Contents
12
Group leaders
Scientific Annual Report 2012
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 80 82 84 86 88 90 92 94 96
Scientific Annual Report 2012
100 102 104 106 108 110 112 114
Neil Aaronson Reuven Agami Roderick Beijersbergen Andre Bergman Rene Bernards Anton Berns Christian Blank Eveline Bleiker Jannie Borst Piet Borst Thijn Brummelkamp Jan-Hermen Dannenberg Karin de Visser John Haanen Michael Hauptmann John Hilkens Metello Innocenti Heinz Jacobs Jacqueline Jacobs Kees Jalink Jos Jonkers Sabine Linn Rene Medema Wouter Moolenaar Sach Mukherjee Jacques Neefjes Huib Ovaa Daniel Peeper Anastassis Perrakis Peter Peters Sven Rottenberg Sanne Schagen Jan Schellens Alfred Schinkel Marjanka Schmidt Ton Schumacher Titia Sixma Arnoud Sonnenberg Fiona Stewart Hein Te Riele Wim Van Harten Flora Van Leeuwen & Matti Rookus Fred Van Leeuwen Maarten Van Lohuizen Bas Van Steensel Marcel Verheij Jelle Wesseling Lodewyk Wessels Rob Wolthuis Wilbert Zwart
2
Introduction Director of Research
8
Board members
118
Division of Diagnostic Oncology
130
Division of Medical Oncology
144
Division of Surgical Oncology
158
Division of Radiotherapy
178
Biometrics Department
184
Research facilities
186
Education in oncology
190
Clinical trials
206
Invited speakers
208
Research projects
226
Personnel index
redactie Suzanne Corsetto C op yrig ht Netherlands Cancer Institute, Amsterdam Ont werp Room for ID’s, Nieuwegein | www.roomforids.nl | f otograf ie Martin Hogeboom, Epe | www.martinhogeboom.nl | Dr uk k er True Colours, Utrecht | www.truecolours.nl |
Netherlands Cancer Institute Plesmanlaan 121 1066 CX Amsterdam The Netherlands www.nki.nl
Scientific Annual Report 2012
We concluded 2012 again with a modest profit for the hospital, but the growth in numbers of patients has resulted in a significant increase in the workload per employee and per square meter. An important challenge for our institute is to accommodate this growth in order to provide the required clinical care for the growing numbers of cancer patients. Yet, this growth must not compromise the high level of quality care that we strive to provide. Unfortunately, the sizeable increase of our clinical activities cannot be matched by a comparable growth in our research program. Due to the dire national economic situation, our research budget from the Ministry of Health, Welfare and Sport will face an annual reduction up to a total of 6% by the end of 2014. In addition, funding options at the Netherlands Organization for Scientific Research have seen a steady reduction 2
table 1 Core research funding the Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital by the Dutch Cancer Society and the Ministry of Health, Welfare and Sport in the period
2012
2011
2010
2009
2008
25
2007
2003 – 2012 in million euros.
2006
The Antoni van Leeuwenhoek is a Comprehensive Cancer Center, combining a dedicated cancer hospital and cancer research institute in a single organization. The hospital has 180 beds, an outpatient clinic, a large radiotherapy department and an extensive infrastructure for clinical research that includes clinical data management, a large array of diagnostic facilities, and a pharmacy with a production unit for experimental drugs and clinical grade biologicals. Over the years the hospital has built a large repository of patient data and a large collection of tumor and normal tissues. Clinical research spans across medical, surgical and diagnostic oncology, radiotherapy, pharmacology, epidemiology and psychosocial oncology and research into cost effectiveness of health care and efficiency of planning and organization. Our hospital has seen steady growth in patient numbers over the last years, with an average annual growth of 7%. This growth is part of a planned expansion that projects a growth of our clinical production by 70% in 2020 (compared to 2010). To facilitate this growth, construction is underway to increase the capacity of our outpatient clinic, operating rooms and intensive care. Construction is planned to be completed in 2015 and will give room to 4 additional operating rooms and the necessary expansion of our intensive care unit. In 2012, building activities for the radiotherapy satellite in Hoofddorp (Spaarne Ziekenhuis) reached completion, and this site will open its doors early in 2013. We have also continued our discussions with the University Medical Center Utrecht (UMC Utrecht) to establish a joint cancer hospital next to the UMC Utrecht, and aim to start reorganizing oncological activities in Utrecht in 2013. The joint activity aims to improve the quality of cancer care, and will bring together a large critical mass in areas such as radiology, radiotherapy and personalized cancer treatment. This will facilitate further innovations in minimally invasive and non-invasive interventions as well as in tailored therapies, areas that hold great promise for further improvements in cancer care. Finally, we aim to establish a proton therapy center next to the Antoni van Leeuwenhoek, in a joint venture with the Academic Medical Center and the Free University Medical Centre and supported in a collaborative effort with the UMC Utrecht and the Princess Máxima Center for Pediatric Oncology (PMC). The proton therapy center is expected to be operational in 2016 and will treat the patients of the PMC requiring moderate to high irradiation, as well as a range of inoperable tumors in adult patients surrounded by radiation-sensitive healthy tissue.
