. Based upon the results of preoperative 131I-MIBG therapy in a mixed group of patients with inoperable neuroblastoma, two new collaborative studies have been initiated. Patients with unresectable neuroblastoma stage II and III will receive two cycles of 131I-MIBG therapy only prior to surgery; if inoperablity persists, chemotherapy will be added. Patients with neuroblastoma stage IV will receive upfront 131I-MIBG therapy combined with Topotecan. Postoperatively, a single high dose of Carboplatin and Melphalan therapy with peripheral blood stem cells rescue will be aimed at treating minimal residual disease, after which 13-cis retinoic acid will be prescribed as a differentiation therapy for two years. For patients with recurrent neuroblastoma the protocol using 131I-MIBG therapy in combination with oxygen therapy under hyperbaric conditions and with vitamine C has been continued. Sentinel node biopsy in breast cancer Mammary lymphoscintigraphy by single intratumoral injection has been found to be highly reproducible in 25 patients with T 1 -T 2 N 0 breast cancer. The first lymphoscintigraphy was performed after injection of 131 MBq (range MBq) 99mTc nanocolloid in a 0.2 ml volume. A mean dose of 133 MBq (range MBq) in a similar volume was re-injected the following day. Both series of lymphoscintigrams were identical with regard to lymphatic flow pattern and draining lymph node basin as well as location and number of sentinel nodes in all 25 patients (reproducibility 100%). Axillary drainage was seen in 24 patients

117 119 DIAGNOSTIC ONCOLOGY Figure XIII.2: Anterior lymphoscintigraphy with 99m Tc-nanocolloid showing drainage from the injection site in the left testis (T) to sentinel lymph nodes of the left para-aortic region. and extra-axillary basins were found in eight patients. In 29 of 400 patients (7,3%) with T 1 -T 3 N 0 breast cancer prone lateral images, performed in addition to standard anterior images, led to the localization of intramammary sentinel lymph nodes. Isolated intramammary metastases were found in three patients and in another three patients they were found together with axillary sentinel node metastases. Intratumoral tracer administration guided by ultrasound or stereotaxis was evaluated in 60 patients with clinically occult breast tumor. Lymphoscintigraphy, performed after administration of a mean dose of 122 MBq 99mTc nanocolloid (range MBq), led to sentinel node visualisation in 56 patients (93%). A high incidence of extra-axillary sentinel nodes (43%) was found. Patients underwent both sentinel node biopsy and gammaprobe guided tumorexcision the day after scintigraphy. Feasibility of testis lymphoscintigraphy The feasibility of lymphoscintigraphy for sentinel node identification was assessed in five patients with clinical stage I testicular cancer. After a funicular block with lidocain 2%, a mean dose of 99mTc nanocolloid in a 0.2 ml volume was injected into the funiculus in the first patient and intratesticular in the following four patients. The funicular administration route led to visualization of only inguinal lymph nodes reflecting lymphatic drainage from the scrotum. Intratesticular administration resulted in visualization of retroperitoneal located lymph nodes in three patients (Figure XIII.3). THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC G Brink, DM Bronk, HE Klaren, D Hahn, CPE Ottenheim, R Pruntel, L Ran, R Regnerus, A Van Rens, G Wigbout, FBL Hogervorst, MA Rookus, LJ Van t Veer, S Verhoef Studies on cancer prone families Since 1995, over 800 families (approximately 2500 individuals) have sought genetic advice in our hospital. DNA testing revealed a germline mutation in 130 breast cancer families (BRCA1/BRCA2) and 35 colon cancer families (mismatch repair genes: hmlh1/hmsh2/hmsh6), including testing for families counselled at the Academic Medical Hospital, Amsterdam (I Kluyt). About 25% of the individuals that initially seek genetic advice decide to discontinue their counseling procedure at an early stage. In a prospective multicenter study, the psychosocial and behavioral impact of the counseling procedure for colorectal cancer is being studied (see Division XII). Furthermore, in a nation-wide cohort study (see Division XII) cancer risk estimates, genotype-phenotype correlations and gene-environment interactions are assessed for BRCA1 and BRCA2 families. In order to prevent cancer, approximately 180 high-risk women have opted for either a prophylactic bilateral mastectomy, an oophorectomy or both. Mastectomy in the group of BRCA1/BRCA2 carriers has reduced their risk for breast cancer with at least 67%. Oophorectomy results in a significant risk reduction for ovarian cancer (HR=0.04; 95%CI ) as well as for breast cancer (HR=0.47; 95%CI ) (International Collaboration, Study Coordinator T Rebbeck, University of Pennsylvania, USA). BRCA1 and BRCA2 carrier assessment and clinical data evaluation in a large unselected series of breast cancer patients (~6000, diagnosed between 1970 and 1995), will provide us with unbiased survival data for gene carriers as compared to non-carriers. At present, DNA testing in archival material and a multi-center protocol have been realized. DEPARTMENT OF RADIOLOGY EE Deurloo, W Klein Zeggelink, S Muller, M Van Vliet, KGA Gilhuijs, LJ Schultze Kool Isolated hepatic perfusion for treatment of liver metastases: optimization and efficacy-testing of a novel, percutaneously applicable perfusion technique

