PATRON HM THE QUEEN BEATRIX

Transcription

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2 Annual Report 1993

3 PATRON HM THE QUEEN BEATRIX

4 Annual Report 1993 THE NETHERLANDS CANCER INSTITUTE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL

5 Annual Report 1993 Illustrations and unpublished data in these reports should not be used without permission of the author. Copyright : The N etherlands Ca neer 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 27 III Cellular Biochemistry 33 IV Immunology 43 V Molecular Biology 51 VI Tumor Biology 61 VII Molecular Genetics 69 VIII Experimental Therapy 77 IX Radiotherapy 87 X Medical Oncology 97 XI Surgical Oncology 109 XII Psychosocial Research and Epidemiology 117 Biometrics Department 125 Biophysics Department 131 Laboratory Animal Department 135 Cancer Hospital 139 Clinical W orking Parties 145 Ongoing Trials 149 Education in Oncology 169 Projects 181 Publications 191 Author Index 219 Personnel-Project Index 229

7 6 Board Members International Scientific Advisory Board Jon J van Rood, Professor ofirnrnuno-hematology, 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, Cam bridge, Mass, USA I Bernard Weinstein, Professor of Medicine and Environmental Sciences, New Vork, USA Charles Weissmann, Professor of Molecular Biology, Zürich, Switzerland National Scientific Advisory Board D Bootsma, Professor of Cell Biology and Genetics, Rotterdam AJ van der Eb, Professor of Fundamental Tumor Virology, Leiden CAM Haanen, Professor of Internal Medicine, Nijmegen WGJ Hol, Professor of Molecular Biology, Groningen (till October 1993) SWJ Lamberts, Professor of Internal Medicine, Rotterdam HL Langevoort, Professor of Histology and Embryology, Amsterdam CJLM Meijer, Professor of Pathological Anatomy, Amsterdam J Oldhoff, Professor of General Surgery, Groningen JJ van Rood, Professor ofirnrnuno-hematology, Leiden E van der Schueren, Professor of Oncology, Leuven GNJ Tytgat, Professor of Gastro-enterology, Amsterdam o Vos, Professor ofcell Biology, Histology and Microscopical Anatomy, Rotterdam (till October 1993)

8 7 Board of Directors P Borst, chairrnan and director of research DHJ van Buren, clinical director a.i. (from ) HM Pinedo, director of clinical research (till ) A Roest, financial director Laboratory Research Coordinator E Roos Board of Governors JD Hooglandt, president ML Frohn-de Winter, vice-president P den Tex, secretary o Hattink, treasurer AC van den Blink JEG de Boer (till ) R Hazelhoff PH Hugenholtz S van der Kooij J van der Meer JHM Temmink 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, he ad J Calafat JG Collard CA FeItkamp A Sonnenberg II Molecular carcinogenesis R Bernards (head) L den Engelse E Kriek (honorary staffmember) E Scherer J Westra III Cel/ular biochemistry WH MooIenaar, (head) WJ van Blitterswijk J Borst A Tulp LN Vernie IV Immunology AM Kruisbeek, head WS Bont (honorary staff member) CG Figdor WR Gerritsen A Hekman EM Rankin Ph Rümke (honorary staff member) H Spits CJM Vennegoor (stationed at the Free University) FA Vyth-Dreese K Weijer V Molecular biology RHA Plasterk, head P Borst APM Jongsma HV Westerhoff VI Tumor biology WJ Mooi, head JH Daams AA van der Gugten PhC Hageman J Hilkens D Ivanyi RJ AM Michalides M Sluyser AA Verstraeten VII Molecular 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 G Baris (deceased ) H Bartelink RW de Boer JH Borger IAD Bruinvis BNFM van Bunningen JMV Burgers EMFDamen LGH Dewit AAM Hart M van Herk RB Keus BJ Mijnheer H Meertens LMFMoonen SH Muller CGJHNiël NS RusselI CCE Schaake-Koning FW Wittkämper

10 9 X M edical oncology R Somers, head JW Baars (from ) P Baas JH Beijnen WW ten BokkeI Huinink JMG Bonfrer W Boogerd H Boot PF Bruning ME Craanen (from 0l ) CC Delprat WR Gerritsen (from ) CA Hoefnagel SP Israëls WJ Nooijen HM Pinedo (till ) EM Rankin DJ Richel (tiu ) S Rodenhuis JH Schornagel BGTaal o van Tellingen RA Valdés Olmos N van Zand wijk Research nurses D Batchelor AC Dubbelman ME Schot Trial office lam Mandjes Scientific administration OB Dalesio XI Surgical oncology JA van Dongen, head EJ Aartsen AJM Balm F van Coevorden E Gortzak RT Gregor FJM Hilgers ThJM Helrnerhorst S HorenbIas BBR Kroon OE Nieweg EJTh Rutgers FAN Zoetrnulder XII Psychosocial research and epidemiology NK Aaronson, head FSAM van Dam FE van Leeuwen Biometrics department OB Dalesio, he ad H van Tinteren JL te Velde (from ) Biophysics department GJF Blommestijn, head LCJMOomen HJ Stoffers Laboratory animal departments RGM ten Berg, head Hospital Divisions Anesthesia M Hellendoorn-Smit, head C Blackburn HVis MKaag Clinical chemistry and hematology WJ Nooijen, head JMG Bonfrer Clinical research manager EVos Dental surgery S Gonggrijp APTimmers Gastro-enterology H Boot BG Taal Gynecology ThJM Helrnerhorst, head EJ Aartsen P Kenemans Hospital pastors PG Kousemaker AH Tönis Internal medicine R Somers, head JW Baars (from ) WW ten Bokkei Huinink PF Bruning WR Gerritsen (from 0l )