2005
I am pleased to present our Scientific Annual Report. It showcases the scientific achievements of the Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL) in 2012. More background information on our research programs and principle investigators can be found on our website (www.nki.nl) or in our Scientific Brochure that is available for download on our website.
2004
Director of Research René Medema
under previous governments. As a result, it is becoming increasingly difficult for bottom-up research initiatives to secure funding, despite the fact that project proposals from the Netherlands Cancer Institute continue to rank well above the national average in funding percentile. On top of this shrinkage in external funding sources, emphasis in our national funding schemes has undergone a dramatic shift towards short-term valorization of scientific research. While our institute performs very well in such projects, and serves as a role model for clinical translation, we do foresee a serious bottleneck to arise in the near future. The cuts we are facing now in fundamental cancer research will inevitably become rate-limiting for further innovation and progress in a few years from now. In recent years, several of our principle investigators have been able to compensate the reductions in national funding of fundamental research by obtaining funding from the European Research Council. It is unclear how the EU research budget will develop in the next years, but the current economic situation makes it likely that this funding source will either also face reductions or not be able to compensate reductions elsewhere.
2003
Introduction
20
15
10
5
0
Dutch Cancer Society Ministry of Health, Welfare & Sport (excluded are the reimbursement for interest and depreciation of buildings) Total
Fortunately, the Dutch Cancer Society has increased their financial support for our research by allowing a gradual increase of the core grant given by the Dutch Cancer Society to the Netherlands Cancer Institute. This funding becomes ever more important for our success, because the continuity of our research activities critically depends on a healthy ratio between core funding and external funding. That ratio has steadily shifted towards external grants, donations and short-term research agreements with third parties. Currently 65% of our total research budget comes from such sources, making it difficult to maintain sufficient manpower in the underlying infrastructure that is increasingly important to deal with the complexity of present-day cancer research. The dependence of our research activities on high-throughput screening facilities, next generation sequencing, proteomics, a state of the art animal facility, bio-informatics, analysis and maintenance of large datasets requires steady investments that cannot be easily accommodated by external grants. In an attempt to deal with this problem, we have, together with the Dutch Cancer Society, established a foundation (NKI-AVL fund) that will raise additional funds for the Netherlands Cancer Institute. In its first full year, this fund managed to raise a little over 1 million euros. Nonetheless, the large majority of those funds were earmarked for specific projects, leaving us with the continued problem of limited core funding.