118 120 DIAGNOSTIC ONCOLOGY Director of Research Anton Berns (PIHP) This project is a fruitful collaboration with Tollenaar and Van de Velde from Leiden University Medical Center (LUMC). At LUMC, there is extensive experience in treating patients with isolated liver metastases of colorectal carcinomas not amenable to surgical resection with isolated liver perfusion after laparatomy. This project aims to develop percutaneous techniques for this procedure, thus avoiding laparotomy. As a first step in developing the treatment for clinical purposes, we have studied the uptake of melphalan after different time points in the rat model with CC531 tumors implanted in the liver, when melphalan was infused in the hepatic artery during either retrograde or normal single pass liver perfusion. It was shown that tumor uptake was unaffected by perfusion direction. Uptake in hepatic tissue was much lower than in tumor tissue, and strongly dependent on perfusion direction: the level after retrograde perfusion was only 15-20% of normal perfusion direction. It was concluded that infusion of melphalan via the hepatic artery during retrograde perfusion of the liver is expected to cause less damage to the whole liver than perfusion in the normal direction. In order to further develop the percutaneous technique, experiments in pigs were performed. It was decided to introduce the catheters in the portal vein with an open surgical approach. The bypass catheters were introduced in the groin and neck region. Alternatively, a direct insertion in the inferior vena cava at the level of the renal veins was used instead of the groin incision. When the caval vein was occluded by means of a long balloon, flowrates through the 5 french catheter in the hepatic artery were low and higher rates could only be achieved with high line pressures. The high line pressures would not allow the use of oxygenation and therefore this technique was abandoned. As an alternative, a simultaneous infusion through both hepatic veins and the hepatic artery was tested, with outflow through the portal vein. The technique allowed high flow rates through the hepatic vein/portal circuit and a low flow through the hepatic artery/portal circuit and fulfilled our criteria. However, it was found that substantial leakage (>20%) to the circulation occurred, due to congestion of the liver during the perfusion with leakage through collaterals. A solution for this was found by control of the portal pressures during the perfusion. Experiments using application of the principle of portal pressure control showed that leakage rates could be kept as low as 1%. Development of a clinical system for computerized assessment of breast lesions in MR screening images of women at increased lifetime risk of breast cancer The purpose of this study was to provide objective and reproducible guidelines to interpret magnetic resonance (MR) screening images of asymptomatic women at high lifetime risk to develop breast cancer: 1) to characterize screeningdetected breast lesions, 2) to achieve prior selected accuracy in excluding presence of malignancy (negative-predictive-value: NPV) and in avoiding biopsy of frequently occurring benign lesions (positive-predictive-value: PPV). To achieve these aims, a clinical system was developed for computerized segmentation, rating and classification of breast lesions (Figure XIII.4). Eighty focal lesions (40 benign, 40 malignant: histopathologically confirmed or >1 year follow-up) from 66 patients were included for training. Taking into account the expected prevalence of malignancy, prospective performance in high-risk screening was estimated by cross-validation, and receiver-operating-characteristics analysis of the computer results derived by two independent operators. It was shown that combination of computer-rated washout, smoothness of uptake, mean and variation of margin sharpness yield significant contribution to characterization (Az=0.95, p<0.001). Estimated NPV exceeds 98% for suspicious lesions at 50% PPV. Inter-operator differences in computer-derived likelihood of malignancy were insignificant (p>0.9): <5% for 90% (72/80) of the lesions. Multi-modality analysis and radiological guidance in breast conserving therapy Our ongoing study aimed at improving the pre-operative estimation of tumor margins, providing better histological and radiological guidance to the surgeon and improving the accuracy in delivery of the radiation boost field, is critically dependent on the formation of a comprehensive database of radiological and histological images. This year, the database has increased to include the data of 283

