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2 Annual Report 1995

3 PATRON HM THE QUEEN BEATRIX

4 Annual Report 1995 THE NETHERLANDS CANCER INSTITUTE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL

5 Annual Report 1995 Illustrations and unpublished data in these reports should not be used without permission ofthe author. Copyright : The N etherlands Cancer Institute Antoni van Leeuwenhoek Huis PIesmanlaan CX Amsterdam The N etherlands Phone Fax ISSN

6 Contents Board Members 6 Research and Hospital Divisions 8 Introduction 13 I Cell Biology 19 II Molecular Carcinogenesis 29 III Cellular Biochemistry 35 IV Immunology 45 V Molecular Biology 55 VI Tumor Biology 65 VII Molecular Genetics 73 VIII Experimental Therapy 81 IX Radiotherapy 89 X Medical Oncology 97 XI Surgical Oncology 107 XII Psychosocial Research and Epidemiology 115 Biometrics Department 123 Biophysics Department 129 Laboratory Animal Department 133 Cancer Hospital 135 Clinical W orking Parties 141 Ongoing Trials 145 Education in Oncology 167 Projects 177 Publications 187 Author Index 217 Personnel-Project Index 229

7 6 Board Members International Scientific Advisory Board Jon J van Rood, Professor ofimmunohematology, Leiden, president Joseph R Bertino, American Cancer Society Professor of Medicine and Pharmacology, New Haven, USA George Klein, Professor of Tumor Biology, Stockholm, Sweden Hilary Koprowski, Former Director of the Wistar Institute, Philadelphia, USA Susumu Tonegawa, Professor of Biology, MIT, Center of Cancer Research, Cambridge, Mass, USA I Bernard Weinstein, Professor of Medicine and Environmental Sciences, New York, USA Charles Weissmann, Professor of Molecular Biology, Zürich, Switzerland N ational Scientific Advisory Board LA Aarden, Professor of Molecular Immunology, Amsterdam D Bootsma, Professor of Cell Biology and Genetics, Rotterdam AJ van der Eb, Professor of Fundamental Tumor Virology, Leiden SWJ Lamberts, Professor of Internal Medicine, Rotterdam HL Langevoort, Professor of Histology and Embryology, Amsterdam B Löwenberg, Professor of Hematology, Rotterdam CJLM Meijer, Professor of Pathological Anatomy, Amsterdam CJM Melief, Professor of Immunohematology, Leiden J Oldhoff, Professor of General Surgery, Groningen HM Pinedo, Professor of Clinical Oncology, Amsterdam 11 van Rood, Professor of Immunohematology, Leiden E van der Schueren, Professor of Oncology, Leuven GNJ Tytgat, Professor of Gastroenterology, Amsterdam PC van der Vliet, Professor of Physiological Chemistry, Utrecht

8 7 Board of Directors P Borst, chairrnan and director of research L Neeleman, director organization and management A Roest, financial director clinical director, vacancy Laboratory Research Coordinator AJM Berns Board of Governors JD Hooglandt, president ML Frohn-de Winter, vice-president P den Tex, secretary (ti ) JF Visser, secretary (fr om ) o Hattink, treasurer AC van den Blink R Hazelhoff P van der Heyden PH Hugenholtz S van der Kooij J van der Meer JHMTemmink GNJ Tytgat MWM Vos-Van Gortel Ladies Committee MC Sickinge-van Eeghen, president YJ Engelsman-Prins, secretary D Jurgens-Kupsch, treasurer

9 8 Research and Hospital Divisions Research Divisions I Cel! Biology E Roos (head) J Calafat JG Collard CA FeItkamp A Sonnenberg II Molecular Carcinogenesis R Bernards (head) L den Engelse (till ) E Kriek (honorary staff member) M van Lohuizen H te Riele E Scherer T Sixma III Cel!ular Biochemistry WH Mooienaar (head) WJ van Blitterswijk J Borst JJ Neefjes A Tulp LN Vernie IV Immun%gy AM Kruisbeek (head) WR Gerritsen A Hekman EM Rankin H Spits CJM Vennegoor (stationed at the Free University) FA Vyth-Dreese K Weijer V M olecu/ar Bi%gy RHA Plasterk (head) P Borst APM Jongsma (stationed at the Free University) VI Tumor Biology WJ Mooi (head till ) J Hilkens (head from ) JH Daams AA van der Gugten PhC Hageman D Ivanyi RJ AM Michalides M Sluyser AA Verstraeten VII Mo/ecu/ar Genetics AJM Berns (head) P Demant M Snoek MA van der Valk VIII Experimental Therapy AC Begg (head) H Bartelink WJ Nooijen S Rodenhuis JH Schornagel LA Smets FA Stewart IX Radiotherapy JV Lebesque (head) BMPAleman H Bartelink RW de Boer J Borger IAD Bruinvis BNFM van Bunningen JMV Burgers EMFDamen LGH Dewit AAM Hart M van Herk RB Keus EAH Masselink BJ Mijnheer LMF Moonen SH Muller VJ de Ru NS RusselI CCE Schaake-Koning (till ) FW Wittkämper X Medica/ Onc%gy S Rodenhuis (head) JW Baars P Baas EM Bais JH Beijnen WW ten Bokkei Huinink