3
Highlights It is impossible to provide a complete overview of the total impact generated by our Institute in 2012 in this introduction. Many of the highlights can be found in the reports of the individual group leaders further on in this annual report or on our website. I have chosen to mention just a few highlights here. Let me begin with an unprecedented success of clinical translation. In March of 2012, the Bernards group published a manuscript explaining how colon carcinomas carrying a B-Raf mutation fail to respond to B-Raf inhibitory drugs that are successfully used in the treatment of melanomas carrying the same mutations. They subsequently showed how the resistance to B-Raf inhibitors could be overcome in colon cancer in tissue culture and in mice. In November 2012, the first colon carcinoma patient entered a clinical trial in our hospital to validate this conceptual advance, an unprecedented timeline between discovery and clinical trial. This example very nicely demonstrates how crosstalk between research and clinic can lead to rapid translation at The Netherlands Cancer Institute. The Bernards group recently identified another mechanism of resistance that might soon lead to new clinical trials. They identified MED12, a component of the transcriptional MEDIATOR complex, as a determinant of response to ALK and EGFR inhibitor drugs in lung cancer. Inhibition of TGFβR signaling can restore drug responsiveness, suggesting a strategy to treat tumors that have acquired drug resistance as a result of activated TGFβ signaling. Liesbeth Vredeveld and Patricia Possik in the group of Daniel Peeper showed that targeted inhibition of PI(3) Kinase restores senescence features in melanoma cells, providing another concept that can be tested for clinical applicability. Janneke Jaspers in the groups of Jos Jonkers and Sven Rottenberg, found that Brca1-mutated mouse mammary tumors acquire resistance to PARP inhibitors through inactivation of 53BP1, suggesting that 53BP1 may play an important role in therapy response and resistance of BRCA1-mutated and BRCA1-like cancers. Piet Borst and Henri van Luenen elucidated the long sought-after function of a modified base in the DNA (called base J). With the help of our deep sequencing facility and the group of Peter Myler in Seattle, they showed that Base J is clustered at chromosomeinternal RNA polymerase II termination sites. These findings have broad implications for our understanding of the formation of repressive chromatin and how chromatin marks can define transcriptional boundaries. Mathias Jenal, Ran Elkon and Fabricio Loayza-Puch in the group of Reuven Agami identified the PABPN1 gene as a regulator of alternative cleavage and poly-adenylation of messenger RNAs (mRNAs), a process linked to proliferation and cancer. Intriguingly, PABPN1 is causal of oculopharyngeal muscular dystrophy (OPMD), and cellular and mouse model systems showed induction of alternative polyadenylation by mutant PABP1, linking this for the first time with a genetic disease. In the group of Arnoud Sonnenberg, Coert Margadant identified a novel missense mutation in the gene encoding the integrin a3 subunit that causes interstitial lung disease and nephrotic syndrome. Francesca Mattiroli in the group of Titia Sixma studied the role of ubiquitination in the DNA damage response, showing that RNF168, not RNF8 ubiquitinates H2A after ionizing radiation. Her studies revealed that this mark is positioned at a novel site on the histone protein, and that this modification, rather than the RNF168-dependent chain formation, is essential for recruitment of downstream effectors such as Brca1 and 53BP1. Karim Nacerddine and colleagues in the group of Maarten van Lohuizen showed that the key Polycomb E3 ubiquitin ligase Bmi/Ring1B is regulated by phosphorylation, coupling Pi3K/AKT signaling to epigenetic gene regulation. Phosphorylation of Bmi1 correlates with PTEN-loss and PI3K activation in prostate cancer with high grade and poor prognosis, whereas mutation of the phosphorylation sites diminishes the oncogenic effects of Bmi1 in prostate cancer cells.
4
Jonathan Coquet and coworkers in the group of Jannie Borst identified novel signaling pathways that suppress the function of pathogenic T-helper 17 cells, a class of T cells that promotes inflammation and auto-immunity. Nienke van Rooij and Marit van Buuren in the Schumacher lab provided the first evidence that information on the mutations present within individual tumors can be used to successfully predict mutant T cell epitopes that are recognized by cytotoxic T cells. This approach may in future studies be utilized for the development of personalized immunotherapies. Metamia Ciampricotti from the group of Karin de Visser demonstrated that -against the prevailing dogmaadaptive immune cells do not affect the outcome of chemotherapy treatment of established spontaneous mammary tumors. The division of Radiotherapy reported the long-term results of the EORTC 10801 breast cancer trial. This multi-centric trial was the first to investigate the efficacy of breastconserving therapy in early breast cancer up to 5 cm. Importantly, the study concluded that survival outcomes are similar in patients treated with breast-conserving therapy or modified radical mastectomy, confirming that breast-conserving therapy can be safely applied in patients with early breast cancer up to 5 cm. The group also showed that local recurrence in prostate cancer is significantly lower when patients are irradiated with a higher local dose. The Epidemiology Group showed that diagnostic radiation exposure of BRCA1/2 mutation carriers is associated with an increased breast cancer risk. Any exposure to diagnostic radiation before the age of 30 was associated with a 1.9-fold increased risk of breast cancer, with a dose-response pattern. The results of this study support the use of nonionizing radiation imaging techniques (such as magnetic resonance imaging) as the main tool for surveillance in young women with BRCA1/2 mutations. The psychosocial research group of Neil Aaronson demonstrated the efficacy of cognitive behavioral therapy and physical exercise in helping to alleviate endocrine symptom burden in young women with breast cancer who experience chemotherapyinduced menopause. Such behavioral approaches to treating menopausal symptoms are important, as available medicines are either contraindicated for women with breast cancer or have significant side effects.