11 10 SP Israëls HM Pinedo (til 0l ) EM Rankin DJ Richel (till ) S Rodenhuis JH Schornagel Nuc/ear medicine CA Hoefnagel, head RA Valdés Olm os SH Muller Nursing HIM Camerik, acting head Otolaryngology - head & neck surgery FJM Hilgers, head AJM Balm RT Gregor Pain physicians C Mattern APE Vielvoye-Kerkmeer Pathology WJ Mooi, head MPWGallee P van Heerde D de Jong BM Loftus-Coll JL Peterse L van 't Veer Patient counsel/or M Keessen-van Luyken Psych ia try LM Gualthérie van Weezei Psychosocial service DHalm Pulmonology P Baas N van Zand wijk Radiotherapy H Bartelink, head G Baris (deceased ) 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 Masse1ink H Meertens LMF Moonen BJ Mijnheer CGJHNiël NS Russel1 CCE Schaake-Koning FW Wittkämper Diagnostic radiology P Cohen, head a.i. AP Besnard W Koops R Kröger F de Leeuw Surgery JA van Dongen, head F van Coevorden E Gortzak BBR Kroon OE Nieweg EJTh Rutgers FAN Zoetrnulder Urology S Horenbias Consulant Staff Bacteriology WPauw Dermatology H Neering General practitioner CC Delprat Neurology W Boogerd JJ van der Sande N eurosurgery ZD Goedhart B Matricali

12 II Ophthalmology L Koornneeff Orthopedie surgery JW van der Eijken MWFidler Safety, health and welfare P de Lange Technical service TCM Wilmering Pediatrics PA Voûte Pharmacy JH Beijnen ACA Paalman Plastic surgery JB de Boer KBos Rehabilitation ELD Angenot Heads of General Services Audiovisual service JM Lomecky Central cancer library MBA Wil helm-de Gouw Custodian research laboratory CA Feitkamp Financial administration JK Koppenol Housekeeping services RMD Schellens Medical administration JH Helversteyn Outpatient clinic R Pet, manager Personnel department JR de Bruijn

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14 13 Introduction 'Le cancer est d'un consentement unanime Ie plus horrible de tous les maux qui attaquent l'homme '. M Dionis, Cours d'opérations de chirurgie, 4th édition, d'houry, Paris The year 1993 was an eventful one for the NKII AvL: the plans for a new institute were brought a big step closer to realization and a major reorganization of the hospital part of the institute was set in motion. An important change at the pers on nel level was the departure of our (part-time) clinical research director, HM (Bob) Pinedo. Also in 1993 the Netherlands Cancer Institute/ Antoni van Leeuwenhoek Hospital (NKII AvL) reached the age ofthe very strong: 80. We celebrated this anniversary with a smashing party highlighted by a steil ar performance of the famous NKII AvL cabaret group. Our science also tlourished in 1993 and this will be the focus ofthis annual report. Research highlights The list of research highlights in this annual report represents a limited and somewhat arbitrary selection. Additional highlights can be found in the introductions to each of the 12 research divisions. J Collard and his coworkers (Division I) fully characterized the tiam-l gene that they had discovered in 1992 as a major gene involved in invasion and metastasis in mouse lymphomas. Whereas this gene is hardly expressed in normal tissues, the human homolog of tiam-l is active in virtually all human tumor types, suggesting that this gene could play a role in tumor progression and metastasis of human cancer. WH Mooienaar (Division lil) and his coworkers discovered th at their newly identified transmembrane proteintyrosine phosphatase, RPTPJ-l, functions as a cell-cell adhesion molecule. By coupling cell recognition (adhesion) to the regulation of protein phosphorylation (protein phosphatase action), RPTPJ-l may transduce signals that control critical aspects of cell beha vi or important for tumorigenesis, such as contact-mediated growth inhibition or differentiation. CG Figdor and his associates (Division IV) demonstrated that peptides derived from the antigen recognized by the famous monoclonal anti body NKI-beteb, one of the best diagnostic markers of melanocytic cells, are recognized by cytotoxic T cells infiltrating tumor tissue in patients with melanomas. With the gene for this melanocyte-specific antigen in hand, it will be possible to set up mouse model systems to optimize cellular immune responses against this antigen. After a five-year quest, P Borst and coworkers (Division V) finally solved the structure of an unusual base they had previously detected in trypanosome DNA. This proved to be fj-4 glucosyl-hydroxymethyluracil, a base never found before in DNA. The specific location of this base in the trypanosome DNA has raised the possibility that it is involved in shutting off gene expression. In a joint effort of the groups of P Borst (Division V) and AJM Berns (Division VII), a knock-out mouse was generated unable to make the P glycoprotein encoded by the mdr2 gene. These mice develop a cholestatic liver disease, which was analyzed in detail with the help of pathologists in the NKII AvL (WJ Mooi, Division VI) and pathologists and biochemists of the Academic Medical Center ofthe University of Amsterdam. This led to the unexpected discovery that the knock-out mice completely lack phospholipid in their bile, showing that the mdr2 P glycoprotein is essential for secretion of phospholipid by the liver cell, presumably because this P glycoprotein is a specialized phospholipid translocater. It seems likely that defects in the MDR3 gene, the human homolog of the mouse mdr2 gene, will be responsible for some human inborn and acquired liver diseases, but these remain to be identified. AJM Berns and his coworkers (Division VII) have further characterized the function of the bmi-l gene, discovered by them as an oncogene involved in mouse lymphomagenesis. The bmi-l gene has a remarkable effect on mouse development. When the gene is overexpressed the resulting mice show anteriorization along the axial skeleton, whereas elimination of the gene results in posterization. The bmi-l gene is therefore another example of a proto-oncogene that turns out to be a master gene with major effects on embryonic development. The first gene in this class is int-1 (now wnt-1), discovered by R Nusse while he was working in the NKII AvL. Overexpression of the bmi-l gene also leads to an expansion of lymphoid progenitor cells, explaining its role in lymphomagenesis. Promising results were obtained by FA Stewart, N van Zandwijk and coworkers (Division VIII) with a new photosensitizer, mthpc. In animal studies, this compound was found to have a therapeutic gain 2-4 times higher than the Photofrin presently used for photodynamic therapy of cancer patients. As the use of photodynamic therapy for the palliative treatment of lung cancer patients is on the increase, better photosensitizers are urgently required. The innovative image processing procedures developed by M van Herk and associates (Division IX) are finding increasing clinical applications, such as the 3-D image correlation of CT and MRI data of the head to enable a more reliable definition of brain tumor localization. By analysis of radiation-induced lung damage, J Lebesque and coworkers (Division IX) could determine dose-effect relations for local changes of perfusion, ventilation and

