The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan 121 1066 CX Amsterdam www.nki.nl T HE NE T HE RLA NDS C A NC ER INSTITU TE SCIENTIFIC ANNU AL R E PO R T 2 0 06 T HE N E T H E R LANDS C A NCE R I N ST I T UT E S C IE NT I FI C A N NUA L R E PO R T 2 0 06 THE NETHERLAND S CANCER I NSTI TUTE SCI ENTI FI C ANNUAL REP ORT 20 0 6 3 S C IE NT I FI C ANNU AL REP OR T 2 0 0 6 4 Patron HM The Queen Beatrix 5 SC I E NT I FI C ANNU AL REPO R T 2 0 0 6 THE NE T HERL ANDS CANCER INS TITU TE CANCER RESEARCH LABORATORY AND CANCER HOSPITAL www.nki.nl 6 COPY R IGHT Scientific Annual Report 2006 Illustrations and unpublished data in these reports may not be used without permission of the author. Copyright В©: The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan 12 1 1066 CX Amsterdam The Netherlands Phone +3 1 20 5 12 9 1 1 1 Fax +3 1 20 6 17 2625 ISSN 1387-86 1 1 7 CONT E NTS Board Members Research Divisions Introduction Education in Oncology I Division of Cell Biology II Division of Molecular Carcinogenesis III Division of Cellular Biochemistry IV Division of Immunology V Division of Molecular Biology VI Division of Tumor Biology VII Division of Molecular Genetics VIII Division of Experimental Therapy IX Division of Radiotherapy X Division of Medical Oncology XI Division of Surgical Oncology XII Division of Psychosocial Research and Epidemiology XIII Division of Diagnostic Oncology Biometrics Department Clinical Trials Invited Speakers Projects Personnel Index 8 9 11 16 17 24 32 41 49 60 69 76 88 102 1 14 124 132 143 147 161 164 176 8 B OARD MEMBERS President of Board of Governors W Kok BO A R D MEMB ERS INTERNATIONAL SCIENTIFIC ADVISORY BOARD T De Lange, Leon Hess Professor, Rockefeller University, New York, USA RA Flavell, Professor of Immunobiology, Yale University School of Medicine, New Haven, USA S Hellman, Pritzker Distinguished Service Professor, The University of Chicago, Chicago, USA WGJ Hol, Professor of Biochemistry and Biological Structure, University of Washington, Seattle, USA J Mendelsohn, President, MD Anderson Cancer Center, The University of Texas, Houston, USA P Nurse, President, The Rockefeller University, New York, USA R Nusse, Professor of Developmental Biology, Stanford University, USA HL Ploegh, Professor of Biology MIT, Whitehead Institute of Biomedical Research, Cambridge, USA K Vousden, Director, Beatson Institute for Cancer Research, Glasgow, UK NATIONAL SCIENTIFIC ADVISORY BOARD LA Aarden, Professor of Molecular Immunology, Amsterdam SWJ Lamberts, Professor of Internal Medicine, Rotterdam B LГ¶wenberg, Professor of Hematology, Rotterdam CJLM Meijer, Professor of Pathology, Amsterdam CJM Melief, Professor of Immunohematology, Leiden HM Pinedo, Professor of Clinical Oncology, Amsterdam GNJ Tytgat, Professor of Gastroenterology, Amsterdam AJ Van der Eb, Professor of Fundamental Virology, Leiden BOARD OF DIRECTORS AJM Berns, chairman and director of research S Rodenhuis, director clinical research and development WH Van Harten, director organization and management BOARD OF GOVERNORS W Kok, president HCJ Van der Wielen, vice-president PJ Kalff, treasurer FH SchrГ¶der D Sinninghe DamstГ© MC Smeets PF Van der Heijden J Van der Meer PC Van der Vliet GP Vooijs SCIENTIFIC ADVISORY COUNCIL AJM Berns, chairman W Moolenaar, secretary S Rodenhuis T Schumacher T Sixma M Van de Vijver 9 RESEARCH DIVISIONS R E S E A R CH DIVISIONS I Cell Biology Arnoud Sonnenberg (head) John Collard Kees Jalink Ed Roos Natasja Borsje-Maeyer (office manager) II Molecular Carcinogenesis RenГ© Bernards (head) Roderick Beijersbergen Anastassis Perrakis Titia Sixma Suellen Boonen (office manager) III Cellular Biochemistry Wouter Moolenaar (head) Nullin Divecha Huib Ovaa Wim Van Blitterswijk Fred Van Leeuwen Marcel Verheij Suzanne Corsetto (office manager) IV Immunology Jannie Borst (head) John Haanen Heinz Jacobs Ton Schumacher Florry Vyth-Dreese JosГ© Overwater (office manager) V Molecular Biology Hein Te Riele (head) Piet Borst (honorary staff member) Jos Jonkers Sabine Linn Henri Van Luenen Bas Van Steensel Lodewyk Wessels Tom De Knegt (office manager) Linda RГ¶mer (secretary) VI Tumor Biology Jacques Neefjes (head) Reuven Agami Maarten Fornerod Rob Michalides Peter Peters Marieke Van der Velde (office manager) VII Molecular Genetics Maarten Van Lohuizen (head) Anton Berns John Hilkens Paul Krimpenfort Daniel Peeper Margriet Snoek Marion Rustenberg (office manager) Marie Anne Van Halem (secretary) VIII Experimental Therapy Alfred Schinkel (head) Adrian Begg Petra Nederlof Jan Schellens Fiona Stewart Marc Van de Vijver Laura Van вЂ™t Veer Thea Eggenhuizen (office manager) IX Radiotherapy Harry Bartelink (head) Berthe Aleman JosГ© Belderbos IaГЇn Bruinvis Eugene Damen Roel De Boer Luc Dewit Rick Haas Guus Hart Wilma Heemsbergen Frank Hoebers Edwin Jansen Joos Lebesque Corrie Marijnen Harry Masselink Ben Mijnheer Luc Moonen Floris Pos Coen Rasch Peter Remeijer Nicola Russell Govert Salverda Christoph Schneider Joep Stroom Jacqueline Theuws Bart Van Bunningen Marcel Van Herk Corine Van Vliet Marcel Verheij Conny Vrieling Frits WittkГ¤mper Patricia Haye-Fewer (management assistent) X Medical Oncology Sjoerd Rodenhuis (head) Joke Baars Paul Baas Evert Bais Jos Beijnen Willem Boogerd Henk Boot Sjaak Burgers Annemieke Cats Jan Paul De Boer Bert De Gast Leo Gualtherie van Weezel 10 RESEARCH DIVISIONS John Haanen Alwin Huitema Martijn Kerst Sabine Linn Anne Lukas Herman Neering Jan Schellens Jan Schornagel Marianne Smits Babs Taal Wim Ten Bokkel Huinink Jaap Van der Sande Marchien Van der Weide Nico Van Zandwijk MariГ«lle De Kwant (secretary) XI Surgical Oncology Bin Kroon (board; head) Fons Balm (board) Omgo Nieweg (board) Axel Bex Dick Buitelaar MatthГ© Burger Marcel Copper Menno De Rie Guus Fons Steven Gonggrijp Joris Hage Frans Hilgers Simon Horenblas Hans Huitink Christiaan Keijzer Houke Klomp Frans Kroon Peter Lohuis Lottie Lubsen Wim Meinhardt Hester Oldenburg May Ronday Emiel Rutgers Peter Schutte Michael ScrГЎmek Marian Slot Ludi Smeele Tino Stoppa Bing Tan Julia Ten Cate Adriaan Timmers Marc Van Beurden Frits Van Coevorden Michiel Van den Brekel Wietze Van der Veen Henk Van der Poel Willemien Van Driel Arjen Van Turnhout Vic Verwaal Marie-Jeanne Vrancken Peeters Leonie Woerdeman Frans Zoetmulder Joke Van der Veen (office manager) XII Psycosocial Research and Epidemiology Neil Aaronson (head) Matti Rookus Frits Van Dam Wim Van Harten Flora Van Leeuwen Marian Chin-A-Kwie (office manager) XIII Diagnostic Oncology Marc Van de Vijver (head) Tanja Alderliesten Philippe Baars Peter Besnard Hans Bonfrer Daphne De Jong Kenneth Gilhuijs Cees Hoefnagel Frans Hogervorst Irma Kluijt Wim Koops Robert KrГ¶ger Claudette Loo Saar Muller Petra Nederlof Willem Nooijen Frank Pameijer RenГ©e Van Pel Hans Peterse Warner Prevoo Michiel Sinaasappel Ferida Sivro Jelle Teertstra Renato Valdes Olmos Hester Van Boven Fijs Van Leeuwen Olaf Van Tellingen Laura Van вЂ™t Veer Loes Van Velthuysen Senno Verhoef Jelle Wesseling Christine Arkes (secretary) Carla Van Tiggelen (secretary) Biometrics Department Otilia Dalesio (head) Harm Van Tinteren Financial Administration Vacancy General Facilities Vacancy Personnel Department Eric De Wilde General Research Coordination Wouter Moolenaar, laboratory research coordinator Henri Van Luenen, laboratory research manager 11 INTRODUCTION Our clinical activities showed a steady expansion, with further growth occurring in the Divisions of Surgery and Radiotherapy. A major upgrading of equipment took place in our Radiology department, with the installation of a 3 Tesla MRI and a PET-CT scanner. In the Radiotherapy department two advanced linear accelerators were put into action permitting imageguided radiotherapy (IGRT) as a regular treatment modality. One of our operating theaters was equipped with a Da Vinci robot allowing highly refined endoscopic surgical interventions, such as prostate resections. On December 8, the Division of Surgical Oncology opened the Photodynamic Therapy Center for function preserving treatment of early staged carcinomas of the head and neck, skin and cervix. Director of Research Ton Berns I N TR O DUCTION I am pleased to present our Scientific Annual Report 2006. It provides an overview of the scientific activities at The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital (NKI-AVL). Additional information can be found at www.nki.nl. A more lucid description of our activities with illustrations can be found in our new brochure that came out in November 2006. In 2006 the hospital budget was under even more strain than in the previous year, due to only partial compensation of rising expenses and additional budget cuts by the Ministry of Health. Our research also had great difficulty to balance the budget. The effects of the earlier 10% budget cut by the Ministry of Health, which is funding roughly half of our infrastructure including the salaries of our permanent staff, were made worse by a lower contribution of the Dutch Cancer Society as a result of lower revenues from legacies. Fortunately, the contribution of the Dutch Cancer Society will increase again in 2007, and this effect is expected to become more substantial in 2008. We hope that a new government will appreciate the important contributions of the NKI-AVL to the battle against cancer and agree on a funding arrangement that does justice to the contribution of the NKI-AVL to the public cause. In November 2006 we held our first institute-wide two-day long retreat in which staff and postdoctoral fellows participated. This meeting stimulated discussions among staff and investigators of both clinical and basic research divisions that do not regularly interact in person. The meeting was a success and we envision that this will become a bi-annual event. The NKI-AVL is a Comprehensive Cancer Center, combining hospital and research laboratories under one roof in a single independent organization. The hospital comprises 180 beds, an outpatient clinic and a large radiotherapy department. Facilities for clinical research include a large patient database, clinical data management, extensive diagnostic facilities, a pharmacy with a production unit for experimental drugs, and active research groups in pharmacy, epidemiology and psychosocial oncology. The laboratory covers all major areas of cancer research, with special emphasis on cell-based screens, mouse tumor models, cell biology, structural biology, immunology and translational research. There is close collaboration between clinical and basic scientists. This scientific report deals mainly with the basic and clinical science in the NKI-AVL. Information on patient-care is described in our General Report. 2006 brought a number of major renovations and expansions of the research and clinical activities of the NKI-AVL. Our renovated auditorium was put into use. The auditorium with its 300 seats compares with the best auditoria elsewhere providing the NKI-AVL with a state-of-the-art facility for seminars, teaching courses and scientific meetings. The old hospital building has been completely stripped and will be transformed into a modern laboratory facility in the course of 2007. Highlights The scientific output of the Institute is again well above par. 2006 was a productive year with important new findings and scientific publications that received wide recognition in top scientific journals. Table 1 Research funding NKI-AVL from the different sources in period 1990-2006 in million EuroвЂ™s. Year Cancer Society Ministry of Health Project Grants Other income Total 1990 1993 1996 1999 2001 2002 2003 2004 2005 2006 2.5 7.3 6.4 2.6 4.1 7.3 8.1 2.5 5.6 8.1 8.6 2.9 7.0 8.8 12.0 2.4 8.7 9.4 12.6 3.8 8.7 9.9 14.4 3.3 9.1 10.1 18.4 1.0 8.9 9.1 18.0 0.8 8.6 9.6 17.4 1.7 8.3 9.4 21.9 1.2 18.8 22.0 25.2 30.2 34.0 36.3 38.6 36.8 36.8 40.8 An additional 2.5 million Euro/year is received from the вЂ�Stichting FondsenвЂ™ of the NKI for specific investments in equipment. 12 INTRODUCTION It is always difficult to estimate the impact of research when the results are still fresh. The research highlights summarized here serve primarily as a sampling of interesting work currently on-going in the Institute. A more complete and detailed account of specific projects can be found in the reports of individual group leaders in this SAR and on the website (www.nki.nl). Norman Sachs and colleagues in the group of Arnoud Sonnenberg (Division of Cell Biology) showed that mice lacking the cell surface molecule known as tetraspanin Cd151 develop a similar kidney failure as mice with a podocyte specific knockout of the integrin a3 subunit, indicating the existence of functional integrin-tetraspanin complexes in vivo. This supports the notion that CD151 plays a key role in strengthening integrin-mediated adhesion, a feature that makes these molecules relevant for keeping cells properly positioned in their microenvironment. John Collard and co-workers (Division of Cell Biology) found that the Rac activator Tiam 1, in conjunction with the Par polarity complex, regulates the polarization and directional migration of epithelial cells as well as chemokine-induced polarization and migration of normal T cells. Rianne Oosterkamp in the group of Rene Bernards (Division of Molecular Carcinogenesis) tested the efficacy of salicylic acid for the treatment for cylindromatosis, a rare genetic disorder causing neoplasms of the skin appendages. This clinical study was based on previous RNA interference studies in which his group had shown that loss of the cylindromatosis tumor suppressor gene leads to activation of NF-kappaB, a transcription factor having anti-apoptotic activity. Cell-based assays indicated that salicylate can prevent NF-kappaB activation caused by loss of the cylindromatosis gene. Salicylate might then also benefit patients with cylindromatosis, a prediction that was supported by the pilot clinical study by Oosterkamp. This work nicely illustrates how basic research can sometimes be quickly translated into tangible clinical results and in general highlights why NKI-AVL has both research and patient care under one roof. Roderik Kortlever from the group of Rene Bernards identified the p53 target gene Plasminogen Activator Inhibitor-1 (PAI-1) as a critical mediator of the p53-dependent senescence response. His data indicate that PAI-1 is not merely a marker of senescence, but both necessary and sufficient for the induction of replicative senescence downstream of p53. The groups of Huib Ovaa and Wouter Moolenaar (both Division of Cellular Biochemistry) have evaluated Autotaxin (ATX) as a therapeutic target. ATX is a secreted phospholipase that generates the lipid growth factor LPA, which has an important signaling function in tumor progression and angiogenesis. In collaboration with David Egan (Division of Molecular Carcinogenesis), they identified three distinct classes of potent small-molecule inhibitors of ATX, which will be further optimized for evaluation in mouse models. Defective cell-cell communication is a hallmark of malignancy. The groups of Wouter Moolenaar, Kees Jalink (Division of Cell Biology) and Nullin Divecha (Division of Cellular Biochemistry) discovered that cellcell communication through gap junctions is tightly regulated by the levels of phosphatidylinositol bisphosphate (PIP2) in the plasma membrane. Using state-of-the-art live-cell imaging techniques, they showed that a fall in PIP2 is necessary and sufficient to shut down gap junctional communication. This new finding may suggest strategies as to how to restore defective cell-cell communication in tumor cells. In an approach combining organic synthesis and immune technology, the groups of Huib Ovaa and Ton Schumacher (Division of Immunology) have developed conditional MHC ligands that can be cleaved by UV irradiation thereby allowing the production of вЂ�emptyвЂ™ MHC complexes. They have used this strategy for the high-throughput production of MHC complexes and MHC tetramers for T cell detection. This approach has led to the identification of highly immunogenic avian flu epitopes and is now being applied to identify T cell epitopes relevant for immunotherapy. Wim van Blitterswijk (Division of Cellular Biochemistry) and Marcel Verheij (Division of Radiotherapy) and collaborators modified the liposomal formulation of the anti-cancer drug doxorubicin (CaelyxВ®) by incorporating N-octanoyl-glucosylceramide in the liposomal bilayer. This sphingolipid significantly facilitated the uptake of doxorubicin in tumor cells xenografted into nude mice. This new delivery method looks promising for clinical application. Matthijs Voorhoeve and Carlos Le Sage in the group of Reuven Agami (Division of Tumor Biology) have set up systems to evaluate the function of individual microRNAвЂ™s and identified two as being involved in formation of testicular germ cell tumors through targeting the tumor-suppressor LATS2. Jacques Neefjes (Division of Tumor Biology), in collaboration with Eric Reits (AMC, Amsterdam), and James Hodge and Jeffrey Schlom (NCI, Washington) identified a novel anti-cancer immunotherapy combining the specificity of adaptive immunotherapy and the spatial application of (low dose) radio-immunotherapy. This combined radio-immunotherapy proved superior over the individual therapies in eliminating local tumors and is currently further developed for application. Helen Pickersgill in the group of Maarten Fornerod (Division of Tumor Biology) in collaboration with Bas Van Steensel (Division of Molecular Biology) defined the DNA segments interacting with the nuclear lamina coating the nuclear envelop. These appear to be transcriptionally silent areas and indicate that DNA is packaged in the nucleus in functionally different areas. Nico Dantuma and Tom Groothuis in the group of Jacques Neefjes have studied the Ubiquitin equilibrium in living cells through a series of biochemical and biophysical experiments. Ubiquitin is a small protein modifying others and shared in many cellular processes. Ubiquitin appears to be present in limiting amounts 13 INTRODUCTION and removed from histones for polyubiquitin under conditions of acute stress. Also anti-cancer drugs induce this, and the mechanisms and consequences are further studied. Sven Rottenberg, a postdoc working in the groups of Piet Borst and Jos Jonkers (Division of Molecular Biology), showed that the Brac1-/-; p53-/- mouse model for mammary cancer is suitable for studying chemotherapy resistance. He was able to generate resistance against natural product drugs, such as doxorubicin and paclitaxel, which had never been obtained before in an animal tumor model. The tumors also respond to cisplatin, but they do not become resistant and can be treated successfully over and over again. The вЂ�remnantsвЂ™ from which tumors regrow after an initial response are of great interest, as these have never been accessible in patient samples or tumor models before. Characterization of these вЂ�remnantsвЂ™ may offer new possibilities for characterizing and tackling the tumor stem cell fraction, held responsible for relapse after chemotherapy. Bas van SteenselвЂ™s group (Division of Molecular Biology) co-authored three papers in Nature Genetics, each reporting important new insights into chromatin structure and composition. Together with the labs of Maarten Van Lohuizen (Division of Molecular Genetics), Maarten Fornerod (Division of Tumor Biology), and Wouter De Laat (Erasmus Medical Center, Rotterdam), they uncovered genome-wide patterns of respectively Polycomb protein binding, nuclear lamina interactions, and three-dimensional chromosome folding. Paul Krimpenfort and Annemieke IJpenberg in the group of Ton Berns (Division of Molecular Genetics) uncovered an unexpected backup function of the Cdkn2b tumor suppressor for Cdkn2a, the latter being the most frequently inactivated tumor suppressor after p53. Whereas loss of one of these two suppressors in mouse models gives rise to only a slightly enhanced tumor predisposition, co-deletion shows a dramatic tumor-prone phenotype. Both genes map close together on the genome and are co-deleted in many human tumors. Since both suppressors appear to preferentially inhibit one specific cyclin-dependent kinase, this offers interesting perspectives for treating tumors in which this locus is deleted or silenced. In search of genes that preserve the senescence growth arrest of human nevi, Daniel PeeperвЂ™s group (Division of Molecular Genetics) has identified an interleukin network as a critical mediator of senescence. John Hilkens and coworkers (Division of Molecular Genetics) finished the first large scale MMTV insertional mutagenesis screen for mammary cancer genes, which resulted in the identification of 17 novel candidate oncogenes. Two of them belong to a novel family of secreted proteins, R-spondins, that stimulate the expression of a unique set of downstream genes in addition to the Wnt pathway. Stephen Tait in the group of Jannie Borst (Division of Immunology) discovered a novel mode of ubiquitination that is essential for mitochondrial permeabilization by the BH3-only protein Bid, which is a key mediator in apoptosis induction by death receptors and DNA damaging anti-cancer regimens. Chiel Maas in the same group has performed a successful RNA interference screen for Trail-induced apoptosis, using the entire NKI RNAi library. This resulted in the identification of a dozen, both known and unknown, mediators of the Trail apoptosis pathway. The image-guided radiotherapy (IGRT) developments based upon the Cone-Beam CT as explored in the Division of Radiotherapy have already led to better results: for example in prostate cancer and breast cancer our own trials demonstrated that by delivering a higher radiation dose, a better local control is achieved. At the same time it was shown that in prostate cancer the toxicity could be reduced with this sophisticated radiotherapeutic approach. The Epidemiology group (Division of Psychosocial Research and Epidemiology) coordinated a nationwide collaboration of Clinical Genetic Centers and brought together 139 families, comprising 1811 family members, with pathogenic mutations in the BRCA2 gene. They assessed the risk of cancers other than breast or ovarian cancer in this database and observed that risk compared to the general population was increased for four cancer sites: pancreas (Relative risk (RR)=5.9), prostate (RR=2.5), bone (RR=14.4) and pharynx (RR=7.3). Families with mutations outside the previously defined ovarian cancer cluster regions tended to have a higher cancer risk. The group of Neil Aaronson reported the results of a nationwide observational study of 846 women at increased risk of hereditary ovarian cancer, 44% of whom had opted for prophylactic bilateral salpingooophorectomy (PBSO) and 56% for periodic gynecologic screening (GS). Women in the PBSO and GS groups reported similar levels of generic quality of life. Compared with GS, PBSO was associated with fewer breast and ovarian cancer worries (p < 0.001) and more favorable cancer risk perceptions (p < 0.05). However, the PBSO group reported significantly more endocrine symptoms (p < 0.001) and worse sexual functioning (p < 0.05). The favorable effects of PBSO in terms of reduced cancer worries and risk perception need to be weighed against the increase in endocrine and sexual symptoms. Balanced information will help clinicians and high-risk women in making informed decisions about the optimal preventive health strategy. The Radiology department (Division of Diagnostic Oncology) has extended its work on evaluating the added value of MRI to guide optimal breast conserving therapy for breast cancer patients. MRI led to a change in treatment in approximately 20% of the patients, showing that MRI is a valuable addition to the diagnostic work-up of breast cancer patients. 14 INTRODUCTION Work of the Molecular Pathology group (Division of Diagnostic Oncology) has previously led to the identification of a 70 gene prognosis profile in tumors from patients with lymph node negative breast cancer. To assess whether this 70-gene MA test can be implemented in daily clinical practice, a multi-center study (вЂњRASTERвЂќ study) was performed successfully in 17 Dutch community hospitals. The results of this study have been very helpful in setting up the MINDACT trial, that will prospectively evaluate the prognostic value of the 70 gene prognosis profile. The pharmacology group of the Division of Medical Oncology has performed a phase I study with the PARP1 (poly(ADP-ribose)polymerase-1) inhibitor KU36-92, in which the agent was administered to cancer patients for the first time. Theoretically, PARP1 inhibitors should represent вЂ�targeted therapyвЂ™ for tumors that lack certain DNA-repair mechanisms, such as in patients with hereditary breast cancer syndromes. Major clinical responses were indeed observed in 2 patients known to be BRCA2 mutation carriers. Quality of research The quality of research can be monitored in several ways. Our scientific productivity as based on objective bibliometric parameters (citations and impact of scientific articles published by the NKI staff) has shown a steady increase since the beginning of the 1980вЂ™s, although in recent years the number of citations and impact has shown some fluctuation (Table 2). It is satisfying to note that the Institute has acquired an internationally prominent position in cancer research. While a high citation score is gratifying, it is only one measure of scientific productivity. The quality of our work should also be gauged by the invitations of our staff to present at prestigious scientific meetings, and from our success in obtaining competitive grant awards. We score highly on all those items. We are fairly successful in securing grant support, scoring on average twofold better than our colleagues in The Netherlands. Quality is also measured by external site visits. International leaders in a particular field of research review the work of a division or research groups with a similar theme, on a quinquennial basis. These site visits require that group leaders prepare a report and reflect on their research activities. This by itself is a useful exercise that helps to focus the research. Early 2006 the work of the Division of Molecular Carcinogenesis was evaluated in a one-day site visit by a site visit team consisting of Bob Weinberg (Whitehead Institute, Cambridge), Ray Stevens (Scripps, La Jolla), Bill Kaelin (Dana Farber, Boston), and John Kuriyan (UC Berkeley). In November 2006, the Division of Psychosocial Research and Epidemiology was evaluated by Patricia Ganz (UC Los Angeles), Bruce Ponder (Hutchison/MRC Research Centre, Cambridge UK), and Christoffer Johansen (Research Institute of Cancer Epidemiology, Copenhagen). The site visitors were overall positive about the activities of both divisions, but also made a number of recommendations to be considered by some of the individual groups. These recommendations will be discussed and, when applicable, implemented to further improve science at the NKI-AVL. Honors and appointments The NKI-AVL cannot award university degrees. However, many of our staff members hold special part-time chairs at Dutch universities. This facilitates awarding degrees to graduate students receiving their training at The Netherlands Cancer Institute. In 2006 27 staff members had professorships at one of the Dutch Universities. No new professorships were granted in 2006. Daniel Peeper, appointed to permanent staff member in 2005, obtained a prestigious VICI award, providing him with 1.2 million euros of funding. VIDI awards were given to Jacqueline Jacobs and Rob Wolthuis, and of the 5 fellowships made available by the Dutch Cancer Society Table 2 Short-term citations and impact of scientific articles published by the NKI research staff 1982 - 2006 Publication year 1982 1983 1984 1985 1986 1987 1988 1989* 1990 1991 1992 1993 1994 1995 1996 1997*** 1998 1999 2000 2001 2002 2003 2004 2005 2006 Citations* 560 779 1340 1286 1366 1839 1775 1273 2127 2199 2074 2221 3455 2896 3324 3617 3240 3727 3551 3991 7432 5094 4884**** Impact** 295 365 616 549 650 765 742 764 854 910 911 958 1292 1415 1520 1811 1392 1766 1664 1573 2455 2122 1882 2461 2913 * Citations in the two years after publication, excluding self-citations. Starting 1989 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 average number of citations per year of an article in a given journal. The total impact is the sum of the impact of all articles published that year. *** As from 1997 publication year of articles is the criterion, instead of Scientific Report-year listing. **** Preliminary data available on Jan 5, 2007 15 to young investigators, 4 were awarded to NKI scientists. There were also prizes awarded to our staff. Piet Borst became Commander of the British Empire and Harry Bartelink was recipient of the bi-annual Regaud Medal Award of the European Society of Therapeutic Radiation and Oncology (ESTRO). He also received the Kilroe Award at the meeting of the Coloprotology Section of Royal Medicine Society. I received the first Massachusetts General Hospital Award in Cancer Research. Jacques Neefjes was elected to EMBO member. The AvL-prize 2006 for young, promising NKI-scientists was awarded to Matthijs Voorhoeve for his work on the role of specific microRNAs in cancer. There are also changes to report in our scientific staff. Bas Van Steensel, AvL fellow in the institute was promoted to associate professor. Rob Ten Berg, director of our animal facility from 1985 to 2002, retired after having served us during the last 4 years as supervising veterinarian and secretary of the Animal Experiment Committee. Staff of the NKI-AVL fulfilled numerous functions in national and international organizations, on scientific boards of scientific journals, as members of study sections, and as organizers or co-organizers of scientific meetings, workshops and congresses. Outlook and acknowledgements I am confident that research at the NKI-AVL will continue to flourish, especially given the perspectives that our funding will again increase in the years to come. This increase will come from a number of sources, including the participation in large nation-wide initiatives that will stimulate joint projects between academia and industry. Contracts for one of these initiatives, the Top Institute Pharma, have been signed in December and will provide funding for several projects that will be executed together with Dutch industries. A new substantial initiative, the Centre for Translational Molecular Medicine, will stimulate development of biomarkers and imaging. We will submit proposals in the course of 2007. We will continue to participate in the Top Research School CBG and the Cancer Genomics Centre CGC. In addition, we hope to increase our income from intellectual property. We have now an effective Technology Transfer Office in place and this will undoubtedly contribute to our income. A range of other initiatives, some of which I have outlined above should provide us with a sound basic funding with a healthy yearly growth. Wise and cost/effective spending of the funds given to us is a recurrent priority. In addition, it is of critical importance that we retain the capacity to swiftly respond to new developments and opportunities, required to perform internationally recognized cuttingedge research. Research is the only way to improve cure rates and the quality of life of cancer patients. I am convinced that breakthroughs in cancer diagnosis and treatment can be realized with approaches that are now broadly applied in the NKI. We are particularly grateful to those who continue to support us: The Dutch Cancer Society, the most reliable and significant sponsor of our research; the Ministry of Health, which provides a substantial core grant to the Institute; and, last but not least, the many individuals who support us directly with their gifts and also with their moral and practical support. Only with their help can we continue to develop new ideas that can improve the perspectives of cancer patients. I hope you are inspired by this report. Ton Berns Director of Research 16 EDUCATION IN ONCOLOGY Theoretical and practical training in research are regular activities in the institute. Several senior staff members have joint appointments as professors at Dutch universities and contribute to the regular curriculum at these universities. In addition, many staff members teach in courses for undergraduate and graduate students, either within the institute or at universities. The research departments attract Master students from universities throughout the country, who contribute to ongoing scientific projects and receive in house theoretical training. The NKI has a formal connection with the Science faculty of the University of Amsterdam (UvA) and is committed to contribute to courses for Master students of this university. The institute participates in the Oncology Graduate School Amsterdam, together with the UvA and the Free University (VU). The medical staff is actively involved in medical and surgical oncology fellowship programs, teaching programs for undergraduate medical students and (inter)national postgraduate courses. The departments of Surgical Oncology and Head and Neck Oncology participate in the residency training program of the Academic Medical Center. Lectures are read by staff members for nurses in training for their oncology certificate. All educational activities are supervised by the Teaching Committee, which consists of J Borst (president and dean undergraduate school), H Te Riele (general affairs), R Beijersbergen (undergraduate courses), J Hilkens and J Collard (HLO students and publicity), T Sixma (dean graduate students) and F Balm (clinical teaching). Specialized tasks include organization of the lecture course, guided tours to research departments, contact with technician training schools, promotional activities at universities. UNDERGRADUATE STUDENTS The undergraduate program in Experimental Oncology attracts students of all national universities in partial fulfillment of the obligations of their MasterвЂ™s curriculum. Students generally have a background in (Medical) Biology, Health Sciences, Chemistry, Pharmacology, Medicine, or Psychology. The undergraduate program offers combined practical and Table 1 вЂ“ Courses in Experimental Oncology theoretical training in various aspects of experimental oncology. Practical training includes participation in ongoing research projects for a minimum of 4 months, after which the student delivers oral and written accounts of the results obtained. The majority of the undergraduate students came from the Amsterdam Universities (UvA and VU), others from Wageningen, Utrecht, Leiden and Nijmegen. The institute also provides practical training opportunities to trainee technicians. The core element of theoretical training is the course in Experimental Oncology, given twice a year by staff members of the institute (Table 1). This course includes lectures and tutorials. The book Cancer Biology by RJ King is recommended for background reading. GRADUATE STUDENTS For the graduate students the NKI-AVL participates in the Oncology Graduate school Amsterdam (OOA) together with the oncology departments of the VUmc and the AMC. The institute has ~130 graduate students registered for the OOA graduate school. Students participate in the research of their group and in the interdepartmental work discussions. In addition they participate in the OOA training program, which consists of courses (Table 2) and an annual retreat in Texel. This retreat focuses entirely on the research of the students themselves. At this retreat students not only present their work in the form of a poster in the first year and an oral presentation in subsequent years, they are also in charge of chairing sessions, discussions and in recent years also in peer review, giving a prize for the best poster and best presentation. In this manner the retreat trains important skills in presentation and interaction but it also provides an overview of the research in the OOA at an early stage of the studentвЂ™s career. This provides a good opportunity for translational interaction and bottom-up research, where the students themselves can contribute significantly to the interaction between different groups. In 2006, 21 graduate students received a PhD degree at a Dutch university. Table 2 вЂ“ OOA graduate student courses Jan 30 вЂ“ Feb 10 Feb Mar 21-24 Will we cure cancer? Epidemiology Surgery Pathology* Gene array Molecular diagnostics Conventional sustemic cancer therapy Radiotherapy* DNA damage response Apoptosis Cancer therapy and apoptosis Cell adhesion Angiogenesis Cell senescence Cell cycle Genomic instability J Borst D Voskuijl F Zoetmulder D De Jong R Kerkhoven L Van вЂ�t Veer J Schornagel M Verheij A Begg J Borst J Borst E Roos E Boven R Beijersbergen R Bernards H Te Riele June 6-16 Sep 25-Oct 6 Oct 11-13 Nov 6-11 Nov 8, 15, 22, 25 Nov 20, 27, Dec 4 Dec 11,12 * including tour Discovery of cancer genes and networks in model organisms. F Van Leeuwen, J Jonkers Stam cell differentiation GJ Schuurhuis, F van Millegen The Macroscopic, microscopic and pathologic anatomy of the mouse R Van Noorden, W Lamers In the footsteps of Antoni van Leeuwenhoek R Van Noorden, G Meijer, P Peters, E Reits, J Belien, K Jalink Protein structure and function T Sixma, A Perrakis Annual retreat T Sixma, M Van der Velde, S Boonen Oncogenomics: A sampler dish of genomic techniques and applications B Ylstra Writing & presenting scientific English A Bless Introduction in the histopathology of tumors N Van Grieken en E Thunissen Biology of colorectal cancer risk R Fijneman 17 CELL BIOLOGY I DI VI S I O N OF CEL L B IOL O G Y RECEPTORS FOR MATRIX ADHESION Integrins connect the extracellular matrix (ECM) with the cell interior and transduce signals by interactions of their cytoplasmic domain with cytoskeletal and signaling molecules. In our group we study the mechanisms by which integrins provide signals for the regulation of important cellular processes such as cell migration and proliferation. Division head, Group leader Arnoud Sonnenberg Arnoud Sonnenberg PhD Group leader Johan De Rooij PhD Post-doc Kevin Wilhelmsen Post-doc Role of the integrin a6b4 and plectin in hemidesmosome formation Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the underlying basement membrane. Modulation of their function is of crucial importance in a variety of biological processes, such as differentiation and migration of keratinocytes during wound healing and carcinoma invasion, in which cells become detached from the substrate and acquire a motile phenotype. The binding of a6b4 to plectin plays a central role in HD assembly and it is becoming increasingly clear that disrupting this association through phosphorylation events is necessary to disassemble HDs. We have defined two regions on b4 that harbor all the serine/threonine phosphorylation sites. These areas are not directly involved in the primary interaction with plectin, which is mediated through its actin-binding domain (ABD), but rather enforce the interaction between the two proteins. Interestingly, however, we have identified three serines (S1356, S1360 and S1364) within the b4 connecting segment that regulate the interaction with the plectin-ABD when they are phosphoylated, suggesting that these phosphoresidues somehow act remotely, possibly through an intramolecular folding event, to block binding. We have also recently established that activation of the EGF receptor, which is known to function upstream of HD disassembly, results solely in the phosphorylation of these three residues in vivo and that S1360 and S1364 are the only PKC and PKA phosphorylation sites, respectively. However, other kinases may also phosphorylate these sites and, in fact, we have evidence that GSK-3b may be involved. Taken together, our studies indicate that multiple kinases act in concert to abrogate the structural integrity of HDs in keratinocytes and that phosphorylation of the b4 subunit at residues S1356, S1360 and/or S1364 is the primary mechanism by which this is achieved. The localization of nesprin-3 at the nuclear envelope depends on an interaction with SUN proteins The position of the nucleus is important for several cellular processes such as mitosis, meiosis, cell migration and polarization. Although the general mechanisms of nuclear positioning are known to require a connection between the nucleus and actin filaments or microtubules, the proteins involved in this connection have only recently been identified. Nesprin-1 and -2, members of the nesprin protein family, are responsible for connecting the nucleus to actin filaments, while nesprin-3 connects the nucleus with intermediate filaments via the cytoskeletal linker protein plectin. Previous studies by other investigators have demonstrated that the localization of nesprin-1 and -2 at the outer nuclear membrane, is dependent on an interaction with SUN proteins present at the inner nuclear membrane. We have now provided evidence that nesprin-3 is retained at the outer nuclear membrane via a similar mechanism. The C-terminal KASH domain of nesprin-3 interacts with SUN proteins and the last four amino acids of nesprin-3 are important for this interaction. In addition, nesprin-3 can associate with itself, forming dimers. Dimers of plectin will interact with dimers of nesprin-3. Since SUN proteins can interact with nuclear lamins and plectin can interact with intermediate filaments, there is a direct connection between the nucleoskeleton and the cytoskeleton, which is potentially important for signaling and cell integrity. Iman Van den Bout Graduate student Mirjam Ketema Graduate student Coert Margadant Graduate student Norman Sachs Graduate student Maaike Kreft Technical staff Ingrid Kuikman Technical staff Figure I.1: Model indicating an indirect connection between the nuclear lamina and the intermediate filament (IF) system. The interaction between nesprin-3 and SUN New insight into integrin-regulated proteins Previously, we have identified MacMARCKS (MRP) as a protein that is downregulated by overexpression of the b3 subunit. We found that this downregulation is strongly correlated with increased cell proteins in the perinuclear space (PNS) links nuclear lamins to cytoplasmic plectin, which in turn can bind IFs. 18 CELL BIOLOGY Publications Bajanca F, Luz M, Raymond K, Martins GG, Sonnenberg A, Tajbakhsh S, Buckingham M, ThorsteinsdГіttir S. Integrin a6b1-laminin interactions regulate early myotome formation in the mouse embryo. Development 2006; 133:1635-1644 Gawlik KI, Mayer U, Blomberg K, Sonnenberg A, Ekblom P, Durbeej M. Laminin a1 chain mediated reduction of laminin a2 chain deficient muscular dystrophy involves integrin a7b1 and dystroglycan. FEBS Lett 2006; 580:1759-1765 Lebbink RJ, De Ruiter T, Adelmeijer J, Brenkman AB, Van Helvoort JM, Koch M, Farndale RW, Lisman T, Sonnenberg A, Lenting PJ, Meyaard L. Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1. J Exp Med 2006; 203:1419-1425 Litjens SHM, De Pereda JM, Sonnenberg A. Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol 2006; 16:376-383 Sachs N, Kreft M, Bergh Weerman MA, Beynon AJ, Peters TA, Weening JJ, Sonnenberg A. Kidney failure in mice lacking the tetraspanin CD151. J Cell Biol 2006; 175:33-39 Wilhelmsen K, Litjens SHM, Sonnenberg A. Multiple functions of the integrin a6b4 in epidermal homeostasis and tumorigenesis. Mol Cell Biol 2006; 26:2877-2886 Wilhelmsen K, Ketema M, Truong H, spreading and the reorganization of the actin cytoskeleton. Furthermore, we showed that the Ras/MAPK pathway is directly involved in the regulation of MRP in normal cells but b3 overexpression inhibits the ability of this pathway to regulate MRP. Since a precise function for MRP is unknown we continued our investigations into the mechanism that controls MRP localization. Our studies show that the attachment of MRP to membranes depends on the myristoyl moiety present at the N-terminus of the protein. Interestingly, in contrast to other reports, we show that the positively charged central effector domain (ED) only targets the protein for binding to PIP2 in the plasma membrane but is not essential for membrane binding per se. Phosphorylation of the ED inhibits this binding and results in the loss of MRP from the plasma membrane. We further show that the overexpression of MRP increases dextran uptake, suggesting that MRP plays a role in endocytosis. This is underlined by our observations that MRP becomes localized to endocytotic vesicle/lysosomes as well as the transgolgi network when it is phosphorylated in the ED.s directly responsible for the downregulation of cell-cell contacts in our model cell line. Regulation of cell-cell adhesion during metastasis Recently, we have shown that the disruption of cell-cell adhesion by HGF, a notorious metastasis-promoting growth factor, depends on actomyosin-induced tension. E-cadherin, the main cell-cell adhesion receptor in these cells, is not affected at the protein level, nor is its ability to form homotypic interactions impaired. We concluded that E-cadherin is regulated by cytoskeletal tension, that there must be a direct link between E-cadherin and the cytoskeleton and that modification of the complex of proteins maintaining this link mediates this regulation. In a recent publication it was, however, questioned whether a direct link between E-cadherin and the actin cytoskeleton really exists. To clarify this discrepancy and to understand the mechanism of E-cadherin disruption, we set out to study the link between E-cadherin and the cytoskeleton and its regulation by tension in detail. We find that both the formation and the turnover of E-cadherin adhesions depend on cytoskeletal tension. During both phases of adhesion, there is no detectable change in the presence of the members of the core E-cadherin complex, a- and b-catenin. We find, however, a change in the presence of several proteins, including vinculin and zyxin, that have been less clearly associated with E-cadherin adhesions and whose precise functions have remained largely unclear. In time-lapse microscopy experiments, we find that these proteins localize to junctions during their formation but are lost during their maturation. Induction of junction-disruption by HGF results in re-localization of these proteins to E-cadherin adhesions. Furthermore, in cells depleted of vinculin the formation of E-cadherin adhesions is strongly impaired. From these data, we hypothesize that a complex of proteins including vinculin and zyxin, mediates a transient link between E-cadherin and the cytoskeleton, which is regulated by cytoskeletal tension and is important for the formation and turnover of E-cadherin-based cell-cell junctions. This unifies the different observations in the literature and explains how tension results in the disruption of cell-cell adhesion needed for tumor metastasis. Sonnenberg A. KASH-domain proteins in nuclear migration, anchorage and other processes. J Cell Sci 2006; 119:5021-5029 Deciphering the functions of Cd151 The overexpression of the tetraspanin Cd151 in tumors of epithelial origin is strongly correlated with enhanced metastasis and poor prognosis. In order to investigate the contribution of Cd151 to tumor formation and metastasis, we have generated conditional Cd151 knockout mice on a p53 null background. Keratinocyte cell lines, established from these mice, are currently being used to determine the role of Cd151 in cell adhesion, migration, proliferation and invasion. Furthermore, we are in the process of generating a mouse model for skin cancer using the conditional Cd151 knockout mice. The physiological role of Cd151 in the kidney was studied using Cd151-null mice. It was shown that these mice develop the same renal pathology of human patients, i.e., with age they develop massive proteinuria caused by focal glomerulosclerosis, disorganization of the glomerular basement membrane, and tubular cystic dilation. Notably, these abnormalities are strikingly similar to those seen in mice with a conditionally inactivated integrin a3 subunit in podocytes, thereby supporting the existence of functional integrin-Cd151 complexes in vivo. 19 CELL BIOLOGY GENETIC CONTROL OF INVASION AND METASTASIS The aim of our research is to identify and characterize genes that play an essential role in the acquisition of an invasive and metastatic phenotype of tumorigenic cells. Insight into the signaling pathways involved in the formation and progression of tumors may lead to the development of novel diagnostic tools or improved therapeutic strategies. Group leader John Collard Function-based screens for invasion-inducing genes In earlier studies we have identified the invasion-inducing Tiam 1 gene by retroviral insertional mutagenesis in combination with in vitro selection of invasive T-lymphoma cells. Additional functional screens for genes involved in T-lymphoma invasion revealed a number of novel genes, including the a5- subunit of the a5b1 integrin, the LPA2 receptor and Rap1, which have been implicated in cell-matrix adhesion and/or signaling pathways mediated by Rho GTPases. For these novel screens, retroviral cDNA libraries were used in combination with in vitro selection of invasive T-lymphoma cells. Currently, the molecular mechanisms are being studied how these genes are involved in the acquisition of an invasive phenotype. Rho family proteins are controlled by various regulatory proteins The invasioninducing Tiam 1 gene encodes an activator or guanine nucleotide exchange factor (GEF) for the Rho-like GTPase Rac. Rho GTPases, which include Cdc42, Rac and RhoA, control a wide range of biological processes such as the adhesion, polarity, and motility of cells. In particular, they act in signaling pathways that regulate the reorganization of the actin cytoskeleton in response to receptor stimulation. The principal regulators of the activities of Rho proteins are the GEFs and GTPase activating proteins (GAPs). GEFs induce activation of the small GTPases by exchanging GDP for GTP, whereas GAPs inactivate the small GTPases by enhancing the intrinsic rate of hydrolysis of bound GTP to GDP. Guanine nucleotide dissociation inhibitors (GDIs) control the activity of Rho proteins. In the cytoplasma, Rac is kept in its GDP-bound form by association with RhoGDI. RhoGDI binds and masks the hydrophobic C-terminal region of Rac, which is responsible for targeting Rac to the plasma membrane. We found that increased intracellular [Ca2+] and the activation of PKC induces phosphorylation of RhoGDI leading to its dissociation from Rac. Cytosolic Rac translocates to the plasma membrane where it can be activated by membrane associated GEFs such as Tiam 1. John Collard PhD Group leader Audrey Gerard PhD Post-doc Michiel Pegtel PhD Post-doc Kristin Strumane PhD Post-doc Saskia Ellenbroek MSc Graduate student Amra Hajdo-Milasinovic MSc Graduate student Sander Mertens MSc Graduate student Tomasz Rygiel MSc Graduate student Rob Van der Kammen Technical staff Publications Engers R, Mueller M, Walter A, Collard JG, Willers R, Gabbert HE. Prognostic relevance of Tiam 1 protein expression in prostate carcinomas. Br J Cancer 2006; 95:1081-1086 Hajdo-Milasinovic A, Mertens AE, Hamelers IHL, Collard JG. Rho GTPases in cell motility and tumorigenesis. In: Wells A, editor. Cell Motility in Cancer Invasion and Metastasis. Berlin [etc]: Springer, 2006: 189-220 Malliri A, Rygiel TP, Van der Kammen RA, Song JY, Engers R, Hurlstone AFL, GEFs and downstream signaling GEFs are regulated at four different levels in the cell, i.e. by intra-molecular inhibition, changes in intracellular localization, posttranslational modifications and interaction with other proteins. All of these regulatory mechanisms seem to be involved in Tiam 1 regulation. In particular, complex formation with various scaffold proteins is an important mechanism to determine signaling towards- and downstream of Rac. For instance, oncogenic Ras controls Rac signaling through direct interaction with the RBD domain of Tiam 1. Tiam 1 may also determine the nature of the signaling downstream of Rac by interacting with various effector or scaffold proteins. We found that Tiam 1 is indispensable for a3b1-mediated Rac activation. In contrast to wild-type keratinocytes, Tiam 1-deficient cells do not adhere to and spread on a glass substrate, because they are unable to deposit their own laminin-5 substrate. Tiam 1-deficient keratinocytes are impaired in activation of Rac upon adhesion to an exogenous laminin-5 substrate, whereas other integrinmediated signaling pathways are intact. Tiam 1-deficiency thereby prevents keratinocyte migration in vitro and re-epithelialization of excision wounds in mouse skin in vivo. It is well known that Rho proteins regulate cell adhesion and migration through their control of actin cytoskeleton dynamics mediated by the Arp2/3 complex. We found that Tiam 1 interacts with the p21-Arc subunit of the Arp2/3 complex. As a result, Tiam 1 co-localizes with the Arp2/3 complex at sites of actin polymerization such as epithelial cell-cell contacts and membrane ruffles. The Arp2/3 complex acts as a scaffold to localize Tiam 1 and thereby Rac-activity, which both are required for activation of the Arp2/3 complex. The Arp2/3 complex drives actin polymerization preliminary at the leading edge of cells. Clevers H, Collard JG. The Rac activator Tiam 1 is a Wnt-responsive gene that modifies intestinal tumor development. J Biol Chem 2006; 281:543-548 Mertens AEE, Pegtel DM, Collard JG. Tiam 1 takes PARt in cell polarity. Trends Cell Biol 2006; 16:308-316 Strumane K, Rygiel TP, Collard JG. The Rac Activator Tiam 1 and Ras-Induced Oncogenesis. In: Balch WE, Der CJ, Hall A, editors. Regulators and Effectors of Small GTPases: Ras Family. Academic Press, 2006: 269-281 Ten Kooster JP, Evers EE, Janssen L, Machesky LM, Michiels F, Hordijk P, Collard JG. Interaction between Tiam 1 and the Arp2/3 complex links activation of Rac to actin polymerization. Biochem J 2006; 397:39-45 20 CELL BIOLOGY Figure I.2: Primary hippocampal neuron isolated from a Tiam 1-deficient mouse. Dendrites are visualized by anti-tyrosine tubulin mAb. Tiam 1-Rac signaling and cell polarity Tiam 1 also associates with components of the Par polarity complex and thereby regulates polarity processes. Tiam 1 localizes to adherens junctions in epithelial cells and ectopic expression of Tiam 1 promotes Ecadherin-based cell-cell adhesions. Conversely, knock down of Tiam 1 leads to loss of cell-cell adhesions and epithelial-mesenchymal transition. Tiam 1 also controls tight junction (TJ) formation, which is required for the establishment of apical-basal cell polarity in epithelial cells. We found that TJ maturation is severely impaired in Tiam 1-deficient keratinocytes. Interestingly, Tiam 1 interacts with two components of the Par polarity complex (Par3 and atypical PKCz) and Tiam 1-mediated Rac activation controls TJ biogenesis by activation of the Par polarity complex. In the absence of cell-cell adhesions, keratinocytes develop front-rear polarization and migrate in a directional persistent fashion. Tiam 1 and the Par complex also function in directional migration. Knock down of Par3, lack of Tiam 1, or reduced PKCz activity impairs front-rear polarization, persistent migration and chemotaxis of keratinocytes. Apparently, in the absence of cell-cell contacts, epithelial cells divert Par polarity signaling and Tiam 1 to promote persistent cell migration. Tiam 1 in conjunction with the Par complex also affects directional protrusion outgrowth in astrocytes and controls Rap1- and chemokine-induced polarization and chemotactic migration of T-cells. Tumorigenicity in Tiam 1 mutant mice Although Tiam 1 is expressed during embryonic development, Tiam 1-deficient (Tiam 1-/-) mice develop, grow, and reproduce normally. In mouse skin, Tiam 1 is present in basal and suprabasal keratinocytes of the epidermis and in hair follicles. We found that Tiam 1-/- mice display resistance to DMBA/TPA induced skin tumor formation. Although the number and growth of tumors is strongly reduced in these Tiam 1-/- mice, Tiam 1-deficiency increases the frequency of malignant conversion of the skin tumors that do develop. Currently we are studying the role of Tiam 1 in tumorigenesis induced by different oncogenic signaling pathways i.e. Ras, Wnt, Myc, Neu and PI3-kinase. We found that Tiam 1 is a Wnt-responsive gene and that Tiam 1 protein levels are increased in intestinal tumors produced in APC min mice as well as in human colon tumors. In addition, initiation and growth of intestinal tumors was reduced in Tiam 1-deficient APC min mutant mice, suggesting that Tiam 1 is required for b-catenin/TCF-induced tumor formation. In contrast, we do not find differences in T-lymphomagenesis induced by constitutive activation of the PI3-kinase pathway in wild-type and Tiam 1-deficient mice. Mammary tumors induced by MMTV-c-neu appeared dependent on Tiam 1 whereas mammary tumors induced by MMTV-myc were not. Together, these studies so far indicate that Tiam 1 may contribute to the formation and progression of tumors induced by various -but not all-oncogenic signaling pathways. Opposing functions of Rac1 and Rac3 Rac1 and Rac3 are highly homologous proteins that share 92% of the amino acid sequences. Most differences are found in the carboxy-terminus of these proteins. Rac1 is ubiquitously expressed and regulates adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in the brain. We found opposing functions for Rac1 and Rac3 in neuronal cells. Knock down of Rac1 by siRNA leads to decreased cell-matrix adhesions and cell rounding. In contrast, knock down of Rac3 induces stronger cell adhesions and increases the outgrowth of neurites in neuronal cells. The intracellular localization of Rac1 and Rac3 is different, which is determined by the different sequences in the carboxy-terminus. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. Our data indicate that Rac3 opposes Rac1 in the regulation of cell-matrix adhesions and differentiation of neuronal cells. Figure I.3: The organization of the microtubule cytoskeleton is disturbed is Tiam 1-deficient astrocytes (bottom) compared to wild type astrocytes (top). 21 CELL BIOLOGY MEC HANISMS IN METASTASIS Metastasis, the spread of tumor cells to distant sites in the body, is the main cause of cancer lethality. We study mechanisms of metastasis of lymphomas and carcinomas. Role of the chemokine receptor CXCR4 in carcinoma metastasis We showed previously that CXCR4, the receptor for the chemokine SDF-1 (CXCL12), is required for metastasis of a colon carcinoma but, surprisingly, not involved in invasion. We next observed that CXCR4 is required for continuation of growth when micrometastases are 500-1000 cells in size. The same was found for dispersed single cells in a s.c. Matrigel plug. The cells had no CXCR4 in vitro but it was upregulated in vivo. The CXCL12 is supposed to be derived from the tumor microenvironment but it was not clear whether it is present in the plugs. To demonstrate involvement of CXCL12, we transfected the dominant-negative K1R-CXCL12 mutant that should inhibit CXCL12induced signals. Surprisingly, however, these cells as well as control cells transfected with intact CXCL12 proliferated much faster, even though they did not express CXCR4. We assume that this is due to the recently discovered receptor CXCR7 that is expressed by the cells and also binds CXCL12. The method we used to block CXCR4 function in vivo should also affect CXCR7. We are currently investigating which of the two is involved in the in vivo phenomena. The role of chemokine receptor CXCR5 in carcinoma metastasis to the liver We detected CXCR5 in many carcinoma cell lines and in 30% of human pancreatic carcinomas. This was surprising since CXCR5 was thought to be only expressed by B- and some T-lymphocytes. We found that the ligand, BCA-1 (CXCL13) enhanced proliferation of CXCR5-positive cells. CXCL13 is present in the liver. Inhibition of CXCR5 reduced growth of liver metastases and after some time led to growth arrest. Preliminary results with a pancreatic carcinoma in CXCL13 knockout mice indicate that CXCL13 is required to resist an anti-tumor response of probably NKT cells in the liver. In the knockout mice the liver metastases grow for seven days and then regress, whereas in +/- litter mates growth is only transiently impeded. Synaptotagmin: role in chemotaxis? Our original hypothesis was that rapid reactions of lymphoid (and lymphoma) cells to chemokines involved rapid fusion of vesicles with the plasma membrane. We thought that this was controlled, as in nerve cells, by synaptotagmins (Syts), and indeed found that inhibition of Syts impaired migration. Syt3 was next shown to be involved, and to be required for CXCL12-induced migration signals. Unexpectedly, however, rapid reaction to chemokines, in particular the induction of adhesion in flow, did not require Syt3. Furthermore, Syt3 was found intracellularly, in multivesicular bodies (MVB), and not at the plasma membrane. Figure 1.4: Regression of pancreatic carcinoma liver metastases in CXCL13 knockout mice. Panc02 pancreatic carcinoma cells, expressing luciferase, were injected into the spleen, which was subsequently removed. This led to the formation of liver metastases, the growth of which was measured by bioluminescence upon injection of luciferin. Note logarithmic scale. Metastases grew rapidly for 7 days in CXCL13-/- knockout mice and heterozygous CXCL13+/- litter mates. After that, growth stopped for a week in CXCL13+/- mice and resumed thereafter. In contrast, liver metastases regressed completely in CXCL13-/- mice. This shows that CXCL13, the ligand of CXCR5, allows the cells to resist a host response, probably of liver NKT cells. Group leader Ed Roos Ed Roos PhD Group leader Agnieszka Masztalerz PhD Post-doc Joost Meijer MSc Graduate student Yvonne Wijnands Technical staff Janneke Ogink Technical staff 22 CELL BIOLOGY Publications Masztalerz A, Zeelenberg IS, Wijnands YM, De Bruijn R, Drager AM, Janssen H, Roos E. Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration. J Cell Sci 2007; 120:219-228 Meijer J, Zeelenberg IS, Sipos B, Roos E. The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver. Cancer Res 2006; 66:9576-9582 Stroeken PJM, Alvarez B, Van Rheenen J, Wijnands YM, Geerts D, Jalink K, Roos E. Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane. J Cell Physiol 2006; 208:620-628 This argued against our hypothesis and suggested that Syt3 has another function. CXCR4 surface levels were much lower in cells in which Syt3 function was blocked, whereas the total amount of CXCR4 protein was not different. Furthermore, recycling of CXCR4 after CXCL12-induced endocytosis was impaired. Finally, overexpression of CXCR4, resulting in increased surface levels, restored migration. Thus, Syt3 is required for recycling of CXCR4 in these cells, and when recycling is abrogated, this leads to CXCR4 surface levels that are insufficient for a migratory response but apparently high enough for arrest of cells in flow. A possibility being considered is that Syt3 controls, in a calcium-dependent fashion, the recruitment of a highly active CXCR4 receptor from MVB to the surface. This might have higher affinity or be associated with particular signaling molecules, and rapidly lose activity. Interestingly, we found that Syt3 is expressed in many carcinomas whereas it is apparently absent in the corresponding normal epithelial cells. We are presently investigating whether this Syt3 is required for the function of chemokine receptors in carcinomas, as described above. 23 CELL BIOLOGY BIOPHYSICS IN CELL SIGNALING Biophysical techniques are exquisitely suited to study cell signaling events with high spatial and temporal resolution. We employ advanced electrophysiological (e.g. patch clamping) and bio-photonic techniques (e.g. Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross Correlation Spectroscopy (FCCS) and photorelease of caged compounds) in research projects in our group as well as in a number of collaborations within and outside our institute. Our lab also contributes to the development hardware, software and FRET sensors for various intracellular messengers. Group leader Kees Jalink Kees Jalink PhD Group leader Michiel Langeslag PhD Post-doc Spatiotemporal aspects of phosphatidylinositol bisphosphate (PIP2) as a membranedelimited messenger Phosphoinositides in the plasma membrane regulate a wide variety of cellular responses. We developed FRET-based assays for the lipids PIP2 and PIP3, and use these to study the regulation of agonist-induced PIP2 breakdown and its subsequent resynthesis by PIPkinases. In collaboration with members from the division of Cellular Biochemistry we have addressed how the concentration of PIP2 in the plasma membrane regulates gap junction communication, and involvement of this lipid in protecting cells against apoptosis. Bas Ponsioen MSc Graduate student Gerard Van der Krogt PhD Technical staff Janneke Ogink Technical staff Publications Altelaar AFM, Klinkert I, Jalink K, De Lange RPJ, Adan RAH, Heeren RMA, Mechanism of activation of TRPM7, a cat ion channel that regulates cell adhesion TRPM7 is a non-selective cation channel with inherent kinase activity that is involved in sensing mechanical forces. TRPM7 mediates agonist-induced cell adhesion through Ca2+ influx and myosin heavy chain phosphorylation. We discovered that PLC-activating agonists strongly stimulate TRPM7 channels. Currently, we study TRPM7 activation in detail by combining biophysical readout techniques with mutational analysis of the C terminus. Our results indicate involvement of a feedback loop that incorporates Ca2+ influx-mediated PLC activation in TRPM7 gating. We also study regulation of the closely related channel TRPM6, which is involved in Mg2+ homeostasis. Piersma SR. Gold-enhanced biomolecular surface imaging of cells and tissue by SIMS and MALDI mass spectrometry. Anal Chem 2006; 78:734-742 Clark K, Langeslag M, Van Leeuwen B, Ran L, Ryazanov AG, Figdor CG, Moolenaar WH, Jalink K, Van Leeuwen FN. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J 2006; 25:290-301 Scope of cyclic nucleotide messengers in coupled cells The cyclic nucleotide cAMP is an important messenger with numerous biological effects. Using live cell imaging with FRET sensors for cAMP we have shown that cAMP readily permeates through Gap Junctions to influence the fate of neighboring cells. In addition, we have generated an improved family of cAMP FRET sensors with superior signal-to-noise ratio and cellular inertia. Halstead JR, Van Rheenen J, Snel M, Meeuws S, Mohammed S, DвЂ™Santos CS, Heck A, Jalink K, Divecha N. A Role for PtdIns(4,5)P2 and PIP5Ka in Regulating Stress-Induced Apoptosis. Curr Biol 2006; 16:1850-1856 Jalink K. Spying on cGMP with FRET. Nature Methods 2006; 3:11-12 Langeslag M, Clark K, Moolenaar WH, Van Leeuwen FN, Jalink K. Activation of TRPM7 Channels by Phospholipase C-coupled Receptor Agonists. J Biol Chem 2007; 282:232-239 Figure I.5: 3-step model for the involvements of TRPM7 in mechano-regulated cell adhesion. Ponsioen B, Van Zeijl L, Moolenaar WH, A) Vesicles containing TRPM7 channels localize at Jalink K. Direct measurement of cyclic AMP or close to the membrane in resting cells. diffusion and signaling through connexin43 B) Through cell adhesion structures, cells sense gap junctional channels. Exp Cell Res mechanical stress such as increased fluid flow. 2007; 313:415-423 As a consequence, TRPM7 vesicles fuse with the plasma membrane and the channels become active. Stroeken PJM, Alvarez B, Van Rheenen J, C) Increased local Ca2+ influx through the TRPM7 Wijnands YM, Geerts D, Jalink K, Roos E. channel pore causes association of the kinase domain Integrin cytoplasmic domain-associated with its substrates at the cell adhesion site. This leads protein-1 (ICAP-1) interacts with the to the formation of podosomes and the further ROCK-I kinase at the plasma membrane. recruitment of other proteins in the complex. J Cell Physiol 2006; 208:620-628. 24 MOLECULAR CARCINOGENESIS II D IV IS ION OF M OLECU LA R CAR CIN OG ENES IS FUNCTIONAL GENOMICS Division head, Group leader RenГ© Bernards RenГ© Bernards PhD Group leader Katrien Berns PhD Post-doc My group uses functional genomics technologies to identify novel cancer-relevant genes. We use both high-throughput gain-of-function genetic screens and loss-offunction genetic screens to identify additional components of the major cancerrelevant pathways. We also use these functional genetic approaches to understand the mechanisms of action of anti cancer drugs. Luis Borlado PhD Post-doc Annette Dirac PhD Post-doc Pieter Eichhorn PhD Post-doc Sidong Huang PhD Post-doc Rianne Oosterkamp MD Clinical fellow Benjamin Rowland PhD Post-doc Miguel Rubio PhD Post-doc Linda Smit PhD Post-doc Mirjam Epping MSc Graduate Student Ernst-Jan Geutjes MSc Graduate student Roderik Kortlever MSc Graduate Student Jasper Mullenders Msc Graduate Student Functional identification of cancer-relevant genes One of the major remaining deficits in our understanding of the human genome is that the function of only one quarter of the approximately 30,000 genes is known to date. Many of these hitherto anonymous genes are potential targets for the development of new anti-cancer drugs. It is therefore important to functionally annotate the thousands of genes for which this information is currently lacking. My laboratory has developed functional genetic approaches to obtain information regarding gene function using high-throughput screens in mammalian cells. We have developed both gain-of-function genetic screens (using retroviral cDNA expression libraries) and loss-of-function genetic screens (using RNA interference libraries) to carry out large-scale genetic screens in mammalian cells. Miranda Van Dongen Technical staff Marielle Hijmans MSc Technical staff Mandy Madiredjo Technical staff Publications Bernards R. Exploring the uses of RNAi Gene knockdown and the nobel prize. N Engl J Med 2006; 355:2391-2393 Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics. Nat Meth 2006; 3:701-706 Brummelkamp TR, Fabius AWM, Gain-of-function genetic screens These screens involve the infection of a cell population with a high-complexity retroviral cDNA expression library, selection of cells with altered phenotype, followed by identification of the cDNA responsible for the phenotype. In the past years, we have used gain-of-function genetic screens to identify genes that confer resistance the anti-proliferative effects of a group of experimental anti cancer drugs: histone deacetylase inhibitors (HDACIs). The aim of this study was the identification of biomarkers that predict resistance to HDACI therapy. Using this approach, we have identified cDNAs that confer resistance to this group of anti-cancer drugs. A remarkable common feature of all three cDNAs identified to date is their ability to inhibit transactivation by retinoic acid receptor in response to its natural ligand, retinoic acid. Conversely, suppression of these genes by RNA interference increases sensitivity to the anti-proliferative effects of HDACI in vitro and in vivo. Importantly, we found that RA and HDACI act synergistically in the inhibition of tumor growth in a mouse xenograft model. Thus, this study identified biomarkers of HDACI resistance and suggests strategies to increase the efficacy of HDACIs in a clinical setting. Mullenders J, Madiredjo M, Velds A, Kerkhoven RM, Bernards R, Beijersbergen RL. An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol 2006; 2:202-206 Creyghton MP, RoГ«l G, Eichhorn PJA, Vredeveld LC, DestrГ©e O, Bernards R. PR130 is a modulator of the Wnt-signaling cascade that counters repression of the antagonist Naked cuticle. Proc Natl Acad Loss-of-function genetic screens Using a mammalian expression vector, which directs the synthesis of short hairpin transcripts (shRNAs), we have inhibited all members of a conserved family of Protein Phosphatase 2A (PP2A) regulatory subunits. These regulatory subunits are thought to act as bridging factors between the PP2A catalytic subunit and its substrate. Using this gene family knockdown library, we identified PR55g as an inhibitor of c-Jun N terminal kinase (JNK) activation by UV irradiation (Figure II.1). We found that PR55g binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we found to be required for JNK activation by c-SRC. We also found that the physical interaction between PR55g and c-SRC is sensitive to UV irradiation, suggesting a novel pathway of c-SRC activation in response to cellular stress. Sci U S A 2006; 103:5397-5402 Kortlever RM, Higgins PJ, Bernards R. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence. Nat Cell Biol 2006; 8:878-884 The tumor suppressor protein p53 limits the proliferative capacity of most primary cells in culture through the induction of a state of growth arrest named replicative senescence. However, little is known concerning the downstream target genes of p53 in this growth-limiting response. In a second study, we asked which (if any) of the transcriptional targets of p53 is responsible for the induction of replicative senescence. We found that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ 25 MOLECULAR CARCINOGENESIS Publications (continued) fibroblasts. Our data indicate that PAI-1 knockdown results in sustained activation of the PI3K-PKB-GSK3b pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signaling. In agreement with this, we found that the PI3K-PKB-GSK3b-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, we found that ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induced a phenotype displaying all the hallmarks of replicative senescence. These data indicate that PAI-1 is not merely a marker of senescence, but both necessary and sufficient for the induction of replicative senescence downstream of p53 (Figure II.2). Unresponsiveness to therapy is a persistent problem in the treatment of cancer. It is therefore important to identify the molecular pathways that contribute to unresponsiveness to cancer therapeutics. We also use loss-of-function genetic screens to identify genes that contribute to drug resistance, in particular to new classes of targeted therapeutics. For these studies, we use our vast collection of RNA interference vectors, which at present consists of 55,000 shRNA vectors that together target over 23,000 mouse and human genes for suppression. We use this vector set in conjunction with a new way to rapidly screen shRNA libraries: siRNA bar code screening (in collaboration with R Beijersbergen, division of Molecular Carcinogenesis, see below). In a first proof-of-concept study, we identified genes required for the cytotoxicity of a promising new cancer drug named Nutlin-3. Nutlin-3 is a small molecule inhibitor of MDM2, which can activate the p53 pathway. We have identified that a component of the DNA damage-induced signaling network, 53BP1, is a critical mediator of Nutlin-3 induced cytotoxicity. This result suggests that Nutlin-3вЂ™s cytotoxic effect results from its ability to turn a cancer cell specific property, activated DNA damage signaling, into a weakness that can be exploited therapeutically. We are currently using a similar approach to identify genes whose inactivation causes resistance to new classes of anti cancer drugs, such as Herceptin, Iressa and Velcade. We have also used our genome-wide shRNA library to identify genes whose suppression can cause resistance to growth arrest induced by the retinoblastoma tumor suppressor protein pRB. We generated human BJ fibroblasts that express a mutant form of pRB in which 15 of the CDK phosphorylation sites are mutated. This pRB-О”CDK is constitutively active as a growth suppressor. However, the growthinhibitory capacity of this protein can be overruled by a temperature sensitive mutant of SV40 T antigen. This mutant T antigen protein binds to pRB at 32В°C, but not at 39В°C. As a result, cells co-expressing the pRB-О”CDK and the temperature sensitive mutant of SV40 T antigen proliferate at 32В°C, but not at 39В°C, when the constitutively active pRB is released from T antigen to inhibit proliferation. Using this cell system, we searched for genes whose suppression by RNA interference causes a bypass of the pRB-induced growth arrest. To date, we have identified one gene, RB1CC1, whose suppression allows cells to proliferate, in the presence of active pRB. We are currently studying the mechanism of action of RB1CC1 in the pRB pathway. Interestingly, RB1CC1 is mutated in a significant fraction of human primary breast cancers, suggesting that loss of this gene may also contribute to resistance to pRB growth arrest in vivo. Nijman SMB, Hijmans EM, El Messaoudi S, Van Dongen MMW, Sardet C, Bernards R. A functional genetic screen identifies TFE3 as a gene that confers resistance to the anti-proliferative effects of the retinoblastoma protein and transforming growth factor-b. J Biol Chem 2006; 281:21582-21587 Oosterkamp HM, Neering H, Nijman SMB, Dirac AMG, Mooi WJ, Bernards R, Brummelkamp TR. An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosis. Br J Dermatol 2006; 155:182-185 Rowland BD, Bernards R. Re-Evaluating Cell-Cycle Regulation by E2Fs. Cell 2006; 127:871-874 Figure II.1: RNAi-based PP2A вЂњBвЂќ subunit genetic screen. (Top): Schematic of the PP2A holoenzyme and outline of the b regulatory subunit families. (Bottom): U2-OS cells were co-transfected with a set of PP2A shRNAs targeting a single PP2A вЂњBвЂќ subunit or a control vector. Levels of phosphorylated JNK (a- pJNK) or JNK1 and JNK2 (a-JNK) are shown in cell lysates for the samples 60 minutes after UV An exciting new concept in the development of cancer drugs is the genotype-specific drug targetвЂ”the protein whose inactivation is toxic only to cells carrying a defined (cancer-specific) genetic lesion. In theory, drugs targeting the products of such genes would be more selective for cancer cells than the current generation of broadly acting cytotoxic drugs, as they require the presence of a cancer-specific lesion to exert their cytotoxic effects. RNAi technology is exquisitely suited for uncovering such genetic interactions in mammalian cells. A new goal is to identify such interactions in mammalian cells, as this may ultimately lead to the generation of вЂњgenotype-specificвЂќ drugs, which act with a considerable degree of specificity on tumor cells. Figure II.2: Proposed model of senescence in fibroblasts. p53 induces PAI-1 and p21CIP1 during ageing in culture. PAI-1 antagonizes uPA/GF (growth factor) signaling to cyclin D1 via PI3K-PKB-GSK3b and p21CIP1 blocks cyclin D1 activity directly. The PAI-1-cyclin D1 connection is dominant over p21CIP1 activity and controls induction of the senescence response downstream of p53 and upstream of pRb. treatment of the cells. 26 MOLECULAR CARCINOGENESIS RNA INTERFERENCE BASED LOSS-OF-FUNCTION SCREENING Group leader Roderick Beijersbergen Roderick Beijersbergen PhD Group leader David Egan PhD Post-doc Armida Fabius MSc Graduate student Johan Kuiken MSc Graduate student Our research is focussed around the development and application of technologies based on RNA interference to perform loss-of-function genetic screens. We have completed our human and mouse collections of shRNA expression vectors for cell based loss of function screens. In addition we have, within the RNAi global consortium, acquired a full genome synthetic siRNA collection (21.500 genes) that is used for high throughput arrayed format screens. This collection enables us to develop screening systems based on more complex phenotypes using high content imaging. We have continued to improve and apply our bar code screening technology to identify novel components in disease-associated signaling pathways and to achieve a more complete understanding of drug action, enabling more rational decisions on drug application. Jeroen Nijwening MSc Graduate student Wouter Nijkamp Technical staff Bar code screens The bar code screening technology has been developed to monitor the effect of the inactivation of gene expression in large complex populations of cells. In this way the effect of gene inactivation on the phenotype of individual cells can be followed. The design of the NKi shRNA library allows for the identification of individual shRNA vectors in a large pool of shRNA vector containing cells. Each hairpin vector contains a unique 19-mer sequence (вЂњbar codeвЂќ) designed to target one specific cellular gene. This 19-mer sequence can be used to determine the relative abundance of a shRNA vector by hybridization to DNA micro-arrays containing the 19-mer bar code sequences. By using this technique, large complex populations of shRNA expressing cells can be monitored for the abundance of each individual shRNA expressing cell (Figure II.3) reflecting the effect of specific gene inactivation on cellular behavior. The bar code screening technology facilitates the identification of genes that contribute to drug resistance in cancer cells, in particular to new classes of targeted therapeutics. An example of this strength is the identification of genes required for the cytotoxicity of a new cancer drug named Nutlin-3. Nutlin-3 is a small molecule inhibitor of MDM2, which can activate the p53 pathway. We have identified that a component of the DNA damage induced signaling network, 53BP1, is a critical mediator of Nutlin-3 induced cytotoxicity. This result suggests that Nutlin-3вЂ™s cytotoxic effect results from its ability to turn a cancer cell specific property, activated DNA damage signaling, into a weakness that can be exploited therapeutically. At this moment several cancer drugs are used to identify genes that either enhance the drugвЂ™s effect or result in resistance to drug treatment. Figure II:3: Large populations of shRNA expressing cells are analyzed in parallel using molecular barcode screens. Each shRNA vector is tagged with a unique вЂњbar codeвЂќ sequence (the 19mer targeting sequence) that is used to monitor the representation of each vector in a complex population by hybridizing to an oligonucleotide microarray containing the 19-mer bar code sequences. When a population of cells is subjected to a selective pressure, there will be selection for those cells that express a shRNA that modulates the cellular response. As a result the representation of individual shRNA constructs is expected to change. This change in the relative abundance of individual shRNA vectors can be determined by comparison of a control population versus the population exposed to selection. The signal intensity of shRNAs that confer resistance to the selection will increase compared to the control population. 27 MOLECULAR CARCINOGENESIS Publications Synthetic lethal interactions For the effective treatment of cancer, there is a great need for drugs that specifically target tumor cells without affecting normal cells. With the use of RNA interference, we have begun to explore synthetic lethal phenotypes in mammalian cells. Synthetic lethal phenotypes; a combination of two mutations which by themselves are non-lethal but together result in a lethal phenotype. These interactions can lead to the identification of novel cancer drug targets that are only cytotoxic in the background of a tumor specific alteration and represent вЂњgenotype specificвЂќ drugs. We have generated multiple isogenic cell lines that contain single or multiple defined genetic alterations in oncogene вЂ“ or tumor-suppressor pathways. These are used in high throughput single well assays in combination with genome wide siRNA collections to identify siRNAs that result in enhanced lethality only in the background of these tumor specific genetic alterations. The identification of the proteins or pathways that are essential for the survival of tumor cells with specific genetic alterations could result in a novel class of cancer therapeutics. Bernards R, Brummelkamp TR, Beijersbergen RL. shRNA libraries and their use in cancer genetics. Nat Meth 2006; 3:701-706 Brummelkamp TR, Fabius AWM, Mullenders J, Madiredjo M, Velds A, Kerkhoven RM, Bernards R, Beijersbergen RL. An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors. Nat Chem Biol 2006; 2:202-206 Kedde M, Le Sage C, Duursma A, Zlotorynski E, Van Leeuwen B, Nijkamp W, Beijersbergen R, Agami R. Telomerase-independent Regulation of ATR by Human Telomerase RNA. J Biol Chem 2006; 281:40503-40514 Figure II.4: Automated cell based assay system and automated confocal fluorescence microscope for high content imaging of the NKI Robotics and Screening Center. NKI Robotics and Screening Center The NRSC was started early 2005 with the goal to develop an efficient technology platform to discover gene function, to unravel molecular pathways and mechanisms, to discover novel drug targets and to support the identification of small molecules both for biological tools and novel drug leads. The NSRC is also a resource center that provides the technology for medium to high throughput applications, provides support and expertise for automated cell and noncell based assays and is used for the development, production and maintenance of large screening reagent collections (Figure II.4). Recently, the NSRC has also acquired an automated fluorescence confocal microscope to enable high content screening at single cell level with multiple cellular markers (Figure II.5). Figure II.5: Automated image analysis of a cell based assay Images obtained with different fluorescent markers are used to identify the region of interest (nucleus and cytoplasm) followed by quantification of the fluorescent signal in both compartments. Results are used to calculate the cytoplasmic to nuclear ratio as measure for pathway activation. 28 MOLECULAR CARCINOGENESIS STRUCTURAL BIOLOGY Group leader Titia Sixma Titia Sixma PhD Group leader Development of cancer is generally due to errors that occur in cellular pathways. Structural biology can help to understand these errors at the atomic level, by studying the proteins and the DNA involved. We use X-ray crystallography as a tool to provide three-dimensional structures and we interpret the structural data using a variety of biochemical and biophysical techniques. These studies provide more insight in the molecular processes and they can also provide targets for drug design studies. In our group we focus mainly on proteins involved in ubiquitin conjugation in chromatin regulation, on DNA mismatch repair and nicotinic receptor signaling. Joyce Lebbink PhD Senior Post-doc Gretel Buchwald PhD Post-doc Alex Fish PhD Post-doc Valerie Notenboom PhD Post-doc Chris Ulens PhD Post-doc Rick Hibbert PhD Post-doc Alex Faesen MSc Graduate student Puck Knipscheer MSc Graduate student Mark Vargas MSc Graduate student Annet Reumer MSc Graduate student Francesca Mattiroli MSc Graduate student Pim Van Dijk Technical staff Sari Van Rossum Technical staff Herrie Winterwerp Technical staff Publications Aricescu AR, Assenberg R, Bill RM, DNA mismatch repair DNA mismatch repair plays a crucial role in ensuring genomic stability. Defects in the mismatch repair cascade in humans predispose to hereditary non-polyposis colorectal cancer and are associated with a variety of sporadic cancers. DNA mismatch repair is initiated by the protein MutS (in Escherichia coli) or its MSH homologs. MutS recognizes and binds to mismatches or unpaired bases that have escaped the proofreading capacity of the DNA replication machinery or are present in the genome during recombinatorial events between nonfully complementary DNA strands. Mismatch binding triggers the uptake of ATP in the MutS nucleotide binding domain and it is this mismatch-dependent ATP state that authorizes repair by recruitment of additional mismatch repair components. In collaboration with Robert van den Heuvel in Utrecht we have used native mass spectrometry to study MutS DNA complexes. One persistent problem in our structural studies of MutS complexes has been that we could only crystallize it in a single crystal form. Recently, we have managed to produce a new crystal form that shows that our previous conclusions were correct that the DNA adapts to the protein, since we see the exact same protein-DNA interaction. Nevertheless this structure was surprising as it gave us new insight in the ATPase cycle of the MutS protein, which we are currently studying. Busso D, Chang VT, Davis SJ, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U, Jones IM, Ksiazek D, Lang C, Maskos K, Messerschmidt A, Macieira S, Peleg Y, Perrakis A, Poterszman A, Schneider G, Sixma TK, Sussman JL, Sutton G, Tarboureich N, Zeev-Ben-Mordehai T, Jones EY. Eukaryotic expression: Developments for structural proteomics. Acta Crystallogr D Biol Crystallogr 2006; 62:1114-1124 Ubiquitin and SUMO conjugation Ubiquitin conjugation processes have emerged as a critical signaling system that is essential for cell stability, by controlling degradation of short-lived proteins and signaling processes. Because of its importance for regulating apoptosis, cell cycle, chromatin regulation and DNA repair deregulation of ubiquitin-dependent processes often leads to cancer. Structure analysis of the proteins involved in ubiquitin and SUMO conjugation could be important in the development of novel drugs inhibiting critical pathways. The process of conjugation by ubiquitin(-like) proteins involves covalent linking of one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. Although the overall mechanism has been defined, the atomic details have been lacking and the specificity determining factors are unclear. We study several E2/E3 complexes involved in conjugation of ubiquitin and the related modifier SUMO as well as proteins involved in de-ubiquitination. Polycomb complexes are transcriptional repressors involved in gene silencing and maintenance of stem cell character. Together with the group of Maarten van Lohuizen we studied the Ring-Ring complex of Bmi1-Ring1b (Figure II.6). We showed in vitro E3 ubiquitin ligase activity for Ring1b against Histone2A in nucleosomes and found that Bmi1 enhances this activity. We determined the minimal Ring-Ring domain that is still active and solved its crystal structure. This showed that the N-terminal arm of Ring1b embraces the Bmi1 Ring-domain, which is essential for dimer formation and activity. We found that Ring1a has a similar interaction with Bmi1. With point mutants we could show that activity in the heterodimer is limited to Ring1b and not Bmi1. Currently we are expanding our studies to the full length Ring1b/Bmi1 complex. Figure II.6: The Ring1b-Bmi1 complex showing the minimal Ring-Ring domain that is active in ubiquitin conjugation of Histone2A in nucleosomes Nicotinic Acetylcholine receptor homolog AchBP In collaboration with Guus Smit at the Free University in Amsterdam we study the Acetylcholine Binding Proteins (AChBP) as tools for understanding the ligand binding in pentameric 29 MOLECULAR CARCINOGENESIS Publications (continued) ligand-gated ion-channels. AChBP has strong sequence similarity to the -subunits of the nicotinic acetylcholine receptor ligand-binding domain and our AChBP crystal structures are the established high-resolution model for the ligand-binding domains in this class of ion channels. We have determined the 2.2-Г… crystal structure of a homolog of the ligand-binding domain of the nAChR, Aplysia californica AChBP (Ac-AChBP), in complex with a-conotoxin ImI (Figure II.7). Banci L, Bertini I, Cusack S, De Jong RN, Heinemann U, Jones EY, Maskos K, Kozielski F, Owens R, Perrakis A, Siebold C, Silman I, Messerschmidt A, Poterszman A, Schneider G, Sixma T, Sussman JL, Stewart-Jones G, Thierry JC, Fig II.7: Crystal of the complex of the Aplysia Moras D. First steps towards effective californica Acetylcholine binding protein complexed methods in exploiting high-throughput to a-conotoxin ImI. technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr D Biol Crystallogr 2006; 62:1208-1217 Buchwald G, Van der Stoop P, Weichenrieder O, Perrakis A, This conotoxin is unique in its selectivity toward the neuronal a3b2 and a7 nicotinic acetylcholine receptors, a feature that is reflected in its selective binding to Ac-AChBP compared with other AChBP homologs. We observe a network of interactions between the residues of the ligand-binding site and the toxin, in which ImI Arg-7 and Trp-10 play a key role. The toxin also forms interactions in the ligand-binding site that we did not see in the complex of Ac-AChBP with PnIA(A10L D14K), a conotoxin variant that lacks binding selectivity to AChBP homologs. In collaboration with the group of Daniel Bertrand we could show on the basis of electrophysiological recordings obtained using the wild-type a7 nAChR and L247T mutant, that conotoxin ImI inhibits ion conduction by stabilizing the receptor in a desensitized conformation. Comparison of the AcAChBPвЂ“ImI crystal structure with existing AChBP structures gave new structural insight into the extent of flexibility of the interface loops and how their movement may couple ligand binding to channel gating in the context of a nAChR. Van Lohuizen M, Sixma TK. Structure and E3-ligase activity of the Ring-Ring complex of Polycomb proteins Bmi1 and Ring1b. EMBO J 2006; 25:2465-2474 Knipscheer P, Sixma TK. Divide and conquer: The E2 active site. Nat Struct Mol Biol 2006; 13:474-476 Lebbink JHG, Georgijevic D, Natrajan G, Fish A, Winterwerp HHK, Sixma TK, De Wind N. Dual role of MutS glutamate 38 in DNA mismatch discrimination and in the authorization of repair. EMBO J 2006; 25:409-419 Natrajan G. MutS: Recognition of DNA mismatches and initiation of repair. Rotterdam: Erasmus Universiteit, 2006 Romier C, Ben Jelloul M, Albeck S, Buchwald G, Busso D, Celie PHN, Christodoulou E, De Marco V, Van Gerwen S, Knipscheer P, Lebbink JH, Notenboom V, Poterszman A, Rochel N, Cohen SX, Unger T, Sussman JL, Moras D, Sixma TK, Perrakis A. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: Experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 2006; 62:1232-1242 Ulens C, Hogg RC, Celie PH, Bertrand D, Tsetlin V, Smit AB, Sixma TK. Structural determinants of selective a-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP. Proc Natl Acad Sci USA 2006; 103:3615-3620 30 MOLECULAR CARCINOGENESIS STRUCTURAL BIOLOGY Group leader Anastassis Perrakis Anastassis Perrakis PhD Group leader Oliver Weichenrieder PhD Senior Post Doc Patrick Celie PhD Post doc Serge Cohen PhD Post Doc Valeria De Marco PhD Post Doc Mark Hilge Post Doc Dene Littler Post Doc Our main research themes continued to keep us occupied and are discussed below. Dr. Oliver Weichenrieder who was leading the team studying function/structure relationships in retrotransposition mechanisms - the first and longest lasting project of our group - has accepted a group leader appointment at the Max Plank Institute in Tubingen and he will continue his research on retrotransposons there. A couple of new projects have been started and are ongoing in a collaborative effort; details for these can be found in the reports of the groups of T SchumacherвЂ™s (Division of Immunology) and M Fornerod (Division of Tumor Biology). While X-ray crystallography remains the work horse for structure determination, new structural techniques are being established in my laboratory: Small Angle X-ray Scattering (SAXS) measurements and analyses are now done routinely; we are proceeding with NMR structure determination of two protein domains; single particle reconstruction Electron Microscopy (EM) pictures have also been obtained at high resolution for one of our projects. Krista Joosten Post Doc Kostas Repanas MSc Graduate Student Evangelos Christodoulou Technical Staff Angelina Huseinovic Technical Staff Suzan Van Gerwen Technical Staff Mobien Kassiem Technical Staff Marouane Ben Jelloul Software Engineer Diederick De Vries Software Engineer The role of Geminin in DNA replication licensing Geminin inhibits the action of Cdt 1, a necessary factor for the formation of the pre-replication complex or вЂ�replication licenseвЂ™, which is necessary to be assembled prior to DNA replication in eukaryotic cells. We have crystallized various truncation constructs of the complex between Cdt 1 and Geminin (Figure II.8) and determined the crystal structure of the human truncated geminin/Cdt 1 complex at a resolution of 3.3 Г… from crystals grown in conditions very close to physiological (pH 7.5, and about 50 mM NaCl). This structure allowed us to establish important structural differences with previously published data. To strengthen and validate our findings, the same molecules were studied in solution by SAXS. Finally, the structure of the full-length geminin/ Cdt 1 complex has been characterized by SAXS and we have obtained high-resolution EM micrographs. We clearly show that the вЂ�core complexвЂ™ of two geminin molecules and one Cdt 1 (2Gem-1Cdt 1) forms a higher order dimer 2x(2Gem-1Cdt 1) under physiological conditions in solution (Figure II.8). The dimerization interface of geminin has been shown in literature to be crucial for function and our structural data clearly explains its role and the biochemical data; the corresponding dimerization region of Cdt 1 has been вЂ�unchartedвЂ™ and we are in the process of characterizing its role in vivo. Geminin also interacts with members of the Hox family of transcription regulators, and with the Hox homologue Six3, a protein important for forebrain development. This dual role of geminin has fundamental implications on how regulatory solutions are вЂ�re-usedвЂ™ in nature, besides the obvious questions about the mode of molecular recognition of Hox proteins. We have cloned and purified Gemin and Six3 from human, mouse and medaka fish and made complexes of Six3 with its target DNA. We have obtained 2D NMR spectra for Six3 (Figure II.7) and are trying to improve the spectra quality to determine the structure of Six3 alone and in complex with DNA. In parallel we are trying to crystallise these complexes and perform affinity measurements of the system components to elucidate how Six3 interacts with geminin and how DNA affects that interaction. Figure II.8: A model of the truncated Geminin (grey cartoon) and Cdt 1 (darker-grey cartoon); Geminin forms a parallel coiled coil, strongly attached to one Cdt 1 molecule. Two such hetero-trimers dimerize to form the biologically active complex. The light-grey cloud is the low resolution envelope of the full length Geminin-Cdt 1 complex, as determined by SAXS, depicting the additional domains. In the background various crystal forms of various truncation constructs of the complex. Structural studies of the plk1 Polo kinase Polo-like kinases play an important but highly controversial role in cell division. If the molecular determinants for the different roles of Plk- 1, 2, 3 and 4 are understood at the structural level we can evaluate better the potential of Polo like kinases potential for the design of specific drugs, targeting for example only Plk- 1 in tumor cells. We focus our efforts towards two goals. Firstly, we want to characterize the full-length human Plk- 1; no crystals have been obtained yet. Secondly, we have purified the polo domain of all four Plk homologues. We have performed pull-down experiments that in combination with in vivo results of our collaborators, and we demonstrate a partial functional overlap but also distinct roles of the different PBDs: PBD2 appears to be more similar to PBD1 function, while PBD3 and PBD4 can only mimic PBD1 function in early mitotic events. 31 MOLECULAR CARCINOGENESIS Publications (continued) Structural studies of Autotaxin Autotaxin (NPP 1) hydrolyzes circulating lysophosphatidylcholine. The large-scale culture of human cells deficient in glycosylation (HEK293 derivatives) and use of antibiotics have lead to production of homogenous protein for crystallization experiments. We are still unable to obtain crystals and provide structural information towards the collaborative screening effort for drug discovery, which is currently ongoing in the NKI. Albeck S, Ben Jelloul M, Perrakis A, Henrick K. SPINE bioinformatics and data-management aspects of highthroughput structural biology. Acta Crystallogr D Biol Crystallogr 2006; 62:1184-1195 Structural studies of JBP 1 The JBP 1 protein binds to DNA that contains base J (J-DNA). It was discovered by the P. Borst group, and has been shown to be essential for survival in many major protozoan human pathogens such as T. brucei (sleeping sickness), T. cruzi (ChagasвЂ™ disease) and Leishmania species (various types of Leishmaniasis). We aim to elucidate the structure of JBP 1 and contribute to an international drug discovery effort. We have worked out procedures to produce from E. coli mg amounts of soluble purified Crithidia fasciculata and Leishmania tarentolae JBP 1 that selectively bind J-DNA. Moreover, we have very recently identified a ~19 kD domain of JBP 1 that binds J-DNA selectively (mini-JBP 1), and we can produce mg amounts of it in purified soluble form. Surface plasmon resonance (SPR) experiments have shown mini-JBP 1 to bind J-DNA with the same affinity as the fulllength protein and NMR experiments have shown it to be well folded and suitable for structure solution by NMR. The shape of both mini-JBP 1 and JBP 1 has been calculated by SAXS experiments. That allows us to establish that mini-JBP 1 is a distinct small domain of the 100 kD JBP 1 protein, that is sufficient for binding JDNA in a specific manner. Alzari PM, Christodoulou E, Perrakis A, Owens RJ. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr D Biol Crystallogr 2006; 62:1103-1113 Aricescu AR, Perrakis A, Jones EY. Eukaryotic expression: Developments for structural proteomics. Acta Crystallogr D Biol Crystallogr 2006; 62:1114-1124 Bahar M, Cohen SX, Perrakis A, Wilson KS. SPINE workshop on automated X-ray analysis: a progress report. Acta Crystallogr D Biol Crystallogr 2006; Structural characterisation of the human L1 retrotransposition machinery The human L1 endonuclease (L 1EN) is encoded by the L 1 non-LTR retrotransposon that is responsible for more than 1.5 million genomic re-integration events in the history of the human genome. This has resulted in more than a quarter of our genomic DNA, suggesting important roles for L1 elements for the вЂњfluidityвЂќ and evolution of genomes. Based on our structure of L1 endonuclease, we have designed a series of L 1EN site and deletion mutants that were tested in a variety of in vitro DNA cleavage and binding assays and in vivo assays by our collaborators. Several structure of such mutants have been determined by X-ray crystallography. We have shown a 7-residue loop to be important for target site recognition and we have managed to manipulate DNA specificity by loop-exchange mutants. This brings us one step closer to modulate the sequence specificity of L1 endonucleases and could help convert the respective retrotransposon into a genetic tool for i.e. gene therapy. 62:1170-1183 Banci L, Maskos K, Perrakis A, Moras D. First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr D Biol Crystallogr 2006; 62:1208-1217 Berry IM, Perrakis A, Messerschmidt A. SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new Methods for high throughput Structural Biology The end of the EU SPINE project and the start of SPINE-2 marked this year. Many of the methodological contributions of this collaborative research project, in most of which our lab had an active contribution, are summarized in a collection of publications that span diverse but necessary fields for efficient structure determination: Protein (co)-expression in eukaryotic and prokaryotic hosts, crystallization, information data management and computational methods. SPINE (technology development for HTP structural characterization) is dead! Long live SPINE-2 (macromolecular complexes of biomedical interests)! technologies and future aspects. Acta Crystallogr D Biol Crystallogr 2006; 62:1137-1149 Haramis APG, Perrakis A. Selectivity and promiscuity in Eph receptors. Structure 2006; 14:169-171 Mamely I, Van Vugt MA, Smits VA, Semple JI, Lemmens B, Perrakis A, Our lab keeps coordinating Crystallization research in the BIOXHIT EU program, focusing on automation as well as new concepts. Our activity for devising laboratory information management systems for structural biology is continuing within the PIMS-LIMS European initiative. Medema RH, Freire R. Polo-like Kinase-1 Methods for X-ray crystallography After the successful launch and establishment of ARP/wARP version 6.1 early last year, time for new science to improve our software was available again! Our latest endeavors include the implementation of a Naive Bayess and a Multilayer Perceptron pattern classifier that allow better assignment of sequence information to structures through prior knowledge from the databases; algorithms to build more disordered regions in protein structures; and an вЂ�intelligentвЂ™ decision system or driving better the ARP/wARP modules. Romier C, Ben Jelloul M, Celie PHN, Controls Proteasome-Dependent Degradation of Claspin during Checkpoint Recovery. Curr Biol 2006; 16:1950-1955 Christodoulou E, De Marco V, Van Gerwen S, Cohen SX, Perrakis A. Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 2006; 62:1232-1242 32 CELLULAR BIOCHEMISTRY III D IV IS ION OF CELLU LA R B IO CHEM IS TR Y LIPID GROWTH FACTORS AND THEIR ROLE IN DEVELOPMENT AND CANCER Division head, Group leader Wouter Moolenaar Wouter Moolenaar PhD Group leader Nicolai Savaskan PhD Post-doc Catelijne Stortelers PhD Post-doc Anna Houben MSc Graduate student Laurens Van Meeteren MSc Graduate student Bas Ponsioen MSc Graduate student Leonie Van Zeijl MSc Graduate student Trudi Hengeveld Technical staff Adrian Rzadkowski Technical staff Our group has a longstanding interest in lipid growth factors, particularly lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S 1P), their signaling properties and their role in health and disease. LPA and S 1P signal through specific G protein-coupled receptors to stimulate cell migration, proliferation and survival. LPA receptor signaling has been implicated in wound healing, embryonic development and cancer. LPA is produced by a secreted lysophospholipase D (lysoPLD), named autotaxin (ATX), an autocrine motility factor for tumor cells and implicated in tumor progression and angiogenesis. Our current research focuses on ATX, the regulation of its activity as well as its role in embryonic development and tumor progression. We found that ATX knockout mice die at midgestation, apparently due to defective vascular development. In addition, we have developed sensitive ATX activity assays, which have successfully been used for high-throughput screening of small-molecule compound libraries. Since ATX is found overexpressed in certain cancers and LPA promotes cancer progression, ATX qualifies as a potential target for therapy. Our work should lead to novel ways of interfering with LPA receptor signaling in tumor cells and with inappropriate LPA production in their microenvironment. A second line of research is devoted to the regulation of cell-cell communication via gap junctions, which is often defective in tumor cells. LPA receptor signaling: gene transcription To examine how LPA receptor signaling influences global gene transcription, we subjected mouse embryonic fibroblasts to microarray analysis using 32K oligo-arrays. About 900 PA-regulated genes could be clustered into groups based on their temporal patterns and biological functions. We find that LPA induces many genes encoding secreted factors, including growth factors, pro-angiogenic factors, pro-inflammatory cytokines and metalloproteases, as confirmed by quantitative RT-PCR. We are currently analyzing the upstream receptor-linked signaling pathways and to what extent the LPAassociated gene signature shows overlap with that induced by peptide growth factors. These studies highlight the importance of LPA in generating fibroblast-derived mediators of wound healing, angiogenesis and tumor progression. Figure III.1: Abnormalities in ATX-deficient embryos compared to heterozygous embryos. Homozygous knockouts die at midgestation due to severe vascular defects in yolk sac (A) and embryo proper (B; arrows; note also defective closure of the neural tube). Knockout embryos show severe malformation and dorsal effusions (C). Autotaxin, a secreted lysophospholipase D implicated in vascular development and tumor progression Regulation of ATX and discovery of small-molecule inhibitors ATX is highly expressed in brain as well as in malignant glioblastoma cells. In adult rat brain, ATX was detected mainly in leptomeningeal cells and oligodendrocyte precursor cells, but not in mature neurons. Following lesion-induced neurotrauma, ATX is strongly upregulated in reactive astrocytes adjacent to the lesion, suggesting a role for the ATX-LPA axis in brain injury. ATX is unique among the ecto-nucleotide phosphodiesterases in that it primarily functions as a lysoPLD, using a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters. We recently discovered that ATX is potently inhibited by LPA and S 1P in a mixed-type manner. Thus, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment. Furthermore, these results reveal a novel role for S 1P as an inhibitor of ATX, in addition to its well-established role as a receptor ligand. To identify non-lipid inhibitors of ATX activity, we have performed high-throughput screening of chemical compound libraries (in collaboration with David Egan and Huib Ovaa). Screening of a 20,000 small-molecule library yielded several positive вЂњhitsвЂќ that inhibit ATX activity as well as ATX-mediated tumor cell migration by more than 70% (see the report by Huib Ovaa). Among these hits are several new chemical structures with IC(50) values in submicromolar range. Specificity, selectivity and toxicity of these pharmacological inhibitors are now under investigation and will be further evaluated in cell-based and animal studies. 33 CELLULAR BIOCHEMISTRY Publications ATX in vascular development and tumor progression ATX is widely expressed, but its in vivo role is unknown. We find that ATX-deficient mice die at midgestation (E9.5) with profound vascular defects in yolk sac and embryo. Additional abnormalities in the ATX-deficient embryos include allantois malformation, neural tube defects, dorsal effusions and asymmetric headfolds. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development and indicate that ATX is the major LPA-producing enzyme in vivo. The ATX-deficient phenotype is reminiscent of the Ga13 knockout. Since LPA receptors couple strongly to Ga13 the ATX-deficient phenotype might be explained by loss of LPA signaling through Ga13. Ex vivo yolk sac studies are now being done in attempt to explain the ATX-deficient phenotype in further mechanistic detail. Having shown that ATX is essential for vascular development, our next step is to establish the importance of ATX in metastasis and tumor angiogenesis, making use of various cancer-prone mouse models. Furthermore, by deleting ATX in specific tissues we hope to learn more about ATX function in adult life and to uncover the origin of ATX in the circulation. Newly discovered small-molecule inhibitors of ATX will be tested in experimental animals for their anti-tumor potential. Clark K, Langeslag M, Van Leeuwen B, Ran L, Ryazanov AG, Figdor CG, Moolenaar WH, Jalink K, Van Leeuwen FN. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J 2006; 25:290-301 Ferguson CG, Bigman CS, Richardson RD, Van Meeteren LA, Moolenaar WH, Prestwich GD. Fluorogenic Phospholipid Substrate to Detect Lysophospholipase D/Autotaxin Activity. Org Lett 2006; 8:2023-2026 Langeslag M, Clark K, Moolenaar WH, Van Leeuwen FN, Jalink K. Activation of TRPM7 Channels by Phospholipase Regulation of gap junctional communication Direct cell-cell communication through gap junctions is essential for coordinated cell behavior; it is usually defective in tumor cels. Gap junctions consist of clustered connexin subunits that form cell-to-cell channels for small molecules, with connexin43 (Cx43) being the most widespread connexin. In fibroblasts, Cx43 gap junctional communication is inhibited by LPA and other G-protein-coupled receptor agonists, such as endothelin, thrombin and ATP. The mechanism underlying G protein-regulated Cx43 gating has remained elusive to date. Our studies reveal that the closure and reopening of Cx43 channels is tightly controlled by phosphatidylinositol 4,5-bisphosphate (PIP2). Phospholipase C (PLC)-mediated depletion of PIP2 results in Cx43 channel closure, whereas knockdown of PLC or overexpression of the PIP2-synthesizing enzyme, phosphatidylinositol-4-phosphate 5-kinase, keeps Cx43 channels open. Similarly, knockdown of the Cx43-associated scaffold protein ZO-1 renders gap junctional communication refractory to receptor stimulation. Our results identify PIP2 as a key regulator of Cx43 gap junctional communication, and suggest that ZO-1 serves to assemble PLC and Cx43 into a signaling complex to allow exquisite regulation of cell-cell coupling by localized changes in PIP2. C-coupled Receptor Agonists. J Biol Chem 2007; 282:232-239 Ponsioen B, Van Zeijl L, Moolenaar WH, Jalink K. Direct measurement of cyclic AMP diffusion and signaling through connexin43 gap junctional channels. Exp Cell Res 2007; 313:415-423 Savaskan NE, Rocha L, Kotter MR, BГ¤r A, Lubec G, Van Meeteren LA, Kishi Y, Aoki J, Moolenaar WH, Nitsch R, BrГ¤uer AU. Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation during development and after neurotrauma. Cell Mol Life Sci 2007;Online publication Sayas CL, Ariaens A, Ponsioen B, Moolenaar WH. GSK-3 Is Activated by the Tyrosine Kinase Pyk2 during LPA1mediated Neurite Retraction. Mol Biol Cell 2006; 17:1834-1844 Van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, Van Rooijen MA, PradГЁre JP, Pettit TR, Wakelam MJO, Saulnier-Blache JS, Mummery CL, Moolenaar WH, Jonkers J. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Mol Cell Biol 2006; 26:5015-5022 Van Rossum AGSH, Moolenaar WH, Schuuring E. Cortactin affects cell migration by regulating intercellular adhesion and cell spreading. Exp Cell Res 2006; 312:1658-1670 34 CELLULAR BIOCHEMISTRY LIPID METABOLISM IN SIGNAL TRANSDUCTION Group leader Wim Van Blitterswijk Wim Van Blitterswijk PhD Group leader Marcel Verheij MD PhD Academic staff Menno Van Lummel PhD Postdoc Membrane lipids not only have a structural function but also can serve as signaling molecules. Our current studies focus on the production, function and conversion of three types of lipids. First, the sphingolipids that associate with cholesterol in microdomains known as lipid rafts. Biosynthesis of the raft constituent sphingomyelin (SM) plays a crucial role in the sensitivity of tumor cells to undergo apoptosis by alkyl-lysophospholipids and other stimuli. Short-chain sphingolipids can enhance the cellular uptake of the anti-cancer agent doxorubicin. Two other lipids, diacylglycerol (DAG) and phosphatidic acid (PA), can function as second messengers and are the respective substrate and product of diacylglycerol kinases. The latter enzymes are the subject of our ongoing research. Jurgen Van Baal PhD Postdoc Alrik Los MSc Graduate student John De Widt Technical staff Shuraila Zerp Technical staff Jeffrey Klarenbeek Technical staff Sphingomyelin synthase 1 determines tumor cell sensitivity to alkyllysophospholipid and other apoptotic stimuli Synthetic alkyl-lysophospholipids such as Edelfosine (ALP; 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine) and Perifosine (a piperidine analog) induce apoptosis in tumor cells, such as S49 lymphoma; they are used as anti-cancer agents in the clinic. ALP rapidly inserts into the plasma membrane, interferes with lipid signaling, accumulates in lipid rafts and then undergoes endocytosis. Prolonged culturing of S49 cells in the presence of ALP resulted in ALP-resistance (S49AR cells) and, intriguingly, cross-resistance to other apoptotic stimuli, such as DNA damage (gв€’radiation, etoposide) and Fas/CD95 death-receptor activation. Resistant cells were devoid of the raft constituent sphingomyelin (SM) due to downregulated expression of SM synthase (SMS 1). SiRNA-induced suppression of SMS 1 in S49 cells blocked SM synthesis, causing resistance to all apoptotic stimuli. The resistance to Fas ligation was overcome by exogenous short-chain SM, which enhanced Fas surface expression, and by a proteasome inhibitor that prevented Fas degradation. Resistance to ALP and DNA damage was due to lack of SMS 1 activity, not to the mere lack of SM content. SMS 1 activity is important for raft formation and membrane vesicular trafficking. Inhibition of Nieman-Pieck-C1 protein-dependent vesicular transport of cholesterol from endosomes to the Golgi, blocked apoptosis and SM synthesis (Figure III.2). We propose that ALP internalization via rafts represents the retrograde route of vesicular recycling, and that the resistance to ALP and DNA damage relates to a defect in recycling. SMS 1 deficiency was accompanied by downregulation of SHIP 1, a PIP3 phosphatase. This resulted in upregulation of the PI3-kinase/PKB survival pathway. We are now examining how apoptotic cross-resistance relates to SMS 1- and SHIP 1deficiency. For clinical applications of ALP-related lipids, see the report by M Verheij, Division IX, Radiotherapy. Short-chain glucosylceramide facilitates uptake of liposomal doxorubicin by tumor cells in vitro and in vivo The anti-cancer agent doxorubicin (DOX) is often administered as a liposomal formulation (CaelyxВ®), from which it is gradually released into the interstitial space and then taken up by tumor cells. We discovered that short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) accelerate the cellular uptake of DOX. GC also enhanced DOX accumulation in A431 carcinoma cells. We tested these liposomes in nude mice that were subcutaneously inoculated with A431 cells. In a dose-escalation study, the anti-tumor efficacy of GC-modified liposomal DOX was superior to standard liposomal DOX (Figure III.3). This new liposomal formulation of DOX, which we have patented, undergoes further testing and seems promising for clinical application. Figure III.2: Biosynthesis of sphingomyelin (SM) in the Golgi is blocked when vesicular transport of cholesterol from endosomes to the Golgi is abrogated by U18666A (2 Вµg/ml). Radiolabeled lipids were extracted and separated by thin-layer chromatography. PC, phosphatidylcholine; LPC, lysoPC. Diacylglycerol kinases (collaboration with N Divecha). Diacylglycerol kinase (DGK) phosphorylates the second messenger DAG, activator of protein kinase C (PKC), to yield PA. We have identified DGKВЈ and reported on its translocation to the plasma membrane in response to noradrenaline. We investigated how the localization of DGKВЈ is controlled by G protein-coupled receptors. In A431 cells, hormone-induced activation of PKCe and -h regulated the translocation of DGKВЈ to the plasma membrane. DGKВЈ translocation required additional tyrosine kinase activity of the EGF receptor. Furthermore, during this process there was physical interaction and 35 CELLULAR BIOCHEMISTRY Publications colocalization of DGKВЈ with the EGF receptor. Expression of DGKВЈ inhibited bradykinin-induced, PKC-mediated Thr654 phosphorylation of the EGF receptor, whereas knockdown of DGKВЈ increased phosphorylation. Our results suggest that G protein-coupled receptor-mediated DGKВЈ translocation can modulate PKC-mediated events. Los AP, De Widt J, Topham MK, Van Blitterswijk WJ, Divecha N. Protein kinase C inhibits binding of diacylglycerol kinase-[zeta] to the retinoblastoma protein. Biochimica et Biophysica Acta (BBA) Molecular Cell Research 2007; In Press Los AP, Vinke FP, De Widt J, Topham MK, Van Blitterswijk WJ, Divecha N. The Figure III.3: N-octanoyl-glucosylceramide (GC) Retinoblastoma Family Proteins Bind to incorporated in doxorubicin-containing liposomes and Activate Diacylglycerol Kinase{zeta}. (GC-DOX) results in more effective reduction of J Biol Chem 2006; 281:858-866 tumor size in vivo compared to regular liposomes (CaelyxВ®) (DOX). Nude mice bearing A431 Van der Luit AH, Budde M, Zerp S, tumors received 6 mg liposomal DOX/kg Caan W, Klarenbeek JB, Verheij M, bodyweight intravenously, after which tumor size Van Blitterswijk WJ. Resistance was measured daily. to alkyl-lysophospholipid-induced apoptosis due to downregulated The DGKz isotype localizes to the nucleus, where it binds the retinoblastoma protein pRb. DGKz interacts with pRb in vitro and in vivo. PKC-mediated phosphorylations of DGKz negatively regulated DGKz binding to pRB. DGKz did not affect the ability of pRB to regulate transcription; however, pRB stimulated DGKz activity in vitro. Overexpression of DGKz in pRB-null fibroblasts reconstituted a cell cycle arrest induced by g-irradiation. pRB-negative cells show increased Ras activity, suggesting that DGKz may stimulate cell cycle arrest by inhibiting Ras signaling. sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesteroldeficiency in lipid rafts. Biochem J 2007; 401:541-549 Vink SR, Lagerwerf S, Mesman E, Schellens JHM, Begg AC, Van Blitterswijk WJ, Verheij M. pRB is known to regulate muscle differentiation. In C2C12 myoblasts, DGKz enhanced the expression of muscle-specific genes. Microarray analysis revealed that DGKz increased expression of diverse muscle genes, especially targets of the myogenic transcription factor MyoD. Indeed, DGKz stimulated promoter activity of the MyoD target genes. DGKz knock-out cells were unable to differentiate or to express muscle-specific genes. Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts. Clin Cancer Res 2006; 12:1615-1622 Vink SR, Van Blitterswijk WJ, Schellens JHM, Verheij M. Rationale and clinical application of alkylphospholipid analogues in combination with radiotherapy. Cancer Treatment Rev 2007; in press. Zerp SF, Vink SR, Ruiter GA, Koolwijk P, Peters E, Van der Luit AH, De Jong D, Budde M, Bartelink H, Van Blitterswijk WJ, Verheij M. Alkylphospholipids inhibit capillary-like endothelial tube formation in vitro: Antiangiogenic properties of a new class of antitumor agents. Anti-Cancer Drugs 1997; in press. 36 CELLULAR BIOCHEMISTRY SIGNALING THROUGH INOSITOL PHOSPHOLIPIDS INTRODUCTION Group leader Nullin Divecha Nullin Divecha PhD Group leader Jonathon Halstead PhD Post-doc David Jones PhD Post-doc Jurgen Van Baal PhD Post-doc Alrik Los MSc Graduate student Cells constantly experience various forms of cellular stress such as hypoxia and oxidative imbalance, which can lead to genetic changes and eventually to the onset of tumorigenesis. Cells therefore have a myriad of mechanisms which sense cell stress and induce processes to counteract them. In extreme cases elimination (apoptosis) of the cell benefits the whole organism. Furthermore the process of tumorigenesis inevitably leads to the selection of cells with upregulated signalling pathways that can confer increased resistance to cell stress, increased proliferation in the absence of extracellular hormones and an increase in the ability of cells to migrate. We are interested in the role of phosphoinositides, a family of phospholipid second messengers, in the regulation of cellular stress pathways and the process of tumorigenesis. Willem-Jan Keune MSc Graduate student Dalila Elouarrat MSc Graduate student Yvette Bultsma Ing Technician Mireille Snel Ing Technician Figure III.4: The figure shows that PIP4KII-beta is phosphorylated at five different sites (P) and is acetylated at nine different lysine residues (Ac). We generated antibodies specific for phosphorylated S326 and showed that S326 becomes phosphorylated in a stress (UV) dependent manner (Inset top panel). RNAi-mediated suppression of p38 map kinase expression (pRetro-RNAi p38) showed that s326 phosphorylation in response to UV irradiation was dependent on p38 activity (inset). Antibodies to other phosphorylation and acetylation sites have been generated and are being to used to study their role in vivo. Phosphoinositides and Stress induced signalling PIP4KII-beta is a nuclear lipid kinase that is able to phosphorylate the phosphoinositide PtdIns5P. PIP4KII-beta is upregulated in breast tumours and is a component of a stress pathway that can ultimately impinge on the function of the human tumour suppressor protein p53. Upon exposure to various cellular stressors PIP4KII-beta regulates the levels of nuclear PtdIns5P. How stress dependent changes in nuclear PtdIns5P are translated into cellular phenotypes is beginning to be unravelled. Plant Homology Domains (PHD) are zinc fingers that are able to interact with phosphoinositides and are present in many proteins that are involved in regulating gene transcription, DNA methylation and DNA repair. The PHD containing protein, Inhibitor of growth-2 (ING2), interacts with PtdIns5P and can regulate numerous nuclear functions through its interaction with chromatin remodelling complexes and histone acetylases and deacetylases. We now show that the intra-nuclear localisation of ING2 is subject to control by cellular stress dependent changes in nuclear PtdIns5P and is an in vivo sensor for changes in nuclear phosphoinositides. To understand how the level of nuclear PtdIns5P is controlled we have identified numerous stress dependent changes in post-translational modifications of PIP4KIIbeta using mass spectrometry (in collaboration with Prof. AJR Heck, Department of Biomolecular Mass Spectrometry Utrecht and Dr C DвЂ™Santos, Probe Bergen Norway). PIP4KII-beta is phosphorylated at five different positions and acetylated at nine different lysine residues. We have generated antibodies specific to two of these phosphorylation sites and show that one of them, serine 326 (S326), is phosphorylated in response to cellular stress by the stress dependent protein kinase p38 (Figure III.4). Furthermore phosphorylation negatively regulates PIP4KII-beta activity and modulates its interaction with other proteins. How these various posttranslational modifications are coordinated into regulation of PIP4KII-beta activity and localisation is under investigation. PtdIns(4,5)P 2 : a role in regulating stress induced apoptosis How apoptotic pathways are engaged in response to cell stress is not well characterized. We have found that different types of cell stress lead to a rapid and irreversible decrease in the cellular level of PtdIns(4,5)P2. PtdIns(4,5)P2 is a key intracellular signaling lipid and is required for the regulation of many important cellular pathways including cell survival, vesicle trafficking and growth factor signaling. Stress induced decrease in PtdIns(4,5)P2 occurs in part via the delocalisation from the plasma membrane and catalytic inactivation of PIP5K, an enzyme that synthesizes PtdIns(4,5)P2. Importantly overexpression of PIP5K prevents the decrease in PtdIns(4,5)P2 and attenuates the induction of apoptosis in response to various stressors. We postulate that cells constantly monitor their PtdIns(4,5)P2 levels and that an irreversible decrease in PtdIns(4,5)P2, induced by cellular stress, may act as a trigger to initiate apoptosis. Interestingly, tumors often show enhanced PIP5K activity, which may be a mechanism to avoid stress induced apoptosis. How increased PtdIns(4,5)P2 synthesis attenuates stress induced apoptosis is not clear, however we have found that overexpression of PIP5K stimulates and maintains activation of the p42/44 mapkinase signaling pathway after cellular stress induction. The p42/44 37 CELLULAR BIOCHEMISTRY Publications mapkinase is intimately involved in stimulating both cell survival and proliferation. Further studies will define how PtdIns(4,5)P2 can regulate Map kinase signaling. Bins A, Van Rheenen J, Jalink K, Halstead J, Divecha N, Spencer D, PIP5Ks and cytoskeletal dynamics The ability to migrate from their natural environment and to invade other tissues is a hallmark of metastatic cancer cells and its onset often defines a poor prognosis for the patient. Understanding signal transduction pathways involved in migration is of the utmost importance in defining novel anti cancer therapeutics. A key player in migration is the small molecular weight G protein Rac, which regulates cytoskeletal dynamics. Rac can interact with PIP5K although the consequence of this interaction is not clear. We have defined specific regions of PIP5K-alpha that interact with Rac and have established a mutant of PIP5K that no longer interacts with Rac (PIP5K-RacM). Interestingly this mutant no longer localizes to the membrane and is also compromised in its ability to synthesize phosphoinositides in vivo, but not in vitro. We have previously demonstrated that PIP5K is required for inducing changes in neuronal morphology in response to numerous signaling receptors such as lysophosphatidic acid (LPA). We have used this cell system to demonstrate that both PtdIns(4,5)P2 synthesis and interaction with Rac are required for remodeling of focal adhesions complexes by PIP5K (Figure III.5). Haanen J, Schumacher T. Intravital imaging of fluorescent markers and FRET probes by DNA tattooing. BMC Biotechnology 2007; 7:2 Guillou H, Lecureuil C, Anderson KE, Suire S, Ferguson GJ, Ellson CD, Gray A, Divecha N, Hawkins PT, Stephens LR. Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach. J Lipid Res 2006;D600038-DJLR200 Halstead JR, Van Rheenen J, Snel M, Meeuws S, Mohammed S, DвЂ™Santos CS, Heck A, Jalink K, Divecha N. A Role for PtdIns(4,5)P2 and PIP5Ka in Regulating Stress-Induced Apoptosis. Curr Biol 2006; 16:1850-1856 Jones DR, Bultsma Y, Keune WJ, Halstead JR, Elouarrat D, Mohammed S, Heck AJ, DвЂ™Santos CS, Divecha N. Nuclear PtdIns5P as a Transducer of Stress Signaling: An In Vivo Role for PIP4Kbeta. Mol Cell 2006; 23:685-695 Figure III.5: Neuronal cells (N1E-115) were transfected with either a control vector or a mutant of PIP5K unable to interact with Rac (PIP5K-RacM) or a Kinase inactive PIP5K (PIP5K-KD) which is unable to Los AP, De Widt J, Topham MK, generate PtdIns(4,5)P2 and acts to inhibit morphological changes in cells in response to LPA. Cells were Van Blitterswijk WJ, Divecha N. Protein maintained as controls (control) or stimulated with LPA. The cells were then fixed after 15 minutes and kinase C inhibits binding of diacylglycerol stained for the focal adhesion protein vinculin (shown as white areas). Stmulation with LPA leads to a kinase-[zeta] to the retinoblastoma protein. rapid dissolution of vinculin from focal adhesion complexes, which is blocked by the expression of both BBA Mol Cell Res 2006; In Press PIP5K mutants. This suggests that both PtdIns(4,5)P2 synthesis and the interaction of PIP5K with Rac is required for regulating focal adhesion disassembly. Vermeer JEM, Van Leeuwen W, Tobena-Santamaria R, Laxalt AM, Our demonstration of the role of PIP5K in regulating apoptosis and cytoskeletal dynamics has led to the development of a targeted screen for small molecule inhibitors of PIP5Ks. We hope that in the future these inhibitors may be useful in cancer therapy. Jones DR, Divecha N, Gadella TW, Jr., Munnik T. Visualization of PtdIns3P dynamics in living plant cells. Plant J 2006; 47:687-700 Weinkove D, Halstead JR, Gems D, Divecha N. Long-term starvation and ageing induce AGE-1/PI 3-kinase-dependent translocation of DAF-16/FOXO to the cytoplasm. BMC Biology 2006; 4:1 38 CELLULAR BIOCHEMISTRY ORGANIC SYNTHESIS AND CHEMICAL BIOLOGY Group leader Huib Ovaa Huib Ovaa PhD Group leader Danny Burg PhD Post-doc The organic synthesis and chemical biology laboratory at the Netherlands Cancer Institute specializes in the design, synthesis and development of diagnostic-, imaging- and proteomics tools. The group has extensive experience in organic synthesis, intein chemistry, expressed protein ligation, mass spectrometry, protein purification, fluorescence-based techniques, FACS analysis, confocal microscopy and various standard biochemical techniques. Research is centered around one central theme: chemical biology of protein degradation and antigen presentation, and divided into three different topics: Silvia Cavalli PhD Post-doc Boris Rodenko PhD Post-doc Fijs Van Leeuwen PhD Post-doc Erica Van Tilburg PhD Post-doc Topic 1: Ubiquitin proteomics Topic 2: Proteasome activity Topic 3: Antigen presentation Celia Berkers MSc Graduate student Rieuwert Hoppes MSc Graduate student Henk Hilkmann Technical staff Publications Crawford LJA, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TCM, Irvine AE. Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132. Cancer Res 2006; 66:6379-6386 Groll M, Berkers CR, Ploegh HL, Ovaa H. Crystal Structure of the Boronic Acid-Based Proteasome Inhibitor Bortezomib in Complex with the Yeast 20S Proteasome. Structure 2006; 14:451-456 Hol EM, Fischer DF, Ovaa H, Scheper W. Ubiquitin proteasome system as a The group develops techniques to profile and interfere with enzymatic activities associated primarily with ubiquitin-mediated proteasomal degradation and antigen presentation and uses an organic synthesis and mass spectrometry driven approach in order to gain further understanding of the biochemical processes under investigation. Antigen production and antigen presentation by MHC complexes leading to T cell activation T lymphocytes serve to recognize and eliminate cells that express foreign (e.g. virus-derived or tumor-specific) proteins. The recognition of antigen presenting cells is based on the binding of the T cell receptor to MHC complexes that have bound a peptide derived from a foreign protein (Figure III.6). Major Histocompatibility Complexes (MHCs) can be divided into MHC class I and class II complexes that are specialized to present antigens on the cell surface originating from different sources. Whereas the class II pathway is responsible for the presentation of extracellular proteins, the class I pathway presents endogenously synthesized antigens, e.g. viral protein fragments, sequestered within the infected cell, although some crosstalk between these pathways exists. Recognition of displayed pMHC class I complexes by cytotoxic T cells leads to death of the antigen-expressing cell whereas pMHC class II complexes are recognized by helper T cells, resulting in immune activation. We currently study the loading of MHC class I molecules and MHC class I binding to T cells. pharmacological target in neurodegeneration. Expert Rev Neurother 2006; 6:1337-1347 Kraus M, Ruckrich T, Reich M, Gogel J, Beck A, Kammer W, Berkers CR, Burg D, Overkleeft H, Ovaa H, Driessen C. Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells. Leukemia 2007; 21:84-92 Figure III.6: MHC class I-mediated immunity: antigen processing, presentation and T cell activation. The Risseeuw MDP, Berkers CR, Ploegh HL, proteasome is responsible for protein degradation, assisted by other (endo)-proteolytic activities, affording Ovaa H. Synthesis of tritium labeled KRN7000 epitopes that can be loaded into MHC class I complexes. Recognition of the presented pMHC complex by Tetrahedron Lett 2006; 47:3677-3679 the TCR results in a T cell-mediated kill of the antigen presenting cell. Rodenko B, Toebes M, Hadrup SR, Ubiquitin-mediated protein degradation by the proteasome Ubiquitin modification is a post-translational modification at the core of proteasome-mediated protein degradation. The C-terminal residue of Ub is conjugated most often to the e-amino group of a lysine residue of another protein forming a ubiquitin polymer linked through isopeptide bonds. These polymers regulate proteasome-mediated destruction and hence stability of most proteins. The action of Ub specific ligases is counterbalanced by the action of Ub specific proteases (USPs) similar to the way phosphorylation, mediated by the action of kinases, is counterbalanced by the action of phosphatases. Malfunction of individual members of these enzyme classes are Van Esch WJE, Molenaar AM, Schumacher TNM, Ovaa H. Generation of peptide-MHC class I complexes through UVmediated ligand exchange. Nat Protocols 2006; 1:1120-1132 39 CELLULAR BIOCHEMISTRY Publications (continued) known to cause disease, which underscores the importance of assessing actual catalytic activity profiles of USPs in different cell types and in cells corresponding to discrete stages of differentiation, activation, and malignant transformation. A variety of USPs act either as oncoprotein or as tumor suppressor protein and USPs also play important regulatory roles by determining the conjugation status of substrate proteins. The combined action of ligases and proteases is responsible for remodeling the shape and size of вЂњtreesвЂќ such as seen in polyubiquitin chains, in which Ub and probably ubiquitin-like molecules are conjugated onto other Ub molecules via isopeptide bonds to form a polymer. Size, shape and composition of such trees likely determine the half-life of a protein. We develop tools that can provide information on the specificity, activity and identity of factors involved in ubiquitinmediated protein degradation. Studies aimed at a functional characterization of proteolytic activity profiles under different physiological and pathological conditions have been boosted by the development of such probes. RolГ©n U, Kobzeva V, Gasparjan N, Ovaa H, Winberg G, Kisseljov F, Masucci MG. Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines. Mol Carcinog 2006; 45:260-269 Toebes M, Coccoris M, Bins A, Rodenko B, Gomez R, Nieuwkoop NJ, Van de Kasteele W, Rimmelzwaan GF, Haanen JBAG, Ovaa H, Schumacher TNM. Design and use of conditional MHC class I ligands. Nat Med 2006; 12:246-251 Conditional MHC class I ligands In collaboration with the Schumacher lab (Division of Immunology) we recently developed a chemical strategy to load MHC class I molecules on command. This strategy takes advantage of epitopes that can be triggered to fragment, by UV irradiation (Figure III.7). Resulting in in situ-generated peptide-receptive protein complexes that can be readily loaded with appropriate ligands, enabling broad epitope scans. This strategy will be used in the rapid identification of both MHC ligands and cytotoxic T cell responses and the value of this strategy was demonstrated by the definition of an avian flu epitope. Figure III.7: Principle of MHC class I exchange: A UV cleavable MHC ligand is triggered to fragment, in situ generating an unstable, peptide receptive MHC molecule. In the presence of a rescue peptide, an efficient ligand exchange takes place allowing the generation of large collections of MHC complexes. High throughput small molecule screening In collaboration with the Dr David Egan (Division of Molecular Carcinogenesis) and the group of Wouter Moolenaar we recently completed the first high throughput small molecule screen carried out so far at an academic institution in the Netherlands. This screen was carried out to find inhibitors of autotaxin (ATX), a widely expressed phosphodiesterase that promotes tumor cell motility, experimental metastasis and angiogenesis, leading to the discovery of a series of low nanomolar ATX inhibitors. Several other small molecule screens are planned for next year. Verdoes M, Berkers CR, Florea BI, Van Swieten PF, Overkleeft HS, Ovaa H. Chemical Proteomics Profiling of Proteasome Activity. In: Nedelkov D, Nelson RW, editors. New and Emerging Proteomic Techniques. Totowa, NJ: Humana Press, 2007: 51-70 40 CELLULAR BIOCHEMISTRY EPIGENETIC REGULATION OF GENE EXPRESSION Group leader Fred van Leeuwen Fred Van Leeuwen PhD Group leader Alex Faber PhD Post-doc Floor Frederiks MSc Graduate student In eukaryotic cells the DNA is packaged into chromatin by histone proteins. Posttranslational modifications of the histone proteins and methylation of DNA can result in heritable changes in gene expression without changes in the actual genetic code. These epigenetic changes have been shown to contribute to tumorigenesis. The mechanisms by which epigenetic imprints are established or prevented are still poorly understood. Many chromatin modifiers are conserved from yeast to humans. Our group uses the budding yeast Saccharomyces cerevisiae as a powerful model system to identify new epigenetic regulators and to unravel the molecular mechanisms by which chromatin-modifying enzymes affect chromatin structure and gene expression. Kitty Verzijlbergen MSc Graduate student Tibor Van Welsem Technical staff Figure III.8: Telomeric silencing in budding yeast; red (dark) and white (light) sectors in a yeast colony indicate epigenetic inheritance of active and silent chromatin. Figure III.9: Interdependence between active and silent chromatin. Histone H3 acetylation (ac) and methylation (me) in active chromatin introduced by methyltransferases (MT) and histone acetyltransferases (HAT) affect assembly of the silencing complex (Sir2,3,4) at silent chromatin. Interdepence between active and silent chromatin We are studying how histones and their post-translational modifications help set up and propagate domains of active and silent chromatin. In yeast we previously identified a novel histone methyltransferase Dot1, which methylates lysine 79 on histone H3 (H3K79) on the top and bottom of the nucleosome core. This unusual enzyme is conserved from yeast to humans and has been shown to be involved in certain human leukemias. In yeast Dot1 is critical for gene silencing but is mainly present in active regions of the genome. Our findings suggest that histone H3K79 methylation in active chromatin prevents promiscuous binding of silencing proteins, and thereby enhances targeting of silencing proteins to silent chromatin. Recent results showed that, in addition to histone H3K79, several other histone modifications associated with active regions of the genome positively affect silencing and that they do so by pathways independent of Dot1. This proposes an important general interdependence between the active and silent chromatin domains in the nucleus. The role of histone H3 lysine 79 methylation in gene silencing Dot1 methylates ~90% of histone H3 in yeast and can add one, two, or three methyl groups to its target lysine. In general, different forms of lysine methylation tend to have specific localizations or functions in chromatin. We recently isolated a mutant of Dot1 that lacks the ability to trimethylate H3K79 in vivo or in vitro. Characterization of this mutant showed that trimethylation of H3K79, which is present on as much as 50% of all histone H3, is not critical for silencing. Our data suggest that the overall degree of H3K79 methylation rather than the amount of one specific methyl form determines the degree of gene silencing. We are currently running genome-wide genetic screens to determine the pathways and molecular mechanisms by which H3K79me affects chromatin organization. An assay to study the role of histones and their modifications in cellular memory One of the main goals of our group is to understand how chromatin modifications can have long-term effects on gene expression. When a cell divides, parental histones (containing the epigenetic marks) and newly synthesized histones (unmodified or in a ground state) are somehow assembled onto the daughter DNA strands in a manner that faithfully reproduces the transcriptional states of chromatin that existed prior to chromosome duplication. It is thought that the post-translational modifications on histones can serve as epigenetic memory marks during cell division. However, the exact mode of histone inheritance is still unclear and recent studies have shown that chromatin can be very dynamic. We have developed a novel assay in yeast to determine protein turnover and protein-complex dynamics in vivo. The assay involves differential labeling of existing and newly synthesized proteins. We are now applying this assay to determine how parental histones and newly synthesized histones and their post-translational modifications are assembled into chromatin. 41 IMMUNOLOGY IV DI VI S I ON OF IMMU NO LOG Y LYMPHOCYTE ACTIVATION AND SURVIVAL Our interest is to determine how cells decide between living and dying. We focus on the mechanism of action of TNF receptor family members, since these govern such decisions. Lymphocytes are our main cell type of interest, since throughout their existence, they mostly live вЂњon the edgeвЂќ between life and death. Our work is inspired by the desire to improve cancer immunotherapy. Sustaining survival of activated lymphocytes is expected to improve anti-tumor immunity by enlarging the tumorspecific lymphocyte pool and by enhancing long-term maintenance of tumor-specific lymphocytes (immunological memory). The second goal of our work is to contribute to the design of novel therapies aimed at killing cancer cells by activating apoptotic pathways. Division head, Group leader Jannie Borst Jannie Borst PhD Group leader Sabine Middendorp PhD Post-doc Esther Wissink PhD Post-doc Anna Keller MSc Graduate student Chiel Maas MSc Graduate student Victor Peperzak MSc Graduate student Elise Veraar MSc Graduate student Inge Verbrugge MSc Graduate student Evert De Vries Technical staff TNF receptor family members and control of the immune response From our work, TNF receptor family member CD27 and its ligand CD70 have emerged as interesting targets to improve anti-tumor immunity. This costimulatory system promotes generation and maintenance of effector and memory T cells, which is accomplished at least in part by rescue of activated T cells from apoptosis. To determine by which molecular mechanisms CD27 directs the T cell response, we use as a prime approach micro-array analysis of CD27-induced gene expression. We employ an in vitro system of T cell stimulation, but are also able to analyze expression profiles of T cells taken ex vivo from mice that are undergoing an immune response. Findings are corroborated at the protein level and by functional assays. In this way, we have determined that one of the key effects of CD27 is directing the synthesis of and responsiveness to certain pro-survival cytokines. Its anti-apoptotic effect seems to be primarily indirect, via these cytokines. We have also obtained the novel insight that CD27 directs the synthesis of certain chemokines that have been implicated in the recruitment of effector T cells. This ties in with the potential of CD27 to promote accumulation of T cells at tissue sites. We have demonstrated that CD27 improves the potential of CD8+ memory T cells to expand upon secondary stimulation. Imprinting of memory characteristics into CD8+ T cells has been reported to require the help of CD4+ T-cells during the primary response. We have established that CD4+ T cells need CD27 to deliver such help to the memory CD8+ T cell response. By protein and gene expression analysis of CD4+ T cells undergoing an immune response in vivo, we have established that CD27 promotes the capacity of CD4+ T cells to synthesize certain cytokines that have been implicated in CD4+ T cell help. The ligand for CD27, CD70, is expressed on activated lymphocytes and dendritic cells (DC). Recent findings in the field suggest that the functional capabilities of T cells are to a large extent imprinted in the priming phase, during T cell-DC contact. To determine the exact impact on T cell function of CD27/CD70 interactions that take place during this contact, we have made novel genetic tools. In the first place, we have generated transgenic mice constitutively expressing CD70 under control of the DC-selective CD11c promoter. While CD70 is normally acquired during DC maturation, these mice express CD70 on immature DC (Figure IV.1). It is known that the maturation stage of DC is decisive for the T cell response. Whereas immature DC maintain T cell tolerance and give abortive responses to foreign antigen, mature DC that express the full array of costimulatory ligands, induce T cell responsiveness. Interestingly, we have found that the mere expression of CD70 on immature DC suffices to induce T cell expansion to foreign peptide antigen. The activated T cells in addition acquire full functional activity, sufficient to eliminate aggressive tumors. This finding emphasizes the potential of exploiting CD27/CD70 interactions to promote anti-tumor immunity. We have recently generated CD70-deficient mice, which will be used as recipients to further address the potential of CD70 transgenic DC. Gerda Van der Horst Technical staff Figure IV.1: Characterization of CD70 transgenic mice. Flow cytometric analysis indicated constitutive expression of CD70 on immature dendritic cells (CD11c+) of CD70 transgenic (tg), but not wild-type (WT) mice (A Keller, unpublished results). 42 IMMUNOLOGY Publications Aulwurm S, Wischhusen J, Friese M, Borst J, Weller M. Immune stimulatory effects of CD70 override CD70-mediated immune cell apoptosis in rodent glioma models and confer long-lasting antiglioma immunity in vivo. Int J Cancer 2006; 118:1728-1735 Keller AM, Borst J. Control of Peripheral T Cell Survival: A Delicate Division of Labor between Cytokines and Costimulatory Molecules. Hum Immunol 2006; 67:469-477 Ortiz F, Tait SW, Robledo G, De Vries E, Borst J, LГіpez-Rivas A. The mitogenactivated protein kinase pathway can inhibit TRAIL-induced apoptosis by prohibiting association of truncated Bid with mitochondria. Cell Death Differ 2006; 13:1857-1865 Wieland CW, Kerver ME, Florquin S, Nolte MA, Borst J, Van Lier R, Van Oers MHJ, Van der Poll T. CD27 contributes to the early systemic immune response to Mycobacterium tuberculosis infection but does not affect outcome. Int Immunol 2006; 18:1531-1539 Wissink EHJ, Verbrugge I, Vink SR, Schader MB, Schaefer U, Walczak H, Borst J, Verheij M. TRAIL enhances efficacy of radiotherapy in a p53 mutant, Bcl-2 overexpressing lymphoid malignancy. Radiother Oncol 2006; 80:214-222 Figure IV.2: Proof of principle of RNA interference library screen. Barcode analysis identified siRNA constructs enriched in MCF-7 cells selected for resistance to TRAIL-induced apoptosis. Among these was siRNA construct for Caspase-8, a known component of the pathway (C Maas, unpublished results). The cell biology of TNF family members CD70 is a type II transmembrane molecule, like most other ligands of the TNF receptor family. Cell surface expression of CD70 is transient and fully dependent on conditions of immune activation. We have revealed that in cells that contain the machinery for MHC class II-restricted antigen presentation, such as DC, CD70 is sorted exactly like MHC class II to the inner vesicles of late endocytic compartments. In cells that lack MHC class II presentation function, CD70 travels by default to the cell surface. We propose that the specific intracellular trafficking of CD70 provides professional antigen-presenting cells, such as DC, with a mechanism to coordinate CD70 cell surface expression both temporally and spatially with antigen presentation. Indeed, when maturing DC were exposed to specific T cells in the presence of the appropriate antigen, all newly synthesized CD70 protein was routed via MHC class II compartments to the immunological synapse formed between the T cell and DC. This work is carried out in close collaboration with the group of J Neefjes (Division of Tumor Biology). Apoptosis signaling and cancer therapy Death receptors, such as the TRAIL receptors and CD95 are TNF receptor family members that share the ability to induce apoptosis. We aim to further unravel pro-apoptotic signaling by death receptors and to exploit this knowledge for cancer therapy. We have performed a functional genetic screen for mediators of TRAIL-induced apoptosis in MCF-7 breast cancer cells. The complete NKI RNA interference library was transduced into these cells, which were subsequently selected for resistance to TRAIL-induced apoptosis. SiRNA inserts were recovered and their identity was determined by bar code array (collaboration with the Division of Molecular Carcinogenesis). The system has worked reliably and revealed proof of principle, since siRNA for Caspase-8 and Bid were selected (Figure IV.2). A dozen of novel candidate regulators for TRAIL-induced apoptosis have been identified in this way, which will be subject of further functional analysis. After death receptor stimulation, Bid is cleaved by Caspase-8 and its carboxy-terminal fragment subsequently translocates to mitochondria where it induces permeability of the outer membrane and release of pro-apoptotic mediators. After DNA damage, Bid can also be activated, according to our data, but by a different mechanism that is currently under investigation in mouse embryo fibroblasts. We have found that the amino-terminal fragment of Bid, upon its generation by Caspase-8 is ubiquitinated in an unconventional way and subsequently degraded by the proteasome (work of postdoc S Tait, currently in Memphis TN). Extensive combined genetic and biochemical analysis has allowed us to define the region in Bid that is subject to ubiquitination and to reveal the likely target residues. Functional experiments have proven that ubiquitination and consequent proteasomal degradation of the amino-terminal fragment of Bid liberates its carboxy-terminal fragment for apoptosis-induction. In collaboration with M Verheij of the Division of Radiotherapy we carry out a project aimed to exploit death ligands to enhance the efficacy of radiotherapy. As proof of principle, we have used human lymphoid tumor cells. In these cells, radiation (and DNA damaging drugs) can sensitize for TRAIL receptor- and CD95 induced apoptosis when the mitochondrial pathway is blocked and p53 is lacking. Sensitization is not due to death receptor upregulation at the cell surface but appears to entail increased caspase activity, resulting from a higher capacity of engaged receptors to generate pro-apoptotic signals. The efficacy of single and combined treatment was determined in a subcutaneous xenotransplant setting of the lymphoid tumor. Tumor growth and regression was determined by bioluminescence as well as by caliper measurement. For results of this study, the reader is referred to the report of M Verheij in this issue. Our ultimate goal is to test efficacy of combined treatment in solid tumor models, particularly those generated by A Berns and coworkers. These models will be used to explore both TRAIL and a novel formulation of recombinant CD95 ligand in combined modality treatment. 43 IMMUNOLOGY DISSECTING VIRUS AND TUMOR-SPECIFIC T CELL IMMUNITY The aim of our research is straightforward 1) to design novel technologies that can be used to examine and modify antigen-specific T cell immunity 2). To use these tools to unravel and manipulate the molecular processes underlying immune recognition by T lymphocytes. Within these projects, a main focus is on the design and testing of novel concepts for adoptive immunotherapy. Group leader Ton Schumacher In vivo visualization of T cell immunity (collaboration with Haanen lab) Aim of this line of research is to define the rules that control the efficiency of vaccineinduced T cell responses and of effector T cell function, both through in vivo imaging. In the past year, focus of this work has mostly been on the evaluation of the DNA tattoo approach developed in the Haanen lab by confocal laser scanning microscopy and the development of model systems in which in vivo T cell responses can be traced by CLSM. These studies have demonstrated that a combination of DNA tattoo and CLSM can be utilized to monitor cellular processes such as apoptosis in skin. Furthermore, ongoing experiments suggest that it will be feasible to trace anti-viral T cell responses by imaging, using a Herpes Simplex virus skin infection model. In the coming year we will set out to utilize this latter model to visualize how anti-viral T cells combat a viral infection in a physiological setting. Ton Schumacher PhD Group leader Ramon Arens PhD Post-Doc Anna Calogero PhD Post-doc Arne Bakker MSc Graduate student Miriam Coccoris MSc Graduate student Carmen Gerlach MSc Graduate student Jeroen Van Heijst MSc Graduate student Koen Schepers MSc Graduate student Moniek De Witte MD Graduate student Marly Van den Boom Technical staff Jeanine Joling Technical staff Erwin Swart Technical staff MHC exchange technology (collaboration with Ovaa and Perrakis labs) MHC tetramers and other types of MHC multimers have become an essential tool for the analysis of T cell immunity both in animal model systems and in the clinic. However, a major obstacle for the effective application of MHC tetramers is the inefficiency of the classical strategy for MHC generation, and this precludes the generation of large libraries of MHC multimers, or of a collection of clinical grade MHC multimer reagents for cellular therapy. In collaboration with H Ovaa (Division of Cellular Biochemistry) we have set out to address this issue, by designing MHC class I molecules occupied with a general producer peptide that can be exchanged with an antigenic peptide of interest at will. We have now shown by X-ray crystallography that MHC molecules form the intended complex with these designed conditional ligands (collaboration with T Perrakis and P Celie, Division of Molecular Carcinogenesis). Furthermore, we have demonstrated that 1) Recombinant MHC molecules can be produced with designed peptide variants that are sensitive to UV exposure for a variety of MHC alleles 2) MHC complexes created in this manner can then be utilized to screen collection of thousands of potential MHC ligands by fluorescence anisotropy or ELISA. In addition we have begun the development of MHC micro-arrays and technology for clinical grade production of conditional MHC complexes for subsequent use in cellular therapy. One specific area in which we consider the prospects of this type of technology significant is that of allogeneic hematopoietic stem cell transplantation for hematological malignancies. Dissection of T cell immunity through retroviral barcoding The ability to visualize antigen-specific T cell responses and to determine the differentiation pathways of different subsets of T cells is essential for our understanding of pathogen- and vaccine-induced immunity. MHC tetramer technology makes it possible to follow the development of immunity at the T cell population level. However, it doesnвЂ™t allow the analysis of cell fate and cellular differentiation pathways. To address this issue, we have developed an approach in which individual T cells are tagged with a genetic barcode. For this purpose we have created an oncoretroviral and lentiviral library containing some 5,000 different barcodes, and the same set of barcodes has been used to generate a barcode microarray. Infection of cell populations by these libraries of retroviral vectors and subsequent micro-array analysis can then be used to trace the progeny of individual cells. In a set of proof-ofprinciple experiments we have first demonstrated that the technology that we have developed can reveal kinship (or lack thereof) between T cell populations that are formed in vivo. We have now used this approach to determine whether T cell populations that migrate to different effector sites are derived from a single or from distinct naГЇve T cells, the latter being consistent with the increasingly popular notion Mireille Toebes Technical staff Jos Urbanus Technical staff Figure IV.3: Retroviral barcoding supports a diaspora model for T cell migration. Barcode analysis of T cells in lung and tumor tissue and the same analysis of the draining lymph nodes of the two sites earlier during antigen encounter. Note that while distinct barcodes are recovered within the draining lymph nodes (left), the T cells ending up at the effector site share the same set of barcodes (right). 44 IMMUNOLOGY Publications Crawford A, MacLeod M, Schumacher T, Corlett L, Gray D. Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells. J Immunol 2006; 176:3498-3506 De Witte MA, Coccoris M, Wolkers MC, Van Den Boom MD, Mesman EM, Song JY, Van Der Valk M, Haanen JBAG, of imprinting of T cell function by DC. Remarkably, although T cells that are activated within the draining lymph nodes contain a distinct set of barcodes, the T cells that are found at the site of infection are fully kin (Figure IV.3). These data strongly support a вЂњdiaspora modelвЂ™ for T cell migration, in which T cells randomly migrate to different sites of inflammation, irrespective of the site of original T cell priming. In addition to these experiments that are aimed at analyzing T cell migration, we are currently setting up model systems to analyze naГЇve T cell differentiation, hematopoietic stem cell and DC differentiation and a host of other issues related to blood cell differentiation. A long-term aim in this line of research is the development of technologies that will allow cellular barcoding in vivo. Schumacher TNM. Targeting self-antigens through allogeneic TCR gene transfer. Blood 2006; 108:870-877 Kessels HWHG, Schepers K, Van Den Boom MD, Topham DJ, Schumacher TNM. Generation of T cell help through a MHC class I-restricted TCR. J Immunol 2006; 177:976-982 Lukens MV, Claassen EAW, De Graaff PMA, Van Dijk MEA, TCR gene therapy (collaboration with Haanen lab) In the past years, our group has developed the retroviral introduction of antigen-specific T cell receptors into peripheral T cells as a means to induce virus- and tumor-specific immunity in vivo. In this strategy, autologous or donor-derived T cell populations are equipped with a TCR of defined reactivity in short-term ex vivo procedures, and re-infusion of the redirected cells is used to supply T cell reactivity against defined antigens. In the past year, we have examined the value of TCR gene therapy in a mouse spontaneous tumor model where the endogenous tumor-specific T cell repertoire is lacking, as is the case for the majority of human tumor-associated self antigens. Importantly, data obtained in these studies indicate that TCR gene transfer can successfully be used to suppress tumor formation in a clinically relevant model (Figure IV.4). Hoogerhout P, Toebes M, Schumacher TN, Van der Most RG, Kimpen JLL, Van Bleek GM. Characterization of the CD8+ T cell responses directed against respiratory Figure IV.4: Suppression of tumor growth in a syncytial virus during primary and spontaneous prostate tumor model by TCR gene secondary infection in C57BL/6 mice. therapy. Mice were either left untreated, treated Virology 2006; 352:157-168 by vaccination, or by vaccination plus TCR gene transfer. Tumor progression was analyzed by MacLeod M, Kwakkenbos MJ, Crawford A, histopathological analysis at 28 wks and Brown S, Stockinger B, Schepers K, compared to mice at the age treatment was Schumacher T, Gray D. CD4 memory T started. Note the marked reduction in tumor cells survive and proliferate but fail to progression upon TCR gene therapy. differentiate in the absence of CD40. J Exp Med 2006; 203:897-906 Rodenko B, Toebes M, Hadrup SR, Van Esch WJE, Molenaar AM, Schumacher TNM, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protocols 2006; 1:1120-1132 Schepers K. Dissection and manipulation of antigen-specific t cell responses. Leiden: Universiteit Leiden, 2006 Toebes M, Coccoris M, Bins A, Rodenko B, Gomez R, Nieuwkoop NJ, Van De Kasteele W, Rimmelzwaan GF, Haanen JBAG, Ovaa H, Schumacher TNM. Design and use of conditional MHC class I ligands. Nat Med 2006; 12:246-251 In addition, we have tested the value of a series of treatments, such as high dose IL-2, TGF-b inhibitors and lymphodepletion, as strategies to enhance the persistence and anti-tumor effect of TCR gene modified T cells. These studies have been highly informative with regard to future clinical trial design. With respect to such trials, from a collection of four gene-optimized high affinity T cell receptors specific for human melanoma antigens, we have selected the most potent TCR. This TCR will be used in a planned phase I clinical trial of TCR gene therapy in patients with metastatic melanoma. 45 IMMUNOLOGY IMMUNOTHERAPY The main objective of this line of research is the development of novel T cell immunity-based strategies that can be translated to clinical application (in close collaboration with T Schumacher). The focus is on treatment of patients with solid tumors, especially melanoma and renal cell carcinoma. A second line of research is focusing on immunomonitoring to evaluate specific and cytokine-based immunotherapies, using advanced technologies for characterization of immune responses in peripheral blood and at the tumor site. Group leader John Haanen John Haanen MD PhD Group leader DNA vaccination for the treatment of cancer Naked plasmid DNA when injected into the skin or muscle is taken up and translated into protein. Although the process of DNA uptake does not seem to be an efficient process, immune response against antigens encoded in the naked DNA plasmids can be found, mostly upon in vitro antigen-specific restimulation. Since DNA vaccines are relatively easy to produce, cheap and appear to be safe, this mode of vaccination is explored for both protection against infectious diseases and treatment of high risk cancer patients. Induction of immunity following DNA vaccination is generally considered a slow process. We have shown that DNA delivery to the skin results in a highly transient pulse of antigen expression. Based on this information, we have developed a novel, rapid and potent intradermal DNA vaccination method. By short-interval intradermal DNA delivery, robust T cell responses, of a magnitude sufficient to reject established subcutaneous tumors, are generated within 12 days. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations where immunity is required in the shortest possible time. These results were confirmed in a non-human primate model (in collaboration with BPRC, Rijswijk). We were able to show that DNA tattooing results in 10-100-fold increase in vaccine-specific T cells compared to intramuscular vaccination. These positive results have convinced us that this strategy should be tested in early phase clinical trials in humans. For this purpose a clinical grade DNA vaccine has been produced in the recently built GMP DNA plasmid production unit at the NKI pharmacy department for a phase I clinical study in HLA-A*0201-positive advancedstage melanoma patients. Florry Vyth-Dreese PhD Senior staff scientist Carina Lotz PhD Post-doc Esther Tjin PhD Post-doc Adriaan Bins MD Graduate student Annelies Jorritsma MSc Graduate student Yeung-Hyen Kim MSc Graduate student Koen Oosterhuis Msc Graduate student Trees Dellemijn Technical staff Raquel Gomez MSc Technical staff Willeke Van de Kasteele Technical staff Johan Sein Technical staff Tineke Vendrig Technical staff DNA vaccination for the treatment of human papilloma virus associated cancers Recently, ZonMW funding was obtained for a phase I/II clinical trial with HPV 16+ penile and cervical cancer patients. Human papilloma virus infection (serotypes 16 and 18) is strongly associated with the development of squamous cell cancer of the penis and cervix. Because the persistence of oncogenic HPV proteins E6 and E7 is required for carcinogenesis, these viral antigens are exquisite targets for immunotherapeutic interventions. Indeed, therapeutic vaccinations targeting these viral antigens have shown some promise in women suffering from cervical cancer. In the next years we will perform a phase I/II study in patients with HPV 16-positive squamous cell cancer of the penis and cervix using our novel and potent intradermal DNA vaccination strategy. In preclinical studies, this strategy was shown to be much more potent in the induction of E6/E7-specific T cell immunity than existing nonviral vaccination strategies, providing a strong rationale for its clinical evaluation. Gene therapy with melanoma-specific human T cell receptor genes In close collaboration with the group of T Schumacher and several foreign groups, we have collected a number of T cell receptor (TCR) a and b genes coding for melanomaspecific TCRs, amongst others the MART-126-35 and gp100209-217-specific receptors. These genes have been cloned into Moloney-based retroviral vectors. These retroviral vector systems enabled us to transduce human peripheral blood T-lymphocytes (see Fig. IV.5). Presently, we have chosen a novel MART-1-specific TCR displaying high avidity towards melanoma tumor cells upon recognition of cognate antigen in both cytolytic activity assay and IFN-g production assay, no detectable alloreactivity against a large panel of cell lines expressing different HLA antigens and superb transduction efficiency of both CD8+ and CD4+ peripheral blood T cells. Figure IV.5. Virus titration of a panel of optimized, melanoma-specific TCRs. The Mart-1-specific 1D3 TCR is codon optimized. Four days post transduction, TCR expression was determined by staining with PE anti-CD8 and APC-MART-1 (26-35. 27 A>L) tetramer. 46 IMMUNOLOGY Publications De Witte MA, Coccoris M, Wolkers MC, A large translational gene therapy grant will allow us to perform a phase I clinical study in melanoma patients in the next years. Van Den Boom MD, Mesman EM, Song JY, Van Der Valk M, Haanen JBAG, Schumacher TNM. Targeting self-antigens IMMUNOMONITORING through allogeneic TCR gene transfer. Blood This work is performed under supervision of F Vyth-Dreese, staff scientist in the group. The primary aim of this work is to evaluate immunotherapy clinical trials, using advanced technologies for characterization of immune responses in peripheral blood and at the tumor site. Work is performed in close collaboration with G de Gast (section X), A Bex (section XI) and W Nooijen (section XIII). 2006; 108:870-877 Haanen JBAG, Baars A, Gomez R, Weder P, Smits M, De Gruijl TD, Von Blomberg BME, Bloemena E, Scheper RJ, Van Ham SM, Pinedo HM, Van Den Eertwegh AJM. Melanomaspecific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells may predict survival in resected advancedstage melanoma patients. Cancer Immunol Immunother 2006; 55:451-458 Overwijk WW, De Visser KE, Tirion FH, De Jong LA, Pols TWH, Van Der Velden YU, Van Den Boorn JG, Keller AM, Buurman WA, Theoret MR, Blom B, Restifo NP, Kruisbeek AM, Kastelein RA, Haanen JBAG. Immunological and antitumor effects of IL-23 as a cancer vaccine adjuvant. J Immunol 2006; 176:5213-5222 Immunotherapy of ongoing and future clinical trials in advanced-renal cell carcinoma and melanoma Tools have been developed that allow us to monitor immunotherapy-based studies in a state-of-the-art fashion. Recent studies of IL-2 and IFN-a based therapies have enhanced insight into the mechanisms and potential of immune responses in renal cell carcinoma patients. Routinely, peripheral blood samples are stained for the presence of subtypes of T cells, using CD4, CD8, Foxp3 and additional markers to distinguish helper, cytotoxic and regulatory T cells, but also naГЇve, memory and effector T cells, NK cells and NKT cells. Tumor-specificity is measured by ELIspot (IFN-g), intracellular cytokine staining (ICS), conventional 51Cr release assays and LAMP-assays. Tumor biopsies and biopsies from vaccination sites are analyzed by culturing infiltrating T cells and their analysis using 6-color flow cytometry. In addition, tissue biopsies are studied using immunohistochemistry and multicolor confocal laser scanning microscopy for the presence and localization of T cells and dendritic cells in relation to tumor cells and the expression of immunomodulating molecules on tumor cells, including B7 family members (Figure IV.6). Tumor cell lines are generated from clinical material for stimulation of T cells. This allows for detection of tumor-specific T cells in an autologous fashion. Toebes M, Coccoris M, Bins A, Rodenko B, Gomez R, Nieuwkoop NJ, Van de Kasteele W, Rimmelzwaan GF, Haanen JBAG, Ovaa H, Schumacher TNM. Design and use of conditional MHC class I ligands. Nat Med 2006; 12:246-251 Vuylsteke RJCL, Molenkamp BG, Figure IV.6: Foxp3 positive regulatory Van Leeuwen PAM, Meijer S, T cells in renal cancer tissue. Wijnands PGJT, Haanen JBAG, Scheper RJ, De Gruijl TD. Tumor-specific CD8+ T cell reactivity in the sentinel lymph node of GM-CSF - Treated stage I melanoma patients is associated with high myeloid dendritic cell content. Clin Cancer Res 2006; 12:2826-2833 Vyth-Dreese FA, Kim YH, Dellemijn TAM, Schrama E, Haanen JBAG, Spierings E, Goulmy E. In situ visualization of antigen-specific T cells in cryopreserved Detection of antigen-specific T lymphocytes in experimental and human tissues Immunohistological techniques have been developed to visualize tetrameric peptide/MHC class I and class II complexes binding tumor- and virus-specific T lymphocytes in situ. Recently, the in situ tetramer technique has been applied successfully for the detection of minor Histocompatibility Antigen-specific T cells in a human ex vivo in situ skin explant model of Graft versus Host reactivity (in collaboration with the group of E Goulmy, LUMC). We have developed a method to detect minor antigen specific T cells, not only in fresh, but also in cryopreserved tissues. This enabled in situ visualization of their functional status as cytotoxic and cytokine producing effector lymphocytes in the skin microenvironment. human tissues. J Immunol Methods 2006; 310:78-85 Development of a human ex vivo in situ skin model for vitiligo and melanoma In collaboration with R Luiten (SNIP and Dermatology, AMC, Amsterdam) and C Melief (LUMC) a human in situ skin model is being developed to study immune and environmental factors involved in the development and potential therapy of vitiligo and melanoma skin cancer. Using melanocyte specific T cell clones cocultured with normal skin tissues, we were able to mimic the induction of vitiligo ex vivo. In addition, bulk T cells obtained from vitiligo lesions were shown to induce severe lesions in this skin model. Separate studies are aimed to set up a similar model in which melanoma explants can be visualized ex vivo in situ. 47 IMMUNOLOGY PROGRAMMED MUTAGENESIS IN B CELLS Antigen receptor diversification, genetic instability and lymphoma development Lymphoma is the most common blood cancer and the third most common cancer in childhood. Principally lymphoma is - like most other cancer types - a genetic disease that causally relates to the accumulation of somatic mutations in dominant and recessive cancer genes. Aberrant targeting of the Immunoglobulin (Ig) remodeling machinery in B cells of the germinal center, seems to boost the accumulation of oncogenic mutations in B cells. In fact, germinal center derived lymphomas like Follicular Lymphoma and most Diffuse large B-cell lymphomas account for more than 50 percent of all lymphomas. Given the mutagenic potential of secondary Ig gene diversification, we focus on its molecular mechanism, regulation and impact on genome stability. Our approaches involve basic and advanced molecular genetics, biochemistry, immunology and recombinant mouse genetics. Germinal center B cells - the physiological equivalent of the above mentioned lymphomas вЂ“ diversify the antigen specific Ig gene repertoire by somatic hypermutation (SHM) and class switch recombination (CSR). While SHM efficiently introduces point mutations into the Ig variable region, CSR is a process enabling isotype switching within the Ig heavy chain constant region. Both SHM and CSR are transcription-controlled and require the Activation Induced Deaminase (AID) to deaminate cytosine in the variable- and switch-regions of Ig genes, respectively. The resulting uracil can be removed by an uracil glycosylase, generating an abasic site. Processing by the MRN complex (MRE11, Rad50 and NBS1) or mismatch repair is thought to prohibit error free base excision repair, favoring mutagenic translesion DNA synthesis as well as the generation of single and double strand DNA breaks. Based on the translocation break points and somatic mutations in dominant and recessive cancer genes, SHM and CSR have been implicated in the molecular pathogenesis of germinal centre-derived lymphoma. A genome wide binding profile of AID Given the mutagenic potential of AIDmediated DNA deamination, we address whether aspecific targeting of AID and the Ig gene remodeling machinery explains why particularly B cells of the germinal center are prone to malignant transformation. As oncogenic translocations provide a selective growth advantage, do they just resemble the tip of the iceberg? Are translocation hotspots targets of AID? To address these and related questions we have set out to derive a genome wide chromatin-binding profile of AID. These profiles are established under physiological (germinal center B cells) and patho-physiological conditions (lymphomas with a germinal center signature). Putative binding sites are verified by sequencing and the binding profile is related to other parameters known to favor mutagenesis. This map will provide information on the mutability of non-Ig genes and their contribution to malignant transformation of germinal center derived lymphomas. Error free versus error prone repair: Posttranslational modification of PCNA Numerous DNA lesions are generated continuously by environmental factors and by endogenous damage, including deamination of cytosine by AID. During replication these DNA lesions can block high fidelity DNA polymerases, causing replication forks to arrest. To increase the fitness of replicating cells in the presence of DNA lesions, i.e. to continue replication without an a priori repair of the initial lesion, two alternative pathways of DNA damage tolerance can be triggered: polymerase switching or template switching. Yeast genetics has led to the identification of the Rad6 pathway, a ubiquitin (Ub) conjugating-system that catalyses monoubiquitination of a single ultra-conserved lysine, K164 of the proliferating nuclear antigen (PCNAK164) to stimulate polymerase switching and polyubiquitination of PCNAK164 to stimulate template switching. PCNA is a ring shaped homo-trimer, that encircles the DNA and serves as a DNA sliding clamp and molecular interaction platform enabling cross talk between proteins involved in DNA replication, DNA repair, DNA modification, chromatin modeling, cell cycle control and apoptosis. Mono-ubiquitination at PCNAK164 is catalyzed by the ubiquitin conjugating/ligating complex Rad6/Rad18. Monoubiquitinated PCNA triggers switching from a damage Group leader Heinz Jacobs Heinz Jacobs PhD Group leader Silke Schnell MSc Graduate student Marinus Heideman MSc Graduate student Peter Krijger MSc Graduate student Petra Langerak MSc Graduate student Paul Van den Berk Technical Staff 48 IMMUNOLOGY Publications Jansen JG, Langerak P, Tsaalbi-Shtylik A, Van den Berk P, Jacobs H, De Wind N. Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice. J Exp Med 2006; 203:319-323 Langerak P, Nygren AOH, Schouten JP, Jacobs H. Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA. Nucleic Acids Res sensitive, high fidelity DNA polymerase (d or e) to one of the damage tolerant, low fidelity translesion DNA synthesis (TLS) polymerases. TLS polymerases can continue replication across non-Watson Crick base pairs. Owing to the reduced accuracy of TLS polymerases, TLS is the major cause of DNA-damage-induced mutations. The finding that DNA damage-inducible ubiquitination of PCNA occurs at identical positions in yeast and humans suggest that this process may also determine the choice between polymerase switching and template switching in mammals. To establish this, we have generated ES cell clones carrying a conditional PCNA allele as well as a mutant K164R allele. Recombinant mouse strains and cell lines thereof will establish the role of posttranslational K164-linked PCNA modification in controlling genetic stability. As TLS lies at the heart of mutagenesis, our investigations will provide not only insights in to the control of SHM and lymphoma development but cancer in general. 2005; 33:e188 Schnell S, DГ©molliГЁre C, Van den Berk P, Kirberg J, Jacobs H. Constitutive expression of the pre-TCR enables development of mature T cells. Int Immunol 2006; 18:911-920 Schnell S, DГ©molliГЁre C, Van den Berk P, Jacobs H. Gimap4 accelerates T-cell death. Blood 2006; 108:591-599 Schnell SAC. Life and death in the T cell lineage. Amsterdam: Universiteit van Amsterdam, 2006 Somatic hypermutation in B cells lacking highly conserved K164 modifications of PCNA Somatic hypermutation of Ig genes enables B cells of the germinal center to generate high affinity Ig variants. B cells make use of damage tolerant, error prone TLS to generate somatic mutations at and around genetic lesions caused by AID. To determine the role of PCNAK164 modifications in mouse development and somatic hypermutation, PCNAK164R knock in mice have been generated. PCNAK164R mutant mice are viable, somatic cells develop normally but are highly sensitive to genotoxic stress. Moreover, PCNAK164R fails to recruit TLS polymerase h, explaining the defective mutagenesis at template A/T in PCNAK164R mutant B cells. Establishing the role of TLS polymerases in SHM To address the role of the TLS polymerase Rev1 in SHM, two recombinant Rev1 mutant mouse strains either lacking the regulatory BRCT domain or the catalytic SCDE domain, respectively have been analyzed. While the mutation frequencies in both mouse strains appear to be normal, the base exchanges are significantly altered in the SCDE mutant Rev1 mice. While mice lacking the BRCT domain show a normal base exchange pattern, mice lacking the SCDE domain however, show an increase in A -> T and T -> C mutations and a decrease in G -> C and C -> G mutations. The decrease in G -> C and C -> G mutations can be explained by the proposed CMP-transferase activity of Rev1, the increase in A -> T and T -> C mutations likely relates to a compensatory activity of the A/T mutator polymerase h. 49 MOLECULAR BIOLOGY V DI VI S I ON OF MOL ECU L A R BIOLOG Y Genetic instability and deregulated cell cycle control are hallmarks of human cancer. Our research involves both aspects focusing on (1) the role of DNA mismatch repair in mutation avoidance, (2) the role of cell cycle checkpoints in tumor suppression and (3) control of mitosis. Division head, Group leader Hein Te Riele Hein Te Riele PhD Group leader DNA MISMATCH REPAIR Camiel Wielders PhD Post-doc Genetic defects in DNA mismatch repair (MMR) underlie the cancer syndrome hereditary non-polyposis colorectal cancer (HNPCC). The primary function of MMR is correction of DNA replication errors. This is initiated by either MSH2/MSH6 or MSH2/MSH3 protein complexes, which recognize base.base mismatches and small loops of unpaired bases, respectively. In addition, two other MMR functions have been identified. The first is suppression of recombination between homologous but not perfectly identical DNA sequences. We have recently found that this activity of MMR also imposes a strong impediment to subtle gene modification by short single-stranded DNA oligonucleotides. The second is suppression of DNA damage-induced mutagenesis by elimination of damaged cells. Rob Wolthuis PhD Post-doc Marieke Aarts MSc Graduate student Sietske Bakker MSc Graduate student Elena Bardina MSc Graduate student Floris Foijer MSc Graduate student Wouter Van Zon Graduate student Tinke Vormer MSc Graduate student Eva Wielders Msc Graduate student Marleen Dekker Technical staff Elly Delzenne-Goette Technical staff Sandra De Vries MSc Technical staff Anja Van der Wal Technical staff Oligonucleotide-directed gene modification Oligonucleotide-directed gene modification (вЂ�oligo targetingвЂ™) is emerging as a powerful novel tool to subtly modify the genome of mouse embryonic stem (ES) cells and the generation of mutant mouse strains. We found that sequence-specific single-stranded oligonucleotides consisting of ~38 deoxyribonucleotides can restore the activity of a disabled neo reporter gene by deletion, insertion or substitution of one or a few base pairs. However, the success of oligo targeting is severely hampered by DNA mismatch repair activity (Dekker et al., NAR 2003;31:e27). Dependent on the type of alteration, oligo targeting was suppressed by MSH2/6 or MSH2/3 activity, or both. Based on this knowledge we have developed procedures for oligonucleotide-directed gene inactivation and modification and shown that mutant alleles can be introduced into the mouse germ line. Publications Aarts M, Dekker M, De Vries S, Van der Wal A, Te Riele H. Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells. Nucleic Acids Res 2006; 34:e147 Oligo-directed insertion of three or four nucleotides can readily be achieved in Msh3-deficient ES cells. As these cells do not show an overt mutator phenotype and Msh3-deficient mice are not cancer prone, we are using oligonucleotide-directed frameshift mutagenesis as an alternative to current knockout procedures. We have thus far generated disruptions in Fancf, Fen1 and Esco2. Oligo-directed base substitution is largely suppressed by MSH2/6 and therefore relies on the absence of MSH2. When we transiently suppress MSH2 activity by RNA interference, cells become permissive for oligonucleotide-mediated substitution of three or four nucleotides, allowing modification of virtually any codon in the genome. Under this condition the level of spontaneous mutagenesis remains low. We are exploiting this procedure to study the pathogenicity of allelic variants found in the human population, focusing on RB, p53 and MSH2/6. Uncoupling of DNA MMR functions A tenfold reduced level of MSH2 protein leads to uncoupling of MMR functions: replication errors are still largely corrected, but DNA-damage-induced mutagenesis is no longer suppressed. In mice, ten percent MSH2 level was sufficient to suppress spontaneous tumorigenesis in the thymus and small intestine. However, in a sensitized background, reduced MSH2 level did accelerate tumorigenesis. We are currently investigating whether this is the result of the slightly reduced efficacy of mismatch correction or the full loss of appropriate DNA damage responses. Furthermore, we are generating, by oligo targeting, specific codon substitutions in Msh2 to uncouple MMR functions and to study the role of each in tumor suppression. Figure V.1: Extending the restriction point. A Cell cycle progression is dependent on Cyclin-CDK activity. B Mitogen starvation results in cell cycle arrest in G1 through degradation or suppression of Cyclin D and CKI-mediated inhibition of Cyclin E-CDK2. C Passage through the G1 restriction point in the absence of mitogens (e.g., through RB loss) results in cell cycle arrest in G2. 50 MOLECULAR BIOLOGY Publications (continued) CELL CYCLE CHECKPOINTS Dannenberg J-H, Te Riele HPJ. The Retinoblastoma Gene Family in Cell Cycle Regulation and Suppression of Tumorigenesis. In: Kaldis P, editor. Cell Cycle Regulation. Berlin; Heidelberg; New York: Springer, 2006: 183-225 Dekker M, Brouwers C, Aarts M, Van der Torre J, De Vries S, Van de Vrugt H, Te Riele H. Effective oligonucleotide-mediated gene disruption in The retinoblastoma suppressor protein pRB and its homologs p107 and p130 (collectively called вЂњpocket proteinsвЂќ) impede the G1-S transition of the cell cycle by binding to and inhibiting E2F transcription factors. G1 arrest is relieved by phosphorylation of pocket proteins by Cyclin D- and E-dependent kinases, whose activity is dependent on mitogens. However, mitogenic signaling is only required before the G1 restriction point after which cells can progress into S-phase in the absence of mitogens. The G1 restriction point mechanistically coincides with the requirement for pocket protein phosphorylation for S-phase entry. Thus, mouse embryonic fibroblasts (MEFs) with defects in the retinoblastoma gene family (TKO MEFs, lacking expression of Rb, p107 and p130) can pass through G1 and enter S phase with similar kinetics in the presence or absence of mitogens. ES cells lacking the mismatch repair protein MSH3. Gene Ther 2006; 13:686-694 Foijer F, Te Riele H. Check, double check: the G2 barrier to cancer. Cell Cycle 2006; 5:831-836 Foijer F. Braking news: The G2 restriction point. Amsterdam: Vrije Universiteit, 2006 Foijer F, Te Riele H. Restriction beyond the Growth-factor independence We found that after passage through S-phase, proliferation of TKO MEFs was still restricted by: (1) massive apoptosis and (2) a G2 arrest that became apparent when apoptosis was suppressed by ectopic expression of Bcl2. The G2 arrest was mediated by upregulation of the cyclin-dependent-kinase inhibitors p21CIP1 and p27KIP1 that inhibited the G2 cyclins Cyclin A and Cyclin B1 through direct interaction. The arrest was reversible: re-addition of serum for 3-6 h led to mitotic entry about 15 h later, thus defining a second restriction point operating in G2 (Fig. V.1). Strikingly, inactivation of p53, overexpression of c-MYC or mutational activation of RAS alleviated the G2 arrest response, providing a rationale for the synergism between loss of pocket proteins and these oncogenic events in tumorigenesis. restriction point: mitogen requirement for G2 passage. Cell Division 2006; 1:8 Foiry L, Dong L, Savouret C, Hubert L, Te Riele H, Junien C, Gourdon G. Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice. Hum Genet 2006; 119:520-526 Zienolddiny S, Ryberg D, Svendsrud DH, Eilertsen E, Skaug V, Hewer A, Anchorage independence We previously showed that ablation of Rb and p107 or Rb and p130 was sufficient to cause immortality and to reverse the growth inhibitory effect of RASV12 into a proliferative stimulus. However, Rb-/-p107-/- or Rb-/-p130-/MEFs were not oncogenically transformed by RASV12: apparently, a cell cycle mechanism still operates to restrict proliferation of these cells in soft agar. We therefore performed screens for genetic events that permit anchorage-independent growth of RASV12-expressing Rb-/-p130-/- MEFs. In one screen we identified the immortalizing oncogene Tbx2 that has previously been shown to interfere with the p53 pathway. As over-expression of Tbx2 and RASV12 in primary wild-type MEFs did not result in anchorage-independent growth, this result indicates that concomitant ablation of the p53 and pRB pathway facilitates oncogenic transformation. Phillips DH, Te Riele H, Haugen A. Msh2 deficiency increases susceptibility to REGULATION OF MITOSIS benzo[a]pyrene-induced lymphomagenesis. In 2005, Rob Wolthuis joined the group of Hein Te Riele to continue his studies of mitosis in normal and cancer cells. During mitosis, the duplicated genome is irreversibly distributed over two newly formed cells. This process requires timely inactivation of mitotic regulators by proteolysis. A multi-subunit assembly, the Anaphase-Promoting Complex (APC/C), ubiquitinates key mitotic proteins such as Cyclin B1 and Securin at highly defined mitotic time points, which targets them for destruction by the 26S proteasome. By precisely regulating mitotic proteolysis, the APC/C coordinates cell division with segregation of sister chromatids, which is essential to guarantee cellular viability and genomic integrity. The question remains how the APC/C recognizes a critical substrate at the right time. To address this important issue in human cells, we established an innovative, combinatorial approach of gene silencing, biochemistry and live-cell imaging (Figure V.2). Hereby, we identified a novel role for Cdk2 in controlling Cyclin A destruction. Furthermore, we discovered that Cyclin B1 and Securin are differentially recruited by the APC/C in metaphase, a process that involves phosphorylation of the subunit APC3. Furthermore, we identified a novel de-Ubiquitinating enzyme, which helps to check APC/C-dependent destruction. Int J Cancer 2006; 118:2899-2902 Figure V.2: Measuring APC/C activity by time-lapse fluorescence microscopy. A plasmid encoding fluorescent Cyclin B1 was injected into G2-phase osteosarcoma cells, a few hours before they started mitosis. The top panel shows a cell undergoing mitotic division, followed over time (min) by differential interference contrast (DIC). The bottom panel shows the localisation and quantitative levels of Cyclin B1 in the fluorescence channel of the same cells. The cell undergoing division rapidly degrades Cyclin B1, as measured by a decrease in fluorescence signal. This assay allows the quantitation of APC/C-dependent protein destruction in live cells while they divide. 