119 DIAGNOSTIC ONCOLOGY Figure XIII.4: Lesion detected in processed contrast-enhanced MR images. Top row: uptake images. Bottom row: washout images. From left to right: saggital, transverse and coronal reconstructions of the processed MR images. A lesion (IDC) with uptake and corresponding washout has been selected for computer-aided characterization at the center of the cross hairs. All six viewers are linked to maintain correspondence. patients with breast cancer, 234 received breast-conserving therapy. As of yet, the database includes mammograms (283), ultrasound (271), MRI (136), x-ray specimen photographs (176) and extended pathology reports on tumor extent and excision volume (214). In preparation of the implementation of image guidance, multivariate analysis was performed on repeat MR data sets of breasts in supine position. The dependence of breast volume and tumor location (anterior, posterior, left, right) was investigated on the magnitude of internal tissue shifts caused by reposition between diagnostic imaging and treatment. Margins have been quantified (~5 mm) to account for these tissue shifts. Ultrasonographic examination of the axilla in the preoperative screening of breast cancer patients to reduce the number of sentinel lymph node procedures Currently, most breast cancer patients with a clinically negative axilla will undergo a sentinel lymph node (SN) procedure. A study was performed: 1) to investigate if the combined use of preoperative axillary ultrasound and Fine Needle Aspiration (FNA) reduces the number of SN procedures; 2) to identify ultrasonographic characteristics that predict metastatic invasion of a lymph node. All breast cancer patients with a clinically negative axilla eligible for a SN procedure (N=268) were included. FNA was performed on lymph nodes with the smallest diameter 5 mm. Appearance of cortex and hilus were noted; length, width and cortex thickness of all aspirated nodes were measured and the shape of these nodes (length/width ratio) was calculated. In 93 patients (35%) at least one lymph node was detected in the axilla with ultrasound. FNA was performed on 66 nodes; thirty-seven (56%) showed tumor cells. For the whole group a reduction in sentinel node procedures of 14% was obtained. Linear discriminant analysis and step-wise selection of nodal features showed that cortex thickness is the main feature in predicting the presence of metastasis (AZ=0.89). Prospective estimates indicate that performing FNA on all lymph nodes with a cortex thickness of at least 2.7 mm reduces the number of SN procedures with 14%, thereby performing FNA in only 18% of the patients.

120 122 BIOMETRICS Director of Research Anton Berns BIOMETRICS DEPARTMENT CLINICAL STUDIES AND OVERVIEWS Division Head, Group leader OB Dalesio OB Dalesio MSc Head H Van Tinteren MSc Academic staff D Baars Technical staff A Boucher Technical staff W Busschers BSc Technical staff W Deurloo Technical staff MAM De Waal MSc Technical staff L Frenken Technical staff AL Hiemstra Technical staff K Hogema Technical staff I Jansen Technical staff JA Lieverst MSc Technical staff J Maaskant Technical staff M Mahn-Schaefers MSc Technical staff IAM Mandjes MSc Technical staff C Modder Technical staff A Ndoye Technical staff A Reinders-Som Technical staff J Remmelzwaal Technical staff D Roberts Technical staff D Storm Technical staff R Valdes Olmos Technical staff LHM Valkenet MD Technical staff E Van der Donk Technical staff I Van der Schilde Technical staff A Van der Velde Technical staff T Van der Velde Technical staff E Van Oosterwijk Technical staff W Van Waardenberg Technical staff M Velthuis MSc Technical staff A Wals Technical staff LDV Wever Technical staff E Willemse Technical staff L Ziblat Technical staff J Oosting PhD Guest A Van der Kwaast Student J Van Gulik Secretary Overview of randomized trials in prostate cancer Since 1989, we coordinate, in collaboration with R Peto and his group at the University of Oxford, an overview of all randomized trials of the treatment of prostate 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 formal overview 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 produce an overall estimate of the differences between the treatments assessed. The findings are discussed at the meetings of the PCTCG that we organize every five years. The initial cycles of this collaboration assessed the use of maximum androgen blockade (MAB). During 2001 we studied the effect of the addition of chemotherapy on mortality in comparison to the standard treatment. Thirty trials were identified: 24 first line (three of these in patients with localized disease) and six after failure to hormonal treatment. For 14 trials data were not available and in a majority of cases not recoverable (the studies were too old and data were not kept.). These trials had included 1381 patients. Individual patient data were obtained for 3000 patients (69%) included in 16 trials. A report of the result is being discussed among the members of PCTCG group and should be published in the near future. Collaboration with National and International trial groups The results of a randomized trial of adjuvant therapy of 5FU plus levamisole in Dukes C colorectal were published in This study was designed as a confirmative trial of a similar one in USA and was conducted in the Netherlands under coordination of the NKI patients with colorectal cancer stage II or III were randomized to receive 5FU plus levamisole for one year or no further treatment following curative surgery. With a median follow-up of nearly five years the study showed significant benefit for Figure 1: Mortality by treatment arm in different subgroups of patients