10 9 JMG Bonfrer W Boogerd H Boot PF Bruning ME Craanen CC Delprat WR Gerritsen CA Hoefnagel SP Israëls C Mattern WJ Nooijen EM Rankin 11 van der Sande JH Schornagel R Somers BGTaal o van Tellingen RA Valdés Olmos APE Vielvoye-Kerkmeer N van Zandwijk Research Nurses D Batchelor AC Dubbelman ME Schot Xl Surgical Oncology JA van Dongen (head til! ) BBR Kroon (head from ) AJMBalm F van Coevorden E Gortzak RT Gregor (till ) FJM Hilgers ThJM Helmerhorst S HorenbIas W Meinhardt OE Nieweg EJTh Rutgers N van der Vange (from ) FAN Zoetmulder XII Psychosocial Research and Epidemiology NK Aaronson (head) FSAM van Dam FE van Leeuwen Biometrics Department OB Dalesio (he ad) B Schaefer H van Tinteren JL te Velde Biophysics Department GJF Blommestijn (head) LCJM Oomen HJ Stoffers Laboratory Animal Department RGM ten Berg (head) Hospital Divisions Anesthesia M Hellendoorn-Smit (head) C Blackburn MKaag Clinical Chemistry and Hematology WJ Nooijen (head) JMG Bonfrer Clinical Research Manager EJ Vos Gastroenterology H Boot ME Craanen BGTaal Gynecology ThJM Helmerhorst (head) P Kenemans N van der Vange (from ) Hospital Pastors PG Kousemaker JE Ringrose-Wegman AH Tönis Internal M edicine S Rodenhuis (head) JW Baars EM Bais WW ten BokkeI Huinink PF Bruning CC Delprat WR Gerritsen SP Israëls H Neering EM Rankin JH Schornagel R Somers Management Team SW van Bergen (Surgery) RK Pet (Diagnostics) PM Terwijn (lnternal Medicine) L van der Velde (Radiotherapy) Neurology W Boogerd 11 van der Sande Nuc/ear M edicine CA Hoefnagel (head) SH Muller RA Valdés Olmos

11 10 Otolaryngology - Head & Neck Surgery F JM Hilgers (head) AJM Balm R Fontijn S Gonggrijp RT Gregor (ti ) APTimmers Pain Physicians C Mattern APE Vielvoye-Kerkmeer Pathology WJ Mooi (head till ) MPWGa11ee P van Heerde D de Jong BM Loftus-Coll JL Peterse L van 't Veer Patient Counsel/or HIM Camerik Plastic Surgery JB de Boer KEBos RP Noordanus Psych ia try LM Gual thérie van Weezei Psychosocial Service DEE Hahn Pulmonology P Baas N van Zandwijk Radiotherapy H Bartelink (head) BMP Aleman RW de Boer JH Borger IAD Bruinvis BNFM van Bunningen JMV Burgers EMFDamen LGH Dewit AAM Hart M van Herk RB Keus JV Lebesque EAH Masselink BJ Mijnheer LMF Moonen SH Muller VJ de Ru NS Russell CCE Schaake-Koning (ti ) FW Wittkämper Diagnostic Radiology R Kröger (he ad) AP Besnard W Koops F de Leeuw Surgery JA van Dongen (head ti l995) BBR Kroon (head from ) JB de Boer KBos F van Coevorden E Gortzak OE Nieweg RP Noordanus EJTh Rutgers FAN Zoetmulder Urology S Horenbias W Meinhardt Consultant Staff Bacteriology WC van Dijk WPauw General Practitioner CC Delprat M axillo-facial Surgery FHM Kroon Ophthalmology L Koornneeff Orthopedic Surgery JW van der Eijken MW Fidler Pediatrics PA Voûte Pharmacy JH Beijnen ACA Paalman Rehabilitation ELD Angenot

12 11 Heads of General Services Audiovisual Service JM Lomecky Central Cancer Library MBA Wilhelm-de Gouw Custodian Research Laboratory CA FeItkamp Financial Administration JKKoppenol Housekeeping Services RMD Schellens M edical Administration JH Helversteyn Out-patient Clinic R Pet (manager) Personnel Department GH van Aller Safety, Health and Welfare P de Lange Technical Service TCM Wil me ring