51 MOLECULAR BIOLOGY DNA BASE J Base J b-glucosyl-hydroxymethyluracil (base J), which we discovered in African trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993; 75:1129-36), is a base present in all kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in DNA and is predominantly located in repetitive sequences, such as telomeric repeats. We have characterized a J-binding protein (JBP 1) that binds with high specificity to J-containing duplex DNA (Cross et al., EMBO J 1999; 18:6573-81). A JBP 1 gene KO in T. brucei has the remarkable effect of reducing the J content of trypanosome DNA to 5% of wild type, without any detectable physiological consequences. In the related kinetoplastid Leishmania, a JBP 1 KO is lethal. Our recent work indicates that JBP 1 is a hydroxylase that catalyses the first step of J biosynthesis, the conversion of T in DNA into hydroxymethyluracil. JBP 1 appears to belong to the family of Fe2+ and 2-oxoglutarate-requiring dioxygenases, as does a second putative hydroxylase, JBP2. We have shown that JBP2 is dispensable in Leishmania under the growth conditions tested thusfar. To determine the function of J in Leishmania we are constructing a conditional KO of the JBP 1 gene. We are also continuing attempts to set up assays for hydroxylase activity in vitro and for the putative glucosyl transferase catalyzing the second step in J biosynthesis. With Anastassis Perrakis (NKI-AVL) we are trying to determine the structure of JBP 1-J-DNA complexes by crystallography; with Charles Weissmann (Scripps Florida), we are trying to find inhibitors of the binding of JBP 1 to DNA and of its hydroxylase function. Group leader Piet Borst Piet Borst MD PhD Group leader Henri Van Luenen PhD Academic staff Jan Wijnholds PhD Academic staff, honorary Sjofn Gunnarsdottir PhD Post-doc Marina Pajic PhD Post-doc Sven Rottenberg DVM PhD Dipl. ECVP Post-doc Saara Vainio PhD Post-doc Koen Van de Wetering PhD Post-doc Cocky De Wolf PhD Post-doc Paul-AndrГ© Genest MSc Graduate student Marcel De Haas Technical staff Liesbeth Van Deemter Technical staff Wouter Feddema Technical staff Ariena Kersbergen Technical staff Bas Ter Riet Technical staff MULTIDRUG RESISTANCE OF CANCER CELLS We are interested in mechanisms of drug resistance in cancer cells and focus on resistance caused by increased ATP-dependent transport of drug out of the cell, mediated by ATP-binding cassette (ABC) transporters. We have isolated genes for these transporters and are characterizing their substrate specificity and sensitivity to inhibitors in transfected cells. We use the baculovirus system to produce insect cell membrane vesicles containing high amounts of transporter protein and suitable for vesicular transport studies. We also use transfected polarized kidney cell monolayers in which the transporters either route to the apical or the basolateral membrane, allowing the study of vectorial transport through the cell layer. To study the physiological function in metabolism and defense of the body against drugs and xenotoxins of these transporters, we have inactivated genes for several drug transporters by targeted gene disruption in mice. Initially we looked at P-glycoproteins (ABCB family); most recently we have studied the Multidrug Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 9. Publications Borst P, Zelcer N, Van de Wetering K. MRP2 and 3 in health and disease. Cancer Lett 2006; 234:51-61 Borst P, De Wolf CJF, Van de Wetering K. Multidrug resistance-associated proteins 3, 4, and 5. Pflugers Arch 2006;Published online Borst P, Zelcer N, Van de Wetering K, Poolman B. On the putative co-transport of drugs by multidrug resistance proteins. FEBS Lett 2006; 580:1085-1093 MRP2 (ABCC2) and MRP3 (ABCC3) are organic anion transporters contributing to the cellular export of endogenous or exogenous (toxic) compounds, conjugated to glutathione, sulphate or glucuronate. Using KO mice, we have previously shown that murine MRP3 is the major transporter mediating export of morphine-3-glucuronide (M3G) from the liver into the blood (Zelcer et al., Proc. Natl. Acad. Sci. USA 2005;102:7274-9). In collaboration with the Schinkel group (NKI-AVL) we have now shown that MRP2 is responsible for extrusion of M3G into bile. As long as either MRP2 or MRP3 is present, the liver can efficiently dispose of M3G. In the Mrp2-/- / Mrp3-/- double KO, M3G accumulates to high levels in the liver, but can still be slowly exported by a transporter that remains to be identified. MRP3 is also important for basolateral export of glucuronated compounds from the gut epithelium into blood. An interesting example is provided by resveratrolglucuronide (R-gluc), which mainly enters the body via MRP3. We have used murine microsomes to generate radioactive R-gluc and shown that this is a high-affinity substrate for MRP3. We have initiated a systematic search for other glucuronides transported by MRPs by comparing the glucuronide derivatives in plasma/urine of WT and KO mice using Mass Spectometry. We have identified several potential novel MRP3 substrates by this approach and anticipate that it may also be helpful to find substrates of other MRPs. Borst P, Genest PA. Parasitology: Switching like for like. Nature 2006; 439:926-927 Borst P, Wielinga P. Pumping Out Drugs: The Potential Impact of ABC Transporters on Resistance to Base, Nucleoside, and Nucleotide Analogs. In: Peters GJ, editor. Deoxynucleoside Analogs in Cancer Therapy. Totowa, NJ.: Humana Press, 2006: 109-118 Borst P. How I became a biochemist. IUBMB Life 2006;58:177-82 Borst P. From cancer cells to trypanosomes and back again. Cell Mol Life Sci 2006;63:745-54 52 MOLECULAR BIOLOGY Publications (continued) Minich T, Riemer J, Schulz JB, Wielinga P, Wijnholds J, Dringen R. The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes. J Neurochem 2006; 97:373-384 Zelcer N, Van de Wetering K, De Waart R, Scheffer GL, Marschall HU, Wielinga PR, Kuil A, Kunne C, Smith A, Van der Valk M, Wijnholds J, Oude Elferink RO, Borst P. Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides. J Hepatol 2006; 44:768-775 MRP4 (ABCC4) and MRP5 (ABCC5) are organic anion transporters with the unusual ability to transport nucleotide analogs and the cyclic nucleotides cGMP and cAMP. Using intact transfected cells and vesicular transport assays, we have found that the affinity of MRP4 and MRP5 for nucleotide analogs and cyclic nucleotides is low, making a major role in drug resistance or cyclic nucleotide disposition doubtful. In search for other functions, we found that MRP4 is able to transport some conjugated bile acids and steroids with high affinity. Interestingly, MRP4 (but not MRP1, 2 or 3) can also transport prostaglandins E1 and E2, and that transport is inhibited by some non-steroidal anti-inflammatory drugs, such as indomethacin, which are also known to block synthesis of these prostaglandins. We are studying whether MRP4 is a limiting factor in the cellular excretion of endogenously made prostaglandin using Mrp4 KO mice. While studying the nature of the transporter(s) responsible for cGMP transport by murine erythrocytes, we found a contribution by the Breast Cancer Resistance Protein (ABCG2). This led us to look at resistance against nucleoside analogs in BCRP-transfected cells. We found a high level of resistance to cladribine, but unexpectedly this is due to transport of cladribine itself rather than transport of cladribine-monophospate. This is the first example of a nucleoside analog being transported by a mammalian ABC-transporter. Drug resistance in вЂњspontaneousвЂќ mouse tumors We have started a new project in collaboration with Jos Jonkers (NKI-AVL) to study resistance mechanisms in вЂњspontaneousвЂќ tumors arising in genetically modified mice. Initial experiments were done in mice conditionally defective in p53 and Brca1. These mice contain floxed alleles of these two genes and a Cre recombinase gene driven by a Keratin14 promotor, active only in epithelial cells, resulting in breast (and skin) cancer. When treated with the maximum tolerable dose of doxorubicin, docetaxel or topotecan, the breast tumors initially respond but eventually always develop resistance. Resistance is often associated with upregulation of the Mdr1a and Mdr1b genes, which encode drug-transporting P-glycoproteins. In addition to gene expression analysis by oligoarrays, we developed a reverse transcriptase-multiplex ligation-dependent probe amplification (RT-MLPA) analysis in collaboration with MRC-Holland to measure tumor mRNA of selected drug target and drug transporter genes more precisely. Our results suggest that the available methods to detect Multidrug Resistance (MDR) caused by P-glycoprotein in clinical material are inadequate. In contrast to the results obtained with MDR drugs, we have been unable to obtain cisplatin resistance in this tumor model. As shown in figure V.3 the tumors respond to each new treatment with cisplatin, but are never fully eradicated. We are studying the вЂњremnantsвЂќ from which the tumors regrow to see whether these represent вЂњstemвЂќ cells resistant to cisplatin. In addition we are using this mouse model to test new anticancer drugs and drug combinations (in collaboration with KuDOS) and we are also crossing in drug transporter knock out mice. Figure V.3: Two typical examples of cisplatin responses of Brca1- and p53-deficient mammary tumors. (A) shows the volume (0.5 x length x width2) of a mammary tumor in a K14Cre; Brca1flox/flox; p53flox/flox female mouse which was treated with the maximum tolerated dose of cisplatin (6mg/kg i.v., arrows). The same treatment was given to a syngeneic wild-type mouse into which a Brca1-/-; p53-/- mammary tumor fragment was transplanted orthotopically (B). Treatment was started when the tumor size was about 200 mm3. After the initial administration on day 0, additional treatments were given when the tumor relapsed to 100%. 53 MOLECULAR BIOLOGY MOUSE MODELS OF BREAST CANCER The focus of our laboratory is on the genetic dissection of human breast cancer through the use of genetically engineered mouse models. Thus far, we have developed models for p53-induced breast cancer, BRCA1- and BRCA2- associated hereditary breast cancer, and E-cadherin-associated metastatic breast cancer. These models are used to (i) investigate genotype-phenotype relations in mammary tumorigenesis; (ii) identify genetic changes underlying breast tumorigenesis; (iii) study the role of innate and adaptive immunity in breast cancer development; (iv) perform therapeutic intervention studies. Group leader Jos Jonkers Jos Jonkers PhD Group leader Peter Bouwman PhD Post-doc Conditional mouse models of BRCA-associated breast cancer In addition to the previously published mouse model for BRCA2-associated breast cancer (Jonkers et al., Nat Genet. 2001;29:418) we have established a mouse model of BRCA1-associated hereditary breast cancer. To this end, we have generated conditional mutant mouse strains with (K14-Cre induced) epithelium-specific loss of Brca1 and p53. Female mice of this strain develop significantly faster mammary tumors than females with epithelium-specific loss of p53 alone, demonstrating collaboration between Brca1 inactivation and p53 loss in mammary tumorigenesis. The Brca1-/-;p53-/- mammary tumors have similar histomorphologic features as basal-like human breast cancers: they are highly proliferative, poorly differentiated, ERBB2/HER2- and estrogen receptor (ER)-negative mammary adenocarcinomas with pushing borders and increased expression of basal epithelial markers (CK5/6). Brca1-/-;p53-/- mammary tumors are highly aneuploid and their gene expression signatures resemble those of human BRCA1-mutated breast cancers. Thus, they display the hallmarks of familial BRCA1-deficient breast cancer in women. This mouse BRCA1 mammary tumor model might therefore be helpful in predicting chemotherapeutic responses of human BRCA1-deficient tumors and basal-like breast cancers with BRCA-like phenotypes (see below). Conditional mouse models for E-cadherin-deficient metastatic breast cancer While metastatic disease is the main cause of death in breast cancer patients, the underlying mechanisms are poorly understood. Loss of E-cadherin is strongly associated with tumor invasion and metastasis, as well as with invasive lobular carcinoma (ILC), which accounts for 10-15% of all breast cancers. To study the role of E-cadherin in breast oncogenesis, we have generated a mouse model for invasive lobular breast carcinoma (ILC) based on tissue-specific inactivation of the E-cadherin tumor suppressor gene. Hereto, we have produced conditional E-cadherin mutant mice and crossed these to a conditional mouse mammary tumor model based on epithelium-specific knockout of p53. Combined loss of E-cadherin and p53 causes a phenotypic change from non-invasive to highly invasive and metastatic mammary tumors, a morphological switch from ductal to lobular carcinomas and a marked reduction in tumor latency, showing collaboration between E-cadherin inactivation and p53 loss in mammary tumorigenesis. The compound mutant female mice develop mammary tumors with striking resemblance to human ILC, showing invasion into the surrounding tissue and metastasis to various sites including gastrointestinal tract and bone. Moreover, loss of E-cadherin suppresses anoikis and facilitates angiogenesis, thus promoting the growth, survival and dissemination of tumor cells. These data establish a causal role for E-cadherin in breast cancer suppression and show that loss of E-cadherin impacts on different stages in breast oncogenesis, including tumor initiation, invasion and metastasis. We are currently performing biochemical and functional genetic studies to identify the pathways downstream of E-cadherin, which mediate the in vitro and in vivo tumor phenotypes associated with E-cadherin loss. Array-CGH analysis of mouse mammary tumors In collaboration with the Wellcome Trust Sanger Institute (Hinxton, UK), we have developed mouse tile path BAC arrays for comparative genomic hybridization (CGH) analysis of mouse mammary tumors. In agreement with the published roles of BRCA1 and BRCA2 in maintenance of genome integrity, CGH analysis revealed significantly more DNA copy number alterations in Brca1-/-;p53-/- and Brca2-/-;p53-/- Michiel De Bruin PhD Post-doc Patrick Derksen PhD Post-doc Karin De Visser PhD Post-doc Gilles Doumont PhD Post-doc Xiaoling Liu PhD Post-doc Rinske Drost Msc Graduate student Bastiaan Evers Msc Graduate student Henne Holstege Msc Graduate student Hermien Boerhout Technical staff Eva Kregel Technical staff Eline Van der Burg Technical staff Hanneke Van der Gulden Technical staff Ellen Wientjens Technical staff 54 MOLECULAR BIOLOGY Publications Alberici P, De Pater E, Cardoso J, Bevelander M, Molenaar L, Jonkers J, Fodde R. Aneuploidy Arises at Early Stages of Apc-Driven Intestinal Tumorigenesis and Pinpoints Conserved Chromosomal Loci of Allelic Imbalance between Mouse and Human. Am J Pathol 2007; 170:377-387 Boesten LSM, Zadelaar ASM, Van Nieuwkoop A, Hu L, Jonkers J, Van de Water B, Gijbels MJJ, Van der Made I, De Winther MPJ, Havekes LM, Van Vlijmen BJM. Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development mammary tumors compared to p53-/- control tumors. We are also using CGH profiling to identify novel oncogenic mutations that cooperate with loss of BRCA1, BRCA2, p53 or E-cadherin in mammary tumorigenesis. To find significantly recurrent regions with DNA copy number aberrations in our array-CGH data sets, we have developed an objective, statistical method for multi-experiment analysis of arrayCGH data. This method, called KC-SMART (Kernel Convolution вЂ“ a Statistical Method for Aberrant Region deTection) uses kernel convolution to generate a Kernel Smoothened Estimate (KSE) of copy number alterations across the genome, aggregated over all tumors. The peaks in the KSE are tested against randomly permutated data to obtain statistically significant aberrant regions. Using this approach, we have identified in our panels of mouse mammary tumors several regions with recurrent copy number alterations, including a region on chromosome 10 that is amplified in over 50% of the Brca2-/-;p53-/- tumors. These regions, when combined with gene expression microarray data, point to various known cancer genes and several new candidate cancer genes. We are currently validating the candidate genes within the amplified region on chromosome 10. in ApoE-deficient mice. FASEB J 2006; 20:953-955 Clark-Knowles KV, Garson K, Jonkers J, Vanderhyden BC. Conditional inactivation of Brca1 in the mouse ovarian surface epithelium results in an increase in preneoplastic changes. Exp Cell Res 2007; 313:133-145 Derksen PWB, Liu X, Saridin F, Van der Gulden H, Zevenhoven J, Evers B, Van Beijnum JR, Griffioen AW, Vink J, Krimpenfort P, Peterse JL, Cardiff RD, Berns A, Jonkers J. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma The role of innate and adaptive immunity in breast cancer development Studies in a transgenic mouse model for skin carcinogenesis revealed an unexpected role for B lymphocytes and their soluble mediators in initiating chronic inflammation during premalignancy and subsequent progression to invasive skin cancer (De Visser et al., Cancer Cell 2005;7:411). We are currently investigating whether a similar link between adaptive and innate immunity contributes to breast cancer development, utilizing conditional and transgenic mouse models for mammary carcinogenesis. Similar to human breast cancer lesions, neoplastic mammary glands of these mice are characterized by leukocyte infiltration. To assess the functional significance of innate and adaptive immune systems during breast cancer development, these mice are being crossed with various immune-deficient mice and consequences for chronic inflammation, angiogenesis, premalignant progression, cancer development and metastasis formation will be analyzed. We believe that identification of the mechanisms underlying inflammation-associated cancer development will reveal drug targets that can be used to design novel therapeutic strategies for arresting cancer development in humans. through induction of anoikis resistance and angiogenesis. Cancer Cell 2006; 10:437-449 Evers B, Jonkers J. Mouse models of BRCA1 and BRCA2 deficiency: Past lessons, current understanding and future prospects. Oncogene 2006; 25:5885-5897 Van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, Van Rooijen MA, re JP, Pettit TR, Wakelam MJO, Saulnier-Blache JS, Mummery CL, Moolenaar WH, Jonkers J. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Mol Cell Biol 2006; 26:5015-5022 Tumor intervention studies In close collaboration with the groups of Piet Borst and Sabine Linn, we are utilizing the BRCA1 and E-cadherin mammary tumor models for preclinical studies. For this purpose we have derived panels of clonal, luciferasemarked tumor cell lines from our вЂњspontaneousвЂќ tumor models, and shown that these cell lines reproduce the disease upon orthotopic grafting in recipient mice. Together with the spontaneous models, these reagents provide a valuable platform for validation of candidate drug targets and anticancer drugs in a physiologically relevant setting. Moreover, synergistic antineoplastic effects of combinations of targeted and conventional therapeutics may be effectively tested. We are currently testing a number of cytotoxic drugs and targeted therapeutics on our panels of Ecad-/-;p53-/- and p53-/- mammary tumor cell lines and the corresponding orthotopic grafting models. The central role of BRCA1 and BRCA2 in DNA repair via homologous recombination (HR) has suggested that BRCA-deficient tumors will be especially sensitive to chemotherapeutics that induce DNA double-strand breaks. Indeed, intervention studies performed by Sven Rottenberg in the group of Piet Borst show that mammary tumors in our BRCA1 model are highly sensitive towards DNAcrosslinking agents such as cisplatin. This chemosensitivity profile is consistent with in vitro responses of BRCA-deficient cells and clinical responses of BRCA-linked ovarian cancers, indicating that our BRCA1 tumor model provides a useful preclinical platform for testing novel therapeutics, such as PARP1-inhibitors, which specifically target HR-deficient breast cancers. 55 MOLECULAR BIOLOGY MOLECULAR DISSECTION OF BREAST CANCER BY DIFFERENTIAL DRUG SENSITIVITY In the clinic, we only use anticancer drugs selected by clinical trials to perform best for the whole group of breast cancer patients, whereas little is known about the molecular mechanisms underlying differential drug sensitivity of breast cancer. The focus of our research line is to unravel these mechanisms in order to develop tests that may guide treatment decisions in the clinic and ultimately improve survival. For this purpose we use several genome-wide approaches and molecular techniques, in order to dissect the mechanisms that divide clinically well-defined cohorts of breast cancer patients into resistant and sensitive to a particular drug. In addition, we use conditional mouse models for breast cancer, and derived clonal tumor cell lines, to study differential chemosensitivity in a controlled fashion. Group leader Sabine Linn Sabine Linn MD PhD Group leader Michiel De Bruin PhD Post-doc Jolien Bueno de Mesquita MD Graduate student Marleen Kok MD Graduate student Ingrid Van der Heijden Technical Staff Feasibility of microarray technology as a diagnostic tool in community hospitals In collaboration with the van de Vijver group, we have assessed whether microarray technology as a diagnostic tool is feasible in daily clinical practice in general hospitals. In a pilot study, from 2004 - 2006, we have demonstrated the feasibility of this technique in general hospitals (see also Division X). Development of a predictive test for tamoxifen (TAM) resistance in breast cancer Based on the assumption that breast cancer patients who benefit from1 adjuvant TAM treatment have the same molecular breast cancer subtype as those patients who did not receive any form of adjuvant systemic treatment, but show long lasting responses on first line TAM in the metastatic setting, we have started a retrospective cohort study in over 500 patients who have been treated with TAM, either in the adjuvant or metastatic setting. Molecular analysis of this series is ongoing (collaboration with Division VIII). In collaboration with the Erasmus Medical Center we validated their predictive gene expression profile for TAM resistance (Jansen et al., J Clin Oncol. 2005; 23:732-40) in a pilot study of 69 metastatic breast cancer patients. Molecular mechanisms underlying sensitivity for high dose alkylating agents We have used Bacterial Artificial Chromosome (BAC) arrays to generate comparative genomic hybridization (CGH) profiles of primary breast cancers of 31 breast cancer patients who had been treated with high dose alkylating agents with hematopoietic stem-cell rescue in the metastatic setting. Ten patients had a progression-free survival (PFS) of over 24 months, while the others progressed within that time. In collaboration with the Wessels group we identified a classifier of 31 BAC clones that correctly classified the patients with a favorable PFS with a sensitivity of 80% and a specificity of 81%. Figure V.4 shows the heatmap of amplifications and deletions of the 31 BAC clones for the 31 patients. We are currently validating this CGH classifier on a larger series. Screening for drugs active against lobular breast cancer In collaboration with the Jonkers group we have generated and characterized a panel of 27 murine breast cancer cell lines derived from tumors with a Trp53-/- or Cdh1-/-;Trp53-/- genotype, modeling ductal and lobular breast carcinoma, respectively. Sofar, growth inhibition assays with conventional chemotherapeutics did not show any differential chemosensitivity. Gene expression profiling of all cell lines has uncovered interesting differences between the two genotypes that may point to drugs active against lobular breast cancer. These results are used to select additional compounds for further testing. We will repeat part of the cell line experiments in orthotopic grafting and spontaneous mouse models, to verify whether the cell lines correctly reflect the in vivo relative chemosensitivity. Figure V.4: Heatmap of amplifications and deletions of 31 BAC clones (horizontal axis) for 31 patients (vertical axis). On the far right side the log2 ratios of amplifications are shown in white and deletions in black, respectively from 0 to 1 and from 0 to -1. The last column of the heatmap shows favorable PFS in white and unfavorable PFS in black. 56 MOLECULAR BIOLOGY CHROMATIN GENOMICS Group leader Bas Van Steensel Bas Van Steensel PhD Group leader Lars Guelen PhD Post-doc Celine Moorman PhD Post-doc Helen Pickersgill PhD Post-doc Ulrich Braunschweig MSc Graduate student Elzo De Wit MSc Graduate student Frauke Greil MSc Graduate student Daniel Peric-Hupkes MSc Graduate student Maartje Vogel MSc Graduate student Greg Hogan MSc Guest researcher Ludo Pagie PhD Bioinformatician Introduction The main goal of our laboratory is to understand how gene expression programs in higher eukaryotes are regulated. In particular, we focus on mechanisms of chromatin-mediated gene regulation in the living cell, in the context of the entire genome. Using Drosophila and mammalian cells as model systems, we develop and apply new whole-genome approaches to study the structure and composition of chromatin and the mechanisms of gene regulation. Over the past years, we developed a novel technique (named вЂњDamIDвЂќ) for the genome-wide mapping of in vivo target loci of chromatin proteins and transcription factors. This microarray-based method allows us to test thousands of genomic sequences for in vivo binding of a chromatin protein or transcription factor of interest. We now routinely use DamID to generate whole-genome maps of protein binding in Drosophila and mammalian cell lines. These binding maps provide a wealth of new insights into the roles of each protein in determining chromatin structure and gene regulation. Gradually, we are building a large database of genomewide binding maps of chromatin proteins and other regulatory proteins. This database is being analyzed using state-of-the-art bioinformatics approaches to obtain insight into the interplay between various regulatory proteins. Francesco Russo MSc Technical staff Wendy Talhout Technical staff Transcription factor hotspots In collaboration with the laboratories of Dr. Kevin White (Yale University) and Dr. Harmen Bussemaker (Columbia University) we have used DamID in combination with genomic tiling arrays to obtain a highly detailed view of the binding patterns of a broadly selected set of 14 regulatory proteins, including transcription factors, co-factors, and chromatin-modifying proteins. Surprisingly, we found that many functionally unrelated transcription factors all show strongly overlapping binding patterns, and that the genome contains many вЂњhotspotsвЂќ that are targeted by all of these proteins. Several control experiments show that the strong overlap is not an artefact of the techniques used. Colocalization hotspots are 1-5kb in size, spaced on average by ~50 kb, and preferentially located in regions of active transcription. We provide evidence that protein-protein interactions play a role in the hotspot association of some transcription factors. Colocalization hotspots constitute a new type of feature in the genome of Drosophila that we intend to study in more detail in the near future. Nuclear lamina вЂ“ genome interactions The nuclear lamina (NL) has long been thought to be an anchoring site of chromatin and to participate in the regulation of gene expression, but genomic sequences that interact with the NL in vivo have remained unknown. In close collaboration with the laboratory of Dr. Maarten Fornerod (Division of Tumor Genetics, NKI), we have used DamID to identify nearly 500 Drosophila genes that interact in vivo with the NL component Lamin. We found that genes that interact with Lamin are transcriptionally silent, devoid of histone modifications typical of active chromatin, and late replicating, yet they do not bind the classical heterochromatin markers HP1 and Su(var)3-9. Interestingly, Lamin target genes tend to be flanked by very large intergenic regions. This suggests that large intergenic regions can play a role in the nuclear organization of the genome. Furthermore, Lamin target genes cluster in the genome, and we provided evidence that these clusters act as units of co-regulation during development. These results provide global insights into the organization of the genome in the cell nucleus. We are currently investigating the dynamics of lamina-genome interactions during differentiation. Polycomb domains Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. In a close collaboration with the laboratory of Dr. Maarten van Lohuizen (Division of Molecular Genetics, NKI), we used a genomewide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as вЂ�Pc domainsвЂ™. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at 57 MOLECULAR BIOLOGY Publications histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains. Choksi SP, Southall TD, Bossing T, Edoff K, De Wit E, Fischer B, Van Steensel B, Micklem G, Brand AH. Prospero Acts as a Binary Switch between Self-Renewal and Differentiation in Drosophila Neural Stem Cells. Dev Cell Heterochromatin Classical heterochromatin is generally believed to be a repressive type of chromatin. However, this notion is mostly based on studies with artificial reporter genes; virtually no natural target genes were known. We have mapped HP1 binding sites on chromosome 2 and 4 in Drosophila Kc cells using high-density oligonucleotide arrays. The resulting high-resolution maps show that HP1 binds along entire transcription units, except for 5вЂ™ regions. Comparison with expression data shows that most of these genes are actively transcribed. HP1 target genes are also marked by the histone variant H3.3 and dimethylated histone 3 lysine 4, which are both typical of active chromatin. Interestingly, H3.3 deposition, which is usually observed along entire transcription units, is limited to the 5вЂ™ ends of HP1-bound genes. Thus, H3.3 and HP1 are mutually exclusive marks on active chromatin. These results demonstrate that HP1-chromatin is transcriptionally active and has extensive links with several other chromatin components. We also employed DamID to identify four novel heterochromatin proteins in Drosophila. A yeast-2-hybrid screen indicated that three of these proteins (HP3, HP4, and HP5) interact directly with HP1, while HP6 in turn binds to each of these three proteins. Immunofluorescence microscopy and DamID mapping of in vivo binding sites confirmed that all four proteins are components of heterochromatin. Depletion of HP1 causes redistribution of these proteins, indicating that HP1 is essential for their heterochromatic targeting. Finally, mutants of HP4 and HP5 are dominant suppressors of position effect variegation, demonstrating their importance in heterochromatic gene silencing. We propose that HP1 acts as a docking platform for several mediator proteins that contribute to heterochromatin function. We have adapted the DamID technology for use in human cells and used it to identify human target genes of heterochromatin proteins. The three HP1 homologs (HP1a, b and g) show a very similar binding pattern, consistent with their ability to form heterodimers. Surprisingly, the HP1 proteins bind to most members of a very large family of genes that encode KRAB domain zinc finger (KRAB-ZNF) proteins. Because most of these KRAB-ZNF genes in the human genome are located on Chromosome 19, we constructed a high-density oligonucleotide tiling array with 60mer probes arranged every 200bp along all non-repetitive regions of this 64Mb chromosome. DamID mapping with these arrays revealed that HP1b covers very large chromosomal domains (some more than 1Mb in size) that harbor clusters of KRAB-ZNF genes (Figure V.5). We suggest that heterochromatin has played a key role in the evolution of this unusually large family of regulatory genes. 2006; 11:775-789 Gilfillan GD, Straub T, De Wit E, Greil F, Lamm R, Van Steensel B, Becker PB. Chromosome-wide gene-specific targeting of the Drosophila dosage compensation complex. Genes Dev 2006; 20:858-870 Greil F, Moorman C, Van Steensel B. DamID: Mapping of In Vivo ProteinGenome Interactions Using Tethered DNA Adenine Methyltransferase. Methods Enzymol 2006; 410:342-359 Moorman C, Sun LV, Wang J, De Wit E, Talhout W, Ward LD, Greil F, Lu XJ, White KP, Bussemaker HJ, Van Steensel B. Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster. Proc Natl Acad Sci USA 2006; 103:12027-12032 Pickersgill H, Kalverda B, De Wit E, Talhout W, Fornerod M, Van Steensel B. Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet 2006; 38:1005-1014 Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, De Wit E, Van Steensel B, De Laat W. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet 2006; 38:1348-1354 Tolhuis B, Muijrers I, De Wit E, Teunissen H, Talhout W, Van Steensel B, Van Lohuizen M. Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster. Figure V.5: DamID binding map of human HP1b (CBX1) along entire chromosome 19 in human MCF7 Nat Genet 2006; 38:694-699 cells. Note that CBX1 forms very large domains, which coincide almost perfectly with clusters of KRABZNF genes. Vogel MJ, Guelen L, De Wit E, Peric-Hupkes D, LodГ©n M, Talhout W, Bioinformatics Bioinformatics tools are essential for the detailed understanding of our genome-wide datasets (now amounting to millions of datapoints). We are actively developing new algorithms and approaches for the systematic analysis of the protein binding patterns that we obtained. In particular, we focus on predicting interactions between chromatin components by comparative analysis, and on the identification of multi-gene chromatin domains. Integration with external datasets (gene expression profiles, genome annotation, sequence motifs) allows us to make functional predictions that can be further tested in our laboratory. In addition, we design our own custom oligonucleotide microrarrays. Feenstra M, Abbas B, Classen AK, Van Steensel B. Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res 2006; 16:1493-1504 58 MOLECULAR BIOLOGY BIOINFORMATICS AND STATISTICS Group leader Lodewyk Wessels Lodewyk Wessels PhD Group leader Nicola Armstrong PhD Academic staff Michael Hauptmann PhD Academic staff Miranda Mandjes PhD Post-doc The research focus within our group is to develop novel computational approaches to exploit a wide variety of data sources in order to improve stratification of cancers into biologically distinct types that are well correlated with outcome and therapy response. In order to achieve this goal, significant effort is devoted to a better understanding of disease development and progression by identification of genes and pathways involved in tumorigenesis. Challenging computational problems that are being addressed include (i) construction of efficient cross-species mappings to translate results obtained in mouse model systems to human tumors; (ii) developing algorithms to efficiently integrate heterogeneous data sources and (iii) extracting structural pathway information from measured data sources and annotation data bases. Carmen Lai MSc Graduate student Wouter Meuleman MSc Graduate student Jeroen De Ridder MSc Graduate student Martin Van Vliet MSc Graduate student Publications Armstrong NJ, Mc Peek MS, Speed TP. Incorporating interference into linkage analysis for experimental crosses. Biostatistics 2006; 7:374-386 Armstrong NJ, Brodnicki TC, Speed TP. Mind the gap: Analysis of marker-assisted breeding strategies for inbred mouse strains. Mamm Genome 2006; 17:273-287 Extracting oncogenes and oncogenic pathways from insertional mutagenesis screens An effective method for identifying candidate oncogenic loci is retroviral insertion mutagenesis. Within a large set of tumors induced by retroviral infection, a subset of viral insertions from independent tumors that map to the same genomic locus is referred to as a Common Insertion Site (CIS). CISs often represent lesions that have been selected for during tumorigenesis. To automatically detect CISs we have developed a computational approach, the kernel convolution framework. This approach finds CISs using a predefined significance level while controlling the family-wise error and takes bias stemming from preferential viral insertion sites into account. In contrast to existing approaches, our method operates at any biologically relevant scale, providing new insights in the behavior of CISs across multiple scales. To find oncogenic lesions which are collaborating events in tumorigenesis, we extended the kernel convolution framework to detect the occurrence of multiple independent insertions within one tumor at a higher frequency than expected by chance (Fig. V.6). A novel, fast biclustering algorithm which finds subsets of tumors with similar insertion profiles will also be employed to extract pathway structures from the insertion data. Prioritizing candidate oncogenes based on genomic data Gene expression data of tumor series have been extensively employed to predict outcome and response to therapy in breast cancer. To uncover the underlying mechanisms that drive these expression signatures, comparative genomic hybridization and microarray gene expression data has been collected for a series of breast tumors representing the intrinsic subtypes. We developed SIRAC, a computational approach which detects genomic regions that are significantly enriched for BAC clones highly correlated with a particular subtype. We also developed KC-SMART, a generalization of the kernel convolution framework to detect genomic regions that are more frequently aberrated in a set of tumors, than one would expect by chance. In contrast to SIRAC, this approach is unsupervised, i.e. it does not require a labeling of the tumor set according to subtypes. Both approaches employ expression data to prioritize genes in a given aberrated region. Application of KC-SMART to a set of p53 deficient mouse tumors resulted in the detection of well-known oncogenes such as c-Met as well as promising new candidates. Figure V.6: Co-occurence plot for all the data in the Retroviral Tagged Cancer Gene Database (RTCGD). The green dots indicate the insertion co-occurences, the red dots mark the locations of the common cooccurrences of insertions. The arrows indicate the gene pairs accociated with a selection of the top-scoring co-occurerneces, Knowledge-based approaches for outcome prediction Algorithms which exploit biological knowledge, such as the functional grouping of genes, could lead to more accurate predictors and enlarge the possibility of generating novel insights in the disease. Over-fitting can also be overcome by constructing larger datasets by collecting related gene expression data sets in a compendium. We employed an unsupervised approach to derive a set of modules (groups of functionally related genes with coordinated expression across a subset of tumors). By employing the module activity as input for training breast cancer outcome predictors, we revealed functional modules associated with breast cancer outcome. We studied modules extracted from several compendia: single breast cancer datasets, breast cancer compendia, and a human cancer compendium. We validated these predictors on independent data from the same institution and other institutions. Modules derived from single breast cancer datasets achieve outperform regular gene-based predictors. 59 MOLECULAR BIOLOGY Publications (continued) Modules derived from a single breast cancer dataset and a cancer specific compendium performs better compared to those derived from a human cancer compendium. De Ridder J, Uren A, Kool J, Reinders M, Wessels L. Detecting Statistically Significant Common Insertion Sites in Mass spectrometry-based response prediction Poly(ADP-ribose) polymerase (PARP)-inhibitors in combination with the DNA cross-linker, cisplatin, have recently been shown to have great potential as a treatment for patients carrying germline BRCA1 or BRCA2 mutations. The research questions we address include whether we are able to find markers for вЂњBRCAnessвЂќ and PARP-inhibitor treatment response. We are doing so by following an incremental procedure. First, we performed a comprehensive comparison of currently used, as well as novel mass spectrometry normalization approaches involving six mass spectrometry datasets, three classification approaches and 17 normalization techniques. Based on the results of this comparison, we constructed a computational workflow to process mass spectra. Next, using SELDI-TOF mass spectrometry, we study the proteomic contents of cell lysates and growth media of both BRCA(1,2)/p53-/- and p53-/- cell lines. When appropriate biomarkers have been identified, we will also analyze cell lysates from spontaneous tumors from BRCA1 and BRCA2 deficient mouse models. Simultaneously, we will study sera from these tumor bearing mice, to investigate the relationship between the markers obtained from tumor tissue and markers present in serum. If all these steps have been successfully completed, the approach can be tested on human tissue from BRCA1 and BRCA2 carriers. In addition to this, we hope to gain a better understanding of the pathways affected by PARP-inhibitors by integrating proteomic data with transcriptomic data, yielded by microarray expression profiling. Retroviral Insertional Mutagenesis Screens. Evaluating effects of timing of exposure on cancer risk The effects of timing of exposure are rarely evaluated in detail in epidemiologic studies of chronic exposures. Cumulative exposure is usually used as the major exposure metric, while age at exposure, time since exposure, or intensity of exposure are often ignored. Using advanced statistical risk modeling, we showed a significantly increased breast cancer risk from smoking before a first full-term pregnancy, particularly before menarche, in a large cohort study. These results suggest that the sensitivity of the female breast to tobacco carcinogens is increased during adolescence and early adulthood but decreases after first childbirth when most breast tissue has terminally differentiated. In an analysis of smoking data from ten case-control studies, including cancers of the lung, bladder, oral cavity, pancreas and esophagus, we demonstrated enhanced carcinogenic potency of smoking at lower intensities, but narrow ranges for packyears increased uncertainty, precluding definitive conclusions. At higher intensities, there was a consistent reduced potency effect across studies. Intensity patterns were comparable across the diverse cancer sites. Lai C, Reinders M, Van вЂ�t Veer L, PLoS Comput Biol 2006; 2:e166 Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DSA, Nobel AB, Van вЂ�t Veer LJ, Perou CM. Concordance among geneexpression-based predictors for breast cancer. N Engl J Med 2006; 355:560-569 Glas AM, Floore A, Delahaye LJMJ, Witteveen AT, Pover RCF, Bakx N, Lahti-Domenici JST, Bruinsma TJ, Warmoes MO, Bernards R, Wessels LFA, VanвЂ™t Veer LJ. Converting a breast cancer microarray signature into a highthroughput diagnostic test. BMC Genomics 2006; 7:278 Knijnenburg TA, Reinders MJT, Wessels LFA. Artifacts of Markov blanket filtering based on discretized features in small sample size applications. Pattern Recogn Lett 2006; 27:709-714 Wessels L. A comparison of univariate and multivariate gene selection techniques for classification of cancer datasets. BMC Bioinformatics 2006; 7:235 Linet MS, Hauptmann M, Freedman DM, Alexander BH, Miller J, Sigurdson AJ, Morin Doody M. Interventional radiography and mortality risks in U.S. radiologic technologists. Pediatr Radiol 2006; 36 Suppl 14:113-120 Van den Broek GB, Balm AJM, Van den Brekel MWM, Hauptmann M, Schornagel JH, Rasch CRN. Relationship Crossover interference in linkage analysis During meiosis, chromosomes replicate, pair and then synapse. Once pairing is complete, crossing over, the reciprocal exchange of chromosomal segments among non-sister chromatids begins. Crossover interference is the non-random placement of the crossover points along the length of the chromosome. While many models for this process have been proposed, the majority of genetic mapping studies still ignore the phenomenon of interference and assume the crossover locations come from a Poisson process. We have implemented the chi2 model for interference in both simulation studies for breeding programs and in an extension of the Lander-Green algorithm for genetic mapping. In simulation studies, this model was shown to be more accurate than the other methods for estimating genetic distances. between clinical factors and the incidence of toxicity after intra-arterial chemoradiation for head and neck cancer. Radiother Oncol 2006; 81:143-150 Van Houwelingen HC, Bruinsma T, Hart AAM, VanвЂ™t Veer LJ, Wessels LFA. Cross-validated Cox regression on microarray gene expression data. Stat Med 2006; 25:3201-3216 60 TUMOR BIOLOGY VI DIV IS ION OF TU M OR BIOLOG Y ANTIGEN PRESENTATION BY MHC CLASS I AND MHC CLASS II MOLECULES Division head, Group leader Jacques Neefjes Jacques Neefjes PhD Group leader Tom Groothuis PhD Post-doc Christoph Lippuner PhD Post-doc Marije Marsman PhD Post-doc Victoria Menendez PhD Post-doc Veronica Monserrat PhD Post-doc Alexander Griekspoor MSc Graduate student Tineke Van den Hoorn MSc Graduate student Coen Kuijl MSc Graduate student Joost Neijssen MSc Graduate student Baoxu Pang MSc Graduate student Petra Paul MSc Graduate student Nuno Rocha MSc Graduate student Izhar Salomon MSc Graduate student Lennert Janssen BSc Technical staff Publications Bergink S, Salomons FA, Hoogstraten D, Groothuis TAM, De Waard H, Wu J, Yuan L, Citterio E, Houtsmuller AB, Neefjes J, Hoeijmakers JHJ, Vermeulen W, Dantuma NP. DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A. Genes Dev 2006; 20:1343-1352 Dantuma NP, Groothuis TAM, Salomons FA, Neefjes J. A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling. J Cell Biol 2006; 173:19-26 Elliott T, Neefjes J. The Complex Route to MHC Class I-Peptide Complexes. Cell 2006; 127:249-251 Griekspoor AC. Single cell biochemistry to visualize antigen presentation and drug resistance. Leiden: Leiden University, 2006 Groothuis T, Neefjes J. The ins and outs MHC class I molecules alert the immune system for intracellular infections or cellular alterations related to tumour formation. The killer cells in the immune system can recognize a small fragment of an altered or foreign protein presented in the context of MHC class I molecules at the cell surface and subsequently respond by eliminating such cells. This system is central in tumour vaccination studies and we study the basic machinery. For successful presentation of fragments of tumour proteins by MHC class I molecules, first fragments have to be generated and these have to be transferred to MHC class I molecules after transport over the ER membrane. We have defined most steps in this process. Proteins are degraded into fragments by the proteasome. Therefore proteins have to be tagged by a small protein called Ub and the polyUb form acts as a recognition signal by the proteasome. We have studied the dynamic nature of the Ub modification in living cells using GFP-tagged Ub. We show that free mono-Ub is in fact rate limiting and the majority of Ub is observed associated to histones2 and associated to large protein complexes. Acute need of Ub (following proteasome inhibition or heat shock) results in more poly-Ub modified proteins, but the Ubs required for this can not be delivered through the rate limiting small Ub pool. Instead, histones are de-Ub resulting in altered chromatin structure and transcription. We are currently studying the details of an analogous event, histone deUb following exposure to topo-isomerase inhibitors like doxorubicin, an often-used anti-cancer drug. Histone-deUb can be an additional mechanism of tumour cell growth control. We have studied other factors controlling MHC class I presentation. One major factor appears to be ionizing radiation that upregulate MHC class I expression. We have identified the molecular basis for this effect and shown that tumours are subsequently better-recognized following radiation. This вЂ�radioimmunotherapyвЂ™ is a new combination therapy for better anti-tumour immune responses and will be further studied in model systems for further improvements. MHC class II molecules work complementary to MHC class I molecules by sampling fragments of proteins degraded in the endocytic track. We have determined and visualized most steps in this process including the control of motor protein-driven MHC class II vesicle transport, interactions with intracellular pathogens and interactions with lysosomal chaperones. We have developed small molecular compounds that affect intracellular growth of pathogens and identified the molecular targets. This is the first successful application of вЂ�reciprocal chemical geneticsвЂ™ and in fact integrates biology, genetics and chemistry to arrive at lead compounds for defined biological effects and corresponding targets. Similar approaches are followed to define molecular targets affecting antigen presentation by MHC class I- and MHC class II molecules. This year we have developed a new combination therapy combining radio- and immunotherapy that strongly improves anti-tumour responses. This combination therapy will be further developed. In addition, we have developed strategies combining chemistry, biology and genetics to identify novel compounds, define corresponding targets and define new signalling pathways, in this case in host responses controlling pathogen growth. This approach will be developed further. of intracellular peptides and antigen presentation by MHC class I molecules. Curr Top Microbiol Immunol 2006; 300:127-148 Groothuis TAM, Dantuma NP, Neefjes JJ, Salomons FA. Ubiquitin crosstalk connecting cellular processes. Cell Division 2006; 1:21 Antigen presentation by MHC class I molecules MHC class I molecules present a sample of the peptides produced intracellularly by the proteasome. But how is the proteasome degraded? We, in collaboration with Dr Fred van Leeuwen (Div of Cellular Biochemistry), have established a method to genetically switch colours on yeast proteasomes and this will be used to establish proteasome half-life. By combining this with the yeast mutant libraries, we will identify proteins determining proteasome half-life. Many factors probably determine the efficiency of antigen presentation by MHC class I molecules. Ionizing radiation induces the level of 61 TUMOR BIOLOGY Publications (c0ntinued) intracellular peptides by either oxidizing proteins resulting in their degradation or by activating the mTOR pathway. Radiated cells are better recognized and killed by the immune system. Other factors could also contribute to MHC class I antigen presentation. We have generated and directly conjugated monoclonal antibodies detecting two conformations of MHC class I molecules at the cell surface. This is combined with selective inactivation of defined proteins by siRNA followed by automated FACS. Potential targets modifying MHC class I expression, can be used for isolation of small molecular compounds to manipulate MHC class I expression and many immune responses. For a proper cytotoxic T cell response, antigen information has to be delivered to professional antigen presenting cells like dendritic cells, a process called crosspresentation. We have recently identified gap junctions as being able to transfer such information in the form of peptides from one cell to its neighbour. However, apoptosis conditions may be ideal for the induction of cross-presentation. We have first confirmed whether antigen presentation continues during apoptosis and that long gap-junction communication persues after apoptosis induction. We are currently testing whether cells in apoptosis are able to transfer peptides through gap junctions to healthy neighbouring cells for cross-presentation. Our approach is to understand the details of MHC class I antigen presentation and, identify targets and develop methods to improve immune responses to tumour cells. Groothuis TAM. Manipulations of the ubiquitin proteasome system and their effects on antigen presentation. Leiden: Leiden University, 2007 Herberts CA, Neijssen JJ, De Haan J, Janssen L, Drijfhout JW, Reits EA, Neefjes JJ. Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: Coupling cytosolic peptide stability to antigen presentation. J Immunol 2006; 176:2697-2701 Jordens I, Westbroek W, Marsman M, Rocha N, Mommaas M, Huizing M, Lambert J, Naeyaert JM, Neefjes J. Rab7 and Rab27a control two motor protein activities involved in melanosomal transport. Pigment Cell Res 2006; 19:412-423 Antigen presentation by MHC class II molecules and the control of endocytosis MHC class II molecules meet antigens and their degradation fragments in the endocytic track followed by transport to the plasma membrane before presentation of the antigenic fragments to the immune system. We are studying various aspects of this process. We have used FRET and FLIM technology to visualize the interaction between MHC class II molecules and the lysosomal chaperone HLA-DM in living cells. These data revealed that HLA-DM and MHC class II interacted only in the internal vesicles rather than the limiting membrane of the late endosomal structures called MIIC. Bacterial-induced phagosomes are devoid of internal vesicles and we showed (both by FRET and by biochemistry) that MHC class II molecules fail to acquire antigen in phagosomes. We are currently studying the molecular basis of this spatial specialization of MIIC with respect to antigen loading of MHC class II molecules. Using FRET, we have visualized the organization of tetraspannin networks that also acquire MHC class II and HLA-DM molecules and determined their redistribution within MIIC structures. This is further studied in detail. We have design a вЂ�reciprocal chemical geneticsвЂ™ approach to identify leads and targets that вЂ“in this study- determine intracellular growth of pathogens like Salmonella and M.tuberculosis. Therefore we synthesized chemical kinase inhibitor homologue libraries to identify a вЂ�chemical profileвЂ™ and combined this with siRNA libraries for the human kinome and automated microscopy to identify possible targets. This revealed the PKB/Akt pathway as controlling intracellular growth of pathogens acting via a novel pathway that is currently studied in detail. The вЂ�reciprocal chemical geneticsвЂ™ approach allows rapid identification of lead and targets and allowed the identification of new signalling pathways downstream of the relevant anti-cancer target, PKB/Akt. To identify other targets controlling antigen presentation by MHC class II molecules, we establish a screen where antigen presentation was followed with two antibodies recognizing two conformations of MHC class II molecules; the successfully peptide loaded form and the form containing the invariant chain remnant CLIP. A kinome library was used followed by automated FACS analyses with the two antibodies. Of the 779 targets tested, only three reproducibly affected antigen presentation by MHC class II molecules. These are now tested in more detail to reveal the molecular mechanism. We have studied the molecular mechanism that control transport of MHC class II molecules from MIIC to the plasma membrane. We had already identified a Rab7 effector protein called RILP that recruited the dynein motor complex to MIIC preventing transport to the plasma membrane. We now have shown that the small GTPase Rab7 interacts with two proteins simultaneously: RILP to recruit the dynein motor by interacting with the stalk p150glued and the oxysterol-binding protein ORP1L that transfers the Rab7-RILP-dynein motor complex to the general dynein receptor spectrin b3. Only then, active minus-end driven transport of late endosomes is observed. Reits EA, Hodge JW, Herberts CA, Groothuis TA, Chakraborty M, Wansley EK, Camphausen K, Luiten RM, De Ru AH, Neijssen J, Griekspoor A, Mesman E, Verreck FA, Spits H, Schlom J, Van Veelen P, Neefjes JJ. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J Exp Med 2006; 203:1259-1271 Figure VI.1: Model for regulation of microtubule-based dynein motor-driven minus-end transport of late endocytic compartments. Active Rab7 localizes to membranes of late endocytic compartments and recruits the RILP and ORP1L effectors to form a tripartite complex. The dyneindynactin motor is recruited to this complex, via an interaction between RILP and p150Glued, and subsequently transferred to the membrane-associated spectrin receptor, a process facilitated by ORP1L. 62 TUMOR BIOLOGY microRNAs, RNA INTERFERENCE AND CANCER Group leader Reuven Agami Reuven Agami PhD Group leader Mathijs Voorhoeve PhD Senior post-doc Geetha Achanta PhD Post-doc Qing Chang PhD Post-doc Suresh Nair PhD Post-doc Eitan Zlotorynski PhD Post-doc Jarno Drost MSc Graduate student Anja Duursma MSc Graduate student Martijn Kedde MSc Graduate student Remco Nagel MSc Graduate student Carlos Le Sage MSc Graduate student Josyanne Van Duijse MSc Graduate student Mariette Schrier PhD Technical staff Joachim Oude Vrielink BSc Technical staff Figure VI.2: Identification of miR-372 and miR-373 that bypass oncogene-induced senescence. A. A flow-chart of the screen. Cells transduced with the miR-Lib were Our main research objective is to understand the cancerous process in humans and identify essential genes. As functional units, the knowledge obtained on these genes allows us to design novel therapeutic ways. Most human tumors harbor multiple genetic alterations that activate oncogenes, inhibit tumor suppressors and induce genomic instability. As each tumor contains many genetic alterations, the study of the contribution of each alteration to the cancerous phenotype was obscured. In the past, we developed a vector-based system that mediates suppression of gene expression through RNA interference (pSUPER). We have successfully applied this gene-inactivation system to study the importance of tumor-suppressors and DNAdamage checkpoints in protecting humans from cancer. As a result, the pSUPER RNAi system is very frequently used in the lab to address emerging inquiries. For example, lately we used pSUPER vectors to characterize a telomerase independent regulation of ATR by human telomerase RNA (hTR). We also investigated with this system the Cdc6- dependent effect exerted by p53 on DNA replication. Last, we recently developed and applied high-throughput pSUPER-RNAi libraries to uncover novel tumor suppressors and DNA-damage checkpoint genes, and characterized their role in cancer prevention. This year we began to study the connection between microRNAs (miRNAs) the new emerging family of genes and cancer. We developed a novel miRNA expression vector (miR-Vec), miRNA library (miR-Lib) and a corresponding barcode array (miR-Array). We used these functional-genetic tools to uncover and describe cancerous miRNAs. At present, a large body of the group is devoted to the development and usage of novel genetic approaches to identify and characterize miRNAs involved in cell cycle control, cell migration and tumor metastasis. Functional genetic screens identify miR-372&3 as potential oncogenes in Testicular Germ Cell tumors To identify cancer-related functions of microRNAs (miRNAs) we performed functional genetic screens. Initially, we constructed miRVec, a retroviral vector for miRNA expression. Based on that vector, we created a human miRNA expression library (miR-Lib) containing the majority of annotated human miRNAs (~450), and produced a corresponding microarray (miR-Chip) containing all miR-Lib inserts. We used these tools to detect miRNAs that bypass oncogene-induced senescence. In response to mitogenic signals from oncogenes, primary human cells irreversibly arrest in a phenotype coined senescence. Primary human cells lacking functional p53 and p16 proteins efficiently escape this arrest and acquire a transformed phenotype. Importantly, this escape from oncogene-induced senescence is a prerequisite for full transformation into tumor cells. We found that the expression of either miR-372 or miR-373 granted cells with tumorigenic growth in the presence of oncogenic stress, while still harboring wild type p53. This effect was due to a miR372/3-induced blockade of the ability of the p53 pathway to inhibit cell cycle dependent kinases (CDK) activities in response to oncogenic stress. This effect was partly through direct miR-372/3-induced suppression of LATS2 (LArge Tumor Suppressor gene) expression. Finally, we provided evidence that miR-372&3 are potential novel oncogenes participating in the development of human Testicular Germ Cell tumors. We found mutual exclusive expression of miR-372&3 and p53 mutations. For details on our recent work see Voorhoeve et al 2006. Altogether, beyond demonstrating the potential of miRNAs to support cellular transformation, our results validated the feasibility and applicability of miRNA-genetic screens and ascertained our capability to identify relevant miRNA target genes and relevant cellular pathways. We are now exploiting other miRNA genetic screens to identify additional cancerous miRNAs. grown for two to three weeks in the presence or absence of RASv12. Subsequently, the population of inserts in each condition was recovered and compared using a barcode miR-array. B. Three independent miR-Array experiments were performed. The position of the reproducibly upregulated miR-Vecs is indicated for each experiment. An RNAi screen identifies PITX1 as a suppressor of RAS activity and human tumorigenicity In the past, we have developed a vector-based system termed pSUPER that mediates stable suppression of gene expression through RNAi. With this system we demonstrated that persistent inactivation of a dominant-RAS oncogene results in the loss-of tumorigenicity of late-stage cancer-cell lines. We also studied the tumor-suppressive functions of the human INK4A locus, which encodes for the p16INK4A and p14ARF genes and is inactivated in many cancers. We showed 63 TUMOR BIOLOGY Publications that p16INK4A, but not p14ARF, is the major tumor suppressor of the human INK4A locus. Recently, a large collection of pSUPER vectors for the knockdown of up to 8000 genes in human cells was assembled in our institute. We used this tool to identify and characterize novel human-tumor suppressor genes. Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or another pathway is deregulated. To uncover novel tumour-suppressor genes, we screened the NKI-RNAi library for knockdown constructs that transform human primary cells in the absence of ectopically introduced oncogenic RAS. We identified PITX1 whose inhibition induces the RAS pathway and tumorgenicity. Interestingly, we observed low expression of PITX1 in prostate and bladder tumours and in colon cancer cell lines containing wild-type RAS. Restoration of PITX1 in the colon cancer cells inhibited tumorigenicity, in a wild-type RAS dependent manner (Figure 2). Finally, we identified RASAL1, a RAS-GTPase-activating protein, as a transcription target through which PITX1 affects RAS function. Thus, PITX1 suppresses tumorgenicity by down-regulating the RAS pathway through RASAL1. Kedde M, Le Sage C, Duursma A, Zlotorynski E, Van Leeuwen B, Nijkamp W, Beijersbergen R, Agami R. Telomerase-independent Regulation of ATR by Human Telomerase RNA. J Biol Chem 2006; 281:40503-40514 Le Sage C, Agami R. Immense promises for tiny molecules: Uncovering miRNA functions. Cell Cycle 2006; 5:1415-1421 Voorhoeve PM, Le Sage C, Schrier M, Gillis AJM, Stoop H, Nagel R, Liu YP, Van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LHJ, Agami R. A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors. Cell 2006; 124:1169-1181 Voorhoeve PM, Agami R. Classifying microRNAs in cancer: The good, the bad and the ugly. BBA Rev Cancer 2006; Figure VI.3: Mutual occurrence of miR-372&373 expression In Press and p53 mutation in testicular germ cell tumors. A summary of miR-372 and miR-373 expression as well p53 status in several TGCT cell lines and primary seminomas (in the latter only exons 5 to 8 were examined). Telomerase independent regulation of ATR by human telomerase RNA Using the pSUPER technology we recently uncovered a novel, telomerase independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation. The human telomerase RNA (hTR) together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. We therefore set to investigate the effects of changes in hTR cellular levels. Surprisingly, inhibition of hTR expression by RNAi triggered a rapid, telomerase independent, growth arrest that was associated with p53 and CHK1 activation. Moreover, increased expression of hTR neutralized activation of these DNA damage pathways. The hTR effects were mediated through ATR and were sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. p53-dependent regulation of Cdc6 protein stability controls cellular proliferation Probably all tumors harbor a certain degree of genomic instability, underling the tight connection between DNA damage responses (repair and checkpoint) and tumorigenesis. In the past, we have shown that the DNA-damage response to ionizing radiation is composed of two processes: initiation and maintenance. DNA damage causes a fast G1-arrest by rapidly degrading cyclin-D1. Later, this initial G1-response to DNA damage is maintained and further strengthened by the stabilization of p53, leading to the induction of p21cip1. Activation of the tumor suppressor p53 in response to genotoxic stress imposes cellular growth arrest or apoptosis. We identified Cdc6, a licensing factor of the pre-replication complex (pre-RC), as a novel target of the p53 pathway. We show that activation of p53 by DNA damage results in enhanced Cdc6 destruction by the anaphase-promoting complex (APC). This destruction is triggered by inhibition of CDK2 mediated CDC6 phosphorylation at serine 54. Conversely, suppression of p53 expression results in stabilization of Cdc6. We show that loss of p53 results in more replicating cells, an effect that can be reverted by reducing Cdc6 protein levels. Our data suggest that initiation of DNA replication is regulated by p53 through Cdc6 protein stability. Figure VI.4: hTR enhances the expression of fragile sites. (A) Quantification of the expression of fragile sites in U2OS transfected with expression vectors for hTR, shRNA for ATR, and ctrl. The number of fragile sites expressed in 400 chromosomes is shown, data are representative of three independent experiments. (B) Pictures exemplifying the extent of fragile sites in the various transfected U2OS cells. Pictures were made with 1000x magnification. 64 TUMOR BIOLOGY DYNAMIC INTERACTIONS AT THE NUCLEAR ENVELOPE Group leader Maarten Fornerod Maarten Fornerod PhD Group leader Stijn Heessen PhD Post-doc Helen Pickersgill PhD Post-doc Noelia Valle PhD Post-doc The nuclear envelope arguably is the most important border within the eukaryotic cell. Borders in general are stable, but can reflect the dynamics of an entire organization. This makes them an attractive platform from which to study a complex system вЂ“ such as the eukaryotic cell. The current focus of the lab is nuclear transport, chromatin dynamics at the nuclear envelope and connections between these two processes. Nuclear transport Nucleocytoplasmic transport is accomplished by distinct classes of soluble transport receptors that interact with transport signals and the nuclear pore complex. We investigate how this fundamental process is used in the regulatory circuits of the cell. Rafael Bernad-Fernandez MSc Graduate student Dieuwke Engelsma MSc Graduate student Jolita Hendriksen MSc Graduate student Bernike Kalverda MSc Graduate student Hella Van der Velde BSc Technical staff CRM1-mediated nuclear export Exportins are nuclear export receptors that bind their cargoes cooperatively with RanGTP. They are responsible for multiple protein and ribonucleoprotein export pathways. Present evidence suggests that most exportins export one or a few structurally related groups of substrates. The main exception is CRM1/Exportin1, that exports multiple functionally unrelated protein and ribonucleoprotein cargoes, and can be thought of as the only вЂњmulti-purposeвЂќ exportin identified to date. Structure-function of the nuclear pore complex The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. So far, very few FG nucleoporins have been described to be essential for specific transport pathways: mutation mostly results in a slight inhibition of a subset of pathways. One of the most stable interactions between a nucleoporin and a nuclear transport receptor is the Nup214 interaction with CRM1/exportin 1, which occurs at the C-terminal FG repeat region. This led to the idea that this interaction is important for CRM1-mediated nuclear export. We tested this by depletion of Nup214 in cultured cells. Perhaps surprisingly, no major defect in nuclear export of simple CRM1 cargoes was observed. In contrast, depletion of Nup214 results in a dramatic reduction of 60S preribosomal nuclear export. However, the stable CRM1/Nup214 interaction is not involved in this: the central coiled-coil domain of Nup214 is able to rescue 60S nuclear export. This suggests that nucleoporin Nup214 plays two distinct roles at the NPC: it participates redundantly in the hydrophobic barrier with its FG-rich domain, and plays a non-redundant structural role that is essential for 60S preribosomal export. From the inside looking in: chromatin dynamics at the nuclear lamina Since the late 19th century, studies of interphase nuclei have indicated that the arrangement of chromosomes within the nucleus is non-random. Many observations suggest that the nuclear envelope plays a key role in the spatial and functional organization of chromatin. In close collaboration with the lab of Bas van Steensel (Dept. of Molecular Genetics), we are analyzing the behaviour of genes in respect to various nuclear envelope components. The nuclear lamina (NL) has long been thought to be an anchoring site of chromatin and to participate in the regulation of gene expression, but genomic sequences that interact with the NL in vivo have remained unknown. We used a genome-wide approach to identify nearly 500 Drosophila genes that interact in vivo with the NL component Lam, a B-type lamin. 65 TUMOR BIOLOGY Publications Bernad R, Engelsma D, Sanderson H, Pickersgill H, Fornerod M. Nup214-Nup88 Figure VI.5: Genome-wide analysis of Lamin/ nucleoporin subcomplex is required for chromatin interactions. 1. Lam binding genes CRM1-mediated 60 S preribosomal nuclear identified by Lamin DamID are in dynamic contact export. J Biol Chem 2006; 281:19378-19386 with the nuclear periphery, as revealed by fluorescence in situ hybridization FISH. 2. Large Pickersgill H, Kalverda B, de Wit E, intergenic regions, transcriptional inactivity and lack Talhout W, Fornerod M*, Van Steensel B*. of active histone marks and late replication are Characterization of the Drosophila contributing factors in Lam binding, as indicated by melanogaster genome at the nuclear lamina. genome-wide comparisons to various chromatin Nat Genet 2006; 38:1005-14. factors. * co-corresponding authors Genes that interact with Lam are transcriptionally silent, devoid of histone modifications typical of active chromatin, late replicating, and widely spaced (Figure VI.5). These factors collectively account for a large proportion of Lamin binding behavior, suggesting the NL acts as an integrator of different types of genomic and chromatin features. Lam target genes cluster in the genome, and genes within each cluster are coordinately activated at specific stages of development. These results provide global insights into the dynamic organization of the genome at the NL, and imply that intergenic or вЂњjunkвЂќ DNA functions in the global organization of chromatin in the nucleus. 66 TUMOR BIOLOGY ESTROGEN RECEPTOR AND BREAST CANCER Group leader Rob Michalides Rob Michalides PhD Group leader Mariska Rondaij PhD Senior Post-doc Wilbert Zwart MSc Graduate Student Desiree Verwoerd Technical staff Cristiane Toaldo Technical staff Publications Griekspoor A, Margarido TC, Zwart W, Michalides R. Review of: BRCA1 and cyclin D1: Gate keepers in hormone responsive tissues? Breast Cancer Online 2006; 9 Griekspoor AC. Single cell biochemistry to visualize antigen presentation and drug resistance. Leiden: Leiden University, 2006 Estrogen Receptor a (ERa)-positive breast cancer patients are commonly treated with tamoxifen. Resistance to tamoxifen treatment is observed in about half of the recurrences in breast cancer where the anti-estrogen tamoxifen acquires agonistic properties for transactivation of ERa. Tamoxifen resistant breast cancer is still sensitive to other anti-estrogens, all with compound specific properties and behavior. The clinical benefits of clarifying pathways underlying resistance can therefore be profound. To study the molecular mechanism of anti-estrogen resistance, we measured conformational changes in ERa by fluorescence resonance energy transfer (FRET). Using an YFP-ER -CFP construct, interaction between the CFP and YFP can be measured by FRET, where a change in fluorescent ratios implies a conformational change of the receptor. When anti-estrogens bind the receptor, it adapts an alternative conformation, which can be visualized using our FRET approach. Using this assay, we studied the efficacy of anti-estrogens to (in)activate ER under different experimental conditions, and have found that Protein Kinase A- (Michalides et al, Cancer Cell 2004) or PAK1-mediated (Rayala et al, Cancer Res, 2006) phosphorylation of Serine 305 of ERa is responsible for resistance to tamoxifen. Activation of PAK1, a downstream mediator of Rac, is therefore also involved in tamoxifen resistance. In addition, we have shown that this phosphorylation of Serine 305 in ERa leads to an altered orientation between ERa and its coactivator SRC-1, which renders the transcription complex active in the presence of tamoxifen. This altered orientation is measured by a change in intermolecular FRET between the C-termini of ERa and SRC-1 and requires interaction between the AF-1 domain at the N-terminus of ERa and SRC-1. An altered orientation between a transcription factor and its cofactor due to phosphorylation of a specific site within a tamoxifen-bound ERa provides a novel mechanism of resistance. Figure VI.6: Model for resistance to tamoxifen by PKA or PAK-1 mediated phosphorylation of ERa. The phosphorylation alters orientation between ERa and cofactor SRC1 in such a way that RNA polymerase II is recruited and transcription is sustained. Since tumors that are resistant to tamoxifen might still be sensitive to treatment with other anti-estrogens, we determined a profile of resistance to 10 different antiestrogens based on accumulation of specific modification(s) of ERa by PKA, either or not in combination with increased expression of cofactors cyclin D1 and SRC-1. Tamoxifen and EM-652 appeared in this profile as the most sensitive to resistance development and Fulvestrant and ICI 164.384 as the most stringent anti-estrogens in this analysis. These FRET findings are supported by transcriptional activation of ER. This approach reveals modifications in ERa that induce resistance to anti-estrogens and defines as such conditions for consecutive application of anti-estrogens. These FRET-based approaches are not directly applicable to clinical specimen, since they require transfection of life cells. Also, only few cells in a tumor may have become resistant to tamoxifen, which would easily be undetermined by FRET techniques. We therefore developed, in collaboration with Upstate Inc, an antibody that specifically detects the phosphorylated Serine 305 ERa in clinical breast tumor samples and which could detect tamoxifen resistance in a minority of the tumor cells. Together with M.Kok and S.Linn, dept of Molecular Pathology, we will evaluate whether staining with this antibody can predict tamoxifen resistance in breast cancer patients. 67 TUMOR BIOLOGY CRYO-IMMUNOGOLD ELECTRON MICROSCOPY AND 3D CRYO-ELECTRON TOMOGRAPHY IS OUR METHODOLOGY TO EMBARK ON AN EXPEDITION TOWARDS A PSEUDOATOMIC ATLAS OF THE ENDOCYTIC SYSTEM Supramolecular architecture in an unperturbed cellular context We now know that assemblies of ten or more proteins carry out almost all processes in a cell. As it performs its functions, each of these assemblies interacts with several other large protein complexes. Indeed, the cell can be viewed as a factory that contains a complex network of interlocking assembly lines, each of which is composed of a set of large protein machines. In order to understand the machinery of life and the mechanisms of disease the three-dimensional structures of many proteins are resolved at an increasing pace by X-ray crystallography and NMR. However, it is the assembly of proteins into molecular machines and the dynamic interactions between proteins, which determine their function and activity. The next great challenge will therefore be to understand the three-dimensional architecture of cells, in terms of the spatial arrangement of organelle-bound protein complexes, and to describe quantitatively how this arrangement alters as the cells respond to stimuli. On a scale of a few nanometers the cell with very dynamic massive supramolecular assemblies is largely an uncharted territory. Just as high-resolution 3D structures of macromolecules provide valuable insights into their working, a better understanding of cellular functions will arise from the ability to visualize supramolecular architecture in an unperturbed cellular context. Our ultimate goal is to interpret the structure and dynamics of the molecular networks. We are now working on new methods to provide 3D maps of cellular components at molecular resolution. There is currently immense excitement among us and our collaborators, since ongoing advances in cryo-sectioning of native material at ultra low temperature as well as cryo-EM instrumentation and computational methods may ultimately make it possible to obtain resolutions in the range of 3 nm, i.e. potentially high enough to locate individual proteins in a cell. To arrive at this technology, we are leading an intense and international group of collaborators that include Helmut Gnaegi (Diatome, Biel Switzerland), Felix de Haas (FEI electron optics, Eindhoven), Nico Sommerdijk (University of Eindhoven), Henny Zandbergen (University of Delft), Peter Frederik (University Maastricht) and Bram Koster (University Leiden). Group leader Peter Peters Peter Peters PhD Group leader Bea Krenn PhD Project manager Sue Godsave PhD Post-doc Pekka Kujala PhD Post-doc Pieter Res PhD Post-doc Nicole Van der Wel PhD Post-doc Diane Houben Graduate student Jason Pierson Graduate student Erik Bos MSc Technical staff Hans Jansen BSc Technical staff Martijn Romeijn BSc Technical staff Maaike Van Zon BSc Technical staff Nico Ong Research assistant Prions Prion diseases are fatal and presently incurable neurodegenerative diseases caused by the abnormally folded form (PrPSc) of the cellular prion protein (PrPc). In collaboration with Stanley PrusinerвЂ™s lab in San Francisco, we have been developing methods to specifically detect PrPSc and PrPc, using cryo-immunogold EM. Quantification of labeling with an antibody that recognizes only PrPc suggests that the level and distribution of PrPc is relatively unchanged in prion-infected hippocampus, while another antibody that recognizes both PrPc and (a subset of) PrPSc gives increased labeling in the neuropil, both on the plasma membrane and in early endocytic vesicles of axons and dendrites. Our results of last year support the idea that PrPc to PrPSc conversion may occur at one of these sites. Synapses may be an early target of prion disease and synapse numbers are reduced by more than fifty per cent during the disease incubation period. Interestingly, we have not found any evidence for PrPSc accumulation at synapses, although PrPC is present. PrPSc has been reported to exist both in protease-sensitive and relatively insensitive forms. We have found that a large proportion of the disease-associated labeling detected in our experiments can be removed by trypsin. The significance of these different PrPSc forms for disease is unknown, but it is likely that protease sensitive PrPSc exists in lower aggregation states and it may be the most relevant for both toxicity and infectivity. Early stages of prion pathogenesis. In the earliest phase orally acquired PrPSc must cross the gut epithelium to reach the germinal centres (GCs) of gut associated lymphoid tissues. We show by microscopical techniques that PrPSc can be detected as a short transient pulse in follicle associated epithelium (FAE) of PeyerвЂ™s patches already one day after infection. Most PrPSc was found in large endosomes of FAE enterocytes, but not in the M cells. After crossing the epithelia PrPSc was found accumulating Figure VI.7: FAE endosomes visualized by cryo immunogold electronmicroscopy. Ultrathin cryosection was labeled against lamp-1 and detected with Protein A conjugated to 10 nm colloidal gold. Bar 500 nm. 68 TUMOR BIOLOGY Publications Klingenstein R, LГ¶ber S, Kujala P, Godsave S, Leliveld SR, Gmeiner P, Peters PJ, Korth C. Tricyclic antidepressants, quinacrine and a novel, synthetic chimera thereof clear prions by destabilizing detergent-resistant membrane compartments. J Neurochem 2006; 98:748-759 Korth C, Peters PJ. Emerging pharmacotherapies for Creutzfeldt-Jakob disease. Arch Neurol 2006; 63:497-501 Figure VI.8: Follicle associated epithelium (FAE) enterocytes have larger endosomes compared to the enterocytes in the villus. Dotted lines represent the approximate location of brush borders both in the FAE and in the villus. Endosomes of wt mouse are labeled against late endosomal marker lamp-1 and the nuclei with dapi. intracellularly in large vacuoles of macrophage/dendritic type cells in the subepithelial dome (SED) just underneath the FAE. In the course of infection some of the DC/macrophages were later found in increasing numbers at the GCs. In the few following days the PrP expression gradually increased at the surface of follicular dendritic cells in the GCs of scrapie infected wild type mice, but not in the non-infected controls, or infected PrPcвЂ“deficient mice. Interestingly, increase of PrP expression on the FDCs was not limited to the GCs of PeyerвЂ™s patches, but was simultaneously also observed in GCs of mesenteric lymph nodes, whereas in the spleen no changes during the first 21 days post infection were observed. In the PeyerвЂ™s patches finding of PrP together with traces of epithelial membrane marker A33 suggests that the infectious agent makes itвЂ™s journey from gut lumen into the GCs of PeyerвЂ™s patches at least partially membrane bound, probably in association with membrane domains originating from the FAE. Macrophages/DCs appear to be strong candidates for assisting the PrP transport from FAE to GC. Mycobacteria In the last year we discovered that one of the most successful human bacterial pathogens, Mycobacterium tuberculosis, has a different localization than the vaccine strain M. bovis BCG used world wide to prevent tuberculosis infections and bladder cancer. When BCG infects cells the bacteria remain localized in a membrane enclosed compartment which the bacteria can modify in such a way that it can survive and replicate. The current dogma is that M. tuberculosis localizes in a similar compartment that is thought to be optimized for the persistence and replication of the infectious bacteria. However we have recently established that at later in infection the bacteria leave such a compartment to enter the cytosol of the host cell. Here it has access to a nutritious environment in which replication appears to occur at a higher rate than in the membrane enclosed compartments in which it resides at the earlier phase of the infection. We have determined which proteins of the pathogenic M. tuberculosis are facilitating the translocation (patented discovery). Importantly M. bovis BCG lacks those proteins and consequently remains localized in the cellular phagosome. This has implications for the recognition by the immune system. The immune system loads parts of the bacteria (antigens) in specialized antigen presenting molecules (MHC I, MHC II and CD1) that with the antigen can trigger T-lymphocytes that then can activate an immune response. These antigenpresenting molecules load antigens in different compartments of a cell. MHC II and CD1 load their antigens in membrane-enclosed compartments of the endocytic pathway while MHC I obtains its antigens from the cytosol. M. bovis BCG is unable to present its antigens via the cytosol as it remains restricted in a membrane-enclosed compartment. M. tuberculosis however traffics through all the compartments where loading occurs and thus can trigger immune responses via all classes of antigen presentation. In conclusion, we can now explain the discrepancy between the immune response induced by M. bovis BCG and M. tuberculosis, which was for a long time one of the most important questions in the field of vaccine development. This at the same time may give us a tool to improve the BCG vaccine by redirecting the bacteria to the cytosol. This will be tested with modified BCG strains. POSTDOCTORAL AFFAIRS Peter Peters has also been active as dean of postdoctoral affairs. For more details see: http://www.mekentosj.com/postdocs. Our work and our institute was highlighted in: Aschwanden C. Professionalizing the postdoctoral experience. Cell. 2006; 124:445-7 and Aschwanden C. Learning to lead. Cell. 2006; 125:407-9. 69 MOLECULAR GENETICS VI I DI VISION OF MOL E CU LA R GE NE T ICS ROLE OF POLYCOMB-GROUP GENES IN TRANSCRIPTIONAL REPRESSION, STEM CELL FATE AND TUMORIGENESIS Our lab has a long-standing interest in epigenetic gene regulation dictated by chromatin modifications. We study the mechanism of stable inherited transcriptional repression by Polycomb-group (Pc-G) protein complexes, and the effects of deregulation of Pc-G genes on development, Cell cycle control, cancer formation and stem cell maintenance. In addition, we are performing large-scale genetic screens in primary cells and in cancer-predisposed mice to identify cancer-relevant combinations of collaborating oncogenes and tumor-suppressor genes. Model organisms comprise Mouse, Drosophila and Zebrafish. Division head, Group leader Maarten Van Lohuizen Maarten Van Lohuizen PhD Group leader Anna Pavlina Haramis PhD Research associate Elisabetta Citterio PhD Senior Post-doc Jean Bernard Beaudry PhD Post-doc Koen Braat PhD Post-doc Jaap Kool PhD Post-doc Karim Nacerdine PhD Post-doc Functional characterization of Pc-G protein complexes Repressive Pc-G proteins and counteracting Trithorax-group (Trx-G) of nucleosome remodeling factors are involved in maintenance of proper gene expression patterns during development at the level of chromatin structure. Pc-G protein complexes control large sets of genes including Hox gene clusters and the INK4a/ARF tumor suppressor locus. At least two biochemical distinct evolutionary highly conserved Pc-G protein complexes can be distinguished. The first (PRC2) contains EzH1/EzH2 (SET domain proteins acting as Histone H3 methylases), Su(z)12, Eed and histone deacetylases. The second large complex (PRC1) encompasses Bmi1/Mel18, M33/MPc2, Mph1/Mph2 and Ring1b/ Ring1a together with other more loosely associated proteins is required throughout development. To study Pc-G function we focus on representative members of PRC1 and PRC2 in gain- and loss-of-function studies in mice, zebrafish and Drosophila cell lines. In genetic and biochemical experiments we recently identified the Bmi1/ Ring1b heterodimer as an E3 ubiquitin ligase for monoubiquitination of histone H2A (collaboration with G Buchwald and T Sixma, Division II). This enzymatic activity is part of the core PRC1 complex and conditional Ring1b loss-of-function experiments indicate an essential role for maintenance of Pc-G repression in development and stem cell maintenance. In cell biological experiments encompassing live cell microscopy, FRET and FLIP of tagged Pc-G proteins we revealed the highly dynamic and cell cycle regulated nature of Pc-G proteins in sub-nuclear domains called Polycomb bodies (collaboration with T Groothuis and J Neefjes, Division VI). In addition, we demonstrated the requirement for the PRC2 EZH2 methyltransferase and the DNA methylase DNMT1 to maintain PRC1 proteins properly localized. This provides new insights in how Pc-G repression is propagated. A major unresolved question is where and how Pc-G complexes bind to chromatin. We recently completed a genome-wide survey of where PRC1 and PRC2 complexes bind to the Drosophila genome using DAMid profiling on cDNA and tilling arrays (collaboration with B Van Steensel, Division V). Combined with ChIP and unbiased quantifiable gene onthology analyses this highlights binding of both PRC1 and PRC2 to distinct domains of 10-140 Kb containing В± 400 target genes. These comprise developmental regulators, transcriptional repressors as well as key stem cell signaling pathways such as Wnt, Hh, Notch as well as several new distinct functional categories and provides unprecedented new insight in Pc-G function. Alexandra Pietersen PhD Post-doc Anke Sparmann PhD Post-doc Bas Tolhuis PhD Post-doc Bart Westerman PhD Post-doc Sophia Bruggeman MSc Graduate student Panthea Taghavi MSc Graduate student Petra Van der Stoop MSc Graduate student Yme Van der Velden MSc Graduate student Marleen Blom Technical staff Danielle Hulsman Technical staff Sonja Noback Technical staff Asheeta Prasad Technical staff Ellen Tanger Technical staff Hans Teunissen Technical staff Els Verhoeven Technical staff Joep Vissers Technical staff Huub Van Vugt Technical staff Publications Bruggeman SWM, Van Lohuizen M. Controling stem cell proliferation: CKIs at work. Cell Cycle 2006; 5:1281-1285 Buchwald G, Van der Stoop P, Weichenrieder O, Perrakis A, Van Lohuizen M, Sixma TK. Structure and E3-ligase activity of the Ring-Ring complex of Polycomb proteins Bmi1 and Ring1b. EMBO J 2006; 25:2465-2474 Fujimura YI, Isono KI, Vidal M, Endoh M, Kajita H, Mizutani-Koseki Y, Takihara Y, Van Lohuizen M, Otte A, Jenuwein T, Connections between Pc-G gene repression, control of stem cell fate and Cancer formation We originally identified Bmi1 as an oncogene that cooperates with cMyc in the induction of B and T cell lymphomas in mice, underscoring the connection between deregulation of Pc-G repression and cancer. In contrast, Bmi1 knockout mice show severe progressive proliferation defects and increased apoptosis of lymphoid and myeloid cells, resulting in severe lymphopenia. In addition, also primary embryo fibroblasts (MEFs) and neurons in the cerebellum of Bmi1 knockouts show proliferation defects. We demonstrated that these defects are due to increased levels of the tumor suppressors p16INK4a and p19ARF, that in turn are critical regulators of the RB and the p53 tumor suppressor pathways. As such, the INK4a/ARF locus acts as an important tumor-prevention mechanism in normal cells Deschamps J, Koseki H. Distinct roles of Polycomb group gene products in transcriptionally repressed and active domains of Hoxb8. Development 2006; 133:2371-2381 Haramis AP, Hurlstone A, Van der Velden Y, Begthel H, Van den Born M, Offerhaus GJA, Clevers HC. Adenomatous polyposis coli-deficient zebrafish are susceptible to digestive tract neoplasia. EMBO Rep 2006; 7:444-449 70 MOLECULAR GENETICS Publications (continued) Haramis AP, Perrakis A. Selectivity and promiscuity in Eph receptors. Structure 2006; 14:169-171 Hosokawa H, Kimura MY, Shinnakasu R, Suzuki A, Miki T, Koseki H, Van Lohuizen M, Yamashita M, Nakayama T. Regulation of Th2 cell development by Polycomb group gene bmi-1 through the stabilization of GATA3. J Immunol 2006; 177:7656-7664 Oguro H, Iwama A, Morita Y, Kamijo T, Van Lohuizen M, Nakauchi H. Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice. J Exp Med 2006; 203:2247-2253 Pirrotta V, Van Lohuizen M. Differentiation and gene regulation. Genomic programs and differentiation. Curr Opin Genet Dev 2006; 16:443-446 Sparmann A, Van Lohuizen M. Polycomb and stem/precursor cells. Our recent results highlight the relative importance of p19ARF and p16INK4a in different cell types. In addition, using the well-established mammary fat pad transplantation model, we revealed the essential role of Bmi1 in mammary epithelial precursor cell proliferation and ductal tree development, again at least in part implicating INK4a/ARF as a critical target in this developmental cell system. A key characteristic of cancer cells is their unlimited self-renewal. In this respect, cancer cells resemble stem cells, and accumulating evidence suggests that many forms of cancer may indeed contain cells carrying stem cell markers. In studying the proliferation defects and progressive loss of cells in Bmi1 deficient mice we discovered that Bmi1 is required for proliferation and self-renewal of neural stem cells. Importantly, loss of the INK4a/ARF locus rescues the proliferation & renewal defects, indicating it also is a critical Pc-G target in neural stem cells. Using a transplantable Glioma model we recently demonstrated a critical role for Bmi1 in maintenance of brain tumor formation (Figure VII.1). Interestingly, Bmi1 acts in this tumor setting in an Ink4a/ARF-independent manner on cell adhesion and migration. These results, together with the recently established role of Bmi1 in hemapoeitic stem cells and leukaemic stem cells, suggest a common conserved role for Bmi1-containing Polycomb complexes in maintenance and expansion of stem cells or committed progenitors and in the pathogenesis of tumors originating from the neoplastic transformation of these cells. The possible broader relevance of these findings for human cancer is further underscored by the amplification of BMI1 in Mantle cell lymphomas and the overexpression of BMI1 in various tumor types including non-small cell lung cancer, breast cancer and liver cancer. Conditional transgenic- and knockout models are currently used to investigate the role of Pc-G genes in various tissue stem/progenitors and in solid cancers that develop in these tissues. silencers control cell fate, development and cancer. Nat Rev Cancer 2006; 6:846-856 Steele JC, Torr EE, Noakes KL, Kalk E, Moss PA, Reynolds GM, Hubscher SG, Van Lohuizen M, Adams DH, Young LS. The polycomb group proteins, BMI-1 and EZH2, are tumour-associated antigens. Br J Cancer 2006; 95:1202-1211 Taghavi P, Van Lohuizen M. Developmental biology: Two paths to silence merge. Nature 2006; 439:794-795 Tolhuis B, Muijrers I, De Wit E, Teunissen H, Talhout W, Van Steensel B, Van Lohuizen M. Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster. Nat In vivo and in vitro genetic screens to identify new groups of collaborating oncogenes or tumor suppressors In close collaboration with A Berns (this Division), J Jonkers (Division V) and A Bradley (The Sanger Center, Hinxton, UK) we have developed high-throughput insertional mutagenesis techniques and are now extending and optimizing these types of screens to other cancer relevant models such as breast cancer. We have also developed in vitro suppressor screens, to screen for collaborating sets of oncogenes and tumor suppressor genes. One such a screen is aimed at identifying genes contributing to tumor-progression. Hereto, combinations of predisposing oncogenes such as TBX2, Myc or RasV12 are introduced in MEFs or conditionally immortalized human primary epithelial cells prior to transduction with tumor-derived retroviral cDNA libraries or RNAi-hairpin pRETROSUPER libraries. Transformed clones are subsequently selected in semi-solid medium, and the relevant genes are isolated using efficient PCR strategies. So far we have isolated several known and novel candidate oncogenes that cooperate with Myc, one novel transcription factor that transforms in combination with RasV12 and two RNAiвЂ™s directed at chromatin-regulating genes. The role and mechanism of action of these new putative oncogenes or tumor suppressors, and their interference with adhesion and invasion of primary mouse and human cells is under investigation. Genet 2006; 38:694-699 The zebrafish as a new model to study cancer and development The zebrafish provides a powerful animal model for genetic analysis of vertebrate embryogenesis, organ development and disease and is recently beginning to be exploited as a cancer model. Furthermore, its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. We use LKB1-deficient zebrafish as a model to investigate if and how LKB1 function may cause neoplastic transformation through disruption of epithelial polarization in a vertebrate organism. In addition, through overexpression and knock-down or knock-out methodologies we will investigate the roles of Polycomb genes during embryonic development, stem cell maintenance and cancer in zebrafish. Figure VII.1: Severely reduced Glioma formation of Bmi1-/- transformed astrocytes. Survival curves indicate that astrocytes oncogenically transformed by loss of INK4a/ARF and activation of EGF-receptor signaling rapidly form agressive gliomas whereas tumor formation is delayed upon transplantation of Bmi1-deficient transformed astrocytes ortholopically transplanted in the forebrain of recipient mice. 71 MOLECULAR GENETICS MOUSE MODELS FOR CANCER The mouse is used as a model organism for establishing the role of oncogenes and tumor suppressor genes in tumor development. Using Cre/Lox mediated switching, expression of multiple oncogenes and tumor suppressor genes can be regulated in a tissue-specific and spatial-temporal fashion. This permits both a more accurate modeling of tumorigenesis as it occurs in man as well as the analysis of specific genotype-phenotype correlations. These models also provide an excellent experimental tool to test prevention and intervention strategies especially when combined with sensitive in vivo imaging techniques. Finally, these models permit us to identify new oncogenes and tumor suppressor genes involved in tumor progression using a variety of techniques, such as array CGH, expression profiling and proviral insertional mutagenesis. Some of the gene families identified in our models are being studied in depth. Group leader Anton Berns Anton Berns PhD Group leader Paul Krimpenfort PhD Academic staff Margriet Snoek PhD Academic staff Jan Hermen Dannenberg PhD Research associate Jacqueline Jacobs PhD Research associate Joan Boren PhD Post-doc Functional analysis of oncogenes and tumor suppressor genes We utilize mice carrying combinations of different oncogene and conditional tumor suppressor gene alleles to model a range of tumors. Our current focus is on lung cancers, mesotheliomas and prostate cancer. To achieve (sporadic) activation of oncogenes and inactivation of tumor suppressor genes we use Adeno-Cre virus to switch the conditional alleles. Subsequently, tumor growth is monitored by noninvasive imaging using a conditional luciferase reporter. Joaquim Calbo PhD Post-doc Hilda De Vries PhD Post-doc Annemieke IJpenberg PhD Post-doc Johan Jongsma PhD Post-doc Martijn Nawijn PhD Post-doc AndrГ© Bergman MD PhD KWF fellow Kate Sutherland PhD Post-doc Anthony Uren PhD Post-doc Lung tumors We focus on small cell lung cancer (SCLC). When Rb and p53 are inactivated specifically in lung, SCLC ensues. The marker profile of these tumors is indistinguishable from that of human SCLC. The tumors also metastasize to the same sites. Array CGH showed frequent amplification of L-Myc further supporting their resemblance with the human counterpart. Additional small amplicons were found and the genes in these amplicons are under study. We have established a series of cell lines from these SCLC that show either neuroendocrine or stem cell like features. Interestingly, these cell lines show common lesions indicating that they share a common progenitor. The cell lines with neuro-endocrine features reproduce SCLC upon orthotopic grafting whereas the cell lines with stem cell features do not. We are following up several transcriptional regulators that are critical for the induction for neuroendocrine differentiation. The cell lines appear relative resistant to cytotoxic drugs. Therefore we are now investigating whether this is also the case for primary tumor cells that have not been propagated extensively in vitro. Mesotheliomas Inactivation of Nf2 and Ink4a has been reported in a subset of human mesotheliomas. This has served as a basis to design a mesothelioma mouse model. We have inactivated Nf2 in combination with either Ink4a/p19Arf, p53, or p53/ Ink4a loss by Adeno-Cre infection of the mesothelial lining of the thoracic and intraperitoneal cavity. Mesotheliomas rarely occurred upon Nf2 inactivation. Concomitant loss of Ink4a/p19Arf or p53 strongly accelerated mesothelioma development. We have derived luciferase expressing cell lines from these tumors, which upon intrathoracic inoculation, reproduce mesotheliomas. We have investigated whether the nature of the introduced defects (p53 or Ink4a/p19Arf loss) results in a different resistance profile of the various cell lines. So far we found no evidence for this. A substantial variation in drug resistance was found. Currently we are investigating the basis of this. RenГ©e Van Amerongen PhD Post-doc Andrej Alendar MSc Graduate student Jaco Van der Torre MSc Graduate student Erwin Van Montfort MSc Graduate student Colin Pritchard MSc Research assistant Rahmen Bin Ali Technical staff Ruben Gaasbeek Technical staff Jules Gadiot Technical staff Jan Paul Lambooij Technical staff Natalie Proost Technical staff Fina Van de AhГ© Technical staff John Zevenhoven Technical staff Figure VII.2: Dysfunctional telomeres impact on DNA damage signaling. Prostate cancer Overexpression fo Myc is observed in a subset of human prostate tumors, and is associated with elevated expression levels of Pim kinases. Concurrent overexpression of Pim1 and cMyc induces a uniform atypical hyperplasia in the mouse prostate, indicating that Pim kinase activity is rate-limiting for the early stages of Myc-driven transformation. We are currently testing the effect of Pim-deficiency on Myc-driven prostate cancer. Ink4 proteins We have shown previously that loss of Ink4a accelerates carcinogeninduced melanomas and that Ink4a deficiency synergizes with hemizygosity of p19Arf and p53. We have now disrupted Ink4a, Ink4b and p19Arf together, a situation often Figure VII.3: Some of requently found common insertion sites in MuLV-induced mouse lymphomas. 72 MOLECULAR GENETICS Publications Berns A. Cancer biology: Can less be more for p53? Nature 2006; 443:153-154 Calbo J, Meuwissen R, Van Montfort E, seen in human tumors. The resulting mice show no developmental abnormalities but appear highly tumor prone. Mice show a preponderance of squamous cell carcinomas and mesotheliomas, next to a low incidence of other tumors. The dramatic tumor predisposition of loss of both Ink4a and Ink4b can most likely be attributed to a backup function that Ink4b appears to fulfill for Ink4a. Van Tellingen O, Berns A. GenotypePhenotype Relationships in a Mouse Model for Human Small-Cell Lung Cancer. Cold Spring Harb Symp Quant Biol 2005; 70:225-232 De Ridder J, Uren AG, Kool J, Reinders MJT, Wessels LFA. Detecting Statistically Significant Common Insertion Sites in Retroviral Insertional Mutagenesis Screens. PLoS Comput Biol 2006; 2:e166 Derksen PW, Liu X, Saridin F, Van der Gulden H, Zevenhoven J, Evers B, Van Beijnum JR, Griffioen AW, Vink J, Krimpenfort P, Peterse JL, Cardiff RD, Berns A, Jonkers J. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 2006; 10:437-449 Huijbers IJ, Krimpenfort P, Chomez P, Van der Valk MA, Song JY, Inderberg-Suso EM, Schmitt-Verhulst AM, Berns A, Van den Eynde BJ. An inducible mouse model of melanoma expressing a defined tumor antigen. Cancer Res 2006; 66:3278-3286 Peeper D, Berns A. Cross-Species Identification of new oncogenic pathways Proviral insertional mutagenesis screens in normal and tumor prone mice marks genes instrumental in the tumorigenesis. Using this approach a large range of oncogenes have been found. In collaboration with the groups of Maarten Van Lohuizen, Lodewyk Wessels, Jos Jonkers, and John Hilkens our current focus is saturating proviral insertional mutagenesis in tumor suppressor knockout mice. With the help of the Sanger Centre we have sequenced the insertion sites of approximately 1000 tumors. This yielded close to 600 significant common insertion sites that mark oncogenes and tumor suppressor genes. Bioinformatical analysis identified a large number of new potentially haploinsufficient tumor suppressor genes and a vast number of specific co-mutations suggesting that there are strict requirements for the activation of cooperating pathways. In addition we continue to study two gene families that were found in previous screens: Pim and Frat. Pim family kinases are highly expressed in strong collaboration with Myc in a range of human malignancies, including prostate carcinoma and lymphomas. Germline deletion of Pim kinases induces a minimal phenotype, making these kinases potentially suitable for therapeutic intervention. The redundancy of Pim kinases and other intracellular signaling pathways in the context of Myc-driven transformation is being analyzed in both lymphoid and epithelial cell context. In addition, we are characterizing one of the novel genes with putative remodeling function that was found in a complementation tagging experiment in Pim1/2 knockout mice. FRAT proteins (FRAT1-3) bind to and inhibit the GSK3b kinase and have therefore been implied as critical mediators of canonical Wnt signaling, resulting in the activation of b-catenin. Indeed, overexpression of FRAT proteins induces b-catenin/ TCF signaling, with FRAT1 and 3 being more potent inducers than FRAT2. Remarkably, mice, lacking all three frat genes are healthy and fertile and do not display gross abnormalities. In line with this, Wnt signaling in primary cells of these tumors appears unaffected. Therefore, Frat is dispensable for mammalian development and not essential for canonical Wnt-signaling in higher organisms. We are currently putting emphasis on the role of the FRAT proteins in non-canonical Wnt signaling, in which the three Frat genes show different activities. Oncogenomics in Cancer Gene Identification. Cell 2006; 125:1230-1233 Shakhova O, Leung C, Van Montfort E, Berns A, Marino S. Lack of Rb and p53 delays cerebellar development and predisposes to large cell anaplastic medulloblastoma through amplification of N-Myc and Ptch2. Cancer Res 2006; 66:5190-5200 Telomeres Loss of telomere function triggers a DNA damage response that results in apoptosis or growth arrest that depends on both p53 and p16INK4a. In addition, dysfunctional telomeres result in genome instability due to DNA repair activities that act on chromosome ends. We are interested in what cellular activities act on chromosome ends, how telomeres control or protect against these activities and what the precise nature of the telomere damage signal is. To investigate this we take both a candidate approach as well as a genome-wide screening approach using cDNA and RNAi libraries to identify and functionally characterize novel genes with a role in the telomere damage response. Central in these approaches is the disruption of telomere function by inhibiting the telomere binding protein TRF2. Van Amerongen R, Berns A. Knockout mouse models to study Wnt signal transduction. Trends Genet 2006; 22:678-689 Histone deacetylases The identification of drug targets whose inhibition will result in selective killing of tumor cells is highly desirable in new anti-cancer therapies. Histone deacetylases (HDACs) seem to fulfill the criteria for such drug targets. Despite promising results obtained in clinical trials using HDAC-inhibitors (HDACi) many of these drugs are targeting several HDACs and show off-target effects resulting in undesired toxicities. Using (tumorprone) mice carrying HDAC1 and HDAC2 conditional knockout alleles we wish to discover anti-tumor relevant HDAC targets and study HDAC1/2 specific and redundant functions in transcription regulation, normal and tumor development and tumor maintenance. Studies aimed at unraveling the transcriptome of these transcriptional co-repressors in combination with the identification of new components of the HDAC1/2 mediated pathways may reveal new drug targets for anti-cancer therapy. 73 MOLECULAR GENETICS CELLULAR PROTECTION AGAINST ONCOGENIC TRANSFORMATION AND METASTASIS Introduction The Peeper laboratory is dedicated to resolving mechanisms that protect mammalian cells against oncogenic transformation, aiming to identify novel cancer drug targets. Non-malignant cells require multiple genetic mutations to transform into tumor cells. With both classical biochemical and genetic approaches, as well as with functional screens (based on both cDNA expression and RNA interference genome-wide libraries), we have been focusing on two of these events: bypass of oncogene-induced growth arrest (вЂ�premature senescenceвЂ™, which may limit the proliferative capacity of cells) and suppression of вЂ�anoikisвЂ™ (apoptosis resulting from lack of adhesion, suppression of which may contribute to metastasis). In addition, we are studying the potential role of senescence as an in-vivo mechanism protecting against tumorigenesis. We anticipate that these approaches, together, will lead to the identification of a network of gene products, deregulation of which allows for tumorigenesis and metastasis. In turn, these proteins may represent novel therapeutic drug targets. Group leader Daniel Peeper Daniel Peeper PhD Group leader Christophe Desmet PhD Post-doc Cornelia HГ¶mig PhD Post-doc Alexandre Prieur PhD Post-doc Remco Van Doorn MD PhD Clinical fellow Thomas Geiger MSc Graduate student Thomas Kuilman MSc Graduate student Chrysiis Michaloglou BSc Graduate student Marjon Smit MSc Graduate student Function-based genomic screens for metastasis genes Metastasis commonly underlies the malignancy of cancers, representing the principal cause of cancertreatment failure. Tumor colonization is prevented by several physiologic barriers, including вЂ�anoikisвЂ™: apoptosis resulting from lack of adhesion. Indeed, acquiring resistance to anoikis has been proposed to represent a general prerequisite for survival of metastases during circulation. In an attempt to identify metastasisassociated oncogenes we designed an unbiased, functional genome-wide screen solely based on anoikis suppression. With this approach, we previously identified TrkB, a neurotrophic tyrosine kinase receptor, as a potent suppressor of anoikis. Whereas non-malignant intestinal epithelial cells underwent caspase-associated anoikis in vitro, this was completely prevented by TrkB, allowing formation of spheroid aggregates growing in suspension. Mice readily cleared parental cells, but TrkB-expressing cells formed many lung and heart metastases. Co-expression of BDNF, the prototypic TrkB ligand, caused highly invasive metastases in virtually every tissue, with an extremely short latency. We and others recently identified cancer-associated mutations of TrkB of which we are currently analyzing the biological significance. We have also begun to identify downstream signaling targets of this receptor, as they, in addition to TrkB, may serve as targets for therapeutic intervention. RNAi knockdown of some of these newly identified targets impairs the tumorigenic potential of TrkB. We have also performed a structure-function analysis of TrkB, identifying specific domains involved in anoikis suppression and metastasis. Finally, we are setting up mouse models, to further explore the role of TrkB in cancer and metastasis, and to serve as a model system for TrkB in cancer, enabling the development and testing of TrkB-inhibitory therapeutic drugs. Function-based genomic screens to identify novel oncogenes In approximately 30% of all human tumors, the RAS GTPase is mutated at a specific residue (e.g., G12V), as a result of which it is locked into a constitutively active state. Surprisingly, rather than proliferating indefinitely upon overexpression of RASV12, non-malignant cells undergo proliferative arrest. RASV12-induced arrest resembles вЂ�senescenceвЂ™, which may act as a tumor-suppressing mechanism that protects cells against oncogenic transformation. We have designed functional genomic screens to identify novel oncogenes that allow cells to bypass RASV12-induced senescence, as well as to identify oncogenes that cooperate with RASV12 to achieve full transformation. This successful approach has led to the independent identification of a number of new oncogenes, some of which are discussed below. One of the genes we identified in this manner encodes DRIL1, a transcription factor of the ARID-domain family of retinoblastoma protein-interacting proteins. We have observed that DRIL1 immortalizes primary murine fibroblasts, rendering them unresponsive to by p19ARF, p53, p21CIP1 and p16INK4a. Furthermore, we have observed that, in contrast to what is seen in mouse cells, DRIL1 induces cell cycle arrest in primary human fibroblasts. We have carried out a structure-function analysis, Liesbeth Vredeveld MSc Graduate student Sirith Douma MSc Technical staff Figure VII.4: A schematic representation of structure-function TrkB mutants. Several mutations were introduced into TrkB, which were examined for their effects on anoikis suppression in vitro and tumor formation in vivo. 74 MOLECULAR GENETICS Publications Desmet CJ, Peeper DS. The neurotrophic receptor TrkB: a drug target in anti-cancer therapy? Cell Mol Life Sci 2006; 63:755-759 Mooi WJ, Peeper DS. Oncogene-induced cell senescence - Halting on the road to cancer. N Engl J Med 2006; 355:1037-1046 Peeper DS, Berns AJ. Cross-Species Oncogenomics in Cancer Gene Identification. Cell 2006; 125:1230-1233 Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 2006; 6:11-23 Figure VII.5 : Genome-wide identification of novel tumor suppressor genes. Primary human cells were transduced with retrovirus encoding the mutant, melanoma-associated BRAFE600 gene. Subsequent changes in the expression pattern of cellular genes were used to identify genes antagonizing the growth-stimulatory action of BRAFE600. This led to the identification of the interleukin IL-6, which was upregulated by BRAFE600. RNA interference of IL-6 abrogated BRAFE600-induced senescence, causing cells to proliferate. identifying the domains in DRIL1 that contribute to growth arrest and oncogenic transformation. We have also identified a set of cell cycle proteins that are associated with DRIL1induced arrest in human cells. Oncogene-induced senescence in melanocytic nevi as a melanomasuppressing mechanism Most normal mammalian cells have a finite lifespan, thought to constitute a protective mechanism against unlimited proliferation. This phenomenon (вЂ�senescenceвЂ™) is driven by telomere attrition, which triggers the induction of tumor suppressors, including p16INK4a. In cultured cells, senescence can be elicited also prematurely, by oncogenes. However, whether such OncogeneInduced Senescence (OIS) represents a physiological process has long been debated. Human nevi (вЂ�molesвЂ™), benign tumours of melanocytes, frequently harbor oncogenic mutations in BRAF (predominantly V600E). Nonetheless, nevi typically remain growth-arrested for decades and only rarely progress into malignancy (melanoma). This raises the question whether nevi undergo BRAFV600E-induced senescence. In a collaborative study with Drs. Mooi (VUmc, Amsterdam), Soengas (Michigan) and Shay (Texas), we found that sustained BRAFV600E expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of p16INK4a as well as of a commonly used senescence marker, Senescence-Associated acidic b-Galactosidase (SA-b-Gal) activity. Validating these results in vivo, congenital nevi are invariably SA-b-Gal-positive, showing for the first time this classical senescence-associated marker in a - largely growth-arrested - neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observe a striking mosaic induction of p16INK4a, suggesting that factors other than p16INK4a also contribute to protection against BRAFE600-driven proliferation. Finally, nevi do not appear to suffer from telomere attrition, arguing in favor of an active oncogenedriven senescent process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAFV600E-expressing melanocytes display classical hallmarks of senescence, suggesting that OIS represents a genuine protective physiological process. Currently, we are using a functional genomic approach to identify novel, p16INK4A independent, melanocyte-specific pathways whose deregulations contribute to melanoma. In addition to taking a candidate gene approach, we are combining gene expression analysis, RNAi and unbiased functional вЂ�barcodeвЂ™ screens. For example, we postulated that putative tumor suppressors, similar to p16INK4A, are transcriptionally upregulated during the senescence response. Microarray expression analysis revealed the induction of various signaling networks that are induced specifically in BRAFE600-expressing, senescent human cells. Among the top outliers was interleukin 6 (IL-6). RNAi-mediated knockdown of IL-6 abrogated the senescence response and led to continuous proliferation of BRAFE600-expressing cells. Similar findings were made for other interleukins, which prompted us to search for a common denominator of BRAFE600-regulated interleukins. We found that C/EBPb acts as a major player in this pathway. Consistently, knockdown of C/EBPb effectively bypassed BRAFE600-induced senescence. Our data suggest that the C/EBPb-IL6 axis acts as a critical mediator of BRAFE600-induced senescence. This integrative genomics approach may identify novel tumor suppressors involved in melanoma and other (BRAFE600-associated) cancers. 75 MOLECULAR GENETICS GENES INVOLVED IN BREAST CANCER PROGRESSION AND METASTASIS Transformation of a normal cell into a fully malignant cell requires several sequential genetic changes affecting key genes controlling various essential cellular pathways involved in growth and development. This multistep process of gradual increase in malignancy is called tumor progression. Complete knowledge of the genes and the pathways they control is essential for the development of more effective novel therapeutic strategies. Although a large number of genes that are involved in breast and other cancers has been discovered, the picture is far from complete. Retroviral insertional mutagenesis (IM) in mouse models is one of the most efficient tools to uncover cancer genes. The aim of our laboratory is to identify and study novel genes involved in breast cancer and unravel the collaborating genetic pathways leading to breast cancer and breast cancer progression using insertional mutagenesis in mouse models. Group leader John Hilkens John Hilkens PhD Group leader Vassiliki Theodorou MSc Graduate student Annabel Zwaagstra MSc Graduate student Mandy Boer Technical staff Publications Identification of novel genes involved in mammary tumor progression by MMTV tagging Mouse mammary tumor virus (MMTV) proviral insertion in the genomic DNA of murine mammary epithelial cells can activate flanking protooncogenes leading to mammary tumor induction by insertional mutagenesis. The availability of the almost complete mouse genome sequence and high-throughput PCR and sequencing technologies has greatly facilitated the identification of retrovirally-activated oncogenes. By using these technologies, we have searched for common MMTV integration sites (CIS) in primary tumors of MMTV infected BALB/c mice and a BALB/c knockout strain that conditionally lacks active p53 in the mammary gland. A CIS indicates the presence of a nearby oncogene involved in mammary tumorigenesis. We have performed a high-throughput retroviral insertional mutagenesis screen in more than 300 MMTV-induced mammary tumors that developed in both mouse strains and identified over 50 CIS. In wild type Balb/c mice we identified 33 CIS and in the p53 deficient mice we found an additional 10 CIS, while combining both studies led to a further 9 CIS. Although Wnts and Fgfs were frequently affected by proviral insertions, almost all novel genes are not members of these families. Approximately 50% of the tagged genes are novel candidate cancer genes not previously implicated in mammary or other cancers. Interestingly, a comparison of all our MMTV tagged genes in mammary tumors (including those tagged in only a single tumor) with all genes tagged by MoMuLV in lymphomas and brain tumors deposited in the Mouse Retrovirus Tagged Cancer Gene Database showed that both viruses targeted mostly different genes acting predominantly in distinct pathways. Indeed, our screen for MMTV common insertion sites reveals a novel repertoire of candidate oncogenes involved in mammary tumorigenesis. Rspo-genes and cancer Two genes, Rspo2 and Rspo3, that belong to a novel gene family, designated R-spondins, were among the most frequently tagged genes, after the Wnt and Fgf genes, in the mammary tumors. R-spondins belong to a highly conserved four member gene family. All four Rspo family members encode secreted molecules with a single thrombospondin type 1 and two furin domains, but the function of these molecules has not been established. RT-PCR analysis revealed that approximately 25% of all MMTV induced mammary tumors in BALB/c mice expressed one of the two former genes. Both genes were also frequently activated in human breast cancers. Rspo1 and Rspo2 are not expressed in normal resting mammary gland cells, in contrast Rspo1 is widely expressed in normal tissues including normal mammary cells and mammary tumors. In murine tumors Rspo2 or Rspo3 were often co-expressed with Rspo1. Moreover, Rspo2 or 3 were frequently activated in the same tumors that also overexpressed Wnt and Fgf genes suggesting that the Rspo genes collaborate with these oncogenes. Preliminary results show that immortalized normal mouse mammary epithelial cells (HC-11 cells) retrovirally transduced with Rspo3 cDNA were tumorigenic in nude mice (Figure VII.6), validating Rspo3 as a novel oncogene. Hilkens J. Recent translational research: oncogene discovery by insertional mutagenesis gets a new boost. Breast Cancer Res 2006; 8:102-5 Yu LG, Andrews N, Zhao Q, McKean D, Williams JF, Connor LJ, Gerasimenko OV, Hilkens J, Hirabayashi J, Kasai K, Rhodes JM. Galectin-3 Interaction with Thomsen-Friedenreich Disaccharide on Cancer-associated MUC1 Causes Increased Cancer Cell Endothelial Adhesion. J Biol Chem 2007; 282:773-781 Figure VII.6: HC11 cells (derived from normal mammary epithelium) retrovirally transduced with Rspo3 or Wnt1 cDNA or an empty retroviral construct (designated HC-Rspo3, HC-Wnt1 and HC-zeo respectively) were subcutaneously injected into the flanks of 5 to 7 BALB/c nu/nu mice per test group. The average latency time until the tumors reached a size of 1 cm3 (end point) and the standard deviation are indicated. Rspo3 expression rendered the HC11 cells highly tumorigenic as compared to the cells transduced with the empty vector, however, less tumorigenic than Wnt1 transduced HC11 cells. 76 EXPERIMENTAL THERAPY VIII D IV IS ION OF EXP ER IM EN TA L THER A PY GENES AND PROTEINS INVOLVED IN ANTICANCER DRUG RESISTANCE AND PHARMACOKINETICS Division head, Group leader Alfred Schinkel Alfred Schinkel PhD Group leader Christian Zimmermann PhD Post-doc Jurjen Lagas MSc Graduate student Zeliha Pala MSc Graduate student Evita Van de Steeg MSc Graduate student Antonius Van Herwaarden MSc Graduate student Robert Van Waterschoot MSc Graduate student Our research focuses on genes and proteins that cause drug resistance in tumors, or influence the pharmacological and toxicological behavior of anticancer and many other drugs and toxins, including carcinogens. Insight into these systems may: i) improve chemotherapy approaches for cancer and HIV/AIDS, as well as pharmacotherapy in a broader sense; ii) increase insights in factors determining susceptibility to carcinogens, and; iii) allow elucidation of physiological functions. To study the physiological, pharmacological and toxicological roles of the proteins involved, and their interactions, we generate and analyze knockout or transgenic mice lacking or overexpressing the relevant genes. Marijn Vlaming MSc Graduate student Corina Van der Kruijssen MSc Technical staff Anita Van Esch Technical staff Els Wagenaar Technical staff Figure VIII.1: Putative structure of a prototypic ABC drug transporter. Figure VIII.2: Hepatobiliary excretion of Impact of active drug transporters We have a long-standing interest in plasma membrane proteins of the ATP binding cassette (ABC) transporter family, including P-glycoprotein (P-gp, ABCB1), MRP2 (ABCC2) and BCRP (ABCG2) (Figure VIII.1). These proteins actively export a wide range of anticancer, anti-HIV/AIDS, and many other drugs from cells. This ATP-dependent drug extrusion can cause multidrug resistance (MDR) in tumor cells. P-gp, MRP2 and BCRP all localize to the apical membrane of polarized epithelial cells, resulting in apically directed export of drug substrates, and there is considerable overlap in substrate specificity between these transporters. Previous experiments in P-gp and Bcrp1 knockout mice indicated that these transporters can protect an organism against exogenous toxins and drugs by limiting penetration of substrates into brain, testis, and fetus, by restricting uptake of orally administered substrates, and by mediating excretion of substrates via liver and intestine. To extend these analyses we have initiated generation of Mrp2 knockout mice, which are crossed with the existing P-gp and Bcrp1 knockout mice in order to elucidate the separate and combined contributions of these transporters to pharmacological, toxicological and physiological functions. Generation and analysis of Mrp2 knockout mice The ABC transporter and multidrug resistance protein MRP2 (ABCC2) forms a natural barrier and efflux system for numerous (conjugates of) drugs, other xenotoxins and many endogenous compounds. We generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. These mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of the multidrug transporters Mrp3 (Abcc3) in liver and Mrp4 (Abcc4) in kidney of Mrp2-/- mice were about 2-fold increased. 30 min after oral administration of the food-derived carcinogens PhIP and IQ, plasma values were 1.9-fold and 1.7-fold higher in Mrp2 knockout mice vs. wild-type mice respectively, demonstrating the role of Mrp2 in restricting exposure to these compounds. At a high dose of 50 mg/kg of the anticancer drug methotrexate, the plasma exposure after intravenous administration was 1.8-fold higher in Mrp2 knockout mice. In contrast, no clear plasma concentration difference arose at low dose (1 mg/kg), suggesting that Mrp2 primarily affects methotrexate (MTX) pharmacokinetics at high plasma concentrations. Using an Mrp2/P-gp compound knockout mouse, we could further show that hepatobiliary excretion of doxorubicin is mediated by both P-gp and Mrp2 (Figure VIII.2). The Mrp2 knockout mouse thus provides a valuable tool for studies of the impact of Mrp2 on behavior of drugs, carcinogens and other xenotoxins, and on liver physiology. Combination with other transporter knockout mice will further allow systematic analysis of a plethora of research questions in these areas. doxorubicin in wild-type (fvb), Mrp2, Mdr1a/1b, and Mdr1a/1b/Mrp2 knockout mice. Excretion during 60 min after intravenous administration of 5 mg/kg doxorubicin was measured. Studies of paclitaxel in Mrp2/P-gp compound knockout mice The widely used anticancer drug paclitaxel is an excellent substrate for P-glycoprotein. We recently demonstrated that also MRP2 transports paclitaxel and docetaxel in vitro. To investigate whether MRP2 affects the pharmacokinetics of paclitaxel, we used Mrp2, 77 EXPERIMENTAL THERAPY Publications Mdr1a/1b, and Mrp2/Mdr1a/1b combination knockout mice. Disruption of the Mrp2 gene had a clear impact on paclitaxel plasma levels after intravenous administration, which was quantitatively similar to absence of P-glycoprotein. The area under the plasma concentration-time curve (AUC) was 1.3-fold higher in both Mrp2 and Mdr1a/1b knockout mice and 1.7-fold higher in Mdr1a/1b/Mrp2 knockout mice, when compared to wild-type mice. In contrast to P-glycoprotein, Mrp2 had no significant role in limiting intestinal absorption of paclitaxel. In Mrp2 knockout mice the oral AUC was not altered compared to wild-type mice, whereas in Mdr1a/1b knockout mice a 8.5-fold increase was found, the latter consistent with our previous results. Interestingly, however, in Mdr1a/1b/Mrp2 knockout mice the oral AUC was 14.2-fold increased compared to wild-type and 1.7-fold compared to Mdr1a/1b knockout mice. The impact of Mrp2 on plasma pharmacokinetics could be explained by our finding that Mrp2 dominates the hepatobiliary elimination of paclitaxel and its principal metabolites. In Mrp2 knockout mice the hepatobiliary excretion of paclitaxel was reduced by 80%, whereas excretion of its monohydroxylated metabolites was almost totally abolished. Therefore, disruption of Mrp2 has a marked impact on paclitaxel plasma levels after intravenous administration and on plasma levels after oral administration when absorption is not limited by P-glycoprotein. Our findings may be of importance for pharmacotherapy, as in current clinical practice taxanes are administered by intravenous infusion. As a consequence, mutational alterations of the MRP2 gene in patients may result in increased plasma levels of paclitaxel and thereby unexpected toxicity. Kummer W, Wiegand S, Akinci S, Wessler I, Schinkel AH, Wess J, Koepsell H, Haberberger RV, Lips KS. Role of acetylcholine and polyspecific cation transporters in serotonin-induced bronchoconstriction in the mouse. Respir Res 2006; 7:65 Lagas JS, Vlaming ML, Van Tellingen O, Wagenaar E, Jansen RS, Rosing H, Beijnen JH, Schinkel AH. Multidrug resistance protein 2 is an important determinant of paclitaxel pharmacokinetics. Clin Cancer Res 2006; 12:6125-6132 Merino G, lvarez AI, Pulido MM, Molina AJ, Schinkel AH, Prieto JG. Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion. Drug Metab Dispos 2006; 34:690-695 Bcrp1, P-gp and Mrp2 compound knockout mice We are currently in the process of generating compound knockout mice for Bcrp1, P-gp and Mrp2. We expect that these mice will yield very useful tools for studying the separate and combined impact of these ABC transporters on shared drug and carcinogen substrates. Moreover, both Bcrp1 and P-gp appear to be expressed in a range of tissue stem cells, and transport overlapping carcinogenic substrates. Exposure of these compound knockout mice to such carcinogens may elucidate the impact of the ABC transporters in the protection against dietary and environmental carcinogens. Finally, we may be able to elucidate additional physiological functions of these transporters, as some may be obscured by partial redundancy in the single knockout mouse strains. Ogasawara M, Yamauchi K, Satoh YI, Yamaji R, Inui K, Jonker JW, Schinkel AH, Maeyama K. Recent advances in molecular pharmacology of the histamine systems: Organic cation transporters as a histamine transporter and histamine metabolism. J Pharmacol Sci 2006; 101:24-30 Van Herwaarden AE, Wagenaar E, Karnekamp B, Merino G, Jonker JW, CYP3A transgenic and knockout mice The cytochrome P450 3A (CYP3A) complex is a major factor in the metabolic breakdown of most drugs used today, and of various endogenous substrates, but also for the activation and inactivation of many (pre-)carcinogens. As CYP3A activity shows great inter- and intra-patient variability, it can have a profound influence on variable drug behavior (pharmacodynamics) and drug toxicity. Moreover, its substrates overlap extensively with those of the drug transporters P-gp, BCRP and MRP2. To study this important system and its interactions with drug transporters in well-defined models, we are generating CYP3A transgenic and knockout mice. We have obtained CYP3A4 transgenic mice with substantial, physiologically relevant and stable CYP3A4 levels in liver, but not in small intestine. These transgenic mice displayed clearly accelerated kinetics of the widely used drugs midazolam and cyclosporin A. We further recently obtained mice with substantial expression of CYP3A4 in the intestine. These mice, together with additional strains currently under construction, will allow highly informative studies on drug behavior and (pre)carcinogen susceptibility. Schinkel AH. Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk. Carcinogenesis 2006; 27:123-130 Van Herwaarden AE, Schinkel AH. The function of breast cancer resistance protein in epithelial barriers, stem cells and milk secretion of drugs and xenotoxins. Trends Pharmacol Sci 2006; 27:10-16 Vlaming MLH, Mohrmann K, Wagenaar E, De Waart DR, Oude Elferink RPJ, Lagas JS, Van Tellingen O, Vainchtein LD, Rosing H, Beijnen JH, Schellens JHM, Schinkel AH. Carcinogen and anticancer drug transport by Mrp2 in vivo: Studies using Mrp2 (Abcc2) knockout mice. J Pharmacol Exp Ther 2006; 318:319-327 78 EXPERIMENTAL THERAPY MECHANISMS AND PREDICTION OF TUMOR RESPONSE TO RADIATION Group leader Adrian Begg We have continued to pursue two lines of research, one aiming at clinical translation and the other on fundamental aspects of the radiation response. The first line focuses on expression microarrays, tissue microarrays and cisplatin adducts to predict therapy outcome. The second involves how the cell handles DNA base damage and single strand breaks, an aspect of radiation damage that may well provide novel targets for radiotherapy for cancer. Adrian Begg PhD Group leader Frank Hoebers MD Radiation Oncologist Conchita Vens PhD Post-doc Sari Neijenhuis MSc Graduate student Jimmy Pramana MD Graduate student Christie Vermeulen MSc Graduate student Ben Floot Technical staff Ingrid Hofland Technical staff Manon Verwijs Technical staff Figure VIII.3: Prediction of outcome after RadPlat (combined radiotherapy and cisplatin-based chemotherapy) by gene expression profiling in advanced head and neck cancer. A 7-gene profile, found by supervised hierarchical clustering, correlates with loco-regional recurrence probability in the training series (top) and in a separate validation series (bottom). Prediction of outcome Microarrays: We are measuring expression profiles of head and neck cancers prior to treatment, to search for genes which predict outcome in patients treated with concurrent radiotherapy cisplatin. (collaboration with the NKI departments of Head and Neck Surgery, Radiotherapy and Pathology). We have now completed a study on 92 tumors which satisfied the criteria of >50% tumor cells in the biopsy, high RNA quality and sufficient follow-up time. Amplified and labeled RNA was hybridized to 35k oligo arrays from our Central Microarray facility. Patients were split into training and validation series. Supervised hierarchical clustering using a nearest centroid algorithm produced genes sets significantly associated with outcome for the endpoints of local control, locoregional control and disease free survival. These signatures contained 31, 8, and 2 genes, respectively. In the validation series, only the locoregional control signature remained significantly predictive (Figure VIII.3). Gene Set Enrichment Analysis (GSEA) on 311 gene sets showed some were associated with recurrence, but false discovery rates were high. Of interest was that genes and gene sets correlating with locoregional recurrence were not found in those correlating with distant metastases, implying locoregional recurrence signatures predict treatment sensitivity (predictive) rather than being a general monitor of malignancy (prognostic). Three tissue microarrays have been made from this patient material, comprising more than 200 patients. To date, these have been stained with antibodies for 8 candidate markers; 12 more are planned before analysis. Some markers were chosen from the expression microarray study. Cisplatin adducts: We are continuing to look at the predictive value of cisplatin-DNA adducts in buccal cells of patients taken early in their treatment course. We have applied an immunofluorescence technique developed by us to a series of 85 cervix cancer patients in a Gynaecology and Oncology Group (GOG) trial of cisplatin plus concurrent radiotherapy. Pre- and post-treatment samples were obtained during the first 2 courses of chemotherapy. In the 53 patients having sufficient data from both first and second courses, high buccal cell adducts were significantly associated with a worse progression free survival. This is in the opposite direction to that found in previous lung cancer studies, and not consistent with the notion that buccal cell adducts are a monitor of integral cisplatin exposure, which should be reflected in tumor response. Further analyses are continuing. Mechanisms and modulation of radiosensitivity DNA polymerase beta is known to play a central role in base excision repair (BER) and single strand break repair (SSBR). We previously showed that expression of a dominant negative to DNA polymerase beta (polbetaDN) resulted in radiosensitization of human tumor cell lines, and also radiosensitizes polbeta-deficient cells, implying interference with polbeta-independent pathways. In contrast, new data show that expression of the DN in polbeta deficient cells protects against the alkylating agent MMS. This is consistent with a dRP lyase activity of the DN, which removes potentially toxic intermediates. This further implies that removal of dRP groups are not important for ionizing radiation. We have now also shown that the polbetaDN does not sensitize cells to H2O2, an agent producing oxidative base damage and SSB. This indicates that the DN is a specific sensitizer for ionizing radiation, and further suggests an involvement in repair of clustered damage, which occurs after radiation but not after H2O2. We further investigated polbetaвЂ™s role by studying polbeta deficient cells. We previously showed that the short patch polymerase inhibitor methoxyamine (MX) sensitized only polbeta deficient cells to ionizing radiation. We now show that MX sensitizes both wildtype and beta deficient cells to H2O2. These and other data 79 EXPERIMENTAL THERAPY Publications indicate polbeta is critical for alkylating damage, but not for H2O2, and that both polbeta and a backup polymerase must act together in the response to ionizing radiation. We have shown that confluent cells, in contrast to log phase cells, are hypersensitive when lacking polbeta. To study the replication dependence of this effect, we irradiated cells in different cell cycle phases using cells synchronized by mitotic shake-off. DNA polymerase b deficient cells were found to be more sensitive to ionizing radiation than wild-type cells in G1, while being equally sensitive after progressing into S. These data were confirmed using a polbeta-deficient line complemented with polbeta. This indicates that there is an important S-phase specific repair pathway after ionizing radiation, which is independent of polbeta, and also confirms polbetaвЂ™s role in the radiation response in G0/G1 cells. To further characterize possible backup pathways, we are studying two candidate polymerases, iota and lambda, shown to participate in BER reactions in vitro. Studies on using both knockout and knock-down strategies for these two polymerases are continuing. Finally, we are investigating a third translesion synthesis polymerase, DNA polymerase kappa. So far, we have shown that kappa knockout cells are sensitive to cisplatin, and we are now investigating its role in ionizing radiation, in both oxic and hypoxic cells. A project is continuing to study the role of apoptosis and senescence in a sporadic tumor model in mice (collaboration with Verheij, Jongsma, Berns; NKI). Experiments with the original lung tumor model (cre-adenovirus instillation activating oncogenic K-ras) have stopped for technical reasons, and re-started with a mesothelioma model with higher tumor incidence and shorter latencies (Nf2-induced tumors, with or without p53 and/or ink4aARF deficiencies). To date, cells from tumors from each genotype have shown little radiosensitivity differences in culture. Experiments on further tumor clones in culture and in animals are underway. Begg AC. Can the severity of normal tissue damage after radiation therapy be predicted? PLoS Medicine 2006; 3:1713-1714 De Schutter H, BarbГ© B, Spaepen M, Begg AC, Balm A, Gregoire V, Haustermans K, Mahy P, Poorten VV, Nuyts S. Microsatellite alterations in head and neck squamous cell carcinoma and relation to expression of pimonidazole, CA IX and GLUT-1. Radiother Oncol 2006; 80:143-150 Goethals L, Debucquoy A, Perneel C, Geboes K, Ectors N, De Schutter H, Penninckx F, McBride WH, Begg AC, Haustermans KM. Hypoxia in human colorectal adenocarcinoma: Comparison between extrinsic and potential intrinsic hypoxia markers. Int J Radiat Oncol Biol Phys 2006; 65:246-254 Hoebers FJP, Pluim D, Verheij M, Balm AJM, Bartelink H, Schellens JHM, Begg AC. Prediction of treatment outcome by cisplatin-DNA adduct formation in patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin-radiation (RADPLAT). Int J Cancer 2006; 119:750-756 Sprong D, Janssen HL, Vens C, Begg AC. Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status. Int J Radiat Oncol Biol Phys 2006; 64:562-572 Vermeulen C, Verwijs-Janssen M, Cramers P, Begg AC, Vens C. Role for DNA polymerase beta in response to ionizing radiation. DNA Repair 2007; 6:202-212 Vink SR, Lagerwerf S, Mesman E, Schellens JHM, Begg AC, Van Blitterswijk WJ, Verheij M. Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts. Clin Cancer Res 2006; 12:1615-1622 80 EXPERIMENTAL THERAPY VASCULAR MEDIATED TISSUE DAMAGE AND THERAPY Group leader Fiona Stewart Fiona Stewart PhD Group leader Improvements in detection and treatment of cancer have lead to increased numbers of long-term survivors at risk for developing treatment related morbidity. Vascular injury is the major cause of late radiation morbidity and may seriously impair the quality of life, or even be life threatening, to cancer patients treated with radiotherapy. In large vessels this manifests as atherosclerosis, whereas in small vessels it manifests as telangiectasia. The aim of our studies is to investigate mechanisms underlying the development and progression of radiation-induced vascular damage with a view to identifying intervention strategies. Paul Baas MD PhD Academic staff Nicola Russell MD PhD Academic staff Jacqueline Kruse PhD Senior Post-doc Saske Hoving PhD Post-doc Martijn Triesscheijn MSc Graduate student Ben Floot Technical staff Marjan Ruevekamp Technical staff Debbie Sprong Technical staff Johannes Te Poele Technical staff Figure VIII.4: A: Lawson staining of irradiated H&N artery (left) and the donor artery of the same patient (right). B: IMT of unirradiated and irradiated H&N arteries corrected for the IMT of the donor arteries (* p<0.05). Figure VIII.5: The TGFГџ receptor kinase inhibitor SB431542 significantly inhibits radiation-mediated (and TGFГџ induced) increases in PAI1 mRNA in HMVECs. Cells were irradiated with a single dose of 0 or 10 Gy in combinations with or without DMSO or SB431542. Stimulation with TGFГџ was used as a positive control. Radiation induced atherosclerosis (in collaboration with Mat Daemen, CARIM, Maastricht) Radiation is an independent risk factor in vascular disease after radiotherapy for HodgkinвЂ™s disease, breast and head and neck cancer. The aim of this work is to investigate mechanisms of development of radiation-induced atherosclerosis and to compare the phenotype of the resulting lesions with those in age-related atherosclerosis. We established a mouse model for studying radiation-induced atherosclerosis in the carotid arteries of ApoE-/- mice. These mice have increased cholesterol levels compared with wild type mice and develop atherosclerosis spontaneously with age. Mice were given single dose (8-14 Gy) or clinically relevant fractionated irradiation (20 x 2 Gy in 4 weeks) to the neck. Already at 1-4 weeks after treatment, early atherosclerotic lesions were seen in irradiated carotid arteries but not in the arteries of control animals. From 22-34 weeks after treatment, there were significantly more lesions in irradiated carotid arteries (single and fractionated doses) compared to age matched controls. The results showed that irradiation accelerated development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. We are currently evaluating the potential of anti-platelet, anti-inflammatory and anti-cholesterol drugs (aspirin, nitric oxide-releasing-aspirin, clopidogrel and atorvastatin) for their potential to inhibit early endothelial cell (EC) damage and the subsequent development of radiation-induced atherosclerosis. In parallel clinical studies, we are characterizing radiation-induced vascular damage in resection material collected from head and neck (H&N) and breast cancer patients undergoing free-flap reconstructive surgery. Biopsies from H&N cancer patients were obtained from a branch of the carotid artery (facial artery) at <1 to 26 years after radiotherapy or surgery. Internal mammary artery biopsies were obtained from breast cancer patients 1 to 19 years after radiotherapy or surgery. Control biopsies were obtained from the free flap donor arteries of each patient (radial or deep inferior epigastric perforator, DIEP, respectively). In the H&N patients we found a 1.7-fold increase in intima-media thickness (IMT) for the irradiated compared to the unirradiated arteries, after correction for the IMT of the donor artery from the same patient (p<0.008) (Figure VIII.4). There was a marked inflammatory component in the intima and adventitia of some or these irradiated arteries, which was not seen in donor arteries from the same patients or in arteries from unirradiated H&N cancer patients. The IMT ratios of the mammarian arteries from breast cancer patients were generally < 50% of that seen in arteries from H&N cancer patients. Although it is known that internal mammary arteries are resistant to the development of age-related atherosclerosis, we saw a trend for increased IMT in irradiated compared to unirradiated patients (1.4-fold, p=0.09). Further analysis is required to determine how co-morbidity factors, e.g. age and smoking, as well as the irradiation dose and time since treatment, affect radiation-induced vascular damage. Radiation induced telangiectasia (in collaboration with Peter Ten Dijke, LUMC, Leiden) Radiation-induced telangiectasia is characterized by abnormal dilated postcapillary vessels. This compromises the functional integrity of tissues, leading to severe morbidity (bleeding). It can occur in all tissues, but has been best reported in skin or rectum after treatment for breast or prostate cancer. Members of TGFb and Notch families are important regulators of vascular development and perturbations in their signaling have been implicated in vascular disorders similar to those seen after radiotherapy. The aim of this study is to investigate the influence of radiation on 81 EXPERIMENTAL THERAPY Publications TGFb/Notch signaling in ECs, in relation to the development of telangiectasia in irradiated tissue. TGFb activates two distinct type I receptors (ALK1 and ALK5), which have opposite effects on EC migration and proliferation: ALK1 signaling stimulates, whereas ALK5 inhibits these processes. Irradiation of human microvascular ECs (HMVECs) in vitro resulted in reduced ALK1 signaling, with decreased production of downstream target Id1, and stimulation of ALK5 signaling, with increased phosphorylation of Smad2 and production of PAI1. This increased PAI1 expression was, at least partly, dependent on ALK5 signaling, since exposure of cells to an ALK5 inhibitor almost completely abolished radiation-induced increase in PAI1 (Figure VIII.5). Irradiation of ECs also resulted in marked increases in the Notch ligand, Jagged1, and downstream target Herp2. Herp2 is known to function as a mediator of BMP signaling in ECs, where it inhibits Id1 expression and promotes degradation of the protein, thereby antagonizing Id1 induced EC migration. Taken together, the radiation-induced changes would be expected to inhibit EC migration and proliferation. We are currently evaluating the functional consequences of radiationinduced changes in TGFb and Notch signaling using assays of cellular migration. To elucidate long-term effect of ionizing radiation on TGF /ALK and Jagged/Notch signaling in vivo, mice were irradiated with 16-20 Gy to the kidneys or rectum. Tissues were harvested at 1 to 30 weeks after irradiation and used for RNA isolation and quantitative real time PCR or immunohistochemical analyses. Kidney irradiation resulted in a progressive increase in PAI1, reaching 6-7 fold at 30 weeks. A transient elevation in Id-1 was found at early time intervals after irradiation (1-10 weeks), whereas at late time intervals (20-30 weeks), when telangiectasia is established, Id-1 expression levels returned to control values. In addition, significant increases in Smad7 and CTGF mRNA were found at 1 week after irradiation, which persisted during the observation period. These in vivo results are in agreement with in vitro findings and demonstrated that irradiation shifts the balance between ALK1 and ALK5 signaling towards a resolution phase (inhibition of migration and proliferation). It has been postulated that CTGF, a profibrotic factor, enhances the TGF /ALK5 signaling pathway by transcriptional suppression of inhibitory Smad 7. Blocking the negative feedback loop provided by Smad 7 might lead to continued activation of the TGF signaling pathway. In the irradiated mouse rectum, a reduction of both ALK1 and ALK5 signaling (mRNA) was found from 10-30 weeks after irradiation. In order to confirm these changes at the protein level and to determine localisation of the proteins in irradiated tissues, immunohistochemical stainings are now being performed. Aalders MCG, Triesscheijn M, Ruevekamp M, De Bruin M, Baas P, Faber DJ, Stewart FA. Doppler optical coherence tomography to monitor the effect of photodynamic therapy on tissue morphology and perfusion. J Biomed Optics 2006; 11:044011 Baas P, Triesscheijn M, Burgers S, Van Pel R, Stewart F, Aalders M. Fluorescence detection of pleural malignancies using 5-aminolaevulinic acid. Chest 2006; 129:718-724 Dorresteijn LD, Stewart FA, Boogerd W. Stroke As a Late Treatment Effect of HodgkinвЂ™s Disease. J Clin Oncol 2006; 24:1480 Stewart FA, Heeneman S, Te Poele J, Kruse J, Russell NS, Gijbels M, Daemen M. Ionizing radiation accelerates the development of atherosclerotic lesions in ApoE-/- mice and predisposes to an inflammatory plaque phenotype prone to hemorrhage. Am J Pathol 2006; 168:649-658 Triesscheijn M, Baas P, Schellens JHM, Stewart FA. Photodynamic therapy in oncology. Oncologist 2006; 11:1034-1044 Triesscheijn M, Ruevekamp M, Out R, Van Berkel TJC, Schellens J, Baas P, Stewart FA. The pharmacokinetic behavior of the photosensitizer meso-tetra-hydroxyphenyl-chlorin in mice and men. Cancer Chemother Pharmacol 2007; In Press Photodynamic Therapy (PDT) In a separate project we developed a pre-clinical program for optimizing treatment protocols for clinical use of photodynamic therapy (PDT). PDT is an effective treatment for small, localized cancers that is now routinely used in the NKI-AVL for small oral cavity tumors and multiple basal cell carcinomas. Treatment involves systemic delivery of a photosensitizer followed by local tumor illumination with light of a specific wavelength, which generates free radicals and singlet oxygen. The penetration depth of the laser light used to excite the photosensitizer limits damage to normal tissues. Over the past 10-years we have used animal models to investigate how sensitizer pharmacokinetics and drug/light scheduling and timing affects the mechanism of PDT-mediated tumor destruction. Our work has demonstrated that vascular mediated damage, and not just tumor cell kill, is an essential component of curative PDT. This has had a major influence on the design of optimal clinical protocols. Our pre-clinical PDT program has resulted in the successful introduction and use of clinical PDT for oral cavity and skin tumors. A dedicated PDT unit will open in December 2006 at the NKI-AVL; laboratory investigations on this topic have now stopped. Triesscheijn ML. mTHPC-mediated photodynamic therapy; from bed to bench and back again. Utrecht: Universiteit Utrecht, 2006 Triesscheijn M, Ruevekamp M, Antonini N, Neering H, Stewart FA, Baas P. Optimizing Meso-Tetra-Hydroxyphenyl-Chlorin Mediated Photodynamic Therapy for Basal Cell Carcinoma. Photochem Photobiol 2006; 82:1686-1690 Van Veen RLP, Nyst H, Rai Indrasari S, Adham Yudharto M, Robinson DJ, Tan IB, Meewis C, Peters R, Spaniol S, Stewart FA, Levendag PC, Sterenborg HJCM. In vivo fluence rate measurements during FoscanВ©-mediated photodynamic therapy of persistent and recurrent nasopharyngeal carcinomas using a dedicated light applicator. J Biomed Optics 2006; 11:a041107 82 EXPERIMENTAL THERAPY PHARMACODYNAMICS OF ANTICANCER DRUGS The pharmacology group Schellens has a continued interest in studies exploring anticancer drug disposition, development of methods to improve oral pharmacokinetics (PK) of anticancer drugs and development of novel pharmacodynamic (PD) methods to support early clinical trials with anticancer drugs. Group leader Jan Schellens Jan Schellens MD PhD Group leader Jos Beijnen PhD Academic staff Irma Meijerman PhD External staff Serena Marchetti MD External staff Maarten Deenen MSc Graduate student Roos Oostendorp MSc Graduate student Sander Veltkamp MSc Graduate student Stefan Vink MSc Graduate student Marije Bolijn Technical staff Dick Pluim Technical staff Monique Van Eijndhoven Technical staff Publications Appels NMGM, Rosing H, Stephens TC, Hughes A, Schellens JHM, Beijnen JH. Absolute quantification of farnesylated Ras levels in complex samples using liquid chromatography fractionation combined with tryptic digestion and electrospray tandem mass spectrometry. Anal Biochem 2006; 352:33-40 Appels NMGM, Rosing H, Stephens TC, Schellens JHM, Beijnen JH. Quantification of farnesylmethylcysteine in lysates of peripheral blood mononuclear cells using liquid chromatography coupled with electrospray tandem mass spectrometry: Pharmacodynamic assay for farnesyl transferase inhibitors. Anal Chem 2006; 78:2617-2622 Breedveld P, Pluim D, Cipriani G, Dahlhaus F, Van Eijndhoven MAJ, De Wolf CJF, Kuil A, Beijnen JH, Scheffer GL, Jansen G, Borst P, Schellens JHM. The Effect of Low pH on Breast Cancer Resistance Protein (ABCG2)-Mediated Transport of Methotrexate, 7-Hydroxymethotrexate, Methotrexate Diglutamate, Folic Acid, Mitoxantrone, Topotecan, and Resveratrol in In Vitro Drug Transport Models. Mol Pharmacol 2007; 71:240-249 Role of ABC drug transporters in absorption and disposition of anticancer drugs The research of the group is focused on assessment of the role of the ABC drug transporters and other transporters on the absorption and disposition of anticancer drugs. We continued the in vivo studies with the novel lipophilic camptothecin derivative gimatecan. We identified that this molecule is transported by BCRP, although at lower affinity than the control drug topotecan. In vivo, area under the curve (AUC) of gimatecan after oral dosing (2 mg/kg) in Bcrp1-/-/Mdr1a/1b-/(TKO) mice was 1.5-fold higher than in wildtype mice (p<0.001). Intravenous PK were not significantly different in TKO and wildtype mice. However, CNS accumulation was significantly increased in wildtype mice upon co-administration of elacridar. Elacridar did not affect the biotransformation of gimatecan. In TKO mice, elacridar further increased the systemic exposure after oral administration of gimatecan, clearly indicating that other drug transporters are involved in the disposition of the drug. We continued studies with the orally bioavailable taxane analog BMS-275183 and established the oral and (i.v.) PK and tissue distribution in vivo in wildtype mice and in the MDR1 knockout mice (Mdr1a/1b-/-). In vivo, after an oral dose of 10 mg/kg, the AUC of BMS-275183 was 4665 + 789 (mean + SE; ng.h/ml) in wt and 8761 + 406 (ng.h/ml) in mdra/b KO mice (p<0.01). Co-administration of one dose of 40 mg/kg of oral pantoprazole did not affect the AUC values in wt mice. CNS concentrations of BMS in wt mice were below the LLQ of the assay, but were 851 + 442 and 932 + 429 ng/g tissue at 1 and 4 h respectively in the KO mice. Pantoprazole had no significant effect on CNS concentrations in wt and KO mice. In vitro and in vivo, BMS-275183 is a good substrate drug for MDR1. In vitro, it is a moderate substrate for MRP2 and not a substrate for BCRP. It is warranted to further explore the in vivo interaction between BMS-275183 and PPIs in patients. Development of resistance to imatinib is observed in many patients. Imatinib has high oral bioavailability (98%), but preliminary data revealed reduced systemic exposure to imatinib over time. In previous in vitro and in vivo experiments, we and others showed that the drug efflux transporters P-glycoprotein (P-gp) and BCRP; (ABCG2) play an important role in the PK of imatinib. We hypothesized that in combination with a P-gp/BCRP inhibitor the variability in the oral PK of imatinib may be further reduced, the tissue distribution improved and PK mechanisms of clinical resistance to imatinib prevented. We investigated in vivo the absolute bioavailability in wildtype mice and combined Mdr1a/1b and Bcrp1 knockout mice (Mdr1a/1b-/-/Bcrp1-/-) and the effect of the Pgp/BCRP inhibitor elacridar on the absorption and disposition of imatinib and its main metabolite CGP74588 after oral administration of imatinib. The absolute oral bioavailability of imatinib in wildtype and Mdr1a/1b-/-/Bcrp1-/- mice was 71 and 49%, respectively, and 107 and 102 % when elacridar is co-administrated. The plasma area under the curve (AUC)oral was not significantly different in Mdr1a/1b-/-/Bcrp1-/- and wildtype mice. The (AUC)i.v. in Mdr1a/1b-/-/Bcrp1-/- mice was 1.6-fold higher versus wildtype mice (p<0.05). Co-administration of elacridar increased the (AUC)oral and (AUC)i.v. 3.3- and 2.0-fold, respectively, in wildtype mice and 2.7- and 1.3-fold in Mdr1a/1b-/-/Bcrp1-/- mice (P<0.001 and P<0.05). This suggests that besides Mdr1a/1b and Bcrp1 also other drug transporters (e.g. efflux/ uptake transporters) or inhibition of biotransformation are involved in the interaction between imatinib and elacridar. Oral [14C]imatinib treated wildtype mice resulted in an overall recovery of 83.2%. Unchanged imatinib and CGP74588 excreted mainly in the feces 24.8 and 1.3% of the dose, respectively, and urinary excretion accounts for 1.4 and 0.10% of the dose, indicating that imatinib is extensively metabolized in other metabolites besides CGP74588. The fecal and biliary excretion of unchanged imatinib was reduced by the absence (5.2 and 8.3-fold, respectively) or inhibition by 83 EXPERIMENTAL THERAPY Publications (continued) elacridar (3.3- and 4.0-fold) of Mdr1a/1b and Bcrp1 (P<0.001), and a role for both Mdr1a/1b and Bcrp1 in these processes was revealed. There is no indication that elacridar inhibited the biotransformation of imatinib. No significant differences were found in liver and kidney levels of imatinib to wildtype and Mdr1a/1b-/-/Bcrp1-/- mice in the presence or absence of elacridar. In vitro studies are ongoing to investigate the outlined interaction between these two drugs at the level of other efflux/uptake transporters. Hoebers FJP, Pluim D, Verheij M, Balm AJM, Bartelink H, Schellens JHM, Begg AC. Prediction of treatment outcome by cisplatin-DNA adduct formation in patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin-radiation New insights into the pharmacology and toxicology of gemcitabine Gemcitabine (2вЂ™,2вЂ™-difluorodeoxycytidine, dFdC), a pyrimidine antimetabolite, is actively taken up into the cell and intracellularly phosphorylated by deoxycytidine kinase (dCK) to its monophosphate (dFdCMP) and subsequently to its active diphosphate (dFdCDP) and triphosphate (dFdCTP) metabolites. Furthermore, dFdC is converted to 2вЂ™,2вЂ™-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA), which is ubiquitously expressed, such as in liver and intestine. Oral dFdC in a phase I study resulted in preferential formation of dFdU and incidental severe liver toxicity. We hypothesized that dFdU is phosphorylated and toxic after liver accumulation following multiple oral dFdC administration. We assessed the in vitro toxicity, uptake, and intracellular activation of dFdC and dFdU in human hepatocellular (HepG2), human lung cancer (A549) and MDCK cells containing high expression of either the human equilibrative or concentrative type 1 nucleoside transporter (hENT1 and hCNT1). Cytototoxicity was assessed at several exposure times using SRB and colony assays. The total cellular uptake plus incorporation into DNA/RNA of radiolabeled dFdC (0-20ВµM) and dFdU (0-1000ВµM) was determined at different time points. Furthermore, intracellular and extracellular concentrations of dFdC, dFdU and metabolites were quantified by HPLC-UVscintillation counting. In addition, quantitative RT-PCR was used to determine gene expression of dCK, CDA, hENT1 and hCNT1. Compared to A549 cells, HepG2 cells had a 4-fold and 7-fold higher sensitivity to dFdC and dFdU, respectively. Furthermore, HepG2 cells showed higher expression of both hENT1 and dCK compared to A549 cells. MDCK cells with hENT1 and hCNT1 overexpression showed increased sensitivity to dFdC and dFdU compared to wildtype cells. dFdC was actively taken up into HepG2 and A549 cells. dFdU was detected in DNA/RNA. Interestingly, we discovered that dFdU was substrate for hCNT1 but not for hENT1 and that dFdUTP was formed intracellularly, suggesting phosphorylation of dFdU and/or dFdUMP. Conclusions: dFdU has high toxicity in HepG2 cells and is a good substrate for hCNT1. Moreover, dFdUTP is formed intracellularly and dFdU seems to be incorporated into DNA/RNA. This could be relevant for the in vivo activity and toxicity of dFdC and dFdU, especially in tissues with high expression of hCNT1, such as liver and kidney. Currently, in vivo mice studies are performed to further assess the relevance of dFdU in the activity and toxicity of dFdC. (RADPLAT). Int J Cancer 2006; Pharmacogenetics of fluoropyrimidines About 3-5% of patients treated with fluorouracil (5-FU) and its oral prodrug capecitabine develop severe hematological and/or gastrointestinal toxicity, often requiring a long of hospitalization, caused by DPD-deficiency. DPD (dihydropyrimidine dehydrogenase) is the enzyme responsible for 85% of the metabolism of 5-FU into the inactive compound FUH2. Polymorphisms in the gene of DPD, DPYD, is held (partially) responsible for a decreased activity of the enzyme. The mutation IVS14+1G>A (DPYD*2A) leads to complete skipping of exon 14, resulting in a truncated protein with major decreased activity. Patients exhibiting this polymorphism are at great risk of developing severe fluoropyrimidine-associated toxicity after receiving a full-dose treatment. We developed a validated, rapid, inexpensive rt-PCR assay, with almost 100% sensitivity and specificity, for detection of DPYD*2A, which is applicable for routine screening prior to therapy. We recently identified three heterozygotes for DPYD*2A based on significant clinical toxicity, which resulted in the death of one of the three patients. We implemented screening for DPYD*2A in all patients who will start fluoropyrimidine therapy and prospectively establish a dosing algorithm in these patients and the cost-effectiveness of this approach. Vink SR, Lagerwerf S, Mesman E, 119:750-756 Liedert B, Pluim D, Schellens J, Thomale J. Adduct-specific monoclonal antibodies for the measurement of cisplatininduced DNA lesions in individual cell nuclei. Nucleic Acids Res 2006; 34:e47 Veltkamp SA, Alderden-Los C, Sharma A, Rosing H, Beijnen JH, Schellens JHM. A pharmacokinetic and safety study of a novel polymeric paclitaxel formulation for oral application. Cancer Chemother Pharmacol 2007; 59:43-50 Veltkamp SA, Thijssen B, Garrigue JS, Lambert G, Lallemand F, Binlich F, Huitema AD, Nuijen B, Nol A, Beijnen JH, Schellens JH. A novel selfmicroemulsifying formulation of paclitaxel for oral administration to patients with advanced cancer. Br J Cancer. 2006; 95: 729-34 Veltkamp SA, Hillebrand MJ, Rosing H, Jansen RS, Wickremsinhe ER, Perkins EJ, Schellens JH, Beijnen JH. Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry. J Mass Spectrom. 2006; 41: 1633-42 Schellens JHM, Begg AC, Van Blitterswijk WJ, Verheij M. Radiosensitization of squamous cell carcinoma by the alkylphospholipid perifosine in cell culture and xenografts. Clin Cancer Res 2006; 12:1615-1622 Vlaming MLH, Mohrmann K, Wagenaar E, De Waart DR, Oude Elferink RPJ, Lagas JS, Van Tellingen O, Vainchtein LD, Rosing H, Beijnen JH, Schellens JHM, Schinkel AH. Carcinogen and anticancer drug transport by Mrp2 in vivo: Studies using Mrp2 (Abcc2) knockout mice. J Pharmacol Exp Ther 2006; 318:319-327 84 EXPERIMENTAL THERAPY MOLECULAR PATHOLOGY AND MOLECULAR EPIDEMIOLOGY OF BREAST CANCER Group leader Laura Van вЂ™t Veer Laura Van вЂ™t Veer PhD Group leader Petra Nederlof PhD PI and Academic staff Frans Hogervorst PhD Academic staff Sabine Linn MD PhD Academic staff Flora Van Leeuwen PhD Academic staff Senno Verhoef MD PhD Academic staff Annegien Broeks PhD Post-doc Dorien Voskuil PhD Post-doc Marjanka Schmidt PhD Post-doc Erik Van Beers PhD Post-doc Britta Weigelt PhD Post-doc Tim Molloy PhD Post-doc Molecular profiling for breast cancer classification and therapy response The classification of breast tumors by the World Health Organization distinguishes 30 different morphological types of breast carcinomas. The rare вЂ�special typeвЂ™ breast cancers exhibit favorable prognostic features, but are often not recognized and classified as invasive ductal carcinoma, not otherwise specified (IDC NOS). We identified by hierarchical clustering of 113 tumors genetically related histological subtypes, such as medullary and metaplastic carcinomas, but also genetically distinct types, like micropapillary carcinomas. We show that most special type breast cancers only belong to one of the molecular intrinsic types. Intrinsic вЂ�basal-likeвЂ™ tumors, apparently comprise subtypes with an excellent prognosis, such as adenoid cystic carcinomas. (see Figure VIII.6) Half of the recurrences of ERa+ breast tumors respond to tamoxifen while the other half is resistant. We validated on a more advanced whole genome platform and in an independent set of tumors (including stable disease) a recently identified 81-gene tamoxifen response profile (Jansen et al., JCO 2005). The profile is outperforming other parameters in a multivariate response prediction analysis. Currently, this profile is optimized for clinical use in the adjuvant setting. The ability to accurately predict tamoxifen treatment outcome would constitute a significant advance in the management of breast cancer. Sjoerd Bruin Clinical fellow Marleen Kok MD Graduate student Marielle Ruijs MD Graduate student Alina Vrieling MSc Graduate student Stella Mook MD Graduate student Astrid Bosma Technical staff Arnout Van der Plas Technical staff Linde Braaf Technical staff Renske Fles Technical staff Simon Joosse Technical staff Renate De Groot Technical staff Renate Udo MSc Research assistant Richard Van Hien Technical staff Radiation-associated breast tumors display a particular gene expression profile Approximately 90% of the breast carcinomas in women who received radiation treatment following Hodgkin Lymphoma is estimated to be a result of the radiation treatment, which makes this series extremely appropriate to determine a radiation-associated molecular profile. We have used microarray gene expression profiling to assess specific molecular changes in radiation-associated breast tumors (BfHL) and unsupervised hierarchical clustering (23 BfHL case- and 19 control breast tumors) resulted in a clustering of the radiation-associated tumors. A supervised classification revealed 430 significant genes that can differentiate radiation-associated breast tumors from control breast tumors. These preliminary results indicate that radiation-associated tumors can be distinguished from other breast tumors, indicating that there is a different etiology for radiation-induced breast tumors. Ferdi Van der Horst Technical staff Anke Nooijen MSc Research assistant Genetic determinants of breast cancer risk and breast cancer outcome Evidence to date strongly suggests that the majority of the familial clustering of breast cancer is unexplained and, therefore, that many breast cancer susceptibility genes still remain to be identified. One candidate is the single nucleotide polymorphism (SNP309) in MDM2. We confirmed that Li-Fraumeni TP53 germline mutation carriers have earlier onset of cancer if they carry the SNP309. However, within unselected breast cancer patients, MDM2 SNP309 does not predispose to an earlier age of onset (study performed within large international collaboration: the Breast Cancer Association Consortium). Germline mutations in genes of the DNA damage repair pathway like BRCA1/2, CHEK2 and MDM2, may affect breast cancer outcome. Our consecutive series of breast cancer patients aged <50 years, representative for the premenopausal breast cancer population, reveals approximately 4% CHEK2 1100delC carriers who have an increased risk of second breast cancer and worse long-term recurrence-free survival, which does not seem to be confounded by other prognostic factors. More research is being performed to explore tumor characteristics of CHEK2 1100delC and gene-treatment interactions. Figure VIII.6: Histology. Panel A shows four invasive ductal carcinomas not otherwise specified. Panel B shows a of special type breast cancers: invasive lobular carcinoma (a), tubular (b) mucinous A (c), mucinous B (d), neuroendocrine (e), invasive ductal carcinoma with osteoclastic giant cells (f), micropapillary (g), apocrine (h), metaplastic (i), medullary (j) and adenoid cystic carcinoma (k). The Insulin-like Growth Factor (IGF) system and its relation to breast and colorectal cancer Meta-analysis of epidemiological studies has shown that the risk of several epithelial cancers for individuals with relatively high serum concentrations of IGF-I. We are evaluating the effects of several dietary strategies to lower components of the circulating IGF-system, and its association with breast and colorectal tissue IGF-systems. In a randomized placebo-controlled cross-over trial, we find that lycopene supplementation does not result in significant changes in serum IGF-system in pre-menopausal women (n=60) at increased risk of breast cancer. Isoflavone supplementation in men at increased risk of colorectal cancer (n=37) did 85 EXPERIMENTAL THERAPY Publications not affect IGF levels overall. However, our results suggest that this may differ depending on isoflavone metabolism. The expression of IGF-system components in normal colorectal biopsies was found to differ markedly between locations in the colon and rectum and between individuals. The association with serum levels and the effects of dietary strategies on tissue IGF expression are under study. The proposed research is expected to provide a better fundamental understanding of whether and how the IGF-system could be the target of cancer preventive measures. Bogaerts J, Cardoso F, Buyse M, Braga S, Loi S, Harrison JA, Bines J, Mook S, Decker N, Ravdin P, Therasse P, Rutgers E, Van вЂ�t Veer LJ, Piccart M. Gene signature evaluation as a prognostic tool: Challenges in the design of the MINDACT trial. Nat Clin Pract Oncol 2006; 3:540-551 Gene expression profiling to predict the presence of disseminated tumor cell in breast cancer patients The presence of disseminated tumor cells (DTCs) in the peripheral blood and/or bone marrow of breast cancer patients is correlated with an increased risk of metastasis. This work aims to use microarray gene expression profiling to develop models with the ability to identify primary tumors at high risk of producing DTCs. Work has already been undertaken in early- and late-stage breast cancer patient cohorts to estimate the disseminated tumor cell load in the peripheral blood by QPCR analyses. RNA from primary breast tumors from these patients is used to interrogate whole-genome microarrays. Bioinformatics techniques will be used to build a predictive model with the ability to distinguish tumors which are likely give rise to high numbers of disseminated tumor cells from those that do not. The ability to predict which tumors are likely to give rise to high numbers of disseminated tumor cells would be of use in the clinic as it would optimize the decision-making process for the appropriate application of adjuvant therapy. Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DSA, Nobel AB, Van вЂ�t Veer LJ, Perou CM. Concordance among geneexpression-based predictors for breast cancer. N Engl J Med 2006; 355:560-569 Ruijs MWG, Verhoef S, Wigbout G, Pruntel R, Floore AN, De Jong D, Van вЂ�t Veer LJ, Menko FH. Late-onset common cancers in a kindred with an Arg213Gln TP53 germline mutation. Fam Cancer 2006; 5:169-174 Ruijs MWG, Schmidt MK, Nevanlinna H, Clinicopathologic classification and genetic profiling of colorectal metastasis Specific cases of colorectal carcinoma may have affinity for peritoneal spread in contrast with more usual lymphatic and haematogenous spread. The NKI-AVL is a center for treating this peritoneal spread with hyperthermic intraperitoneal chemotherapy, first as an experimental but now as an established treatment in this clinical context, although with many side effects. Using histology, gene expression profiling and CGH analysis we will try to predict response to this treatment. Furthermore, we will try to find the molecular and histological characteristics that discriminate tumours with peritoneal spread from those with the more usual lymphatic and haematogenous metastasis. Tommiska J, Aittomaki K, Pruntel R, Verhoef S, Van вЂ�t Veer LJ. The singlenucleotide polymorphism 309 in the MDM2 gene contributes to the Li-Fraumeni syndrome and related phenotypes. Eur J Hum Genet 2006; 15:110-114 Schmidt MK, Tollenaar RA, De Kemp SR, Broeks A, Cornelisse CJ, Smit VT, Peterse JL, Van Leeuwen FE, V an вЂ�t Veer LJ. Breast Cancer Survival and Genome-wide chromosome analysis in hereditary breast cancer (PI Petra Nederlof ) We have shown that BRCA1 and BRCA2 tumors show specific chromosomal changes, and that these somatic aberrations can be used to classify individual tumors. We use a genome-wide array-CGH analysis containing 3500 1 Mb spaced BAC clones (www.sanger.ac.uk) with routinely formalin-fixed paraffin embedded (FFPE) tumor material. In collaboration with Dr. P Devilee (Leiden University), we analyzed a series of 62 breast tumors from high-risk BRCA1/2 negative breast cancer only (HBC) families. Within these BRCAx families, different subgroups of tumors were identified on the basis of their aCGH profile. In a series of 40 breast tumors from high-risk BRCA1/2 negative breast and ovarian cancer (HBOC) families, only three tumors showed a BRCA1-like profile, one was proven to be a splice-site mutant by RNA analysis and one patient showed BRCA1 inactivation through promoter hypermethylation. In addition, so-called unclassified variants were tested, and showed that aCGH profiling could classify them as BRCA1, which is clear additional proof of significance of the variation. Tumor Characteristics in Premenopausal Women Carrying the CHEK2*1100delC Germline Mutation. J Clin Oncol 2007; 25:64-69 Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FBL, Verhoef S, Nederlof PM. A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer 2006; 94:333-337 Van Beers EH, Nederlof PM. Array-CGH and breast cancer. Breast Cancer Res 2006; 8:210 Van Houwelingen HC, Bruinsma T, Molecular characteristics of uterine corpus cancers subsequent to use of tamoxifen for breast cancer (PI Petra Nederlof ) Long-term use of tamoxifen (TAM) after breast cancer is associated with an unexplained increased risk of uterine corpus cancer. We analyzed FFPE endometrioid, serous and carcinosarcomas tumor samples after long-term exposure (в‰Ґ3 years) to TAM and TAM- (age- and morphology) matched control patients by 1Mb array-CGH. Results show that there is no correlation between TAM use and the number and type of genomic aberrations detected. Endometrioid tumors show fewer aberrations than the other tumor types, and frequently show MSI. Fresh-frozen tissue of 170 patients has been collected and RNA isolated from these samples will be hybridized on 30K oligo-microarrays to study differentially expressed genes in TAM+ and TAM- tumors. Hart AAM, Van вЂ�t Veer LJ, Wessels LFA. Cross-validated Cox regression on microarray gene expression data. Stat Med 2006; 25:3201-3216 86 EXPERIMENTAL THERAPY MOLECULAR ANALYSIS OF BREAST CANCER Group leader Marc Van de Vijver Our lab is studying breast cancer, with the aim to understand the genetics of the development of these tumors and use this knowledge for the clinical management of breast cancer patients. Breast cancer is a heterogeneous disease and based on morphological and genetic characteristics, various subtypes can be identified. The main aim of our research is to refine the classification of breast cancer; to achieve this goal, we are combining histological examination, genetic analysis and gene expression profiling of tumors and correlate these features to clinical parameters. Marc Van de Vijver MD PhD Group leader Harry Bartelink MD PhD Academic staff Hans Peterse MD Academic staff Sjoerd Rodenhuis MD PhD Academic staff Emiel Rutgers MD PhD Academic staff Juliane Hannemann MSc Graduate student Hugo Horlings MD Graduate student Bas Kreike MD Graduate student Dimitry Nuyten MD Graduate student Hans Halfwerk Technical staff Stephanie Hundscheid Technical staff Petra Kristel Technical staff Lennart Mulder Technical staff Publications Abdullah-Sayani A, Bueno de Mesquita JM, Van de Vijver MJ. Technology insight: Tuning into the genetic orchestra using Molecular subtypes of breast cancer and amplification of Topoisomerase II : predictive role in dose intensive adjuvant chemotherapy Benefit from chemotherapy treatment in breast cancer patients is determined by the molecular make-up of the tumor. In a retrospective analysis of patients treated in a randomized clinical trial comparing high dose chemotherapy with standard chemotherapy. In addition, the topoisomerase IIa amplification status was determined by FISH and CISH. 411 of the 753 tumours (55%) were classified as luminal-like, 137 (18%) as basal-like and 205 (27%) as HER2 amplified. The 137 basal-like tumors were defined as having no expression of ER and HER2; 98 of them did express EGFR and/or cytokeratin 5/6. The luminal-like tumors had a significantly better recurrence free and overall survival than the other two groups. From the 194 HER2 positive tumors, 47 (24%) were shown to harbor an amplification of TOP2A. Patients with a HER2amplified tumor randomized to the high-dose therapy arm did worse than those in the conventional treatment arm, possibly caused by the lower cumulative anthracycline dose in the high-dose arm. The tumors with a TOP2A amplification contributed hardly to this difference, suggesting that TOP2A amplification is not the cause of the steep dose-response curve for anthracyclines in breast cancer. The difference of the cumulative dose of only 25% between the treatment arms was possibly insufficient to yield a survival difference. microarrays - Limitations of DNA microarrays in clinical practice. Nat Clin Pract Oncol 2006; 3:501-516 Adler AS, Lin M, Horlings H, Nuyten DSA, Van de Vijver MJ, Chang HY. Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet 2006; 38:421-430 Chi JT, Wang Z, Nuyten DSA, Rodriguez EH, Schaner ME, Salim A, Wang Y, Kristensen GB, Helland A, Predicting the response to specific forms of systemic therapy An ongoing research project investigates whether the gene expression profile of the primary breast tumor can predict response to specific regimens of neoadjuvant chemotherapy. Gene expression data of the primary tumor before chemotherapy are now available from 63 patients and recruitment of patients is ongoing. 27% of the patients showed a (near) pathologic complete remission, 40% of the patients had a partial remission and 33% of the patients did not respond to chemotherapy. Based on gene expression profiling, we identified the molecular subtypes of breast cancer in our data. Basal-like and ERBB2-like tumors responded frequently to chemotherapy, whereas the luminal tumors were often resistant to treatment. We are exploring the predictive value of several gene expression signatures identified by others; and are using supervised classification approaches to find chemotherapy response classifiers. BГёrresen-Dale A-L, Giaccia A, Longaker MT, Hastie T, Yang GP, Van de Vijver MJ, Brown PO. Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers. PLoS Medicine 2006; 3:395-409 Hannemann J, Velds A, Halfwerk JBG, Kreike B, Peterse JL, Van de Vijver MJ. Classification of ductal carcinoma in situ by gene expression profiling. Breast Cancer Res 2006; 8:R61 Classification of ductal carcinoma in situ by gene expression profiling Ductal carcinoma in situ (DCIS) is characterized by the intraductal proliferation of malignant epithelial cells. Several histological classification systems have been developed, but assessing the histological type/grade of DCIS lesions is still challenging, making treatment decisions based on these features difficult. To obtain insight into the molecular basis of the development of different types of DCIS and its progression to invasive breast cancer, we have studied differences in gene expression between different types of DCIS and between DCIS and invasive breast carcinomas. Gene expression profiling using microarray analysis has been performed on 40 in situ and 40 invasive breast cancer cases. DCIS cases were classified as well (n=6), intermediately (n=18) and poorly (n=14) differentiated type. Of the 40 invasive breast cancer samples, 5 samples were grade I, 11 samples were grade II and 24 samples were grade III. Using two-dimensional hierarchical clustering, the basal-like type, ERB-B2 type, and the luminal type tumors originally described for invasive breast cancer could also be identified in DCIS. Using supervised classification, we identified a gene expression classifier of 35 genes, which differed between DCIS and invasive breast cancer; a classifier of 43 genes could be identified separating between well and poorly differentiated DCIS samples. 87 EXPERIMENTAL THERAPY Publications Do specific DNA copy number changes regulate prognostic gene expression signatures in breast cancer? Our goal is to identify candidate functional regulators of expression signatures by comparing genome-wide DNA copy number with gene expression data. We want to test if these candidate functional regulators can act as potential prognostic and/or predictive markers in breast cancer patients. In order to reach this goal we carried out Comparative Genomic Hybridization (CGH) in 67 human breast carcinomas for which gene expression data and clinical data were available. Statistical analysis linked distinct regions of DNA copy numbers alterations to different prognostic gene expression signatures. For example: the previously identified poor prognosis 70 gene signature is associated with gain of 3q and the 70 genes good prognosis profile with loss at 16q; the activated wound signature is associated with gain at 8q and 12q; genomic grade 3 is associated with genetic gain at 3q, 8q; basal type tumors show losses at 4p, 5q, 14q, 15q and a gain of 6p, 10p and 12p; HER2 positive breast tumors gain at 17q (where the HER2 gene is located), but no specific additional alterations. These findings help establish a link in the interaction between genetic changes and gene expression profiles that determine tumor cell behavior in breast cancer. Hannemann J, Kristel P, Van Tinteren H, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van Der Wall E, Hupperets P, De Vries EGE, Rodenhuis S, Van de Vijver MJ. Molecular subtypes of breast cancer and amplification of topoisomerase IIa: Predictive role in dose intensive adjuvant chemotherapy. Br J Cancer 2006; 95:1334-1341 Kreike B, Halfwerk H, Kristel P, Glas A, Peterse H, Bartelink H, Van de Vijver MJ. Gene expression profiles of primary breast carcinomas from patients at high risk for local recurrence after breast-conserving therapy. Clin Cancer Res 2006; Gene expression profiles of primary breast carcinomas from patients at high risk for local recurrence after breast conserving therapy Several risk factors for local recurrence of breast cancer after breast conserving therapy (BCT) have been identified. The identification of additional risk factors would be very useful in guiding optimal therapy and also improve understanding of the mechanisms underlying local recurrence. We used cDNA microarray analysis to identify gene expression profiles associated with local recurrence. Gene expression profiles were obtained from 50 patients who underwent BCT. Of these 50 patients 19 developed a local recurrence; the remaining 31 patients were selected as controls as they were free of local recurrence at least 11 years after treatment. For 9/19 patients also the local recurrence was available for gene expression profiling. Unsupervised and supervised methods of classification were used to classify patients in groups corresponding to disease outcome and to study the overall gene expression pattern of primary tumors and their recurrences. No significant differences in gene expression between primary breast cancer tumors in patients with or without local recurrence after breast conserving therapy were identified. Furthermore, analyses of primary tumors and the paired local recurrences show a preservation of the overall gene expression pattern in the local recurrence, even after radiotherapy. From this work we conclude that there are no major differences in the gene expression profile of tumors that do or do not develop a local recurrence development, but that it is likely that subtle differences are present. To identify such subtle differences, larger tumor series will need to be studied. We have therefore expanded our study group to 170 primary breast carcinomas by collaborating with several other hospitals (J. Foekens, Erasmus Medical Center; P. Bult (UMC St. Radboud) and S. Veltkamp, Amstelland Hospital, Amstelveen). 12:5705-5712 Sotiriou C, Wirapati P, Loi S, Harris A, Fox S, Smeds J, Nordgren H, Farmer P, Praz V, Haibe-Kains B, Desmedt C, Larsimont D, Cardoso F, Peterse H, Nuyten D, Buyse M, Van de Vijver MJ, Bergh J, Piccart M, Delorenzi M. Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 2006; 98:262-272 Figure VIII.7a: Figure VIII.7b: Survival curves: Recurrence-free and overall survival among Forest Plot: Correlation of the recurrence free the HER2-expressing, ER-expressing and the basal-like survival in these subgroups with response to subtype of invasive breast cancer. conventional anthracycline-based or alkylating high-dose chemotherapy, respectively. In this Forest-plot, the closed squares represent the point estimate of the effect of high-dose chemotherapy (HD) vs conventional-dose therapy (CONV) within that particular subgroup (with the size of the square corresponding to its variance) and the lines correspond to the 99% confidence interval of that effect. The open figure at the bottom shows the overall effect of high-dose chemotherapy vs CONV for this particular cohort of patients. 