15 14 lnlroduclioll density, using a sophisticated 3-D analysis of CT and SPECT data. FE van Leeuwen and her coworkers (Division XII) extrapolated data on passive smoking and lung cancer recently published by the US Environmental Protection Agency to the Dutch population. Their conclusion is that passive smoking is responsible for lung cancer deaths per year in the Netherlands. The announcement of these disturbing figures resulted in moderate publicity in the general press, and in a promise from the Dutch Secretary of Health to take additional steps to restrict tobacco advertising and smoking in the workplace. Nevertheless, it remains remarkable that the Dutch are so complacent about tobacco smoke, a class A carcinogen, in their environment. Honors H Bartelink received a special award from the American Society of Therapeutic Radiation Oncology for his contributions to the improvement of treatment outcome of breast cancer patients. A Berns shared the prestigious Prix Antoine Lacassagne 1993 with D Hanahan, San Francisco, USA. This 200,000 FF prize of the 'French Ligue contre Ie Cancer' was given to A Berns for unraveling mechanisms of tumor progression in transgenic mice. P Borst received the Muntendam Prize of the Dutch Cancer Society for his work on mechanisms of drug resistance in cancer cells and the Gold Medal of the Robert Koch Foundation. CR Lincke was the recipient of the 1992 Antoni van Leeuwenhoek Prize for his elegant studies on the function of P glycoproteins in mammals and in the nematode worm C elegans. The AvL prize is awarded every year to the young investigator(s), student or post doc, who make(s) the most important contribution to cancer research in the NKI. The money for this prize is generously provided by Boehringer Mannheim Ltd. ved the Student Research Award from the 'American Association for the Study ofliver Diseases'; FJM Hilgers became an honorary member of the 'Association des Rééducateurs des Mutilés de la Voix' in France. Research quality Good research should lead to major discoveries that improve our understanding of nature and our ability to diagnose, treat and prevent disease. Major discoveries are rare, however, and of ten it takes years to recognize that an interesting result is actually a major discovery. In the meantime, the public needs some indication that research money is being weil spent and research directors need objective parameters to measure achievement and distribute money accordingly. Two parameters that have found widespread acceptance are the citation frequency and impact of research publications. The impact measures the quality of the journal in which an investigator publishes. High-quality journals have a high impact. The citations measure the number of times an article is cited by other investigators. If an article is never cited, it is unlikely to contain a seminal discovery. The citation score of an article is like some wines: it gets better with age. Citations over a 30-year period are obviously more informative than citations only in the year following publication. For practical purposes, however, only short-term citations can be used for judging active (rather than retired) scientists. Since 1982 we have recorded the short-term citations and impact of scientific articles published by the NKI/ AvL research staff. Table 1 presents the results of this analysis. The trend is up, confirming other indications that the NKI/ A v L is on the right track and lives up to its reputation as a center of excellence. Table 1 Short-term citations and impact of scientific articles published by the NKI research staff : Other honors inc/ude: J Borst was the 'Litchfield lecturer 1993' at the University of Oxford (UK); JJM Smit recei- Pub/ication year Citations* Impact** Professor Piet Borst (right) receives the gold medal of the Robert Koch Foundation from the German Minister of Health, Horst Seehofer (left). * In the two years after publication, excluding self 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 of an article in a given journal. The total impact is the sum of the impact of all articles published that year.