Quality of Research The quality of our research can be monitored in several ways. First of all, objective bibliometric parameters (citations and impact of scientific articles published by NKI staff) demonstrate that our scientific productivity continues to be outstanding (see table 2). The year 2012 is no exception to this trend and it is gratifying to note, that despite the difficulties to obtain sufficient funds for our research, we manage to maintain our position at the international forefront of cancer research. Secondly, our prominent international standing in cancer research is reflected by the frequency with which our staff members are invited to present at international meetings and in the awards and grants that they obtain. We score high on all of these accounts. Several NKI-postdocs have received competitive grants from national and international organizations. In addition, a number of our principle investigators received prestigious (inter)national research grants. Jacqueline Jacobs and Thijn Brummelkamp were awarded an ERC Starting Grant, Reuven Agami received an ERC Advanced Grant. Fred van Leeuwen received a prestigious VICI grant and Rene Bernards received the Queen Wilhelmina Research Award. Anton Berns coordinated the Mouse Clinic for Cancer and Ageing, together with the European Institute for the Biology of Ageing (ERIBA). They received 18 million euros from the National Roadmap to build a national research facility for the generation of mouse models for cancer and ageing. The ERC Synergy proposal of Anton Berns, Daniel Peeper, Lodewyk Wessels and groups of the Sanger Institute was selected out of over 700 proposals and was awarded with 15 million euros for the next 5 years. This project, entitled COMBATCANCER, will seek to optimize personalized medicine for lung and melanoma cancer. Finally, Rene Bernards coordinated a Zwaartekracht proposal that was awarded 30 million euros for the next 10 years. In this project several other groups from the NKI, as well as groups from several academic institutions, will 5
work together to study (targeted) drug responses and optimize targeted therapies. The quality of research within each division is assessed every 5 years by external site visit teams. In 2012 we have organized several site visits. On March 5th and 6th, a site visit team composed of Hidde Ploegh (MIT, Boston), Reinhard Faessler (MaxPlanck Institute, Martinsried), Vivek Malhotra (CRG, Barcelona), Rienk Offringa (DKFZ, Heidelberg) and Benjamin Geiger (Weizmann Institute, Rehovot) evaluated the divisions of Cell Biology I, Cell Biology II and Immunology. On May 14th, a site visit committee formed by Johann De Bono (ICR, London), Antoni Ribas (Jonsson Comprehensive Cancer Center, UCLA), Rolf Stahel (UZH, Zurich) and Ian Tannock (The Princess Margeret, Toronto) evaluated the research program of the division of Medical Oncology. Finally, on December 18th and 19th, a site visit team formed by Roel Nusse (Stanford, CA), Kristian Helin (BRIC, Copenhagen), David Livingston (DFCI, Boston), Simon Boulton (Clare Hall Laboratories, London) and Michael Gottesman (NCI, Center for Cancer Research, Bethesda) evaluated the research of the divisions of Molecular Pathology, Molecular Oncology, Molecular Genetics and Biological Stress Response. The overall impression of each of these committees was very positive and the ranking of the majority of the evaluated principle investigators was outstanding. All committee members were impressed with the international standing of our research programs, considering the relatively small size of our institute. Several valuable suggestions were made; how to further strengthen the ties between fundamental and clinical research, how to provide more support for our junior investigators. We will implement these suggestions where possible in the next years.