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14 13 Introduction The year 1995 was a good one for the Netherlands Cancer Institute. Financial stability provided the basis for a high output in research and patient care. The new outpatient clinic with over 2000 m 2 net additional space was a boost to patient care and created space for new initiatives, such as a familial cancer clinic. Plans to build a new hospitaion the premises moved forward smoothly and a final decision is expected in Part of the old hospital will then become available for research, giving us breathing space for the next decades. Research highlights The list of research highlights that follows is a limited selection of the most significant discoveries of 1995 suitable for presentation in simplified form. More complete and balanced overviews of all highlights of our research efforts can be found in the introductions of each of the 12 research divisions. Transgenic mice are becoming one of the most powerful and indispensable too1s of basic cancer research and medical biological research in genera!. We are fortunate to have at our disposal a large and wellequipped animal house with astrong tradition in mouse genetics going back to the thirties. Experts on transgenic and knock-out mice, such as A Berns (Division VII) and H te Rie1e (Division 11), allow us to make optimal use of these facilities. Not only scientists in the institute, but also elsewhere in the Netherlands, profit from this expertise. In , some 250 new transgenic lines were generated (with additional genes added to the germline) and about 30 knock-out lines (with a gerrnline gene disruption). Many of these we re generated in collaboration with investigators outside the institute. I mention here some of the most interesting resu1ts obtained by NKI staff: A Sonnenberg and coworkers (Division I), in collaboration with the Berns group (Division VII), generated a mouse unab1e to make the a6/ /34 integrin, a cell surface molecule involved in adhesion of epithelial cells to the extracellular matrix. These knock-out mice develop severe disease: skin detachment and also (unexpectedly) poor lung development. Such mice, and celllines derived from them, provide important new models for the study of causes of hereditary skin blistering in humans and ofthe role ofthe a6/ /34 integrin in adhesion and metastasis of tumor cells, in which it is of ten overexpressed. The group of H te Riele generated a mouse model to study the role of DNA mismatch repair in genome stability and tumor evolution by targeted disruption of the DNA mismatch repair gene Msh2. They found th at Msh2-deficient ceus have a mutator phenotype and reduced gen ome stability, whereas Msh2-deficient mice are predisposed to cancer. This mouse should provide an important new model to study the way in which inherited mismatch repair defects in humans lead to a familial form of colon cancer. Simple eukaryotic 'model' systems continue to yield important new insights relevant to cancer. The group of Plasterk (Division V) studied the sm all nematode C. elegans. In 1995 his group succeeded in reconstituting the transposition of the DNA transposon Tel in a cell-free system, and derived a model for the mechanism of this reaction. Using the transposon as a tooi for gene interruption, they analyzed the role ofg proteins in signal transduction in C. elegans, and found th at Go and several other G proteins primarily act in the nervous system and affect different behaviors. Research on metastasis, cell-cell communication and intracellular signaling is yielding new insights into the causes of the remodeling of the cytoskeleton associated with malignant transformation. Collard and coworkers (Division I) found that the invasion- and metastasisinducing Tiam-J gene is an activator of the small GTPbinding protein Rac and plays a key role in Rac-mediated signaling pathways that regulate the organization of the cytoskeleton in response to extracellular signais. They also found that aberrations in the Tiam-l-Rac signaling pathway may contribute to both tumor formation and metastasis. This is only one of many examples of basic research leading into the clinic. Other examples came from tumor immunology and oncogene research. W Mooienaar and associates (Division 111) continued their investigations on Iysophosphatidic acid (LPA), a mitogenic serum phospholipid discovered by Mooienaar that activates aspecific G protein-coupled receptor. In 1995, they found that LPA rapidly shuts off cell-cell communication, apparently due to phosphorylation of gap-junctional proteins via a novel pathway involving the Src proto-oncogene product. Inhibition of cell-cell communication is a hall mark of malignant transformation and probably anormal physiological event d uring wound healing. The Mooienaar group, together with S Rodenhuis, A Westermann and J Beijnen (Division X), also found th at LPA-like molecules are major mitogens in ascitic fluid of ovarian cancer patients. This finding indicates that there is a previously unsuspected link between LP A production and certain human cancers. Ph ase I clinical trials with LPA antagonists are underway. G van Bleek and A Kruisbeek and their associates

15 14 (Division IV) found how stress protein gp96 isolated from certain tumor cells can serve as an efficient anti-tumor vaccin. When virus-infected cells are used as a source of gp96, dominant viral antigens are 'carried', in the form of small peptides, by the gp96 molecules. These peptidecarrying gp96 molecules could be used to prime anti-virus specific T cells both in vivo and in vitro. It is therefore possible that gp96 molecules isolated from tumor cells present an array of tumor-associated antigenic peptides, thus providing a means to vaccinate against a given tumor without first having to identify the antigen ic peptides fr om the tumor expresses. Encouraging clinical responses we re se en by E Rankin and coworkers (in collaboration with T Boon, Brussels) in a peptide vaccination study in melanoma patients. Upon treatment with one of the peptides derived from a melanoma-associated antigen (the MAGE-3 peptide), one patient had a complete remission. This result shows that the weak immunogenicity of melanomas can in principle be used for an immunological attack on the tumor, using the sophisticated knowledge gained in recent years about the ins and outs ofthe immune system. Berns and coworkers (Division VII), in collaboration with De Jong (Division VI) showed that the pim-l and pim-2 oncogenes, identified earlier as potent collaborators of c-myc in hematopoietic tumors in mice, are likely to be involved in hematopoietic malignancies in humans as evidenced by the high level of expression of pim-l, pim-2 or both in a substantial fraction of human lymphoma specimens. The group of R Michalides (Division VI) showed that overexpression of cyclin Dl triggers autonomous growth ofbreast tumor cells by preventing exit from the cell cycle. Precision is the essence of good radiotherapy and a reproducible positioning of the patient in the be am is a prerequisite for precision. K Gilhuis and M van Herk (Division IX) developed two new methods for the analysis of electronic portal images in three dimensions. By these methods, the patients' setup during treatment can be determined in three dimensions (including rotations), making use of the CT data of the patient and two portal images of the patients' setup. Two clinical trials coordinated by NKII AvL clinicians yielded remarkable results generating nation-wide discussion. Zoetrnulder and coworkers (Division XI) are coordinating a prospective randomized trial to test the value of adjuvant 5-Fluorouracil and levamisol in patients with colorectal cancer. Although this adjuvant therapy is now considered standard treatment in the US, no significant effect has been observed thus far in the Dutch situation. Rodenhuis (Division X) is coordinating a Dutch multicenter trial ofhigh dose chemotherapy with stem cell transplantation as adjuvant therapy in breast cancer. Accrual in this study is even more rapid than in the American trials in which more than 100 centers participate, showing the great interest for this new modality. A potentially serious side effect of this treatment was noted by F van Dam (Division XII) and S Rodenhuis (Division X) when they found significant long-term impairment in cognitive function in treated patients. This needs verification in a 1arger prospective study. Honors P Borst was e1ected as foreign honorary member of the second American Academy, the American Academy of the Arts and Sciences. He was a1ready a member of the National Academy ofsciences U.S. MEssers received the ESTRO-V ARIAN award in radiation physics for her excellent contribution in the field of in vivo dosimetry. B Kapteijn, received the Malinckrodt award for her study on the superiority of gamma probe detection over patent blue in localizing sentinellymph nodes in patients with me1anoma. F van Leeuwen received the Henny C Dirven prize for breast cancer research of the State University of Leiden for her work on identifying risk factors associated with the development of breast cancer, and on the long-term effects of hormona1 treatment of breast cancer. B Mijnheer became an honorary member of the Hungarian Society for Radiation Oncology. A Schinkel received the Antoni van Leeuwenhoek prize 1994 for his elegant and thorough analysis of mice 1acking the ability to make one or more P-glycoproteins, membrane proteins involved in drug resistance of cancer cells. A Sonnenberg received the quinquennial prize of the Izaak Korteweg and Anna Ida Overwater Fonds for his research on the nature and structure of cell surface adhesion molecules and the defects in these molecules in human disease. Table 1 Short-term citations and impact of scientific articles published by the NKI research staff Publication year Citations* rl.l.l.( Impact** * In the two years af ter publication, excluding se1f citations. Starting with 1989, the citation analysis has been carried out on-line. This allows detection (and elimination) of all self citations. Before 1989 this pruning was limited to first authors. ** The impact factor is the ave rage number of citations per year of an article in a given journal. The total impact is the sum of the impact of all articles published that year.