16 Introduction 15 The Oneology Graduate School Amsterdam, European post-does and new NKI Professors Although the NKII AvL has no formal teaching obi i gations, education is an essential part of the activities of the institute as detailed elsewhere in this report. In 1993, 75 graduate students were doing research towards their PhD in the institute. Since 1989, the in-house teaching of these students has been formalized, initially in the NKI Graduate School, the first fully operational Graduate School in the Netherlands, and since 1991 in the Oncology Graduate School Amsterdam in which we collaborate with our oncological colleagues ofthe University of Amsterdam and the Free University (also located in Amsterdam). In 1993, this Graduate School was formally recognized by the Royal Academy of the Arts and Sciences of the Netherlands and its activities we re stimulated by a fl 1 million grant from the Netherlands Organization for Scientific Research (NWO). We were also recognized this year by the European Community as an institute for training post-docs from other European countries in the framework ofthe Human Capital and Mobility program. The NKI/ AvL cannot award degrees, but many NKII A v L staff members hold special part- time chairs in one of the Dutch universities, taking about 10% of their time. In 1993, RHA Plasterk, head of the Division of Molecular Biology and one of the three directors of the Oncology Graduate School Amsterdam, was appointed part-time Professor of Molecular Microbiology at the Free University of Amsterdam. Some new developments in the NKII AvL One of the unique advantages of an integrated cancer institute, such as the NKII AvL, is the ease of organizing multi-disciplinary activities. Patients are seen by surgeons, radiotherapists and medical oncologists in jointly run outpatient clinics; research projects requiring coope- ration between cell biologists, biochemists, immunologists and geneticists are unencumbered by rigid departmental segmentation; couaborative projects between basic scientists and clinicians, often so difficult to organize in universities, develop naturally in an integrated cancer institute. As improvements in clinical oncology are more and more science driven, we cherish the opportunities th at the NKII AvL offers for clinical research with substantial input from the research laboratory. In the past two years we have further improved the interface between hospital and research laboratory in three areas. 1) Molecular pathology was strengthened by an increase in the pathology staff and by the setting up of a molecular pathology laboratory as part ofthe Pathology Department. 2) Clinical immunology was strengthened by the hiring of a medical oncologist WR Gerritsen, and acellular immunoiogist H Spits, both with experience in the use of growth factors. Together with existing staff they form astrong team to exploit new opportunities in immunotherapy, as indicated in the introduction to the report ofdivisions IV and X. 3) Although it is too early to judge whether gene therapy wiu make a contribution to cancer therapy, this is certainly an area that requires vigorous exploration in the coming years. The NKI/ AvL, with strong groups in molecular genetics, immunology and clinical research, is in a good position to make a significant contribution in this area. Our entry into gene therapy is being facilitated by the experience gained by staffmember AlM Berns during a sabbaticalleave at Somatix Therapy Corporation USA, in which he is helping to work out protocols for culturing ceus from human tumors and introducing new genes into these cells. Promising results have already been obtained in mouse model systems with melanoma ceus transformed with the gene for the growth factor GM-CSF. When these transformed cells are given back to mice, they evoke a strong cellular immune response that can also kiu the non-transformed melanoma cells present in the recipient mouse. We are setting up a clinical trial exploiting this approach in NKI/ AvL patients with melanoma, in collaboration with Somatix Therapy Corporation. Table 2 NKi research budgets * Budgets (x 10 6 HFL) 1. Annual program grants a. Department of Health: b. Dutch Cancer Society (KWF) - structural incidental Large equipment and special facilities Project grants Yield bond issue Deficit * Estimate

17 16 IIllroduction Research budgets In July 1992, the Secretary of Health announced his intention to cut the government core grant to the NKII A vl by 8%. In December 1992, however, we were successful in convincing the members of parliament th at the cut should not be carried through. In July 1993, the Secretary of Health inforrned us of his plan to cut the NKI/ AvL co re grant by 5% in 1994, and by an additional 10% in The core grants from the government and the Dutch Cancer Society pay for our staff, our research costs and our infrastructure. We also attract a large number of competitive project grants, as illustrated in Table 2, but these grants contain no overhead component. All overheads must be paid from core grants. Between 1983 and 1992 our government core grant rose from fl 15.9 to fl16.7 million, a me re 5% (Tabie 11). Note that these are nominal figures uncorrected for inflation. The purchasing power of this grant has fallen to less than 70% of its original value in these 10 years. We have only survived by rigorous cost-cutting and, in recent years, by the increasing support from our national cancer charity, the Dutch Cancer Society (KWF). Like most governments, the Dutch government struggles to balance the budget. Government spending rose by 47% between 1983 and This was clearly not caused by lavish support for cancer research. Hence, the cut in our core grant is unreasonable. It is also not justified by lack of performance (but this argument has never been used in this unfortunate affair). As we cannot handle a 15% cut in our core grant without serious damage to the institute, we are again putting our case before the members of parliament. As this report goes to press, we just learned that the members of parliament have unanimously voted to support our request to maintain our core grant at the 1993 level. A change in the organizational structure of the hospital Unti11993, the clinical activities ofthe NKII AvL hospital were organized in many small departments, and the responsibility for clinical research was separate from responsibility for patient care. In 1993, a new structure was created, which subdivides clinical activities into four clusters: surgical oncology, radiotherapy, medical oncology and diagnostics. Each cluster is led by a medical cluster head, with final responsibility for both care and research in the cluster. We expect this structure to further improve the quality and visibility of clinical research, to diminish tensions between care and research, and to improve the efficient management of tight budgets. Changes in personnel In early December we were shocked and saddened by the sudden death of Gertrude Baris, who had worked as a radiotherapist in the institute since Gertrude was an energetic, cheerful and much respected radiotherapist who will be greatly missed by her colleagues and patients alike. In September 1993, HM Pinedo decided to step down as clinical research director. We had induced Bob Pinedo to come to the NKII A vl in 1989 and for four years we profited from his profound knowledge ofmedical oncology, his keen eye for new developments in clinical research, his reputation as a clinician, his fame and his world-wide connections as one of the leading European oncologists, and rus remarkable talents in attracting money for research. For four years Bob Pinedo combined his job as clinical research director ofthe NKII AvL with hisjob as head of Medical Oncology (which includes the Clinical Cancer Pharmacology Unit) at the Free University. In the long run, however, the combination of these two highly demanding jobs became too much, even for somebody with the energy and stamina of Bob Pinedo. In the end he opted for his department in the Free University, which allowed him a c10ser involvement in patient care and research than a directorship at the NKII AvL could possibly offer. We we re very sorry to see him go, we shall miss his insights, gentie sense of humor and sparkling ideas, but we shall remain most grateful for his many contributions to research and patient care in our institute. Fortunately, we shall continue to be in close contact with Bob Pinedo, as he has ag reed to accept a post as special adviser to the board of di rectors of the NKII AvL and as an honorary staff member of our Medical Oncology group. Two new AvL fellows joined the institute in 1993, the cell biologist JJ Neefjes (Division 111) and the physicist M van Herk (Division IX). The AvL fellowship is a 5-year research career award for exceptionally gifted young scientists. It offers an independence rarely available to junior scientists in the Netherlands. Early in 1993, H Spits, a former staff member of the institute, returned as a new staff member of Division IV af ter a highly productive period at DNAX, initially in France and later in the USA. He brings to the institute his experience in T -cell differentiation and cytokine actions. W Gerritsen, a young medical oncologist hematologist, joined the institute after his training in the Memorial Sloan Kettering Cancer Center New York, and the University of Utrecht. He has a joint appointment in our Divisions of Immunology and Medical Oncology and he will be one of the clinicians who will help us to strengthen the interface between clinical and basic research. The Pathology Department sawa substantial increase of its staff in 1993 by the recruitment of a young clinical pathologist, D de Jong, trained at Leiden University, and L van 't Veer, an experienced molecular biologist who worked with R Bernards in our Division of Molecular Carcinogenesis. D Richel, one ofthemedical oncologists who set up the very successful peripheral stem cell transplantation program in our institute left and was rep!aced by J Baars, who received her training with HM Pinedo at the Free University and at the Dr Daniel den Hoed Cancer Center. Our medica! gastroentero!ogy group was strengthened by the recruitment of M Craanen, who recent!y comp!eted his training with G Tytgat in the Academic Medica! Center of the University of Amsterdam. NS RusselI, a young radiation oncologist working in the institute as a fellow of the European Cancer Center, joined the Department of Radiotherapy. She will continue her work on predictive assays of radiation-induced