121 BIOMETRICS patients receiving 5FU/ levamisole with regard to both recurrence and overall survival for stage II as well as stage III colorectal cancer (Figure 1). The data have also been included in the Colorectal Cancer Collaborative Group Overview. New randomized studies in this disease are being planned. Indeed, currently, we coordinate a multi center phase I/II study of the combination of capecitabine and irinotecan, which would be the starting point of a large phase III trial run by our group in collaboration with the British colorectal group. We have developed collaborations with several cooperative groups, industry and clinical research organizations and we function as Data Center for clinical trials generated by them, providing statistical expertise, randomization services and data management. In 2001 we collaborated with the New Drug Development Office (NDDO) handling the data of the Early Clinical Studies Group (ECSG) of the EORTC, analyzing and reporting the studies on LU in Melanoma (Dolastatin analogue), ISIS 5132 (C-Raf kinase inhibitor) in non-small cell lung cancer and ISIS 3521 (PKC-a mrna inhibitor) in melanoma and in non-small cell lung cancer. The collaboration with the Dutch Chest Physician Association (NVALT) resulted in two randomized studies in oncology. 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. Already 140 patients of the intended 440 patients have been included. The second study, also performed in collaboration with the British Medical Research Council, is aimed to evaluate the benefit of neo-adjuvant chemotherapy in operable NSCLC. Prognostic factors and markers studies An immuno histo-chemical study of p27kip1 expression in breast carcinomas was performed on 512 patients with longterm follow-up. The total sample included an Italian series of 270 patients (Trento, Milan) and a Dutch series of 242 patients from the NKI together. The median followup was nine years. Although reduced p27 expression was associated with adverse prognostic factors, in a multivariate regression analysis we did not identify p27 as an independent factor in contrast to what was claimed in the literature. We also collaborated in a study of cyclin A as a prognostic indicator in early stage breast cancer with and without tamoxifen treatment. The staining of markers was performed in collaboration with the department of pathology of the Free University Hospital. Special interest was focused on overexpression of G1-S regulators cyclin D1 and cyclin A in relation to ER values and other cell cycle regulators including cyclin E, p53, p21, p27, Neu, EGF-R, Ki-67, ER and PgR. Cyclin A proved to be an independent marker for recurrence in a model including generally established clinical parameters (Lymph node status, age, T classification, grade and ER values). ICT PROJECTS For several years we have developed information and communication technology (ICT) projects and we have obtained funding under the various framework programs of the European Commission (EC). A common denominator of all current project activities is the extensible Markup Language (XML), which is the most important emerging technology in the Information Technology world. Whereas HTML defines the markup of web pages and therefore the layout when such a page is displayed in a browser, XML enables the communication between applications by allowing specific tags to describe the content of a file or message. It is expected that within the coming years XML will bring a new breed of web applications on the market, eventually replacing most of the current client / server based systems by web enabled applications. However, before applications can communicate, standards need to be defined for the representation of data in XML. The EC is currently actively stimulating the involvement of European Healthcare providers and industry in the standardization processes by umbrella organizations such as CENTC251, CDISC, HL7 and Medbiquitous. RUBIS Projects RUBIS is an Acronym for Reseau Ubiquitaire á Integration de Services. In a previous project a service pilot platform under the namespace oncon- Key publications Barbareschi M, Van Tinteren H, Mauri FA, Veronese S, Peterse H, Maissoneuve P, Caffo O, Scaioli M, Doglioni C, Galligioni E, Dalla Palma P, Michalides R. p27kip1 expression in breast carcinomas: an immunohistochemical study on 512 patients with long-term follow-up. Int J Cancer 2000; 89: Ferreira CG, Van der Valk P, Span SW, Ludwig I, Smit EF, Kruyt FA, Pinedo HM, Van Tinteren H, Giaccone G. Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected nonsmall cell lung cancer patients. Clin Cancer Res 2001; 7(8): Kruijtzer CMF, Verweij J, Schellens JH, Beijnen JH, Pronk L, Bo M, Lustig V, Van Tinteren H, Mackay M, Ten Bokkel Huinink WW. Docetaxel in 253 previously treated patients with progressive locally advanced or metastatic breast cancer: results of a compassionate use program in The Netherlands. Anticancer Drugs 2000; 11: Malingre MM, Beijnen JH, Rosing H, Koopman FJ, Van Tellingen O, Duchin K, Ten Bokkel Huinink WW, Swart M, Lieverst J, Schellens JH. A phase I and pharmacokinetic study of bi-daily dosing of oral paclitaxel in combination with cyclosporin A. Cancer Chemother Pharmacol 2001; 47: Meerum Terwogt JM, Ten Bokkel Huinink WW, Schellens JH, Schot M, Mandjes IA, Zurlo MG, Rocchetti M, Rosing H, Koopman FJ, Beijnen JH. Phase I clinical and pharmacokinetic study of PNU166945, a novel water-soluble polymer-conjugated prodrug of paclitaxel. Anticancer Drugs 2001; 12: Op de Coul BM, Hilgers FJ, Balm AJ, Tan IB, Van den Hoogen FJ, Van Tinteren H. A decade of postlaryngectomy vocal rehabilitation in 318 patients: a single Institution s experience with consistent application of provox indwelling voice prostheses. Arch Otolaryngol Head Neck Surg 2000; 126: Taal BG, Van Tinteren H, Zoetmulder FAN. Adjuvant 5FU plus levamisole in colonic or rectal cancer: improved survival in stage II and III. BJC (in press).