16 15 Research quality Since 1982 we havc recorded thc short-term citations and impact of scientific articles published by the NKII AvL research staff. Table 1 presents the results of this analysis. The trend is up and the considerable increase in the impact of our articles in 1994 is especially satisfactory. This confirms other indications th at the NKI/ AvL is living up to its reputation as a center of excellence. Good research should not only lead to good articles in good journais, but also to important discoveries. What is important is not always easy to define, but one objective criterion is the number of publications in the so-called high impact journais. Table 2 gives an overview of the number of publications of NKI/ A vl scientists in such high impact journais, arbitrarily defined as non-review journals with an impact factor of more than 10. Clearly, both our basic research and our clinical research contribute important discoveries to the scientific literature. Table 2 Number of NKI publications in journals with an impact factor above 10 in the period 1985 to See Table I for definition of impact factor. IF Journal Total Cell N Engl J Med I Nature Science Lancet Genes Dev J Exp Med EMBOJ J Cell Biol Proc Natl Acad Sci USA 1 20 Total Translational research In the past 15 years we have had the privilege to witness an explosion of knowledge ab out cancer. Basic research has outlined how cells in our body are instructed by molecular messages from outside to divide, move, or differentiate, commensurate with the needs of the entire organism. We also know now, in outline, how cancer cells escape these instructions and become independently replicating and migrating cells that eventually destroy their host. We are also rapidly increasing our understanding of the differences between cancer cells and normal ceus. These differences are primarily genetic and alterations in less than 0.01 % of the human gene complement suffice to turn a normal cell into a metastasizing highly malignant cancer cello This is why cancer is such a formidable problem: the cancer cell is more than 99.9% identical to the normal ceu from which it arose. Small wonder that the specific eradication of cancer cells without also damaging normal cells is so difficult. Small wonder also that successes in cancer diagnosis and treatment come only slowly and in small steps. This is difficult to accept, as many of our patients are dying today. What use are the splendid new insights in the detailed workings of the cell to them? Can we not speed up the translation of our new knowledge of oncogenes and tumor suppressor genes, of cell cycle regulation and signal transduction, of radiosensitivity and drug resistance, into more effective therapies? In short, should we not do more translational research? This pressure to do more applied 'translational' research is mounting worldwide in cancer research. The motives for this pressure are understandable and often nobie, but nevertheless this pressure is threatening the very basis on which the new therapies must be built. It is true that we know in outline what is wrong in cancer cells, but it is also true that details count in biology. We know some 100 genes involved in the regulation of cell division and cell differentiation but it is also clear that there should be at least one thousand genes involved in these complex cellular processes and we still have a long way to go before we really understand how cells operate in detail. There is not yet a single tumor in which au genetic defects that contribute to the cancerous state have been identified. We are still very far from an inventory of all genetic alterations that can potentially give rise to multi-step oncogenesis of a single type of cells in our body. How can we be expected to simply translate this very incomplete knowledge into effective new therapies? To use an oversimplified analogy: in the library of nature we now know which book contains the key to cancer, i.e. the book describing the regulatory genes that con trol cell multiplication and differentiation. We have even identified many keywords in this book, giving us a strong idea what the main message will beo However, we know only one word of every sentence of this text. Is that sufficient for an all-out effort to translate the text? Should we not spend a large fraction of our time in completing the text of the cancer book? In the past 15 years the methods to decipher this book have become increasingly powerful and sophisticated. We know where to look and we have the tools to find what we are looking for. This is why we are convinced that basic research should remain a high priority in cancer research, also today. Does this mean second priority for translational research? Of course not. We are fortunate to work in an integrated cancer center in which clinical scientists and basic scientists work under the same roof and interact intensively. The pressure for better treatment and earlier, more precise diagnosis is pervasive and every scientist in the institute is aware of the need to find applications for the results of our basic research. We have stepped up our efforts in translational research in recent years. We have increased our investments in clinical research (mainly carried out in Divisions VI and IX-XII) and translational research using animal models (mainly, but not exclusively, in Division VIII) considerably over the past 7 years, as can be seen in Table 3. The senior research staff of the institute spent a Saturday in December to develop new projects and strengthen existing collaborations between lab and clinical scientists. I think that all these efforts are paying off.a recent site visit of our Division of