88 RADIOTHERAPY Division head, Group leader Harry Bartelink Berthe Aleman MD Academic staff Harry Bartelink MD PhD Academic staff Jose Belderbos MD Academic staff Iain Bruinvis PhD Academic staff EugenГЁ Damen PhD Academic staff Roel De Boer PhD Academic staff Luc Dewit MD PhD Academic staff Rick Haas MD Academic staff Guus Hart MSc Academic staff Wilma Heemsbergen MSc Academic staff Edwin Jansen MD Academic staff Joos Lebesque MD PhD Academic staff Corrie Marijnen MD PhD Academic staff Harry Masselink MD Academic staff Ben Mijnheer PhD Academic staff IX D IV IS ION OF R A D IOTHER A PY Our previous research results, leading to the development of a Cone Beam CT guided linear accelerator and the creation of software for image guided radiotherapy (IGRT), are now in full operational use in our and many other clinics. These developments have led to new approaches in several tumor sites, for example adaptive radiotherapy for prostate cancer. Here, treatment plans for individual patients are refined based upon repetitive imaging of prostate and normal tissues during the first week of treatment. Another example is frameless stereotactic radiotherapy for lung cancer: by localizing the tumor just prior to each daily treatment fraction, a very high radiation dose can be delivered safely in a few fractions to the tumor process. A complimentary development is in vivo dosimetry with electronic portal imaging devices, which allows us to check for each individual patient the proper delivery of the radiation dose. This is especially important since more complex and sophisticated treatments are being introduced in the clinic with intensity-modulated radiotherapy (IMRT). These developments have already led to better results in improving the cure rate: for example in prostate cancer and breast cancer our own trials demonstrated that by delivering a higher radiation dose, a better local control is achieved. At the same time in prostate cancer it was shown that the toxicity could be reduced with these approaches. Safe execution of these complex techniques is, however, hardly possible without the aforementioned developments in patient positioning and quality assurance. Luc Moonen MD PhD Academic staff Coen Rasch MD PhD Academic staff Peter Remeijer PhD Academic staff Nicola Russell MD PhD Academic staff Govert Salverda MD Academic staff Christoph Schneider PhD Academic staff Joep Stroom PhD Academic staff Jacqueline Theuws MD PhD Academic staff Bart Van Bunningen MD Academic staff Marcel Van Herk PhD Academic staff Marcel Verheij MD PhD Academic staff Corine Van Vliet-Vroegindeweij PhD Academic staff Fritts WittkГ¤mper PhD Academic staff Conny Vrieling MD PhD Academic staff Nina Bijker MD PhD Temporary staff Monique Bloemers MD Temporary staff Research aimed at combined modality treatment with new novel agents is ongoing to maximally profit from the progress with sophisticated radiotherapy with IMRT and IGRT. Translational research is focused at discovery of new targets; novel agents directed against these targets are now brought into the clinic. Especially, improving the results of radiation by increasing the amount of apoptosis by novel agents is explored extensively. To predict treatment outcome and monitor the response during therapy, several approaches are investigated in the clinic. Examples of successful strategies to predict treatment outcome are: in vivo annexin V scintigraphy to demonstrate the amount of apoptosis after radiation, or measuring cisplatin DNA adducts in patients receiving cisplatin-based chemoradiotherapy. Recently, a clinical trial has been completed comparing the intra-arterial versus intra-venous administration of cisplatin concomitant with radiation for head- and neck tumors, aiming at delivering a higher dose of the cisplatin drug into the tumors without unacceptable side effects. Finally, a large multicenter randomized phase III trial has been launched this year investigating the role of post-operative chemoradiotherapy in gastric cancer. Gerben Borst MD Temporary staff Desiree Van den Bongard MD Temporary staff Patricia De Haan MD Temporary staff IMAGE ACQUISITION AND PROCESSING Frank Hoebers MD Temporary staff Anja Betgen, Jose Belderbos, Josine De Bois, Eugene Damen, Jurjen Van Dijk, Joop Duppen, Joost Knegjens MD Temporary staff Michel Frenay, Joris Van der Geer, Frank Hoebers, David Jaffray1, Rianne de Jong, Simon Van Kranen, Bas Kreike MD Temporary staff Vannesa Mexner, Danny Minkema, Tonnis Nuver, Jasper Nijkamp, Erik-Jan Rijhorst, Tezontl Rosario, Dimitry Nuyten MD Temporary staff Floris Pos, Christoph Schneider, Monique Smitsmans, Roel Steenbakkers, Joost Voogt, Vera Opperdijk MD Temporary staff Corine Van Vliet-Vroegindeweij, Marnix Witte, Jochem Wolthaus, Lambert Zijp, Coen Rasch, Floris Pos MD Temporary staff Joos Lebesque, Peter Remeijer, Jan-Jakob Sonke, Marcel Van Herk Roel Steenbakkers MD Temporary staff Jan-Jakob Sonke PhD Post-doc Leah McDermott Ba/Bs Graduate student Agnieszka Olszewska MSc Graduate student Marco Schwarz MSc Graduate student Monique Smitsmans MSc Graduate student Anja Betgen Technical staff Bob Brand Technical staff Josine De Bois Technical staff The continuing remodeling of the department necessitated a temporary displacement of the research group to the 3rd floor. With the increased distance the communication between the clinic and research is more difficult. In the next year the final housing should be finished and we are eager to renormalize the situation. This year saw a large increase in the clinical application of the cone-beam CT (CBCT) guided radiotherapy: now available on four machines in our department. The software used in this equipment is for a large part developed in our department, and is now in use in over 100 departments worldwide. Our leading role in this field was recognized by Joop Duppen Technical staff Michel Frenay Technical staff 1 University of Toronto/Princess Margaret Hospital, Ontario, Canada 89 RADIOTHERAPY invitations for lectures and teaching courses and also in the form of our chairman receiving the Regaud award at the 2006 ESTRO. With the increased clinical application there is also a hugely increased data-production. There is now around 3TB cone beam data stored. The help of our research developers was invaluable to manage this flow. First clinical results of an adaptive radiation scheme using CBCT for prostate cancer An adaptive scheme for prostate cancer radiotherapy based on kV cone-beamCT (CBCT) images obtained in the first six treatment days is in large-scale use. In this way, better estimates of the average prostate position and rectum shape are obtained, and the PTV margin was safely reduced from 10 to 7 mm. Weekly CBCTscans, used for patient set-up correction, are also used to verify that the prostate is inside the new PTV. So far, 46 patients were treated with our adaptive treatment scheme. For 92% of the CBCT-scans a successful grey-value match was obtained, the other scans were discarded. Only in a few cases the weekly scans show a marginal miss (1 mm) of the CTV. We estimate that our scheme reduces rectal toxicity by 30%. This is the first routine clinical application of soft tissue image guidance for the prostate using kV CBCT. Contrary to adaptive schemes that use implanted markers, our method is non-invasive and improves localization of the rectum, as well as the prostate. The influence of a dietary protocol on automatic prostate localization in cone-beam CT scans The success rate of the automatic prostate registration process described above depends on the image quality of the CBCT scans. The image quality of these scans is deteriorated when gas in the rectum moves during CBCT acquisition. We have evaluated the impact of a dietary protocol on the presence of moving gas and automatic grey value registration. To that end we analyzed 271 scans of 21 patients that were subjected to the dietary protocol (DP data set) and 190 scan of 18 patients that were not subjected to the protocol (NDP data set). The percentage of scans with feces, gas and moving gas were significantly reduced from 67%, 61% and 45% in the NDP data set to 47%, 30% and 26% in the DP data set (p<0.001). The success rate of 3D gray-value registration improved significantly from 83% in the NDP to 92% in the DP data set (p<0.001). This suggests that a dietary protocol is also important to reduce intra-fraction prostate motion during treatment delivery. Therefore, the dietary protocol is currently prescribed to all our prostate cancer patients, independent of the imaging protocol (CBCT or EPID) used. (Figure IX.1a and 1b) Figure IX.1: The impact of a large, moving gas pocket during cone-beam computed tomography (CBCT) image acquisition on the CBCT reconstruction result. (a) Projection images (PI) showing a large, moving gas pocket (bright structure) emerging in the rectum in a time span of 17 s at a position where the prostate would be projected in the projection images. (b) The reconstruction result, showing the streak artifacts in a transverse slice of the CBCT scan. High precision CBCT guided hypofractionated radiotherapy We implemented high dose hypofractioned regimes for solitary brain metastases and small lung tumors with on-line patient position verification and correction using CBCT and investigated the accuracy of these techniques. First, a CBCT scan was acquired and registered to the planning CT (using bone for the head, and soft tissue for the lung) Tom Minderhoud Technical staff Kenneth Pengel Technical staff Maddalena Rossi Technical staff RenГ© Tielenburg Technical staff Peter Van de Ven MSc Technical staff Lambert Zijp Technical staff 90 RADIOTHERAPY to check the patient setup. Deviations larger than 1 mm were corrected by a manual couch shift and verified by a 2nd CBCT. The stability of the patient position was checked by an additional scan after treatment. Patient stability was excellent and no errors larger than 2 mm were observed in the follow-up scans for the brain. For the lung cancer patients, however, larger shifts were observer, probably due to instability of the matting used for patient comfort. By using on-line CBCT we can correct for setup errors and verify the patientвЂ™s position during treatment, thereby providing a similar accuracy and safety as stereotactic techniques but more efficient and patient friendly. However, patient stability during hypofractionated lung radiotherapy must be improved. Figure IX.2: Schematic overview of systematic (arrows) and random (ellipses) base-line deviations and their locations found in a group of 32 lung cancer patients. Analysis of baseline shifts in lung cancer patients With four-dimensional (4D) CT it is possible to measure the tumor trajectory and mean position relative to the bony anatomy. A single 4DCT scan, however, captures just a snapshot movie-loop of the patientвЂ™s respiratory motion pattern. We investigated the variability of respiratory motion over the course of radiotherapy using repeat 4D CBCT scans. It was found in a group of 32 patients that the shape of the tumor trajectory (averaged over the course of 4D CBCT acquisition taking approximately 4 min) hardly changes over the course of treatment (1 mm SD). The center of mass of the trajectory, on the other hand, exhibited substantially baseline shifts, i.e., variations relative to the bony anatomy. Systematic deviations were 1 mm (LR), 3 mm (CC) and 2 mm (AP) and similar random deviations 1 mm (LR), 3 mm (CC) and 3 mm (AP). These baseline shifts should be incorporated in an appropriate PTV margin or be corrected for using softtissue imaging and guidance. (Figure IX.2) Anatomical variability of head and neck cancer patients Head and neck cancer patients in our clinic receive radiotherapy while immobilized by a 5-point mask. Geometrical deformations of the anatomy due to patient setup were analyzed. In CBCT scans of 15 patients (7 scans per patient), local rigid body registration was used to measure displacements of each bone from CBCT to planning CT. To separate setup error and deformation, vertebrae C3-C5 were used a reference. Deformations far away from the reference are often larger than the setup errors of C3-C5. Causes of these deformations are bending if the neck and instability of the mandibula in patients using a biteblock. In conclusion, head and neck cancer patients often show anatomical variability that cannot be corrected with a simple couch shift. Careful registration and selection of the reference point is required, as well as adjustment of the margins, to avoid underdosage of the target and overdosage of organs at risk. (Figure IX.3) Figure IX.3: Local geometrical uncertainties (AP, CC) after corrections for patient setup vertebrae C3-C5 taken as correction point. Effect of respiratory monitoring on tumor motion 4DCT is used to decrease geometrical uncertainties induced by respiratory tumor motion. During 4DCT, we use a thermocouple mounted on a standard oxygen mask to register the respiratory cycle. We investigated the impact of this monitoring system on tumor amplitude and mean position by applying it during 4DCBCT acquisitions for routine set-up verification. The respiration signal required for 4DCBCT reconstruction was directly obtained from the CB projection data making the oxygen mask unnecessary. Half the scans were performed with the oxygen mask in place. There was no significant difference in the mean tumor position between the 4D CBCT scans with or without the respiratory monitoring system. Tumor amplitude, however, increased on average with 29% (9-64%) for scans with the oxygen mask. We conclude that the use of this monitoring system has an impact in tumor amplitude, potentially leading to an overestimation of treatment margins. Software development for image-guided radiotherapy The implementation of on-line corrections based on soft tissue imaging of lung tumors required a method for inline 4D reconstruction, i.e. while the scan is acquired. This is problematic as the phase-sorting algorithm interpolates over many projection images to estimate the motion. To solve this problem, a new algorithm was developed in which the projection images are temporarily cached until sufficient neighbors are available. In practice this mean that a small fraction of projection images (50 out of 1000) must be processed after acquisition is complete. The delay between image acquisition and 91 RADIOTHERAPY Publications availability of the 4D data is, however only a few seconds. We also implemented automatic grey-value based registration of the lung tumor in each phase of the 4D CBCT scan. This means that currently on-line corrections for lung treatments applying full 4D analysis are feasible. The technique has just been introduced clinically. Image acquisition and processing Keall PJ, Mageras GS, Balter JM, Emery RS, Forster KM, Jiang SB, Kapatoes JM, Low DA, Murphy MJ, Analysis of inter-patient variability in setup motion It is generally assumed that the statistics of geometric errors differs from patient to patient. We developed a method to estimate the distribution variability based on analysis of a large patient population and investigated the effect of detected variability on population based PTV-margins. Setup data of 470 prostate cancer patients (11 fractions per patient), were analyzed for random setup errors. The SD of the SD per patient contains the real distribution variability diluted by вЂњmeasurement error in the SDвЂќ due to the limited number of samples. After correction is applied for this вЂњmeasurement errorвЂќ, the true variability is about 25% of the SD. This only slightly exceeds the SD measurement error and is therefore difficult to detect for individual patients. Interpatient distribution variability does lead to larger PTV-margins, because the range of dose blurring becomes patient dependent. Assuming normality and the same SD variability in random and systematic errors, the margin for systematic errors increases from 2.5SD to about 2.8SD, maintaining the same margin for random errors. Murray BR, Ramsey CR, Van Herk MB, Vedam SS, Wong JW, Yorke E. The management of respiratory motion in radiation oncology report of AAPM Task Group 76. Med Phys 2006; 33:3874-3900 Koshani R, Balter JM, Hayman JA, Henning GT, Van Herk M. Short-term and long-term reproducibility of lung tumor position using active breathing control (ABC). Int J Radiat Oncol Biol Phys 2006; 65:1553-1559 Lotz HT, Pos FJ, Hulshof MCCM, Van Herk M, Lebesque JV, Duppen JC, Remeijer P. Tumor motion and Modeling tumour control probability including geometrical uncertainty For over 500 high-risk prostate cancer patients the delineated CTV (prostate + SV) and rectum contours were available, as well as the dose distribution from the treatment planning system. We simulated many treatment histories by sampling the distributions of systematic and random set-up errors and organ motion, translating and rotating the CTV volume relative to the dose. The expected TCP (Webb model) was estimated and fitted with the clinical data. The effect of the geometrical uncertainties is a slight reduction of the estimated inter-patient variability in radiosensitivity. This is the first TCP model fit, using a moving CTV as tumor, instead of a static PTV. Future work will focus on the impact of inhomogeneous clonogen distributions and incorporation of rectum distention subgroup data. deformation during external radiotherapy of bladder cancer. Int J Radiat Oncol Biol Phys 2006; 64:1551-1558 Ruan D, Fessler JA, Balter JM, Sonke JJ. Exploring breathing pattern irregularity with projection-based method. Med Phys 2006; 33:2491-2499 Steenbakkers RJHM, Duppen JC, Van Herk M, Rasch CRN. In response to Dr. Rodriguez de Dios. Int J Radiat Oncol The effect of undersampling pathology on safety margins for microscopic disease To accurately define the clinical target volume for radiotherapy, pretreatment imaging is be correlated with histopathology. In clinical practice, however, only a limited number of histopathological slices can be analyzed. In this study, we investigate the influence of this undersampling on the accuracy of detecting microscopic disease spread. The microscopic disease is assumed to consist of islets spread randomly around a spherical tumor, with a probability that decreases exponentially with distance from the gross tumor. Possible undersampling due to limited number of slices is calculated for various values of the GTV size, the number and the size of the microscopic islets, and the number of microscopic slices analyzed. For instance, using 10 slices, the maximum distance found would on average be only 60% of the actual distance (i.e. 0.84 cm). We conclude that pathology findings of microscopic spread around tumors must be corrected for possible undersampling effects. Otherwise, the margin for microscopic disease may be seriously underestimated. Figure IX.2: Schematic overview of systematic (arrows) and random (ellipses) base-line deviations and their locations found in a group of 32 lung cancer patients. Biol Phys 2006; 65:1276-1277 Steenbakkers RJHM, Duppen JC, Fitton I, Deurloo KEI, Zijp LJ, Comans EFI, Uitterhoeve ALJ, Rodrigus PTR, Kramer GWP, Bussink J, De Jaeger K, Belderbos JSA, Nowak PJCM, Van Herk M, Rasch CRN. Reduction of observer variation using matched CT-PET for lung cancer delineation: A threedimensional analysis. Int J Radiat Oncol Biol Phys 2006; 64:435-448 92 RADIOTHERAPY Publications (continued) DOSIMETRY Dosimetry Leah McDermott, Anton Mans, Jan-Jakob Sonke, Joep Stroom, Marcel Van Herk, Markus Wendling, Ben Mijnheer Bloemen-van Gurp EJ, Minken AWH, Mijnheer BJ, Dehing-Oberye CJG, Lambin P. Clinical implementation of MOSFET detectors for dosimetry in electron beams. Radiother Oncol 2006; 80:288-295 McDermott LN, Wendling M, Sonke JJ, Van Herk M, Mijnheer BJ. Anatomy changes in radiotherapy detected using portal imaging. Radiother Oncol 2006; 79:211-217 Portal dosimetry Portal imaging devices (EPIDs) are now used in our department for the dosimetric verification of external beam, megavoltage radiation therapy вЂ“ both pre-treatment and in vivo. Many studies have reported dosimetric characteristics of these devices. Some studies ascribed a non-linear signal to gain ghosting and image lag. Other reports, however, state the effect is negligible. This study compared the signal-to-monitor unit (MU) ratio for three different brands of EPID systems. The signal was measured for a wide range of monitor units (5-1000), dose-rates and beam energies. All EPIDs exhibited a relative under-response for beams of few MUs; giving 4 to 10% lower signal-to-MU ratio relative to that of 1000 MUs as shown in Figure IX.4 this under-response is consistent with ghosting effects due to charge trapping. McDermott LN, Wendling M, Van Asselen B, Stroom J, Sonke JJ, Van Herk M, Mijnheer BJ. Clinical experience with EPID dosimetry for prostate IMRT pre-treatment dose verification. Med Phys 2006; 33:3921-3930 Figure IX.4: Signal-to-MU ratios for Elekta, McDermott LN, Nijsten SMJJ, Sonke JJ, Varian and Siemens a-Si EPIDs, averaged Partridge M, Van Herk M, Mijnheer BJ. over 2 to 3 dose-rate settings for different Comparison of ghosting effects for three energies, with 1 or 2 detectors for each brand. commercial a-Si EPIDs. Med Phys 2006; All points are normalised at 1000 MUs. 33:2448-2451 Different scintillators employed by different brands will exhibit slight variation in ghosting Van Elmpt WJC, Nijsten SMJJ, Schiffeleers effects; however there is a consistent under- RFH, Dekker ALAJ, Mijnheer BJ, Lambin response for fields of fewer MUs for all three P, Minken AWH. A Monte Carlo based brands. three-dimensional dose reconstruction method derived from portal dose images. Med Phys 2006; 33:2426-2434 Wendling M, Louwe RJW, McDermott LN, Sonke JJ, Van Herk M, Mijnheer BJ. Accurate two-dimensional IMRT verification using a back-projection EPID dosimetry method. Med Phys 2006; 33:259-273 In order to investigate the feasibility of replacing pre-treatment with in vivo EPID dosimetry for IMRT prostate cancer treatments, dose distributions of 75 patient plans were reconstructed from EPID images, inside a patient or phantom. Planned and EPID dose values were compared at the isocentre and in 2D using the g index (3%/3mm). The number of measured fractions required to detect the same errors as pre-treatment verification was determined, and the time required to perform both methods was compared. Average planned and EPID isocentre dose values agreed within 1% for both pre-treatment verification and 5 in vivo fractions. An alert was raised for 10 out of these 75 pre-treatment checks, however, with no clinically relevant errors. Multiple in vivo fractions were combined by assessing gamma images comprising median, minimum and low pixel values of 1 to 5 fractions. Eleven plans had discrepancies using the low gamma values of 3 fractions. Additional time for pretreatment checks was ~2.5 h per plan. For in vivo dosimetry, only an additional 15 min + 10 min/fraction were required. It can be concluded that in vivo EPID dosimetry is an efficient alternative to pre-treatment verification for prostate IMRT treatments. The number of in vivo fractions required to replace pre-treatment verification is a balance between accurate detection and workload. Checking three fractions in vivo appears to be optimal, in order to distinguish systematic errors from random errors, with very little additional time required for verification. The g-evaluation method is a tool by which dose distributions can be compared in a quantitative manner combining dose-difference and distance-to-agreement criteria. One major disadvantage is, however, its long computation time, which especially applies to the comparison of 3D dose distributions. In this study, we present a fast algorithm for a full 3D g-evaluation at high resolution. Both the reference and evaluated dose distributions are first resampled on the same grid. For each point of the reference dose distribution, the algorithm searches for the best point of agreement according to the g method in the evaluated dose distribution, which can be done at a sub-voxel resolution. Speed, computer memory efficiency, and high 93 RADIOTHERAPY Publications (continued) spatial resolution are achieved by searching around each reference point with increasing distance in a sphere. The smaller the sample step size and the larger the differences between the dose distributions, the longer the g-evaluation takes. Two clinical examples were investigated using 3% of the maximum dose as dosedifference and 0.3 cm as distance-to-agreement criteria. For 0.2 cm grid spacing, the change in g indices was negligible below a sample step size of 0.02 cm. Comparing the full 3D g-evaluation and slice-by-slice 2D g-evaluation for these clinical examples, the g indices improved by searching in full 3D space, with the average g index decreasing by at least 7%. Treatment planning Schwarz M, Van der Geer J, Van Herk M, Lebesque JV, Mijnheer BJ, Damen EMF. Impact of geometrical uncertainties on 3D CRT and IMRT dose distributions for lung cancer treatment. Int J Radiat Oncol Biol Phys 2006; 65:1260-1269 TREATMENT PLANNING Van Asselen B, Schwarz M, Jose Belderbos, Suzanne Van Beek, Martijn Eenink, Marcel Van Herk, Katrien De Jaeger1, Vliet-Vroegindeweij C, Lebesque JV, Annemarie. Lakeman-Groot, Emmy Lamers, Joos Lebesque, Ben Mijnheer, Tom Minderhoud, Mijnheer BJ, Damen EMF. Intensity- Anke Van Mourik, Coen Rasch, Christoph Schneider, Marco Schwarz, Jan-Jakob Sonke, modulated radiotherapy of breast cancer Corine Van Vliet-Vroegindeweij, Maddalena Rossi, Jochem Wolthaus, Eugene Damen using direct aperture optimization. Radiother Oncol 2006; 79:162-169 Evaluation of different strategies for integration of 4D CT scans into the treatment planning of lung cancer patients. For 45 patients with lung tumors, tumor size and tumor motion were assessed from 4D CT scans. The average tumor volume was 40 В± 47 cm3, while the average tumor motion was 1.9 В± 1.3 mm, 7.2 В± 5.5 mm, and 3.1 В± 2.3 mm in left-right, cranial-caudal, and anterior-posterior direction, respectively. The GTV was delineated in the first phase of the 4D scan by an experienced radiation-oncologist. The delineation was copied to the other phases by applying the transformation matrix resulting from a phase-to-phase tumor match. Three different strategies to incorporate tumor motion and setup uncertainties into the PTV were evaluated: 1) the concept of Internal Target Volume (ITV); 2) gating at the exhale phase of the breathing cycle; 3) the use of a mid-ventilation scan. For each approach, the GTV to PTV margin was calculated using the recipe of van Herk: margin = 2.5S + 0.7s, where S and s are the standard deviations of systematic and random errors, respectively. The resulting PTV volumes were compared to the conventional approach, i.e. without using a 4D CT scan. The ITV approach resulted in an average increase of the PTV volume with 5.5% В± 4.5%. Gating and the mid-ventilation concept resulted in an average decrease of PTV volume with 12.7% В± 10.8%, and 8.9% В± 8.6%, respectively. Based on the results it was concluded that the ITV concept leads to grossly overestimated PTV volumes compared to the mid-ventilation and gating approaches and may therefore lead to an increased probability of normal tissue complications. Motion Estimation in 4D CT scans for assessment of internal motion, image enhancement and construction of the optimal CT scan for treatment planning Currently we use a mid-ventilation CT scan (MidV CT) for treatment planning of lung tumors. This MidV CT is reconstructed from a 4D CT scan and represents the tumor and surrounding lung tissue in its average position over the breathing cycle. However, by choosing the MidV CT frame from the 4D data set, a geometrical error ( eh ) with respect to the mean position is introduced due to hysteresis in the tumor motion. To overcome this problem, software was developed to warp each frame of the 4D CT scan and project all structures on their time-weighted mean position. The software uses an adaptation of an existing motion estimation method. The resulting CT scan depicts the tumor in its exact time-averaged position and yields less noisy images since the transformed CT frames are averaged (see Figure IX.5). The influence of heterogeneity corrections on the dose distribution of stereotactic treatment of lung tumors A protocol for stereotactic irradiation of medically inoperable stage I NSCLC has been introduced, closely following the RTOG 0236 phase II trial. The RTOG protocol required dose calculation without correction for tissue heterogeneity. Since this is not the current clinical practice in our institution, we investigated the influence of heterogeneity corrections on the prescription dose and the dose to organs at risk. The planning CT scans of 8 patients with NSCLC were used to compare different radiation plans. The IMRT plans were 1 Catharina Ziekenhuis, Eindhoven, The Netherlands Wolthaus JWH, Schneider C, Sonke JJ, Van Herk M, Belderbos JSA, Rossi MMG, Lebesque JV, Damen EMF. Mid-ventilation CT scan construction from four-dimensional respiration-correlated CT scans for radiotherapy planning of lung cancer patients. Int J Radiat Oncol Biol Phys 2006; 65:1560-1571 94 RADIOTHERAPY Figure IX.5: Results of motion estimation software. The upper row shows in column 1-3 coronal reconstructions of phase 0, 3, and 7 of the original 4D CT scan (10 phases). The last column shows the average over all phases. The middle row shows the deformation fields from phase 3 and 7 to phase 0. The lower row shows the result of deforming phase 3 and 7 to phase 0 (column 2 and 3). The last column of the lower row shows the average of the deformed images, illustrating the reduction in image noise that can be achieved by this method. optimized in Pinnnacle with direct machine parameter optimization (DMPO, 10-20 beams, 1-2 segments per beam), with and without heterogeneity correction in the dose calculation. Dose prescription for the homogeneous plans (i.e. calculated without heterogeneity correction) was according to RTOG 0236 criteria, i.e. 60 Gy (20 Gy per fraction) was prescribed to the isodose surface encompassing 95% of the PTV volume. For the heterogeneous plans (i.e. calculated with heterogeneity correction), dose calculation was performed while maintaining the monitor units of the homogeneous plan. Compared to the homogeneous plan, heterogeneity correction resulted on average in a lower dose to the PTV: average = 54.9 Gy (range 46.6 to 61.7 Gy). Due to decreased attenuation and broadening of the beam penumbra in low-density lung tissue, the relative maximum dose (Dmax) in the PTV was higher for the heterogeneous plans (i.e. the dose in the PTV was less homogeneous), while dose fall-off outside the PTV was less steep. The latter is reflected in a higher value of V20 for lung. Intensity-modulated versus conformal radiotherapy of parotid gland tumors: potential impact on hearing loss The purpose of this study was to design and evaluate an IMRT class solution in order to prevent hearing loss due to irradiation of parotid gland tumors. Twenty patients with parotid gland cancer were retrospectively planned with two different techniques using the original planning target volume (PTV). First a conventional technique using a wedged beam pair was applied yielding a dose distribution conformal to the shape of the PTV. Next an IMRT technique with pre-defined optimization constraints was designed. A dose of 66 Gy in the PTV was given at the ICRU dose prescription point. Dose distributions were compared with respect to PTV coverage and dose to organs at risk (OAR). With similar PTV coverage, the dose to OARs was lower in the IMRT plans. The mean volume of the middle ear receiving a dose higher than 50 Gy decreased from 66.5 to 33.4%. The mean dose in the oral cavity decreased from 19.4 Gy to 16.6 Gy. The auditory system can be spared if the distance between the inner ear and the PTV is 0.6 cm or larger, and if the overlap between the middle ear and the PTV is smaller than 10%. The maximum dose in the spinal cord was below 40 Gy in all treatment plans. It can be concluded that the auditory system as well as the oral cavity can be spared with IMRT. 95 RADIOTHERAPY Publications (continued) Evaluation of a single-isocenter technique for axillary radiotherapy in breast cancer A planning study was performed in 20 breast cancer patients, comparing a conventional AP-PA technique with a single-isocenter technique for irradiation of the axilla. The target volume of the axillary treatment encompassed the periclavicular and axillary lymph node areas. The lymph node areas and the organs at risk (OAR, i.e., the esophagus, spinal cord, brachial plexus, and lung) were delineated on CT scans. The conventional technique consisted of an AP-PA field with a transmission plate placed in the AP beam. The single-isocenter technique consisted of AP-PA fields using a gantry rotation of + or -20 degrees and a medial AP segment. Dose distributions were analyzed with respect to target coverage and dose to OARs. The field borders and humeral shielding were re-defined based on the 3D anatomical references. Adapting the humeral shielding reduced the irradiated volume by 19% and might contribute to a reduction of the incidence of arm edema and impairment of shoulder function. With similar PTV coverage and maximum dose to the brachial plexus, the maximum dose in the esophagus and spinal cord was reduced by more than 50% using the single-isocenter technique. Moreover, the single-isocenter technique allowed a fast and easy treatment preparation and reduced the execution time considerably (with approximately 10 minutes per fraction). Studies of radiation response in tumors and organs at risk Belderbos JSA, Heemsbergen WD, De Jaeger K, Baas P, Lebesque JV. Final results of a Phase I/II dose escalation trial in non-small-cell lung cancer using threedimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 2006; 66:126-134 Heemsbergen WD, Peeters STH, Koper PCM, Hoogeman MS, Lebesque JV. Acute and late gastrointestinal toxicity after radiotherapy in prostate cancer patients: Consequential late damage. Int J Radiat Oncol Biol Phys 2006; 66:3-10 Heemsbergen WD, Hoogeman MS, STUDIES OF RADIATION RESPONSE IN TUMORS AND ORGANS AT RISK Witte MG, Peeters S, Incrocci L, Paul Baas, Jose Belderbos, Michel Dielwart1, Katrien De Jaeger2, Guus Hart, Wilma Heemsbergen, Lebesque JV. Increased risk of biochemical Mischa Hoogeman3, Peter Koper3, Stephanie Peeters, Wim Van Putten3, Annerie Slot4, and clinical failur for prostate patients with Joos Lebesque a large rectum at radiotherapy planning: results from the Dutch trial of 68 Gy Prostate cancer: The Dutch dose escalation randomized trial of 68 Gy versus 78 Gy with 669 prostate cancer patients showed a significantly improved freedom from failure in patients treated to 78 Gy, at the cost of a very modest increase in gastrointestinal toxicity. Different aspects of this toxicity were studied in detail. versus 78 Gy. Int J Radiat Oncol Biol Phys 2007; In press Nuver T, Hoogeman MS, Remeijer P, Van Herk MB, Lebesque JV. An adaptive Localized volume effects for late rectal and anal toxicity after radiotherapy for prostate cancer In this analysis 641 patients from the Dutch dose escalation randomized trial (68 Gy vs. 78 Gy) were included. Toxicity was scored with adapted RTOG/EORTC criteria and five specific complications. The variables derived from DVH of anorectal, rectal and anal wall were: % receiving в‰Ґ 5 to 70 Gy (V5-V70), maximum (Dmax) and mean dose (Dmean). The anus was defined as the most caudal 3 cm of the anorectum. Statistics were done with multivariate Cox regression models. Median follow-up was 44 months. Anal dosimetric variables were associated with RTOG/EORTC grade в‰Ґ 2 (V5-V40, Dmean), and incontinence (V5-V70, Dmean). Bleeding correlated most strongly with anorectal V55-V65, and stool frequency with anorectal V40 and Dmean. Use of steroids was weakly related to anal variables. No volume effect was seen for RTOG/ EORTC grade в‰Ґ 3 and pain/cramps/tenesmus. off-line procedure for radiotherapy of prostate cancer. Int J Radiat Oncol Biol Phys 2007; In press Peeters STH, Heemsbergen WD, Koper PC, Van Putten WL, Slot A, Dielwart MF, Bonfrer JM, Incrocci L, Lebesque JV. Dose-response in radiotherapy for localized prostate cancer: results of the Dutch multicenter randomized phase III trial comparing 68 Gy of radiotherapy with 78 Gy. J Clin Oncol 2006; 24:1990-1996 Peeters STH, Lebesque JV, Rectal bleeding, fecal incontinence and high stool frequency after conformal radiotherapy for prostate cancer: normal tissue complication probability modeling This study included 468 prostate cancer patients participating in the above mentioned trial. We fitted the probability of developing late toxicity within 3 years (rectal bleeding, high stool frequency and fecal incontinence) with the original, and a modified LKB-model, in which a clinical feature (e.g. history of abdominal surgery) was taken into account by fitting subset specific TD50вЂ™s. The ratio of these TD50вЂ™s is the dose-modifying factor for that clinical feature. Dose distributions of anorectal (bleeding and frequency) and anal wall (fecal incontinence) were used. The modified LKB-model gave significantly better fits than the original LKB-model. Patients with a history of abdominal surgery had a lower tolerance to radiation than patients without previous surgery, with a dose-modifying factor of 1.1 for bleeding, and of 2.5 for fecal incontinence. The dose-response curve for bleeding was Heemsbergen WD, Van Putten WLJ, Slot A, Dielwart MFH, Koper PCM. Localized volume effects for late rectal and anal toxicity after radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys 2006; 64:1151-1161 Peeters STH, Hoogeman MS, Heemsbergen WD, Hart AAM, Koper PCM, Lebesque JV. Rectal bleeding, fecal incontinence, and high stool frequency after conformal radiotherapy for prostate cancer: Normal tissue complication probability modeling. Int J Radiat Oncol 1 Zeeuws Radiotherapeutic Institute, Vlissingen, The Netherlands 2 Catharina Ziekenhuis, Eindhoven, The Netherlands 3 Erasmus MC-Daniel Hoed den Cancer Center, Rotterdam, The Netherlands 4 Radiotherapeutic Institute Friesland, Leeuwarden, The Netherlands Biol Phys 2006; 66:11-19 96 RADIOTHERAPY Publications (continued) Breast cancer Antonini N, Jones H, Horiot JC, Poortmans P, Struikmans H, Den Bogaert WV, Barillot I, Fourquet A, Jager J, Hoogenraad W. Effect of age and radiation dose on local control after breast conserving treatment: EORTC trial 2288110882. Radiother Oncol 2006; In Press Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, Gennaro M, Rouanet P, Avril A, Fentiman IS, Bartelink H, Rutgers EJ. approximately two times steeper than that for frequency, and three times steeper than that for fecal incontinence Acute and late gastrointestinal toxicity after radiotherapy in prostate cancer patients: consequential late damage We studied whether there was a direct relationship between acute and late GI toxicity for the patients in the trial. We defined three outcomes for acute reactions: 1) maximum RTOG (Radiation Therapy Oncology Group) acute toxicity, 2) maximum acute mucous discharge (AMD) and 3) maximum acute proctitis. At multivariate analysis, AMD and acute proctitis were strong predictors for overall late toxicity, вЂ�intermittent bleedingвЂ™ and вЂ�incontinence padsвЂ™ (pв‰¤0.01). For вЂ�stools в‰Ґ 6/dayвЂ™ all three were strong predictors. No significant associations were found for вЂ�severe bleedingвЂ™ and вЂ�use of steroidsвЂ™. The predictive power of the dose parameters remained at the same level or became weaker for most late endpoints. These results suggest a significant consequential component in the development of late GI toxicity. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853--a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006; 24:3381-3387 Lung Final results of a phase I/II dose escalation trial in non-small cell lung cancer using three-dimensional conformal radiotherapy A phase I/II trial was performed including inoperable non-small cell lung cancer patients. Patients were irradiated once or twice daily, with 2.25 Gy per fraction and a fixed overall treatment time of 6 weeks. According to the relative mean lung dose (rMLD) 5 risk groups were defined. The rMLD was defined as the ratio of the MLD and the NTD normalized prescribed dose. The rMLD boundaries of these risk groups were: group 1, 0.0-0.12; group 2, 0.12-0.18; group 3, 0.18-0.24; group 4, 0.24-0.31; and group 5, 0.31-0.40. The dose was escalated with 6.75 Gy after a follow-up of 6 months without DLT. Eighty-eight patients included had tumor stage I or II in 53%, IIIA in 31% and IIIB in 17%. The maximum tolerated dose (MTD) was not achieved in the lowest risk group (reached dose 94.5 Gy). For risk groups 2 and 3 the MTD was 81 Gy. The 74.3 Gy was safe for group 4. In group 5 the starting dose level of 60.8 Gy was evaluated and safe. In 2 patients (5%) an isolated nodal relapse occurred. The dose delivered did have an impact on failure-free interval (FFI) and overall survival (OS), especially for small tumor volumes in multivariable analysis. In conclusion, dose escalation is safe up to 94.5 Gy in 42 fractions in 6 weeks in patients with a MLD в‰¤ 13.6 Gy. Higher doses are associated with a better FFI and OS for smaller tumor volumes. Involved-field irradiation results in a low percentage of isolated nodal relapses. BREAST CANCER Ninja Antonini, Liesbeth Boersma1, Eugene Damen, Guus Hart, Heather Jones, Bas Kreike, Ben Mijnheer, Nicola Russell, Laura Van вЂ™t Veer, Marc Van De Vijver, Conny Vrieling, Harry Bartelink Figure IX.6: Rish on local recurrence by age. The 10 years result of an EORTC trial were analyzed to investigate the long term impact of a boost radiation dose of 16 Gy on local control, risk of developing fibrosis and overall survival for patients with stage I and II breast cancer who underwent lumpectomy and whole breast irradiation as part of their breast conserving therapy (BCT). In this trial 5318 patients with microscopically complete excision followed by whole breast irradiation of 50 Gy were randomized between a boost dose of 16 Gy (2661 patients) and no boost dose (2657 patients) and follow-up for a median of 10.8 years. The median age was 55 years. Local recurrence was reported as the first failure in 278 with boost vs. 165 patients without boost; at 10 years, the cumulative incidence of local recurrence was 10.2% vs. 6.2 % for the no boost and the boost group, respectively (P<0.0001). The hazard ratio of local recurrence was 0.59 (0.46 вЂ“ 0.76) in favour of the boost, with no statistically significant interaction per age group. The absolute risk reduction at 10 year per age group was the largest in patients 40 years or less 23.9% to 13.5% (P=0.0014). Severe fibrosis was statistically significantly (P< 0.0001) increased in the boost group with 10-year rate of 4.4% versus 1.6% in the no boost group (P<0.0001). Survival at 10 years was 82% in both arms. We concluded that a boost irradiation of 16 Gy improved local control in all age groups at 1 MAASTRO Clinic, Maastricht, The Netherlands 97 RADIOTHERAPY Publications (continued) the cost of a small increase of severe fibrosis, while no difference in survival was observed. Mechanisms, modulation and prediction of radiation-induced cell death MECHANISMS, MODULATION AND PREDICTION OF RADIATION-INDUCED CELL DEATH Den Dulk M, Verheij M, Cats A, Jannie Borst, Daphne De Jong, Rick Haas, Frank Hoebers, Marina Kartachova, Jeffrey Klarenbeek, Jansen EPM, Hartgrink HH, Menno Van Lummel, Jan Schellens, Renato ValdГ©s Olmos, Wim Van Blitterswijk, Inge Verbrugge, Van de Velde CJH. The essentials of Stefan Vink, Esther Wissink, Shuraila Zerp, Harry Bartelink, Marcel Verheij locoregional control in the treatment of gastric cancer. Scand J Surg 2007; Our line of research continues to focus on (1) the identification of novel targets and agents to increase the cytotoxic effect of radiation and on (2) the validation of new endpoints to quantify and predict the efficacy and toxicity of new combination therapies. The ultimate goal is to translate these strategies from the lab into the clinic. 95:242-247 Hoebers FJP, Pluim D, Verheij M, Balm AJM, Bartelink H, Schellens JHM, Begg AC. Prediction of treatment outcome by cisplatin-DNA adduct formation in Alkyl-lysophospholipids (ALPs) ALPs such as Perifosine represent a first group of compounds that have become available for clinical use along this approach. These synthetic anti-tumor agents are known to accumulate in dynamic, sphingomyelin (SM) and cholesterol-enriched plasma membrane microdomains, also known as вЂњlipid raftsвЂќ. Once taken up via these membrane portals, ALPs interfere with lipid metabolism, inhibit survival signaling and stimulate apoptosis induction. In combination with radiation, ALPs cause a synergistic apoptotic effect and reduce clonogenic cell survival. Besides these radiosensitizing properties, ALPs show inhibition of cell cycle progression and potent anti-angiogenic effects in vitro. In a separate project (in collaboration with W Van Blitterswijk, Division III) we investigate mechanisms of cellular ALP uptake and transport, and address the functional implications of an altered membrane lipid composition on cellular drug uptake, signal transduction and radiosensitivity. We found that the uptake of ALPs via lipid rafts is essential for their cytotoxic effect, because disruption of these membrane structures by pharmacological (SM degradation/cholesterol depletion) or genetic (SM synthase knock down) approaches, results in resistance to ALP. Interestingly, these cells that lack SM and thus have defective rafts, show cross-resistance to a variety of other, unrelated apoptotic stimuli, including death receptor ligands and DNA-damaging regimens. The common factor of this вЂњmulti-stress resistanceвЂќ is a defect in SM synthesis by downregulation of the enzyme SMS1. The functional consequences, however, are different for the various apoptotic stimuli and are topic of current investigation. Following their in vitro characterization, ALPs were subsequently tested in vivo. In a mouse xenograft KB squamous cell carcinoma model the combination of oral Perifosine and radiation resulted in complete and sustained tumor regression. Histopathological examination demonstrated a prominent apoptotic tumor response. These in vitro and in vivo observations constituted the basis for a clinical phase I study that was completed in 2004. This study defined the MTD, toxicity profile and optimal scheduling of the combined treatment consisting of oral administration of Perifosine and radiotherapy in patients with locally advanced inoperable solid tumors. Next, we initiated a multicenter randomized placebo-controlled phase II study in locally advanced NSCLC, randomizing patients between radiation + placebo and radiation + daily Perifosine. patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin-radiation (RADPLAT). Int J Cancer 2006; 119:750-756 Horsman MR, Bohm L, Margison GP, Milas L, Rosier JF, Safrany G, Selzer E, Verheij M, Hendry JH. Tumor radiosensitizers - Current status of development of various approaches: Report of an International Atomic Energy Agency meeting. Int J Radiat Oncol Biol Phys 2006; 64:551-561 Jansen EPM, Saunders MP, Boot H, Oppedijk V, Dubbelman R, Porritt B, Cats A, Stroom J, ValdГ©s Olmos R, Bartelink H, Verheij M. Prospective study on late renal toxicity following postoperative chemoradiotherapy in gastric cancer. Int J Radiat Oncol Biol Phys 2007; In Press, Corrected Proof Kartachova MS, ValdГ©s Olmos R, Haas RLM, Hoebers FJP, Van den Brekel MW, Van Zandwijk N, Herk MV, Verheij M. Mapping of treatment-induced apoptosis in normal structures: 99mTc-Hynic-rh-annexin V SPECT and CT image fusion. Eur J Nucl Med Mol Imaging 2006; 33:893-899 Van de Velde CJH, Verheij M, Cats A. Other radiosensitizers in preclinical development In a separate project with J Borst (Division IV) we study the interaction between the death receptor ligand TRAIL and radiation. TRAIL induces apoptosis in a wide variety of tumor tissues, but lacks normal tissue toxicity in preclinical animal models. Although of interest as a therapeutic modality in itself, TRAIL is also an attractive candidate for combination therapy, since TRAIL and radiation or chemotherapy activate partially distinct apoptosis signaling pathways, while a molecular basis for synergy lies in the sensitization of cells by radiation to TRAIL-induced apoptosis. For proof of principle, we use lymphoid cells as model system, because these cells effectively die by apoptosis upon treatment with either radiation or TRAIL. In both EВµ myc and Jurkat cells, we demonstrated combined (additive to synergistic) effects of TRAIL and radiation, which were independent of the p53 status of the cells. In Jurkat cells, the Gastric cancer: a personal view. Adv Gastrointest Canc 2006; In press Van der Luit AH, Budde M, Zerp S, Caan W, Klarenbeek JB, Verheij M, Van Blitterswijk WJ. Resistance to alkyllysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelinand cholesterol-deficiency in lipid rafts. Biochem J 2007; 401:541-549 98 RADIOTHERAPY Publications (continued) Vink SR, Schellens JHM, Beijnen JH, Sindermann H, Engel J, Dubbelman R, Moppi G, Hillebrand MJX, Bartelink H, Verheij M. Phase I and pharmacokinetic study of combined treatment with perifosine and radiation in patients with advanced solid tumours. Radiother Oncol 2006; 80:207-213 Vink SR, Lagerwerf S, Mesman E, Schellens JHM, Begg AC, Van Blitterswijk WJ, Verheij M. Radiosensitization of squamous cell carcinoma by the apoptotic response to radiation or TRAIL is blocked by Bcl-2 (indicating dependence on the mitochondrial signalling pathway). Interestingly, upon treatment of the cells with radiation, TRAIL can effectively induce apoptosis independent of the mitochondrial pathway. This results in synergistic cell kill upon combined treatment. The effectiveness of the combined treatment modality was subsequently evaluated in vivo in collaboration with Dr. Henning Walczak, DKFZ, Germany. Jurkat T cells overexpressing Bcl-2 were grown as xenografts in mice and treated with radiation, IZ-huTRAIL i.p and the combination. Tumor growth delay was measured by caliper and luciferin-based in vivo bioluminescence. While radiation or TRAIL alone caused significant tumor growth delay, the combination was much more effective as tumors regressed and showed no regrowth (Figure IX.7). Normal tissue toxicity measured as changes in body weight and in liver enzymes, was minimal. These experiments are currently expanded with another death receptor ligand (FasL) and using spontaneous solid tumor mouse models for mesothelioma. alkylphospholipid perifosine in cell culture and xenografts. Clin Cancer Res 2006; 12:1615-1622 Wissink EHJ, Verbrugge I, Vink SR, Schader MB, Schaefer U, Walczak H, Borst J, Verheij M. TRAIL enhances efficacy of radiotherapy in a p53 mutant, Bcl-2 overexpressing lymphoid malignancy. Radiother Oncol 2006; 80:214-222 Figure IX.7: Inhibition of tumor growth by radiation (IR) and/or TRAIL. Sequential bioluminescence images of animals treated with vehicle (control) or with IR + TRAIL. Figure IX.8: Chemical structure of racemic Gossypol, a small molecule inhibitor of antiapoptotic Bcl-2 family members. Sequential bioluminescence images of animals treated with vehicle (control) or with IR + TRAIL In collaboration with Drs. Lippman and Yang from the University of Michigan and the division of Radiobiology at the Free University of Amsterdam, we have initiated preclinical efficacy testing of (-) Gossypol, a potent small-molecule inhibitor of Bcl-XL and Bcl-2. Gossypol binds to the BH3 domains of Bcl-2, Bcl-XL, and possibly other family members, and induces apoptosis. Because overexpression of these antiapoptotic molecules confers radioresistance and is often associated with poor prognosis, inhibition of Bcl-2/Bcl-XL represents a promising strategy to increase tumor response to radiation. A panel of human leukemic and head and neck cancer cell lines has been selected based on their expression pattern of the most relevant Bcl-2 family members. Gossypol was found to induce apoptosis in a time- and dosedependent fashion and to increase radiation-induced cell death. Isobolographic analysis revealed a synergistic interaction between radiation and Gossypol in these cell systems. In addition, activation of the pro-apoptotic SAPK/JNK pathway appeared to play an important role in this response as blockade of this signaling cascade by kinase inhibitor or overexpression of a dominant negative mutant of c-Jun (TAM-67) abrogated Gossypol-induced apoptosis. (Figure IX.8) Prediction of tumor response (1) In vivo apoptosis imaging. To evaluate whether early treatment-induced apoptosis can be visualized in vivo and whether it predicts clinical outcome, we evaluated a novel in vivo apoptosis imaging technique in collaboration with the department of nuclear medicine (ValdГ©s Olmos, Division XIII). This 99mTc-Annexin V scintigraphy (TAVS) is based on the exposure of phosphatidylserine (PS) at the outer leaflet of the plasma membrane lipid bilayer during the early phase of apoptosis. The human endogenous protein annexin V has a high affinity for PS, providing a detection method for apoptotic cells in vitro and in vivo. We have gained experience with this imaging technique in a variety of tumor types, including NHL, NSCLC, SCLC, H&NSCC and sarcomas treated by 99 RADIOTHERAPY Publications (continued) radiotherapy and/or chemotherapy. A total of 65 patients underwent TAVS before and early after the start of treatment. A statistically significant correlation was found between the increase in annexin V tumor uptake on the post-treatment scan and tumor response. These results indicate that TAVS might be useful as a non-invasive predictive test for treatment outcome. (2) Cisplatin-DNA adducts. Levels of cisplatin-DNA adducts have been shown to correlate with treatment outcome in patients with NSCLC. Quantification of adduct levels may provide a valuable tool to select patients for cisplatin-based combined modality treatment. In collaboration with the Division VIII (Begg and Schellens) we measure levels of cisplatin-DNA adducts in tumor and normal tissue obtained from different patient populations treated with cisplatin-based chemoradiation. In patients with stage IV head and neck cancer treated by conventional i.v (100 mg/m2), supradose i.a (150 mg/m2), or daily low dose i.v. (6 mg/m2) cisplatin-based chemoradiation (RADPLAT) and in cervical cancer patients treated by 40 mg/m2 cisplatin-based chemoradiation adduct levels have been measured. Samples for cisplatin-DNA adduct determination were obtained from 77 patients. Normal tissue samples were taken from all patients, tumor biopsies from 28 patients. Adduct-levels in tumor were 2.5-5 times higher than those in WBC for all 4 treatment regimes (p < 0.001). Analysis by schedule showed that increasing doses of cisplatin resulted in higher levels of GG-adducts in primary tumor (Spearman rank-correlation, r = 0.70, p < 0.001). For normal tissue (WBC and buccal cells) a similar pattern was seen for the i.v. schedules. However, the i.a. 150 schedule resulted in decreased GGadduct formation in normal tissue compared to the i.v. 100 schedule, indicative of more systemic exposure to cisplatin after i.v. cisplatin compared to selective highdose i.a. cisplatin administration. In multivariate analysis the relative contribution of cisplatin-dose and mode of administration (i.v. vs i.a.) were calculated separately. The level of GG-adducts was higher with higher cisplatin-dose (increase about 1.5% per mg cisplatin; p < 0.001). Adduct-levels in normal tissue were lower after i.a. administration than expected from this dose effect (by about 65 - 70%; p < 0.001). The effect of i.a. administration on primary tumor adducts showed a non-significant effect compared to i.v. treatment. In a subgroup of 35 head and neck cancer patients treated with conventional i.v. or supradose i.a. cisplatin-based chemoradiation, high tumor GG-adduct levels predicted better disease free survival than low adduct levels. Combination of radiotherapy and chemotherapy Van den Broek GB, Balm AJM, Van den Brekel MWM, Hauptmann M, Schornagel JH, Rasch CRN. Relationship between clinical factors and the incidence of toxicity after intra-arterial chemoradiation for head and neck cancer. Radiother Oncol 2006; 81:143-150 Van den Broek GB, Rasch CRN, Pameijer FA, Peter E, Van den Brekel MWM, Balm AJM. Response measurement after intraarterial chemoradiation in advanced head and neck carcinoma: Magnetic resonance imaging and evaluation under general anesthesia? Cancer 2006; 106:1722-1729 Zuur CL, Simis YJW, Lansdaal PEM, Rasch CRN, Tange RA, Balm AJM, Dreschler WA. Audiometric patterns in ototoxicity of intra-arterial cisplatin chemoradiation in patients with locally advanced head and neck cancer. Audiol Neurootol 2006; 11:318-330 99mTc Hynic-rh-Annexin V scintigraphy (TAVS) imaging is a non-invasive technique to demonstrate apoptosis in vivo. It was previously shown that the degree of Annexinuptake correlated with response to therapy in lymphoma patients (Kartachova et. al, 2004). We now used this technique in patients with head and neck squamous cell carcinoma, treated with concurrent cisplatin-based chemoradiotherapy and wanted to evaluate patterns of Annexin-uptake in normal tissue and tumor. TAVS was performed before and within 48 hours after the 1st course of cisplatin-based chemoradiation. SPECT data were co-registered to planning CT-scan. A complete set of these data was available for 13 patients. Regions of interest (ROI) were delineated: primary tumor, involved lymph nodes and salivary glands. The radiation-dose at follow-up TAVS was calculated for each ROI. Annexin-uptake was determined in each ROI, and the difference between follow-up and baseline TAVS represented the absolute Annexin-uptake: Delta-uptake (О”U). Annexin V scintigraphy showed a radiation-dose-dependent uptake in parotid glands, indicative of early apoptosis during treatment. The interindividual spread in Annexin-uptake in primary tumors could not be related to differences in treatment schedule or tumor volume, but the Annexin-uptake in tumor and lymph nodes were closely correlated. This effect might represent a tumor-specific apoptotic response. Other Adler AS, Lin M, Horlings H, Nuyten DSA, Van de Vijver MJ, Chang HY. Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet 2006; 38:421-430 Aleman BMP, Van den Belt-Dusebout AW, De Bruin ML, VanвЂ™t Veer MB, Baaijens MH, De Boer JP, Hart AA, Klokman WJ, Kuenen MA, Ouwens GM, Bartelink H, Van Leeuwen FE. Late cardiotoxicity after treatment for HodgkinвЂ™s lymphoma. Blood 2006;[Epub ahead of print] COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY Baas P, Belderbos JSA, Senan S, Kwa HB, Berthe Aleman, Jose Belderbos, Henk Boot, Edwin Jansen, Corrie Marijnen, Annemiek Cats, Van Bochove A, Van Tinteren H, Floor Van Leeuwen, Marcel Verheij, Harry Bartelink, Coen Rasch Burgers JA, Van Meerbeeck JP. Concurrent chemotherapy (carboplatin, paclitaxel, Head and neck cancer Cisplatin and irradiation for inoperable head and neck cancer: a multi-institutional trial comparing intra-arterial with intravenous cisplatin chemo-radiation was closed early November 2004. A total of 240 patients with stage III/IV head and neck cancer were included. Median follow-up was 20 months. No etoposide) and involved-field radiotherapy in limited stage small cell lung cancer: A Dutch multicenter phase II study. Br J Cancer 2006; 94:625-630 100 RADIOTHERAPY Publications (continued) Belderbos JSA, Heemsbergen WD, difference was found in the loco-regional recurrence rate between the treatment arms (Figure IX.9). De Jaeger K, Baas P, Lebesque JV. Final results of a Phase I/II dose escalation trial Figure IX.9: Locoregional control as first in non-small-cell lung cancer using three- event by treatment arm. dimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 2006; 66:126-134 Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, Gennaro M, Rouanet P, Avril A, Fentiman IS, Bartelink H, Rutgers EJ. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853--a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006; 24:3381-3387 Boyle P, Yasantha Ariyaratne M, Barrington R, Bartelink H, Bartsch G, Berns A, De Valeriola D, Dinshaw KA, Eggermont AM, Gray N, Kakizoe T, Singh Karki B, Kaslar M, Kerr DJ, Khayat D, Khuhaprema T, Kim IH, Martin-Moreno J, McVie G, Park JG, Philip T, Ringborg U, Rodger A, Seffrin JR, Semiglazov V, Soo KC, Sun Y, Thomas R, Tursz T, Veronesi U, Wiestler O, Yoo KY, The effect of the intra-arterial versus intravenous regimen on loss of hearing was measured. The aim of the study was to determine the difference between the treatment arms and to determine prognostic factors. Earlier study only demonstrated that the largest predictive factor for hearing loss was the pretreatment hearing loss for intra-arterial chemo-radiation treated patients. The mean hearing loss at 4 kHz was 20dB. There was a 10 dB (15 versus 25) difference in hearing loss at 4 kHz between the treatment arms favoring the intra-arterial arm, the mean dose to the inner ear was 14 Gy. Prediction of hearing loss remained predominantly determined by the pre-treatment audiogram (figure IX.10). For 101 patients treated with radiation alone the hearing damage due to radiation was measured by comparing pre- and posttreatment audiograms. The median dose to the inner ear was 11.4 (0.2 to 69 Gy). The hearing loss at 1,2-4 kHz was dependent on dose to the target and pretreatment hearing loss. For the 153 ears receiving <25 Gy no damage was measured. For the 22 ears receiving 26 to 35 Gy the mean damage was 5.5 dB. For the 19 ears receiving 3669 Gy the mean damage was 9 dB. Hearing loss due to chemo-radiation is predominantly determined by pretreatment hearing loss, thereafter cisplatin (intravenous more than intra-arterial) and thereafter by the radiation dose. Zatonski W, Zhao P. Tobacco: deadly in any form or disguise. Lancet 2006; 367:1710-1712 Chi JT, Wang Z, Nuyten DSA, Rodriguez EH, Schaner ME, Salim A, Wang Y, Kristensen GB, Helland A, BВ°rresen-Dale AL, Giaccia A, Longaker MT, Hastie T, Yang GP, Van de Vijver MJ, Brown PO. Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers. PLoS Figure IX.10: Hearing loss for Intra-arterial chemoradiation (left) versus intravenous chemoradiation Medicine 2006; 3:395-409 (right) after each administration of cisplatin. De Bock GH, Van der Hage JA, Putter H, Bonnema J, Bartelink H, Van de Velde CJ. Isolated loco-regional recurrence of breast cancer is more common in young patients and following breast conserving therapy: Long-term results of European Organisation for Research and Treatment of Cancer studies. Eur J Cancer 2006; 42:351-356 Gastroenterology Combined radiotherapy and chemotherapy (CRT) has improved treatment outcome and organ preservation in a variety of solid tumors (uterine cervix, lung, esophagus, head and neck, anorectal). More recently, a significant survival benefit in postoperative CRT was reported in gastric cancer. Based on these results we designed two adjuvant chemoradiotherapy phase I-II trials in gastric cancer in collaboration with our department of Gastroenterology and the Christie Hospital in Manchester, UK. In the first trial, that was finished recently, postoperative radiotherapy was combined with escalating doses of daily cisplatin and capecitabine. The maximal tolerated dose (MTD) was established as 5 mg/m2 for cisplatin and 650 mg/m2 bid for capecitabine. We are currently testing cisplatin in a weekly schedule in this combination. In the second trial radiotherapy is combined with escalating doses of capecitabine only. In this study, with a dose of 1000 mg/m2 the MTD is not reached and accrual continues. 101 RADIOTHERAPY Publications (continued) Until now, we have treated over 100 gastric cancer patients with CRT. In the first 91 patients that had an adequate follow up in only 16% in-field recurrences were found, which compares favorably with historical surgical series. Furthermore, with longterm follow up using serial renography to monitor kidney function, a 27% and 52% decrease in left renal function is observed at 18 and 24 months, respectively. This has prompted us to introduce more sophisticated radiotherapy strategies (IMRT; cone beam CT scan on the linear accelerator) in the postoperative treatment of gastric cancer to minimize normal tissue toxicity. We have designed a large international phase III study (CRITICS) which has been granted by the Dutch Cancer Society (CKTO) and has been approved by the protocol review board, in which patients with operable gastric cancer will receive preoperative chemotherapy, surgery and then randomized between continuation of chemotherapy or postoperative CRT in a schedule based on the above-mentioned phase I-II studies. Deurloo EE, Klein Zeggelink WFA, Teertstra HJ, Peterse JL, Rutgers EJT, Muller SH, Bartelink H, Gilhuijs KGA. Contrast-enhanced MRI in breast cancer patients eligible for breast-conserving therapy: Complementary value for subgroups of patients. Eur Radiol 2006; 16:692-701 Kreike B, Halfwerk H, Kristel P, Glas A, Peterse H, Bartelink H, Van de Vijver MJ. Gene expression profiles of primary breast carcinomas from patients at high risk for In locally advanced rectal cancer, preoperative CRT with 5FU has demonstrated superiority in downsizing, downstaging and local recurrence rates compared to radiotherapy alone. Capecitabine, an oral prodrug of 5FU, mimics infusional 5FU and has provided similar response rates with acceptable acute toxicity. In the last year, we treated 43 patients with preoperative concurrent capecitabine (825 mg/m2 bid, day 1-33) and radiotherapy, because of a locally advanced tumor. A retrospective analysis of 33 patients demonstrated that this treatment was feasible with minor acute toxicity and promising efficacy (TRG в‰Ґ 2 in 64%). Serious peri-operative toxicity occurred in 10%, mainly following a low anterior resection. To further optimize the effect of CRT on rectal tumors, without increasing the toxicity, a new study has been initiated with an antibody against the VEGF receptor (bevacizumab). For patients with a locally advanced rectal carcinoma, preoperative radiotherapy will be combined with capecitabine (825 mg/ m2 bid, day 1-33) and bevacizumab (AvastinВ®, 5 mg i.v. on day вЂ“14, 1, 15, 29). Surgery will be performed 6-8 weeks after the last chemoradiotherapy. This combination has shown to be effective in a phase I study, performed in the US. The primary goal of this study is to evaluate the feasibility, with special attention for the surgical complications. So far, 11 patients have been included in this multicenter study. An interim analysis for surgical complications will take place after the first 15 APR patients have been included. When the procedure seems feasible in these first 15 patients, another 45 patients will be included. local recurrence after breast-conserving therapy. Clin Cancer Res 2006; 12:5705-5712 Nuyten DSA, Kreike B, Hart AAM, Chi JTA, Sneddon JB, Wessels LFA, Peterse HJ, Bartelink H, Brown PO, Chang HY, Van de Vijver MJ. Predicting a local recurrence after breast-conserving therapy by gene expression profiling. Breast Cancer Res 2006; 8:R62 Sotiriou C, Wirapati P, Loi S, Harris A, Fox S, Smeds J, Nordgren H, Farmer P, Praz V, Haibe-Kains B, Desmedt C, Larsimont D, Cardoso F, Peterse H, Nuyten D, Buyse M, Van de Vijver MJ, Bergh J, Piccart M, Delorenzi M. Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 2006; 98:262-272 Van den Belt-Dusebout AW, Nuver J, De Wit R, Gietema JA, Ten Bokkel Huinink WW, Rodrigus PT, Schimmel EC, Aleman BMP, Van Leeuwen FE. Long-term risk of cardiovascular disease in 5-year survivors of testicular cancer. J Clin Oncol 2006; 24:467-475 102 MEDICAL ONCOLOGY X D IV IS ION OF M ED ICA L ON COLOG Y CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS Jos Beijnen, Jan Schellens, Henk Boot, Annemieke Cats, Helgi Helgason, Alwin Huitema, Bastiaan Nuijen, Hilde Rosing, Wim Ten Bokkel Huinink Division head, Group leader Sjoerd Rodenhuis Sjoerd Rodenhuis MD PhD Head Joke Baars MD PhD Academic staff Paul Baas MD PhD Academic staff Evert Bais MD Academic staff Background and objectives The Division of Pharmacology is involved in research in different phases of anticancer drug development. These include: I) Pharmaceutical formulation, II) Bioanalytical method development and implementation in PK studies and III) Early clinical phase I/II studies. A new research line was initiated which is supportive care of patients with breast cancer (IV). Jos Beijnen PhD Academic staff Willem Boogerd MD PhD Academic staff Henk Boot MD PhD Academic staff Wieneke Buikhuisen MD PhD Academic staff The research activities of the subdivision of Pharmacology of the department of Medical Oncology, the department of Pharmacy & Pharmacology and the department of Experimental Therapy (group Schellens) are closely integrated. Sjaak Burgers MD PhD Academic staff Annemieke Cats MD PhD Academic staff Jan Paul De Boer MD PhD Academic staff Helga Droogendijk MD Academic staff Bert De Gast MD PhD Academic staff John Haanen MD PhD Academic staff Helgi Helgason MD PhD Academic staff Alwin Huitema PhD Academic staff Agnes Jager MD PhD Academic staff Martijn Kerst MD PhD Academic staff Marjolein Klous PhD Academic staff Sabine Linn MD PhD Academic staff Anne Lukas MD PhD Academic stafff Herman Neering MD PhD Academic staff Bastiaan Nuyen PhD Academic staff Hilde Rosing PhD Academic stafff Jan Schellens MD PhD Academic staff Jan Schornagel MD PhD Academic staff Marianne Smits MD PhD Academic staff Gabe Sonke MD Academic staff Babs Taal MD PhD Academic staff Wim Ten Bokkel Huinink MD PhD I. Pharmaceutical formulation Formulation research and clinical manufacture of small molecule experimental anticancer agents has continued with the compounds EO-9 (EOquinTM), Kahalalide F, Imexon, and C1311 (SymadexTM). All compounds are currently in worldwide clinical trials in patented formulations designed by us. Recently, a formulation project with the anti-metastatic ruthenium complex NAMI-A was initiated. The clinical manufacture (thalidomide tablets) or development (5-MI tablets) of oral formulations of anticancer agents has continued. A pharmaceutical development program of oral taxanes has been initiated. The Amsterdam BioTherapeutics Unit (AmBTU) is now fully operational for the manufacture of a GMP-grade DNAplasmid. A lyophilized pharmaceutical formulation of pDERMATT (plasmid DNA Encoding Recombinant Mart-1 And Tetanus Toxin fragment-c) for the intradermal administration by tattooing was developed for the upcoming phase I clinical trial in our institute. Also, a research program in collaboration with Utrecht University has been initiated, aimed at the pharmaceutical development of non-viral vectors (e.g. lipoplexes and polyplexes) for improvement of DNA-plasmid transfection. For this, an in vitro human skin screening model is being developed in close collaboration with the Division of Immunology. The ultimate aim of this program is the translation of selected, promising vectors to the clinic. By the end of 2006, the clean room facilities of the AmBTU have been expanded in order to enable GMP-preparation of T-lymphocytes for T-cell receptor gene therapy. Academic staff Jaap Van der Sande MD PhD Academic staff Annette Van der Velden MD Academic staff Marchien Van der Weide MD PhD Academic staff Roel Van Gijn PhD Academic stafff Rianne Van Maanen PhD Academic staff Nico Van Zandwijk MD PhD Academic staff David Boss Graduate student Elke Brouwers Graduate student Carola Damen Graduate student Maarten Deenen Graduate student Corine Ekhart Graduate student Judith Engwegen Graduate student Suzanne Frankfort Graduate student Claudia Heijens Graduate student Marie-Christine Gast Graduate student Robert Jansen Graduate student Markus JГ¶rger Graduate student Ron Keizer Graduate student Stijn Koolen Graduate student Roos Oostendorp Graduate student II. Bioanalytical method development and implementation in PK studies A new laboratory dedicated to the bioanalysis of samples using Liquid Chromatography coupled with tandem quadrupole Mass Spectrometry (LC-MS/MS) has been built in the department of Pharmacy & Pharmacology. In total, 5 LC-MS/MS systems have been transferred to this laboratory. A new assay has been validated for gemcitabine and its (phosphorylated) metabolites to support a clinical study with the gemcitabine prodrug LY2334737. A quantitative assay for the determination of the bioreductive alkylating aziridinyl-quinone EO9 and its metabolites EO5a and EO9-Cl in urine has also been validated. The drug is administered intravesically for the treatment of superficial bladder cancer. We also found a new potential metabolite, EO9-OH. The structure of this compound remains to be elucidated. E7389 is a synthetic analogue of the natural product halichondrin B and it is currently being developed as an anti-cancer agent. For the bioanalysis of E7389, the compound is extracted from plasma and an LC-MS/MS method has been validated for the quantitative analysis. We continued to measure drug levels to support several clinical trials in and outside the Institute. This concerns clinical studies with: topotecan, ES-285, trabectedin, kahalalide F, D-24851, paclitaxel, and docetaxel. In addition, clinical trials with the farnesyl transferase inhibitor lonafarnib are supported. 103 MEDICAL ONCOLOGY Trastuzumab is a humanized monoclonal antibody and inhibits the proliferation of human tumor cells that over express HER2. For the determination of trastuzumab concentrations in human serum, an Enzyme-Linked Immuno Sorbent Assay (ELISA) has been described in literature. Our laboratory is involved in the pharmaceutical characterisation of trastuzumab and the bioanalysis of the drug using LC-MS/MS. III. Early clinical phase I/II studies III-1 Novel approaches to improve oral bioavailability We previously demonstrated that the oral applicability of commonly used anticancer drugs is limited by affinity for one or more of the ABC transporters expressed in the epithelial layer of the gastro-intestinal tract, especially P-glycoprotein (Pgp; ABCB1), Breast Cancer Resistance Protein (BCRP; ABCG2), MRP2 (ABCC2) and/or affinity for cytochrome P450 3A (CYP3A). This year we have shown that the systemic exposure to oral docetaxel can be significantly improved by co-administration of oral docetaxel and one single oral dose of 100 mg of ritonavir. The systemic exposure of 100 mg oral docetaxel is comparable to exposure after iv administration of the same dose. The phase I study with the novel orally applied taxane derivative BMS-275183, revealed significant interpatient variability in pharmacokinetics. The oral bioavailability is low and variable (mean and SD are 24 В± 11%). Based on our observation that the co-administration of pantoprazole (a benzimidazole proton pump inhibitor) and BMS-275183 resulted in a significant two-fold increase in the systemic exposure to BMS-275183 and also in an increase of clinical toxicity, we embarked on a drug-drug interaction study with BMS-275183 and esomeprazole. We continued development of oral formulations of paclitaxel in capsules. [Supported by a six-year program grant of the Dutch Cancer Society] Susanne Quaak Graduate student Rob Ter Heine Graduate student Ly Tran Graduate student Liia Vainchtein Graduate student Joost Van den Berg Graduate student Jolanda Van den Hoven Graduate student Sabien Van der Schoot Graduate student Annemieke Van Winden Graduate student Anthe Zandvliet Graduate student Carolien Alderden Technical staff Valerie Doodeman Technical staff Abadi Gebretensae Technical staff Michel Hillebrand Technical staff Ciska Koopman-Kroon Technical staff Lianda Nan-Offeringa Technical staff MariГ«t Ouwehand Technical staff Mathijs Tibben Technical staff III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs Currently, we perform 22 phase I/II, pharmacological and proof of concept studies. We recruited close to 200 new patients in these studies in 2006. Thirteen of the current phase I studies are two- or multicenter studies. Representative examples are described below. Marja Roelvink Clinical trial manager Sandra Adriaansz Nurse practitioner Annelies Boekhout Nurse practitioner Ria Dubbelman Nurse practitioner Joke Foekema Nurse practitioner Marjo Holtkamp Nurse practitioner a. Studies with cell cycle inhibitors The phase I study with AZD5438, given orally four times per day on one day every week has been completed. The starting dose was 4 times 10 mg, which could safely be increased to 4 times 90 mg. At this dose-level, asthenia and general malaise developed. As major toxicities and wide pharmacokinetic variability have been observed at other schedules, the development of the drug has been discontinued. Marianne Keessen Nurse practitioner Maria Kuiper Nurse practitioner Henk Mallo Nurse practitioner Annemarie Nol Nurse practitioner Albert Olijve Nurse practitioner Margaret Schot Nurse practitioner Jana Van der Sar Nurse practitioner b. PK/PD support of a phase I study with oral gemcitabine Gemcitabine is an attractive candidate for oral application. The phase I study with oral gemcitabine every other day for 21 days followed by one week of rest was completed. One patient with metastatic leiomyosarcoma which had been rapidly progressive on doxorubicine as well as on ifosfamide therapy, showed tumor regression followed by disease stabilization for three years. She had to be taken off study because of moderate, but reversible, liver dysfunction. Clinical and laboratory studies continue with a novel prodrug formulation of gemcitabine, which is not extensively metabolized presystemically. Marja Voogel-Fuchs Nurse practitioner Publications Aalders MCG, Triesscheijn M, Ruevekamp M, De Bruin M, Baas P, Faber DJ, Stewart FA. Doppler optical coherence tomography to monitor the c. Studies with angiogenesis inhibitors The three phase I studies with the orally available angiogenesis inhibitors E7080, Bay 57-9352 and AZD2171, that were started last year are ongoing. The main mechanism of action of these three molecules is inhibition of VEGFR 1 and 2. In dose-escalating studies, E7080 is employed as single agent and Bay 57-9352 in combination with irinotecan and capecitabine. AZD2171 is given at a fixed dose in a randomized study with or without upfront antihypertensive medication to test whether the best treatment for hypertension induced by this molecule is either upfront co-administration of antihypertensives or treatment when hypertension develops during treatment. All three studies have revealed hypertension and proteinuria as the main side-effects. Significant antitumor activity has been observed in all three studies. effect of photodynamic therapy on tissue morphology and perfusion. J Biomed Optics 2006; 11:044011 Aleman BMP, Van den Belt-Dusebout AW, De Bruin ML, Van вЂ�t Veer MB, Baaijens MH, De Boer JP, Hart AA, Klokman WJ, Kuenen MA, Ouwens GM, Bartelink H, Van Leeuwen FE. Late cardiotoxicity after treatment for HodgkinвЂ™s lymphoma. Blood 2006; [Epub ahead of print] 104 MEDICAL ONCOLOGY Publications (continued) Appels NMGM, Tung KO, Rosing H, Schellens JHM, Beijnen JH. A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors. Biomed Chromatogr 2006; 20:161-165 Appels NMGM, Rosing H, Stephens TC, Hughes A, Schellens JHM, Beijnen JH. Absolute quantification of farnesylated Ras d. Other phase I studies We embarked on a three-center phase I study with the orally available irreversible HER-1, -2 and -4 inhibitor PFS-00299804. This is a first-in-man study initiated in our institute. The dose-level of 0.5 mg/day could be increased to 60 mg/day for 21 days q day 28. Moderate gastro-intestinal toxicity was observed. A first-in-man phase I study with an orally available PARP1 (poly(ADP-ribose) polymerase-1) inhibitor (KU36-92) was initiated. The study is especially of interest for patients with BRCA2 mutated tumors. Two significant tumor regressions were observed in BRCA2 mutated breast and ovarian cancer patients. PD studies in white blood cells, skin biopsies and tumor biopsies revealed up to 60 % inhibition of PARP1 activity, compatible with active drug levels in these cells and tissues. levels in complex samples using liquid chromatography fractionation combined with tryptic digestion and electrospray tandem mass spectrometry. Anal Biochem 2006; 352:33-40 Appels NMGM, Rosing H, Stephens TC, Schellens JHM, Beijnen JH. Quantification of farnesylmethylcysteine in lysates of peripheral blood mononuclear cells using liquid chromatography coupled with electrospray tandem mass spectrometry: Pharmacodynamic assay for farnesyl transferase inhibitors. Anal Chem 2006; 78:2617-2622 Baas P, Belderbos JSA, Senan S, Kwa HB, Van Bochove A, Van Tinteren H, Burgers JA, Van Meerbeeck JP. Concurrent III-3 Advanced data analysis employing population analysis by NONMEM Non-linear mixed effects modeling (NONMEM) has been applied for population pharmacokinetic and pharmacodynamic data analysis. Using this technique, the complex interaction between capecitabine and indisulam, as observed in a phase I study, was described. It was shown that the observed excessive hematological toxicity of the combination was caused by time-dependent inhibition of indisulam elimination by capecitabine. Subsequently, the developed pharmacokinetic/ pharmacodynamic model was used in a simulation study to establish a feasible regimen for the combination of these two drugs. Population pharmacokinetic analyses were performed to investigate the influence of relevant SNPs in metabolic enzymes and drug transporters on the disposition of several (experimental) anticancer agents, such as irinotecan, docetaxel, epirubicine, indisulam, thiotepa and cyclophosphamide. By linking these pharmacokinetic models to models describing the dose-limiting hematological toxicity of these agents, the influence of the polymorphic forms on treatment outcome could be assessed. For instance for indisulam, it was shown that considerable dose reductions are necessary for safe administration of this drug in patients with polymorphic alleles of CYP2C9 and 2C19. chemotherapy (carboplatin, paclitaxel, etoposide) and involved-field radiotherapy in limited stage small cell lung cancer: A Dutch multicenter phase II study. Br J Cancer 2006; 94:625-630 Baas P, Triesscheijn M, Burgers S, Van Pel R, Stewart F, Aalders M. Fluorescence detection of pleural malignancies using 5-aminolaevulinic acid. Chest 2006; 129:718-724 III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment Pharmacogenetic screening of patients treated with 5-FU/capecitabine The genotype of the DPYD gene in patients treated with 5-FU or capecitabine was determined, in order to relate the genotype and phenotype to severe 5-FU-induced toxicity. A diagnostic real-time PCR assay was developed, suitable for highthroughput screening of this mutation in order to prevent severe 5-FU-related toxicity. Recently, we identified three heterozygotes for DPYD*2A that showed severe toxicities, resulting in the death of one of the three patients. We now consider the assessment of this SNP as the standard of care in all patients with planned fluoropyrimidine therapy and we have finalized a protocol with the aim to show that adaptive dosing in DPYD*2A patients is cost-effective. Baas P, Van вЂ�t Hullenaar N, Wagenaar J, Kaajan JPG, Koolen M, Schrijver M, SchlГ¶sser N, Burgers JA. Occupational asbestos exposure: How to deal with suspected mesothelioma cases - The Dutch approach. Ann Oncol 2006; 17:848-852 Belderbos JSA, Heemsbergen WD, De Jaeger K, Baas P, Lebesque JV. Final III-5 Proteomics We aim to identify and prospectively validate unique protein profiles in serum of patients with breast cancer, colorectal cancer and other solid malignancies that can be used as biomarkers to predict treatment outcome (antitumor activity and toxicity) after (adjuvant) chemotherapy, surgery and/or radiotherapy. The ProteinChip SELDI-TOF MS system (Ciphergen) is used for this purpose in our group. We have performed proteomics studies in breast cancer, colorectal cancer (CRC) and renal cancer (RCC). results of a Phase I/II dose escalation trial in non-small-cell lung cancer using threedimensional conformal radiotherapy. Int J Radiat Oncol Biol Phys 2006; 66:126-134 Breast cancer: In a group of 114 breast cancer patients, we have identified the haptoglobin alpha-1 protein to be predictive for the recurrence-free survival after surgery and adjuvant chemotherapy. The expression of this protein was consecutively proven to be dependent on the three major haptoglobin phenotypes. Haptoglobin phenotype appeared to be highly predictive of recurrence free survival (global Log Rank test; p=0.01, test for trend; p= 0.0025). Cross-institute validation studies revealed that, although the haptoglobin phenotype initially showed a significant relation with recurrence free survival in high-risk primary breast cancer patients, this significant relationship was not found upon expansion of the study population to 105 MEDICAL ONCOLOGY Publications (continued) n=432. However, at explorative analysis in specific subgroups of patients, a significant relation of haptoglobin phenotype on recurrence free survival again was observed. A prospective study is ongoing in which serum and tissue is collected from patients diagnosed with either primary breast cancer or benign breast diseases and from healthy women undergoing breast reducing surgery. Belderbos J, Uitterhoeve L, Van Zandwijk N, Belderbos H, Rodrigus P, Van de Vaart P, Price A, Van Walree N, Legrand C, Dussenne S, Giaccone G, Koning C, on behalf of the In 2006, we started a collaboration with the Department of Molecular Biology in the investigation of the effects of PARP-inhibitors, which are potential drugs against breast cancer. To this end, cultured BRCA1- and BRCA2-deficient murine cell lines as well as control cell lines are grown. We are currently analyzing the protein content of both the cultured cells and the culture medium to investigate the influence of PARP inhibitors on cell behavior. Results permitting, the effects of PARP inhibitors will also be investigated in mouse models and in human cell lines. Microarray analysis will be performed, which allows comparisons between our findings on the proteomic and the genomic levels. EORTC LCG and RT Group. Randomised trial of sequential versus concurrent chemoradiotherapy in patients with inoperable non-small cell lung cancer (EORTC 0897222973). Eur J Cancer 2007; 43:114-121 Beumer JH, Beijnen JH, Schellens JHM. Mass balance studies, with a focus on anticancer drugs. Clin Pharmacokinet 2006; 45:33-58 Colorectal cancer: Using a previously developed SELDI ProteinChipВ® protocol, we assessed changes in protein expression in patients with advanced CRC treated with first-line oxaliplatin (130 mg/m2 day 1, q 21)-capecitabine (1000 mg/m2 d1-14, q21). By comparing t=0 sera from these patients with those of healthy persons, we identified the same discriminating peaks in this prospective analysis as in previous retrospective ones. Moreover, some of these proteins (e.g. apolipoprotein A-I) were found to change during chemotherapy depending on response. We were also able to discriminate responders from non-responders based on the t=0 protein profile. A prospective study is ongoing (currently n=400) in which serum and tissue is collected from patients undergoing colonoscopy. This will yield a control group of persons without polyps and tumor, a group with adenomatous polyps and a group with (mostly early-stage) CRC. An analytical protocol will be set up for analysis of tissue lysates and serum. Tissue protein profiles of these three groups will be compared and analyzed for discriminating proteins. Beumer JH, Rademaker-Lakhai JM, Rosing H, Hillebrand MJX, Bosch TM, Lopez-Lazaro L, Schellens JH, Beijnen JH. Metabolism of trabectedin (ET-743, YondelisTM) in patients with advanced cancer. Cancer Chemother Pharmacol 2007; Published online: 20 September 2006 Beumer JH, Buckle T, Ouwehand M, Franke NEF, Lopez-Lazaro L, Schellens JHM, Beijnen JH, Van Tellingen O. Trabectedin (ET-743, Yondelisв„ў) is a substrate for P-glycoprotein, but only high expression of P-glycoprotein Renal cancer: The stability and reproducibility of previously obtained SELDI-TOF MS protein profiles for RCC were confirmed one year after the original analyses and they were subsequently validated with an independent method, i.e. ELISA. As only one protein cluster (serum amyloid- and fragments) showed biomarker potential in these analyses, we developed a new protocol for RCC serum protein profiling with SELDI-TOF MS to yield more biomarker candidates. Several new protein peaks were shown to discriminate patients from healthy controls in two populations from different institutes. The identity of these proteins is currently under investigation. The prognostic potential of our RCC serum protein profiles was also evaluated and a model was constructed that could reliably distinguish short-term from long-term survivors (sensitivity and specificity 70-80%), independent of the commonly used prognostic factor models (collaboration with the UMC Utrecht). confers the multidrug resistance phenotype. Invest New Drugs 2007; 25:1-7 Bex A, Kerst M, Mallo H, Meinhardt W, Horenblas S, De Gast GC. Interferon alpha 2b as medical selection for nephrectomy in patients with synchronous metastatic renal cell carcinoma: A consecutive study. Eur Urol 2006; 49:76-81 Boekhout AH, Beijnen JH, Schellens JHM. Symptoms and treatment in cancer therapyinduced early menopause. Oncologist 2006; In conclusion, we have identified the haptoglobin phenotype to be predictive for recurrence free survival after surgery and adjuvant chemotherapy in specific subgroups of breast cancer patients. In renal cancer, we found prognostic serum protein profiles as well and in colorectal cancer we found protein profiles predictive of response as well as chemotherapy-induced changes in protein profiles. The research that is performed in collaboration with several departments of the NKI will likely result in a better understanding of the molecular development of cancer. We expect that such proteomic analyses will benefit patients with breast cancer and CRC and possibly other tumors by improving prediction of treatment outcome and possibly early detection. 11:641-654 Boogaerts M, Oberhoff C, Ten Bokkel Huinink W, Nowrousian MR, Hayward CRW, Burger HU. Epoetin beta (NeoRecormonВ«) therapy in patients with solid tumours receiving platinum and nonplatinum chemotherapy: A meta-analysis. Anticancer Res 2006; 26:479-484 Bosch TM, Meijerman I, Beijnen JH, IV. Supportive care studies in patients with breast cancer We initiated a single center study to identify the best therapeutic intervention in female patients with premature menopausal complaints induced by treatment for breast cancer. It concerns a prospective double-blind randomized placebo-controlled intervention study with placebo, venlafaxine (EfexorВ®) and clonidine hydrochloride. The primary endpoint is reduction in intensity and number of flushes. Secondary Schellens JHM. Genetic polymorphisms of drug-metabolising enzymes and drug transporters in the chemotherapeutic treatment of cancer. Clin Pharmacokinet 2006; 45:253-285 106 MEDICAL ONCOLOGY Publications (continued) Bosch TM, Huitema ADR, endpoints are effect on sexual dysfunction and side-effects of therapy. The total study population is 100 and a total of 39 patients has been included. Doodeman VD, Jansen R, Witteveen E, Smit WM, Jansen RL, Van Herpen CM, Soesan M, Beijnen JH, Schellens JHM. CLINICAL IMMUNOTHERAPY AND TARGETED THERAPY Pharmacogenetic screening of CYP3A and John Haanen, Gijsbert De Gast, Willem Boogerd, Henk Mallo ABCB1 in relation to population pharmacokinetics of docetaxel. Clin Cancer Res 2006; 12:5786-5793 Bosch TM, Doodeman VD, Smits PHM, Meijerman I, Schellens JHM, Beijnen JH. Pharmacogenetic screening for polymorphisms in drug-metabolizing enzymes and drug transporters in a Dutch population. Mol Diagn Ther 2006; 10:175-185 Bosch TM, Deenen M, Pruntel R, Smits PHM, Schellens JHM, Beijnen JH, Meijerman I. Screening for polymorphisms in the PXR gene in a Dutch population. The clinical immuno- and targeted therapy group focuses on the treatment of melanoma and renal cell carcinoma patients. In the past years, a series of immunotherapy trials have been conducted, including peptide and TLR agonist vaccination trials, cytokine-based immunotherapy and concurrent chemo- and immunotherapy trials. Recently, due to the development of small molecule receptor tyrosine kinase inhibitors (RTKI) the group has participated in several trials in which these RTKIвЂ™s have been studied as single agent therapy or in combination with chemotherapy. Based on the exciting results, investigator-inititated studies have been designed to explore the combination of small molecules and cytokines, such as IL-2 in patients with metastatic disease. Melanoma A phase I clinical study has been performed in stage IV melanoma patients with a multiple peptide vaccine. Although no clinical responses were observed immunological efficacy was detected (Figure X.1). Eur J Clin Pharmacol 2006; 62:395-399 Figure X.1: Peripheral blood samples from patient 1 Bottomley A, Gaafa R, Manegold C, and 2 after MART-1 peptide vaccination in Burgers S, Coens C, Legrand C, Montanide ISA 51, GM-CSF and tetanus toxoid. Vincent M, Giaccone G, Van Meerbeeck J. Short-Term Treatment-Related Symptoms and Quality of Life: Results From an International Randomized Phase III Study of Cisplatin With or Without Raltitrexed in Patients With Malignant Pleural Mesothelioma: An EORTC Lung-Cancer Group and National Cancer Institute, Canada, Intergroup Study. J Clin Oncol 2006; 24:1435-1442 Brandon EFA, Sparidans RW, Guijt KJ, LГ¶wenthal S, Meijerman I, Beijnen JH, Schellens JHM. In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (YondelisВ«, TrabectidinВ«), a novel marine anti-cancer drug. Invest New Drugs 2006; 24:3-14 Brandon EFA, Sparidans RW, Van Ooijen RD, Meijerman I, Lopez-Lazaro L, Manzanares I, Beijnen JH, Schellens JHM. In vitro characterization of the human biotransformation pathways of aplidine, a novel marine anti-cancer drug. Invest New Drugs 2007; 25:9-19 Participations in three phase III clinical studies have been initiated: Randomized, placebo-controlled multicenter trial for second-line treatment of unresectable stage III or IV melanoma patients. In this study a total of six patients were enrolled. Patients were randomized to receive carboplatin/paclitaxel chemotherapy either in combination with placebo or with sorafenib. Sorafenib, a small molecule RTKI, was originally developed as a B-Raf inhibitor. B-Raf kinase is mutated in the majority of melanomas. It is involved in the Ras-Raf-MEK-MAPK pathway which is important for cell proliferation. Iinhibition of B-Raf kinase appears to be a rational target for this patient population. We further participate in two phase III clinical trials in which a fully human monoclonal antibody against вЂ�cytotoxic lymphocyte antigen-4вЂ™ (CTLA4) is the study drug. Blockade of CTLA4, a molecule expressed on activated T lymphocytes 107 MEDICAL ONCOLOGY Publications (continued) with immunomodulating effects on the immune response (safe guard against overstimulation) has been shown to increase cancer-specific immune responses in a number of malignancies, either as a single agent or in combination with vaccination. Due to a more vigorous immune system, the typical side effects of these drugs are socalled immune breakthrough events, such as immune colitis, dermatitis, uveitis, vitiligo, pancreatitis and hypophysitis. These syndromes can be managed by administration of topical or systemic corticosteroids. The first of these studies is a randomized multicenter phase III trial of first line treatment for metastatic melanoma. Patients are randomized between standard chemotherapy (dacarbazine) and ticilimumab. The clinical endpoint of this study is overall survival. The second trial is a randomized placebo-controlled multicenter trial of second line treatment for metastatic melanoma. Only HLA-A*0201-positive patients can be enrolled since the treatment comprises two gp100 peptide vaccines that have binding affinity only for HLA-A2 molecules. Patients are randomized between peptide vaccine only, ipilimumab only or the combination. Brandon EFA, Bosch TM, Deenen MJ, Levink R, Van Der Wal E, Van Meerveld JBM, Bijl M, Beijnen JH, Schellens JHM, Meijerman I. Validation of in vitro cell models used in drug metabolism and transport studies; Genotyping of cytochrome P450, phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines. Toxicol Appl Pharmacol 2006; 211:1-10 Brandsma D, Voest EE, De Jager W, Bonfrer H, Algra A, Boogerd W, Korse T, Reijneveld JC, Verbeek MM, Rijkers G, Renal cell carcinoma In the renal cell carcinoma program we closely collaborate with Axel Bex from the urology-oncology group and with Florry Vyth-Dreese who supervises the monitoring of clinical immunotherapy studies (in collaboration with the Clinical Laboratory, headed by Wim Nooijen). The development of investigatorinitiated study to define the optimal time point for tumor nephrectomy in metastatic renal cell carcinoma patients is under way. We have also participated in a treatment-use program of the small molecule sunitinib, a multiple receptor tyrosine kinase inhibitor with high affinity for VEGF-R, PDGF-R, c-KIT and FLT3. Since VEGF and PDGF play a prominent role in the pathogenesis of sporadic clear cell renal cell cancer, inhibition of kinase activity of their receptors appeared to be a rational target in this patient population. Indeed, phase II studies showed impressive objective clinical responses and durable stable disease in patients treated with single agent sunitinib. In total we enrolled over 50 patients in this program and we observed objective clinical responses in a substantial number of patients, and durable stable disease lasting over 12 months in others. The overall results are being analyzed in collaboration with investigators from the Free University Medical Center. Taphoorn MJB. CSF protein profiling using Multiplex Immuno-assay: A potential new diagnostic tool for leptomeningeal metastases. J Neurol 2006; 253:1177-1184 Brandsma D, Taphoorn MJB, De Jager W, Bonfrer H, Algra A, Reijneveld JC, Boogerd W, Korse T, Verbeek MM, Rijkers GT, Voest EE. Interleukin-8 CSF levels predict survival in patients with leptomeningeal metastases. Neurology 2006; 66:243-246 Breedveld P, Beijnen JH, Schellens JHM. Use of P-glycoprotein and BCRP inhibitors to improve oral bioavailability and CNS penetration of anticancer drugs. Trends Pharmacol Sci 2006; 27:17-24 BREAST CANCER THERAPY AND RESPONSE PREDICTION Breedveld P, Pluim D, Cipriani G, Sjoerd Rodenhuis, Sabine Linn, Joke Baars, Jos Beijnen, Jan Paul De Boer, Corine Ekhart, Dahlhaus F, Van Eijndhoven MAJ, Juliane Hannemann, Helgi Helgason, Marjo Holtkamp, Alwin Huitema, Marc Van de Vijver, De Wolf CJF, Kuil A, Beijnen JH, Jan Schornagel Scheffer GL, Jansen G, Borst P, Schellens JHM. The Effect of Low pH The objective of this program is to develop and improve potentially curative therapy for patients with early or oligometastatic breast cancer. For all these studies, close collaborations are maintained with other departments and research divisions. The Pharmacy has a key role in the clinical pharmacology studies of the high-dose chemotherapy program and the Molecular Pathology Department is central in the response prediction studies. on Breast Cancer Resistance Protein (ABCG2)-Mediated Transport of Methotrexate, 7-Hydroxymethotrexate, Methotrexate Diglutamate, Folic Acid, Mitoxantrone, Topotecan, and Resveratrol in In Vitro Drug Transport Models. Mol Pharmacol 2007; 71:240-249 Intensive chemotherapy Autologous hematopoietic progenitor cell transplantation continues to be studied, despite the negative attitude towards this treatment modality that has developed in recent years. In addition to the encouraging results of the Brouwers EEM, Tibben MM, Rosing H, Hillebrand MJX, Joerger M, Schellens JHM, Beijnen JH. Sensitive inductively coupled plasma mass spectrometry assay for the determination of platinum originating from cisplatin, carboplatin, and oxaliplatin in human plasma ultrafiltrate. J Mass Spectrom 2006; 41:1186-1194 Figure X.2: Relapse-free survival in 621 patients with HER-2/neu negative tumors. 