122 124 BIOMETRICS Director of Research Anton Berns Key publications (continued) Van Tinteren H, Hoekstra OS, Smit EF, Verboom P, Boers M. Toward less futile surgery in non-small cell lung cancer? A randomized clinical trial to evaluate the cost-effectiveness of positron emission tomography. Control Clin Trials 2001; 22: nect.net, with national boundaries nl.onconnect.net, fr.onconnect.net, etc was created. We have been operating the Dutch component (nl.onconnect.net), hosting a wide range of services from national sources. Our task in the 4th framework project RUBIS 2, was an extension of that project and included the setting up of web portals. The project was performed with partners in ICSF and Institut Bergonié in Bordeaux (FR) the Centre Joseph Strauss in Alsace (FR), and the Olou University (FI). In the Aquitaine region we were involved in the set up of a regional Cancer Network (Reseau Cancerologie Aquitaine). In addition, we developed two important services in close collaboration with ICSF (Integrated Care Systems France). These services were required to allow running multiple web applications in a single context: RUBIS Single Sign On (RSSO) and RUBIS Distributed Searching (RDS). Both services are the intellectual property of the NKI/AvL and they are redistributed under Open Source licensing terms. RUBIS Single Sign On A Health portal is an access point on the Internet to different web based resources related to health. The nl.onconnect.net portal is a pilot service of such a portal and it provides access to several services related to cancer such as the pharmacy database of the Slotervaart Hospital, the ALEA software for randomization of patients in clinical trials, the TRION Trial Information system, Hematology website of the AMC. Most of these services require a username and password to be entered before access is granted to these services. In order to avoid multiple usernames and passwords for doctors using these services, a mechanism providing Single Sign On was developed in RUBIS in close collaboration with the French partners in Bordeaux. This mechanism asks a username and password only once, and all services mentioned may be accessed without additional entry of usernames and passwords. RUBIS Distributed Searching Another major problem for services available on a portal is searching. If services available through a portal are running on different servers it is impossible to search all services simultaneously, through a single interface. In order to support distributed searching, the RUBIS consortium adopted DCM (Dublin Core Metadata), a standard for indexing and searching of electronic resources. We have developed a search dispenser and a generic RDS responder for all applications on nl.onconnect.net (NL) and RCA (FR) in order to support distributed searching. Effectively, a user may now enter search criteria in a single browser form, and find hits to these search terms on all services to which the user was granted access on the portal. Flash animations explaining the mechanism of RSSO and RDS are available in the technology sections of (Figure 2). Figure 2 Onconnect distributed searching

123 126 RESEARCH FACILITIES Director of Research Anton Berns RESEARCH FACILITIES The research divisions within the NKI/AvL are supported by a number of indispensable research service facilities. Each facility comprising a group of experts who provide information, instruction and service assistance and housing state of the art equipment. Besides the facilities described below, the institute has a central cryogenic storage facility, a glassware kitchen, a (electro-) technical workshop and an audiovisual department. BIOMETRICS OB Dalesio MSc, D Baars, A Boucher, W Busschers BSc, W Deurloo, MAM De Waal MSc, L Frenken, AL Hiemstra, K Hogema, I Jansen, JA Lieverst MSc, J Maaskant, M Mahn-Schaefers MSc, IAM Mandjes MSc, C Modder, A Ndoye, J Oosting PhD, A Reinders-Som, J Remmelzwaal, D Roberts, D Storm, R Valdes Olmos, LHM Valkenet MD, E Van der Donk, A Van der Kwaast, I Van der Schilde, A Van der Velde, T Van der Velde, J Van Gulik, E Van Oosterwijk, H Van Tinteren MSc, W Van Waardenberg, M Velthuis, A Wals, LDV Wever, E Willemse, L 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 biostatistical 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 in national and international multicenter studies.