17 16 Experimental Therapy (VIII) showed th at our translational research is 'among the best in the world'. Our Radiotherapy Division provides clear proof that an effective integration of clinical radiotherapy research and more basic research in radiophysics and radiobiology can lead to substantial improvements in diagnosis and therapy of cancer that have been adopted by other top institutes the world over. The new molecular pathology laboratory, opened 3 years ago, has strengthened the interface between laboratory and clinical research. The new familial cancer clinic, opened early in 1995, provides another example of the clinical application of new knowledge of genes altered in cancer. Table 3 shows the allocation (in million NLG) of untargeted research funds to basic laboratory research, clinical research and general support services in the period Untargeted research funds encompass all non-project money. General support services not only include overhead and cost of heating, electricity, etc., but also services directly linked to research, such as library, animal house, photography, etc. There is no overhead component in Dutch project grants and the general support services also support all project personnel. Note that these figures are not corrected for inflation, formally 19% in the period, but in practice much higher due to a further shift in research programs towards molecular biology and cell culture. Table 3 Activity Basic research Clinical research General support services Total It should be clear, however, that the notion that the trees of basic research be ar ample fruit that only need picking, is erroneous. Possible applications are being explored either in academia or in industry with tremendous vigor and persistence. It is indeed the ideal of most basic investigators to find useful applications for their discoveries and I am convinced that the increasing emphasis on the applied aspects in cancer research is based on a misunderstanding ofthe state ofthe art. There are trees with fruit, but the trees are few and wide apart. We urgently need more trees, not more people trying to get the few fruits available. There is a clear risk that attempts to force more translational research while the science base is still narrow will result in more duplication and more trivial research, rather than benefits to the patient. Empirica! research in cancer is reaching its natura! limits. What we need are solutions based on a thorough understanding of the underlying cell biology. This integrated science-based approach will remain the guiding principle in our research. A site visit of the Division of Experimental Therapy Site visits by outside experts are an essential part of our research policy. In June!995 Division VIII was visited by Or WG McKenna, Henri K Pancoast Associate Professor and Chairman, University of Pennsylvania Medical Center, Philadelphia, Prof. I Tannock, Chief of Medicine, Ontario Cancer Institute/ Princess Margaret Hospital, Toronto, and Prof. NA Wright, Director of Clinical Research, Imperial Cancer Research Fund, London. In a day packed with talks and discussions the visitors evaluated the ongoing research and comrnented on future plans. Thus far site visits were organized somewhat haphazardlyon an ad hoc basis. In view ofthe stimulating effect of these visits on optimizing our research programs, we have started a more systematic site visit schedule in which every research division will be reviewed on a quinquennial basis. N ew NKI professors and other honorable side activities The NKI/ AvL cannot award university degrees, but many NKI/ AvL staff members hold special part- time chairs in one of the Dutch universities, taking about 10% of their time. In 1995, W Mooienaar was appointed parttime professor of Molecular Cell Biology at the U niversity of Leiden and N Aaronson part-time professor of Psychosocial Oncology at the Free U niversity in Amsterdam. A coveted distinction for basic scientists is the elected membership of the European Molecular Biology Organization. In 1995 two additional NKI/ A vl staff members were elected: R Bernards and R Plasterk. Changes in NKI academie staff In September Joop van Dongen left us after a long and distinguished career as oncological surgeon in our institute: he started 39 years ago as junior surgical assistant and he has been an essential and representative part of the institute for nearly half of its 82 year history, for 10 years also serving as head of surgery. Although an erudite, amiable gentleman with respect for consensus and tradition, he proved a staunch defender of surgical oncology as the key element in the multidisciplinary approach to cancer treatment. A world authority on breast cancer, he was a master in integrating the multiple treatment modalities and his interest in adjuvant therapy and quality oflife issues made him an obvious conference chairman, consensus reporter and ambassador of the NKI/ AvL approach to cancer treatment. As the special professor in surgical oncology in the University of Amsterdam, Joop van Dongen shared his profound knowledge of cancer with his academic colleagues, interns and medical students. We celebrated Joop's retirement with a festive Symposium annex party, in which art and science were harmoniously integrated, an integration also personified by Joop van Dongen and his family. Joop was

18 17 succeeded in 1993 as he ad of surgery by Bin Kroon, staff surgeon in the NKII AvL since In September we also said goodbye to Wolter Mooi, head of our Pathology Department and also head of the Division of Tumor Biology. In the 9 years th at Wolter worked in the NKII AvL his contributions we re manifold and substantial. In 1988, only 31 years old, he became head of Pathology and in the years that followed the scientific output of the department was more than doubled. He set up a molecular pathology laboratory within Pathology that became a key player in moving research at the cross-road between clinic and basic research. As the first clinician to lead a laboratory division in the NKII AvL, Tumor Biology, Wolter Mooi played an essential role in strengthening translational research in the NKI/ AvL. The 7 years that W Mooi headed Pathology were among the most turbulent in the history ofthe institute. The organisational structure of all clinical activities was drastically modernized and several plans were made for an entirely new NKII AvL. As a member of the council of senior scientists and chairman of the medical staff Wolter Mooi helped to steer the institute through these turbulent waters. He also managed to produce a large number of important papers on lung cancer and a hefty book on Biopsy Pathology of Melanocytic Disorders (together with Krausz), attesting to his brilliance and wide range of activities in the past 9 years. In the Erasmus University and Rotterdam Academic Hospital Wolter Mooi wiu continue his career as Chairman ofthe Pathology Department and although his moving is a loss to our institute it will certainly help to further improve the cordial relations and collaboration between the Erasmus Cancer Center and the NKII AvL. W olter Mooi will be succeeded as head of Pathology by Marc van de Vijver, a pathologist who did his PhD with RoeI Nusse in the Division of Molecular Biology of the NKII AvL and then received his pathology training at the University of Leiden, where he became a staff member after spending a few months with J ohn Mendelsohn in the Memorial Sloan Kettering Institute in New York. Another well-known NKII AvL staff member who retired in 1995 was Mels Sluyser, staff member of the Division of Tumor Biology. Sluyser is known for his studies on murine breast cancer, especially for the mechanism of progression to horrnone independence. He writes weil and he is known to the general public for his popular books on cancer and his editorial work as Editorin-chief of Cancer Drugs, the popular journal Tijdschrift Kanker and as editor of Cancer Letters. Other staff members that left the institute were L den Engelse, C Schaake-Koning, R Gregor and H Vis. Leo den Engelse retired early for health reasons. He is an expert on chemical carcinogenesis who served the institute in many different functions, e. g. as head of the Division of Molecular Carcinogenesis, as Laboratory Research Coordinator and as the special professor in Biochemical Oncology at Leiden University. Caro Schaake-Koning, staff radiotherapist, left the institute to become head of Radiotherapy at the Westeinde Hospital in The Hague. For years she was a prominent member of the Medical Staff Council and the Ethics Committee and her important studies on combination therapy of lung cancer with radiotherapy and cisplatin, reported in the New England J Med, attracted world-wide attention. She will also be remembered for her ability to explain, in simple and elegant terms, complex issues in cancer to the lay public. R Gregor was invited to become the new chairman of the Department of Otolaryngology at the University of Stellen bosch and we were sorry to lose this charming and competent South-African again af ter only three years at our institute. He will be replaced by IB Tan. H Vis retired after serving the institute for more than 25 years as anaesthesist. An important new addition to the institute was the new plastic surgery group that started its work in January The members ofthis group, K Bos, J de Boer and R Noordanus have part-time appointments in our institute and in the Academic Medical Centre in Amsterdam and their addition to our surgical staff has greatly increased our ability to offer sophisticated reconstructive plastic surgery to patients after large oncological operations. Table 4 NKI research budgets, (x10 6 HFL) ) Annual program grants a) Department of health: - structural b) Dutch Cancer Society (KWF) - structural incidental 2) Large equipment and special facilities ) Project grants ) Yield bond issue 5) Members contributions and other income ) Deficit * Estimate