108 MEDICAL ONCOLOGY Publications (continued) Davies AHG, Larsson G, Ardill J, Friend E, Jones L, Falconi M, Bettini R, Koller M, Sezer O, Fleissner C, Taal B, Blazeby JM, Ramage JK. Development of a diseasespecific quality of life questionnaire module for patients with gastrointestinal neuroendocrine tumours. Eur J Cancer 2006; 42:477-484 De Boer S, Dingemans JA, Huijbers EJM, Zych TR, Beijnen JH. Late cardiotoxicity after use of anthracycline: The reverse side of success. Pharm Weekbl 2006; 141:1154-1157 De Bree E, Rosing H, Michalakis J, Romanos J, Relakis K, Theodoropoulos PA, Beijnen JH, Georgoulias V, Tsiftsis DD. Intraperitoneal chemotherapy with taxanes for ovarian cancer with peritoneal dissemination. Eur J Surg Oncol 2006; 32:666-670 Dutch randomized study (Figure X.2) the meta-analysis of the Oxford group (EBCTCG) has now shown that high-dose chemotherapy has a rather substantial beneficial effect on both recurrence and breast cancer death, and that this cannot be explained by a castration effect. Recent evidence has raised the possibility that alkylating agents in breast cancer are specifically active in tumors lacking the ability for homologous recombination. Breast cancers in BRCA1 or BRCA2 carriers have a defect in this DNA-repair pathway and the same is probably true for 20 to 30% of sporadic breast cancers. The intensive chemotherapy program will be continued in this subset of breast cancers. A small clinical study that investigated the feasibility of an intermediate alkylator dose regimen (with cyclophosphamide, thiotepa and carboplatin) has shown that such a regimen can be administered in the outpatient-setting twice with a 3-week interval. A randomized phase II study in locally advanced breast cancer is planned to evaluate the efficacy of the new tandem intermediate dose alkylator regimen in tumors with a homologous recombination defect. The pathological complete response rate will be used as the primary endpoint of this study. The preoperative chemotherapy program in patients with tumors larger than 3 cm has continued in 2006. Preoperative chemotherapy has been shown to be particularly effective in basal-like (triple negative) tumors (50% pathologic complete response rate). A collaborative study with the Molecular Pathology Department has shown that a mRNA-expression pattern associated with complete response to dose-dense AC can be distinguished. This does, however, require prospective validation in the ongoing study. De Bree E, Theodoropoulos PA, Rosing H, Michalakis J, Romanos J, Beijnen JH, Tsiftsis DD. Treatment of ovarian cancer using intraperitoneal chemotherapy with taxanes: From laboratory bench to bedside. Cancer Treat Rev 2006; 32:471-482 De Jong AE, Hendriks YMC, Kleibeuker JH, De Boer SY, Cats A, Griffioen G, Nagengast FM, Nelis FG, Rookus MA, Vasen HFA. Decrease in mortality in Lynch syndrome families because of surveillance. Gastroenterology 2006; 130:665-671 De Jong AE, Nagengast FM, Kleibeuker JH, Van de Meeberg PC, Van Wijk HJ, Cats A, Griffioen G, Vasen FA. What is the appropriate screening protocol in Lynch syndrome? Fam Cancer 2006; 5:373-378 De Jonge ME, Huitema ADR, Beijnen JH, Rodenhuis S. High exposures to bioactivated cyclophosphamide are related to the occurrence of veno-occlusive disease of Microarrays, prognosis and response prediction Microarray technology is gaining credibility as an important diagnostic tool in guiding treatment decisions in breast cancer. To assess the feasibility of this novel technology in community hospitals we have conducted a pilot study from 2004 until December 2006 in seventeen general hospitals that varied in geographical localization, organization and size (RASTER study). After inclusion of 771 early breast cancer patients we obtained a prognostic gene expression profile in 80% of pN0 patients within two weeks after surgery. Fifty percent of the node-negative patients had a poor prognosis signature. Approximately 22% of patients who had a high risk of distant metastases according to the microarray test had a low risk according to the Dutch guidelines (CBO 2004), while 7% had a low risk of distant metastases according to the microarray test, but a high risk according to the Dutch guidelines. In 2004 a randomized, multicenter adjuvant study in node-positive primary breast cancer (MATADOR study; CKTO 2004-04; BOOG 2005-02) was initiated and this study is currently open in 26 centers in the Netherlands. The primary objective of this study is to identify predictive gene expression profiles for a disease-free survival benefit of either a docetaxel-containing regimen (docetaxel-doxorubicincyclophosphamide) or an accelerated doxorubicin-cyclophophamide regimen (dose-dense). Using BAC array comparative genomic hybridization, a predictive profile for a progression-free survival of over 24 months after high dose chemotherapy with autologous stem cell support in the metastatic setting has been identified. This profile will need prospective validation. the liver following high-dose chemotherapy. Br J Cancer 2006; 94:1226-1230 THORACIC ONCOLOGY De Vries NA, Beijnen JH, Boogerd W, Nico Van Zandwijk, Paul Baas, Sjaak Burgers, JosГ© Belderbos, Houke Klomp, Petra Nederlof, Van Tellingen O. Blood-brain barrier and Jan Schellens chemotherapeutic treatment of brain tumors. Expert Rev Neurother 2006; 6:1199-1209 De Witte MA, Coccoris M, Wolkers MC, Van Den Boom MD, Mesman EM, Non Small Cell Lung Cancer (NSCLC) Prognostic micro-array profiles obtained by supervised classification of RNA samples from completely resected NSCLC patients1 can be used as a tool to predict disease-free survival and outperform TNM staging. The confirmation of this result in an independent series of patients has been established in 2006 and the important question regarding selection of NSCLC Song JY, Van Der Valk M, Haanen JBAG, Schumacher TNM. Targeting self-antigens through allogeneic TCR gene transfer. Blood 2006; 108:870-877 1 Microarray Consortium: Netherlands Cancer Institute, VU Medical Center, Thoraxklinik Heidelberg, Medical Academy Gdansk, Medical Academy Bialystok, Agendia 109 MEDICAL ONCOLOGY Publications (continued) patients for adjuvant therapy on the basis of micro-array profile is now ready to be answered in a prospective trial. Prospective evaluation of the mutation status of the Epidermal Growth Factor Receptor (EGFR) and the response to small-molecule Tyrosine Kinase Inhibitors (TKIs) in adenocarcinoma patients has revealed that the majority (90%) of patients harboring an EGFR mutation will experience significant relief of tumor symptoms and tumor volume. In the subgroup of patients with exon 19 deletions, all patients have achieved an objective response and several patients have successfully continued TKI therapy for more than two years. EGFR mutations can thus be employed as a selection criterion for TKI treatment. KRAS and EGFR mutations never coincided, indicating that KRAS mutations can be used as a negative selection criterion for TKI treatment. The pharmacogenomic project for patients with advanced NSCLC correlating Single Nucleotide Polymorphisms (SNPвЂ™s) with gemcitabine and cisplatin metabolism and with clinical outcome is ongoing and has accrued over 40 patients. Regional hospitals have started to contribute. A similar approach is part of a protocol (NVALT 7), a study randomizing patients with recurrent NSCLC to either pemetrexed alone or the combination of pemetrexed and carboplatin. The final analysis of the EORTC study (08972), which compares sequential versus concurrent chemoradiotherapy, revealed more adverse events in the concurrent arm. The efficacy of the two different treatment approaches was comparable. Den Brok MWJ, Nuijen B, GarcГa JL, Miranda E, Calvo P, Manada C, Beijnen JH. Compatibility and stability of the novel anticancer agent ES-285 + HCl formulated with 2-hydroxypropyl-?-cyclodextrin in infusion devices. Pharmazie 2006; 61:21-24 Den Brok MWJ, Nuijen B, Harms R, Buluran JN, Harvey MD, Grieshaber CK, Beijnen JH. Compatibility and stability of the novel anti-cancer agent C1311 in infusion devices and its in vitro biocompatibility. J Oncol Pharm Pract 2005; 11:13-19 Dorresteijn LD, Stewart FA, Boogerd W. Stroke As a Late Treatment Effect of HodgkinвЂ™s Disease. J Clin Oncol 2006; 24:1480 Ekhart C, De Jonge ME, Huitema ADR, Malignant Pleural Mesothelioma (MPM) The randomized phase III study testing the value of thalidomide maintenance therapy after pemetrexed and cisplatin induction chemotherapy (NVALT 5) has now accrued 33% of the projected number of patients. An interim analysis has confirmed that thalidomide dosing according to our previous experience (200 mg/day orally) is rather well tolerated. In parallel to this clinical study, thalidomide is being tested in a conditional knock-out mouse mesothelioma model (Division of Molecular Genetics). Eleven patients with very early MPM were included in a feasibility study of induction chemotherapy followed by surgery and postoperative radiotherapy (EORTC 08031). The focus is now on further treatment for patients, who are apparently disease-free after this demanding combined modality approach, and also on improved patient selection criteria for future trials. Two second-line studies have been activated in 2006: a phase II study of the proteasome inhibitor Bortezomib and in a phase IB/II study of the alkylating agent S 23906. In both studies, serum and tissue is being collected to enable translational research. Schellens JHM, Rodenhuis S, Beijnen JH. Flat dosing of carboplatin is justified in adult patients with normal renal function. Clin Cancer Res 2006; 12:6502-6508 Engwegen JYMN, Gast MCW, Schellens JHM, Beijnen JH. Clinical proteomics: searching for better tumour markers with SELDI-TOF mass spectrometry. Trends Pharmacol Sci 2006; 27:251-259 Engwegen JYMN, Helgason HH, Cats A, Harris N, Bonfrer JMG, Schellens JHM, Beijnen JH. Identification of serum proteins discriminating colorectal cancer patients Optical Coherence Tomography (OCT) 2 The OCT apparatus has been investigated in a mouse model for photodynamic therapy (PDT). Vessel collapse followed by edema formation was an important feature that could be observed in real time (Figure X.3). and healthy controls using surface-enhanced laser desorption ionisation-time of flight mass spectrometry. World J Gastroenterol 2006; 12:1536-1544 Figure X.3: Comparison of histology Geurts TW, Ackerstaff AH, slides and corresponding OCT image in Van Zandwijk N, Hart AAM, Hilgers FJM, a mouse bearing a subcutaneous tumor. Balm AJM. The psychological impact of Within three hours after photodynamic annual chest X-ray follow-up in head and therapy the borders between the different neck cancer. Acta Otolaryngol 2006; layers vanish due to influx of edema and 126:1315-1320 cell swelling. The OCT images were taken in real time a few minutes before Haanen JBAG, Baars A, Gomez R, excision of the tumors. Weder P, Smits M, De Gruijl TD, Von Blomberg BME, Bloemena E, Scheper RJ, Van Ham SM, Pinedo HM, Van Den Eertwegh AJM. Melanomaspecific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells In three patients with basal cell carcinoma that were treated with PDT the OCT set-up has been tested and further improvements are being implemented. may predict survival in resected advancedstage melanoma patients. Cancer Immunol Immunother 2006; 55:451-458 2 Collaboration with Laser Center Academical Medical Center: Maurice Aalders 110 MEDICAL ONCOLOGY Publications (continued) Hannemann J, Kristel P, Van Tinteren H, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van Der Wall E, Hupperets P, De Vries EGE, Rodenhuis S, Van De Vijver MJ. Molecular subtypes of Chemoprevention 3 The final analysis of standard H&E and immunohistochemical stains of bronchial biopsies taken as part of the randomized (phase IIB) fluticasone intervention study in high risk individuals (volunteers) has not revealed an appreciable preventive effect of this inhalational corticosteroid. The ability of assays for HTERT expression to refine the standard criteria for the diagnosis of premalignant bronchial lesions will be further analyzed. breast cancer and amplification of topoisomerase IIa: Predictive role in dose intensive adjuvant chemotherapy. GASTROENTEROLOGY Br J Cancer 2006; 95:1334-1341 Henk Boot, Annemieke Cats, Marianne Smits, Babs Taal, Edwin Jansen, Corrie Marijnen, Marcel Verheij Helgason HH, Kruijtzer CMF, Huitema ADR, Marcus SG, Ten Bokkel Huinink WW, Schot ME, Schornagel JH, Beijnen JH, Schellens JHM. Phase II and pharmacological study of oral paclitaxel (Paxoral) plus ciclosporin in anthracyclinepretreated metastatic breast cancer. Br J Cancer 2006; 95:794-800 Hoebers FJP, Pluim D, Verheij M, Balm AJM, Bartelink H, Schellens JHM, Begg AC. Prediction of treatment outcome by cisplatin-DNA adduct formation in patients with stage III/IV head and neck squamous cell carcinoma, treated by concurrent cisplatin-radiation (RADPLAT). Int J Cancer 2006; 119:750-756 Hooning MJ, Dorresteijn LD, Aleman BMP, Kappelle AC, Klijn JG, Boogerd W, Van Leeuwen FE. Decreased risk of stroke among 10-year survivors of breast cancer. Gastric cancer In the USA, adjuvant chemoradiation has become standard treatment for gastric cancer, whereas in Europe perioperative chemotherapy with epirubicin, cisplatin and capecitabine has gained wide acceptance. Improvement of the chemotherapy regimen used in the USA is desired. From December 2002 till March 2006 we evaluated the feasibility of concurrent chemoradiation with capecitabine and cisplatin as adjuvant treatment in 38 patients with pT2-4N0-3M0 gastric and esophageal adenocarcinoma. Treatment started with two weeks of capecitabine (1000 mg/m2 bid). Subsequently, a fixed dose of 45 Gy was given to the original tumor site and adjacent lymph nodes with concurrent cisplatin and capecitabine in escalating daily doses. In six patients receiving cisplatin 6 mg/m2 and capecitabine 650 mg/m2 bid, three patients developed dose-limiting toxicity (neutropenia grade III and IV, thrombocytopenia grade III, and weight loss/nausea grade III). The MTD therefore was cisplatin 5 mg/m2 i.v. and capecitabine 650 mg/m2 bid orally. This chemoradiation regimen will be the basis for a multicenter nationwide phase III study comparing perioperative chemotherapy with postoperative chemoradiation therapy in resectable gastric cancer. Two additional studies have been performed. Firstly, in the abovementioned chemoradiotherapy regimen cisplatin was administered on a weekly basis (cisplatin 20-30 mg/m2) in a phase I fashion with six patients per dose level. In 12 patients, no grade III/IV toxicity was observed. Secondly, capecitabine monotherapy on radiotherapy days was escalated from 600-1000 mg/m2 bid. In 66 patients, including those treated at the highest dose level, no grade III/IV toxicity has been observed. These regimens can be incorporated in future adjuvant chemoradiotherapy trials. J Clin Oncol 2006; 24:5388-5394 Jansen EPM, Saunders MP, Boot H, Oppedijk V, Dubbelman R, Porritt B, Cats A, Stroom J, ValdГ©s Olmos R, Bartelink H, Verheij M. Prospective study on late renal toxicity following postoperative chemoradiotherapy in gastric cancer. Int J Radiat Oncol Biol Phys 2007; In Press Rectal cancer In locally advanced rectal cancer, preoperative chemoradiotherapy with 5FU is superior in downsizing the tumor and in reducing local recurrences compared with radiotherapy alone. The question is whether similar efficacy without increasing toxicity can be achieved by the use of bevacizumab and capecitabine. We have started a multicenter feasibility study in 60 rectal cancer patients at high risk for a positive resection margin with chemoradiotherapy with capecitabine (825 mg/m2 bid, day 1-33) to which bevacizumab (5 mg/kg, day вЂ“14, 1, 15, 29) is added. The number of treatment failures is the primary endpoint. Currently, 11 patients have been included in the study. Joerger M, Huitema ADR, Van Den Bongard HJGD, Baas P, Schornagel JH, Schellens JHM, Beijnen JH. Determinants of the elimination of methotrexate and 7-hydroxy-methotrexate following high-dose infusional therapy to cancer patients. Br J Clin Pharmacol 2006; 62:71-80 Joerger M, Huitema ADR, Boogerd W, Van Der Sande JJ, Schellens JHM, Beijnen JH. Interactions of serum albumin, valproic acid and carbamazepine with the Neuroendocrine tumors The clinical features of 244 patients admitted to our hospital from 1994 through 2004 have been collected in a database (116 men, 128 women; median age 57 years). Liver metastases were present in 65%, skeletal metastases in 14%. The Octreotide scan was positive in 77% and the MIBG scan in 60% of the patients. An increasing problem is a mesenterial mass, which is present in 44% of the patients (Figure X.4). In patients with a positive Octreotide scan, survival is significantly better than in those with a negative scan (67% versus 40%). A strongly elevated tumor marker, Chromogranin A, is negative predictor of survival. Evaluation of treatment is mainly based on hormonal activity, which is responsible for most symptoms. The tumor size is only rarely reduced by drug therapy. To measure quality of life, an international EORTC questionnaire has been developed that will be tested in a larger group to evaluate the effect of treatment. pharmacokinetics of phenytoin in cancer patients. Basic Clin Pharmacol Toxicol 2006; 99:133-140 3 Chemoprevention Group: Netherlands Cancer Institute, VUmc (Department of Pulmonology): Egbert Smit 111 MEDICAL ONCOLOGY Publications (continued) Figure X.4: Mesenterial mass invading the superior mesenterial artery, in a patient Joerger M, Bosch TM, Doodeman VD, with a neuroendocrine tumor. Beijnen JH, Smits PHM, Schellens JHM. Novel deoxycytidine kinase gene polymorphisms: A population screening study in Caucasian healthy volunteers. Eur J Clin Pharmacol 2006; 62:681-684 Joerger M, Huitema ADR, Van Den Bongard DHJG, Schellens JHM, Beijnen JH. Quantitative effect of gender, age, liver function, and body size on the population pharmacokinetics of paclitaxel in patients with solid tumors. Clin Cancer Res 2006; 12:2150-2157 EUS Radial endoscopic ultrasound is important to select patients with esophageal or gastric tumor for preoperative chemotherapy or chemoradiation. In 2004 the linear endoscopic EUS became available to puncture enlarged lymph nodes for an optimal staging. We currently participate in a multicenter trial to evaluate the effect of mediastinal staging in lung cancer. With linear EUS (n=93) the fine needle aspiration was representative in 86% and a surgical procedure was prevented in 45%, usually following the detection of N2 lymph node metastases. Kartachova MS, ValdГ©s Olmos R, Haas RLM, Hoebers FJP, Van Den Brekel MW, Van Zandwijk N, Herk MV, Verheij M. Mapping of treatment-induced apoptosis in normal structures: 99mTc-Hynic-rh-annexin V SPECT and CT image fusion. Eur J Nucl Med Mol Imaging 2006; 33:893-899 Kemper EM, Leenders W, sters B, Lyons S, Buckle T, Heerschap A, Boogerd W, Beijnen JH, Van Tellingen O. Development of luciferase tagged brain tumour models in mice for chemotherapy intervention studies. Eur J Cancer 2006; 42:3294-3303 Kemper EM, Boogerd W, Beijnen JH, Van Tellingen O. Paclitaxel alone is not useful for the treatment of brain tumors. Pharm Weekbl 2006; 141:380-383 Kramer GWPM, Legrand CL, Van Schil P, Uitterhoeve L, Smit EF, Schramel F, Biesma B, Tjan-Heijnen V, Van Zandwijk N, Splinter T, Giaccone G, Van Meerbeeck JP. Quality assurance of thoracic radiotherapy in EORTC 08941: A randomised trial of surgery versus thoracic radiotherapy in patients with stage IIIA non-small-cell lung cancer (NSCLC) after response to induction chemotherapy. Eur J Cancer 2006; 42:1391-1398 Kreukels BPC, Schagen SB, Ridderinkhof KR, Boogerd W, Hamburger HL, Muller MJ, Van Dam FSAM, Schagen SB. Effects of high-dose and conventional-dose adjuvant chemotherapy on long-term cognitive sequelae in patients with breast cancer: An electrophysiologic study. Clin Breast Cancer 2006; 7:67-78 112 MEDICAL ONCOLOGY Publications (continued) Kroep JR, Smit EF, Giaccone G, Overwijk WW, De Visser KE, Tirion FH, Sparidans RW, Bosch TM, JГ¶rger M, Van der Born K, Beijnen JH, De Jong LA, Pols TWH, Van Der Velden YU, Schellens JHM, Beijnen JH. Liquid Van Groeningen CJ, Van der Vijgh WJF, Van Den Boorn JG, Keller AM, Buurman WA, chromatography-tandem mass spectrometric Postmus PE, Pinedo HM, Peters GJ. Theoret MR, Blom B, Restifo NP, assay for the analysis of uracil, 5,6-dihydrouracil Pharmacology of the paclitaxelв€’cisplatin, Kruisbeek AM, Kastelein RA, Haanen JBAG. and b-ureidopropionic acid in urine for the gemcitabineв€’cisplatin, and Immunological and antitumor effects of IL-23 as measurement of the activities of the pyrimidine paclitaxelв€’gemcitabine combinations in a cancer vaccine adjuvant. J Immunol 2006; catabolic enzymes. J Chromatogr B Analyt patients with advanced non-small cell lung 176:5213-5222 Technol Biomed Life Sci 2006; 839:45-53 Puri R, Palit V, Loadman PM, Flannigan M, Sparidans RW, Dost F, Crommentuyn KML, Shah T, Choudry GA, Basu S, Double JA, Huitema ADR, Schellens JHM, Beijnen JH. Kuppens IELM, Dansin E, Boot H, Lenaz G, Chawla S, Beer M, Van Kalken C, Liquid chromatographic assay for the non- Feger C, Assadourian S, Bonneterre ME, De Boer R, Beijnen JH, Twelves CJ, peptidic protease inhibitor tipranavir in plasma. Beijnen JH, Schellens JHM, Bonneterre J. Phillips RM. Phase I/II Pilot Study of Biomed Chromatogr 2006; 20:671-673 Dose-finding phase I clinical and Intravesical Apaziquone (EO9) for Superficial pharmacokinetic study of orally Bladder Cancer. [Miscellaneous Article]. Sternberg CN, De Mulder P, Schornagel JH, administered irinotecan in patients with J Urol 2006; 176:1344-1348 Theodore C, Fossa SD, Van Oosterom AT, cancer. Cancer Chemother Pharmacol 2006; 58:509-516 Witjes JA, Spina M, Van Groeningen CJ, advanced solid tumors. Clin Cancer Res Rademaker-Lakhai JM, Crul M, Zuur L, Baas P, Duclos B, Roberts JT, De Balincourt C, Beijnen JH, Simis YJ, Van Zandwijk N, Colette L, EORTC Genito-Urinary Cancer Lagas JS, Vlaming ML, Van Tellingen O, Schellens JH. Relationship between cisplatin Group. Seven year update of an EORTC phase Wagenaar E, Jansen RS, Rosing H, administration and the development of III trial of high-dose intensity M-VAC Beijnen JH, Schinkel AH. Multidrug ototoxicity. J Clin Oncol 2006; 24:918-924 chemotherapy and G-CSF versus classic M-VAC 2006; 12:3774-3781 resistance protein 2 is an important in advanced urothelial tract tumours. determinant of paclitaxel pharmacokinetics. Rodenhuis S, Rutgers EJT. Adjuvant therapy for Clin Cancer Res 2006; 12:6125-6132 breast cancer. Br J Surg 2006; 93:1443-1445 Liedert B, Pluim D, Schellens J, Rodenhuis S, Bontenbal M, of 5-fluorouracil in the adjuvant setting: Thomale J. Adduct-specific monoclonal Van Hoesel QGCM, Smit WM, Nooij MA, High-dose, low-dose or no-dose leucovorin? antibodies for the measurement of cisplatin- Voest EE, Van Der Wall E, Hupperets P, Commentary. Nat Clin Pract Oncol 2006; induced DNA lesions in individual cell Van Tinteren H, Peterse JL, Van De Vijver MJ, 3:300-301 nuclei. Nucleic Acids Res 2006; 34 De Vries EGE. Efficacy of high-dose alkylating Eur J Cancer 2006; 42:50-54 Taal BG, Cats A, Van Tinteren H. Modulation Meijerman I, Beijnen JH, Schellens JHM. chemotherapy in HER2/neu-negative breast Toebes M, Coccoris M, Bins A, Rodenko B, cancer. Ann Oncol 2006; 17:588-596 Gomez R, Nieuwkoop NJ, Van De Kasteele W, Rimmelzwaan GF, Haanen JBAG, Ovaa H, Herb-drug interactions in oncology: Focus on mechanisms of induction. Oncologist Scagliotti GV, Szczesna A, Ramlau R, Schumacher TNM. Design and use of 2006; 11:742-752 Cardenal F, Mattson K, Van Zandwijk N, conditional MHC class I ligands. Nat Med Price A, Lebeau B, Debus J, Manegold C. 2006; 12:246-251 Moser EC, Noordijk EM, Van Leeuwen FE, Docetaxel-based induction therapy prior to Le Cessie S, Baars JW, Thomas J, Carde P, radiotherapy with or without docetaxel for non- Triesscheijn M, Baas P, Schellens JHM, Meerwaldt JH, Van Glabbeke M, small-cell lung cancer. Br J Cancer 2006; Stewart FA. Photodynamic therapy in oncology. Kluin-Nelemans HC. Long-term risk of 94:1375-1382 Oncologist 2006; 11:1034-1044 aggressive non-Hodgkin lymphoma. Blood Siegel-Lakhai WS, Crul M, De Porre P, Triesscheijn M, Ruevekamp M, Out R, 2006; 107:2912-2919 Zhang S, Chang I, Boot H, Beijnen JH, Van Berkel TJC, Schellens J, Baas P, Schellens JH. Clinical and pharmacologic study Stewart FA. The pharmacokinetic behavior of Moser EC, Noordijk EM, Van Leeuwen FE, of the farnesyltransferase inhibitor tipifarnib in the photosensitizer meso-tetra-hydroxyphenyl- Baars JW, Thomas J, Carde P, Meerwaldt cancer patients with normal or mildly or chlorin in mice and men. Cancer Chemother JH, Van Glabbeke M, Kluin-Nelemans moderately impaired hepatic function. J Clin Pharmacol 2007; In Press HC. Risk of second cancer after treatment of Oncol 2006; 24:4558-4564 cardiovascular disease after treatment for Triesscheijn M, Ruevekamp M, Antonini N, aggressive non-HodgkinвЂ™s lymphoma; an EORTC cohort study. Haematologica 2006; Sparidans RW, Dost F, Crommentuyn KML, Neering H, Stewart FA, Baas P. Optimizing 91:1481-1488 Huitema ADR, Schellens JHM, Beijnen JH. Meso-Tetra-Hydroxyphenyl-Chlorin Mediated Liquid chromatographic assay for the protease Photodynamic Therapy for Basal Cell inhibitor atazanavir in plasma. Biomed Carcinoma. Photochem Photobiol 2006; Chromatogr 2006; 20:72-76 82:1686-1690 113 MEDICAL ONCOLOGY Publications (continued) Vainchtein LD, Thijssen B, Stokvis E, Van Zandwijk N, Mathy A, Boerrigter L, Vos EJ, Linn SC, Rodenhuis S. [Effects and Rosing H, Schellens JHM, Beijnen JH. Ruijter H, Tielen I, De Jong D, Baas P, costs of adjuvant chemotherapy for operable A simple and sensitive assay for the quantitative Burgers S, Nederlof P. EGFR and KRAS lymph node positive breast cancer with analysis of paclitaxel and metabolites in human mutations as criteria for treatment with tyrosine HER2/neu overexpression] Effecten en plasma using liquid chromatography/tandem kinase inhibitors: retro- and prospective kosten van adjuvante chemotherapie bij mass spectrometry. Biomed Chromatogr 2006; observations in non-small-cell lung cancer. operabele lymfklierpositieve borstkanker met 20:139-148 Ann Oncol 2007; 18:99-103 HER2/neu-overexpressie. Ned Tijdschr Vainchtein LD, Rosing H, Mirejovsky D, Veltkamp SA, Thijssen B, Garrigue JS, Huynh V, Lenaz L, Hillebrand MJX, Lambert G, Lallemand F, Binlich F, Vrieling A, Rookus MA, Kampman E, Schellens JHM, Beijnen JH. Quantitative Huitema ADR, Nuijen B, Nol A, Beijnen JH, Bonfrer JM, Korse CM, Van Doorn J, analysis of EO9 (apaziquone) and its metabolite Schellens JHM. A novel self-microemulsifying Lampe JW, Cats A, Witteman BJ, EO5a in human plasma by high-performance formulation of paclitaxel for oral administration Van Leeuwen FE, Van вЂ�t Veer LJ, liquid chromatography under basic conditions to patients with advanced cancer. Br J Cancer Voskuil DW. Isolated Isoflavones do not coupled to electrospray tandem mass 2006; 95:729-734 Affect the Circulating Insulin-Like Growth Geneeskd 2006; 150:776-780 spectrometry. J Mass Spectrom 2006; 41:1268-1276 Factor (IGF) System in Men at Increased Veltkamp SA, Alderden-Los C, Sharma A, Colorectal Cancer Risk. J Nutr 2007; 137: Rosing H, Beijnen JH, Schellens JHM. accepted Vainchtein LD, Rosing H, Mirejovsky D, A pharmacokinetic and safety study of a novel Lenaz L, Schellens JHM, Beijnen JH. Stability polymeric paclitaxel formulation for oral Vuylsteke RJCL, Molenkamp BG, experiments in human urine with EO9 application. Cancer Chemother Pharmacol Van Leeuwen PAM, Meijer S, (apaziquone): A novel anticancer agent for the 2007; 59:43-50 Wijnands PGJT, Haanen JBAG, Scheper RJ, De Gruijl TD. Tumor-specific intravesical treatment of bladder cancer. Veltkamp SA, Hillebrand MJX, Rosing H, CD8+ T cell reactivity in the sentinel lymph Jansen RS, Wickremsinhe ER, Perkins EJ, node of GM-CSF - Treated stage I Van den Berg JH, Beijnen JH, Balm AJM, Schellens JHM, Beijnen JH. Quantitative melanoma patients is associated with high Schellens JHM. Future opportunities in analysis of gemcitabine triphosphate in human myeloid dendritic cell content. Clin Cancer preventing cisplatin induced ototoxicity. Cancer peripheral blood mononuclear cells using weak Res 2006; 12:2826-2833 Treat Rev 2006; 32:390-397 anion-exchange liquid chromatography coupled J Pharm Biomed Anal 2007; 43:285-292 Van den Broek GB, Balm AJM, with tandem mass spectrometry. J Mass Vyth-Dreese FA, Kim YH, Dellemijn TAM, Spectrom 2006; 41:1633-1642 Schrama E, Haanen JBAG, Spierings E, Van den Brekel MWM, Hauptmann M, Goulmy E. In situ visualization of antigen- Schornagel JH, Rasch CRN. Relationship Vink SR, Schellens JHM, Beijnen JH, specific T cells in cryopreserved human between clinical factors and the incidence of Sindermann H, Engel J, Dubbelman R, Moppi tissues. J Immunol Methods 2006; toxicity after intra-arterial chemoradiation for G, Hillebrand MJX, Bartelink H, Verheij M. 310:78-85 head and neck cancer. Radiother Oncol 2006; Phase I and pharmacokinetic study of combined 81:143-150 treatment with perifosine and radiation in Ye H, Gong L, Liu H, Ruskone- patients with advanced solid tumours. Radiother Fourmestraux A, De Jong D, Pileri S, Oncol 2006; 80:207-213 Thiede C, Lavergne A, Boot H, Caletti G, Van Der Schoot SC, Vainchtein LD, Beijnen JH, Gore A, Mirejovsky D, Lenaz L, WГјndisch T, Molina T, Taal BG, Elena S, Nuijen B. EO-9 bladder instillations: Vink SR, Lagerwerf S, Mesman E, Schellens Neubauer A, MacLennan KA, Siebert R, Formulation selection based on stability JHM, Begg AC, Remstein ED, Dogan A, Du MQ. Strong characteristics and in vitro simulation studies. Van Blitterswijk WJ, Verheij M. BCL10 nuclear expression identifies gastric Int J Pharm 2007; 329:135-141 Radiosensitization of squamous cell carcinoma MALT lymphomas that do not respond to H by the alkylphospholipid perifosine in cell culture pylori eradication. Gut 2006; 55:137-139 Van Der Schoot SC, Nuijen B, Sood P, and xenografts. Clin Cancer Res 2006; 12:1615- Thurmond II KB, Stewart DR, Rice JR, 1622 Beijnen JH. Pharmaceutical development, Zandvliet AS, Schellens JHM, Copalu W, Beijnen JH, Huitema ADR. A semi- quality control, stability and compatibility of a Vlaming MLH, Mohrmann K, physiological population pharmacokinetic parenteral lyophilized formulation of the Wagenaar E, De Waart DR, model describing the non-linear disposition investigational polymer-conjugated platinum Oude Elferink RPJ, Lagas JS, of indisulam. J Pharmacokinet antineoplastic agent AP5346. Pharmazie 2006; Van Tellingen O, Vainchtein LD, Pharmacodyn 2006; 33:543-570 61:835-844 Rosing H, Beijnen JH, Schellens JHM, Schinkel AH. Carcinogen and anticancer drug Zandvliet AS, Copalu W, Schellens JHM, Van Tuyl SAC, Van Noorden JT, Timmer R, transport by Mrp2 in vivo: Studies using Mrp2 Beijnen JH, Huitema ADR. Saturable Stolk MFJ, Kuipers EJ, Taal BG. Detection of (Abcc2) knockout mice. binding of indisulam to plasma proteins small-bowel neuroendocrine tumors by video J Pharmacol Exp Ther 2006; 318:319-327 and distribution to human erythrocytes. capsule endoscopy. Gastrointest Endosc 2006; 64:66-72 Drug Metab Dispos 2006; 34:1041-1046 114 SURGICAL ONCOLOGY X I D IV IS ION OF SU RG ICA L ON COLOG Y PLASTIC AND RECONSTRUCTIVE SURGERY Marieke Van den Berg, J Joris Hage, Arjen Van Turnhout, Leonie Woerdeman Division head, Group leader Bin Kroon Board Bin BR Kroon MD PhD FRCS, Head Alfons JM Balm MD PhD FRCS FACS Omgo E Nieweg MD PhD General Surgical Oncology Bin BR Kroon MD PhD FRCS Academic staff, The clinical activities of the Department of Plastic and Reconstructive Surgery are restricted to reconstructive surgery following oncological resections performed by general surgeons, urologists, and gynaecologists. This reflects on its research that is focused on primary and secondary breast reconstruction, and on the vascular supply and functional anatomy of tissue flaps. Part of the research is conducted in collaboration with the Departments of Radiology and Radiotherapy of the NKI-AVL, with Departments of Plastic and Reconstructive Surgery at the Academic Medical Centre in Amsterdam and the University Medical Centre in Utrecht, and with the Faculty of Movement Sciences at the Vrije Universiteit in Amsterdam. Head Department General Surgical Oncology (till 01-11-2006) Emiel Rutgers MD PhD Academic staff, Head Department General Surgical Oncology (from 01-11-2006) Frans Zoetmulder MD PhD Academic staff Frits Van Coevorden MD PhD Academic staff Omgo Nieweg MD PhD Academic staff Hester Oldenburg MD PhD Academic staff Houke Klomp MD Academic staff Vic Verwaal MD PhD Academic staff Marie-Jeanne BaasвЂ“Vrancken Peeters MD PhD Academic staff Reinie Kaas MD PhD Academic staff Ingrid Kappers MD Academic staff Philip Meijnen MD Academic staff Marjan Piek-den Hartog MD Academic staff Robert Smeenk MD Academic staff Maartje Van Rijk MD Academic staff Johanna Van Sandick MD PhD Fellow Joost Van der Hoeven MD PhD Fellow Immediate breast reconstruction Hardly any criteria are available to preoperatively distinguish patients in whom an eventful postoperative course may be expected after combined skin-sparing mastectomy and immediate prosthetic breast reconstruction. Therefore, we established which factors increase the risk of surgical complications to such a level as to adjust our indications for this procedure. We prospectively studied the clinical relevance of six patient-related and nine procedure-related potential risk factors for a complicated surgical outcome in 400 combined procedures in 309 patients by univariate and multivariate logistic regression analysis. Risk factors that proved significantly correlated with loss of implant by both analyses were accepted as clinical selection criteria. Mild complications occurred significantly more often in patients who were older than the mean age of 43 year, and in breasts that were more than averagely sized or were operated by a fellow in surgical oncology. Implants were significantly more often lost in patients who were obese or smoked (32% risk of loss vs. 6.3% risk for вЂњoptimalвЂќ patients; odds ratio of 5.09), and in breasts that were more than averagely sized (27% risk of loss vs. 12%; odds ratio 2.32). We conclude that the clinically relevant increase of risk of implant loss should lead to reluctance to perform combined skin-sparing mastectomy and immediate prosthetic breast reconstruction in obese patients who smoke, and in those with more than average sized breasts. Nikola Kimmings MD PhD Fellow Head and Neck Oncology and Surgery Frans JM Hilgers MD PhD Academic Staff, Head Annemieke Ackerstaff PhD Academic Staff Alfons Balm MD PhD FRCS FACS Academic staff Marcel Copper MD PhD Academic staff Fenna Ebbens MD Resident Tom Geurts MD Research physician Peter Van Vuuren MD Resident Steven Gonggrijp DDS Academic staff Petra Jongmans MSc Research fellow Desiree Kerkdijk DDS Academic staff Frans Kroon DDS PhD Academic staff Martin Klop MD Fellow Peter Lohuis MD PhD Academic staff Lisette Van der Molen Research fellow Heike Nyst MD Research physician Jimmy Pramana MD Research physician Ludi Smeele MD DDS PhD Academic staff Bing Tan MD PhD Academic staff Adriaan Timmers DDS Academic staff Corina Van As-Brooks PhD Academic staff Michiel Van den Brekel MD PhD Academic staff Microsurgical tissue transplantation The possibility to perform a muscle-sparing procedure and the extra-muscular course of the vascular pedicle may provide suitable features for free transplantation of a segment of the pectoralis major muscle. We investigated the length and diameter of this vascular pedicle to determine its feasibility. Seventeen anatomical dissections were performed in nine human cadavers. The length of the pedicle, its arterial diameter, and its entry point into the muscle were determined. The mean length of the vascular pedicle up to the medial border of the pectoralis minor muscle was 6.6 cm. A mean additional length of 1.7 cm was obtained by proximal dissection of the thoracoacromial trunk to its origin from the axillary artery. The mean external arterial diameter was 1.8 mm and the diameter of the concomitant vein was consistently larger. The vascular pedicle consistently entered the muscle lateral to the midpoint of the clavicular line, a mean of 8.8 cm caudal to this line. We conclude that the vascular pedicle is sufficient for microvascular anastomosis. An anatomic landmark for the entry point of the pedicle and, therefore, the cranial border of the muscle flap could not be defined. The sternocostal part of the pectoralis major muscle has the potential to be used as a segmental free flap. 115 SURGICAL ONCOLOGY ANAESTHESIOLOGY AND INTENSIVE CARE Johannes Huitink, Dirk Buitelaar, Christiaan Keijzer, Tino Stoppa, May Ronday, Julia Ten Cate, Michael SrГЎmek, Peter Schutte Awake fibrecapnic intubation in head and neck cancer patients with a difficult airway: new findings and refinements to the technique Recently we described a novel intubation technique: awake fibrecapnic intubation. The aim of the present study was to evaluate the efficacy and risk for complications of this technique in a consecutive series of head and neck cancer patients with a difficult to intubate airway. Forty intubations in head and neck cancer patients were prospectively studied. After topical anaesthesia, a flexible bronchoscope was introduced into the pharynx. Spontaneous respiration was maintained. A special suction catheter for carbon dioxide measurements was advanced through the suction channel of the bronchoscope, into the airway. All adverse events and complications were documented. There were no complications of the technique. The median (range) time to intubation was three minutes (1.5-15 minutes). All patients were intubated successfully: thirty-nine (98%) with awake fibrecapnic intubation. There was one patient with severe tumour bleeding and acute airway obstruction caused by advancement of the tube over the bronchoscope, but this was not caused by the fibrecapnic intubation technique. It is concluded that awake fibrecapnic intubation is a safe and valuable technique in head and neck cancer patients with a difficult airway. Compound A and carbon monoxide production from sevoflurane and seven different types of carbon dioxide absorbents The degradation of sevoflurane can lead to the production of compound A, carbon monoxide, and increase of temperature of the absorbent. Compound A is known to be nephrotoxic in rats. These reactions depend upon the content of strong bases of the carbon dioxide absorbent used, and its water content. The purpose of this study, that was done in collaboration with the department of Anesthesiology of Vrije Universiteit Medisch Centrum, was to measure the maximum amounts of compound A, carbon monoxide and temperature increase using seven different carbon dioxide absorbents for sevoflurane with differing contents of strong bases. Seven absorbents (AmsorbВ®, DrГ¤gersorb 800plusВ®, MedisorbВ®, SpherasorbВ®, LoflosorbВ®, SuperiaВ®) were used in hydrated and completely desiccated condition in a patient model, using a circle anaesthesia system connected to an artificial lung. A low flow anaesthesia with an oxygen/nitrous oxide mixture was maintained using 0.8% sevoflurane. For quantification of compound A and carbon monoxide, a portable gas chromatograph was used. Temperature was measured inside the absorbent. Most absorbents including desiccated AmsorbВ® produced small amounts of compound A. LoflosorbВ® and SuperiaВ®, which are free of strong bases, did not produce any compound A or carbon monoxide in hydrated or desiccated condition. Only desiccated DrГ¤gersorbВ®, MedisorbВ® and SpherasorbВ® demonstrated small amounts of carbon monoxide accompanied by a significant temperature increase. Guido Van den Broek MD Research fellow Maya Van Rossum PhD Research fellow Charlotte Zuur MD Research fellow Karel Zuur MD Reseach physician Urologic Oncology Simon Horenblas MD PhD FEBU Academic staff, Head Axel Bex MD PhD Academic staff Wim Meinhardt MD PhD Academic staff Henk Van de Poel MD PhD Academic staff Joost Leijte MD Research fellow Remco De Vries MD Research fellow Gynaecology MatthГ© Burger MD PhD Academic staff, Head Marc Van Beurden MD PhD Academic staff Guus Fons MD Academic staff Lotti LubsenвЂ“Brandsma MD Academic staff Willemien Van Driel MD PhD Academic staff Rolien Olivier MD Research fellow Plastic and Reconstructive Surgery Joris Hage MD PhD Academic staff, Head Marieke Van der Berg MD Academic staff Arjen Van Turnhout MD Academic staff Leonie Woerdeman MD PhD Academic staff Brigit Drost MD Academic staff Diederik Hofstede MD Fellow DaniГ«lle Derks MD Fellow Anesthesiology Peter Schutte MD Academic staff, Head Dick Buitelaar MD Academic staff Hans Huitink MD PhD Academic staff Christiaan Keijzer MD Academic staff Gene expression effects on breast cancer and brain tumour cells assessed by microarray analysis The gene expression effects of volatile anaesthetics on breast cancer cells and brain tumour cells were assessed using microarray techniques in collaboration with the Central Microarray Facility. Two volatile anaesthetics (sevoflurane and desflurane) were tested in vitro. All cell lines were tested during one hour exposure to volatile anaesthetics. Preliminary findings show a profound difference in gene-expression between the two vapours. This may be of importance when gene expression profiles are used to predict cancer survival and cancer therapy effects. May Ronday MD Academic staff Michael SrГЎmek MD PhD Academic staff Tino Stoppa MD Academic staff Julia Ten Cate MD Academic staff 116 SURGICAL ONCOLOGY Publications LYMPHATIC MAPPING Bex A, Kerst M, Mallo H, Meinhardt W, Omgo Nieweg, Maartje Van Rijk, Bin K Kroon, Joost Leijte, Pieter Tanis, Renato ValdГ©s Olmos, Horenblas S, De Gast GC. Interferon Johannes Peterse, Emiel Rutgers, Hester Oldenburg, Simon Horenblas, Jelle Teertstra, alpha 2b as medical selection for Augustinus Hart, Saar Muller, Cornelis Hoefnagel, Bin BR Kroon nephrectomy in patients with synchronous metastatic renal cell carcinoma: A consecutive study. Eur Urol 2006; 49:76-81 Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, Gennaro M, Rouanet P, Avril A, Fentiman IS, Bartelink H, Rutgers EJ. Lymphatic mapping Lymphatic mapping with sentinel node biopsy is a staging technique devised to detect early tumour involvement of the regional lymph nodes. This technique enables the identification of patients who may benefit from early regional treatment and from adjuvant systemic therapy. The tumour status of the sentinel node provides prognostic information. The lymphatic mapping research program was initiated in 1993 and the technique has evolved into a generally accepted procedure. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853--a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. The aims of our research program are to outline indications and contra-indications, to refine the technique of lymphoscintigraphy, of surgery and pathology, and to determine implications in terms of more individualized treatment, survival and regional tumour control. Studies are done in cooperation with the Medisch Spectrum Twente, IJsselland Ziekenhuis, Haga Ziekenhuis, Memorial Sloan Kettering Cancer Center and the John Wayne Cancer Center. J Clin Oncol 2006; 24:3381-3387 Bogaerts J, Cardoso F, Buyse M, Braga S, Loi S, Harrison JA, Bines J, Mook S, Decker N, Ravdin P, Therasse P, Rutgers E, Van вЂ�t Veer LJ, Piccart M. Gene signature evaluation as a prognostic tool: Challenges in the design of the MINDACT trial. Nat Clin Pract Oncol 2006; 3:540-551 Broekhuizen LN, Wijsman JH, Peterse JL, Rutgers EJT. The incidence and significance of micrometastases in lymph nodes of patients with ductal carcinoma in situ and T1a carcinoma of the breast. Eur J Surg Oncol 2006; 32:502-506 Breast cancer: Sentinel nodes in unusual locations The preferential lymphatic drainage pathway from the breast is towards the lower axilla but sentinel nodes are also found at other sites. Various aspects of sentinel node biopsy outside level I and II of the axilla and internal mammary chain were investigated. Unusually situated sentinel nodes were visualized on the lymphoscintigrams in 91 of the 785 breast cancer patients (12%). These nodes were located in the internal mammary chain, above and below the clavicle, within the breast and in-between the pectoral muscles (RotterвЂ™s nodes). Such oddly situated sentinel nodes could be retrieved in 80 of these 91 patients (82%). These were tumour-positive in sixteen patients (20%). In eleven patients (14%) this was the only involved node and five patients also had a tumourpositive sentinel node in the axilla. Based on these findings, the treatment strategy was adjusted in twelve individuals with the addition of adjuvant regional or systemic therapy. Axillary clearance was avoided in two patients with drainage limited to sentinel nodes elsewhere. The pursuit of unusually situated sentinel nodes provides additional staging information and this approach results in treatment that is better tailored to the needs of the individual patient. Buitelaar DR, Balm AJM, Antonini N, Van Tinteren H, Huitink JM. Cardiovascular and respiratory complications after major head and neck surgery. Head Neck 2006; 28:595-602 Buyse M, Loi S, Van вЂ�t Veer L, Viale G, Delorenzi M, Glas AM, dвЂ™Assignies MS, Bergh J, Lidereau R, Ellis P, Harris A, Bogaerts J, Therasse P, Floore A, Amakrane M, Piette F, Rutgers E, Sotiriou C, Cardoso F, Piccart MJ, Decker N, Straehle C. Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J Natl Cancer Inst 2006; Sentinel node biopsy and neoadjuvant chemotherapy More and more patients now receive neo-adjuvant chemotherapy. Is sentinel node biopsy reliably possible in these patients? In eighteen studies published about sentinel node biopsy after neoadjuvant chemotherapy, the sentinel node was identified in an average 89% of patients and the false-negative rate was on average 10%. Because of these mediocre results, none of these investigators dares to omit axillary clearance just yet. We studied sentinel lymph node biopsy prior to neoadjuvant chemotherapy in 25 T2N0 patients. Ten of them had a tumour-positive axillary sentinel node. Axillary lymph node dissection was performed in these ten patients after completion of the chemotherapy regimen. The other fifteen patients (60%) were spared axillary lymph node dissection. No recurrences have been observed after a median follow-up of eighteen months. It is concluded that sentinel node biopsy prior to neo-adjuvant chemotherapy appears successful and reliable. This approach spares patients who receive neoadjuvant chemotherapy the unnecessary morbidity of axillary lymph node dissection when the nodes are not involved. 98:1183-1192 Corten EML, Schellekens PPA, Bleys RLAW, Hage JJ, Kon M. The segmental pectoralis major free flap: Anatomical features of its vascular pedicle. Ann Plast Surg 2006; 56:82-86 Penile cancer At The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, dynamic sentinel lymph node biopsy is routinely performed in patients with clinically localized squamous cell carcinoma of the penis. The majority of patients with penile cancer with a tumour positive sentinel node do not benefit from complementary lymph node dissection because the remainder of the nodes are normal. A study was carried out to look for factors that may determine the involvement of additional nodes. A total of 158 patients with clinically node-negative penile carcinoma underwent sentinel node biopsy. Factors analyzed for their 117 SURGICAL ONCOLOGY Publications (continued) association with additional nodal involvement were T-stage, diameter of the primary tumour, malignancy grade, vascular invasion of the primary tumour, and size of the sentinel node metastasis. Tumour-positive sentinel nodes were found in 46 groins and complementary lymph node dissection was performed. Nine of these 46 groins (20%) contained additional involved lymph nodes. The size of the sentinel node metastasis proved to be the only significant prognostic variable for additional lymph node involvement (p=0.02). None of the fifteen groins with only sentinel metastasis smaller than 2 mm in the sentinel node contained additional involved nodes. The conclusion of the study is that additional nodal involvement is related to the size of the metastasis in the sentinel node. Sentinel node metastases smaller than 2 mm are not associated with other involved lymph nodes, suggesting that these patients can be spared complementary lymph node dissection. Deurloo EE, Klein Zeggelink WFA, Teertstra HJ, Peterse JL, Rutgers EJT, Muller SH, Bartelink H, Gilhuijs KGA. Contrast-enhanced MRI in breast cancer patients eligible for breast-conserving therapy: Complementary value for subgroups of patients. Eur Radiol 2006; 16:692-701 Fons G, Hasibuan SM, Van der Velden J, Ten Kate FJ. Validation of tissue micro array technology in endometrioid cancer of the endometrium. J Clin Pathol 2006;[Epub MELANOMA ahead of print] Omgo Nieweg, Eva Noorda, Bart Vrouenraets, Gooike Van Slooten, Bert Van Geel*, Alexander Eggermont*, Jolanda Wittenberg**, Bin BR Kroon Hage JJ, Van Turnhout AAWM. Long-term outcome of metaidoioplasty in 70 female-to- Follow-up of patients with clinically localized melanoma appears useless The purpose was to determine the value of ancillary blood tests and imaging studies for routine follow-up, and to determine the value of follow-up per se in patients who have been treated for clinically localized disease. A systematic literature search in the Medline database and the Cochrane Library was performed. The keywords and medical subject heading terms were вЂ�follow upвЂ™ and вЂ�melanomaвЂ™, starting in 1984. The selected publications were assessed for patient population, methodology, intervention and outcome measures. The methodological quality of every study was appraised and a level of evidence was assigned. Sixty-six publications form the source of information on which this study is based. Randomised studies examining a survival benefit from follow up with or without imaging studies and lab exams are lacking. Few patients with metastases are identified by these routine tests, and even fewer survive because of these tests. Far more often the results are false positive, which invariably causes unnecessary concern and leads to futile additional testing and may even provoke needless operations. The limited evidence that is available indicates that follow up per se is not beneficial. There is no literature on quality of life with and without follow up. The conclusion of the literature study is that there is no convincing evidence that regional disease control, survival and quality of life improve through surveillance (level 3). The present findings challenge the current common practice of routine follow up. As a result, the Dutch national guidelines now recommend follow up only for patients with a melanoma of more than 1 mm Breslow thickness. Follow up visits are limited to a history and physical examination. Additional testing is done only for suspected recurrence. male transsexuals. Ann Plast Surg 2006; 57:312-316 Hage JJ. Reconstruction of the penis. In: Grabb WC, Smith JW, Thorne CH, editors. Philadelphia, Pa.; London: Lippincott Williams & Wilkins, 2006: Hage JJ, Karim RB. Risk of breast cancer among reduction mammaplasty patients and the strategies used by plastic surgeons to detect such cancer. Plast Reconstr Surg 2006; 117:727-735 Hage JJ, Van Turnhout AAWM, Dekker JJML, Karim RB. Saving labium minus skin to treat possible urethral stenosis in female-to-male transsexuals. Ann Plast Surg 2006; 56:456-459 Huitink JM, Burggraaf JP, Rennings JHL, Slagt C. Air medical transport in the Netherlands. In: Blumen I, editor. Principals and Direction of Air Medical Transport. Salt Lake City: Air Medical Repeat isolated limb perfusion for recurrent melanoma The treatment of recurrent limb melanoma after isolated perfusion, occurring in about 70% of the patients, is a continuing challenge. Major amputation is a deterrent option for local control and palliation in these patients, who have a rather poor prognosis. Between 1978 and 2001, 21 patients underwent repeat isolated perfusion with Tumour Necrosis Factor a (TNFa) and melphalan for recurrent or persistent melanoma lesions after failure of previous perfusion. First perfusions had been performed with melphalan alone in 13 patients and with the addition of TNFa in eight, for a median of nine lesions. Repeat perfusion was performed for a median of nine lesions. Median follow-up after repeat perfusion was 18 months. Thirteen patients attained a complete response after repeat perfusion. Nine patients relapsed after complete remission. Median limb recurrence-free interval was 13 months. The limb salvage rate was 95%. Overall survival was 62 months after complete remission, compared to 13 months for those without complete remission (p=0.05). Regional toxicity after repeat perfusion was mild. * Erasmus Medical Center, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands ** Dutch Institute for Healthcare Improvement CBO, Utrecht, The Netherlands Physician Association Publishing, 2006: Huitink JM, Buitelaar DR, Schutte PFE. Awake fibrecapnic intubation: A novel technique for intubation in head and neck cancer patients with a difficult airway. Anaesthesia 2006; 61:449-452 Karim RB, Hage JJ, Van Rozelaar L, Lange CAH, Raaijmakers J. Complications of polyalkylimide 4% injections (BioAlcamidTM): a report of 18 cases. J Plastic Reconstr Aesthet Surg 2006; 59:1409-1414 118 SURGICAL ONCOLOGY Publications (continued) Karim RBM, Brito BLR, Dutrieux RPM, Lassance FPP, Hage JJ. MMP-2 Repeat isolated perfusion with TNF and melphalan is feasible, with a relatively high complete remission rate, a good limb recurrence-free survival and a high limb salvage rate. Assessment as an Indicator of Wound Healing: A Feasibility Study. Adv Skin Wound Care 2006; 19:324-327 BREAST CANCER Emiel Rutgers, Hester Oldenburg, Reinie Kaas, Philip Meijnen, Hans Peterse, Leonie Woerdeman, Keijzer C, Perez RSGM, De Lange JJ. Maartje Van Rijk, Marie-Jeanne Vrancken Peeters, Augustinus Hart, Saar Muller, Claudette Loo, Compound A and carbon monoxide Omgo Nieweg production from sevoflurane and seven different types of carbon dioxide absorbent in a patient model. Acta Anaesthesiol Scand 2007; 51:31-37 Klein S, Hage JJ. Measurement, calculation, Stage of tumours detected in the surveillance of women at high risk for breast cancer The study aimed at analyzing the stage distribution of breast cancers found during surveillance (1994-2006) with annual mammography and bi-annual palpation in women at a life time risk of at least 15%. In a total of 152 women a first or contralateral breast cancer was detected in the period under study. and normal range of the ankle-arm index: A bibliometric analysis and Table XI.1: Stage distribution recommendation for standardization. Ann Vasc Surg 2006; 20:282-292 Stage Goal BOB Non-BRCA n = 102 Kreulen M, Smeulders MJC, Veeger HEJ, O+I Minimal > 50% > 30% > 75% 65/102 38/102 10/50 Stage Goal BOB BRCA1 n = 39 Dev Med Child Neurol 2006; 48:436-441 0+I Minimal pNo > 50% > 30% > 75% 24/39 9/39 33/39 Kreulen M, Smeulders MJ, Veeger HE, Minimal = pTisN0 + pT1abN0 Hage JJ. Movement patterns of the upper BOB: Bevolkingsonderzoek borstkanker extremity and trunk associated with *Statistical analysis was done using the chi-square test and when appropriate the Fisher exact test Hage JJ. Movement patterns of the upper extremity and trunk before and after corrective surgery of impaired forearm rotation in patients with cerebral palsy. 64% 37% 20% 62% 23% 85% BRCA1/2 n = 50 27/50 76/102 36/50 54% 76% 72% BRCA2 n = 11 3/11 1/11 3/11 27% 9% 27% P value 0.25 0.032 0.74 P value 0.044 0.42 0.001* impaired forearm rotation in patients with hemiplegic cerebral palsy compared to healthy controls. Gait Posture 2006;[Epub ahead of print] In conclusion, with annual mammography and (self)palpation the detected breast cancers in non-BRCA carriers comply with the goals for biennial population screening. The BRCA1 carriers failed in the goal for minimal breast cancer, the BRCA2 carriers failed all goals, but the numbers are small. Kriege M, Brekelmans CTM, Boetes C, Muller SH, Zonderland HM, Obdeijn IM, Manoliu RA, Kok T, Rutgers EJT, De Koning HJ, Klijn JGM, Bartels CCM, Besnard APE, Hoogerbrugge N, Meijer S, Oosterwijk JC, Seynaeve C, Tilanus-Linthorst MMA, Tollenaar RAEM. Differences between first and subsequent rounds of the MRISC breast cancer screening program for women with a familial or genetic predisposition. Cancer 2006; 106:2318-2326 Kriege M, Brekelmans CT, Obdeijn IM, Boetes C, Zonderland HM, Muller SH, Kok T, Manoliu RA, Besnard AP, Tilanus-Linthorst MM, Seynaeve C, Bartels CC, Kaas R, Meijer S, Oosterwijk JC, Hoogerbrugge N, Tollenaar RA, Rutgers EJ, De Koning HJ, Klijn JG. Factors affecting sensitivity and specificity of screening mammography and MRI in women with an inherited risk for breast cancer. Breast Cancer Res Treat 2006; 100:109-119 Ductal carcinoma in situ diagnosed on core-needle biopsy: risk of invasion and axillary lymph node metastases The aim of this study was to assess the risk of invasion and axillary lymph node metastases in patients with DCIS diagnosed on preoperative core-needle biopsy to select criteria in what patients SN biopsy might be warranted. One hundred and seventy-two patients diagnosed with DCIS on coreneedle biopsy were analysed. Axillary staging was performed by SN biopsy, basal node sampling, or level 1-2 axillary lymph node dissection. Invasive breast cancer was found in the surgical specimens of 45 (26%) patients. Risk factors for invasion were a palpable lesion (OR: 2.95 (1.20-7.26), p=0.019), presence of a mass on mammography (OR: 3.06 (1.43-6.56), p=0.004), intermediately (OR: 5.81 (1.18-28.57), p=0.030), or poorly differentiated tumour grade (OR: 5.46 (1.17-25.64), p=0.031). Lymph node metastases were found in 10 patients with DCIS and invasion on final pathology. Factors associated with metastases were age up to 55 years (p=0.030), invasion larger than 1.0 cm (p<0.001), and presence of vascular invasion (p=0.001). The presence of invasion in 26% of patients with DCIS diagnosed on core-needle biopsy poses the problem of identifying patients in whom axillary staging by SN biopsy is most warranted. While others suggest to perform SN biopsy in all patients with a core-needle biopsy or at least in all patients who undergo mastectomy, we think that patients could be selected more carefully to prevent axillary staging in patients who stand to gain very little from it. SN biopsy should be considered in patients with an initial diagnosis of DCIS on core-needle biopsy. Patients at risk for invasion may be identified preoperatively by a palpable lesion, a mass on mammography, intermediately, or poorly differentiated tumour grade. 119 SURGICAL ONCOLOGY Publications (continued) Neomamma Database Neoadjuvant administration of chemotherapy (or primary systemic treatment) is widely used in breast cancer patients with locally advanced disease and its use is extending to earlier stages of disease. The goals of this regime are to treat occult systemic disease, decrease the tumour bulk, and to reduce the extent of local surgery. Over the past 5 years we have treated over 300 patients with breast cancer according to different regimes of neoadjuvant chemotherapy. Most patients were enrolled in the two main randomized trials that were active during this time frame. This year we have created a data base : Neomamma, to collect data of these patients to retrospectively evaluate the effect of neoadjuvant chemotherapy on the outcome of patients with breast cancer. The primary endpoint of the study is the pathological complete remission rate. Secondary endpoint of the study is the influence of primary systemic therapy on the extent of local surgery. Data of 326 patients are ready to be collected from their records. Patient characteristics are obtained including age and all results of diagnosis and staging prior the start of chemotherapy. The histological type and grade and receptor status will be defined. In a subset of patients (NOODCD and NO4POM trial patients) tumour samples were subjected to micro-array analysis in an attempt to define geneexpression profiles associated with sensitivity or resistance against chemotherapy. The exact chemotherapy regime is registered. Evaluation of the response by physical examination and by radiological examination (ultrasound, MRI) will be documented. Furthermore, the type of surgery is documented and compared with the type of surgery that was required when no neoadjuvant chemotherapy had been given. The final pathologic result will be obtained and the pathologic response in the breast will be measured. In the year 2007 we expect to gather and analyze all data to answer the primary and secondary end points of the study. Kroon BK, Nieweg OE, Van Boven H, Horenblas S. Size of Metastasis in the Sentinel Node Predicts Additional Nodal Involvement in Penile Carcinoma. J Urol 2006; 176:105-108 Lakhani SR, Audretsch W, Cleton-Jensen AM, Cutuli B, Ellis I, Eusebi V, Greco M, Houslton RS, Kuhl CK, Kurtz J, Palacios J, Peterse H, Rochard F, Rutgers E, on behalf of EUSOMA. The management of lobular carcinoma in situ (LCIS). Is LCIS the same as ductal carcinoma in situ (DCIS)? Eur J Cancer 2006; 42:2205-2211 Leijte JAP, ValdГ©s Olmos R, Mens JWM, Horenblas S. Use of positron emission tomography in spindle cell carcinoma of the penis. Urology 2006; 68 Lont AP, Gallee MPW, Meinhardt W, Van Tinteren H, Horenblas S. Penis Conserving Treatment for T1 and T2 Penile Carcinoma: Clinical Implications of a Local Recurrence. J Urol 2006; 176:575-580 GYNAECOLOGY Matthe Burger, Marc Van Beurden, Willemien Van Driel, Guus Fons, Lotti Lubsen-Brandsma, Lont AP, Kroon BK, Horenblas S, Roelien Olivier, Manon Van Seters Gallee MPW, Berkhof J, Meijer CJLM, Snijders PJF. Presence of high-risk human Hereditary ovarian carcinoma The predicted value of ovarian cancer screening in high risk women has been established in 312 women and has been found to be poor (PPV 40%). The clinical outcome of (PBSO) in BRCA1/2 mutation carriers has been studied in 90 women. In 8.6% an occult ovarian or fallopian tube carcinoma was found. A gene expression profile was found in high stage ovarian cancer which is able to discriminate between poor and good overall-survival with an accuracy of 78%. A specific proteomic pattern is studied in serum and tissue in patients who are either at high risk of developing ovarian cancer, or who are diagnosed with ovarian cancer. Goal is to develop a tumour marker profile in the primary diagnosis of ovarian cancer, to predict the response to treatment, as well as for screening of high risk populations. The psychosocial impact of PBSO has been examined in a nationwide, crosssectional, observational study. Forty-four percent of 846 high-risk women had undergone PBSO, and 56% had opted for gynaecological screening (GS). No significant differences were observed in generic QOL in both groups. PBSO was associated with fewer breast and ovarian cancer worries (P < .001) and more favourable cancer risk perception (P < .05). However, the PBSO group reported significantly more endocrine symptoms (P < .001) and worse sexual functioning (P < .05).(KWF project: NKI 2001-2382) A multicentered randomized trial to investigate the effect of cognitive behavioural therapy (CBT) and physical exercise for climacteric symptoms in breast cancer patients experiencing treatment-induced menopause will be shortly opened. (KWF project: NKI 2006-3470). In a multicenter trial the effect of different hormonal replacement regimen on bone density, breast density and quality of live after prophylactic bilateral salpingo oophorectomy are examined in a randomised control trial (M05HIR Hirise) The effect of hormonal replacement therapy on menopausal complaints related to biochemical changes in surgically and naturally postmenopausal women is currently being investigated in a prospective observational comparative study.M06HRT (Novaria). papillomavirus DNA in penile carcinoma predicts favorable outcome in survival. Int J Cancer 2006; 119:1078-1081 Madalinska JB, Van Beurden M, Bleiker EM, Valdimarsdottir HB, Lubsen-Brandsma L, Massuger LF, Mourits MJ, Gaarenstroom KN, Van Dorst EB, Van der Putten H, Boonstra H, Aaronson NK. Predictors of prophylactic bilateral salpingooophorectomy verus hynecological screening uptake in BRCA1/2 mutation carriers. J Clin Oncol. In press Madalinska JB, Van Beurden M, Bleiker EM, Valdimarsdottir HB, Hollenstein J, Massuger LF, Gaarenstroom KN, Mourits MJ, Verheijen RH, Van Dorst EB, Van der Putten H, Van der Velden K, Boonstra H, Aaronson NK. The impact of hormone replacement therapy on menopausal symptoms in younger high-risk women after prophylactic salpingo-oophorectomy. J Clin Oncol 2006; 24:3576-3582 120 SURGICAL ONCOLOGY Publications (continued) Molenaar S, Sprangers M, Oort F, Rutgers E, Luiten E, Mulder J, Van Meeteren M, De Haes H. Exploring the black box of a decision aid: what information do patients select from an interactive Cd-Rom on treatment options in breast cancer? Patient Educ Couns 2007; 65:122-130 Morton DL, Thompson JF, Cochran AJ, Mozzillo N, Elashoff R, Essner R, Nieweg OE, Roses DF, Hoekstra HJ, Karakousis CP, Reintgen DS, Coventry BJ, Glass EC, Wang HJ. Sentinel-node biopsy or nodal observation in melanoma [Erratum in N Engl J Med 2006; 355:1944]. N Engl J Med 2006; 355:1307-1317 Nieweg OE, Kroon BBR. The conundrum Ovarian carcinoma A randomised, multicentre, phase III study of erlotinib versus observation in patients with no evidence of disease progression after first line, platinum-based chemotherapy for high-risk stage I and stage II-IV ovarian epithelial, primary peritoneal, or fallopian tube cancer. Primary endpoint will be progression free survival; secondary endpoint will be overall survival, safety and quality of life. (EORTC 55041). Preparation are made for a phase III randomised clinical trial for stage III ovarian carcinoma investigating the effect of secondary debulking surgery with or without hyperthermic intraperitoneal cisplatinum which will shortly be opened. Vulva Vulvar cancer and tissue micro array (TMA): COX-2 and Caspase-3 are identified as potential prognostic markers for vulvar cancer in a test set of 50 vulvectomy specimens using immunohistochemical staining of TMA. The additional value of this immunohistochemical profile in relation to well known clinicopathological variables in vulvar cancer is tested in a validation study. The technique of TMA is validated by comparing immunohistochemical staining results of triplicate core biopsies on TMA with the results of full section analysis. Progression of vulvar cancer in waiting time before surgery: Progression in waiting time is determined in 25 patients by comparing the area of the tumour on two digital photographs. The first is taken at first visit and the second prior to operation. of follow-up: should it be abandoned? Surg Oncol Clin N Am 2006; 15:319-330 Noorda EM, Vrouenraets BC, Nieweg OE, Kroon BBR. Isolated limb perfusion in regional melanoma. Surg Oncol Clin N Am 2006; 15:373-384 Noorda EM, Vrouenraets BC, Nieweg OE, Van Geel AN, Eggermont AMM, VIN The histology and human papillomavirus (HPV) status in patients with a history of both LS and VIN without coexistent SCC is studied. VIN related to LS without coexisting SCC is likely to be undifferentiated, unlike what was previously thought. HPV DNA was demonstrated in 31% and strongly related to high-grade VIN. HPV DNA/peptide vaccination. KWF grant is submitted in September 2006 Protec study; Purpose of this study is to assess the benefit of wearing therapeutic elastic stockings in order to prevent lymphedema for patients treated with inguinal lymph node dissection. Secondairy endpoints are effect on body image and quality of life issues. Design of the study is a non-blinded, randomised controlled trial. Kroon BBR. Repeat isolated limb perfusion with TNFa and melphalan for recurrent limb melanoma after failure of previous HEAD AND NECK ONCOLOGY AND SURGERY perfusion. Eur J Surg Oncol 2006; Fons Balm, Jan Paul De Boer, Michiel Van den Brekel, Marcel Copper, Frans Hilgers, Frank Hoebers, 32:318-324 Marina Kartachova, Peter Lohuis, Saar Muller, Coen Rasch, Jan Schornagel, Ludi Smeele, Bing Tan, Renato Valdes Olmos, Karel Zuur Olivier RI, Lubsen-Brandsma MAC, Verhoef S, Van Beurden M. CA125 and transvaginal ultrasound monitoring in high-risk women cannot prevent the diagnosis of advanced ovarian cancer. Gynecol Oncol 2006; 100:20-26 Olivier RI, Van Beurden M. Role of BRCA1/BRCA2 in ovarian, fallopian tube, and peritoneal papillary serous carcinoma. In: Hayat MA, editor. Immunohistochemistry and in Situ Hybridization of Human Carcinomas -molecular genetics, gastrointestinal carcinoma, and ovarian carcinoma. Academic Press, 2006: 347-357 Olivier RI, Van Beurden M, Van вЂ�t Veer LJ. The role of gene expression profiling in the clinical management of ovarian cancer. Eur J Cancer 2006; 42:2930-2938 Intra-tracheal temperature and humidity assessment in laryngectomized individuals Airway climate change is an unavoidable consequence of total laryngectomy. Heat and moisture exchangers (HME) ameliorate pulmonary sequels of total laryngectomy significantly, but the (patho) physiological mechanisms behind this beneficial effect are not well understood. Therefore, an intra-tracheal temperature and humidity analyzer was developed for assessment of airway climate in laryngectomized individuals and assessment of heat and moisture exchanger (HME) influence on this climate. Intratracheal positioning of a sensor is not suitable. Tracheal air has to be sampled with a thin catheter and measured externally and. the sensor should be able to follow normal breathing frequencies. Primary goals of engineering were prevention of condensation within the device and achievement of fast response time characteristics. The airway climate explorer (ACE; see figure XI.1) developed in close collaboration with the departments of вЂ�Medische Technologie OntwikkelingвЂ™ and ENT of the AMC, consists of a small diameter (5 mm), heated airsampling catheter connected to a heated sensor house incorporating a humidity sensor. Air is sucked through the catheter by a pump with controlled flow. Validation was performed in a climate chamber using a calibrated reference sensor and in a twoflow system. The validation revealed that over the clinically relevant range of humidity values (5вЂ“42 mg H2O/L air) sensor output correlated highly to reference sensor readings (R2 >0.99). The 1вЂ“1/e response times were all <0.5 s. In vivo assessment was successful. In conclusion, this newly developed, validated, and fast responding temperature and humidity analyzer is suitable for post-laryngectomy airway climate and HME assessment. Further clinical studies assessing the HME effect under (climatologically) different circumstances are underway. 121 SURGICAL ONCOLOGY Publications (continued) Ortin-Perez J, Van Rijk MC, Valdes-Olmos RA, Vidal-Sicart S, Nieweg OE, Vilalta A, Kroon BB, Pons F. Lymphatic mapping and sentinel node biopsy in MerkelвЂ™s cell carcinoma. Eur J Figure XI.1: Air climate explorer Surg Oncol 2006;[Epub ahead of print] (ACE) for intra-tracheal temperature and humidity Rodenhuis S, Rutgers EJT. Adjuvant assessment in laryngectomized therapy for breast cancer. Br J Surg 2006; individuals. 93:1443-1445 Smeenk RM, Bex A, Verwaal VJ, Evaluation treatment strategies From 2000 to November 2004 236 patients from 5 hospitals with (functionally) inoperable cancer of the oral cavity (19%), oropharynx (63%) and hypopharynx (18%) were randomly assigned to receive 70 Gy/35 fractions/7 weeks combined with either four courses of intra-arterial (IA) cisplatin (150 mg/m2) and intravenous Na-thiosulfate on days 2, 9, 16 and 23 (N=118) or intravenous (IV) cisplatin (100 mg/m2) on days 1, 22 and 43 (N=118). The median follow-up was 17 months. At two years no significant difference was seen between IA and IV chemotherapy in loco-regional control (62 vs 68% ) or overall survival (61% vs 63%) (Figure XI.2). Loco-regional control at 2 years was lower for oral cavity sites (50%) compared to hypopharynx (76%) and oropharynx (68%, p=0.05). Renal toxicity > grade II was more frequent in the intravenous arm (1 vs 10%, p=0.02), neurological toxicity was more frequent in the intra-arterial arm (7 vs 1%, p=0.005). In conclusion: No tumour control difference is seen between the treatment arms. Severe renal toxicity is less frequent in the intra-arterial arm, but in this treatment arm neurological toxicity is more common. Horenblas S, Zoetmulder FAN. Pseudomyxoma peritonei and the urinary tract: Involvement and treatment related complications. J Surg Oncol 2006; 93:20-23 Van Dalen T, Plooij JM, Van Coevorden F, Van Geel AN, Hoekstra HJ, Albus-Lutter C, Slootweg PJ, Hennipman A, Dutch Soft Tissue Sarcoma Group. Long-term prognosis of primary retroperitoneal soft tissue sarcoma. Eur J Surg Oncol 2006; In Press Van der Poel HG. Foreseeing cancer metastases. Eur Urol 2006; 50:648-649 Van der Poel HG. Molecular markers in the diagnosis of prostate cancer. Crit Rev Oncol Hematol 2007; 61:104-139 Van der Poel HG, Antonini N, Hoefnagel CA, Horenblas S, ValdГ©s Olmos RA. Serum hemoglobin levels Figure XI.2: Kaplan Meier overall survival predict response to strontium-89 and curves comparing randomized phase III intra- rhenium-186-HEDP radionuclide treatment arterial and intravenous chemo-irradiation in for painful osseous metastases in prostate stage III/IV head and neck cancer patients. cancer. Urol Int 2006; 77:50-56 In vivo imaging of apoptosis in tumour and normal tissue The purpose of this study is to evaluate if 99mTc Hynic-rh-Annexin V scintigraphy (TAVS), a technique to depict apoptosis, is feasible to predict treatment response in patients with head and neck squamous cell carcinoma (HNSCC) treated with chemoradiation. Sixteen consecutive patients with stage IV HNSCC underwent TAVS using planar images and SPECT before and 48-hours after the start of therapy (Figure XI.3). Treatment-induced changes of tracer uptake (О”U) in the tumour and normal tissues were calculated. Statistically significant correlation was found between radiation dose and Annexin uptake in salivary gland and bone marrow. A positive correlation for maximum U in primary tumour and lymph nodes was observed on an inter-patient level (r = 0.71; p = 0.006). No correlation was observed between the Annexin-uptake in tumour or lymph nodes and initial treatment response. Annexin V scintigraphy showed a radiation-dose-dependent uptake in parotid glands and bone marrow, indicative of early treatment-induced apoptosis. The fact that Annexin uptake in primary tumour and nodal metastases are correlated suggests a tumour specific apoptosis response. Lack of correlation between the tracer tumour uptake and shortterm outcome can be explained by the pre-existing necrosis and lymphocyte infiltration. Van der Poel HG, Beetsma DB, Van Boven H, Horenblas S. Perineal salvage prostatectomy for radiation resistant prostate cancer. Eur Urol 2006; In Press Van der Steen LPE, Hage JJ, Kon M, Monstrey SJ. Intra-rater repeatability of a structured method of selecting abstracts for the annual euraps scientific meeting. Eur J Plast Surg 2006; 29:111-114 Van Duijnhoven FH, Jansen MC, Junggeburt JMC, Van Hillegersberg R, Rijken AM, Van Coevorden F, Van Der Sijp JR, Van Gulik TM, Slooter GD, Klaase JM, Putter H, Tollenaar RAEM. Factors influencing the local failure rate of radiofrequency ablation of colorectal liver metastases. Ann Surg Oncol 2006; 13:651-658 122 SURGICAL ONCOLOGY Publications (continued) Van Rijk MC, Peterse JL, Nieweg OE, Oldenburg HSA, Rutgers EJT, Kroon BBR. Additional axillary metastases and stage migration in breast cancer patients with micrometastases or submicrometastases in sentinel lymph nodes. Cancer 2006; 107:467-471 Van Rijk MC, Tanis PJ, Nieweg OE, ValdГ©s Olmos RA, Rutgers EJT, Hoefnagel CA, Kroon BBR. Clinical implications of sentinel nodes outside the axilla and internal mammary chain in patients with breast cancer. J Surg Oncol 2006; 94:281-286 Van Rijk MC, Nieweg OE, Rutgers EJT, Oldenburg HSA, Olmos RV, Hoefnagel CA, Kroon BBR. Sentinel node Figure XI.3: Pre- (A, C, E) and biopsy before neoadjuvant chemotherapy post-radiotherapy (B, D, F) uptake spares breast cancer patients axillary lymph of Annexin in a base of tongue node dissection. Ann Surg Oncol 2006; carcinoma. 13:475-479 Van Rijk MC, Nieweg OE, UROLOGY Oldenburg HSA, Rutgers EJT, Kroon BBR. Simon Horenblas, Wim Meinhardt, Axel Bex, Henk Van der Poel The sentinel node biopsy in patients with breast cancer; many controversies remain. Eur J Gynaecol Oncol 2006; 27:321-328 Van Rijk MC, Teertstra HJ, Peterse JL, Clinical and scientific activities of the department of Urology comprise a variety of topics in the field of oncological urology. Many aspects of bladder, prostate, kidney, penis, and testicular cancer were studied in both preclinical and clinical evaluations. The most recent developments are mentioned here: Nieweg OE, Olmos RAV, Hoefnagel CA, Kroon BBR. Ultrasonography and fineneedle aspiration cytology in the preoperative evaluation of melanoma patients eligible for sentinel node biopsy. Ann Surg Oncol 2006; 13:1511-1516 Van Rijk MC, Deurloo EE, Nieweg OE, Gilhuijs KGA, Peterse JL, Rutgers EJT, KrГ¶ger R, Kroon BBR. Ultrasonography and fine-needle aspiration cytology can spare breast cancer patients unnecessary sentinel lymph node biopsy. Ann Surg Oncol 2006; 13:31-35 Van Seters M, Ten Kate FJW, Van Beurden M, Verheijen RHM, Meijer CJLM, Burger MPM, Helmerhorst TJM. In the absence of (early) invasive carcinoma vulvar intraepithelial neoplasia associated with lichen sclerosus is mainly of undifferentiated type: new insights in histology and aetiology. J Clin Pathol 2006;Published Online Metastasized renal cancer management In primary metastatic renal cell carcinoma cytoreductive surgery may prolong survival in selected patients. In 2006 we continued to investigate the timing of nephrectomy and the value of initial immunotherapy as selection for surgery in a randomized trial. In collaboration with our department of immunotherapy metastatic patients failing immunotherapy were included in trials with tyrosine-kinase inhibitors. Invasive bladder cancer management During the last 10 years we have witnessed an increase in referrals for the management of muscle infiltrating cancer. This has led to analyze the changes from a low volume hospital to high volume hospital. With the aid of the database of the comprehensive cancer center Amsterdam (IKA) we were able to compare results of the management of bladder cancer. A large number of parameters were compared. We were especially interested in the local recurrence rate as a measure of surgical expertise. The analysis did show a trend towards lower recurrence rates in hospitals with a large patient load of bladder cancer. A renewed analysis of a modified cystectomy with complete anatomic reconstruction, developed at this institute, showed the oncological safety together with the preservation of sexual functions. Prostate cancer sentinel node After the development of sentinel node procedures for penile and bladder cancer, the initiation of a study on the laparoscopic sentinel node procedure for testicular and urachus cancer we initiated a protocol for the laparoscopic sentinel node detection of the prostate (Figure X1.4). The current protocol combines the procedure with an extensive nodal dissection. No falsenegative node sentinel nodes were encountered suggesting that in the future a sentinel node procedure may be sufficient for the detection of nodal metastases in prostate cancer and an extensive node dissection will be superfluous for diagnostic purposes. 123 SURGICAL ONCOLOGY Publications (continued) Verbruggen MBM, Verheijen RHM, Van de Goot FRW, Van Beurden M, Dorsman JCP, Van Diest PJM. Serous borderline tumor of the ovary presenting with cervical lymph node involvement: a report of 3 cases. [Article]. Am J Surg Pathol 2006; 30:739-743 Woerdeman LAE, Kortmann JBJ, Hage JJ. Routine histologic examination of 728 mastectomy scars: Did it benefit our patients? Plast Reconstr Surg 2006; 118:1288-1292 Woerdeman LAE, Hage JJ, Figure XI.4: Laparoscopic sentinel node procedure of the prostate: Injection of technetium-nanocolloid Smeulders MJC, Rutgers EJT, under transrectal ultrasound (1); scintigraphy and CT-scan matching (2); intraoperative sentinel node Van der Horst CMAM. Skin-sparing detection using laparoscopic gamma-probe (3); documentation of removed lymph nodes (4). mastectomy and immediate breast reconstruction by use of implants: An Penile cancer During the last 12 years we developed the world largest experience in sentinel node biopsy in penile cancer. The results were analyzed and updated leading to a thesis by Bin Kroon jr. Prognostic significance of HPV presence was analyzed in collaboration with the department of pathology of the Free University medical center. assessment of risk factors for complications and cancer control in 120 patients. Plast Reconstr Surg 2006; 118:321-330 Wong SL, Morton DL, Thompson JF, Robot-assisted prostatectomy The introduction of the Da Vinci S surgical robot in our hospital changed the approach to prostatectomy. Within the first 5 months since its introduction over 50 robot-assisted laparoscopic prostatectomies (RALPs) were performed succesfully. Conversion to an open procedure because of intraoperative problems was never necessary and no complications for which operative intervention was required occurred. An important advantage of the da Vinci S surgical robot is the option of image integration (Figure X1.5). Intraoperative transrectal ultrasound facilitated the dissection of bladder neck and neurovascular bundles and significantly improved oncological and functional outcome. Figure XI.5: Intraoperative view of da Vinci S system presenting simultaneous transrectal ultrasound, operative view, and operative instrumentation. Gershenwald JE, Leong SPL, Reintgen DS, Gutman H, Sabel MS, Carlson GW, McMasters KM, Tyler DS, Goydos JS, Eggermont AMM, Nieweg OE, Cosimi AB, Riker AI, Coit DG. Melanoma patients with positive sentinel nodes who did not undergo completion lymphadenectomy: A multi-institutional study. Ann Surg Oncol 2006; 13:809-816 124 PSYCHOSOCIAL RESEARCH X II D IV IS ION OF PS YCHOS OCIA L RES EA R CH A ND EPID EM IOLOG Y PSYCHOSOCIAL ONCOLOGY Division head, Group leader Neil Aaronson Neil Aaronson PhD Group leader The subdivision of psychosocial oncology is pursuing three major research lines: (1) quality of life assessment in clinical research and clinical practice (Group Aaronson); (2) psychosocial issues in cancer clinical genetics (Group Aaronson); and (3) symptom perception and management (Group Van Dam). Babs Taal MD PhD Academic staff Marc Van Beurden MD PhD Academic staff Senno Verhoef MD Academic staff Eveline Bleiker PhD Post-doc Kirsten Douma MSc Graduate student Karin Gehring MSc Graduate student Doranne Hilarius MSc Graduate student Rianne Hoopman MSc Graduate student Ruud Knols MSc Graduate student Catharina Korse Msc Graduate student Chantal Lammens MSc Graduate student Joanna Madalinska MA MSc Graduate student Chad Gundy MSc Senior statistical analyst Miranda Gerritsma Research assistant Esther Janssen Research assistant Tanja Nagtegaal Research assistant Publications The use of health-related quality-of-life (HRQL) assessments in daily clinical oncology nursing practice: A community hospital-based intervention study This sequential cohort study is investigating the efficacy of incorporating standardized HRQL information as a routine part of the community hospital-based outpatient adjuvant and palliative chemotherapy treatment in terms of: (1) facilitating nurse-patient communication; and (2) increasing nursesвЂ™ awareness of their patientsвЂ™ HRQL. Additional outcomes include HRQL-related medical record notations and actions, patientsвЂ™ and nursesвЂ™ satisfaction, and patientsвЂ™ self-reported HRQL. The experimental intervention involves having patients complete a computer-assisted (touchscreen) version of the EORTC QLQ-C30 and relevant condition-specific questionnaire modules at 3 points in time. A graphic summary of the HRQL profile is provided to the responsible nurse and to the patient. In total, 11 nurses and 219 patients have been recruited into the study and data collection has been completed. Preliminary analyses indicate that the intervention had a significant, salutary effect on: (1) nurse-patient communication (mean number of HRQL topics discussed per consultation), with largest increases in the discussion of cognitive functioning, dyspnea, sleep, and GI complaints; (2) nursesвЂ™ awareness of their patientsвЂ™ level of activities of daily living, pain, and overall HRQL; (3) HRQL-related medical chart notations; and (4) HRQL-related advice-giving. Efficace F, Horneber M, Lejeune S, Van Dam F, Leering S, Rottmann M, Aaronson NK. Methodological quality of patient-reported outcome research was low in complementary and alternative medicine in oncology. J Clin Epidemiol 2006; 59 Groenvold M, Petersen MA, Aaronson NK, Arraras JI, Blazeby JM, Bottomley A, Fayers PM, de Graeff A, Hammerlid E, Kaasa S, Sprangers MA, Bjorner JB. EORTC QLQ-C15-PAL: the new standard in the assessment of health-related quality of life in advanced cancer? Palliat Med 2006; 20:59-61 Hilarius DL, Kloeg PH, Detmar SB, Muller MJ, Aaronson NK. Level of agreement between patient self-report and An empirical study of proxy viewpoint in the evaluation of health-related quality of life (HRQL) There are situations in which patient self-reported HRQL must be substituted by proxy assessments. Pickard & Knight (2005) recently delineated two proxy perspectives: the so-called вЂњproxy-patientвЂќ (proxy assessment of how a patient would assess himself) and the вЂњproxy-proxyвЂќ perspective (the proxyвЂ™s own assessment of the patient). The purpose of this study was to evaluate patientproxy concordance based on these two alternative approaches. The EORTC-QLQ-C30 was administered to a sample of 224 cancer patients and their proxies (i.e., significant others). The proxies were randomly assigned to either the вЂњproxypatientвЂќ or the вЂњproxy-proxyвЂќ perspective. Contrary to expectations, few differences between conditions were noted in percentage agreement. The only significant difference was a higher level of agreement for the вЂњproxy-proxyвЂќ condition on the role functioning scale. The cognitive functioning scale showed a significantly higher correlation in the вЂњproxy-proxyвЂќ condition, with a similar trend for the physical and role functioning scales. These results suggest that the functional scales of the QLQ-C30 may show an improvement in proxy-patient agreement if the proxy is instructed to report his own opinion rather than attempt to estimate how the patient would respond. observer ratings of health-related quality of life communication in oncology. Patient Educ Couns 2007; 65:95-100 Hoopman R, Muller MJ, Terwee CB, Aaronson NK. Translation and validation of the EORTC QLQ-C30 for use among Turkish and Moroccan ethnic minority cancer patients in the Netherlands. Eur J Cancer 2006; 42:1839-1847 Cognitive rehabilitation of glioma patients: A prospective, randomized study This multicenter, randomized trial is evaluating the effectiveness of a cognitive rehabilitation program on the neuropsychological test performance, self-reported cognitive complaints, and HRQL of patients with low-grade glioma. The rehabilitation program incorporates both a computer-based cognitive retraining component, and a compensation strategy training component. As of November, 2006, 137 patients have been randomized into the study, 119 patients have completed the immediate post-training assessment, and 82 the 6 month follow-up. first follow-up. In 200, 72 patients were recruited into the study. Patient recruitment and follow-up is ongoing. 125 PSYCHOSOCIAL RESEARCH Publications (continued) A randomized controlled trial of exercise training in hematological cancer patients after peripheral blood stem cell transplantation This RCT is being carried out by a PhD student of ours in Zurich, Switzerland. 134 adult hematologic cancer patients who have undergone peripheral blood stem cell transplantation (PBSCT) are randomly assigned to a 12-week ambulatory supervised endurance and repetitive strength exercise program (minimum 3 hours/week) or a usual care group. Primary outcomes include musculoskeletal performance (knee extension and grip strength), 6 minute walk test and 15-meter walking speed, and self-reported physical functioning. Secondary outcomes include self-reported HRQL and fatigue, self-reported and objectively assessed physical walking activity, whole body composition and hematological values. To date, 65 patients have been randomized into the study. Madalinska JB, Van Beurden M, Bleiker EM, Valdimarsdottir HB, Hollenstein J, Massuger LF, Gaarenstroom KN, Mourits MJ, Verheijen RH, Van Dorst EB, Van der Putten H, Van der Velden K, Boonstra H, Aaronson NK. The impact of hormone replacement therapy on menopausal symptoms in younger high-risk women after prophylactic salpingo-oophorectomy. J Clin Oncol 2006; 24:3576-3582 Predictors of prophylactic bilateral salpingo-oophorectomy (PBSO) versus gynecological screening (GS) update in BRCA1/2 mutation carriers As part of a prospective, multicenter study investigating quality of life, menopausal symptoms and sexual functioning among high-risk women undergoing PBSO versus GS, we examined the baseline predictors of PBSO versus GS uptake in a subset of 160 BRCA1/2 carriers. During the 12 month period following the first gynecologic consultation, 74% of women had undergone PBSO. Women with lower educational levels, with poorer general health perceptions, those who view ovarian cancer as an incurable disease, and those who believe more strongly in the benefits of surgery were found to be significantly more likely to undergo PBSO. These results emphasize the need to ensure that high-risk women are well-informed about the low predictive value of gynecologic screening techniques and about the lethal threat posed by ovarian cancer. Mols F, Vingerhoets AJJM, Coebergh JW, Vreugdenhil G, Aaronson NK, Lybeert MLM, Poll-Franse LV. Better quality of life among 10-15 year survivors of HodgkinвЂ™s lymphoma compared to 5-9 year survivors: A population-based study. Eur J Cancer 2006; 42:2794-2801 Mols F, Poll-Franse LV, Vingerhoets AJJM, Hendrikx A, Aaronson NK, Houterman S, Coebergh JWW, Essink-Bot ML. Long-term quality of life among Dutch prostate cancer survivors: Results of a population-based Long-term psychosocial impact of genetic testing among familial adenomatous polyposis (FAP) families This 4-year cross-sectional study began in November 2004, in collaboration with the Netherlands Foundation for the Detection of Hereditary Tumors (STOET; Hans Vasen). The study is investigating the mid-tolong-term (>1 yr) psychosocial impact of a (clinical or genetic) diagnoses of FAP, the social and psychosexual impact of such a diagnosis among those diagnosed before the age of 20 years, the experience with and predictors of compliance with preventive health options (endoscopic screening), and the knowledge of and attitudes toward prenatal diagnosis of FAP and pre-implantation techniques. The study will include all members of families, registered at the STOET, with a high risk for FAP who are currently 16 years of age or older (estimated n = 219 families; n = 750 family members). The partners of individuals with positive test results or clinical diagnoses are also invited to participate (estimated n = 250). Data are collected via self-report questionnaires and personal interviews. To date, questionnaires have been returned by 488 family members (response rate = 66%) and 124 partners (response rate = 84%), and 52 semi-structured interviews have been completed. Psychosocial aspects of genetic testing in families at high risk of multiple tumors at various sites and ages This multicenter study, which is being conducted in collaboration with the Family Cancer Clinic (Senno Verhoef), is investigating: (1) the uptake of genetic testing for Li-Fraumeni Syndrome (LFS) and Von HippelLindau (VHL) and the characteristics of those who do and do not undergo genetic testing; (2) the psychosocial consequences (e.g., quality of life, cancer worries, family relationships) of (not) undergoing genetic testing; (3) compliance with recommended surveillance programs and factors associated significantly with (non) compliance; and (4) the attitudes of at-risk individuals and their partners toward prenatal diagnosis of LFS/ VHL and pre-implantation genetic diagnosis. The study will include members of the 18 families known to have the Li-Fraumeni syndrome (105 adults and approximately 50 partners) and the 40 families with Von Hippel-Lindau syndrome (approximately 160 adults and 110 partners). Data are collected via questionnaire and, for a subset of respondents, via semi-structured interviews. Data collection began in August 2006 and will continue until September 2007. As of November 2006, 53 of 64 distributed questionnaires have been completed (83% response rate). study. Cancer 2006; 107:2186-2196 Scott NW, Fayers PM, Aaronson NK, Bottomley A, De Graeff A, Groenvold M, Koller M, Petersen MA, Sprangers MAG. The use of differential item functioning analyses to identify cultural differences in responses to the EORTC QLQ-C30. Qual Life Res 2006; 16:115-129 Van Luijn HEM, Aaronson NK, Keus RB, Musschenga AW. The evaluation of the risks and benefits of phase II cancer clinical trials by institutional review board (IRB) members: A case study. J Med Ethics 2006; 32:170-176 Figure XII.1: Health-related quality of life profile scores for use in daily clinical oncology practice. 