18 Introduction 17 normal tissue damage in collaboration with Division VIII. EMF Damen a post doc physicist working in a project on the analysis of radiation-induced lung damage, also joined the Department of Radiotherapy. Finally E Vos succeeded SEM Kloezen as the clinical research manager. N ational and international activities In addition to their research and clinical activities, staff members of the Netherlands Cancer Institute fulfijled a large number of functions in internationalorganizations such as AACR, CIBA, EACR, EBCTCG, EMBO, EORTC, ESSO, ESIRO, ICRU, MRC, OECI, WHO. Staff members also served on boards of organizations such as the EORTC cooperative groups, International Association for Breast Cancer Research, Nederlandse Commissie voor Stralingsdosimetrie, Nederlandse Werkgroep Hoofd-Halstumoren, Oog en Orbita Commissie, European Society of Psychosocial Oncology, European Community Committee on Palliative Cancer Care, Comprehensive Cancer Center Amsterdam, European Society for Therapeutic Oncology, the WHO Quality of Life Group, 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 ofthe Cancer Research Campaign, 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. The next 80 years When the NKII AvL was inaugurated in 1913 as one of the first integrated cancer institutes in the world, little was known of the nature or causes of cancer. The methods available for diagnosis and treatment were inadequate, even pathetic when compared to present-day standards. To improve care, research would be required. Hence, a modest research laboratory was already a prominent part of the hospital that was inaugurated and this concept of care and research within one organization has stood the test of time. Improvements in care are more and more science-driven, and even basic cancer research increasingly yields results with direct applications in patient care. Hence, there is a clear need for independent, integrated cancer institutes, such as the NKI/ AvL. Although it is conceivable that such centers could also flourish in a university context, this remains to be proven in the Dutch situation. For the time being, we treasure our independence. To guarantee an illustrious future commensurate with our past, we need room for expansion and we need stateof-the-art facilities. Our present space and facilities are stretched to the limit and offer only very lirnited possibilities for much needed renovation, particularly of our hospital facilities. In the past two years we have worked out two plans for building a new hospital. Plan I would move the entire NKII AvL, hospital and research lab, to a new location adjacent to one of the two academic hospitals in Amsterdam. This would pro vide us with a brand new building with ready access to academic medical facilities. Detailed studies in showed that there is adequate space next to both academic hospitais. Plan II is limited to the construction of a new hospital on the present premises; more room for research would be created by renovating and adapting the old hospital building. Plans land II carry a very different price tag: fl330 versus fl 200 million. The difference is largely explained by the loss of the capital value of the present buildings for radiotherapy, animal care and research if we were to move to another location. These buildings are still relatively new and could not be used for other purposes. Early this year, the Secretary of Health nominated a smajl committee to evaluate the two plans. This committee, consisting of the former Secretary of Health, L Stuyt and the lawyer and former member of parliament, J van der Meer, consulted all medical faculties and governing boards of academic hospitals in the Netherlands and carefully weighed possible alternatives. In November the committee advised the government to follow plan Il. Co st was a major consideration. The committee also believed that the Netherlands needs a strong, independent cancer research institute, and that this independence might be jeopardized by relocating next to an academic hospital. We expect the government to accept this advice, but finding the funds for the bricks and morter wijl not be easy was again a busy year for all the boards and individuals who support and advise us in our efforts to keep the NKII A vl at the cutting edge of cancer research and care. The reorganization ofthe hospital, the planning ofthe future NKII AvL and the threat of a major cutback in our core grant, kept our goveming board intensely occupied during the entire year. Their ad vice and support was indispensable. Our national and international scientific boards helped us to maintain perspective, and the governing board and staff of the Dutch Cancer Society gave us generous financial and moral support. We are grateful to all those individuals for their help and friendship and we hope th at they wiu find inspiration in this report to continue their support of astrong and independent Netherlands Cancer Institute in 1994 and beyond. Piet Borst Director of Research