19 18 We also welcome two new staff clinicians, N van der Vange (gynaecologist) and BMP Aleman (radiotherapist). One AvL-fellow joined the institute in 1995: Maarten van Lohuizen received rus PhD on the basis ofwork done in our Division of Molecular Genetics (Berns) and he completed his training in the laboratory of Ira Herskowitz in UCSF. The AvL-fellowship is a 5-year research career award for gifted young scientists. It offers an opportunity for independent research rarely available to junior scientists in the Netherlands. N ational and international activities In addition to their research and clinical activities, staff members of the NKI/ AvL fulfilled a large number of functions in international organizations such as AACR, CIBA, EACR, EBCTCG, EMBO, EORTC, ESSO, ESTRO, ICRU, MRC, OECI, WFSOS, WHO. Staff members also served on boards of organizations such as the EOR TC cooperative groups, International Association for Breast Cancer Research, Nederlandse Commissie voor Stralingsdosimetrie, Nederlandse Werkgroep Hoofd-Halstumoren, Oog en Orbita Commissie, International Psycho-oncology Society, European Community Committee on Palliative Cancer Care, Comprehensive Cancer Center Amsterdam, European Society for Therapeutic Oncology, the WHO Cancer and Palliative Unit, the International Academy of Pathology, the Gezondheidsraad, het NWO Gebiedsbestuur der Medische Wetenschappen, the European Cancer Center, National Advisory Board on AIDS, Netherlands Health Council Committee on Home Care for Cancer Patients, Scientific Council on Social Oncology (WRSO) of the Dutch Cancer Society, Education and Psychosocial Research Committee of the Cancer Research Campaign, the Dutch and European Societies of Surgical Oncology, the World Federation of Surgical Oncological Societies, etc. Others served as editors ofscientific books or served on editorial boards of journals (38 in total). Staff members were also active in organizing national and international oncology meetings, workshops and congresses, and participated in teaching for the European School of Oncology and the ESTRO teaching course on Radiation Physics for Clinical Radiotherapy and the ESTRO teaching course on Basic Clinical Radiobiology. Changes in Board of Governors and Advisory Boards Af ter serving for 14 years as the secretary of our Board of Governors P den Tex, a lawyer and director of an insurance company, retired in We are most grateful to him for his steady support and advice over this long period. In his place we welcomed JF Visser, a lawyer who brings to the board his long expertise in financial matters as the former director and chief executive officer of the Westland/ Utrecht mortgage and loan bank. Two members of our National Scientific Advisory Board retired from the board in 1995: Professor C Haanen, emeritus professor of Internal Medicine at the University of Nijmegen and Professor J Oldhoff, emeritus professor of General Surgery at the University of Groningen. They were both active members of our advisory board and we are indebted to them for their ad vice and support in critical periods in the life of the institute. Outlook These are excltmg times for cancer institutes. The genetic paradigm for understanding cancer is proving remarkably successful and the secrets of the cancer cell are unveiled at a high pace. We are indeed privileged to be able to participate at the cutting edge of these profound new developments. We are fortunate to receive the support in our work from so many gifted and dedicated individuals, who gave their time to our Governing Board and our National and International Advisory Boards. We are also grateful to the Governing Board and staff of the Dutch Cancer Society for their gene rous and unflinching financial and moral support. We thank all these individuals for their help and friendship and we hope that they will find thejustification in this report to continue their support of astrong and independent Netherlands Cancer Institute in 1996 and beyond. Piet Borst Director of Research

20 19 I Division OF Cell Biology Head E Roos PhD Permanent academie staff J Calafat PhD, JGNM Collard PhD, CA Feitkamp PhD, A Sonnenberg PhD Other academie staff GO Delwel MSc, MHE Driessens MSc, A vd Flier MSc, HAM Geerts PhD, CML Gimond PhD, GGM Habets PhD, PL Hordijk PhD, H Kemperrnan PhD, FN van Leeuwen PhD, M van Leusden MSc, AA de Melker MSc, AML Meijne MSc, GAM Michiels PhD, R van der Neut PhD, CM Niessen MSc, MP Sanchez Aparicio PhD, RDM Soede PhD, JC Stam MSc, PJM Stroeken MSc Permanent technical staff JWRM Janssen, RA van der Kammen, IMM Kuikman, E Noteboom, CH Ong, NF Rodriguez Erena, EMF Ruuls-Van Stalle, YM Wijnands Other technical staff DLA Fles, EHM Huisman, D Kramer, AM Martinez de Velasco, EAM van Rijthoven, PEM van Run, SE VerbakeI Division leader Ed Roos Students and trainee technicians H Kain, JP ten Klooster, JJL Schoofs Guests C Baudoin PhD, C Benassayag PhD, L Borradori MD Secretary SP Smeets