Honors and Appointments The NKI-AVL cannot award university degrees, but many of our staff members hold special part-time chairs at Dutch Universities. This allows them to award PhD degrees to graduate students who receive their training at the Netherlands Cancer Institute. Currently, 33 staff members have professorships at one of the Dutch Universities. In 2012, Huib Ovaa was appointed Professor of Chemical Biology at Leiden University, Lodewyk Wessels as Professor of Bioinformatics at the Technical University in Delft and Sabine Linn as Professor of Translational Oncology at the University Medical Center in Utrecht.
table 2 Short term citations and impact of scientific articles published by the Netherlands Cancer Institute research staff 2000 – 2010/2012
Publication publications citations citations/ impact year publications
6
2000
270
4314
16,0
1699
2001
305
4944
16,2
1554
2002
349
7436
21,3
2324
2003
330
5084
15,4
1963
2004
324
5254
16,2
2018
2005
362
6261
17,3
2442
2006
390
6302
16,2
2584
2007
400
5515
13,8
2590
2008
408
5671
13,9
2553
2009
472
7405
15,7
3122
2010
467
8784
18,8
2847
2011
444
3110
2012
412
2527
There were several changes in our staff. Karin de Visser was appointed as junior group leader, Sven Rottenberg was appointed as staff member and head of the intervention unit of our mouse cancer clinic, and Jacqueline Jacobs was appointed as tenured staff member. With my appointment as Scientific Director, my research group also took up base at the The Netherlands Cancer Institute, in the division of Cell Biology I. Staff of the NKI-AVL fulfilled numerous functions in national and international organizations, on boards of scientific journals, as members of study sections, site visit committees and as organizers or co-organizers of scientific meetings, workshops and conferences. Jannie Borst and Jos Jonkers were elected as members of the European Molecular Biology Organization (EMBO).
Outlook and Acknowledgements For the last decennia our Institute has been at the international forefront in cancer research and innovative cancer treatments. It has demonstrated to be able to maintain that position, despite the difficult economic situation of the last few years. We have been very successful in obtaining external grants for our research and I am convinced that we will continue to do so. Provided that we can match this with a healthy ratio of core funding, I foresee that the Netherlands Cancer Institute can continue to deliver important breakthroughs that will prove beneficial in the treatment of cancer. Particularly in a time when our ever growing molecular understanding of cancer meets up with a new generation of anti-cancer drugs that target well-defined nodal points in the cancer cell. This calls for a more individualized treatment of cancer, in which molecular pathology in the form of a genetic and/or immunological fingerprint of the tumor is extensively used in making clinical decisions how to treat the individual patient. Success in this area will critically depend on a close collaboration between basic and clinical research; where basic research can provide the concepts for new drug combinations that can be taken to the clinic, and vice-versa, where response failure of a genetically and immunologically defined tumor in the clinic can be taken to the lab to identify alternative strategies. The integration of research and clinic in a single Comprehensive Cancer Center provides us with the ideal setting to facilitate this collaboration, and our first concrete steps in this area hold great promise for the future. However, these promising developments bring new demands for our research infrastructure that cannot be financed from external project grants. It is therefore imperative that we raise additional funds through alternative routes to enable us to maximize our research efforts in this area. This will receive high priority in the coming years. Our newly established NKI-AVL fund and our upcoming anniversary provide us with great opportunities to obtain such funds. In 2013, The Netherlands Cancer Institute will celebrate its 100th year anniversary. We will use this year to bring our new ambitions to the attention of a large audience, ranging from individuals to large organizations. By showcasing our past performance and by providing a clear explanation of the opportunities that lie within our grasp, combined with the drive and enthusiasm of our employees, we should be able to convince a wide audience to support our cause. I want to end by thanking all of our employees and everyone that supported us; the Dutch Cancer Society that has been a very significant sponsor of our research for many years; the Ministry of Health Welfare and Sport that provides a substantial core grant to our Institute and has provided the funds to renovate our research facilities; and all of those individuals that provided us with financial, moral and practical support. Their support is making it possible for us to continue to strive for better treatments to improve the outlook of cancer patients. And last but not least, I would like to extend my sincere gratitude to all of our patients willing to participate in our clinical studies; they are vital to the progress that we can make.