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20 19 I Division of Cell Biology Head E Roos PhD Permanent academie staff J Calafat PhD, JG Collard PhD, CA Feitkamp PhD, A Sonnenberg PhD Other academie staff GO Delwel MSc, MHE Driessens MSc, GGM Habets MSc, FBL Hogervorst PhD, H Kemperman MSc, G La Rivière PhD, AML Meijne MSc, AA de Melker MSc, R van der Neut PhD, CM Niessen MSc, Je Stam MSc Permanent technical staff JWRM Janssen, RA van der Kammen, JWTM Klein Gebbinck, IMM Kuikman, E Noteboom, CH Ong, NF Rodriguez Erena, EMF Ruuls-Van Stalle, CA Schipper, YM Wijnands Other technical staff DM Casey, DLA Fles, EHM Huisman, EAM van Rijthoven, AEM Zuurbier, DA TM Zuydgeest Students and trainee technicians V Blok, C Molenaar, B Nagelkerken, E Rots, PStroeken, J van der Velde Guests C Gimond, A Martinez de Velasco Secretary JAM van Niele-Pouw, E Stolk

21 20 Cell Bia/agy Introduction The main research topic in this division is the regulation of adhesion and motility of cells, in relation to tumor progression and metastasis. One of our aims is to identify adhesion molecules, and study the regulation of their activity and their interactions with cytoskeletal components. Furthermore, we aim to identify genes that control the function of adhesion molecules and other elements of the invasive phenotype. Major developments this year were the completion of the coding sequence of the Tiam-l gene and the demonstration that a truncated transcript induces invasive potential in Iymphoma cells, the discovery that adhesion molecules involved in lymphoma invasion require activation upon contact with the invaded tissues and the determination of the ligand specificities of the a3f31 and a6f34 integrins. Adhesion mechanisms in metastasis G la Rivière I, H Kemperman 2, MHE Driessens 3, AML Meijne4, CA Schipper, NF Rodriguez Erena, YM Wijnands 2, JWTM Klein Gebbinck I, EAM van Rijthoven 3, DM Casey 4, AEM Zuurbierl, EMF Ruuls-Van Stalle, PStroeken, CA Feitkamp, E Roos Activation of adhesion molecules during lymphoma metastasis Lymphomas of ten metastasize preferentially to the liver, in addition to many other organs. We study the mechanisms of invasion of lymphoma cells from the blood into tissues in several in vitro modeis: short-term cultures of rat hepatocytes and liver sinusoidal endothelial cells for liver-specific invasion and rat fibroblast cultures for more general invasion mechanisms. Previously, we observed that activated normal T lymphocytes rapidly invaded these monolayers. Fusion of such T cells with non-metastatic Iymphoma cells yielded T-cell hybridomas that were highly invasive and metastasized widely. In addition to these hybrids, the two highly metastatic Iymphoma cell lines ESb and MDAY-D2 were studied. We previously established that the cellular adhesion molecule (CAM) LF A-I is required for invasion and metastasis of T-cell hybridomas. For the ESb and MDA Y -D2 cells, we have now identified three additional CAM that are involved: CD44, VLA-4 and VLA-5. Adhesion to the monolayers, the first step in the invasion process, was reduced bya monoclonal antibody (mab) that blocks the binding of CD44 to hyaluronic acid, indicating that the cells ad here to cell surface-bound hyaluronic acid or another CD44 ligand. Furthermore, adhesion was reduced by a combination of mab against LF A-I and VLA-4 (and for MDAY-D2 also VLA-5). Thus, in addition to the LFA-I ligands ICAM-I and ICAM-2, the cells probably also attach to surface-bound fibronectin, the only known VLA-5 ligand, and one of the counterstructures of VLA-4. Fibronectin is present on hepatocyte surfaces and we have shown that adhesion of certain carcinoma cells to hepatocytes is due to interaction with this fibronectin. For lymphoma cells this remains to be demonstrated. The subsequent step in the invasion process, the migration between cells, appears to depend primarily on LFA-I, particularly in hepatocyte cultures. The major ligand in the liver is ICAM-I, which is abundantly expressed on the lateral hepatocyte surfaces and, in hepatocyte cultures, on the substrate-associated membranes where the invaded cells accumulate. We observed that invading lymphoma cells extensively redistribute their LF A-I to the leading edge inserted between hepatocytes. This suggests that the presence ofan ICAM-I gradient may be one of the factors guiding migration of the Iymphoma cells. The preference of lymphoma cells for the liver and their tendency to spread diffusively between hepatocytes may be due to the high constitutive expression of ICAM-I, which in most other organs is expressed only during inflammation. Surprisingly, we found that the CAM involved in the invasion process were often not in an active state, since the cells usually did not bind spontaneously to purified ligands, i. e. ICAM-I for LFA-I, fibronectin for VLA-4 and VLA-5, and hyaluronic acid for CD44. For adhesion, inducers of the active state were required, like PMA, Mn 2 + or the CD44 mab IRAWB. Nevertheless, the CAM were active during invasion and were th\ls apparently activated during interaction with the monolayer. We obtained evidence that the signal for the activation of LF A-I is transmitted by a G protein that is sensitive to pertussis toxin (PT). Invasion was strongly reduced by PT but this effect was reversed by Mn 2 +, an LFA-I activator, and this Mn 2 +-induced invasion was blocked by LFA-l mab. We postulate that the signal is given by factors associated with the fibroblast and hepatocyte surface. Whether the other CAM are activated by the same pathway is not yet clear. To further establish the role of CAM in vivo, we aim at generating mutants by targeted disruption in the diploid ESb and MDAY-D2 cells. CD44 and LFA-I f3-chain genes have been cloned. Constructs in which the coding sequence is interrupted by a hygromycin- or neomycinresistance conferring cassette were generated, and were recently transfected into MDA Y-D2 cells. Whether disruption has been achieved is not yet clear. Non-invasive (NI) T-cell hybrids express LFA-I levels similar to highly invasive (Hl) cells. Based on indications th at the conformation of LF A-lof the NI cells was different, we have attempted to raise mab against LFA-I that distinguish Hl from NI cells. We obtained mab that did not react with most NI celllines. However, these were not directed against LF A-I but, possibly, against an LF A-I-associated molecule. In preliminary experiments several mab inhibited invasion. Further characterization of the antigen is ongoing. Carcinoma interaction with hepatocytes: the role of rt.6f34 The liver is unique in that it contains no basement membrane underneath the sinusoidal endothelium, where metastasizing tumor cells invade this organ. To move through the endothelium, the tumor cells must therefore adhere to the underlying hepatocyte surface. We have identified the integrin a6f34 as one of the CAM involved,