21 20 Ce!! Bio!ogy Introduction The most dangerous aspect of cancer is metastasis, the spread of ceus from the primary tumor to distant sites, of ten to vitalorgans. The reasons for this aberrant behavior are still poorly understood but most likely include changes in the regulation of cell adhesion and migration. These phenomena are therefore the main focus of our research. One of our recent results was the discovery ofthe Tiaml gene, which encodes a protein that can induce metastatic capacity in Iymphoma cells. The Tiam I protein was demonstrated to be an activator ofthe Rac GTPase. This year we discovered that Tiaml is also an oncogene. It can transform normal fibroblasts to tumor cells. Another major result was the generation of mice that are deficient in the a6[34 integrin. This adhesion molecule is normally mostly confined to specific structures, the hemidesmosomes. However, in tumor cells it is often dispersed and overexpressed. The loss of a6[34 was found to result in multiple defects. The availability of these mice will facilitate the elucidation of the many functions of this molecule. A third important result was the demonstration of a requirement for certain G proteins in metastasis oflymphomas, e. g. to the liver. This implies that factors present in the liver participate in the metastatic process. Adhesion mechanisms in metastasis H Kemperman I, MHE Oriessens 2, AML Meijne, ROM Soede4, C Benassayag 5, NF Rodriguez Erena, YM Wijnands4, EAM van Rijthoven l,3, PEM van Run 2, EMF Ruuls-Van Stalle, P Stroeken3, CA FeItkamp, E Roos Role of adhesion molecules in metastasis studied with knock-out mutants To study the role of proteins in tumor metastasis, we aim to generate tumor cell mutants in which the encoding genes have been disrupted. We obtained C044-negative mutants of MOA Y-02 Iymphosarcoma cells by sequential disruption of both alleles of the CD44 gene. The C044 protein, i.e. the standard form not containing domains encoded by alternatively spliced exons, has been postulated to be essential for s.c. growth and metastasis of Iymphoma and melanoma cells. However, we observed no difference between MOA Y -02 and mutant cells in s.c. growth or metastasis to liver, spleen and bone marrow. Hence, C044 is not required for metastasis of all Iymphomas. The discrepancy might be explained if some tumor cell lines depend on proliferation signals induced upon binding of C044 to its ligand hyaluronic acid and others, such as MOA Y -02, do not. Additional mutants with disruptions in integrin genes are being generated. The role of LFA-l in lymphoma metastasis LFA-I-deficient mutants of TAM202 T-cell hybridoma ceus hardly metastasize, in line with the complete block of in vitro invasion by the anti-lfa-i mab M 17 / 4. However, M does not affect metastasis. This discrepancy may be explained by our recent finding that M 1714, in slightly less than saturating concentrations, stimulates LF A-I-mediated adhesion instead of blocking it. We observed that MI7 14 induced aggregation oftam202 cells and that this was inhibited bya mab against the LFA-lligand ICAM-2, present on these eejls, implying that it was mediated by LF A-I itself. Since aggregation is not induced by Fab fragments, we assume th at cross-linking of LF A-I, and probably signal transmission induced by th is, is responsible for the effect. Strikingly, M18 / 2, a mab against the [32-subunit of LF A-I which does not affect adhesion to purified ICAM-I, blocked this aggregation. It remains to be determined whether this inhibition is due to aspecific block oficam-2 binding, implying that the binding sites for ICAM-l and ICAM-2 are distinct, or to interference with M 1714-induced cross-linking. The lack of effect on metastasis by M 1714 may therefore be due to insufficient in vivo concentrations, which may have caused stimulation rat her than inhibition of invasion. Alternatively, the interaction with ICAM-2, which is present in many tissues, may not have been blocked by MI7 / 4. We have generated a new anti-[32 mab, GAME-46, which blocks both aggregation and ICAM-I binding. We will repeat the metastasis blocking experiments with this mab. These results ijlustrate one of the many pitfajls in the use of antibodies to study the role of surface proteins in metastasis. This is a major reason why we aim to use targeted mutants instead, as described above. G-protein signals are essential for T-cell hybridoma metastasis 10 liver and spleen LFA-l is required for metastasis, but yet it is not in an active state on T-ceJl hybridomas. The cells require stimulators like the phorbol ester PMA or Mn2+ to bind to LFA-l, and thus stimulation apparently occurs during invasion. Pertussis toxin inhibits invasion, probably by blocking LF A-I stimulation, since inhibition is reversed by PMA or Mn2+. This implies that invaded tissues contain LF A-I-activating factors. To demonstrate the relevance of this phenomenon for metastasis, we transfected the cona encoding the SI catalytic subunit of the toxin into T AM202 cells, to inactivate G proteins permanently. In two transfectants, S05 and S09, 88% and 95% of the G j proteins we re inactivated, respectively. In vitro invasiveness of both was greatly reduced. The S09 ceus formed virtually no metastases in liver or spleen. Strikingly, metastasis of S05 cejls was not reduced. However, the metastases formed by S05 cells were found to contain cells that had lost SI expression. This selection demonstrates that G j-transduced signals are essential. G-protein-aclivated adhesion: role of tyrosine kinases and phospholipases The G-protein activator AlF 4 - was found to induce ICAM-l-mediated adhesion of TAM202 cells, supporting the notion that G-protein signals can stimulate integrin function. This was also true for [3I-integrin-mediated adhesion of MOAY-02 cells to fibronectin. This may be particularly relevant for liver