René Medema Director of Research
7
Board members
Chairman of Board of Governors T de Swaan
Board of Directors
RH Medema
Chairman and Director of Research S Rodenhuis
Director Clinical Research and Development
Scientific Advisory Council
RH Medema
Chairman
International scientific advisory board
T De Lange
Leon Hess Professor, The Rockefeller University, New York, USA
National scientific advisory board
DD Breimer
Professor of Pharmacology, Leiden University
TNM Schumacher
Secretary S Rodenhuis
RA Flavell
Professor of Immunobiology, Yale University School of Medicine, New Haven, USA
JL Bos
Professor of Physiological Chemistry, University of Utrecht
WH Van Harten
Director Organization and Management
B Van Steensel (until October 1, 2012) T Ruers J Jonkers ML Van Lohuizen
WGJ Ho
Professor of Biochemistry and Biological Structure, University of Washington, Seattle, USA J Mendelsohn
President MD Anderson Cancer Center, University of Texas, Houston, USA P Nurse
Board of Governors
Professor of Microbiology, President of The Rockefeller University, New York, USA R Nusse
T De Swaan
Professor of Developmental Biology, Stanford University, Stanford, USA
EGE De Vries
Professor of Medical Oncology, University of Groningen JHF Falkenburg
Professor of Experimental Hematology, Leiden University CG Figdor
Professor of Experimental Immunology, Radboud University Nijmegen JHJ Hoeijmakers
Professor of Molecular Genetics, Erasmus University Rotterdam
President HL Ploegh EH Swaab
Vice-president JP Balkenende GH Blijham FH Schröder MC Smeets
Professor of Biology, Whitehead Institute for Biomedical Research, Cambridge, USA S Powell
Chairman, Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, USA
P Lambin
Professor of Radiation Oncology, Maastricht University CJH Van de Velde
Professor of Surgical Oncology, Leiden University
IF Tannock
Daniel E. Bergsagel Professor of Medical Oncology,
Princess Margaret Hospital and University of Toronto, Toronto, Ontario, Canada (from June 1, 2012).
F Sijbesma (from October 1, 2012) K Vousden PC Van der Vliet
Director, Beatson Institute for Cancer Research, Glasgow, UK
MJ Van Mourik RA Weinberg
Professor of Biology, Massachusetts Institute of Technology, Whitehead Institute for Biomedical Research, Cambridge, USA
8
9
10
11
A
A Holzner B, Efficace F, Basso U, Incrocci L, Johnson C, Aaronson N, Arraras J, King M, Chow E, Oberguggenberger A, Bottomley A, Steiner H, Giesinger J. Cross-cultural development of an EORTC questionnaire to assess health-related quality of life in patients with testicular cancer: The EORTC TC26. Qual Life Res, 2012
Group leader Neil Aaronson
Neil Aaronson PhD Group leader Marieke Van Leeuwen PhD Post-doc Chantal Lammens PhD Post-doc Willem Eijzenga MSc PhD student Lisanne Hummel MSc PhD student Hanna Van Waart MSc PhD studentt Marijke Wevers MD MSc PhD student Jacobien Kieffer MSc Senior statistical analyst Marianne Berkhof Research assistant Miranda Gerritsma Research assistant Marianne Kuenen Research assistant Jacoline Melis MSc Research assistant Grace Sidharta Research assistant
Publications
Duijts SFA, van Beurden M, Oldenburg HSA, Hunter MS, Kieffer JM, Stuiver MM, Gerritsma MA, Menke-Pluymers MBE, Plaisier PW, Rijna H, Lopes Cardozo AMF, Timmers GJ, van der Meij S, van der Veen H, Bijker N, de Widt-Levert LM, Geenen MM, Heuff G, van Dulken EJ, Boven E, Aaronson NK. Efficacy of cognitive behavioral therapy and physical exercise in alleviating treatment-induced menopausal symptoms in patients with breast cancer: Results of a randomized controlled multicenter trial. J Clin Oncol 2012;30:4124-33 Gundy CM, Fayers PM, Groenvold M, Petersen MA, Scott NW, Sprangers MAG, Velikova G, Aaronson NK. Comparing higher order models for the EORTC QLQ-C30. Qual Life Res 2012;21:1607-17 Hilarius DL, Kloeg PH, van der Wall E, van den Heuvel JJG, Gundy CM, Aaronson NK. Chemotherapy-induced nausea and vomiting in daily clinical practice: A community hospitalbased study. J Support Care Cancer 2012;20:107-117