126 PSYCHOSOCIAL RESEARCH SYMPTOM PERCEPTION AND MANAGEMENT Cognitive impairment of cancer chemotherapy, a multidisciplinary research line Group leader Frits Van Dam Frits Van Dam PhD Group leader Willem Boogerd MD PhD Academic staff Sanne Schagen PhD Post-doc Baudewijntje Kreukels MSc Graduate student Christien Schilder MSc Graduate student Chad Gundy MSc Senior statistical analyst Anky Caspers Research assistant Tinneke Danhof Research assistant Elke Eggens Research assistant Suzanne Leering Research assistant Annemarie Van Nuland Research assistant Marion Weevers Research assistant A prospective randomized controlled study on the effects of chemotherapy for breast cancer We continued the analyses of our longitudinal randomized study with a group of high-risk breast cancer patients that received either adjuvant high-dose CTC chemotherapy (n=28) or standard-dose FEC (n=39) chemotherapy. The patients were tested neuropsychologically before the start of chemotherapy and one year after this first assessment. The results of these patients were compared to the results of a group of breast cancer patients not treated with chemotherapy (n=57), tested with a similar time interval, and to the results of a group of matched healthy controls (n=60), which also underwent repeated neuropsychological testing. We are currently analyzing a second follow-up assessment for all patients, which took place 6 months after the first follow-up. We also asked the proxies of all patients to assess the patientsвЂ™ cognitive status and compared this with patientsвЂ™ own assessment of her cognitive status. 6 months after completion of chemotherapy, there is a high concordance between the patientsвЂ™ and the proxies ratings. Currently, we are studying changes in cognitive complaints reported by patients and their proxies over time, i.e. from pre-chemotherapy to post treatment. Animal studies on the effects of cytotoxic agents on cognition In follow-up to our experiments in which rats were injected with singles dosages of intraveneous MTX (which showed a significant reduction in cell proliferation in the hippocampus, with a clear dose response relation, which was also reflected in their cognitive behavior), we are currently investigating whether other cytotoxic agents cause similar effects on neurogenesis. 5-FU and cyclophosphamide are the first agents to be studied. Effects of endocrine treatment on cognitive functioning A prospective comparative study into the neuropsychological sequelae of postmenopausal breast cancer patients receiving exemestane or tamoxifen compared to a healthy group of women matched for menopausal status and age is conducted since 2003. Patients are recruited from the TEAM trial; an open label, randomized multicenter study of either exemestane treatment (25 mg once daily) or 2-3 years adjuvant tamoxifen (20 mg once daily) followed by 3-2 years exemestane treatment (25 mg once daily). Patients are being assessed twice: before receiving tamoxifen or exemestane and after 12 months of treatment. Healthy controls are assessed with the same time interval. 38 hospitals are participating in this study. At the moment, patient inclusion is complete: 206 patients and 124 healthy controls are included in the study. The collection of follow-up data will be complete in March 2007. Figure XII.2: Spatial learning in the Morris water maze. Used to study the effects of cytotoxic agents on the cognitive function of rats. Effects of AC-chemotherapy, followed by endocrine treatment on cognitive functioning We conducted a cross-sectional study in a group of postmenopausal breast cancer patients that received endocrine therapy after they completed AC-chemotherapy in the adjuvant setting. Patients were recruited from the TEAM-trial and received either exemestane treatment (AC-EXE group; n=51; mean age = 58,5) or tamoxifen treatment (AC-TAM group, n=30; mean age = 57,9). The patients were tested neuropsychologically on average 2.2 years after completion of AC-chemotherapy. The results of these patients were compared to the results of a group of healthy controls (mean age 60,2; n=48), matched on age and IQ. In both the AC-TAM group as in the AC-EXE group more women reported memory problems in daily life than in the group of healthy controls (resp. 27,6% and 25,5 % versus 6,3%, p=.02). No difference between tamoxifen users and exemestane users with respect to experienced cognitive problems was found. On the neuropsychological measures, both the AC-TAM as the AC-EXE group had significantly lower mean scores on tests of вЂ�information processing speedвЂ™ than healthy controls (p=.023 and .008). In addition, tamoxifen users scored significantly lower on a вЂ�mental flexibilityвЂ™ test 127 PSYCHOSOCIAL RESEARCH Publications (p=.007) and a вЂ�category fluencyвЂ™ test (p=.000) than healthy controls. Tamoxifen-users tend to score worse than exemestane users on several tests, particularly attention tests, a letter fluency test, a verbal memory test and a visual association test. Furthermore, no correlations were found between reported cognitive problems in daily life and cognitive test scores, but cognitive problems in daily life were correlated with fatigue and symptoms of anxiety and depression. Kreukels BPC, Schagen SB, Ridderinkhof KR, Boogerd W, Hamburger HL, Muller MJ, Van Dam FSAM, Schagen SB. Effects of high-dose and conventional-dose adjuvant chemotherapy on long-term Cognitive functioning and endocrine therapy for prostate cancer Endocrine therapy plays an increasing role in the treatment of prostate cancer. Since testosterone seems to influence cognitive functioning, like visuo-spatial functioning and probably also memory, it has been suggested that endocrine therapy for prostate cancer may have an effect on cognitive functioning as well. We performed a cross-sectional study in which the cognitive functioning of patients with localised prostate cancer (n=25) currently undergoing neo-adjuvant endocrine treatment with LHRH agonists was examined using standardized neuropsychological tests. The cognitive performance of these patients was compared with that of prostate cancer patients who had been treated with brachytherapy (n=32) in the last three years. All patients were also interviewed about cognitive problems, health-related quality of life, emotional distress and prostate cancer specific symptoms. A significant higher percentage of patients undergoing endocrine treatment reported cognitive problems compared to the patients previously treated with brachytherapy. Compared with the brachytherapy group, the endocrine therapy group also reported significantly more feelings of emotional distress, more prostate cancer specific symptoms, and they indicated more problems regarding several health-related quality of life aspects. The neuropsychological results showed that 36% of the patients undergoing endocrine treatment were classified as cognitively impaired, compared with 6% of the brachytherapy patients (p=.027). For the brachytherapy patients, time since treatment had no influence on the reported complaints or on the test performance. A larger study with a longer follow-up, that would also include a post endocrine therapy assessment, should point out whether this association between of cognitive impairment and endocrine treatment still holds. Complementary and Alternative Medicine (CAM) A substantial number of patients use one or more alternative treatments in addition to their regular cancer treatment. From surveys in the Netherlands Cancer Institute over more than twenty years with more than 3,500 patients, we noticed that the number of patients using CAM has decreased dramatically over the last years. E.g. the number of patients using вЂ�anti-cancerвЂ™ diets has dropped from about 13% in 1999 to less than 2% in 2006. External factors like media attention for patients who have died because of using solely CAM and the downfall of important alternative therapists seem to have contributed to this decline in the number of patients using CAM. We plan to continue our yearly inventory of the number of patients using CAM. cognitive sequelae in patients with breast cancer: An electrophysiologic study. Clin Breast Cancer 2006; 7:67-78 Schagen SB, Muller MJ, Boogerd W, Mellenbergh GJ, Van Dam FS. Change in cognitive function after chemotherapy: a prospective longitudinal study in breast cancer patients. J Natl Cancer Inst 2006; 98:1742-1745 128 PSYCHOSOCIAL RESEARCH EPIDEMIOLOGY The cancer epidemiology group is currently concentrating on two principal research lines: (1) the etiology of hormone-related cancers; (2) the long-term health consequences of cancer treatment, particularly in terms of the risk of developing second malignancies or cardiovascular disease. Group leader Flora Van Leeuwen Group leader Matti Rookus Flora Van Leeuwen PhD Group leader Matti Rookus PhD Group leader Berthe Aleman MD Academic staff Petra Nederlof PhD Academic staff Nicola Russell PhD Academic staff Emiel Rutgers MD, PhD Academic staff Laura Van вЂ™t Veer PhD Academic staff Senno Verhoef PhD Academic staff Marieke De Bruin PhD Post-doc Lisette Hoogendoorn PhD Post-doc Peggy Manders PhD Post-doc Ina Mulder PhD Post-doc Marjanka Schmidt PhD Post-doc Eric Vermeulen PhD Post-doc Dorien Voskuil PhD Post-doc Richard Brohet MSc Graduate student Mathilde Cardous-Ubbink MSc Senior Graduate student Maartje Hooning MD Graduate student Agnes Olde Damink-Van Rosmalen MSc Graduate student Anouk Pijpe MSc Graduate student Sandra Van den Belt-Dusebout MSc Graduate student Janneke Verloop MSc Graduate student Alina Vrieling MSc Graduate student Willem Klokman MD MSc Senior Statistical analyst Thea Mooij MSc Statistical analyst Gijs Besseling MSc Research assistant Ivo Bisschops Research assistant Steven De Jager Research assistant Geri De Leeuw-Mantel Research assistant Simone Dijkhuis MSc Research assistant Bregje Huisman MSc Research assistant Esther Janssen Research assistant Kiki Jeanson MSc Research assistant Kim Karsenberg MSc Research assistant Marianne Kuenen Research assistant Monica Legdeur Research assistant Gabey Ouwens Research assistant Risk factors for hormone-related cancer In our nationwide cohort study in families with a BRCA1/2 mutation (GEO-HEBON study), we are studying 1) whether hormonal/life-style factors modify cancer risk in BRCA1/2 families, 2) the agespecific cumulative risks of breast, ovarian and other cancers based on full pedigree information. We are now expanding the existing cohort and we also started to collect follow-up information from women who were tested for a BRCA1/2 mutation and were already included in the study in 1996-2000. Presently, the cohort comprises 616 BRCA1 families and 175 BRCA2 families with 37,912 and 11,001 family members, respectively. We received risk factor questionnaires from 3,633 family members and follow-up questionnaires from 457 women who had been tested in the past. To obtain sufficient power for the assessment of gene-environment interactions, our data are incorporated in an international cohort study of BRCA1/2 carriers (IBCCS). In a retrospective cohort of the first 1,601 BRCA1/2 carriers we examined the impact of very low dose diagnostic radiation. Any reported exposure to chest X-rays was associated with an increased risk of breast cancer (HR=1.54). The increased risk was found among carrier women aged 40 and younger (HR=1.97), and in women born after 1949 (HR=2.56). Especially, women exposed only before age 20 showed an increased risk (HR=4.64). Thus, in our series of BRCA carriers we detected a relatively large effect on breast cancer risk with a level of radiation exposure that is at least an order of magnitude lower than in previously studied cohorts exposed to diagnostic radiation. Although part of this increase may be attributable to recall bias, the observed patterns of risk in terms of age at exposure and attained age are consistent with those found in previous studies. If confirmed, the results have important implications for the use of X-ray imaging in young BRCA1/2 carriers. In another IBCCS analysis we examined endogenous hormonal factors. Breast cancer risk was not significantly related to age at menarche nor age at menopause. There was evidence of a protective effect of oophorectomy (HR 0.56) and a significant trend of decreasing risk with increasing time since oophorectomy, but no apparent effect of natural menopause. In 2006, we received funding to expand our cohort of 19,213 women who received ovarian stimulation for in vitro fertilization (IVF) in the period 1983-1995. We will include 8800 women who received IVF more recently (1995-1998) to assess the risk of hormone-related cancers. The control group of 6,017 women will also be expanded (collaboration with CW Burger, Erasmus MC Rotterdam) and follow-up of the full cohort (n=38,000) will be extended. In 2006 we continued data collection for our cohort study on hormone-related cancer risk in women who were exposed to diethylstilbestrol (DES) in utero (DES daughters). In total, 8,557 DES daughters returned a risk factor questionnaire. We are validating 4,520 reported health problems of 3,046 women with clinical medical records. The Epidemiology group is also closely involved in three intervention studies (using lycopene and isoflavone supplements) focusing on the insulin-like growth factor system in relation to breast cancer and colorectal carcinogenesis (see Division Experimental Therapy). Overweight and physical activity as determinants of (second) cancer risk and cancer prognosis are being studied in several projects. Several systematic reviews have been conducted of the epidemiological literature on physical activity and risk of breast, colorectal, and endometrial cancer. We concluded that physical activity decreases the risk of breast cancer, especially in postmenopausal women, and possibly also the risk of cancer of the colon and endometrium. Currently, several pilot studies are being conducted to assess the frequency of overweight and (unintentional) weight gain in breast cancer patients after diagnosis, and to evaluate a combined diet, physical activity and behavioral counseling intervention on weight loss in overweight breast cancer patients after primary treatment. 129 EPIDEMIOLOGY Late effects of cancer treatment Now that curative treatment is available for a substantial group of cancer patients, it is increasingly important to evaluate how the occurrence of late complications of treatment affects their long-term survival. We aim to evaluate the risk of second cancers and cardiovascular disease (CVD) after radioand chemotherapy (RT and CT) for HodgkinвЂ™s lymphoma (HL) (n=3,400), testicular cancer (n=2,707) and breast cancer (n=8,000) over a period of up to 30 years after primary treatment. It has been shown that CVD causes excess mortality in survivors of HL. However, few studies have evaluated the long-term incidence of CVD and quantified the effects of RT and CT. We assessed CVD risk in 1,474 5-year survivors of HL treated before the age of 41 years in the NKI between 1966 and 1995 (median follow-up time, 18.7 years). In all, 233 patients had developed one or more coronary heart diseases while only 58 were expected (standardized incidence ratio (SIR)=4.0). SIRs were strongly elevated for myocardial infarction (MI) (SIR=3.6), angina pectoris (SIR=4.1) and congestive heart failure (HF) (SIR=4.9). The SIRs remained increased with longer follow-up; e.g. for MI, the SIR was 4.0 in the 5-25 year follow-up interval and 2.9 among 25-year survivors. Mediastinal radiotherapy significantly increased the risks of MI, angina pectoris, HF and valvular disorders (2- to 7-fold). Anthracyclines significantly added to the elevated risks of HF and valvular disorders from mediastinal RT (Hazard Ratios (HRs): 2.8 and 2.1, respectively). We also analyzed second malignancy risk in 1155 women treated for HL before age 51. After a median follow-up of 18.2 years we observed 100 cases of second breast cancer (SIR 5.4). Breast cancer risk remained increased up to 30 years after treatment (SIR 8.7). Patients treated before age 21 experienced the highest risk (SIR 16.9), resulting in a cumulative risk of 21% of developing breast cancer before age 45. Patients who received RT including the breast area had a 5-fold increased risk for breast cancer compared to those who received no such irradiation. Among these patients, both chemotherapy and radiotherapy to the ovaries decreased risks for BC (HR 0.5 and 0.3, respectively), probably because these gonadotoxic therapies induce premature menopause. Gene-expression analysis of breast cancer after HL suggests a distinct molecular profile of radiation-induced breast cancer (see Division VIII). Monique Simons MSc Research assistant Renate Udo Research assistant Josette Van As MSc Research assistant Anne Van Dijk Research assistantt Publications Aleman BMP, Van den Belt-Dusebout AW, De Bruin ML, Van вЂ�t Veer MB, Baaijens MH, De Boer JP, Hart AA, Klokman WJ, Kuenen MA, Ouwens GM, Bartelink H, Van Leeuwen FE. Late cardiotoxicity after treatment for HodgkinвЂ™s lymphoma. Blood 2006;[Epub ahead of print] Andrieu N, Easton DF, Chang-Claude J, Rookus MA, Brohet R, Cardis E, Antoniou AC, Wagner T, Simard J, Evans G, Peock S, Fricker JP, Nogues C, Van вЂ�t Veer L, Van Leeuwen FE, Goldgar DE. Effect of chest X-rays on the risk of breast cancer among BRCA1/2 mutation carriers in the international BRCA1/2 carrier cohort study: a report from the EMBRACE, GENEPSO, GEOHEBON, and IBCCS CollaboratorsвЂ™ Group. J Clin Oncol 2006; 24:3361-3366 Andrieu N, Goldgar DE, Easton DF, Rookus M, Brohet R, Antoniou AC, Peock S, Evans G, Eccles D, Douglas F, Nogues C, Gauthier-Villars M, Chompret A, Van Leeuwen FE, Kluijt I, Benitez J, Arver B, Olah E, Chang-Claude J. Pregnancies, breastfeeding, and breast cancer risk in the International BRCA1/2 Carrier Cohort Study (IBCCS). J Natl Cancer Inst 2006; 98:535-544 Hooning MJ, Aleman BMP, Van Figure XII.3: Risk of breast cancer after HodgkinвЂ™s lymphoma by follow-up time. Based on 1155 female Rosmalen AJ, Kuenen MA, Klijn JG, survivors, age at diagnosis <50 yr, 1966-1995 (median follow-up: 18 years) Van Leeuwen FE. Cause-specific mortality in long-term survivors of breast cancer: The effects of treatment on premature menopause were studied separately in 518 female 5-year HL-survivors, younger than 40 years at treatment. Chemotherapy was associated with a 12-fold increased risk for premature menopause (HR 12.2). Increased risks were only found for alkylating agents (HR 14.4), especially procarbazine (HR 12.2) and cyclophosphamide (HR 3.4). The risk associated with procarbazine was dose-related and independent of the effect of mechlorethamine. Per additional 1.4 g/m2 of cumulative exposure to procarbazine (which is equal to the amount given in one cycle of MOPP or two cycles of MOPP/ABV) the risk of menopause increased with 25%. A 25-year follow-up study. Int J Radiat Our study on late effects of testicular cancer treatment focuses on the long-term risk of second malignancies (SMNs) following radiation and the risk of cardiovascular disease (CVD) after chemotherapy (CT). The nationwide cohort comprises 2,707 5-year testicular cancer survivors treated between 1965 and 1995. Monninkhof EM, Elias SG, Vlems FA, Oncol Biol Phys 2006; 64:1081-1091 Hooning MJ, Dorresteijn LD, Aleman BMP, Kappelle AC, Klijn JG, Boogerd W, Van Leeuwen FE. Decreased risk of stroke among 10-year survivors of breast cancer. J Clin Oncol 2006; 24:5388-5394 Van der Tweel I, Schuit AJ, Voskuil DW, Van Leeuwen FE. Physical Activity and Breast Cancer: A Systematic Review. Epidemiology 2007; 18:137-157 130 EPIDEMIOLOGY Publications (continued) Moser EC, Noordijk EM, Van Leeuwen FE, Le Cessie S, Baars JW, Thomas J, Carde P, Meerwaldt JH, Van Glabbeke M, Kluin-Nelemans HC. Long-term risk of cardiovascular disease after treatment for aggressive non-Hodgkin lymphoma. Blood 2006; 107:2912-2919 Schmidt MK, Tollenaar RA, De Kemp SR, Broeks A, Cornelisse CJ, Smit VT, Peterse JL, Van Leeuwen FE, Van вЂ�t Veer LJ. Breast Cancer Survival and Tumor Characteristics in Premenopausal Women Carrying the CHEK2*1100delC Germline Mutation. J Clin Oncol 2007; 25:64-69 Van den Belt-Dusebout AW, Nuver J, De Wit R, Gietema JA, Ten Bokkel Huinink WW, Rodrigus PT, Schimmel EC, Aleman BMP, Van Leeuwen FE. Long-term risk of cardiovascular disease in 5-year survivors of testicular cancer. J Clin Oncol 2006; 24:467-475 Figure XII.4: Cumulative risk of cardiovascular disease after breast cancer by radiotherapy (yes/no) and period (<1980, в‰Ґ1980) Figure XII.5: Uterine corpus cancer (UCC)-specific survival according to use of tamoxifen for breast cancer. Based on 642 women with uterine corpus cancer after breast cancer. After a median follow-up of 18 years, we observed 287 SMNs and 357 CVDs of interest (the most important diagnoses acute myocardial infarction (MI), angina pectoris (AP) and congestive heart failure (CHF)). The risk of SMN overall was 1.7fold increased with an AER of 32.3 excess cases per 10,000 person-years and the risk of CVD was 1.2-fold increased compared with the general population. Tumors from the digestive and genitourinary tract contributed most to the absolute excess risk of SMNs. The risk of SMN was 2.5-fold increased after subdiaphragmatic radiotherapy and 1.8-fold increased after chemotherapy, compared with surgery only. We also analyzed the risk of developing one of multiple adverse outcomes. The risk of developing SMN or CVD was 1.7-fold increased after subdiaphragmatic radiotherapy and was significantly increased (1.7-fold) after chemotherapy. A 1.7-fold increased risk of SMN or CVD was also observed after smoking. We conclude that radiotherapy strongly increases the risk of SMNs but not of CVD, while chemotherapy slightly increases the risks of both SMNs and CVDs. In 2006, we assessed the incidence of CVD in 1601 patients with T1-2N0 breast cancer treated with postlumpectomy irradiation restricted to tangential fields in 5 different Dutch hospitals between 1980 and 1993. Patients treated with radiation fields other than breast tangentials and those treated with adjuvant chemotherapy were excluded. For patients with left-sided breast cancer maximum heart distance (MHD) was measured by two observers on the original simulator films as a proxy for irradiated heart volume. Follow-up was complete for 94% of the patients and median follow-up was 16 years. The incidence of CVD was 16% in patients with left-sided breast cancer versus 11.6% in right-sided cases. The hazard ratio (HR) associated with left-sided versus rightsided breast cancer was significantly increased for ischemic heart disease (HR, 1.41), and other heart disease (HR, 1.53). The risk of CVD did not significantly increase with increasing MHD. Possibly, the higher incidence of CVD in patients irradiated with tangential fields for left-sided breast cancer can be explained by radiation dose-volume aspects or a higher radiation dose to crucial coronary arteries. In a previous study on tamoxifenвЂ™s late effects, we showed that the risk of poorprognosis uterine corpus cancers increased with duration of tamoxifen treatment for breast cancer. We are now conducting a new nationwide study to confirm these findings and to also obtain insight into the mechanism whereby tamoxifen causes uterine corpus cancer. We are investigating whether the clinicopathologic characteristics and prognosis of uterine corpus cancers differ between women with and without long-term tamoxifen treatment. We are also searching for tamoxifenspecific genomic aberrations and differences in gene-expression. Our existing cohort of 309 patients with uterine corpus cancer following breast cancer was expanded with 533 of such patients diagnosed since 1996. Information about breast cancer treatment and follow-up was abstracted from the medical records. Tissue blocks of 98% of uterine corpus cancers were obtained to review histology and to examine genomic aberrations. For 200 prospective cases we also collected fresh-frozen tissue. The 3-year uterine corpus cancer-specific survival was significantly worse for long-term tamoxifen users than for non-users (82% for в‰Ґ2 yr tamoxifen users vs. 93% for non-users). These results confirm our previous findings. Genomic and gene-expression analysis are ongoing. In the Netherlands the law may be changing regarding secondary use of biological samples obtained for diagnostic or therapeutic purposes. At present, secondary use for research is allowed when patients have not objected to such use after having been informed about the possibility of secondary use of samples in a general patient information leaflet. The new law may include written informed consent, with no clarity as to whether the permission can apply to a broad research area. Clearly, this would heavily impact on future research with biological samples. The Cancer Genomics Center funded a study to evaluate various options for asking informed consent. The research aims to find out whether patients give consent for research with their remaining tissue, what information needs of patients are, what kind of decision procedure patients prefer and which choice options they would want to have. Data collections begins at the end of 2006. 131 PSYCHOSOCIAL RESEARCH QUALITY OF ONCOLOGIC CARE Constructive Technology Assessment of the introduction of a 70-gene micro array prognostic test in breast cancer treatment From 2003 to the end of 2006, a study was conducted on the feasibility of introducing a 70-gene microarray test as a prognostic tool in the treatment of node negative breast cancer (the RASTER-study). As the diffusion of this technology is in a very early stage and the course of development is difficult to predict, an evaluation approach, constructive technology assessment (CTA), was chosen that takes the technology dynamics into account. In total, 789 patients from 16 hospitals were recruited into the study, of whom 417 were tested. Preliminary results show that approximately 30% of patients receive treatment advise based on the microarray test that is based on traditional clinical guidelines (discordant cases). Additionally, it was observed that, on average, it takes 6 months for a hospital to decide to make use of and to implement this new technology. Pre- and post introduction measurements concerning logistics, teamwork and patient centeredness have been performed in 2006 and will continue in 2007. A proposal has been prepared to continue the CTA as a side study of the MINDACT study, a large trial in which in one arm the discordant cases are randomised. The proposal includes quality-related aspects such as timeliness, safety and patient centeredness, a cost-effectiveness analysis (in cooperation with MGZ-Rotterdam), a study on legalethical aspects of patients rights concerning banked tissues, and scenario-use as a means to guide implementation processes. Rehabilitation, Physical Activity and Cancer In recent years it has become evident that survivorship care and rehabilitation requires greater attention than it has received to date. In 2006, we have worked on developing a research infrastructure for future studies studies in this area. In collaboration with the Slotervaart Hospital and the Jan van Breemen rehabilitation clinic, a multidisciplinary rehabilitation program was initiated for breast cancer survivors receiving adjuvant treatment. To date, pre- and post measurements of the pilot groups have indicated that there is sufficient improvement in quality of life and functional health to embark on a more formal randomized clinical trial to investigate the programвЂ™s clinical effectiveness. Additionally, a (retrospective) needs assessment survey was performed among 109 breast cancer survivors, indicating that between 20% and 30% of survivors expressed a desire for rehabilitation activities to improve physical and psychological well being, and to reduce fatigue. Finally, a pilot, feasibility study has been initiated on вЂњActive LifestyleвЂќ during chemotherapy. This approach has been demonstrated to be effective in other patient populations. This also includes a literature review of theoretical models used in such interventions with cancer patients. Group leader Wim van Harten Wim van Harten MD PhD Group leader Jolien Bueno De Mesquita MD Research staff Rutger Dahmen MD Academic staff Miranda Van Duijn MSc Academic staff Kirsten Douma MSc Graduate student Nienke Ballast Research assistant Inge Haenen Research assistant Kim Karsenberg MSc Research assistant Wineke Van Lent MSc Research assistant Maaike Van Vliet Research assistant Publications Van Luenen HGAM, Van Harten WH. Quality Management and Stimulation of Technology Transfer in a Research Institute. Creat Innov Manag 2006; 15:207-217 Van der Ploeg H, Streppel KRM, Van Der Beek AJ, Van Der Woude LHV, Vollenbroek-Hutten MMR, Van Harten WH, Van Mechelen W. Counselling increases physical activity behaviour nine weeks after rehabilitation. Brit J Sports Med 2006; 40:223-229 132 DIAGNOSTIC ONCOLOGY X III D IV IS ION OF DIA G NOS TIC ONCOLOG Y DEPARTMENT OF CLINICAL CHEMISTRY Pharmacological studies in mice Division head, Group leader Marc Van de Vijver Nienke Van den Brink-de Vries, Tessa Buckle, Jin Zhao, Roos Oostendorp, Laura Donga, Olaf Van Tellingen DEPARTMENT OF CLINICAL CHEMISTRY Willem Nooijen PhD Academic Staff Hans Bonfrer PhD Academic Staff Olaf Van Tellingen PhD Academic Staff Tessa Buckle Technical staff Tiny Korse Technical staff DorothГ© Linders Technical staff Marian Buning Technical staff Nienke Van den Brink-de Vries Graduate student DEPARTMENT OF NUCLEAR MEDICINE Cornelis A Hoefnagel MD PhD Academic Staff Philippe Baars MD Academic Staff Saar Muller PhD Academic Staff Michiel Sinaasappel PhD Academic Staff Ferida Sivro-Prndelj MD Academic Staff Renato ValdГ©s Olmos MD PhD Academic Staff Remco Knol MD Registrar Lenka Pereira-Arias MD Registrar Albert Lafeber MD Registrar Marina Kartachova MD Clinical Fellow Saskia Baank Technical Staff Martine Bakker Technical Staff Carolien Beers-Bauhuis Technical Staff Natascha Bruin Technical Staff Petra Doodeman Technical Staff Christel Feenstra Technical Staff Bert Pool Technical Staff Lyandra Rooze Technical Staff Mariska Sonneborn Technical Staff DEPARTMENT OF PATHOLOGY Frans Hogervorst PhD Academic Staff Daphne De Jong MD PhD Academic Staff Petra Nederlof PhD Academic Staff RenГ©e Van Pel MD Academic Staff Hans Peterse MD Academic Staff Hester Van Boven MD PhD Academic Staff Marc Van de Vijver MD PhD Academic Staff, Head Division XIII Loes Van Velthuysen MD PhD Academic Staff Laura Van вЂ™t Veer PhD Academic Staff Jelle Wesseling MD PhD Academic Staff Limited blood-brain barrier (BBB) penetration of drugs is generally considered to be a major cause of the overall lack of efficacy of systemic chemotherapy against intracranial malignancies. We and others have shown the important role of drug efflux pumps (P-gp and BCRP) at the BBB. We have now shown that these transporters also limit the BBB penetration of temozolomide (TMZ), the first and only drug with proven activity against malignant glioma. This finding was not expected, since there is a general consensus that this activity is based on the good BBB penetrating properties of this agent. In vitro experiments clearly demonstrated TMZ vectorial translocation by MDCKBcrp1 cells, but not by LLC-Mdr1a or LLC-MDR1 cells, suggesting that BCRP but not P-gp is capable of transporting TMZ. Subsequently, we have studied the brain penetration in wild-type (WT), Bcrp1 KO, Mdr1a/Mdr1b DKO and Mdr1a/Mdr1b/ Bcrp1TKO mice. We found that the accumulation of TMZ in the brain was about 20% higher in KO and DKO mice and about 50% higher in TKO mice compared to wild-type controls. Thus, both P-gp and BCRP are working together in limiting the brain penetration of TMZ. Interestingly, the enhanced brain penetration was achieved without reducing the plasma clearance of this agent. Although the gain in brain penetration of TMZ was more modest than the 10-fold or higher increases previously shown for other drugs, we expect that a 50% enhancement of a drug that is already active against malignant glioma may substantially further improve its clinical efficacy. We are currently generating TKO nude mice that will be used for efficacy studies against intracranial xenografts. More or less similar results have been observed with the tyrosine kinase inhibitors for EGFR (erlotinib) and the PDGFR (imatinib). Both novel molecularly targeted agents, primarily developed for other (more common) tumor types, also interfere in pathways involved in malignant glioma. Therefore, they are being tested in malignant glioma patients. Our studies show markedly (3 to 4-fold) increased brain levels in DKO mice, which was further enhanced in TKO mice. Thus both P-gp and BCRP limit the BBB penetration of these agents. These results highlight the necessity to inhibit drug efflux pumps at the BBB in order to achieve adequate drug delivery of agents directed at potentially drugable targets in malignant glioma. In order to study such novel compounds, better brain tumor models than traditional xenograft models are essential. We have been successful in the development of spontaneous models of malignant glioma, based on Cre-Lox conditional mouse models. We have generated a lentiviral vector containing truncated GFAP promotordriven Cre recombinase that gives rise to grade 3 astrocytomas after stereotactic intracranial injection in compound conditional kRAS transgenic, Ink4a/Arf knockout animals. When either PTEN or P53 conditional genes are also deleted the picture shifts to grade 4 astrocytoma (Glioblastoma multiforme). Concurrent switching of conditional luciferase allows for non-invasive monitoring, which is an essential feature for performing intervention studies. We are currently characterizing these tumors in more detail. Douwe Atsma Technical Staff Jolien Bueno de Mesquita Graduate Student Novel strategies for biomarker discovery using MALDI-MS based proteomics tools Stella Mook Graduate Student DorothГ© Linders, Olaf Van Tellingen, Hans Bonfrer, Wim Nooijen, Theo Luider, Lennard Dekker, Marie-Christine Hermus Technical Staff Markus Meyer, Dagmar Niemeyer Lucie Boerrigter-Barendsen Technical Staff Aafke Wieringa-Ariaens Technical Staff Henrique Ruijter-Schippers Technical Staff Marjanka Schmidt PhD Post-doc Carla Schippers-Gillissen Technical Staff In collaboration with investigators from the Erasmus University and Bruker Daltronics we have undertaken a study to identify potential serum tumor markers from a group of sarcoma patients using ClinProtВ® beads technology combined with 133 DIAGNOSTIC ONCOLOGY MALDI-MS detection. We compared 80 controls versus 167 samples from patients with malignancy using weak cat ion exchange beads to reduce the sample complexity. We were able to show one differentially expressed peak at m/z of 8938, identified by MALDI-MS/MS as C3a anaphylotoxin, a fragment of complement C3 precursor. C3a anaphylotoxin has been identified in other tumor types as well and is a reactive protein and not a genuine tumor-related product. This repeated finding of fragments of moderately to high-abundant reactive proteins show one of the major shortcoming of techniques like SELDI-TOF or MALDI-TOF for biomarker discovery. In serum about 20 proteins make up about 99% of the proteome. Tumor markers such as prostate specific antigen (PSA) or human chorionic gonadotropin ( HCG) are present at much lower quantities. Moreover, the molecular weight of most intact proteins is too high to allow sensitive detection by MALDI-based mass spectrometry as this technique is more suited for peptides with sizes ranging up to 10 kDa. We expect that it will be necessary to invest in sample pretreatment by including comprehensive fractionation to reduce sample complexity and by digestion of proteins to yield peptides of shorter lengths. However, such complex sample handling procedures will destroy quantitative information, making differential profiling by direct sample comparison impossible. We are now trying to address this quantitative issue by implementing a technique called Isotope Coded Protein Labeling (ICPL). In ICPL, protein samples are first derivatised with a primary amine-specific agent (nicotinoyloxy succinimide; Nic) that will covalently attach to the amino acid (AA) of lysines and to the N-terminal AA of the peptide. This agent is available in two isoforms, with 4 deuterium atoms (heavy; d4-Nic) or 4 hydrogen atoms (light; d0-Nic) in the nicotinoyl-ring. The reference sample is derivatised with d4-Nic and the unknown with d0-Nic. Next, the unknown and the reference sample are mixed and can be subjected to extensive pretreatment without loosing quantitative information because all labeled proteins in the reference sample will act as internal standard for those in the unknown sample. They are chemically identical but differ 4 Da in mass per molecule of Nic. After digestion and further reduction of the sample complexity, MALDI-MS will visualize labeled peptides as peak pairs differing 4 Da in mass per substituted Nic moiety, where the peak area ratio of the peaks provide information on the relative quantity of the peptide in the samples. We are currently trying to show the potential applicability of this technique for biomarker discovery by searching for known tumor markers. DEPARTMENT OF PATHOLOGY (Continued) Ivon Tielen Technical Staff Dennis Veldhuizen Technical Staff Tin Wu Technical Staff THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC Frans Hogervorst PhD Academic Staff Laura Van вЂ™t Veer PhD Academic Staff Senno Verhoef MD PhD Academic Staff Irma Kluijt MD Academic Staff Priscilla Axwijk Academic Staff Petra Nederlof Academic staff Marieke Bronk Genetic associate Anja Van Rens Genetic associate Gea Wigbout Genetic associate Mohamed Achachah Technical staff Rob Plug Technical staff-Quality staff Roelof Pruntel Technical staff Paul Van der Voort Technical staff Majella Boutmy-de Lange Technical staff Aafke Wieringa-Arieaens Technical Staff DEPARTMENT OF RADIOLOGY Jelle Teertstra MD Academic Staff Peter Besnard MD Academic Staff Mesomark, novel marker for mesothelioma Kenneth Gilhuijs PhD Academic Staff Hans Bonfrer, Michel Van den Heuvel, Catharina Korse, Paul Baas Wim Koops MD Academic Staff Robert KrГ¶ger MD Academic Staff Mesothelin is a 40 kD cell surface glycosylated phosphatidyl-inositol anchored glycoprotein. Mesothelin is expressed by normal mesothelial cells but highly overexpressed in tumors such as malignant mesothelioma, ovarian carcinoma and lung carcinoma. An assay has been developed to measure soluble mesothelin related peptides (SMRPs) in blood. Increased levels of SMRPs have been found in serum of patients with malignant mesothelioma and ovarian carcinoma. We studied retrospectively the concentration of SMRPs by the Mesomark assay (CisBio, France) in samples from patients with lung cancer. We compared the results with Carcino Embryonic Antigen (CEA) and Cytokeratin 19 fragments (Cyfra) concentrations. The latter assays are more established markers for this tumor type. The median level of SMRP in a healthy group of 50 was 0.56 nmol/l (95% 1.2 nmol/l). In mesotheliomas a median of 1.54 nmol/l was found (p<0.01). The median Cyfra concentration in the mesothelioma group of 74 patients was 2.93 Вµg/l compared to 0.71 in the healthy population. (The 95% reference level is 1.9 Вµg/l.) We also measured CEA in the mesothelioma patients and as we expected, in all (but one) the CEA concentration was normal. Drawing ROC curves revealed that the best discriminating power to diagnose mesothelioma in a group of patients with NSCLC was a combination of Mesomark and CEA. Claudette Loo MD Academic Staff Saar Muller PhD Academic Staff Frank Pameijer MD PhD Academic Staff Warner Prevoo MD Academic Staff Michiel Sinaasappel PhD Academic Staff Tanja Alderliesten PhD Academic Staff Fijs Van Leeuwen PhD Academic Staff Christian Siedschlag PhD Academic Staff William Klein Zeggelink Graduate Student Miret Emanuel Technical Staff Anita Paape Technical Staff Kenneth Pengel Technical Staff Angelique Schlief Technical Staff Marja Van Vliet Technical Staff 134 DIAGNOSTIC ONCOLOGY Publications Technology insight: Tuning into the genetic Novaria-study The effect of hormonal replacement therapy on menopausal complaints related to biochemical changes in surgically and naturally postmenopausal women orchestra using microarrays - Limitations Catharina Korse, Marc Van Beurden, Katja Gaarenstroom, Hans Bonfrer, Matti Rookus, of DNA microarrays in clinical practice. Neil Aaronson Abdullah-Sayani A, Bueno de Mesquita JM, Van de Vijver MJ. Nat Clin Pract Oncol 2006; 3:501-516 Adler AS, Lin M, Horlings H, Nuyten DSA, Van de Vijver MJ, Chang HY. Genetic regulators of large-scale transcriptional signatures in cancer. Nat Genet 2006; 38:421-430 Alderliesten T, Schlief A, Peterse J, Teertstra H, Muller S, Gilhuijs K. Validation of Semiautomatic Measurement of the Extent of Breast Tumors Using Contrast-Enhanced Magnetic Resonance Imaging. Invest Radiol 2007; 42:42-49 Aleman BMP, Raemaekers JMM, Tomisic R, Baaijens MHA, Bortolus R, Lybeert MLM, Van der Maazen RWM, Girinsky T, Demeestere G, Lugtenburg P, Lievens Y, De Jong D, Pinna A, Henry-Amar M. Involved-field radiotherapy for patients in partial remission after chemotherapy for advanced HodgkinвЂ™s lymphoma. Int J Radiat Oncol Biol Phys 2007; 67:19-30 Baas P, Triesscheijn M, Burgers S, Van Pel R, Stewart F, Aalders M. Fluorescence detection of pleural A recent study found that the effectiveness of Hormonal Replacement Therapy (HRT) for alleviating endocrine and/or sexual complaints and quality of life was of limited importance for women who had become postmenopausal as a result of ovarian ablation. An explanation for this effect may be found in the hypothalamicpituitary-adrenal axis (HPA axis) and the hypothalamic-pituitary-gonadal axis (HPG axis). The possible differences on the various hormone concentrations of sex hormones or neurotransmitters between вЂњsurgicalвЂќ and вЂњnaturalвЂќ postmenopausal women could be an explanation for the results of HRT by these women. We have started a prospective study to investigate. 1a: Changes in menopausal symptoms and biochemical changes as result of PBSO in premenopausal women. 1b: Changes in menopausal symptoms and biochemical changes as result of PBSO in postmenopausal women. 2: Difference in intensity of menopausal symptoms and related biochemical parameters between surgically and naturally postmenopausal women (control group 2). 3: The effect of HRT in alleviating menopausal complaints and related biochemical parameters in surgically postmenopausal women compared to naturally postmenopausal women (control group 1). 4: Difference in menopausal symptoms and related biochemical parameters in surgically postmenopausal women between HRT and non-HRT users. 5: The prevalence of serotonin receptor (5-HT2A receptor) and serotonin polymorphism (5-HTTLPR) in surgically postmenopausal women with respect to the effect of HRT use. The study population will be 1: Women who are eligible for PBSO. 2: Women with climacteric complaints visiting a gynecologist. 3: Healthy postmenopausal women >=46 and <56 years. malignancies using 5-aminolaevulinic acid. Chest 2006; 129:718-724 DEPARTMENT OF NUCLEAR MEDICINE Beumer JH, Buckle T, Ouwehand M, Michiel Van den Brekel, Sjaak Burgers, Axel Bex, Marcel Van Herk, Frank Hoebers, Simon Horenblas, Franke NEF, Lopez-Lazaro L, Bin Kroon, Joost Leijte, Wim Meinhardt, Omgo Nieweg, Henk Van der Poel, Maartje Van Rijk, Schellens JHM, Beijnen JH, Annette Van der Velden, Marcel Verheij, Babs Taal, Ly Tran, Nico Van Zandwijk, Philippe Baars, Cees Van Tellingen O. Trabectedin (ET-743, Hoefnagel, Marina Kartachova, Saar Muller, Ferida Sivro, Michiel Sinaasappel, Renato ValdГ©s Olmos Yondelisв„ў) is a substrate for P-glycoprotein, but only high expression of P-glycoprotein confers the multidrug resistance phenotype. Invest New Drugs 2007; 25:1-7 Bijker N, Meijnen P, Peterse JL, Bogaerts J, Van Hoorebeeck I, Julien JP, Gennaro M, Rouanet P, Avril A, Fentiman IS, Bartelink H, Rutgers EJ. Breast-conserving treatment with or without radiotherapy in ductal carcinoma-in-situ: ten-year results of European Organisation for Research and Treatment of Cancer randomized phase III trial 10853--a study by the EORTC Breast Cancer Cooperative Group and EORTC Radiotherapy Group. J Clin Oncol 2006; 24:3381-3387 Imaging of apoptosis in patients receiving radiotherapy and/or chemotherapy Changes of 99mTc Hynic-rh-Annexin V tumor uptake were evaluated using co-registration of SPECT with CT in 16 chemo-naГЇve patients with advanced stage non-small lung cancer receiving platinum-based chemotherapy. Scintigraphy performed before and early after the start of treatment showed increase in tumor uptake varying from 16% to 121% above the baseline in 6 patients with objective response to chemotherapy. In 10 patients classified as non-responders (stable disease or progression) tumor uptake varied from вЂ“30% to 23%. A significant correlation (p-value 0.00001) was found between the tumor tracer uptake and treatment outcome. In another study involving 16 patients with head and neck squamous cell carcinoma treated with concurrent cisplatin-based chemoradiotherapy, scintigraphy, performed before and early after the first course of chemotherapy, showed a radiation-dose-dependent uptake in parotid glands indicative of early apoptosis during treatment. The interindividual spread in Annexin-uptake in primary tumors could not be related to differences in treatment schedule or tumor volume, but the Annexin-uptake in tumor and lymph nodes was closely correlated. 135 DIAGNOSTIC ONCOLOGY Sentinel node detection in prostate cancer Lymphoscintigraphy for pelvic lymphatic mapping and sentinel node identification was evaluated in 26 patients with prostate cancer of the intermediate prognostic group (T3 or PSA>10ug/l or Gleason score >6) scheduled for laparoscopic extended pelvic lymphadenectomy. Tracer administration assisted by transrectal ultrasound enabled injection of approximately 90% of the planned doses with an effective average dose of 211 MBq (range 123-266MBq) 99mTc-nanocolloid. Overall sentinel node visualization rate was 96% with rates of 88% at 15 min and 96% at 2 and 4 hours. A total of 128 sentinel nodes were visualized and at laparoscopy 101 of these nodes were found. In 3 patients operated more than 24 hours after tracer injection no radioactive nodes could be intraoperatively detected. Sentinel node metastases were found in 10 patients. At histology no skip-metastases were seen. SPECT combined with low dose CT was able to identify sentinel nodes inside the area of pelvic lymphadenectomy in 25 patients, and outside in 13 patients (10 in aortic-iliac junction and 3 para-aortic). All these unexpected sentinel nodes mapped by SPECT/CT (Fig. XIII.1) were found using the laparoscopic gamma probe. Inguinal lymphatic mapping in melanoma The images of 199 patients with melanoma draining to the groin area at sentinel node lymphoscintigraphy using 99mTc-nanocolloid were retrospectively evaluated in order to establish the location of second- and higher-echelon lymph nodes. Secondary lymph nodes were observed in 195 patients (98%). Transmission scanning with a 57Co flood source was used to outline the body contour and for anatomical orientation lead bands were placed in the inguinal folds as well as 57Co point sources over the anterior superior iliac spine and on the pubic tubercle. The groin was divided in the superior and inferior regions in relation to the saphenofemoral junction as well as the ilio-obturator basin and the central region connecting the first two regions through the femoral canal to the ilioobturator region. None of the 130 patients with a sentinel node outside the superior region had secondary nodes in this particular area. None of the 42 patients with a sentinel node outside the inferior region had drainage to secondary nodes in this region. This knowledge may allow the surgeon to limit the extent of therapeutic lymph node dissection in case of an involved sentinel node. Positron Emission Tomography The results of 19 18F-FDG PET studies corresponding to 17 patients with penile carcinoma were evaluated and compared to histopathology. PET was true positive in 14 patients with lymph node metastases and false positive in 2 patients without metastasis (sensitivity 100%, specificity 71%). Particularly for the inguinal regions sensitivity of PET was 88% and specificity 100%. In two patients detection of metastases at distance led to change in treatment. Following a previous study which demonstrated prominent bowel activity of 18F-FDG in 57% of Diabetes Mellitus (DM) patients (versus 23% in a control group of patients without DM) the pattern of bowel activity was evaluated in 32 DM patients scheduled for PET in the morning and in 38 scanned in the afternoon. Whereas no differences were observed in blood glucose levels (6.7 mmol/l vs 5.6 mmol/l) in 53% of patients of the afternoon group intense bowel activity was observed (against 28% in the morning group). This led to the recommendation that the FDG-PET evaluation of abdominal malignancies in DM patients should preferentially be planned in the morning. Imaging in neuroendocrine tumors The results of scintigraphy with 111Inpentetreotide and 131I-MIBG as well as 99mTc-medronate bone scintigraphy were evaluated in 52 patients with midgut neuroendocrine tumors. 111In-pentetreotide scan was slightly better in detecting metastases (71 %) compared to 131I-MIBG (60%). A combination of the two investigations in metastastatic disease revealed in 5 of 11 patients with a negative 131I-MIBG to have a positive 111In-pentetreotide. By contrast, in 5 of the 14 patients with the primary tumour negative at the 111In-pentetreotide scintigraphy proved to be positive at the 131I-MIBG scan. 99mTc-medronate bone scintigraphy revealed bone metastases in 20% of the patients; in 40% of the patients with positive bone scan the two other nuclear tests missed the bone abnormalities. Figure IX.2: Schematic overview of systematic (arrows) and random (ellipses) base-line deviations and their locations found in a group of 32 lung cancer patients. Figure XIII.1: A. Lymphatic drainage to the left para-aortic region (black arrow) in a patient with a prostate carcinoma. B. Appearing 15 minutes after transrectal ultrasound assisted injection of 99mTcnanocolloid into the prostate, the para-aortic sentinel node is accurately localized by means of SPECT-CT (white arrow) 136 DIAGNOSTIC ONCOLOGY Publications (continued) DEPARTMENT OF PATHOLOGY Boelens MC, Van den Berg A, Vogelzang I, Wesseling J, Postma DS, Timens W, Groen HJM. Differential expression and distribution of epithelial adhesion molecules in non-small cell lung Research in the department of Pathology is aimed at genetic analysis, including gene expression profiling using microarray analysis, of various malignancies. Part of this research, carried out by Petra Nederlof, Laura Van вЂ�t Veer and Marc Van de Vijver, is described under Division VIII and XII. A number of more clinically oriented projects are carried out in the department of Pathology itself and described here. cancer and normal bronchus. J Clin Pathol 2006;Published online Booman M, Douwes J, Glas AM, Riemersma SA, Jordanova ES, Kok K, Rosenwald A, De Jong D, Schuuring E, Kluin PM. Mechanisms and effects of loss of human leukocyte antigen class II expression in immune-privileged site-associated B-cell lymphoma. Clin Cancer Res 2006; 12:2698-2705 Booman M, Douwes J, Glas AM, De Jong D, Schuuring E, Kluin PM. Primary testicular diffuse large B-cell lymphomas have activated B-cell-like subtype characteristics. J Pathol 2006; EGFR mutation in lung carcinomas and response to Iressa therapy Gefitinib (Iressa, ZD1839), an inhibitor of epidermal growth factor receptor-tyrosine kinase (EGFR-TK), has shown potent anti-tumor effects and improved symptom and qualityof-life of a subset of patients with advanced NSCLC. However, a large portion of the patients does not respond to this agent. Recent publications show a correlation between the presence of a mutation (small in-frame deletion or point mutation) in exon 18, 19, 20 or 21 of the EGFR gene and response to therapy. In collaboration with dr N. van Zandwijk, a retrospective series (N=15) of tumors from NSCLC patients who are/were on Gefinitib therapy as part of a clinical trial was studied. It was shown that the patients with a mutation in EGFR (N=3) all responded to IRESSA therapy, whereas the patients without the mutation did not respond. In a prospective series, Gefinitib eligible patients were screened for mutations. A total of 12 mutations have been identified in 38 patients (2004 and 2005): one point mutation in exon 18, eight deletions in exon 19, and three point mutation in exon 21. In none of the tumors with a EGFR mutations a KRAS mutation could be identified. 210:163-171 Bosga-Bouwer AG, Van den Berg A, Haralambieva E, De Jong D, Boonstra R, Kluin P, Van den Berg E, Poppema S. Molecular, cytogenetic, and immunophenotypic characterization of c-KIT mutation in Gastrointestinal Stromal Tumors (GIST) and response to imatinib therapy Both activating c-KIT mutations and mutations resulting in resistance to imatinib therapy have been described in literature. Mutation analysis of c-KIT and PDGFRA is performed on small tumor biopsies and preliminary results indeed reveal single or multiple mutations in primary tumors and resistant recurrences (Figure XIII.2). follicular lymphoma grade 3B; a separate entity or part of the spectrum of diffuse large B-cell lymphoma or follicular lymphoma? Hum Pathol 2006; 37:528-533 Broekhuizen LN, Wijsman JH, Peterse JL, Rutgers EJT. The incidence and significance Pulmonary squamous cell carcinoma following head and neck squamous cell carcinoma: metastasis or second primary? Of 46 patients with a primary SCC of the Head and Neck followed by a SCC of the lung, clinical data (stage, tumor status, radiology, time interval), histology and PCR-based analysis of loss of heterozygosity (LOH) with 12 markers on 11 chromosome arms were used to distinguish between metastases and second primary tumors. of micrometastases in lymph nodes of patients with ductal carcinoma in situ and T1a carcinoma of the breast. Eur J Surg Oncol 2006; 32:502-506 Chi JT, Wang Z, Nuyten DSA, Rodriguez EH, Schaner ME, Salim A, Wang Y, Kristensen GB, Helland A, BГёrresen-Dale A-L, Giaccia A, Longaker MT, Hastie T, Yang GP, Van de Vijver MJ, Brown PO. Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers. PLoS Medicine 2006; 3:395-409 Cserni G, Bianchi S, Vezzosi V, Peterse H, Sapino A, Arisio R, Reiner-Concin A, Regitnig P, Bellocq JP, Marin C, Bori R, Penuela JM, Iturriagagoitia AC. The value of cytokeratin immunohistochemistry in the evaluation of axillary sentinel lymph nodes Figure XIII.2: Mutation analysis of the c-KIT gene on paraffin embedded tumor biopsy from a patient in patients with lobular breast carcinoma. with a gastrointestinal stromal tumor (GIST), which predicts response to imatinib therapy. A frequent J Clin Pathol 2006; 59:518-522 c-KIT exon-11 deletion is detected. 137 DIAGNOSTIC ONCOLOGY Publications (continued) LOH analysis indicated metastatic disease in 12 cases, probable metastases in 9 cases, second primary SCC in 21 cases and probable second primary disease in 3 cases. In 1 case LOH analysis was inconclusive. Clinical scoring suggested that 39 patients had metastases and 7 had second primaries. Histology gave firm evidence in 1 case for metastasis and in 1 other for a second primary tumor. For 28 patients LOH supported the clinical scoring and in 17 cases it did not. These 17 discordant cases were considered to be second primary tumors by LOH analysis, whereas they were suggested to be metastasis clinically. In this study we developed a novel interpretation strategy for LOH-profiling results to differentiate metastases and second primaries in routine clinical practice. A considerable number of lung lesions clinically interpreted as metastases, are suggested to be second primaries by LOH analysis. In addition, in collaboration with Brakenhof (VUMC) P53 mutation status was assessed in all tumor-pairs. All P53 results were in concordance with the LOH data. De Vries NA, Beijnen JH, Boogerd W, Van Tellingen O. Blood-brain barrier and chemotherapeutic treatment of brain tumors. Expert Rev Neurother 2006; 6:1199-1209 Derksen PWB, Liu X, Saridin F, Van der Gulden H, Zevenhoven J, Evers B, Van Beijnum JR, Griffioen AW, Vink J, Krimpenfort P, Peterse JL, Cardiff RD, Berns A, Jonkers J. Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 2006; 10:437-449 RASTER-STUDY Feasibility of gene expression profiling in community hospitals; preliminary results of a pilot study in N0 breast cancer patients Deurloo EE, Klein Zeggelink WFA, Jolien Bueno de Mesquita, Marc Van de Vijver, Wim Van Harten, Sabine Linn Muller SH, Bartelink H, Gilhuijs KGA. Teertstra HJ, Peterse JL, Rutgers EJT, Contrast-enhanced MRI in breast cancer Recently we have identified a gene expression profile of 70 genes using microarray analysis (70-gene MA test), which was a more powerful prognostic factor for freedom of distant metastases than current clinicopathological features in node negative breast cancer patients up to 55 years of age (van вЂ�t Veer et al., Nature 2002; Van de Vijver et al., New Engl J Med 2002). To assess whether this 70-gene MA test can be implemented in daily clinical practice we aimed to answer the following three questions: I. Is it feasible to collect fresh tumor samples in order to make this test available in pN0 breast cancer patients in community hospitals? II. What is the proportion of a вЂњpoor prognosisвЂќ versus a вЂњgood prognosisвЂќ in current node negative patients? III. What is the concordance between the 70-gene MA risk profile and the metastasis risk as assessed with current Dutch guidelines based on clinicopathological factors (such as age, pT, tumor grade, hormonal receptor-status)? In this study, women younger than 55 years presenting with cT1-2N0M0 unifocal breast cancer were eligible to participate and entered onto this prospective study after informed consent. Fresh tumor tissue samples were collected within one hour after surgery and sent in RNAlaterВ® to our hospital. A 70-gene MA test was performed in node negative patients with a representative tumor tissue sample; pN+ patients were excluded. According to current Dutch guidelines (used for guiding adjuvant systemic treatment) for node negative patients, high risk is defined as age < 35 years, pT any size and grade 2-3 or pT > 1cm and grade 1, or age > 35 years, pT1-2 cm and grade 3, pT2-3cm and grade 2-3 or pT>3cm and any grade; all others are defined as low risk. Thus far, 771 patients were included (average age 48 years), 404 (52%) 70-gene prognostic signatures were obtained and 367 (48%) patients were excluded after surgery. Of these excluded patients, 24% appeared not to be eligible for the test due to pN+, 2% due to wrong inclusion, 3% was postoperatively not eligible, 14% due to sampling failure, 3% due to incorrect procedures, and in 2% the quality of the RNA was insufficient. For 200 (50%) patients, a poor prognosis 70-gene MA-risk profile was found; for 200 (50%) a good prognosis profile. In 29% of the patients the 70-gene risk profile was discordant with risk assessment based on clinicopathological factors according to the Dutch guidelines. These first results show that the implementation of the 70-gene MA test in community hospitals is feasible. Furthermore, the 70-gene MA risk profile is discordant in a substantial proportion of lymph node-negative breast cancer patients compared with metastasis risk assessed with conventional clinicopathological factors according to Dutch guidelines. The accrual will end at December 1st of 2006 after which an international randomized trial, the MINDACT-trial, will start in which the 70-gene signature will be prospectively validated. patients eligible for breast-conserving therapy: Complementary value for subgroups of patients. Eur Radiol 2006; 16:692-701 Green JA, Berns EMJJ, Coens C, Van Luijk I, Thompson-Hehir J, Van Diest P, Verheijen RHM, Van de Vijver M, Van Dam P, Kenter GG, Tjalma W, Ewing PC, Teodorovic I, Vergote I, Van der Burg MEL. Alterations in the p53 pathway and prognosis in advanced ovarian cancer: A multi-factorial analysis of the EORTC Gynaecological Cancer group (study 55865). Eur J Cancer 2006; 42:2539-2548 Hannemann J, Velds A, Halfwerk JBG, Kreike B, Peterse JL, Van de Vijver MJ. Classification of ductal carcinoma in situ by gene expression profiling. Breast Cancer Res 2006; 8 Hannemann J, Kristel P, Van Tinteren H, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van der Wall E, Hupperets P, De Vries EGE, Rodenhuis S, Van de Vijver MJ. Molecular subtypes of breast cancer and amplification of topoisomerase IIa: Predictive role in dose intensive adjuvant chemotherapy. Br J Cancer 2006; 95:1334-1341 Jalving M, Koornstra JJ, Wesseling J, Boezen HM, De Jong S, Kleibeuker JH. Increased risk of fundic gland polyps during long-term proton pump inhibitor therapy. Aliment Pharmacol Ther 2006; 24:1341-1348 138 DIAGNOSTIC ONCOLOGY Publications (continued) Jalving M, De Jong S, Koornstra JJ, THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC Boersma-van Ek W, Zwart N, Wesseling J, Mohamed Achachah, Priscilla Axwijk, Majella Boutmy-de Lange, Marieke Bronk, Daniela Hahn, De Vries EGE, Kleibeuker JH. TRAIL Frans Hogervorst, Irma Kluijt, Petra Nederlof, Rob Plug, Roelof Pruntel, Anja Van Rens, Induces Apoptosis in Human Colorectal Marielle Ruijs, Laura Van вЂ™t Veer, Senno Verhoef , Paul Van der Voort, Gea Wigbout Adenoma Cell Lines and Human Colorectal Adenomas. Clin Cancer Res 2006; 12:4350-4356 Kaas R, Kroger R, Peterse JL, Hart AAM, Muller SH. The correlation of mammographic-and histologic patterns of breast cancers in BRCA1 gene mutation In 2006 the number of families who have sought genetic advice through our hospital since the start of the Family Cancer Clinic in 1995 has passed 2200. The steady increase in number of families seen per year has maintained at about 10% yearly, and this growth is maximised at this percentage as a consequence of limit imposed by the insurance companies. Most families present with a possible genetic predisposition for breast and/or ovarian cancer. carriers, compared to age-matched sporadic controls. Eur Radiol 2006; 16:2842-2848 Kartachova MS, ValdГ©s Olmos R, Haas RLM, Hoebers FJP, Van den Brekel MW, Van Zandwijk N, Herk MV, Verheij M. Mapping of treatment-induced apoptosis in normal structures: 99mTc-Hynic-rh-annexin V SPECT and CT image fusion. Eur J Nucl The DNA-diagnostic laboratory has screened almost 2000 families for germline mutations in the BRCA1/2 genes since the start. These families are obtained through our Family Cancer Clinic and the oncogenetic section of the Department of Clinical Genetics of the Academic Medical Center. In 153 families a BRCA1 mutation and in 82 families a BRCA2 mutation was identified. This year we noticed again an increase in the number of predictive, presymptomatic testing for these genes, from 190 to 250. Furthermore, the laboratory is testing new techniques in order to shorten the through put time of samples. We hope to achieve this using Conformation Sensitive Capillary Electrophoresis combined with automated pipetting and analysis. Med Mol Imaging 2006; 33:893-899 Kemper EM, Leenders W, Sters B, Lyons S, Buckle T, Heerschap A, Boogerd W, Beijnen JH, Van Tellingen O. Development of luciferase tagged brain tumour models in mice for chemotherapy intervention studies. Eur J Cancer 2006; 42:3294-3303 As good results have been obtained with the research into BRCA1 genetic profiles using array CGH, a start has been made to implement this technique as an additional tool for hereditary breast/ovarian cancer screening into clinical practice at the Family Cancer Clinic (in collaboration with E Van Beers and PM Nederlof, Division VIII and XIII). In a series of 38 tumors from HBOC families with a negative test result for BRCA1/2 only 3 cases showed a BRCA1-like aCGH profile, indicating that the routine diagnostic screening is very efficient. In addition, we have started to apply the aCGH BRCA1 classifier on unclassified variants (UVs) to gain additional proof of significance of the variations. Kreike B, Halfwerk H, Kristel P, Glas A, Peterse H, Bartelink H, Van de Vijver MJ. Gene expression profiles of primary breast carcinomas from patients at high risk for local recurrence after breast-conserving therapy. Clin Cancer Res 2006; 12:5705-5712 Kriege M, Brekelmans CTM, Boetes C, Muller SH, Zonderland HM, Obdeijn IM, Manoliu RA, Kok T, Rutgers EJT, The number of requests for microsatellite studies (MSI) and immunohistochemistry of the MMR genes (performed in collaboration with D De Jong) has stabilized at 80 yearly. In about 20% of the colon tumors microsatellite instability was detected, as an indication for possible pathogenic mutations in mismatch repair genes. Germline screening is now offered for hMLH1, hMSH2, hMSH6 and the MutY (MYH) gene. Furthermore, in case of a MSI high profile in a colon tumor without detectable mutations in the MMR genes, methylation status of the hMLH1 promotor and the presence of a specific somatic mutation in the BRAF gene (V600E) have been assessed. Several tumors were shown to have a methylated promoter which result is of use for the clinical interpretation of the family history data. De Koning HJ, Klijn JGM, Bartels CCM, Besnard APE, Hoogerbrugge N, Meijer S, Oosterwijk JC, Seynaeve C, Tilanus-Linthorst MMA, Tollenaar RAEM. Differences between first and subsequent rounds of the MRISC breast cancer screening program for women with a familial or genetic predisposition. Cancer 2006; 106:2318-2326 The Family Cancer Clinic contributes data to several multi-center national and international research projects, e.g. GEO-HEBON (gene environment interactions in hereditary breast and ovarian cancer, see Division XII), a national study of families with Li Fraumeni and variant Li Fraumeni(-like) syndrome, DNA-profiling by classic cGH and array cGH of breast and ovarian cancer patients (see Division VIII), the Breast Cancer Linkage Consortium, the BCAC and CIMBA consortiums, a study into the biological significance of so called unclassified variants (DNA changes of which it is uncertain whether they be pathogenic mutations or polymorphisms) in a national collaboration with other DNA-diagnostic labs, coordinated from LUMC by Dr P Devilee cs, psychosocial studies, in collaboration with the department of psychosocial research and epidemiology (Division XII) and clinical and genetic research in families with gastrointestinal cancer, including stomach cancer (Dr A Cats, Division X). 139 DIAGNOSTIC ONCOLOGY Publications (continued) Progress has been made into the development of guidelines for hereditary breast cancer management, by the participation of the clinical geneticists of the Family Cancer Clinic in the WKO (Werkgroep Klinische Oncogenetica) a working party of the Dutch Clinical Genetics Society, and local guidelines for management of familial colorectal cancer patients in a multidisciplinary team, whilst a national guideline is in development through the WKO. Kriege M, Brekelmans CT, Obdeijn IM, Boetes C, Zonderland HM, Muller SH, Kok T, Manoliu RA, Besnard AP, Tilanus-Linthorst MM, Seynaeve C, Bartels CC, Kaas R, Meijer S, Oosterwijk JC, Hoogerbrugge N, Tollenaar RA, Rutgers EJ, De Koning HJ, DEPARTMENT OF RADIOLOGY Klijn JG. Factors affecting sensitivity and Kenneth Gilhuijs, Tanja Alderliesten, Miret Emanuel, Claudette Loo, Saar Muller, Anita Paape, specificity of screening mammography and Kenneth Pengel, Jelle Teertstra, Fijs Van Leeuwen, Angelique Schlief, Christian Siedschlag, MRI in women with an inherited risk for Marja Van Vliet breast cancer. Breast Cancer Res Treat 2006; 100:109-119 The diagnostic-imaging laboratory at the department of Radiology pursues new imaging techniques, image processing, multi-modality registration, pattern recognition and molecular imaging to improve the sensitivity and specificity of cancer diagnosis and pre-operative assessment of tumor extent. Kroon BK, Nieweg OE, Van Boven H, Horenblas S. Size of Metastasis in the Sentinel Node Predicts Additional Nodal Involvement in Penile Carcinoma. J Urol Impact of preoperative contrast-enhanced MRI in patients eligible for breastconserving therapy: update on 325 patients The purpose of this study was to prospectively assess the incidence and the impact of additional findings in preoperative contrast-enhanced (CE) MRI of patients eligible for breast conserving therapy (BCT). Between November 2000 and December 2005, 325 patients eligible for BCT on the basis of conventional imaging and palpation underwent pre-operative CE-MRI with Gd-DTPA using FLASH 3-D imaging. The images were read by experienced breast MRI radiologists using the BIRADs lexicon. The incidence of additional findings (enhancing lesions separate from the known malignancy or larger extent of the known malignancy than appreciated from conventional imaging) and their impact on treatment were assessed. The gold standard was histology or follow-up. Additional findings were detected in 101 patients (30.9%): more extensive disease was found in 6.4% of patients, additional enhancing lesion(s) in 22.0%, and both in 2.4%. One-hundred additional lesions in 72 patients (72/325=22%) were detected; 47 in the same quadrant (74% of which are malignant), 32 in a different quadrant (41% of which are malignant) and 21 in the contralateral breast (19 of which are malignant). BIRADs scores of additional lesions were benign (n=1), probably benign (n=15), indeterminate (n=19), suspicious (n=30) and highly suggestive of malignancy (n=35). A trend was observed towards decreased reporting of benign findings (BIRADs 2 and 3) from 26% to 9%. Additional lesions referred for further workup (n=74) were visible on ultrasonography in 55.4% of the patients. Benign lesions (pathology-proven or benign by follow up (median 40 months)) occurred in 48/325 (15%) of the patients. Additional malignant lesions (all pathology proven) occurred in 36/325 (11%) of the patients. MRI led to a change of treatment in 71 patients (21.8%): mastectomy in 32 (9.8%), wider excision in 27 (8.3%, 1.2% of which due to benign lesions), contralateral surgery in 3 (0.9%), and neoadjuvant chemotherapy in 9 (2.8%) patients. Infiltrating lobular carcinoma (ILC) and ductal carcinoma in situ (DCIS) were found to be significantly associated with unexpected additional findings at CE MRI compared with infiltrating ductal carcinoma (IDC) (p=0.02). The density of the breast parenchyma was borderline significant (p=0.05). In summary, approximately 50% of the additional lesions detected at preoperative CE MRI in patients eligible for BCT on the basis of conventional imaging are malignant, leading to a change of treatment in 22% of the patients, and mastectomy in 10% of the patients. Treatment changes due to benign findings (wider excision) are uncommon. 2006; 176:105-108 Lakhani SR, Audretsch W, Cleton-Jensen AM, Cutuli B, Ellis I, Eusebi V, Greco M, Houslton RS, Kuhl CK, Kurtz J, Palacios J, Peterse H, Rochard F, Rutgers E, on behalf of EUSOMA. The management of lobular carcinoma in situ (LCIS). Is LCIS the same as ductal carcinoma in situ (DCIS)? Eur J Cancer 2006; 42:2205-2211 Li L, McVety S, Younan R, Liang P, Du Sart D, Gordon PH, Hutter P, Hogervorst FBL, Chong G, Foulkes WD. Distinct patterns of germ-line deletions in MLH1 and MSH2: the implication of Alu repetitive element in the genetic etiology of Lynch syndrome (HNPCC). Hum Mutat 2007; 27:a388 Lopez-Guerrero JA, Riegman PHJ, Oosterhuis JW, Lam KH, Oomen MHA, Spatz A, Ratcliffe C, Knox K, Mager R, Kerr D, Pezzella F, Van Damme B, Van de Vijver M, Van Boven H, Morente MM, Alonso S, Kerjaschki D, Pammer J, Carbone A, Gloghini A, Teodorovic I, Isabelle M, Passioukov A, Lejeune S, Therasse P, Van Veen EB, Dinjens WNM, Llombart-Bosch A. TuBaFrost 4: Access rules and incentives for a European tumour bank. Eur J Cancer 2006; 42:2924-2929 140 DIAGNOSTIC ONCOLOGY Publications (continued) Morente MM, Mager R, Alonso S, Pezzella F, Spatz A, Knox K, Kerr D, Dinjens WNM, Oosterhuis JW, Lam KH, Oomen MHA, Van Damme B, Van de Vijver M, Van Boven H, Kerjaschki D, Pammer J, Lopez-Guerrero JA, Llombart Bosch A, Carbone A, Gloghini A, Teodorovic I, Isabelle M, Passioukov A, Lejeune S, Therasse P, Van Veen EB, Ratcliffe C, Riegman PHJ. TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network. Eur J Cancer 2006; 42:2684-2691 Niessen RC, Sijmons RH, Ou J, Olthof SGM, Osinga J, Ligtenberg MJ, Hogervorst FBL, Weiss MM, Tops CMJ, Hes FJ, De Bock GH, Buys CHCM, Kleibeuker JH, Hofstra RMW. MUTYH and the mismatch repair system: Partners in crime? Hum Genet 2006; 119:206-211 Nuyten DSA, Kreike B, Hart AAM, Chi JTA, Sneddon JB, Wessels LFA, Peterse HJ, Bartelink H, Brown PO, Chang HY, Van de Vijver MJ. Predicting a local recurrence after breast-conserving therapy by gene expression profiling. Breast Cancer Res 2006; 8:R62 Olivier RI, Lubsen-Brandsma MAC, Verhoef S, Van Beurden M. CA125 and Benefit of semi-automatic volumetric measurement of extent of breast tumors from contrast-enhanced MRI Contrast-enhanced (CE) magnetic resonance imaging (MRI) is known to accurately visualize invasive breast tumors. The efficacy of Contrast-enhanced (CE) magnetic resonance imaging (MRI) is currently investigated to monitor response to neoadjuvant chemotherapy for breast cancer. Large inter- and intra-observer variations in the assessment of the largest diameter of the tumor from MRI have, however, been found, leading to reduced ability to accurately determine changes in tumor extent. The aim of this substudy was to assess whether semi-automatic volumetric measurement of breast tumors from CE-MRI decreases measurement variation and increases precision with respect to tumor extent as measured from histopathology. 25 Patients who underwent breast-conserving therapy for 26 tumors (21 invasive ductal, 4 invasive lobular and 1 mucinous carcinoma) were included. Detailed histopathology analysis was performed on the whole excision specimens including measurement of tumor volume and largest diameter of the tumor. Preoperative CE-MRI (3D FLASH) was obtained for all breasts. Two experienced breast-MR radiologists (R1, R2) independently assessed the largest diameter of the tumor from MRI. In addition, a computerized system developed in our laboratory was used to automatically segment the breast tumors in 3D and to determine volume as well as largest diameter of the tumor. Two observers (O1, O2) independently segmented the 26 tumors. Linear regression analysis was used to assess the precision and random variation in volumetric and largest-diameter measurements of tumor extent. Semi-automatic volumetric measurement of tumor volume was more accurately correlated with histopathology than conventional measurement of the largest diameter of the tumor (O1: slope=0.97, intercept=0.26, adjusted R-square=0.79, O2: slope=0.92, intercept=0.26, adjusted R-square=0.76 vs. R1: slope=0.68, intercept=0.72, adjusted R-square=0.52, R2: slope=0.72, intercept=0.47, adjusted R-square=0.68). The measurement precision relative to pathology increased, on average, by 30% using automated volumetric measurement. The variation in measurement dropped from 18% for the manual assessment of the largest diameter of the tumor to 10% for the semi-automatic volumetric measurement. Compared to conventional assessment of the largest diameter of the tumor, semiautomatic volumetric measurement of breast tumors from CE-MRI leads to smaller variations as well as improved precision with respect to tumor extent as measured from histopathology. transvaginal ultrasound monitoring in high-risk women cannot prevent the diagnosis of advanced ovarian cancer. Gynecol Oncol 2006; 100:20-26 Ortin-Perez J, Van Rijk MC, Valdes-Olmos RA, Vidal-Sicart S, Nieweg OE, Vilalta A, Kroon BB, Pons F. Lymphatic mapping and sentinel node biopsy in MerkelвЂ™s cell carcinoma. Eur J Surg Oncol 2006;[Epub ahead of print] Accurate extent definition of non-small cell lung tumors using pathologyvalidated PET-CT A project has been initiated aimed at pre-treatment prediction of the distribution of the tumor cell spread around non-small cell lung cancers (NSCLC) as well as the biological characteristics of the cancers using pre-treatment respiratory-correlated PET and CT. To pursue this aim, the pre-treatment PET and CT images of NSCLC patients is compared in detail with the histopathology of the tumor after surgery. So far, 8 patients have been included in the study. Non-rigid fusion of the pathology data and preoperative CT has been applied to account for differences in deformation of the lung. First results suggest underestimation of the extent of microscopic disease by a factor of 1.8 when deformations are not taken into account. Press RR, Buckle T, Beijnen JH, Van Tellingen O. The effect of P-glycoprotein and cytochrome P450 3a on the oral bioavailability of vinorelbine in mice. Cancer Chemother Pharmacol 2006; 57:819-825 Optimizing functional imaging approaches in a preclinical setting; molecular imaging in mice The functional information provided by molecular imaging may reveal the molecular basis of a tumor in a non-invasive manner-and also enable earlier and more specific tumor detection, all of which will help to tailor therapy approaches. In a joint effort between the departments of radiology and nuclear medicine we are currently using the (advanced) mouse models present at the NKI to develop new clinical molecular imaging strategies. For the visualization of both the tumor extent and the biological processes activated within the tumor, it is very advantageous to register imaging techniques that provide anatomical features (X-ray, CT, and MRI) with those that enable the visualization of molecular processes (Gamma camera, PET, and SPECT). To achieve multimodality imaging in mice, we apply two approaches: 1) imaging immobilized mice in different 141 DIAGNOSTIC ONCOLOGY Publications (continued) Riegman PHJ, Dinjens WNM, Oomen MHA, Spatz A, Ratcliffe C, Knox K, Mager R, Kerr D, Pezzella F, Van Damme B, Van de Vijver M, Van Boven H, Morente MM, Alonso S, Kerjaschki D, Pammer J, Lopez-Guerrero JA, Llombart Bosch A, Carbone A, Gloghini A, Teodorovic I, Figure XIII.3: Tumor specific monitoring of Pgp-mediated efflux in hereditary breast tumors Isabelle M, JaminГ© D, Passioukov A, (in collaboration with Sven Rottenberg). Lejeune S, Therasse P, Van Veen EB, Lam KH, Oosterhuis JW. TuBaFrost 1: modalities using different contrast agents (each modality visualizes a different process) and 2) imaging immobilized mice using a single contrast agent that is detectable in different modalities (modalities visualize the same process). A number of preclinical imaging projects that are currently running: the development of multimodality imaging strategies and contrast agents for the visualization of DCIS, evaluation of mouse models for small cell lung cancer (in collaboration with Tessa Buckle), evaluation of radioactive contrast agents for the detection of metastatic breast cancer (in collaboration with Hermien Boerhout), and determination of drug resistance mechanisms in hereditary breast cancer cancer (in collaboration with Sven Rottenberg). In all these projects we are looking for a tumor specific and functional readout. This is achieved using a combination of different imaging modalities such as: X-ray and gamma camera, and PET/CT and MRI. Uniting local Frozen Tumour Banks into a European Network: an overview. Eur J Cancer 2006; 42:2678-2683 Rodenhuis S, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van Der Wall E, Hupperets P, Van Tinteren H, Peterse JL, Van de Vijver MJ, De Vries EGE. Efficacy of high-dose alkylating chemotherapy in HER2/neu-negative breast cancer. Ann Oncol 2006; 17:588-596 Rudkin TM, Hamel N, Galvez M, Hogervorst F, Gille JJP, MГёller P, Apold J, Foulkes WD. The frequent BRCA1 mutation 1135insA has multiple origins: A haplotype study in different populations. BMC Med Genet 2006; 7 Ruijs MWG, Verhoef S, Wigbout G, Pruntel R, Floore AN, De Jong D, Van вЂ�t Veer LJ, Menko FH. Late-onset common cancers in a kindred with an Arg213Gln TP53 germline mutation. Fam Cancer 2006; 5:169-174 Schmidt MK, Tollenaar RA, De Kemp SR, Broeks A, Cornelisse CJ, Smit VT, Peterse JL, Van Leeuwen FE, Van вЂ�t Veer LJ. Breast Cancer Survival and Tumor Characteristics in Premenopausal Women Carrying the CHEK2*1100delC Germline Mutation. J Clin Oncol 2007; 25:64-69 Sotiriou C, Wirapati P, Loi S, Harris A, Fox S, Smeds J, Nordgren H, Farmer P, Praz V, Haibe-Kains B, Desmedt C, Larsimont D, Cardoso F, Peterse H, Nuyten D, Buyse M, Van de Vijver MJ, Bergh J, Piccart M, Delorenzi M. Gene expression profiling in breast cancer: Understanding the molecular basis of histologic grade to improve prognosis. J Natl Cancer Inst 2006; 98:262-272 142 DIAGNOSTIC ONCOLOGY Publications (continued) Van Apeldoorn MJ, Rustemeijer C, Voerman BJ, Van Rijk MC, Nieweg OE, Rutgers EJT, Peterse J. Mesothelioma of the Tunica Vaginalis Oldenburg HSA, Olmos RV, Hoefnagel CA, Complicated by Chyluria. J Clin Oncol 2006; Kroon BBR. Sentinel node biopsy before 24:5329-5330 neoadjuvant chemotherapy spares breast cancer patients axillary lymph node dissection. Ann Surg Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Oncol 2006; 13:475-479 Hogervorst FBL, Verhoef S, Nederlof PM. A multiplex PCR predictor for aCGH success of Van Rijk MC, Teertstra HJ, Peterse JL, FFPE samples. Br J Cancer 2006; 94:333-337 Nieweg OE, Olmos RAV, Hoefnagel CA, Kroon BBR. Ultrasonography and fine-needle Van den Broek GB, Rasch CRN, Pameijer FA, aspiration cytology in the preoperative evaluation Peter E, Van den Brekel MWM, Balm AJM. of melanoma patients eligible for sentinel node Response measurement after intraarterial biopsy. Ann Surg Oncol 2006; 13:1511-1516 chemoradiation in advanced head and neck carcinoma: Magnetic resonance imaging and Van Rijk MC, Deurloo EE, Nieweg OE, evaluation under general anesthesia? Cancer Gilhuijs KGA, Peterse JL, Rutgers EJT, KrГ¶ger R, 2006; 106:1722-1729 Kroon BBR. Ultrasonography and fine-needle aspiration cytology can spare breast cancer Van der Hout AH, Van den Ouweland AMW, patients unnecessary sentinel lymph node biopsy. Van der Luijt RB, Gille HJP, Bodmer D, Ann Surg Oncol 2006; 13:31-35 BrГјggenwirth H, Mulder IM, Van der Vlies P, Elfferich P, Huisman MT, Ten Berge AM, Van Veen EB, Riegman PHJ, Dinjens WNM, Kromosoeto J, Jansen RPM, Van Zon PHA, Lam KH, Oomen MHA, Spatz A, Mager R, Vriesman T, Arts N, Boutmy-de Lange N, Ratcliffe C, Knox K, Kerr D, Van Damme B, Oosterwijk JC, Meijers-Heijboer H, Van de Vijver M, Van Boven H, Morente MM, Ausems MGEM, Hoogerbrugge N, Verhoef S, Alonso S, Kerjaschki D, Pammer J, Halley DJJ, Vos YJ, Hogervorst F, Ligtenberg M, Lopez-Guerrero JA, Llombart Bosch A, Hofstra RMW. A DGGE system for comprehensive Carbone A, Gloghini A, Teodorovic I, Isabelle M, mutation screening of BRCA1 and BRCA2: Passioukov A, Lejeune S, Therasse P, application in a Dutch cancer clinic setting. Hum Oosterhuis JW. TuBaFrost 3: Regulatory and Mutat 2006; 27:654-666 ethical issues on the exchange of residual tissue for research across Europe. Eur J Cancer 2006; Van der Poel HG, Antonini N, Hoefnagel CA, 42:2914-2923 Horenblas S, ValdГ©s Olmos RA. Serum hemoglobin levels predict response to strontium-89 Van Zandwijk N, Mathy A, Boerrigter L, and rhenium-186-HEDP radionuclide treatment Ruijter H, Tielen I, De Jong D, Baas P, for painful osseous metastases in prostate cancer. Burgers S, Nederlof P. EGFR and KRAS Urol Int 2006; 77:50-56 mutations as criteria for treatment with tyrosine kinase inhibitors: retro- and prospective Van der Poel HG, Beetsma DB, Van Boven H, observations in non-small-cell lung cancer. Horenblas S. Perineal Salvage Prostatectomy for Ann Oncol 2007; 18:99-103 Radiation Resistant Prostate Cancer. Eur Urol 2006; In Press Verdoes M, Florea BI, Menendez-Benito V, Maynard CJ, Witte MD, Van der Linden WA, Van Rijk MC, Peterse JL, Nieweg OE, Van den Nieuwendijk AMCH, Hofmann T, Oldenburg HSA, Rutgers EJT, Kroon BBR. Berkers CR, Van Leeuwen FWB, Groothuis TA, Additional axillary metastases and stage Leeuwenburgh MA, Ovaa H, Neefjes JJ, migration in breast cancer patients with Filippov DV, Van der Marel GA, Dantuma NP, micrometastases or submicrometastases in sentinel Overkleeft HS. A Fluorescent Broad-Spectrum lymph nodes. Cancer 2006; 107:467-471 Proteasome Inhibitor for Labeling Proteasomes In Vitro and In Vivo. Chem Biol 2006; 13:1217-1226 Van Rijk MC, Tanis PJ, Nieweg OE, ValdГ©s Olmos RA, Rutgers EJT, Hoefnagel CA, Zandvoort A, Van der Geld YM, Jonker MR, Kroon BBR. Clinical implications of sentinel nodes Noordhoek JA, Vos JTWM, Wesseling J, outside the axilla and internal mammary chain in Kauffman HF, Timens W, Postma DS. High patients with breast cancer. J Surg Oncol 2006; ICAM-1 gene expression in pulmonary fibroblasts 94:281-286 of COPD patients: a reflection of an enhanced immunological function. Eur Respir J 2006; 28:113-122 143 BIOMETRICS BI OME T R I CS DEPARTMEN T CLINICAL STUDIES AND OTHER COLLABORATIONS We have developed collaborations with several cooperative groups; academic groups, industry and clinical research organizations, and we function as partner for clinical studies and clinical trials, being involved from the generation of the idea, protocol setting, the planning, and providing randomization services, quality assurance, data handling and statistical expertise. We coordinate the overview of randomized trials of the treatment of prostate cancer. The statistical analyses are made centrally in collaboration with R Peto and the Clinical Trials Service Unit at the University of Oxford. This is a continuous effort that is currently in its 2nd cycle. The initial cycles of this collaboration assessed the use of maximum androgen blockade (MAB). The present cycle continued its course in 2006 with the support of the grant by of the Quality of Life program of the European Commission. The Prostate Cancer TrialistsвЂ™ Collaborative Group (PCTCG) was created to bring together these randomized trials and currently counts more than 400 investigators. In the current cycle of the overview trials studying the effect of immediate endocrine therapy as compared to deferred endocrine treatment in asymptomatic patients with no recorded metastases are being overviewed and publication of the effects on prostate cancer mortality and non-prostate cancer mortality is in discussion. Collaboration with the Dutch Colorectal Cancer Group (DCCG) has resulted in several randomized studies in patients with colorectal carcinoma for which we are the Statistical Center. In the first study in advanced previously untreated patients sequential versus combination chemotherapy was compared. Seventy institutes participated randomizing the total accrual planned of 820 patients in 2 years. The final analysis is in preparation and results will be presented during ASCO 2007. The second study (CAIRO 2) is a randomized study in the same patients group studying Cetuximab added to capecitabine, oxaliplatin and bevacizumab.It is expected that the accrual of 800 patients will be completed in April 2007 and a third study is already being evaluated by the ethical committee and by the KWF for funding. An additional study after radical resection of liver metastases of colorectal cancer comparing bevacizumab in combination with capecitabine and eloxatin vs eloxatin alone as adjuvant treatment has started in December 2006 and should include in excess of 600 patients. The collaboration with the Dutch Chest Physician Association (NVALT) already resulted in 6 randomized studies in which in excess of 70 hospitals have already included more than 1200 patients. The first study showed that docetaxel in a weekly schedule had insufficient activity as compared with docetaxel + carboplatin in a three weekly schedule in patients with stage IIIb and IV NSCLC. The second study, performed in collaboration with the British Medical Research Council, is aimed at evaluating the benefit of neo-adjuvant chemotherapy in operable NSCLC. Accrual to this study has been completed and final analysis is being finalized. The NVALT3 is a study in elderly patients with non-small cell lung cancer. In this study 2 chemotherapy regimens are compared in terms of toxicity and quality of life. A battery of tests and questionnaires is being used and special nurses were trained in 12 institutes to support the study. Accrual has been completed in 2006 and the analysis is currently being performed. The NVALT4 study is a randomized placebocontrolled study of docetaxel/carboplatin with celecoxib or placebo in patients with locally advanced or metastatic non-small cell lung cancer. Accrual to the study began end of 2003 but was suspended during several months in 2005 waiting for the evaluation by the FDA of reports of excessive cardiovascular toxicity associated to Cox2 inhibitors in the treatment of pain. In excess of 400 patients have already been randomized. We have obtained support from the Dutch Cancer Society for local data management in the participating sites. We also obtained support for the the NVALT 5, a phase III trial of the antiangiogenic agent Thalidomide in patients with malignant pleural mesothelioma after first line chemotherapy. Participation of Australian institutes is being organized. The NVALT 7 is a study of 2 chemotherapy regimens in non small cell cancer patients with recurrence after first line Department head, Group leader Otilia Dalesio Otilia Dalesio MSc Head Ninja Antonini MSc Academic staff Harm Van Tinteren PhD Academic staff Andrew Vincent PhD Academic staff Danny Baars Technical staff Roman Bohoslavsky MSc Technical staff Monique Carreno Technical staff Vincent Coppen Technical staff Suzanne De Boer MSc Technical staff Marjolijn De Waal MSc Technical staff Jitske Dijkstra Technical staff Brigitte Erven-Dufournij Technical staff Song-Hieng Hau MSc Technical staff Annelies Hiemstra Technical staff Edwin Klerkx Technical staff Jenneke Koorn Technical staff Bettina Kortekaas Technical staff Marianne Mahn-Schaefers MSc Technical staff Ingrid Mandjes MSc Technical staff Tineke Meindertsma Technical staff Carla Modder Technical staff Rick Muussen Technical staff Cecile Paulus van Pauwvliet Technical staff Anneke Reinders-Som Technical staff Jolanda Remmelzwaal Technical staff Daniel Roberts Technical staff Marijke Scholten Technical staff Dea Storm Technical staff Martha Swart Technical staff Laura Vader Technical staff Heleen Vaessen MSc Technical staff Renato Valdes Olmos Technical staff Tjalling Valdes Olmos Technical staff Ludy Valkenet MD Technical staff Marjolijn Van den Haak MSc Technical staff Emile Van der Donk Technical staff Tony Van der Velde Technical staff Gabry Van Netten Technical staff Els Van Oosterwijk Technical staff Wil Van Waardenberg Technical staff Marcel Vlaskamp MSc Technical staff Anneke Wals Technical staff Lidwina Wever Technical staff Els Willemse Technical staff Lonny Ziblat Technical staff 144 BIOMETRICS chemotherapy and a total of 150 patients have already been accrued. Two new studies in low and high risk patients as determined by preoperative PET SUVmax values are being developed and should start in 2007. We collaborate with the radiotherapy department in carrying out several randomized studies with support from the Dutch Cancer Society. More than 300 patients were randomized this year into a study of radiotherapy boost in young women with early breast cancer. In this study tumor material is collected for micro array analysis and profile determination. Further, accrual has been completed into a study in lung cancer that will be analyzed and reported in 2007. The statisticians have collaborated with other departments of the institute, the Free University and other institutes in the region in a variety of studies, many of which resulted in co-authorships. Figure 1: Number of resections in the IKA region by year PET prevents futile surgery in NSCLC Positron Emission Tomography (PET) prevents futile surgery in 1 out of 5 lung cancer (NSCLC) patients. This was the result of a randomized controlled trial with PET that was performed several years ago in the IKA region. PET is a relatively new nuclear medicine imaging technique that allows the visualization of biochemical processes in tissues. Diagnostic accuracy studies had already shown promising results for PET in NSCLC. However, improved accuracy does not necessarily imply clinical usefulness, e.g. better patient management and improved clinical outcome. The assessment of the value of a diagnostic test to patient management best follows a multi-phase hierarchical process, similar to what we are familiar with in therapeutic research. The process encompasses the technical aspects of the test such as image quality and reproducibility, diagnostic accuracy, diagnostic and therapeutic impact, patient outcomes and finally, the cost-benefit analysis of the introduction of the technology. Obviously, demonstration of efficacy at each lower level is logically necessary, but not sufficient, to assure efficacy at a higher level. To assess the value of PET we developed a framework which basically follows the postulated hierarchical approach. The starting point of our studies was a notion of residual inefficiency which might be amenable with the suggested improved accuracy of FDG-PET (in NSCLC). Retrospective cohort analysis substantiated this notion, specified the nature of the errors and it proved to be the main incentive for clinicians to participate in further research. The run-in experiment on the clinical value of PET was the learning curve of diagnosticians and clinicians. In the end, the coherence of this approach contributed to a very fruitful environment for collaboration and has facilitated the successful completion of the two RCTs and the appearance and implementation of working guidelines in our region. Data from the IKA cancer registry suggests that the introduction and implementation of the guidelines effectively reduced the number of resections in the region with an absolute 20% compared to the average over the five preceding years (Figure 1). Anno 2006, the PET-situation in the Netherlands has changed substantially. The PET-scanner capacity has increased dramatically and it is estimated that in about 80% of the patients in the IKA region (2.6 million inhabitants, 1300 NSCLC annually) with suspected operable NSCLC a PET-scan is integrated in their work-up. These studies and results are the core of the PhD thesis presented at the Free University in Amsterdam by Harm van Tinteren. ICT projects During 2006 the Department negotiated with the European Commission Services the Initial Deployment contract of the TENALEA project. A total maximum funding of 3.7 million was obtained for this 42 months project. In addition of the NKI, which is the coordinator of the project, the consortium consists of academic and industrial contractors: the Institut Gustave Roussy, in Paris; the Cochrane Collaboration UK, in Oxford; the UniversitГ¤ts Klinikum KГ¶ln in Germany; the Medical University of Gdansk in Poland; the RГ©seau de CancГ©rologie dвЂ™Aquitaine in Bordeaux; AKZO/ NOBEL NV in The Netherlands and InferMed Ltd in London. TENALEA is an initiative of the Department that attempts to increase harmonization of the Clinical Trials Data Handling tools which are being pushed towards the investigators and study sites by providing best-of-breed solutions at affordable price. Currently TENALEA provides a service for patient randomization in clinical trials, based on ALEA software that was developed within the department. The service is 145 BIOMETRICS Publications hosted in the Department and a QA system for the operational management of the service is in place. ALEA is a randomization service which was evaluated by 25 data centers throughout Europe and was found suitable for the randomization procedure of all clinical trials operated by these data centers. It was successfully tested for a clinical trial selected by a pharmaceutical company (ORGANON NV). In 2006 we have offered randomization services for 33 studies in the Netherlands in which 2200 patients have been entered. In addition to the studies run by the NKI, also studies of the IKA, IKO, IKL and Untrecht University have been supported. Within this new phase of the project a framework to enable controlled extension of the services to fulfill other requirements of clinical trial data centers, such as Remote Data Entry will be developed. With Remote Data Entry, the traditional paper Case Report Forms is replaced by an Electronic system through which the patient data for a specific trial is collected. InferMedвЂ™s MACRO provides a Remote Data Entry service and is used by several centers in the TENALEA consortium. Therefore, MACRO will be operated in the TENALEA Shared Services Infrastructure to provide a hosted Remote Data Entry solution to clinical trial data centers. TENALEA is not a single service but a placeholder for several clinical trials data management services. Therefore in addition to the Randomization and Remote Data Entry other services will be identified and included in the framework as described in Figure 2. The basis of this framework is a secure, fault tolerant, validated, audited and compliant Shared Services Infrastructure. This Shared Services Infrastructure provides a service platform for hosted tools supporting the clinical trials data management process. Baas P, Belderbos JSA, Senan S, Kwa HB, Van Bochove A, Van Tinteren H, Burgers JA, Van Meerbeeck JP. Concurrent chemotherapy (carboplatin, paclitaxel, etoposide) and involved-field radiotherapy in limited stage small cell lung cancer: A Dutch multicenter phase II study. Br J Cancer 2006; 94:625-630 Buitelaar DR, Balm AJM, Antonini N, Van Tinteren H, Huitink JM. Cardiovascular and respiratory complications after major head and neck surgery. Head Neck 2006; 28:595-602 Den Herder C, Van Tinteren H, De Vries N. Hyoidothyroidopexy as a surgical treatment for obstructive sleep apnoea syndrome. Ned Tijdschr Geneeskd 2006; 150:198-203 Hannemann J, Kristel P, Van Tinteren H, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van der Wall E, Hupperets P, De Vries EGE, Rodenhuis S, Van de Vijver MJ. Molecular subtypes of breast cancer and amplification of topoisomerase IIa: Predictive role in dose intensive adjuvant chemotherapy. Br J Cancer 2006; 95:1334-1341 Figure 2: TENALEA service organization Koopman M, Antonini NF, Douma J, Wals J, Honkoop AH, Erdkamp FLG, The TENALEA Service Management manages the service portfolio of the TENALEA Service Framework through the introduction of new services, new products for existing services, periodic auditing of products and services, and the upgrading and phasing out of existing products. Before a new service is deployed on the TENALEA Service Framework, a common standard procedure is applied which will be further detailed in the TENALEA Service Catalogue. The TENALEA Services Framework will adopt the relevant methodologies and concepts from the ITIL Service Level Management and ITIL Service Delivery. The TENALEA Service Catalogue will be an important living document throughout the initial deployment term. In addition, all services will be provided through Service Level Agreements. Not all services from the TENALEA Service Catalogue will become available at the same point in time (Figure 3). Of the foreseen services in the portfolio, the Randomization service will be available in the early phase of the project, while the Remote Data Entry service will start at a later point during the project. Also, the expected number of trials eventually hosted under the TENALEA Service Framework will not be the same for all services. Whereas the TENALEA Service Catalogue includes a Remote Data Entry service based on MACRO, the eventual goal of TENALEA is to provide Electronic Data Capture (EDC). The concept of Electronic Data Capture extends on Remote Data Entry to include the ability to retrieve (part of the) patient data from other electronic sources within the clinical setting, most prominently the Electronic Patient Record. Electronic Data Capture best serves the main objective of TENALEA. Success indicators of the project are: the number of trials which have used one or more services from the TENALEA portfolio for their operational process and the number of data centers which have adopted one It is anticipated that by the end of De Jong RS, Rodenburg CJ, Vreugdenhil G, Akkermans-Vogelaar JM, Punt CJA. Randomised study of sequential versus combination chemotherapy with capecitabine, irinotecan and oxaliplatin in advanced colorectal cancer, an interim safety analysis. A Dutch Colorectal Cancer Group (DCCG) phase III study. Ann Oncol 2006; 17:1523-1528 Lont AP, Gallee MPW, Meinhardt W, Van Tinteren H, Horenblas S. Penis Conserving Treatment for T1 and T2 Penile Carcinoma: Clinical Implications of a Local Recurrence. J Urol 2006; 176:575-580 Rodenhuis S, Bontenbal M, Van Hoesel QGCM, Smit WM, Nooij MA, Voest EE, Van der Wall E, Hupperets P, Van Tinteren H, Peterse JL, Van de Vijver MJ, De Vries EGE. Efficacy of high-dose alkylating chemotherapy in HER2/neu-negative breast cancer. Ann Oncol 2006; 17:588-596 146 BIOMETRICS ONCOLOGY DIAGNOSTIC Publications Smeenk RM, Verwaal VJ, Antonini N, Zoetmulder FAN. Progression of Pseudomyxoma Peritonei after Combined Modality Treatment: Management and Initial Deployment 120 clinical trials will operate one or more TENALEA Services, and between 50 and 70 data centers will be using TENALEA services. Finally, the main success indicator is whether or not the service can be continued without further funding. After Initial Deployment, the service must generate sufficient revenues to cover the operating costs. Outcome. Ann Surg Oncol 2006;Published online Triesscheijn M, Ruevekamp M, Antonini N, Neering H, Stewart FA, Baas P. Optimizing Meso-TetraHydroxyphenyl-Chlorin Mediated Photodynamic Therapy for Basal Cell Carcinoma. Photochem Photobiol 2006; 82:1686-1690 Underberg R, Van Sornsen de Koste J, Lagerwaard F, Vincent A, Slotman B, Senan S. A dosimetric analysis of respiration-gated radiotherapy in patients with stage III lung cancer. Radiation Oncology 2006; 1:8 Van der Poel HG, Antonini N, Hoefnagel CA, Horenblas S, ValdГ©s Olmos RA. Serum hemoglobin levels predict response to strontium-89 and rhenium-186-HEDP radionuclide treatment for painful osseous metastases in prostate cancer. Urol Int 2006; 77:50-56 Figure 3: TENALEA project plan Towards Electronic Data Capture (EDC) Technological trends are changing the current developments in the market in the direction of EDC. Emerging standards for electronic forms, such as PDF, xFORMS and InfoPath, combined with the emerging standards in the field of electronic signatures, XML schemaвЂ™s (ODM, HL7) and ontology are likely to replace вЂ“ probably starting this decade - the proprietary frontend of suite vendors to a forms front-end selected by the study site or investigator. Such forms front-end can be generic, i.e. not for a particular niche segment such as clinical trials data management and therefore be much more economic. This will drastically accelerate the take up of electronic online tools for clinical trials data management. The electronic Case Record Forms (eCRF) Data Entry module is a generic electronic form data entry facility which presents the eCRFвЂ™s as web forms, PDF forms, xForms or InfoPath forms on the userвЂ™s computer device. The second system into which the eCRF definition is fed into is an EPR XML Broker. This is most likely a proprietary system which analyzes the ODM metadata representation and allows the mapping of data available in the EPR to data required in the eCRF. This may be a manual procedure performed after retrieving a metadata eCRF ODM representation or it may be partially, semi or fully automated on the basis of common ontologies. The EPR XML Broker and the eCRF data entry module collaborate in a Just In Time EPR data retrieval mechanism. At the time the user interface of the eCRF Data Entry tool invokes an electronic Case Report Form, it first invokes the EPR XML broker which on the basis of the ODM representation of the eCRF, retrieves all known data from the Electronic Patient Record and inserts it onto the eCRF before it is displayed to the user. Effectively, the clinician completing the form will only need to provide the data which could not be located in the EPR. The introduction of this concept is not restricted to hospitals working with EPR, but may also be based on an HL7 broker which вЂ�listensвЂ™ to HL7 messages referring to patients which are treated in a clinical trial protocol. 