124 RESEARCH FACILITIES The department maintains a Tumor Register database, containing information on patients with benign tumors, pre-malignant and malignant tumors seen in the institute since This database is a valuable resource for research and currently contains more than 100,000 registrations. Between June 30, 2000 and July 1, 2001 a total of 5628 new entries were made, including 4560 malignancies, 63 pre-malignancies and 1065 non-malignancies. Of the malignancies 1163 were seen only for a second opinion, while 3397 malignancies were actually treated. Support of the medical divisions for central administration, patient registration and data collection of clinical trials is provided through the department s Trial Office. Between July 2000 and June 2001, 768 patients were entered into one of the trials open to accrual. There were 139 such studies. Forty percent of these were included in studies sponsored by the institute, 30% in studies supported by the Dutch Cancer Society or by the National Health Insurance, and 30% in studies sponsored by the industry. Half of the patients were entered into early clinical trials (pilot, phase I and phase II). The department data managers also visited 21 national institutes affiliated with the Dutch Chest Physician Organization (NVALT) to collect data on the 136 patients entered into their first randomized trial. Furthermore, central registration and randomization services for eleven other multi-center studies were provided. During 2001, a new version of TRION, the information system on current clinical trials developed and maintained by the department, was made available on the Intranet. This new version provides the insurance form and the informed consent page in one single document to comply with legal requirements. The course on Medical Statistics for researchers and physicians that was so successful previous years was offered for the fifth time. COMPUTERS AND NETWORK SUPPORT T Canrinus, EJ De Kruijf, F Kaslander, J Stoltz, IV Van de Pavert, GJ Van der Stroom The central ICT department provides general IT support for all research divisions as well as specific site wide services (such as facilities) for all personnel. Tasks include: management of routers, Ethernet switches, file servers, job-and-database servers, configuration of network software on PCs and Apple Macintosh computers of end users and, if necessary, the development and maintenance of custom server and/or client side software. We are providing IT services in line with the latest technology standards. An example is the Gigabit Internet technology. To ensure delivery of high bandwidth connectivity for the research personnel, all network connections were migrated to 100Mb switched Ethernet in the fourth quarter of We are also building protected zones in the IT infrastructure to ensure that only authorized persons are able to connect to our sources. DIGITAL MICROSCOPY L Oomen PhD, L Brocks PhD The Digital Microscopy Facility provides four imaging systems: two confocal laser scanning microscopes and two microscope setups with cooled CCD-cameras. This year one of the confocal systems has been upgraded. The new features offered by the system include true 12 bit imaging, a correction method for instability in laser excitation intensity and flexibility in scan modes. Together with the possibility of continuously adjustable wavelength selection bands in all four emission channels, this offers a powerful imaging system very well suited to perform studies on living cells. Special topics herein are the study of protein synthesis, transport and dynamics in living cells, making use of the green fluorescent protein (GFP) and its analogs (YFP, CFP). In addition, the software of both confocal systems has been upgraded. Operating the systems has become more user-friendly and the interface can be finetuned to user specific needs. The analysis functions have been greatly extended (up to 5D [x,y,z,t and wavelength]) and also include new features like analysis and display of colocalization of different markers in cells and tissue. The microscope/ccd-camera systems consist of a highly sensitive black and white camera and a color camera. The black and white camera system has recently been upgraded

125 128 RESEARCH FACILITIES Director of Research Anton Berns in order to keep at pace with developments in hardware (camera, computer, optical components) and software and can operate in a fully automated image acquisition mode, without requiring user intervention. This upgrade has also secured the ongoing use of the system to analyze genetic alterations in human tumors (comparative genomic hybridization [CGH] and fluorescent in situ hybridization [FISH] studies) and to analyze the effects of radiotherapy on human tumors and normal tissue. The color camera system is primarily suited to capture digital color images directly from (immuno-)histochemically stained tissue sections. All studies involving animal histopathology, most noticeably those on transgenic and knock-out mice, can make use of this facility. ELECTRON MICROSCOPY J Calafat PhD, JWRM Janssen The facility consists of a Philips CM10 and a Philips EM410 transmission electron microscope and Leica (cryo)-ultramicrotomes. In addition to its own research activities (see division of Cell Biology), the facility assists researchers in their ultrastructural morphological analysis of macromolecules, viruses, cells and tissues. Immunoelectron microscopy of ultrathin cryosections reveals a great wealth of structural details in the 2 to 100 nm range, providing information that can be obtained in no other way. This technique is used for the following studies: the exact localization of antigens/molecules in the cell organelles, localization of two different antigens simultaneously using colloidal gold of different sizes, trafficking of molecules in the cell, localization of molecules in the cell during signal transduction events and cellcell and cell-matrix interactions. FLOW CYTOMETRY E Noteboom, AS Pfauth The Flow Cytometry Facility has two sorters, four analyzers and two computer systems for data analysis. The operators train every user to work on one of the analyzers independently.