12
Khoshnevisan A, Yekaninejad MS, Ardakani SK, Pakpour AH, Mardani A, Aaronson NK Translation and validation of the EORTC brain cancer module (EORTC QLQ-BN20) for use in Iran. Health Qual Life Outcomes 2012;10:54 Lagerwaard FJ, Aaronson NK, Gundy CM, Cornelis JA, Slotman BJ, Senan S. Patient-reported quality of life after stereotactic ablative radiotherapy for early stage lung cancer. J Thorac Oncol 2012;7:1148-54 Nieuwenhuis MH, Douma KFL, Bleiker EMA, Aaronson NK, Clevers H, Vasen HFA. Clinical evidence for an association between familial adenomatous polyposis (FAP) and type II diabetes. Int J Cancer 2012;131:1488-9 Scott NW, Etta JA, Aaronson NK, Bottomley A, Fayers PM, Groenvold M, Koller M, Kuli D, Marais D, Petersen MA, Sprangers MA. An evaluation of the response category translations of the EORTC QLQ-C30 questionnaire. Qual Life Res 2012 Snyder C, Aaronson N, Choucair A, Elliott T, Greenhalgh J, Halyard M, Hess R, Miller D, Reeve B, Santana M. Implementing patient-reported outcomes assessment in clinical practice: A review of the options and considerations Qual Life Res 2012;21:1305-14 Snyder CF, Blackford AL, Wolff AC, Carducci MA, Herman JM, Wu AW, Aaronson N, Gotay C, Halyard M, Hynes D, Jones JB, Yount S, Velikova G. Feasibility and value of PatientViewpoint: A web system for patient-reported outcomes assessment in clinical practice. Qual Life Res, 2012 Wevers MR, Hahn DE, Verhoef S, Bolhaar MD, Ausems MG, Aaronson NK, Bleiker EM. Breast cancer genetic counseling after diagnosis but before treatment: Treatment consequences and psychological impact. Pat Educ Counsel 2012;89:89-95
Health-related quality of life assessment and behavioral interventions in clinical oncology This research line has two primary foci: (1) development of methods and applications of health-related quality of life (HRQL) assessment in clinical research and clinical practice; and (2) development and testing of behavioral and psychosocial inter ventions to reduce symptom burden and improve the HRQL of patients with cancer.
Methods and measures for assessing the HRQL of long-term survivors of testicular and prostate cancer This study, a collaboration between the EORTC Quality of Life and Genitourinary Cancer Groups, has two primary objectives: (1) to test the logistics required to conduct long-term survivorship studies within the context of the EORTC; (2) to pilot test questionnaires for assessing the HRQL of long-term cancer survivors (> 10 years disease free). We recruited 280 long-term prostate and testicular cancer survivors who had participated in EORTC phase III clinical trials. Patients were drawn from 3 broad geographic/cultural regions: (1) Northern Europe; (2) Southern Europe; and (3) the United Kingdom. HRQL was assessed at 3 levels: (1) generic (the SF-36 Health Survey); (2) cancer-specific (the EORTC QLQ-C30 plus condition-specific modules; and (3) cancer survivor-specific (the Impact of Cancer). Time required to obtain medical ethical approval for the patient survey ranged from 1.5 to 25 months. We encountered most problems with obtaining ethical approval in the UK, Italy and Belgium. The average time to recruit patients and field the survey questionnaire was 3 months. Completed questionnaires were received from 242 patients. Although many survivors scored at the upper extremes of the questionnaires, in general, the SF36, the QLQ-C30 and the IOCv2 exhibited adequate reliability (< 0.70), and known groups validity (e.g., discriminated between subgroups formed on the basis of comorbidity and age). The QLQ-C30 condition-specific modules could benefit from modification for cancer survivor populations (e.g., by deleting acute, treatment-related side effects).