147 CLINICAL TRIALS C LI NI CA L TRIAL S IN THE N E T HE R LANDS CANCER IN S TITU TE type of cancer study title study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M03D24 Dose-finding and pharmacokinetic trial of orally administered D-24851 to patients with solid tumors Schellens, JHM I 02/04/2003 25 M03MEN A phase I study of intravenous MEN 4901/T-0128 administered once every 6 weeks in patients with solid tumors Schellens, JHM I 10/11/2003 14 M04OAZ A phase I, open label, multicenter study to assess the safety, tolerability, pharmacodynamics and pharmacokinetics of AZD5438 given orally, in 2 dosing schedules, in patients with advanced solid malignancies Schellens, JHM I 29/06/2004 11 M05AZA A phase-I open-label, multicenter study to assess the safety, tolerability and pharmacokinetics of AZD1152 given as as 2 hour intravenous infusion on 2 dose schedules in patients with advanced solid malignancies Schellens, JHM I 23/05/2005 20 M05HOP An open label phase I dose escalation study of E7080 Schellens, JHM I 28/06/2005 20 M05PAR A phase I, pharmacokinetic and biological evaluation of a small molecule inhibitor of poly ADP-Ribose Polymerase-1 (PARP-1), KU-0059436, in patients with advanced tumors Schellens, JHM I 15/11/2005 14 M05PFS A phase I, open-label, dose escalation study to evaluate safety, pharmacokinetics and pharmacodynamics of 2 dosing schedules of PF 00299804 in patients with advanced malignant solid tumors Schellens, JHM I 27/09/2005 15 M05RTB A phase III international randomized trial of single versus multiple fractions for re-irradiation of painful bone metastases Oppedijk, V III 10/01/2006 2 M06BBC Phase I study to determine the safety, maximum tolerated dose and pharmacokinetics of BAY57-9352 in combination with bevacizumab in subjects with advanced solid tumors Schellens, JHM I 19/05/2006 6 M06BCI Phase I study to determine the safety, maximum tolerated dose, pharmacokinetics and biomarker status of BAY 57-9352 in combination with capecitabine and irinotecan in subjects with advanced solid tumors Schellens, JHM I 12/10/2006 3 M06ESO Effect of esomeprazole on the pharmacokinetics of BMS-275183 in patients with advanced malignancies Roelvink, M I 12/12/2006 1 ALL SITES 148 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M06FRE Effect of fibrin sealant in reducing REsection surface related complications - The FRESCO trial Zoetmulder, FAN other 06/09/2006 0 M06HYP A phase II randomised factorial double-blind study to investigate the management of AZD2171 induced hypertension and efficacy of AZD2171 at doses of 30 mg and 45 mg in patients with advanced solid tumors Schellens, JHM II 21/08/2006 15 M06OGP Phase I dose escalation study of oral gemcitabine prodrug (LY2334737) alone and in combination with erlotinib (Tarceva) in patients with advanced solid tumors Schellens, JHM I 29/08/2006 5 M06PPI Collection of skin biopsies with hair follicle samples from patients with advanced solid malignancies receiving Irinotecan to develop biomarker assays Schellens, JHM NA 26/01/2006 2 N03DOR Oral bioavailability of docetaxel in combination with ritonavir Schellens, JHM I 07/04/2003 22 N03JJP Relative bioavailability of Paclitaxel in combination with Cyclosporin A. A pilot investigation Schellens, JHM Pilot 26/01/2004 12 N05GEN Genotyping as tool for dose individualization in highdose chemotherapy with cyclophosphamide, thiotepa and carboplatin Huitema, A NA 14/06/2005 9 N06LTN Long term neurotoxicity after treatment with the platinum-containing anti-cancer agents cisplatin and oxaliplatin Schellens, JHM NA 26/04/2006 45 N06PFB Pleural fluid bank Heuvel, MM Van den NA 27/09/2006 * N98CTO A phase I study to determine the maximum tolerated doses of Carboplatin and Topotecan administered intravenously every 28 days to patients with malignant tumors Bokkel Huinink, WW Ten I 19/04/1999 41 P05VVD Behoefte-onderzoek naar voedingszorg/voorlichting op de dagbehandeling DiГ«tetiek 08/12/2005 * P06HRM Psychosocial aspects of genetic testing in families at high risk of multiple tumors at various sites and ages Aaronson, N NA 20/06/2006 0 M04CRG Cognitive rehabilitation of glioma patients: A prospective, randomized study Boogerd, W NA 28/04/2004 (21/12/2006) 9 N05THB Totale hersenbestraling met een eenmalige boost bij patienten met solitaire hersenmetastase van een epitheliale tumor - een registratiestudie Dewit, LGH IV 07/04/2005 5 BRAIN / CNS * = not registered at trialbureau type of cancer study 149 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 title study coordinator in NKI-AVL phase E10021 Trial 10021: An IDBBC randomized, double blind, placebo-controlled, multi-center phase II trial of anastrozole (Arimidex) in combination with Iressa (zd1839) or placebo in patients with advanced breast cancer Schornagel, JH II/II 04/11/2003 0 E10031 A phase III trial evaluating the role of ovarian function suppression and the role of exemestane as adjuvant therapies for premenopausal women with endocrine responsive breast cancer; tamoxifen versus ovarian function suppression + tamoxifen versus ovarian function suppression + exemestane Schornagel, JH III 20/07/2005 1 E10041 Micro-array in node-negative disease may Avoid Chemotherapy: a prospective randomized study comparing the 70-gene signature with the common clinical-pathological criteria in selecting patients for adjuvant chemotherapy in node-negative breast cancer Rutgers, EJTh III 02/01/2007 0 E10981 After mapping of the axilla: Radiotherapy or surgery? Rutgers, EJTh III 21/12/2000 331 E16023 Phase I study of lonafarnib (SCH66336) in combination with herceptin plus paclitaxel in Her 2 neu overexpressing breast cancer Schellens, JHM I 01/08/2003 13 E22051 SUPREMO, an MRC phase III randomised trial to assess the role of adjuvant chest wall irradiation in вЂ�intermediate riskвЂ™ operable breast cancer following mastectomy Russell, NS III 23/11/2006 0 M03ARR The implementation of microarrays in cancer diagnosis Linn, SC Pilot 21/10/2003 * M03HTT Open label, comparative, randomized, multicenter, study of trastuzumab (Herceptin) given with docetaxel (Taxotere) versus sequential single agent therapy with trastuzumab followed by docetaxel as first-line treatment for metastatic breast cancer (MBC) patients with HER2-neu overexpression Schornagel, JH II 22/08/2003 3 M03RBC Radiation dose intensity study in breast cancer in young women: a randomized phase III trial of additional dose to the tumor bed (вЂњYoung BoostвЂќ) Bartelink, GMM III 29/03/2004 53 M04GTB A randomized phase II trial of biweekly dose-dense gemzar plus taxol (GT2) and three weekly gemzar plus taxol (GT3) in patients with metastatic breast cancer Schornagel, JH II 19/08/2004 9 M04MAT Microarray analysis in breast cancer to tailor adjuvant drugs or regimens (MATADOR) Linn, SC III 26/04/2004 32 M05BRI Long term risk of breast cancer following treatment of HodgkinвЂ™s disease Russell, NS 05/01/2006 3 BREAST * = not registered at trialbureau 150 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M05HIR Hormonal substitution after prophylactic adnectomy in women with an increased risk for breast- and ovarian cancer due to a genetic predisposition: HIRISE (highrisk women and hormonal substitution exposure) Beurden, M Van IV 31/05/2005 * M06TM2 A randomised, multicenter, prospective, phase III trial investigating (a) Neoadjuvant horminal therapy with exemestane for three versus six months and/or (b) The efficacy and safety of the addition of ibandronate to adjuvant hormonal therapy in postmenopausal women with hormone receptor positive early breast cancer Linn, SC III 26/09/2006 0 M99BLU Lymphatic drainage analysis through preoperative intradermal injection of blue dye in patients undergoing a modified radical mastectomy Nieweg, OE Pilot 04/05/2000 (18/12/2006) 48 N02ANB Phase II study of anastrozole as neoadjuvant treatment in postmenopauzal women with ER+ locally advanced breast cancer Oosterkamp, R II 05/02/2003 1 N03BCC Proteomic profile and treatment response, in patients, with advanced breast cancer, treated with Capecitabine chemotherapy Helgason, HH II 07/10/2003 92 N03IGF The relation between serum and normal breast tissue Insulin-like Growth Factor (IGF) system components Voskuil, DW IV 24/05/2004 40 N04PBC Proteomic profile in patients with primairy breast cancer Helgason, HH other 28/04/2004 165 N04POM Tailored preoperative chemotherapy in stage II or III breast cancer with either a primary tumor over 3 cm in size or a clinically tumor-positive axilla. Rodenhuis, S II 21/03/2005 64 N04RTB Effecten van bestraling op bloedvaten Russell, NS Pilot 05/10/2004 45 N05ECP Prospectief, dubbelblind, gerandomiseerde placebo gecontroleerde farmacologisch interventieonderzoek: evaluatie van de frequentie, duur en intensiteit van opvliegers na interventie met Venlafaxine (EfexorВ®) en Clonidine versus placebo bij vrouwen met vervroegd postmenopauzale klachten tgv adjuvant chemotherapie en/of hormonale therapie voor een mammacarcinoom Schellens, JHM III 10/10/2005 45 N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and prediction of tumor respons to chemotherapy and/or radiotherapy in cancer patients Valdes Olmos, RA other 07/06/2005 6 N05RHN Feasibility study of intensive alkylating chemotherapy in residual HER2/neu-negative breast cancer after preoperative chemotherapy Rodenhuis, S NA 14/11/2005 13 N05TOM A clinical trial to compare the efficacy of the lorad tomosynthesis mammography system to conventional mammography Teertstra, HJ other 07/02/2006 * * = not registered at trialbureau 151 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 type of cancer study title study coordinator in NKI-AVL phase N06IAA Randomized phase II/III study of intensified alkylating agent chemotherapy with peripheral blood progenitor cell support in the preoperative chemotherapy of breast tumors that are deficient for homologous recombination Rodenhuis, S II/II 08/01/2007 0 N06PMK Meaurement of force of the pectoralis major muscle after subpectoral implantation of a prosthesis for the reconstruction of the breast immediately following a skin sparing mastectomy Hage, JJ NA 30/08/2006 * N06TRZ The effect of Trastuzumab treatment on B-cell kinase activities assessed by microarray derived phosphorylation profiles Schellens, JHM NA 11/12/2006 0 N06VNI Meting van de functie van de arm en de bijdrage daaraan van de grote borstspieren voor en na de tweezijdige implantatie van een endoprothese ten behoeve van een borstreconstructie die direct aansluitend aan de huidsparende borstoperatie wordt uitgevoerd Hage, JJ other 26/09/2006 N96NKB The contribution of ATM heterozygosity to the risk of radiation-induced breast cancer Russell, NS Pilot 12/09/1996 (19/12/2006) 255 P00MRI MRI-scan bij patienten met borstkanker ter bepaling van de afmetingen van de afwijking Gilhuijs, KGA NA 06/10/2000 547 P04PKC Patienteninformatiebrief en bewustbereidverklaring micro-array en proteomics Linn, SC NA 04/11/2004 * P05GEO Onderzoek naar de rol van omgevingsfactoren/ leefgewoonten bij het ontstaan van erfelijke borst- en eierstokkanker (GEO-onderzoek II) Rookus, M NA 23/01/2006 * P05NAZ Behoefteonderzoek nazorg/revalidatie na behandeling voor mammacarcinoom Hesselink, S 22/03/2006 * P06VOL Beslissen over in het verleden opgeslagen lichaamsmateriaal Vermeulen, E 23/11/2006 * P99SHB Impact van regelmatige controle (screening) bij vrouwen met een verhoogd risico op borstkanker vanwege een familiaire predispositie Rutgers, EJTh NA 01/05/2000 676 Verwaal, VJ III 23/06/2003 11 GASTRO INTESTINAL E40004 CLOCC trial (chemotherapy + local ablation versus chemotherapy). Randomized phase III study of local treatment of liver metastases by radiofrequency combined with chemotherapy versus chemotherapy alone in patients with F.A.N. unresectable colorectal liver metastases * = not registered at trialbureau 152 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M01BAX POCASTER trial (J-POuch- Colo-Anale anastomose versus Side-To-End colorectale anastomose na preoperatieve radiotherapie en Totale Mesorectale Excisie (TME) bij Rectumcarcinoom; een multicenter, gerandomiseerd onderzoek Zoetmulder, FAN III 06/06/2002 5 M02COL Een landelijke studie naar de waarde van periodiek colonoscopisch onderzoek bij personen met een positieve familieanamnese voor colorectaal carcinoom Cats, A Pilot 19/08/2003 1 M02PCR Postoperative chemo-radiotherapy after surgical resection of gastric and esophageal cancer. A multicenter phase II study of a fixed radiotherapy regimen with concurrent chemotherapy with capecitabine Boot, H II 16/01/2003 44 M03CRE Chemoradiation for irresectable (T4) esophageal cancer - Phase II multi-center study Aleman, BMP II 26/11/2003 2 M05CRC Colorectale polipe/maligniteit studie GUT club. Onderzoek naar de behoefte planning en wachtlijst problematiek bij dikkedarmkanker Cats, A Pilot 15/11/2005 * M05RAX Pre-operative chemoradiotherapy regimen with capecitabine and bevacuzimab in locally advanced rectal cancer. A feasability study (RAX) Marijnen, C II 20/03/2006 4 M06CRI A multicenter randomized phase III trial of neoadjuvant chemotherapy followed by surgery and chemotherapy or by surgery and chemoradiotherapy in resectable gastric cancer (CRITICS-study: ChemoRadiotherapy after Induction chemo Therapy In Cancer of the Stomach) Verheij, M III 13/11/2006 0 M06SCR A multicenter phase III randomised trial comparing total mesorectal excision with pre-operative radiotherapy with or without post-operative oral capecitabine in the treatment of operable primary rectal cancer Cats, A III 09/05/2006 1 M06SUS Surgical gastrojejunostomy or endoscopic duodenal stent placement for the palliation of malignant gastric outlet obstruction; a randomized study. Surgery versus Stent for malignant gastro-duodenal obstruction; SUSTENT study Cats, A III 06/09/2006 0 N02ECC A single institution phase II study of ECC (Epirubicin, Cisplatin, Capecitabin) in locally advanced or metastatic gastric cancer and adenocarcinoma of the oesophago-gastric junction and distal oesophagus Boot, H II 29/08/2002 115 N02NIG Nutrients and the Insulin-like Growth Factor (IGF) system in men and women at increased risk of colorectal cancer Voskuil, DW III 28/01/2003 (29/12/2006) 208 * = not registered at trialbureau 153 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 type of cancer study title study coordinator in NKI-AVL phase N02RCA Post operative chemo-radiotherapy after resection of gastric and esophageal cancer Boot, H I/II 19/12/2002 50 N03PGE Proteomics profile and treatment response, in patients, with advanced gastric or distal esophageal cancer, treated with first-line Cisplatin, and Capecitabine (ECC) or 5FU/LV (ECF) chemotherapy Schellens, JHM other 23/06/2003 88 N05GEA Saving the gastro-epiploic artery at omentectomy and the effect on post-surgical recovering gastric function Zoetmulder, FAN NA 21/03/2006 8 N05MCT A phase II study of treatment with the combination of unlabelled (вЂњcoldвЂќ) and radioactive (вЂњhotвЂќ) metaiodobenzylguanidin (MIBG) in metastatic carcinoid tumors Smits, ME other 05/12/2005 0 N05STP Serum and tissue protein profiling and tumor genetic analysis in patients with potential premalignant conditions or colorectal cancer Smits, ME NA 19/01/2006 49 N06DCM Feasibility of the analysis of platinum-DNA adducts in tumor tissue in cisplatinum treated with advanced gastric cancer Schellens, JHM NA 12/12/2006 * P05FAP Mid to long-term psychosocial impact of the genetic testing among familial adenomatous polyposis (FAP) on families Aaronson, N NA 31/08/2005 506 P06GIN Clinical and psychometric validation of a diseasespecific questionnaire module in assessing the quality of life of patients with GI-related neuroendocrine tumors Taal, BG NA 02/11/2006 * P06POR Decision making in rectal cancer, the costs and benefits of preoperative radiotherapy Marijnen, C NA 05/10/2006 * GYNAECOLOGICAL E55041 A randomised, multicentre, phase III study of Erlotinib vs observation in patients with no evidence of disease progression after first line, platinum-based chemotherapy for high-risk stage I and stage II-IV ovarian epithelial, primary peritoneal, or fallopian tube cancer Driel, WJ Van III 25/09/2006 1 M01EPO An open-label phase IIa trial evaluating the safety and efficacy of EPO906 in patients with advanced ovarian, primary fallopian, or primary peritoneal cancer Bokkel Huinink, WW Ten I/II 22/01/2002 39 M02MCA Measurement of cisplatinum adduct in patients with cervical cancer treated with chemoradiotherapy Verheij, M Pilot 01/05/2002 (18/12/2006) 4 M05HIR Hormonal substitution after prophylactic adnectomy in women with an increased risk for breast- and ovarian cancer due to a genetic predisposition: HIRISE (HighRisk women and hormonal Substitution Exposure) Beurden, M Van IV 31/05/2005 * * = not registered at trialbureau 154 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M05PPO Proteomic patterns in blood and tissue of ovarian cancer patients Driel, WJ Van other 12/01/2006 * M05YDO An open-label multicenter randomized phase III study comparing the combination of DOXIL/CAELYX and YONDELIS with DOXIL/CAELYX alone in subjects with advanced relapsed ovarian cancer Bokkel Huinink, WW Ten III 20/07/2006 1 M06HRT The effect of hormonal replacement therapy on menopausal complaints related to biochemical changes in surgically and naturally postmenopausal women. A prospective observational comparative study Korse, CM NA 25/09/2006 13 M06OVH Phase III randomised clinical trial for stage III ovarian carcinoma randomising between secondary debulking surgery with or without hyperthermic intraperitoneal chemotherapy (OVHIPEC-1) Driel, WJ Van III 04/01/2006 0 N05CGO Randomized clinical pharmacological dose-escalation study to explore both safety and preliminary efficacy and pharmaco-kinetics, -dynamics and -genomics of fixed dose rate gemcitabine and 30- minute standard gemcitabine infusion both administered in combination with carboplatin as second-line treatment in patients with advanced ovarian cancer Schellens, JHM I 08/09/2005 11 M02STA The effect of a statin on the progression of the intimamedia thickness induced by radiotherapy Boogerd, W III 28/01/2003 43 M05PET FDG-PET for avoidance of futile direct laryngoscopies under general anaesthesia with taking of biopsies in patients with suspicion on recurrent laryngeal carcinoma after radiotherapy Brekel, M Van de NA 11/05/2005 5 N02SNL Early detection of lymph node metastasis of squamous cell carcinoma of the larynx with lymphatic mapping and sentinel node biopsy Lohuis, PJFM Pilot 03/04/2002 7 N04LFV Longfunctie na toediening van inhalatiemedicatie (salbutamol-ipratropiumbromide dosisaerosol) met en zonder voorzetkamer bij gelaryngectomeerden Hilgers, FJM III 07/02/2005 8 N04RTB Effecten van bestraling op bloedvaten Russell, NS Pilot 05/10/2004 45 N05HME De kortetermijninvloed van een Heat and Moisture Exchanger op de endotracheale temperatuur en luchtvochtigheid bij gelaryngectomeerden Zuur, JK Pilot 01/09/2005 * N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and prediction of tumour respons to chemotherapy and/or radiotherapy in cancer patients Valdes Olmos, RA other 07/06/2005 6 HEAD AND NECK * = not registered at trialbureau 155 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 type of cancer study title study coordinator in NKI-AVL phase N05TSP Prevention of trismus, swallowing and speech problems in patients treated with chemo-radiation for advanced head and neck cancer Hilgers, FJM NA 14/11/2005 4 P05MDI Patienteninfobrief en bewustbereidverklaring nader gebruik lichaamsmateriaal bij hoofd-halskankerpatiГ«nten Brekel, M Van de NA 13/04/2005 100 P06AIQ De AnaesthetIQ Intubator in de praktijk Hilgers, FJM NA 18/10/2006 0 LEUKAEMIA / MDS M02H49 Randomised Phase III study in eldery patients with a multiple myeloma on the value of Thalidomide added to Melphalan plus Prednison Kerst, M III 31/01/2003 0 M05H65 A randomized phase III study on the effect of Bortezomib combined with Adriamycin, Dexamethason (AD) for induction treatment, followed by High Dose Melphalan and Bortezomib alone during maintenance in patients with multiple myeloma Baars, JW III 03/10/2005 0 M05H68 A randomized phase III study in previously untreated patients with biological high-risk CLL: fludarabine + cyclophophamide (FC) versus FC + low dose alemtuzumab Kerst, M III 21/03/2006 0 E08021 A randomized phase III study of follow up with or without adjuvant Gefitinib (Iressa TM) following chemotherapy in patients with advanced non-small cell lung cancer Burgers, JA III 11/05/2005 (19/12/2006) 0 E08031 A phase II feasibility trial of induction chemotherapy followed by extrapleural pneumonectomy and postoperative radiotherapy in patients with malignant pleural mesothelioma Baas, P II 24/08/2005 10 M03THA Phase III trial of the antiangiogenic agent Thalidomide in patients with malignant pleural mesothelioma after first line chemotherapy (NVALT5) Baas, P III 18/02/2004 52 M04ZON Implementation of Endoscopic Ultrasound guided Fine Needle Aspiration (EUS-FNA) as a part of the standard work-up in lung cancer patients (ZonMw-Project) Burgers, JA IV 25/01/2005 60 M05ALI A randomised phase II study of pemetrexed compared to pemetrexed-carboplatin in pretreated patients with advanced Non Small Cell lung Cancer Burgers, JA II 05/04/2006 21 M05ATD Accurate target definition of non-small cell lung tumors using pathology-validated PET and CT imaging for the optimization of radiation treatment Stroom, J NA 05/01/2006 * LUNG * = not registered at trialbureau 156 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 M05GCP Pharmacogenomic and pharmacokinetic study in patients with advanced non small-cell lung cancer (NSCLC) treated with first-line gemcitabine / platinum combination chemotherapy Schellens, JHM other 13/09/2005 47 M05OPE Daily oral Perifosine (ZEN-004) in combination with radiotherapy in non-small cell lung cancer patients Verheij, M II 10/01/2006 4 M06BOR An open label phase II multicentre clinical trial of single agent bortezomib in patients with malignant pleural mesothelioma Baas, P II 22/11/2006 1 M06NEL Efficacy of neoadjuvant erlotinib in patients with clinical stage I/II non-small cell lung cancer (NSCLC) Klomp, HM I/II 08/11/2006 1 M06PFN International, randomized, open-label, phase III trial of Gemcitabine/Cisplatin plus PF-3512676 versus Gemcitabine/Cisplatin alone as first-line treatment of patients with advanced non-small cell lung cancer Burgers, JA III 22/05/2006 (03/01/2007) 9 N00ALF Endoscopic detection op pre-neoplastic lesions and carcinoma in the bronchial tree with delta-aminolevunic acid fluorescence Baas, P Pilot 30/06/2000 41 N04LSN Feasibility of lymphoscintigraphy and sentinel node biopsy in patients with non-small lung cancer Klomp, HM Pilot 08/09/2004 5 N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and prediction of tumour respons to chemotherapy and/or radiotherapy in cancer patients Valdes Olmos, RA other 07/06/2005 * N99HIM Cytoreduction and hyperthermic intra-thoracic chemotherapy (HITHOC) in patients with pleural mestastases opf thymoma or limited mesothelioma Zoetmulder, FAN Pilot 26/01/2000 24 P04MMI Optimization of conformal radiation therapy of NSCLC by advanced 3-D plannning and multimodality imaging (NKI projekt 2003-2943) Belderbos, JSA I/II 03/06/2004 51 LYMPHOMA - HODGKINвЂ™S DISEASE E20011 A randomized trial of BEAM plus PBSCT versus single agent high dose therapy followed by BEAM plus PBSCT in patients with relapsed HodgkinвЂ™s disease Baars, JW III 23/01/2002 (31/12/2006) 0 E20012 BEACOPP (4 cycleвЂ™s escalated + 4 cycleвЂ™s baseline) vs ABVD (8 cycleвЂ™s) in Stage III & IV HodgkinвЂ™s Lymphoma. Baars, JW III 29/03/2004 0 E20051 The H10 EORTC/GELA randomized Intergroup trial on early PET-scan guided treatment adaptation versus standard combined modality treatment in patients with supradiaphragmatic stage I/II HodgkinвЂ™s lymphoma Baars, JW III 23/11/2006 1 * = not registered at trialbureau type of cancer study title study coordinator in NKI-AVL phase LYMPHOMA - NON-HODGKINвЂ™S 157 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 E20971 A phase III study on low-dose total body irradiation and involved field radiotherapy in patients with localized, stages I and II, low grade non-hodgkinвЂ™s lymphoma Haas, RLM III 02/02/2004 4 M05H55 Efficacy of maintenance therapy with rituximab after induction therapy (R-CHOP versus R-FC) for elderly patients with mantle cell lymphoma not suitable for autologous stem cell transplantation Boer, JP De III 20/07/2005 0 M05H63 Randomized phase III study of Rituximab with intensified CHOP chemotherapy (R-iCHOP-14) versus Rituximab with High-Dose Sequential Therapy and Autologous Stem Cell Transplantation (R-HDT+ASCT) in adult patients (18-65years) with stage II-IV highintermediate or High Risk Diffuse Large B-cell Lymphoma Baars, JW 21/03/2006 0 M05H69 A phase II study of anti-CD52 monoclonal antibody (alemtuzumab, Mab-Campath) with 2-weekly CHOP chemotherapy (Camp-CHOP 14) in patients with mature T-cell non-HodgkinвЂ™s lymphoma Baars, JW II 11/01/2005 0 M05H73 Primary rituximab and maintenance (PRIMA) study: a multicenter phase III open labeled randomized study in patients with advanced follicular lymphoma evaluating the benefit of the maintenance therapy with rituximab after induction of response with chemotherapy plus rituximab in comparison with no maintenance therapy Baars, JW III 21/03/2006 3 M06CMV Cytomegalovirus (CMV) - specific T cell immunity in B cell malignancies Kerst, M other 28/09/2006 * M06H77 Efficacy and safety of a single dose of 14.8 MBq/kg 90Y-ibritumomab tiuxetan (Zevalin) in elderly patient with diffuse large B-cell lymphoma and FDG-PET positive partial remission following first-line R-CHOP therapy. A phase II clinical trial Baars, JW II 23/05/2006 0 N03OFE A randomized phase II/III study of eradication therapy with additional oral fludarabine versus eradication therapy alone in H.pylori positive gastric MALT lymphoma Boer, JP De II/II 17/06/2003 (18/12/2006) 3 N03OFP A phase II study of eradication therapy with additional oral fludarabine in t(11;18) positive gastric MALT lymphoma Boer, JP De II 06/10/2003 4 N03RIM Radioimmunotherapy (131I-antiCD20 monoclonal antibody) as consolidation treatment after remission induction for patients with relapsed or refractory CD20+ B-cell NHL Baars, JW Pilot 25/06/2003 0 N05MIB 99mTc-methoxyisobutylisonitrile (MIBI) for imaging of apoptosis and prediction of tumor respons to chemotherapy and/or radiotherapy in cancer patients Valdes Olmos, RA other 07/06/2005 * * = not registered at trialbureau 158 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 MELANOMA / SKIN M05MSL A phase III multicenter randomized trial of sentinel lymphadenectomy and complete lymph node dissection versus sentinel lymphadenectomy alone in cutaneous melanoma patients with molecular or histopathological evidence of metastases in the sentinel node Nieweg, OE III 11/09/2006 0 M06MDX A randomized, double-blind, multicenter study comparing MDX-010 monotherapy, MDX-010 in combinatio with a melanoma vaccine monotherapy in HLA-A*0201-positive patients with previously treated unresectable stage III or IV melanoma Haanen, JBAG III 13/09/2006 2 M06TDT A3671009: a phase 3, open label, randomized, comparative study of ticilimumab and either dacarbazine or temozolomide in patients with advanced melanoma Haanen, JBAG III 18/05/2006 16 N03CYL Pilot study of topical application of salicylate in familial cylindromas Oosterkamp, R Pilot 26/06/2003 5 N03LAM Longitudinal analysis of melanoma-specific immunity in stage III and IV melanoma patients Haanen, JBAG II 22/08/2003 1 N06TIS Integrated analyses of melanoma-T cell interactions; relevance for immunotherapy Haanen, JBAG NA 29/08/2006 * M06PRO The efficacy of therapeutic compression hoses for prevention of lymphedema after inguinal lymph node dissection in cancer patients Stuiver, M other 05/09/2006 18 N01RIT Identifications of molecular mechanisms involved in radiation-induced telangiectasia Russell, NS Pilot 07/05/2002 21 N03THY Therapeutic management of thymoma and thymic carcinoma: - a prospective registration study based on preoperative risk assessment of local failure Dewit, LGH Pilot 25/11/2003 11 N05CHO The efficacy of cordotomies in patients with chest pain due to primary localized malignancies of the chest Lukas, A II 17/01/2006 * N05SHB Bestudering van basis van normaal S100B Gast, GC De Pilot 21/04/2005 * N99EOL Endoscopisch echo-onderzoek van de centrale luchtwegen onder narcose: feasibility study Baas, P Pilot 14/12/1999 * P05PDE Prevalentie onderzoek naar het voorkomen van decubitus other 14/11/2005 0 III 09/07/2003 7 MISCELLANEOUS SOFT TISSUE / OSTEOSARCOMA E62012 Randomised trial of single agent doxorubicin versus doxorubicin plus ifosfamide in the first line treatment of advanced or metastatic soft tissue sarcoma Rodenhuis, S * = not registered at trialbureau 159 CLINICAL TRIALS activated no pts. (closed) in NKIAVL per 09/01/ 2007 type of cancer study title study coordinator in NKI-AVL phase E62024 Intermediate and high risk localized, completely resected, gastrointestinal stromal tumors (GIST) expressing KIT receptor:a controlled randomized trial on adjuvant Imatinib mesylate (Glivec) versus no further therapy after complete surgery Rodenhuis, S III 25/11/2004 9 E62027 Phase II study of Glivec (imatinib) in locally advanced and/or metastatic soft tissue sarcoma expressing the t(17;22)(q22;q13)translocation resulting in a COL1A1/ PDGF fusion protein i.e. DermatoFibroSarcoma Protuberans (DFSP) and Giant Cell Fibroblastoma (GSF) Rodenhuis, S II 04/11/2004 0 M01EUR EURO-E.W.I.N.G. 99. EUROpean Ewing tumor Working Initiative of National Groups Rodenhuis, S III 18/09/2001 9 M01ROS Phase II study of rosiglitazone in advanced liposarcoma Rodenhuis, S II 24/08/2001 5 E30983 Randomized phase II/III study of Taxol-BEP versus BEP in patients with intermediate prognosis germ cell cancer Schornagel, JH II/II 07/09/1999 3 E30986 Randomized phase II/III study assessing gemcitabine/ carboplatin and methotrexate/carboplatin/Vinblastine in previously untreated patients with advanced urothelial cancer ineligible for cisplatinum based chemotherapy Schornagel, JH II/II 07/05/2002 19 E30994 Randomized phase III trial comparing immediate versus deferred chemotherapy after radical cystectomy in patients with pT3-pT4, and/or N+ M0 transitional cell carcinoma (TCC) of the bladder Kerst, M III 28/01/2003 17 M00LMT Identification of occult lymph node metastases in testicular cancer to select patients for adjuvant treatment. Feasability of a laparoscopic selective retroperitoneal lymphadenectomy Horenblas, S Pilot 14/11/2000 0 M05AIR Atrasentran combined with interferon-apha in renal cell carcinoma: dose escalation phase I study and efficacy phase II study Haanen, JBAG I/II 20/07/2005 6 M06SIL Phase I dose-escalation trial for the combination of sorafenib with interleukin-2 treatment in patients with clear cell renal carcinoma Haanen, JBAG I 20/11/2006 0 M94SAL Salvage regimen incorporating repeated ablative chemotherapy with autologous PSCT, a phase II study Rodenhuis, S II 04/07/1994 24 N02PBI Feasability of partial bladder irradiation with an adaptive margin strategy Pos, F Pilot 14/03/2003 (15/12/2006) 4 URO-GENITAL 160 CLINICAL TRIALS type of title cancer study study coordinator in NKI-AVL phase activated (closed) no pts. in NKIAVL per 09/01/ 2007 N04ILR Neoadjuvant plus adjuvant interleukine-2 around nephrectomy/metastasectomy for patients with a primary renal cell carcinoma with high-risk of progression or with a localized metastasis Gast, GC De II 06/01/2005 7 N04IRC Initial immunotherapy as selection for nephrectomy versus initial nephrectomy followed by immunotherapy in patients with metastatic renal cell carcinoma and the primary tumor in situ Bex, A II 05/08/2004 16 P04TBB Verkorte procedure Protocol Toetsingscommissie van het patient tevredenheidsonderzoek behandeltraject oppervlakkig blaascarcinoom in het Antoni van Leeuwenhoek Ziekenhuis Blok, W De NA 23/12/2004 (19/12/2006) * P06HCP Het effect van hormonale therapie op het cognitief functioneren van patiГ«nten met prostaatkanker Schagen, SSB 09/05/2006 0 * = not registered at trialbureau 161 INVITED SPEAKERS I N V I T E D S PEAKERS 2006 Reuven Agami, Amsterdam, The Netherlands Genetic screens identify cancer-related functions of microRNAs Peter Cullen, Bristol, UK Switching off signalling through the Ras proto-oncogene Anna Akhmanova, Rotterdam, The Netherlands CLASPing microtubules to the cell cortex Edwin Cuppen, Utrecht, The Netherlands Generation and characterization of knockout models in zebrafish and rat Patrick Aloy, Barcelona, Spain Structure-based systems biology: modelling interactions and complexes Ilan Davis, Edinburgh, UK Intracellular mRNA transport and anchoring in Drosophila Haico van Attikum, Basel, Switserland Chromatin dynamics at a chromosomal DNA double-strand break Haryana Dhillon, Sydney, Australia Antipodean Haze - cognitive function research Fran Balkwill, London, UK Inflammation and cancer: the cytokine link Wolfgang Baumeister, Martinsried, Germany Mapping molecular landscapes inside cells by cryoelectron tomography Peter Becker, Munich, Germany Dosage compensation in flies: the end of generalisations Derk ten Berge, Stanford, USA Separate control of cell proliferation and differentiation by Wnt proteins Wendy Bickmore, Edinburgh, UK Higher-order chromatin structure of the mammalian genome and its relationship to gene expression Mary-Ann Bjornsti, Memphis, USA Mechanisms of resistance to DNA topoisomerase I poisons Doug Easton, Cambridge, UK Finding susceptibility genes for breast cancer Francois Fagotto, Montreal, Canada How are different cell types sorted? Insights from the frog embryo Douglas Fearon, Cambridge, UK A stem cell-like phase of CD8+ T cell clonal expansion? Ronald Germain, Bethesda, USA Building a systems level understanding of adaptive immunity: from molecules to models to movies Dan Gottschling, Seattle, USA Loss of heterozygosity in old cells: A model for ageinduced cancer Sergio Grinstein, Toronto, Canada Rapid remodeling of the plasma membrane during phagocytosis Jeanine Boesen, Petten, The Netherlands Molecular imaging Frank Grosveld, Rotterdam, The Netherlands Transcription factor networks and long range interactions Xandra Breakefield, Boston, USA Following a twisted path: from dystonia to the endoplasmic reticulum Per Hall, Stockholm, Sweden Genetic and environmental factors influence the biological behaviour of breast cancer Harmen Bussemaker, New York, USA Sequence-based modeling of gene regulatory circuitry Peter Hordijk, Amsterdam, The Netherlands Subcellular targeting of Rac1 in cell adhesion and directional motility Michael Clarke, Stanford, USA Normal stem cells and cancer stem cells: two sides of the same coin Eran Hornstein, Rehovot, Israel miRNAs in invertebrate development John Condeelis, New York, USA Discovery and testing of an invasion signature for mammary tumors Sandra Jacob, Basel, Switserland Structural biology contributions to the discovery of nilotinib: overcoming imatinib resistance in CML Vincent Colot, Paris, France Studying epigenetic variation and its phenotypic impact in Arabidopsis using epigenomics Ritsert Jansen, Groningen, The Netherlands Genetical genomics - a multifactorial perturbation strategy 162 INVITED SPEAKERS Peter-Michael Kloetzel, Berlin, Germany The UPS and immunoproteasomes: Defining a function to be non-functional? Geert Kops, Utrecht, The Netherlands Mitotic checkpoint kinases in chromosomal instability and cancer Rik Korswagen, Utrecht, The Netherlands Genetic dissection of conserved signal transduction pathways in C. elegans Eugene Krissinel, Cambridge, UK Inference of macromolecular assemblies from crystalline state Philippe Lambin, Maastricht, The Netherlands Individualized radiation oncology: the example of non small cell lung cancer stage III Meindert Lamers, Berkeley, USA Evolution of DNA replication: the crystal structure of E. coli DNA polymerase III Thomas Look, Boston, USA Upstream and downstream modifiers of the BCL-2 survival pathway in cancer Scott Lowe, Cold Spring Harbor, USA Dissecting the p53 tumor suppressor network Anastassis Perrakis, Amsterdam, The Netherlands Developing methods for structural biology Jacques PouyssГ©gur, Nice, France Hypoxia signaling, angiogenesis and cancer John Quackenbush, Boston, USA Learning biology from gene expression profiles Peter Ravdin, San Antonio, USA Individualizing adjuvant therapy: classical methods and new possibilities; adjuvant! online and beyond Eraz Raz, GГ¶ttlingen, Germany Development and guided migration of primordial germ cells in zebrafish Dietrich Rebholz-Schuhmann, Cambridge, UK Towards integration of automated literature analysis into bioinformatics services Gunter Reuter, Halle, Germany Histone demethylation and the control of gene silencing in Drosophila Al Reynolds, Nashville, USA Novel roles for p120-catenin in Rac-dependent inhibition of Rho Nicholas Luscombe, Cambridge, UK Transcription regulation: a genomic network Jeffrey Rosen, Houston, USA Stem/progenitor cells in the etiology and treatment of breast cancer Hiten Madhani, San Francisco, USA Gene regulation in Saccharomyces cerevisiae: MAP kinases, histone variants and beyond Gunter Reuter, Halle, Germany Histone demethylation and the control of gene silencing in Drosophila Umar Mahmood, Boston, USA Optical molecular imaging techniques in cancer research Michael Sharpe, Edinburgh, UK Can cancer nurses manage depression in cancer outpatients? Results of a randomized efficacy trial of SMaRT oncology depression care Jan Paul Medema, Leiden, The Netherlands Apoptotic cell death in cancer Kim Nasmyth, Oxford, UK Genome propagation: the chemistry of sister chromatid cohesion and its dissolution at anaphase Laurence Pearl, London, UK Structural biology of the HSP90 molecular chaperone system Petra Peeters, Utrecht, The Netherlands European prospective investigation into cancer and nutrition: results for breast cancer Lucas Pelkmans, ZГјrich, Switserland Towards systems analysis of endocytosis Dirk SchГјbeler, Basel, Switserland Epigenetic gene regulation: Insights from chromosomal maps Martin Schwartz, Charlottesville, USA Integrins in signal transduction and mechanotransduction Jerry Shay, Dallas, USA Aging and cancer: are telomeres and telomerase the connection? Holger Stark, Gottingen, Germany 3D structure and dynamics of large macromolecular machines determined by single particle cryo-EM Bas van Steensel, Amsterdam, The Netherlands Chromatin genomics 163 INVITED SPEAKERS John Strouboulis, Rotterdam, The Netherlands Characterisation of hematopoietic transcription factor complexes by in vivo biotinylation tagging Hugues de ThГ©, Paris, France Modeling PML/RARA leukemogenesis through therapy response David Tuveson, Philadelphia, USA Modeling and manipulating ductal pancreatic cancer in mice Geerten Vuister, Nijmegen, The Netherlands NMR in structural biology: possibilities, limitations and prospects Robert Van Waardenburg, Memphis, USA Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress 164 PROJECTS P R OJ E C T S SU P PORTED B Y T HE DUTCH CANCER SOCIETY Number of project Title Div. Principal Investigator Starting Date NKI 99-2035 Quantitative assessment of treatment margins and implementation of IX/XIII K Gilhuijs Sept 1999 multimodality imaging in breast conserving therapy XI E Rutgers IX H Bartelink Ended NKI 99-2042 G protein control of cell proliferation and transformation III W Moolenaar Dec 1999 NKI 99-2055 The role of the nuclear inositide pathway in cell cycle progression III N Divecha Dec 1999 NKI 99-2056 Mechanism of cell cycle regulation and control of cellular lifespan by VII M Van Lohuizen May 1999 VII A Berns Aug 1999 VII M Van Lohuizen Jan 2000 VII M Van Lohuizen March 2000 III W Moolenaar Sept 2000 mTHPC mediated photodynamic therapy (PDT): how do drug uptake VIII F Stewart Sept 2000 Apr 2006 and distribution influence tumour and normal tissue response X P Baas X J Schellens XII N Aaronson Dec 2001 Apr 2006 the Polycomb-group and oncogene Bmi1 NKI 99-2058 Development of a mouse model to study the genetic basis of mesothelioma NKI 00-2251 Genetic screens to identify new components of the senescence/ immortalization pathway NKI 00-2253 Functional analysis of BMI1-RING finger interacting proteins and application of microarray technology to identify BMI1-responsive genes NKI 00-2258 Cell-cell communication and connexin signalling: suppression of cell transformation NKI 00-2282 NKI 01-2382 The impact of prophylactic oophorectormy in women from hereditary breast/ovarian cancer families on psychosocial health and symptom NKI 01-2416 H Boonstra experience XI M Van Beurden Gamma irradiation and the generation of novel peptides for MHC VI J Neefjes Febr 2001 Analysis of vaccine-induced melanoma specific T cell immunity: IV/X J Haanen Oct 2002 a combined human and mouse study IV T Schumacher X G De Gast Cancer susceptibility genes that predispose for radiation induced VIII A Broeks breast cancer XIII L Van вЂ™t Veer XII F Van Leeuwen class I molecules NKI 01-2417 NKI 01-2425 NKI 01-2426 Long-term effects of exposure to DES in utero on the risk of hormone XII related cancers M Rookus Nov 2001 Nov 2001 F Van Leeuwen T Helmerhorst UU 01-2438 Control of cellular proliferation in normal and tumor cells by the V B Burgering Sept 2001 P Borst March 2002 PI3K/PKB/Forkhead pathway NKI 01-2473 MRP5 and resistance to purine analogs V J Wijnholds Nov 2006 165 Number of project Title Div. Principal Investigator Starting Date NKI 01-2474 Characterization of MRP3, a newly identified transporter of anti-cancer V P Borst Febr 2002 VII A Berns July 2001 II T Sixma Jan 2001 July 2001 PROJECTS Ended drug NKI 01-2476 Identification of the PIM regulatory network by microarray analysis and high throughput retroviral tagging in compound mutant mice NKI 01-2479 Structure/function analysis of Muts homologs in HNPCC NKI 01-2489 Cloning of novel mammary tumor progression and metastasis genes VI J Hilkens VII A Berns NKI 01-2562 T cell tolerance and immunity during spontaneous tumor development IV P Krimpenfort Sept 2001 NKI 01-2570 Improvement of tumor response by combined modality treatment: a IX M Verheij Oct 2001 The Insulin-like Growth Factor (IGF) system in breast and colorectal VIII D Voskuil Oct 2001 carcinogenesis: dietary intervention and molecular studies XIII/VIII L Van вЂ�t Veer translational approach NKI 01-2579 E Kampman NKI 02-2575 NKI 02-2586 Microarray analysis of breast cancer as a diagnostic tool to guide XIII M Van de Vijver optimal treatment IX H Bartelink XIII/VIII L Van вЂ�t Veer XII F Van Leeuwen Pathological and molecular characteristics of tamoxifen-induced endometrial tumor NKI 02-2589 Jan 2002 Jun 2002 H Hollema XIII P Nederlof VIII A Begg July 2002 Activated proteolysis, a novel rapid response to DNA damage VI R Agami Febr 2002 A functional genomics approach to identify genes which Influence II R Bernards Jan 2002 I J Collard Dec 2002 F Van Leeuwen Aug 2002 Determinants of radiosensitivity in mammalian cells: elucidating mechanisms using a putative dominant negative to DNA polymerase beta NKI 02-2590 NKI 02-2591 tumor cell behavior in vivo NKI 02-2592 Function-based screening for genes involved in Invasion of epithelial tumor cells NKI 02-2593 Regulation of tumor cell motility and invasion by myosin heavy-chain III kinase NKI 02-2605 NKI 02-2634 W Moolenaar Functional genomics of gene regulation by heterochromatin proteins VIII B Van Steensel May 2002 The identification of oncogenic events collaborating with loss of V H Te Riele Nov 2002 function of the retinoblastoma gene family in tumor development and progression NKI 02-2635 The role of BRCA1 and BRCA2 loss-of-function in breast cancer V J Jonkers Jan 2003 NKI 02-2652 Characterisation of a redox regulated PIP3 synthesis pathway III N Divecha Aug 2002 NKI 02-2654 Inclusion of geometric uncertainties in treatment planning for intensity IX M Van Herk July 2002 modulated radiotherapy J Lebesque C Schneider NKI 02-2764 The role of survivin in cell cycle regulation and lymphomagenesis V R Medema S Lens March 2002 Oct 2006 166 Title Div. Principal Investigator Starting Date NKI 02-2771 Nov 2002 PROJECTS Number of project UU 03-2783 The incidence, nature and etiology of cognitive problems following XII S Schagen chemotherapy for cancer XII F Van Dam Cognitive rehabilitation of glioma patients: A prospective, randomized XII study M Taphoorn Oct 2003 M Sitskoorn N Aaronson UL 03-2804 Ex vivo in situ analyses of Graft versus Host mechanisms by Minor IV F Vyth-Dreese Dec 2003 Role of regulated exocytosis in migration, invasion and metastasis I E Roos Jan 2003 Role of the chemokine SDF-1 and its receptor CXCR4 in carcinoma and I E Roos March 2003 E Danen March 2003 Histocompatibility antigen specific T cells; relevance for stem cell transplantation NKI 03-2854 NKI 03-2856 melanoma metastasis NKI 03-2858 Cooperation between the Src oncogene and integrin v 3 in oncogenic I transformation of epithelial cells NKI 03-2859 Improving anti-tumor immunity via TNF receptor family member CD27 A Sonnenberg IV J Borst Aug 2003 and its ligand NKI 03-2860 Feasibility and limitations of T cell receptor gene transfer IV T Schumacher Nov 2003 NKI 03-2861 Peptidases and improving the MHC class I immune response VI J Neefjes March 2003 Characterization of oncogenic potential and mechanism of gene VII M Van Lohuizen Febr 2003 VII M Van Lohuizen April 2003 VII D Peeper Febr 2003 Compound Mrp2(Abcc2) and other drug transporter knock-out mice to VIII A Schinkel Nov 2003 study determinants of pharmacokinetics, toxicity risks and possible X J Beijnen NKI 03-2933 repression of TBX2 NKI 03-2935 Efficient in vitro genetic screens to identify new oncogenes involved in tumor progression NKI 03-2938 Oncogenic function of human DRIL1, a member of the ARID family of retinoblastoma protein-interacting factors NKI 03-2940 J Schellens therapy optimization for anticancer drugs NKI 03-2943 Optimization of conformal radiotherapy of non-small cell lung cancer IX by advanced 3-D planning and multi-modality imaging E Damen April 2003 J Lebesque L Boersma NKI 03-2960 Isolation and functional analysis of novel genes that act in the II R Bernards Jan 2003 R Agami Jan 2003 p16INK4A-cyclin D-pRB-E2F pathway NKI 03-2962 High throughput functional genetic screens for stable loss-of-function VI phenotypes in mammalian cells NKI 03-2963 NKI 03-2964 Studying tumorigenesis using siRNA-expressing vectors Role of LPA receptor signaling in cell migration, invasion, proliferation R Bernards VI R Agami II R Bernards March 2003 III W Moolenaar May 2003 V R Medema Jan 2003 and tumorigenesis NKI 03-2967 The roles of the human cyclin-dependent kinase subunits HsCks1 and HsCks2 in mitotic progression Ended 167 Number of project Title Div. Principal Investigator Starting Date NKI 03-2977 Psychological and behavioral issues in cancer genetics XII E Bleiker Nov 2003 N Aaronson NKI 04-2987 Long-term psychosocial impact of genetic testing among Familial XII E Bleiker Adenomatous Polyposis (FAP) families XII N Aaronson Nov 2004 H Vasen NKI 04-3062 The role of Tiam1 and Rac in Wnt/beta-catenin-induced tumorigenicity I J Collard Aug 2004 NKI 04-3063 Structure/function analysis of the role of polo-like kinase-1 in cellular V R Medema April 2004 transformation II A Perrakis Function of integrin-CD151 complexes in tumor invasion and I A Sonnenberg Nov 2004 Nov 2004 NKI 04-3067 metastasis NKI 04-3068 NKI 04-3069 Long-term risk of second cancers and cardiovascular disease following XII F Van Leeuwen treatment of HodgkinвЂ™s disease IX B Aleman Molecular bar code screens to identify genetic interactions with protein II R Bernards Jan 2004 VI J Neefjes Jan 2004 IV J Borst May 2005 kinases in mammalian cells NKI 04-3078 On the molecular mechanisms of cross-presentation of primimg for an efficient anti-tumor immune response NKI 04-3079 Tumor therapy with ionizing radiation and death ligand Trail: efficacy and key molecular determinants NKI 04-3080 The role of RanBP3 in beta-catenin dependent nuclear signalling and M Verheij VI M Fornerod Sept 2004 Application of Pathology-correlated Imaging to Improve the Accuracy of IX/XIII K Gilhuijs Oct 2004 Surgery and Radiotherapy in Breast-conserving Therapy XI E Rutgers IX H Bartelink Subtle gene modification to study the role of the mismatch repair V H Te Riele complex MSH2/MSH6 in mutation avoidance and tumor suppression II T Sixma Dissecting the role of the pp21 INK4a genes in development and tumor VII P Krimpenfort oncogenesis NKI 04-3082 NKI 04-3084 NKI 04-3086 suppression in mouse models NKI 04-3087 Manipulation of the CD70 locus to monitor and modulate the anti- Nov 2004 Dec 2004 A Berns IV J Borst Febr 2004 Sept 2004 tumor immune response and tumor development NKI 04-3088 NKI 05-3209 NKI 05-3370 NKI 05-3373 Gene-environment interactions in BRCA1/2 breast/ovarian cancer XII F Van Leeuwen families, a project of the Netherlands Collaborative Group on XII M Rookus Hereditary Breast Cancer XIII L Van вЂ�t Veer Psychosocial aspects of genetic testing in families at high risk of XII E Bleiker multiple tumors at various sites and ages. XII N Aaronson VII S Verhoef The role of TGFb and Notch signaling in the development of radiation- VIII F Stewart induced telangiectasia IX N Russell Radiation-induced atherosclerosis: mechanisms and preventive VIII F Stewart intervention strategies NKI 05-3375 The role of PRAME in oncogenesis Nov 2005 April 2005 June 2005 M Daemen IX H Bartelink II R Bernards May 2005 PROJECTS Ended 168 PROJECTS Number of project Title Div. Principal Investigator Starting Date NKI 05-3377 Lipid rafts as novel targets for anti-cancer therapy and induction of III W Van Blitterwijk Jan 2005 apoptosis IX M Verheij High precision radiotherapy in the presence of anatomical changes IX J-J Sonke NKI 05-3378 Nov 2005 M van Herk C Rasch NKI 05-3379 A study of drug resistance mechanisms using large scale RNA II interference screens P Borst June 2005 R Bernards R Beijersbergen NKI 05-3387 Identification and characterization of human tumor suppressor genes VI in the p53 oathway NKI 05-3388 Molecular basis of resistance to anti-estrogens in estrogen-receptor VI positive breast cancer NKI 05-3391 TPPII and the role in antigen presentation by MHC Class I molecules M Voorhoeve Sept 2005 R Agami R Michalides Jan 2005 J Neefjes VI J Neefjes Nov 2005 E Reits NKI 05-3420 NKI 05-3421 NKI 06-3530 Expression profiling to predict outcome after combined radiotherapy XI M Van den Brekel and chemotherapy in head and neck tumors VIII A Begg IX C Rasch X J Beijnen XIII W Bonfrer X J Schellens IV T Schumache Clinical proteomics in breast cancer Generation of human tumor specific T cells with high affinity T cell March 2005 July 2005 receptors NKI 06-3545 High Image guided correction strategies to optimize the precision of IX radiotherapy JJ Sonke Aug 2006 JV Lebesque M Van Herk NKI 06-3558 Genetic screens to identify cancer related functions of human miRNAs VI NKI 2006-3566 Mechanisms of chemotherapy resistance in spontaneous tumors of V genetically modified mice NKI 2006-3589 Significance of the Fanconi anemia caretaker pathway in development R Agami Nov 2006 P Borst J Jonkers V H Te Riele X S Linn and treatment of cancer NKI 06-3706 Towards patient-tailored systemic therapy in breast cancer: a combined approach of translation research and mouse model systems NKI 06-3714 Tumor metastasis: the role of acto-myosin contraction in the I downregulation of E-cadherinmediated cell-cell adhesion J De Rooij A Sonnenberg VU 04-3046 Second primary tumors in hereditary retinoblastoma patients XII F Van Leeuwen RUG 04-3157 Cardiovascular morbidity in testicular cancer survivors: case-control XII F Van Leeuwen study of risk factors and assessment of pharmacogenomic determinants of toxicity Dec 2006 Ended 169 PROJECTS M A J OR PR OJECTS SUPPOR T ED B Y OTHER OR G A N IZ ATION S Granting agency project number Title Div. Principal investigator AICR Cytoskeletal regulation of cell-cell adhesion in oncogenic transformation I J De Rooij EU-LSHC-CT-2004-503224 BRECOSM I J Collard NWO/ALW 815-02-002 Analysis of the function of a novel nuclear envelope protein, nesprin-3, in anchoring I A Sonnenberg A Sonnenberg the nucleus to the intermediate filament system DEBRA (UK) Generation of a mouse model for EBS-MD I NWO/NW TOP Molecular mechanism of renal magnesium wasting I 40-00812-98-06-110 R Bindels K Jalink N Knoers NWO CW 98016 The Process of transposition: three-dimensional structure analysis of Tc1 and Tc3 II T Sixma R Bernards transposase from Caenorhabditis elegans NWO MW 901-02-191 Genetic screens to identify novel cancer-relevant genes II EU QLRT-2001-00988 Structural proteomics in Europe (SPINE) II T Sixma A Perrakis NWO-CW PIO.01.09- Structural analysis of the mechanism of ubiquitin conjugation, a generalized cellular 2002/01690 addressing system that controls protein and DNA stability NWO-CW Structural and functional analysis of the human L1/Alu retrotransposition machinery II T Sixma II A Perrakis 700.51.012-2002/03653 O Weichenrieder NIH Automatic model building and refinement in crystallography II A Perrakis NWO Identification of cancer-relevant genes using a functional genetic approach II R Bernards NWO Identification of cancer-relevant genes using high-hroughput loss-of-function genetic II R Bernards Cancer Genomics screens in mammalian cells. Center for Biomedical Genetics Consortium EU-FP6-LSHC-CT-2003- Identification of novel targets for cancer therapy (INTACT) 506803 EU-FP6-LSCH-2002-503438 Translational and functional onco-genomics: from cancer-oriented genomic II R Bernards VII M Van Lohuizen VII A Berns II R Bernards scrfeenings to new diagnostic tools and improved cancer treatment (TRANSFOG) N I H-1 P50 CA112970-01 System-based predications of responses to cancer therapy II R Bernards NWO-MW Spinozapremie II R Bernards EU-LSGH-2005-018683 The Role of Ubiquitin and Ubiquitin-like Modifiers in Cellular Regulation (RUBICON) II R Bernards T Sixma 170 PROJECTS Granting agency project Title number Div. Principal investigator NWO CW VENI Structural and functional characterization of DNA mismatch repair protein complexes II J Lebbink STW BBC.6035 Exploring new uses of ChhBP as model for ligand-gated ion channel research II T Sixma EU-MRTN-CT-2006-03455 Signal Transduction by Ubiquitination, a Matter of Location (UbiRegulators) II T Sixma CBG Center for Biomedical Genetics Participation in Top-Research School II T Sixma 700.53.407 R Bernards EU-MTKI-CT-2005-029655 NWO CS VIDI 700.54.427 Breast Cancer Biomarkers and Functional Mediators: Harnessing the New Wealth of II R Bernards -Omic Data (TARGET-BREAST) VIII L Van вЂ�t Veer L1 and Alu elements: Structural and functional analysis of human non-LTR II O Weichenrieder II A Perrakis II A Perrakis retrotransposons EU-FP6-LSHG-CT-2003- Biocrystallography (X) on a highly integrated technology platform for European 503420 structural genomics (BIOXHIT) EU-FW6-LSHG-CT-2004- A multidisciplinary approach to determine the structures of protein complexes in a 512028 model organism (3D-Repertoire) MAX-INF EU Network European Macromolecular Crystallography infrastructure Network, 2004-2008 II A Perrakis EMBO Fellowship The structural and functional basis for coupling of ligand activation to channel opening II C Ulens T Sixma in the nicotinic acetylcholine receptor CBG Lysophospholipid receptor signaling III W Moolenaar EU HPMF-CT2001-01218 Stress mediated regulation of phosphositides III N Divecha The Leukemia and A novel form of gene regulation that provides specificity by preventing promiscuity III F Van Leeuwen Lymphoma Society ((Special Fellow 3409-04) NWO-ALW-VIDI The role of histone methylation in silencing and transcriptional memory III F Van Leeuwen European TMR (864-04-003) EU-FP6-LSGH-CT-2004- NET member of the Epigenome Network of Excellence (EPIGENOME) 503433 III F Van Leeuwen VII B Van Steensel VII M Van Lohuizen NWO Modulation of autotaxin activity by small molecules III H Ovaa NWO Chemical biology of ubiquitin and ubiquitin-like modifications III H Ovaa ZON-MW 912-04-32 The role of CD27/CD70-regulated cell survival in shaping effector and memory T cell IV J Borst responses ZON-MW VIDI 917-056-328 Role of translesion DNA synthesis in immunity and cancer development IV H Jacobs EU-FP6-LSCH-CT-2005- Adoptive engineered T-cell targeting to activate cancer killing (ATTACK) IV T Schumacher 018914 171 Granting agency project number Title Div. PROJECTS Principal investigator EU-FP6-2005- Determining the relationships between haematopoietic lineages using genetic IV T Schumacher Mobility-7-039477 barcoding (Genetic Barcoding) IV S Naik Treatment of HPV positive penile cancer by introduction of HPV E7-specific T cell IV J Haanen ZON-MW 432-00-001 immunity thourgh DNA vaccination T Schumacher XI S Horenblas LSBR MHC multimer-based adoptive T cell therapy IV T Schumacher TI-Pharma project TNF ligands in cancer IV J Borst UVA 2006-3606 Integrated analyses of melanoma-T cell interactions in situ; relevance for IV F Vyth-Dreese immunotherapy NWO-MW Program Phosphoinositide 3-kinase signaling: Regulation and function V 901-28-092 NWO MW Vidi 917.36.347 B Burgering P Coffer Mammary tumorigenesis in conditional tumor suppressor gene knockout mice: V J Jonkers V H Te Riele VI R Agami V L Wessels disease progression, gene discovery and cellular pathways EU FP6 -LSCH-CT-2004- Mutant p53 as target for improved cancer treatment (MUTP53) 502983 NGI-BioRange SP 1.3.2 Developing clinical predictors based on high-dimensional genomics data, pathway information and directed experimentation NGI-BioRange SP 1.2.1 A unifying ramework for data-driven pathway discovery V L Wessels NGI-NWO-Horizon Discovering interactions and functional groups of oncogenic lesions V L Wessels Subtle is the oligo: in vitro and in vivo gene modification by single-stranded DNA V Breakthrough Project 050-71-045 Horizon Project 050-71-051 oligonucleotides ZonMW 912-02-073 The pathofysiological relevance of transporters in the enterohepatic cycle V (2002-2006) R Oude Elferink P Borst Susan G. Komen Breast Identification of collaborating mutations in Brca1 and Brca2 mouse mammary tumors Cancer Foundation BCTR by genome-wide screening for chromosomal imbalances V J Jonkers V P Derksen V C Wielders 0403230 NWO-ZonMw Veni Functional characterisation of E-cadherin signalling in breast cancer progression and 016.056.135 metastasis Nationaal Regie-Orgaan Enzymatic production of low-complexity RNAi libraries for high throughput loss-of- Genomics, Horizon function screening Doorbraakproject 050-71-348 UCSF Prion protein localization VI P Peters NWO 901-02-250 Role of the nuclear pore complex in b type catenin nuclear signalling VI M Fornerod NWO-ALW 811-38-001 Identification of specific transport pathways through the nuclear pore complex using in VI M Fornerod vitro nuclear reconstitution 172 PROJECTS Granting agency project Title number Div. Principal investigator NWO-CW 700.51.013- Function and structure characterisation of the RILP/RAB7 complex controlling VI J Neefjes 2002/02696 lysosomal transport II A Perrakis NWO ZON MW PGS Manipulation of the phagosomal pathway induced by salmonella and mycobacteria VI J Neefjes 912-03-026 and the host immune response HPMF-CT-2002-02146 Investigating the role of proteins associated with the nuclear pore complex in Marie Curie Individual regulating gene expression and DNA double stranded break repair T Ottenhoff VI M Fornerod CD1 trafficking and loading of Mycobacterium leprae lipids in maturing dendritic cells VI P Peters EU-FP5- QLK2-CT- Pre-clinical improvement of combined immunotherapy and chemotherapy for new VI P Peters 2002-81628 variant Creutzfeld-Jacob disease EU-FP5- QLK5-CT- Passage from intestine to brain: assessing the role of dendritic cells in capturing, VI P Peters 2002-01044 expanding and disseminating prions EUвЂ“FP6-LSHB- Development of a pre-clinical blood test for prion diseases (ANTEPRION) VI P Peters Immunological and structural studies of prion diversity (IMMUNOPRION) VI P Peters EUвЂ“FP6-FOOD- Understanding prion strains and species barriers and devising novel diagnostic VI P Peters CT-2006-023183 approaches (STRAINBARRIER) R01 GM58615 National Biogenesis of transport vesicles coated by COPI VI P Peters Genome wide search for DNA damage checkpoint functions in human cells VI R Agami VI R Agami fellowship Dutch Leprosy Foundation 702.02.20 CT2006-019090 EUвЂ“FP6-FOODCT-2006-023144 Institutes of Health Hsu ESF NWO EURYI-award 044.016.001 E Zlotorynski EU-MEIF-CT-2005-514788 Identification and characterization of novel genes involved in DNA damage-response VI pathways in humans (DNA damage RNAi) E Zlotorynski R Agami NIH 2 R01 AI028973-17 Pathways of antigen presentation by CD1 a, b and c VI P Peters EU-MRTN-CT-2004-512385 High throughput development of drugs for immunotherapy of autoimmune diseases VI J Neefjes (Drugs for therapy) EU-MRTN-CT-2003-504227 Microban VI J Neefjes EU-FP6-2005-EIF 041382 The role of the intracellular peptidases in the immune system and in the cellular VI J Neefjes VII M Van Lohuizen metabolism (PROTEUS) NWO Pionier 906-99-001 Mechanism of gene repression by mammalian Polycomb-group proteins: connections to cell cycle regulation and other silencing processes EU QLG1-1999 00622 Inducible Melanoma Model VII A Berns EU 5th framework Molecular mechanisma of senescence and ageing VII M Van Lohuizen QLK6-CT-2001-00616 173 Granting agency project number Title Div. PROJECTS Principal investigator NWO genomics program Genome-wide mapping of oncogenic pathways by high-throughput insertional VII A Berns 2002, 050-10-008 mutagenesis NWO/Vidi 864-02-005 Novel functional genomic screens to identify pathways that protect mouse and human M Van Lohuizen VII D Peeper Identification of novel targets for cancer therapy (MUGEN) VII A Berns Whole-genome analysis of the regulation of chromatin structure and gene expression VII B Van Steensel BSIK program: 03038 Stem cells in development and disease VII M Van Lohuizen Hersenstichting Nederland: Onderzoek naar de rol van het Bmi1 Polycomb groep gen in het reguleren van neurale VII M Van Lohuizen 12F04(2).14 stamcel identiteit Centre of Biomedical Participation in Top-Research School together with Universities of Utrecht, Leiden, VII A Berns Genetics Rotterdam and Hubrecht Laboratory NOW R.O. Cancer Genomic Centre cells against oncogenic transformation by mutant RAS EU Network of Excellence. LSHG-CT-2005-005203 European Young Investigator Award M Van Lohuizen VII A Berns M Van Lohuizen EU-LSGH-CT-2006-018739 Platforms for biomedical discovery with human ES cells (ESTOOLS) VII M Van Lohuizen NWO-Genomics Program Chromatin profiling of human gene silencing complexes VIII B Van Steensel DIRF: An in vivo screen to identify proteins that bind to a genomic region of interest VIII B Van Steensel Multi-gene chromatin domains VIII B Van Steensel Human Frontier Science Whole-genome analysis of the interplay between chromatin context and gene VIII Program RGP56/2003 expression control 050-10-002 Netherlands Genomic Initiative 050-71-026 Netherlands Genomic Initiative 050-71-048 B Van Steensel H Bussemaker K White STW 2004-2008 Generation and validation of cytochrome P450 3A knockout and transgenic mice as VIII A Schinkel (BFA.6165) tools for improved drug development and research X J Schellens J Beijnen J Smit EU-LSCH-CT-2005-018911 Detections of minimal tumor cell admixture in patients with breast cancer (DISMAL) VIII L Van вЂ�t Veer EU-LHC-CT-2004-503426 Translating molecular knowledge into early breast cancer management: building on the VII L Van вЂ™t Veer Breast International Group network BIG for improved treatment tailoring (TRANSBIG) XI E Rutgers EU-LSGH-CT-2004-505785 BioCare IX H Bartelink EU-015997 EUROCAN + PLUS EU DGXII IX H Bartelink VII A Berns A code of practice for dosimetry of boron neutron capture therapy (BNCT) in Europe IX B Mijnheer Application of an electronic portal imaging device for dosimetry in radiotherapy IX B Mijnheer SMT4-CT98-2145 STW BGN 33-3240 174 PROJECTS Granting agency project number Title Div. Principal investigator STW BGN 33-3240 Portal image analysis IX M Van Herk Pfizer and Roche TEAM II: A randomized, multicentre, prospective phase III trial investigating: a. X Neoadjuvant hormonal therapy with exemestane for three versus six months and/or b. S Linn C Van de Velde The efficacy and safety of the addition of ibandronate to adjuvant hormonal therapy in postmenopauzal women with hormone receptor positive early breast cancer NWO 612-066-201 Learning and using understandable prognostic models in medicine X P Lucas B Taal NCI PO1 CA29605-12 Multicenter selective lymphadenectomy trial XI O Nieweg Atos Medical Development and evaluation of a third generation ProvoxTM voice prosthesis XI F Hilgers I Tan A Ackerstaff Erasmus MC, 3M company Clinical, virological and immunological studies of imiquimod in VIN XI M Van Beurde T Helmerhorst Michel Keijzer Foundation Airway pathophysiology after total laryngectomy and the influence of heat and XI F Hilgers XI F Hilgers moisture exchangers Breuning-ten Cate Intelligibility of tracheoesophageal speech Foundation L Pols International Health IVF treatment, unexplained subfertility and number of retrieved oocytes in relation to Foundation age at menopause IARC International BRCA1/2 Carrier Cohort Study XII F Van Leeuwen Nefkens Foundation Effects of chemotherapy on cognitive skills and evoked potentials in women receiving XII F Van Dam adjuvant treatment for breast cancer X W Boogerd An international field study of the reliability and validity of the EORTC QLQ-C30 and a XII N Aaronson EORTC 15011-30011 XII F Van Leeuwen C Burger disease-specific questionnaire module (QLQ-PR25) for assessing the quality of life of patients with prostate cancer Lance Armstrong Long term risk of second cancers and cardiovascular disease following treatment of XII F Van Leeuwen Foundation testicular cancer IX B Aleman Pharmacia BV MO2TEA Assessment of neuropsychological sequelae of tamoxifen and exemestane in XII postmenopausal women with early breast cancer Medical Centre Alkmaar The use of health related quality of life assessments in daily clinical oncology nursing S Schagen F Van Dam XII N Aaronson XII N Aaronson XII N Aaronson XII F Van Leeuwen practice: A community hospital based intervention study Organon Hormonal substitution after prophylactic adnectomy in women with an increased risk of breast and ovarian cancer due to a genetic predisposition: HIRISHE (High-risk women and hormonal substitution exposure) University Hospital Zurich Physical exercise to improve health related quality of life in peripheral stem cell and (UHZ) bone marrow transplantation receipts Cancer Genomics Center The benefits and risks of cancer genomics for society N Aaronson Granting agency project number Title Clinical Research Foundation Effects of chemotherapy on neurogenesis and learning abilities in experimental animal / University of Groningen research EU-2004-012926 Radiation exposure at an early age: Impact of genotype on breast cancer risk (GENE Div. XII 175 PROJECTS Principal investigator S Schagen F Van Dam XII F Van Leeuwen RAD RISK) EU QLG1-CT-2002-30242 PCTCG overview Biom O Dalesio EU eTEN program- C510736 Trans European Network for patient randomization in clinical trials Biom O Dalesio E Van der Donk CKTO 2006-02 NVALT-Oncology A multicenter randomized phase III trial of neo-adjuvant chemotherapy followed by M Verheij surgery and chemotherapy or by surgery and chemoradiotherapy in respectable gastric C Van de Velde cance A Cats Statistical Center NVALT Clinical Trials in oncology Biom O Dalesio 176 PERSONNEL INDEX PE R S O NNEL INDEX Aaronson, Neil 124 Aarts, Marieke 49 Achachah, Mohamed 133 Achanta, Geetha 62 Ackerstaff, Annemieke 114 Adriaansz, Sandra 103 Agami, Reuven 62 Albers, Harald 38 Alderden, Carolien 103 Alderliesten, Tanja 133 Aleman, Berthe 88, 128 Alendar, Andrej 71 Antonini, Ninja 143 Arens, Ramon 43 Armstrong, Nicola 58 Atsma, Douwe 132 Axwijk, Priscilla 133 Baank, Saskia 132 Baars, Danny 143 Baars, Joke 102 Baars, Philippe 132 Baas, Paul 80, 102 BaasвЂ“Vrancken Peeters, Marie-Jeanne 114 Bais, Evert 102 Bakker, Arne 43 Bakker, Martine 132 Bakker, Sietske 49 Ballast, Nienke 131 Balm, Alfons 114 Bardina, Elena 49 Bartelink, Harry 86, 88 Beaudry, Jean Bernard 69 Beers-Bauhuis, Carolien 132 Begg, Adrian 78 Beijersbergen, Roderick 26 Beijnen, Jos 82, 102 Belderbos, Jose 88 Ben Jelloul, Marouane 30 Bergman, AndrГ© 71 Berkers, Celia 38 Bernad-Fernandez, Rafael 64 Bernards, RenГ© 24 Berns, Anton 71 Berns, Katrien 24 Besnard, Peter 133 Besseling, Gijs 128 Betgen, Anja 88 Bex, Axel 115 Bijker, Nina 88 Bin Ali, Rahmen 71 Bins, Adriaan 45 Bisschops, Ivo 128 Bleiker, Eveline 124 Bloemers, Monique 88 Blom, Marleen 69 Boekhout, Annelies 103 Boer, Mandy 75 Boerhout, Hermien 53 Boerrigter-Barendsen, Lucie 132 Bohoslavsky, Roman 143 Bolijn, Marije 82 Bonfrer, Hans 132 Boogerd, Willem 102, 126 Boot, Henk 102 Boren, Joan 71 Borlado, Luis 24 Borst, Gerben 88 Borst, Jannie 41 Borst, Piet 51 Bos, Erik 67 Bosma, Astrid 84 Boss, David 102 Boutmy-de Lange, Majella 133 Bouwman, Peter 53 Braaf, Linde 84 Braat, Koen 69 Brand, Bob 88 Braunschweig, Ulrich 56 Broeks, Annegien 84 Brohet, Richard 128 Bronk, Marieke 133 Brouwers, Elke 102 Bruggeman, Sophia 69 Bruin, Natascha 132 Bruin, Sjoerd 84 Bruinvis, Iain 88 Buchwald, Gretel 28 Buckle, Tessa 132 Bueno de Mesquita, Jolien 55, 131, 132 Buikhuisen, Wieneke 102 Buitelaar, Dick 115 Bultsma, Yvette 36 Buning, Marian 132 Burg, Danny 38 Burger, MatthГ© 115 Burgers, Sjaak 102 Calbo, Joaquim 71 Calogero, Anna 43 Cardous-Ubbink, Mathilde 128 Carreno, Monique 143 Caspers, Anky 126 Cats, Annemieke 102 Cavalli, Silvia 38 Celie, Patrick 30 Chang, Qing 62 Christodoulou, Evangelos 30 Citterio, Elisabetta 69 Coccoris, Miriam 43 Cohen, Serge 30 Collard, John 19 Coppen, Vincent 143 Copper, Marcel 114 Dahmen, Rutger 131 Dalesio, Otilia 143 Damen, Carola 102 Damen, EugenГЁ 88 Danhof, Tinneke 126 Dannenberg, Jan Hermen 71 De Boer, Jan Paul 102 De Boer, Roel 88 De Boer, Suzanne 143 De Bois, Josine 88 De Bruin, Marieke 128 De Bruin, Michiel 55 De Gast, Bert 102 De Groot, Renate 84 De Haan, Patricia 88 De Haas, Marcel 51 De Jager, Steven 128 De Jong, Daphne 132 De Leeuw-Mantel, Geri 128 De Marco, Valeria 30 De Ridder, Jeroen 58 De Rooij, Johan 17 De Visser, Karin 53 De Vries, Diederick 30 De Vries, Evert 41 De Vries, Hilda 71 De Vries, Remco 115 De Vries, Sandra 49 De Waal, Marjolijn 143 De Widt, John 34 De Wit, Elzo 56 De Witte, Moniek 43 De Wolf, Cocky 51 Deenen, Maarten 82, 102 Dekker, Marleen 49 Dellemijn, Trees 45 Delzenne-Goette, Elly 49 Derks, DaniГ«lle 115 Derksen, Patrick 53 Desmet, Christophe 73 Dewit, Luc 88 Dijkhuis, Simone 128 Dijkstra, Jitske 143 Dirac, Annette 24 Divecha, Nullin 36 Doodeman, Petra 132 Doodeman, Valerie 103 Douma, Kirsten 124, 131 Douma, Sirith 73 Doumont, Gilles 53 Droogendijk, Helga 102 Drost, Brigit 115 Drost, Jarno 62 Drost, Rinske 53 Dubbelman, Ria 103 Duppen, Joop 88 Duursma, Anja 62 Ebbens, Fenna 114 Egan, David 26 Eggens, Elke 126 Eichhorn, Pieter 24 Ekhart, Corine 102 177 PERSONNEL INDEX Ellenbroek, Saskia 19 Elouarrat, Dalila 36 Emanuel, Miret 133 Engelsma, Dieuwke 64 Engwegen, Judith 102 Epping, Mirjam 24 Erven-Dufournij, Brigitte 143 Evers, Bastiaan 53 Faber, Alex 40 Fabius, Armida 26 Faesen, Alex 28 Feddema, Wouter 51 Feenstra, Christel 132 Fish, Alex 28 Fles, Renske 84 Floot, Ben 78, 80 Foekema, Joke 103 Foijer, Floris 49 Fons, Guus 115 Fornerod, Maarten 64 Frankfort, Suzanne 102 Frederiks, Floor 40 Frenay, Michel 88 Gaasbeek, Ruben 71 Gadiot, Jules 71 Gast, Marie-Christine 102 Gebretensae, Abadi 103 Gehring, Karin 124 Geiger, Thomas 73 Genest, Paul-AndrГ© 51 Gerard, Audrey 19 Gerlach, Carmen 43 Gerritsma, Miranda 124 Geurts, Tom 114 Geutjes, Ernst-Jan 24 Gilhuijs, Kenneth 133 Godsave, Sue 67 Gomez, Raquel 45 Gonggrijp, Steven 114 Greil, Frauke 56 Griekspoor, Alexander 60 Groothuis, Tom 60 Guelen, Lars 56 Gundy, Chad 124, 126 Gunnarsdottir, Sjofn 51 Haanen, John 45, 102 Haas, Rick 88 Haenen, Inge 131 Hage, Joris 115 Hajdo-Milasinovic, Amra 19 Halfwerk, Hans 86 Halstead, Jonathon 36 Hannemann, Juliane 86 Haramis, Anna Pavlina 69 Hart, Guus 88 Hau, Song-Hieng 143 Hauptmann, Michael 58 Heemsbergen, Wilma 88 Heessen, Stijn 64 Heideman, Marinus 47 Heijens, Claudia 102 Helgason, Helgi 102 Hendriksen, Jolita 64 Hengeveld, Trudi 32 Hermus, Marie-Christine 132 Hibbert, Rick 28 Hiemstra, Annelies 143 Hijmans, Marielle 24 Hilarius, Doranne 124 Hilge, Mark 30 Hilgers, Frans 114 Hilkens, John 75 Hilkmann, Henk 38 Hillebrand, Michel 103 Hoebers, Frank 78, 88 Hoefnagel, Cornelis 132 Hofland, Ingrid 78 Hofstede, Diederik 115 Hogan, Greg 56 Hogervorst, Frans 84, 132, 133 Holstege, Henne 53 Holtkamp, Marjo 103 HГ¶mig, Cornelia 73 Hoogendoorn, Lisette 128 Hooning, Maartje 128 Hoopman, Rianne 124 Hoppes, Rieuwert 38 Horenblas, Simon 115 Horlings, Hugo 86 Houben, Anna 32 Houben, Diane 67 Hoving, Saske 80 Huang, Sidong 24 Huisman, Bregje 128 Huitema, Alwin 102 Huitink, Hans 115 Hulsman, Danielle 69 Hundscheid, Stephanie 86 Huseinovic, Angelina 30 IJpenberg, Annemieke 71 Jacobs, Heinz 47 Jacobs, Jacqueline 71 Jager, Agnes 102 Jalink, Kees 23 Jansen, Edwin 88 Jansen, Hans 67 Jansen, Robert 102 Janssen, Esther 124, 128 Janssen, Lennert 60 Jeanson, Kiki 128 Joling, Jeanine 43 Jones, David 36 Jongmans, Petra 114 Jongsma, Johan 71 Jonkers, Jos 53 Joosse, Simon 84 Joosten, Krista 30 JГ¶rger, Markus 102 Jorritsma, Annelies 45 Kaas, Reinie 114 Kalverda, Bernike 64 Kappers, Ingrid 114 Karsenberg, Kim 128, 131 Kartachova, Marina 132 Kassiem, Mobien 30 Kedde, Martijn 62 Keessen, Marianne 103 Keijzer, Christiaan 115 Keizer, Ron 102 Keller, Anna 41 Kerkdijk, Desiree 114 Kersbergen, Ariena 51 Kerst, Martijn 102 Ketema, Mirjam 17 Keune, Willem-Jan 36 Kim, Yeung-Hyen 45 Kimmings, Nikola 114 Klarenbeek, Jeffrey 34 Klein Zeggelink, William 133 Klerkx, Edwin 143 Klokman, Willem 128 Klomp, Houke 114 Klop, Martin 114 Klous, Marjolein 102 Kluijt, Irma 133 Knegjens, Joost 88 Knipscheer, Puck 28 Knol, Remco 132 Knols, Ruud 124 Kok, Marleen 55, 84 Kool, Jaap 69 Koolen, Stijn 102 Koopman-Kroon, Ciska 103 Koops, Wim 133 Koorn, Jenneke 143 Korse, Tiny 124, 132 Kortekaas, Bettina 143 Kortlever, Roderik 24 Kreft, Maaike 17 Kregel, Eva 53 Kreike, Bas 86, 88 Krenn, Bea 67 Kreukels, Baudewijntje 126 Krijger, Peter 47 Krimpenfort, Paul 71 Kristel, Petra 86 KrГ¶ger, Robert 133 Kroon, Bin BR 114 Kroon, Frans 114 Kruse, Jacqueline 80 Kuenen, Marianne 128 Kuijl, Coen 60 Kuiken, Johan 26 Kuikman, Ingrid 17 Kuilman, Thomas 73 Kuiper, Maria 103 Kujala, Pekka 67 Lafeber, Albert 132 Lagas, Jurjen 76 Lai, Carmen 58 178 PERSONNEL INDEX Lambooij, Jan Paul 71 Lammens, Chantal 124 Langerak, Petra 47 Langeslag, Michiel 23 Le Sage, Carlos 62 Lebbink, Joyce 28 Lebesque, Joos 88 Leering, Suzanne 126 Legdeur, Monica 128 Leijte, Joost 115 Linders, DorothГ© 132 Linn, Sabine 55, 84, 102 Lippuner, Christoph 60 Littler, Dene 30 Liu, Xiaoling 53 Lohuis, Peter 114 Loo, Claudette 133 Los, Alrik 34, 36 Lotz, Carina 45 LubsenвЂ“Brandsma, Lotti 115 Lukas, Anne 102 Maas, Chiel 41 Madalinska, Joanna 124 Madiredjo, Mandy 24 Mahn-Schaefers, Marianne 143 Mallo, Henk 103 Manders, Peggy 128 Mandjes, Ingrid 143 Mandjes, Miranda 58 Marchetti, Serena 82 Margadant, Coert 17 Marijnen, Corrie 88 Marsman, Marije 60 Masselink, Harry 88 Masztalerz, Agnieszka 21 Mattiroli, Francesca 28 McDermott, Leah 88 Meijer, Joost 21 Meijerman, Irma 82 Meijnen, Philip 114 Meindertsma, Tineke 143 Meinhardt, Wim 115 Menendez, Victoria 60 Mertens, Sander 19 Meuleman, Wouter 58 Michalides, Rob 66 Michaloglou, Chrysiis 73 Middendorp, Sabine 41 Mijnheer, Ben 88 Minderhoud, Tom 89 Modder, Carla 143 Molloy, Tim 84 Monserrat, Veronica 60 Mooij, Thea 128 Mook, Stella 84, 132 Moolenaar, Wouter 32 Moonen, Luc 88 Moorman, Celine 56 Mulder, Ina 128 Mulder, Lennart 86 Mullenders, Jasper 24 Muller, Saar 132, 133 Muussen, Rick 143 Nacerdine, Karim 69 Nagel, Remco 62 Nagtegaal, Tanja 124 Nair, Suresh 62 Nan-Offeringa, Lianda 103 Nawijn, Martijn 71 Nederlof, Petra 84, 128, 132, 133 Neefjes, Jacques 60 Neering, Herman 102 Neijenhuis, Sari 78 Neijssen, Joost 60 Nieweg, Omgo 114 Nijkamp, Wouter 26 Nijwening, Jeroen 26 Noback, Sonja 69 Nol, Annemarie 103 Nooijen, Anke 84 Nooijen, Willem 132 Notenboom, Valerie 28 Nuyen, Bastiaan 102 Nuyten, Dimitry 86, 88 Nyst, Heike 114 Olde Damink-Van Rosmalen, Agnes 128 Oldenburg, Hester 114 Olijve, Albert 103 Olivier, Rolien 115 Olszewska, Agnieszka 88 Ong, Nico 67 Oostendorp, Roos 82, 102 Oosterhuis, Koen 45 Oosterkamp, Rianne 24 Opperdijk, Vera 88 Oude Vrielink, Joachim 62 Ouwehand, MariГ«t 103 Ouwens, Gabey 128 Ovaa, Huib 38 Paape, Anita 133 Pagie, Ludo 56 Pajic, Marina 51 Pala, Zeliha 76 Pameijer, Frank 133 Pang, Baoxu 60 Paul, Petra 60 Paulus van Pauwvliet, Cecile 143 Peeper, Daniel 73 Pegtel, Michiel 19 Pengel, Kenneth 89, 133 Peperzak, Victor 41 Pereira-Arias, Lenka 132 Peric-Hupkes, Daniel 56 Perrakis, Anastassis 30 Peters, Peter 67 Peterse, Hans 86, 132 Pickersgill, Helen 56, 64 Piek-den Hartog, Marjan 114 Pierson, Jason 67 Pietersen, Alexandra 69 Pijpe, Anouk 128 Plug, Rob 133 Pluim, Dick 82 Ponsioen, Bas 23, 32 Pool, Bert 132 Pos, Floris 88 Pramana, Jimmy 78, 114 Prasad, Asheeta 69 Prevoo, Warner 133 Prieur, Alexandre 73 Pritchard, Colin 71 Proost, Natalie 71 Pruntel, Roelof 133 Quaak, Susanne 103 Rasch, Coen 88 Reinders-Som, Anneke 143 Remeijer, Peter 88 Remmelzwaal, Jolanda 143 Repanas, Kostas 30 Res, Pieter 67 Reumer, Annet 28 Roberts, Daniel 143 Rocha, Nuno 60 Rodenhuis, Sjoerd 86, 102 Rodenko, Boris 38 Roelvink, Marja 103 Romeijn, Martijn 67 Rondaij, Mariska 66 Ronday, May 115 Rookus, Matti 128 Roos, Ed 21 Rooze, Lyandra 132 Rosing, Hilde 102 Rossi, Maddalena 89 Rottenberg, Sven 51 Rowland, Benjamin 24 Rubio, Miguel 24 Ruevekamp, Marjan 80 Ruijs, Marielle 84 Ruijter-Schippers, Henrique 132 Russell, Nicola 80, 88, 128 Russo, Francesco 56 Rutgers, Emiel 86, 114, 128 Rygiel, Tomasz 19 Rzadkowski, Adrian 32 Sachs, Norman 17 Salomon, Izhar 60 Salverda, Govert 88 Savaskan, Nicolai 32 Schagen, Sanne 126 Schellens, Jan 82, 102 Schepers, Koen 43 Schilder, Christien 126 Schinkel, Alfred 76 Schippers-Gillissen, Carla 132 Schlief, Angelique 133 Schmidt, Marjanka 84, 128, 132 Schneider, Christoph 88 Schnell, Silke 47 Scholten, Marijke 143 179 PERSONNEL INDEX Schornagel, Jan 102 Schot, Margaret 103 Schrier, Mariette 62 Schumacher, Ton 43 Schutte, Peter 115 Schwarz, Marco 88 Sein, Johan 45 Siedschlag, Christian 133 Simons, Monique 129 Sinaasappel, Michiel 132, 133 Sivro-Prndelj, Ferida 132 Sixma, Titia 28 Smeele, Ludi 114 Smeenk, Robert 114 Smit, Linda 24 Smit, Marjon 73 Smits, Marianne 102 Smitsmans, Monique 88 Snel, Mireille 36 Snoek, Margriet 71 Sonke, Gabe 102 Sonke, Jan-Jakob 88 Sonneborn, Mariska 132 Sonnenberg, Arnoud 17 Sparmann, Anke 69 Sprong, Debbie 80 SrГЎmek, Michael 115 Steenbakkers, Roel 88 Stewart, Fiona 80 Stoppa, Tino 115 Storm, Dea 143 Stortelers, Catelijne 32 Stroom, Joep 88 Strumane, Kristin 19 Sutherland, Kate 71 Swart, Erwin 43 Swart, Martha 143 Taal, Babs 102, 124 Taghavi, Panthea 69 Talhout, Wendy 56 Tan, Bing 114 Tanger, Ellen 69 Te Poele, Johannes 80 Te Riele, Hein 49 Teertstra, Jelle 133 Ten Bokkel Huinink, Wim 102 Ten Cate, Julia 115 Ter Heine, Rob 103 Ter Riet, Bas 51 Teunissen, Hans 69 Theodorou, Vassiliki 75 Theuws, Jacqueline 88 Tibben, Mathijs 103 Tielen, Ivon 133 Tielenburg, RenГ© 89 Timmers, Adriaan 114 Tjin, Esther 45 Toaldo, Cristiane 66 Toebes, Mireille 43 Tolhuis, Bas 69 Tran, Ly 103 Triesscheijn, Martijn 80 Udo, Renate 84, 129 Ulens, Chris 28 Urbanus, Jos 43 Uren, Anthony 71 Vader, Laura 143 Vaessen, Heleen 143 Vainchtein, Liia 103 Vainio, Saara 51 ValdГ©s Olmos, Renato 132, 143 ValdГ©s Olmos, Tjalling 143 Valkenet, Ludy 143 Valle, Noelia 64 Van Amerongen, RenГ©e 71 Van As, Josette 129 Van As-Brooks, Corina 114 Van Baal, Jurgen 34, 36 Van Beers, Erik 84 Van Beurden, Marc 115, 124 Van Blitterswijk, Wim 34 Van Boven, Hester 132 Van Bunningen, Bart 88 Van Coevorden, Frits 114 Van Dam, Frits 126 Van de AhГ©, Fina 71 Van de Kasteele, Willeke 45 Van de Poel, Henk 115 Van de Steeg, Evita 76 Van de Ven, Peter 89 Van de Vijver, Marc 86, 132 Van de Wetering, Koen 51 Van Deemter, Liesbeth 51 Van den Belt-Dusebout, Sandra 128 Van den Berg, Joost 103 Van den Berk, Paul 47 Van den Bongard, Desiree 88 Van den Boom, Marly 43 Van den Bout, Iman 17 Van den Brekel, Michiel 115 Van den Brink-de Vries, Nienke 132 Van den Broek, Guido 115 Van den Haak, Marjolijn 143 Van den Hoorn, Tineke 60 Van den Hoven, Jolanda 103 Van der Berg, Marieke 115 Van der Burg, Eline 53 Van der Donk, Emile 143 Van der Gulden, Hanneke 53 Van der Heijden, Ingrid 55 Van der Hoeven, Joost 114 Van der Horst, Ferdi 84 Van der Horst, Gerda 41 Van der Kammen, Rob 19 Van der Krogt, Gerard 23 Van der Kruijssen, Corina 76 Van der Molen, Lisette 114 Van der Plas, Arnout 84 Van der Sande, Jaap 102 Van der Sar, Jana 103 Van der Schoot, Sabien 103 Van der Stoop, Petra 69 Van der Torre, Jaco 71 Van der Velde, Hella 64 Van der Velde, Tony 143 Van der Velden, Annette 102 Van der Velden, Yme 69 Van der Voort, Paul 133 Van der Wal, Anja 49 Van der Weide, Marchien 102 Van der Wel, Wel 67 Van Dijk, Anne 129 Van Dijk, Pim 28 Van Dongen, Miranda 24 Van Doorn, Remco 73 Van Driel, Willemien 115 Van Duijn, Miranda 131 Van Duijse, Josyanne 62 Van Eijndhoven, Monique 82 Van Esch, Anita 76 Van Gerwen, Suzan 30 Van Gijn, Roel 102 Van Harten, Wim 131 Van Heijst, Jeroen 43 Van Herk, Marcel 88 Van Herwaarden, Antonius 76 Van Hien, Richard 84 Van Leeuwen, Fijs 38, 133 Van Leeuwen, Flora 84, 128 Van Leeuwen, Fred 40 Van Lent, Wineke 131 Van Lohuizen, Maarten 69 Van Luenen, Henri 51 Van Lummel, Menno 34 Van Maanen, Rianne 102 Van Meeteren, Laurens 32 Van Montfort, Erwin 71 Van Netten, Gabry 143 Van Nuland, Annemarie 126 Van Oosterwijk, Els 143 Van Pel, RenГ©e 132 Van Rens, Anja 133 Van Rijk, Maartje 114 Van Rossum, Maya 115 Van Rossum, Sari 28 Van Sandick, Johanna 114 Van Steensel, Bas 56 Van вЂ�t Veer, Laura 84, 128, 132, 133 Van Tellingen, Olaf 132 Van Tilburg, Erica 38 Van Tinteren, Harm 143 Van Turnhout, Arjen 115 Van Velthuysen, Loes 132 Van Vliet, Maaike 131 Van Vliet, Marja 133 Van Vliet, Martin 58 Van Vliet-Vroegindeweij, Corine 88 Van Vuuren, Peter 114 Van Waardenberg, Wil 143 Van Waterschoot, Robert 76 180 PERSONNEL INDEX Van Welsem, Tibor 40 Van Winden, Annemieke 103 Van Zandwijk, Nico 102 Van Zeijl, Leonie 32 Van Zon, Maaike 67 Van Zon, Wouter 49 Vargas, Mark 28 Veldhuizen, Dennis 133 Veltkamp, Sander 82 Vendrig, Tineke 45 Vens, Conchita 78 Veraar, Elise 41 Verbrugge, Inge 41 Verheij, Marcel 34, 88 Verhoef, Senno 84, 124, 128, 133 Verhoeven, Els 69 Verloop, Janneke 128 Vermeulen, Christie 78 Vermeulen, Eric 128 Verwaal, Vic 114 Verwijs, Manon 78 Verwoerd, Desiree 66 Verzijlbergen, Kitty 40 Vincent, Andrew 143 Vink, Stefan 82 Vissers, Joep 69 Vlaming, Marijn 76 Vlaskamp, Marcel 143 Vogel, Maartje 56 Voogel-Fuchs, Marja 103 Voorhoeve, Mathijs 62 Vormer, Tinke 49 Voskuil, Dorien 84, 128 Vredeveld, Liesbeth 73 Vrieling, Alina 84, 128 Vrieling, Conny 88 Vyth-Dreese, Florry 45 Wagenaar, Els 76 Wals, Anneke 143 Weevers, Marion 126 Weichenrieder, Oliver 30 Weigelt, Britta 84 Wesseling, Jelle 132 Wessels, Lodewyk 58 Westerman, Bart 69 Wever, Lidwina 143 Wielders, Camiel 49 Wielders, Eva 49 Wientjens, Ellen 53 Wieringa-Ariaens, Aafke 132, 133 Wigbout, Gea 133 Wijnands, Yvonne 21 Wijnholds, Jan 51 Wilhelmsen, Kevin 17 Willemse, Els 143 Winterwerp, Herrie 28 Wissink, Esther 41 WittkГ¤mper, Fritts 88 Woerdeman, Leonie 115 Wolthuis, Rob 49 Wu, Tin 133 Zandvliet, Anthe 103 Zerp, Shuraila 34 Zevenhoven, John 71 Ziblat, Lonny 143 Zijp, Lambert 89 Zimmermann, Christian 76 Zlotorynski, Eitan 62 Zoetmulder, Frans 114 Zuur, Charlotte 115 Zuur, Karel 115 Zwaagstra, Annabel 75 Zwart, Wilbert 66 181 COLOFON COLOFON Coordinators Suzanne Bakker Suzanne Corsetto Henri Van Luenen Photograph HM The Queen Beatrix EnquiryвЂ™s/permission: Rijksvoorlichtingdienst, afd. Pers en Publiciteit/FOTO Postbus 20009 2500 EA Den Haag Photo Ruud Taal/Capital Press Copyright RVD Photograph AJM Berns Loek Zuijderduin Other Photographs and Illustrations Audiovisual Services The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan 12 1 1066 CX Amsterdam The Netherlands Printed by Thieme Amsterdam The Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital Plesmanlaan 121 1066 CX Amsterdam www.nki.nl T HE NE T HE RLA NDS C A NC ER INSTITU TE SCIENTIFIC ANNU AL R E PO R T 2 0 06 T HE N E T H E R LANDS C A NCE R I N ST I T UT E S C IE NT I FI C A N NUA L R E PO R T 2 0 06 THE NETHERLAND S CANCER I NSTI TUTE SCI ENTI FI C ANNUAL REP ORT 20 0 6