126 RESEARCH FACILITIES Many different types of experiments are performed because almost every research group in the institute makes use of the facility. Examples include measurements of apoptosis, cell cycle progression and cell surface markers. Cell sorting experiments include those for transfected cells (co-transfection with surface marker gene), progenitor cells and green fluorescent protein (GFP) and for subcloning cells. Sorting of live as well as fixed cells is routinely carried out, including four way sorts, double laser experiments and sorting into 96-well plates. LABORATORY ANIMAL DEPARTMENT RGM Ten Berg DVM PhD, K Ankema, NH Bosnie, NJ Bovenkerk, ML Breuer PhD, MT Bulale, M Chailan, C Friederich, JJ Greven, HMG Grimminck, M Groen, D Grund, PJ Handgraaf, TL Hetem-Maidment, M Hoffmann, JJ Janssen, WST Kenbeek, J Kleinsma, FC Krekko, M Lageweg-Beumkes, AJL Lagro, FT Nicolaas, B Rijnbout, AJ Schrauwers, C Spaans, HP Starrevelt, MAM Timpico, AJM Tolkamp, R Van den Berg, FH Van der Ahe, D Van der Pijl, HCA Van Hattum, N Van Roosmaelen, MS Voetel, W Wolff, Y Zagmouti, A Zwerver 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 department provides the infrastructure for animal experiments: production and maintenance of mice and rats, rendering of biotechnical 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 animals, with special emphasis on sick animal screening. LABORATORY ANIMAL PATHOLOGY AND HISTOLOGY JC Bulthuis, CCJ De Goeij, J-Y Song, MHJ Tjin-A-Koeng, MA Van der Valk 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,

127 130 RESEARCH FACILITIES Director of Research Anton Berns sectioning and staining, cryosectioning and immune histology. The pathologists perform histopathology on request, either on a regular, project-related basis, or incidentally, for most research groups in the institute. The staff also give advice on subjects involving post-mortem examination, tissue sampling, handling and processing and different staining methods. LIBRARY S Bakker MSc, IS Benne, IN Goede, M Kanhai, GJ Kroeskamp The Central Cancer Library, serving the research, clinical, nursing and paramedical departments of the institute, is offering access to an increasing number of electronic sources and services. In recent years the journal collection expanded with electronic access to the full-text over the Internet under site-license agreements. Current awareness bulletins are available as hypertext links to PubMed (including the expert search strategy). The library has organized many introductory courses on the use of reference management programs for medical, nursing, research and secretarial staff. The library catalog and databases will soon become available through a web-browser interface on the Intranet. Electronic books and application programs on CD-ROM are now a substantial part of the collection; some will be made available on the Intranet, many more will be available on loan to NKI/AvL staff. The library publishes the annual reports of the institute in HTML- and PDF-format on the NKI/AvL Internet website and maintains and updates major parts of the general information about the institute on the website. Electronic discussion lists are in use for communication with library users (e.g. CKB-AANVRAGEN@nic.surfnet.nl is available for literature retrieval or interlibrary loans and CKBINFO@nic.surfnet.nl for news about library services). The library is responsible for the annual scientometric analyses (based on impact factors and citations). MICROARRAY FACILITY RM Kerkhoven PhD, M Daemen, M Heimerikx, D Van Alewijk PhD, A Velds Msc The production of significant numbers of high complexity human and mouse cdna microarrays was the main aim this past year. To accomplish this, the facilities have been improved significantly. The clean room with microarray print robot has been equipped with a separate air conditioning unit for better control of printing conditions, leading to a better spot morphology. We expect that production will increase significantly with the installation of a new and improved printing robot. New protocols for RNA-labeling have been developed and introduced. The sequence verified human library was printed on a total number of 1312 microarrays, 648 of which were high complexity 18K arrays. The first of the two murine sequence-verified libraries that were acquired this year was PCR amplified, purified and spotted onto 242 glass slides, 216 of which contained the complete 15K mouse embryo cdna set. In addition to the human and murine cdna arrays, a start was made to produce murine SNP and BAC arrays. This year the Microarray Facility was upgraded to become a national resource of DNA microarray technology thanks to a grant from the Dutch Cancer Society. As a result, the facility now also produces 18K human and 15K mouse cdna arrays for Dutch Cancer Society-sponsored research nationwide. We have established a productive collaboration with the National Cancer Institute in Bethesda, USA. Through this agreement, software for microarray data analysis developed by NCI s Center of Information Technology became available to users of the Microarray Facility. The installation of computer hardware and software was made possible thanks to a generous grant from the Josephine Nefkens Foundation. Many new tools for basic analysis (quantification, normalization and scaling) and higher order analysis (clustering, SOM, MDS) have been implemented. A database has been created around the cdna clone information, which is used to analyze the microarrays. Gene information can be extracted via a web-based interface (Intranet, Also, a license of the Celera database has been acquired, which is hosted by the Microarray Facility. The NKI community can now search the