22 Cel! Biology 21 metastasis, as we have shown for a carcinoma cellline (see below). Neither AIF 4 --induced adhesion to ICAM-l nor invasion was affected by protein kinase C inhibitors, which blocked PMA-stimulated binding. Conversely, the G-protein pathway was blocked by tyrosine kinase inhibitors, which had much less effect on PMA-induced adhesion. This is consistent with the notion of G-protein stimulation of LF A-I during invasion. Phospholipases are potential downstream targets in the G-protein pathway. Indeed, both the PI-PLC inhibitor U and the PC-PLC inhibitor D609 substantially reduced adhesion and invasion. However, PMA- and Mn2+ -stimulated adhesion were also affected. These reagents may either block common post-ligand-binding events, or affect a basic requirement for adhesion, possibly PIP 2 levels. Modulation ofintegrinfunction by other surface molecules MDAY-D2 cells express both ct4f31 and ct5f31 fibronectin receptors and yet only ct5f31-fibronectin binding is stimulated by PMA, Mn 2 + or the 9EG7 anti-f31 mab, which we found to enhance integrin activity. Binding by ct4f31 was, however, induced in ESb T-Iymphoma cells, which do not express ct5f31. Furthermore, we did observe ct4f31 stimulation in a MDA Y-D2 variant which lacked ct5f31 expression. In contrast, binding of 0:4131 to VCAM-l occurred normally in the presence of 0:5131. The reason for this hierarchy is not clear but it is conceivably due to competition for accessory proteins. Both the 9EG7 mab and Mn2+ stimulated 0:6131- mediated laminin binding of T-cell hybridomas. This resulted in accumulation of ct6f31 and, surprisingly, also LFA-I in vinculin- and ct-actinin-containing structures. These two stimulators act by changing the conformation of ct6f31. PMA did not cause this accumulation, suggestirig that it does not enhance adhesion by inducing similar changes in conformation that increase affinity. The binding of ct4f31 on melanoma cells to the CSI sequenee in fibronectin is influenced by chondroitin sulphate proteoglycans (CSPG), which bind to sites near CS I. One of our graduate students, ALM Meijne, studied th is further in the laboratory of J McCarthy of the University of Minnesota. A mab against a melanoma proteoglycan enhanced ct4f31 function, even when bound elsewhere at the ceb surface, indicating th at dimerization of the proteoglycan induced integrin-activating signais. Removal of chondroitin sulphate GAG-chains reduced adhesion to a recombinant fibronectin fragment containing CS 1 but no CSPG binding site. This inhibition was reversed by an integrin-activating mab, indicating th at direct interactions between ct4f31 and CSPG also occur, which stabilize the active conformation of the integrin. FinaUy, we have shown that the mucin epiglycanin completely prevents adhesion mediated by 0:6134 and also by E-cadherin on suspension-grown TA3/ Ha mammary carcinoma ceus. Adhesion and the formation of an epithelial monolayer was resto red upon removal of the mucin. It is clear that the activity of integrins is influenced by multiple parameters: diverse signaling pathways, association with the cytoskeleton, as weil as other cell surface molecules. The role of integrins in invasion and metastasis of specific tumor cells can therefore only be fully understood when all of these parameters can be taken into account. Fibronectin receptors in carcinoma metastasis to the liver Previously, we found that adhesion of TA3/ St mammary carcinoma cells to hepatocytes is due to interaction of ct5f31 with fibronectin on the hepatocyte surface. The liver is unique in that a basement membrane is lacking under the endothelium of microvessels. Instead, a fibronectin-rich matrix is present. Thus, adhesion to fibronectin may be of major importance for metastasis to the liver. To study this further, we have used a triple knockout (TKO) mutant of F9 murine embryonal carcinoma cells, in collaboration with CA Damsky of the University of California in San Francisco. These F9 cells contained three copies ofthe 131 integrin gene, which have all been disrupted by targeted mutation in the TKO cells. Upon intrasplenic injection, F9 cells formed abundant liver metastases, but TKO eells very few. Both cells grew equally weil when injected directly into the liver parenchyma and remaining viabie cells at the injection site gave ri se to similarly large tumors. Interestingly, the TKO cells are unable to grow subcutaneously. To show that this reduced metastasis is due to the lack of fibronectin binding, we intend to transfect the cdna of the integrin 136 subunit that we have isolated from a keratinocyte library. With the endogenous o:v subunit, this should yield an ctv 136 dimer which is aspecific fibronectin receptor. The ctv 135 and ctv 136 integrins have been discovered as tumor-specific integrins on aggressive carcinomas. Later, they were also found on normal epithelial cells in areas of inflammation and wound healing and they may play specific roles in migration and proliferation in wounds. We found that HT29 human colon carcinoma cells, which lack ct5f31, ad here to fibronectin via ctv 136, even though the levels are very low. Using ctv-specific mab and immune fluorescence, we observed specific surface structures, different from adhesion plaques, th at are possibly required for these special functions. Given the size of the structures and the intensity of the fluoreseence, not only o:v 136, but also the abundant o:v 135 may be located there, even though the latter is not involved in fibronectin binding. We have generated mabs against the 135 and 136 cytoplasmic domains to investigate this further. Given the postulated role in migration and, therefore, invasion, it will be interesting to compare the in vivo malignant phenotype of F9 eells and the f36-transfected TKO ceus. Src transformation of rat fibroblasts requires new gene transcription Previously, we have studied the disruption of adhesion plaques by v-sre in RSV -infected chicken fibroblasts. To use a better defined system, we extended these studies to rat fibroblasts, stably transfeeted with temperaturesensitive mutants ofv-src and also v-fps. The sub st ra te of