22 Cel! Biology 21 at least for T A3 / Ha mammary carcinoma cells. This was surprising since these cells did not adhere to the established ex6[34 counter-structures laminin and kalinin. However, TA3 / Ha cells express high levels ofthe mucin epiglycanin. Wesseling and Hilkens (see Division of Tumor Biology) have shown that such mucins are anti-adhesive and that adhesion can be enhanced upon capping of the mucins with appropriate mab. Among hamster anti-t A3 mab previously raised, we identified several that were directed against epiglycanin. Those mab that capped epiglycanin efficiently, all apparently reacting with oligosaccharide epitopes, induced adhesion to laminin and kalinin and enhanced adhesion to hepatocytes. This shows that ex6[34 is functional and suggests that the antiadhesive effect of epiglycanin is reduced during interaction with hepatocytes, possibly by induced redistribution of the mucin molecules. Immune precipitation showed that ex6[34 is the major integrin on TA3 / Ha cells, and that [31 integrins are hardly expressed, explaining the lack of adhesion to fibronectin and collagens, even af ter epiglycanin capping. The counter-structure for ex 6 [34 in the liver remains obscure, however, since laminin is not produced by hepatocytes, and is not present along liver sinusoids; the same is probably true for kalinin. Attempts to isolate this molecule are ongoing. Carcinoma adhesion to hepatocytes: the role of fibronectin receptors Another variant of the same tumor, the epiglycaninnegative TA3 / St cellline, also adheres to hepatocytes and forms liver metastases. Nevertheless, ex6[34 expression was very low. These cells did express [31 integrins, including VLA-S (exs[31). Adhesion ofta3 / St cells to hepatocytes was strongly inhibited by a mab against murine VLA-S, by antibodies against rat fibronectin and by the RGD peptide known to block binding ofvla-s to fibronectin. T A3 / St adhesion to pure fibronectin was inhibited to the same extent, indicating that fibronectin is the major hepatocyte ligand for binding of T A3 / St cells. Indeed, we found fibronectin to be present on the hepatocyte surface. Furthermore, it is present under the sinusoidal endothelium in the liver where a basement membrane and therefore matrix components like laminin and collagen are lacking. Thus, particularly in the liver, adhesion to fibronectin may be of major importance for metastasis formation, albeit not for all tumor eell types. It is striking that two so closely related cel1lines use quite distinct mechanisms to interact with hepatocytes. We have started to study these adhesion mechanisms for human carcinomas, such as the HT29 human colon carcinoma cell line, which forms liver metastases upon intrasplenic injection into nude mice. The cells express ex6[34 and ad here to fibronectin. However, they do not express VLA-S, which is in fact only rarely found on carcinomas. Most likely, the fibronectin receptor involved is one of the many exv integrins. Carcinoma-hepatocyte adhesion: the role of the OP AR -antigens We have previously generated a mab against rat hepatocytes th at inhibited adhesion of T A3 carcinoma cells. The mab reacts with several bands on Westerns blots of liver plasma membranes. By affinity purification we have isolated different antigens. Peptide sequencing showed that these were serum glycoproteins known to be secreted by hepatocytes, like exl macroglobulin and C4-binding protein. Subsequently, the epitope was detected in lower density on other liver-derived serum proteins like fibronectin. The epitope is sensitive to neuraminidase treatment, and hence appears to be a rat liver-specific sugar moiety. Adhesion of cells to fibronectin derived from the rat, but not from other species, is blocked by the OP AR mab. This explains the effect ofopar on TA3 / St adhesion to rat hepatocytes. Adhesion of TA3 / Ha cells is, however, also effectively inhibited. This indicates that the elusive ex6[34 ligand on hepatocytes is probably also an OPAR epitope-carrying secreted liver glycoprotein. Interactions of adhesion molecules with the cytoskeleton One of the factors that determine the strength of cell adhesion is the assembly of adhesion molecules in supramolecular structures associated with the cytoskeleton. We have studied such structures using immuno-electron microscopy. Cells we re wet-cleaved, i. e. nitrocellulose was attached to the dorsal surface, and then stripped off. This removes most of the cell body and thus yields a substrateattached membrane with associated cytoskeletal structures thin enough to be viewed in the electron microscope. In fibroblasts, integrins are of ten concentrated in adhesion plaques. Formation of plaques is reduced after neoplastic transformation and this is probably one of the factors responsible for the induction of a migratory, invasive phenotype. How the many plaque components interact to form this structure, and how this is affected by transformation, is not clear. We have studied the localization ofthe [31 integrin subunit using antibodies against the cytoplasmic domain in fibroblasts spread on a substrate coated with pure fibronectin. Contrary to expectations based on immune fluorescence observations, the integrin was not found in a homogeneous layer in the plaque area but rather in clusters at the periphery ofthe plaque. These clusters apparently act as anchorage points for the talinvinculin network that is the core ofthe plaque structure. It is not clear how the plaque is attached to the remainder of the membrane in the plaque area, and it is therefore not evident wh at determines the close interaction of the membrane with the substrate at this site. The spacing of the clusters is deterrnined in part by the arrangement of integrin binding sites on fibronectin fibrils formed by the cello Inhibition offibril formation by cycloheximide treatment led to the formation of plaques that we re aberrant in size, structure and location. This phenotype was reversed by addition of soluble fibronectin that was assembied by the cells into fibrils. Thus, fibronectin fibrils seem to be required for normal plaque formation, at least for these cells and conditions. It has been hypothesized that the induction of the active state oflfa-l is due to the formation oflfa-l clusters. We have found no evidence for this in cleaved cells. In T-cell hybrids where LFA-I was not active, since the cells did not adhere to ICAM-I, LFA-l was found in clusters. In this case cells had been fixed in suspension and