128 131 RESEARCH FACILITIES Celera information on the human and mouse annotated genomes and human SNP collection. To provide training and education, a total of eight two-day microarray courses were given for 64 users from a number of different research laboratories in the Netherlands. Most users have started doing microarray hybridizations in their own lab. A website ( has been made for the exchange of technical information. A start has been made to set up user group meetings and an newsletter. MONOCLONAL ANTIBODY UNIT EM Groeneveld The Monoclonal Antibody Unit provides support and advice concerning antibody production, development and characterization. Both mice and rats are used for immunization. Immunochemical techniques, such as different immunoassays, cell staining, immunohistochemistry and Western blotting, are used to characterize the monoclonal antibodies and the antibody-antigen interaction. PEPTIDE SYNTHESIS HAM Hilkmann This year 180 peptides were synthesized. Peptides were mainly 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. To elucidate the cause of a less successful synthesis and for a further control of the peptides, mass spectroscopy

129 132 RESEARCH FACILITIES Director of Research Anton Berns is performed in collaboration with the Free University of Amsterdam and the Department of Pharmacy and Pharmacology of the Slotervaartziekenhuis of Amsterdam. Most of the peptides synthesized are used for biological studies in tissue culture or for raising antisera, 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 of biotinylated or acylated peptides, peptides containing phosphorylated amino acids and peptides existing of two chains of amino acids. Peptides are also being synthesized for clinical use in collaboration with the Department of Pharmacy and Pharmacology of the Slotervaartziekenhuis of Amsterdam, where the extensive testing of these peptides take place. In collaboration with Division IV, small amounts of different peptides are synthesized on the Syro multi peptide synthesizer. RADIONUCLIDE LABORATORY TE Lamers, ALM Luts, H Van Rooij In addition to departmental laboratories licensed for radioactive work (present on most floors of the research building) there is a central radiochemistry facility (class 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 department is equipped with up to date gamma and scintillation counters, gamma analyzers, a tissue oxidizer and HPLC apparatus for on line radiodetection. There are also separate facilities for animal experiments with radioactive tracers for cancer research

130 133 RESEARCH FACILITIES (e.g. multidrug-resistance). The department provides regular courses on Radiation Protection, level 5B, for all new personnel (including students and guests) whose work entails the use of radioactive material and also provides practical courses level 4A/B for radiotherapeutical technicians. In addition to general aspects of education and safety, the staff of the department take an active part in various research activities, including HPLC analysis of experimental pharmaceuticals like 99mTc Bru (Bracco study, imaging of hypoxic tissue in patients) and synthesis of radiolabeled peptides and proteins. SEQUENCE FACILITY LJ Van t Veer PhD, FBL Hogervorst PhD, R Pruntel The Sequence Facility offers a service for DNA sequence and fragment analyses to users in the research departments and the DNA-diagnostic laboratory of the Pathology Facilities. 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. The purchase of the capillary machine has significantly shortened the sample turnover time and abolished the time-consuming and laborious gel-casting and sample loading. At the moment about 2500 sequence samples are analyzed per month. The number of fragment analyses (500) is also increasing as novel applications are implemented on the ABI In its third year following the establishment of the Sequence Facility, two major events took place. First, the facility moved from the Division of Molecular Biology to a small but dedicated lab in the Division of Molecular Carcinogenesis. The second event was the accreditation of the DNA-diagnostic laboratory of the Family Cancer Clinic. The Sequence Facility plays an important role in the diagnostic analyses of this laboratory and therefore the procedures and protocols are qualified by Sterlab. TRANSGENIC MOUSE SERVICE PJA Krimpenfort PhD, L Rijswijk, F Van de Ahé, WK Van t Wout, CJH Van Veen Buurman The activities of the transgenic mouse service are the manipulation of mouse embryos for cryopreservation and the genetic modification of mice. In 2001, we stopped using vitrification as a way of freezing genetically modified mouse embryos and are now acquiring experience in technologies that enable the preservation of mouse lines based on sperm freezing and in vitro fertilization. The sperm based preservation is by far more efficient with respect to cost and number of animals required than the currently applied embryo cryopreservation.

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