23 22 Cell Biology attached to poly-l-lysine before cleavage. When adhesion to ICAM-l was induced in the presence of Mn 2 +, LF A-I redistributed to the ventral surface, accumulated in the periphery of spreading cells, and was present in very large amounts on thin filopodia extended over the surface. However, in all of these locations, LF A-I was present in similar clusters as in the absence of Mn 2 +. Publications La Rivière G et al. J Leukoc Biol 1993; 53: La Rivière G et al. In: Cell Adhesion Molecules. Cellular recognition mechanisms, pp New York: Plenum Press, Kemperman H et al. Cancer Res 1993; 53: Roos E. Semin Cancer Biol 1993; 4: Meijne ALM et al. J Cell Science (in press). La Rivière G et al. J Cell Science (in press). Kemperman H et al. Cell Adhes Commun (in press). Notes I Funding: Dutch Cancer Society, Project NKI Funding: Dutch Cancer Society, Project NKI Funding: Dutch Cancer Society, Project NKI Funding: Dutch Cancer Society, Project NKI cdna into non-invasive BW5147 cells yielded invasive transfectants, demonstrating the invasion-inducing capacity of this gene. Sequence of Tiam-l and the predicted Tiam-l protein The 7,294 nt long Tiam-I cdna sequence contains an open reading frame of 4,773 nt, encoding a protein of 1,591 amino acids with a predicted molecular weight of 177 kd. The Tiam-l protein lacks membrane-spanning or signal peptide sequences, is overall hydrophilic, and has a N-terminal glycine residue at position 2 th at could function as a myristoylation site to anchor the protein into the membrane. Furthermore, the N-terminal end of Tiam-I contains PEST domains th at have been associated with a short half-life ofproteins. Basic domains, present at several positions in the predicted Tiam-I amino acid sequence, could possibly function as nuclear translocation signals. Within the C-terminal end, Tiam-I contains a DBLhomologous (DH-) domain and two Pleckstrin homologous (PH-) domains found in a number ofgdp-dissociation stimulators (GDS) for small GTP-binding proteins of the RAS and RHO subfamilies (see Figure 1.1). Invasion- and metastasis-inducing genes GGM Habets l, J Stam 2, RA van der Kammen, D Zuydgeest, J Blok, J van der Velde, C Molenaar, JG Collard Identification of the invasion-inducing Tiam-l gene By proviral tagging in combination with a functional in vitro selection system for invasive cell variants we have identified a novel invasion- and metastasis-inducing gene, designated Tiam-I. Non-invasive murine BW5147 T-Iymphoma cells we re infected with Moloney murine Leukemia virus and invasive variants were subsequently selected in vitro on monolayers offibroblasts. Thirty independently generated invasive variants were isolated and analyzed for an invasion-relevant proviral insertion cluster. Provirus insertions were found in 40% of these invasive variants, clustered at two sites in a DNA locus th at we termed Tiam-I. All proviral insertions occurred in the same transcriptional orientation, in two coding Tiam-I exons, and resulted in truncated but functional transcripts ofthe 5'- and 3'-end ofthe gene. The 5'-end Tiam-I mrnas are prematurely terminated by the poly-adenylation signal ofthe inserted provirai5'-ltr sequences. The 3'-end Tiam-l mrnas are controlled by the 3'-LTR promoter of the inserted proviruses and are translated from internal initiation sites. Another isolated invasive variant showed a ten-fold amplification of the Tiam-I locus and elevated Tiam-l transcription, suggesting th at invasiveness could be induced either by an increased protein level or by protein truncation as a result of proviral insertion. The generated invasive variants produced metastases upon tail vein injection into nude mice, confirming the previously established correlation between acquired invasiveness and metastatic capacity of these cells. Moreover, transfection of a truncated 3'-end Tiam-l HOMOLOGOUS DOMAlNS IN GDS-PROTEINS U D T1AM-1 M< ;.11;... DSl VAV ect - 2 CDC24 SCR RAS -GRF/CDC25 Mm SOS CDC25 SDC25 ste6 ral - GDS CDC25 Mm i protein interactions RHO-like? RHO -like RAS!RHO -like? ~ RHO -like ~ RHO-like RHO -like (GAP) RAS RHO -like? -iiiiilliiji... I-- -- RAS RHO -like? _ RAS -like _ RAS -like _ RAS-like _ RAL ---ililliiihi- ----iiiliiiiia. _ CDC25 RAS -like Figure 1.1 DBL-homologous (DH) and Pleckstrin-homologous (PH) domains in genes encoding proto-oncogenes and/ or GDP-GTP exchange proteins implicated in signal transduction mediated by small GTPases. On the right: established or supposed (indicated by?) association with specific GTPases. In Tiam-I, the location of the PEST domains ( P), the potential myristoylation site (M ), and the upstream ( U) and the downstream (D) pro virus insertion cluster are indicated. Pro virus insertions interrupt the Tiam-l gene and result in 5 '-end and 3 '-end transcripts that en code N- and C-terminal end Tiam-l proteins. The DH-domain is present in CDC24, DBL, VA V, BCR, Ect-2, SOS and Ras-GRF and has been identified as the transforming and oncogenic region of the proto-oncogenes DBL, VA Vand Ect-2. It has been proposed, but not proven, that the DH-domain is also involved in GDP GTP exchange activity particularly towards RHO- and RAC-like proteins. Proteins that share the CDC25 do-