Identifying symptom clusters in the endocrine scale of the Functional Assessment of Cancer Therapy Measurement System (The FACT-ES) The FACT-ES is an 18-item questionnaire assessing treatmentinduced endocrine symptoms. Currently, the scoring algorithm for the FACT-ES yields a single, overall score. We carried out a psychometric study to identify a potential subscale structure for the FACT-ES, reflecting symptom clusters. We used questionnaire data from 422 breast cancer patients who had experienced chemotherapy-induced menopause. Using exploratory factor analysis, we identified 6 symptom clusters: vasomotor symptoms, gynaecological symptoms, sexual symptoms, mood, weight, and other endocrine symptoms. Confirmatory factor analysis confirmed this subscale structure in a second sample of 141 women who had undergone prophylactic oophorectomy due to a heightened risk of familial breast/ovarian cancer, and who had experienced treatment-induced menopause. These
findings support the tenability of generating both subscale and overall scores for the FACT-ES. This will allow a more detailed assessment and reporting of endocrine symptoms.
Physical exercise during chemotherapy to improve physical fitness and reduce fatigue (PACES) In this multicenter RCT we are evaluating the effectiveness of two physical exercise interventions in maintaining or enhancing physical fitness and minimizing fatigue in patients undergoing adjuvant chemotherapy for breast or colon cancer: (1) a low intensity, home-based, self-management program; and (2) a moderate intensity, structured, supervised program. All partici pants undergo physical performance tests and complete selfreport questionnaires at baseline, at the completion of chemo therapy, and at 6 month follow-up. We completed patient recruit ment in December 2012. In total, we have entered 253 patients (230 and 23 with breast and colon cancer, respectively), of whom 190 have completed the first follow-up (T1) and 140 the 6-month follow-up (T2). Data collection will continue until December 2013.
Behavioral and psychosocial effects of rapid genetic counseling and testing (RGCT) in newly diagnosed breast cancer patients In this multicenter, randomized trial, carried out in collaboration with the University Medical Center Utrecht (Dr. Margreet Ausems), we are investigating the uptake of RGCT when offered routinely to newly diagnosed breast cancer patients who, prior to receiving primary treatment, are identified as having at least a 10% risk of carrying a mutation in the BRCA1/2 gene, and the impact of RGCT on choice of surgery and psychosocial well-being. Women were recruited from 12 hospitals in the Amsterdam and Utrecht regions of the Netherlands and were randomized to either the RGCT group or a usual care group (2:1 ratio). The study endpoints include: (1) uptake of RGCT; (2) choice of clinical management strategy, including direct bilateral mastectomy or delayed preventive contralateral mastectomy; (3) cancer risk perception and cancer-related distress; (4) knowledge of genetic aspects of breast cancer; (5) decisional satisfaction; (6) HRQL; and (6) satisfaction with RGCT. Between 2008 and 2010, 265 women were randomized into the study. Questionnaires were administered at study entry, and at 6 month (response 92.5%) and 12 month follow-up (response 90.6%). A subset of women has been interviewed to obtain supplementary, qualitative data. Follow-up was completed in 2012 and data analysis is currently ongoing.
Online cognitive behavioral therapy (CBT) for climacteric symptoms in breast cancer patients experiencing treatment-induced menopause Menopausal symptoms are common, and may be particularly severe in younger women with breast cancer who undergo treatment-induced menopause. Effective and safe treatment options for these symptoms in breast cancer patients are needed. In a recently completed RCT, we demonstrated the efficacy of a group-based, CBT program that included psychoeducation, cognitive restructuring, behavioral strategies and relaxation training. However, compliance with the program was problematic. In attempt to increase the accessibility to and compliance with this CBT program, we are developing a web-based version in collaboration with MindDistrict and King’s College, London. The program is currently under development and will be tested iteratively for feasibility and effectiveness.
Wolf SL, Barton DL, Qin R, Wos EJ, Sloan JA, Liu H, Aaronson NK, Satele DV, Mattar BI, Green Nb, Loprinzi CL. The relationship between numbness, tingling and shooting/burning pain in patients with chemotherapy induced peripheral neuropathy (CIPN) as measured by the EORTC QLQ-CIPN20 instrument. Support Care Cancer 2012;20:625-32
Smith EML, Barton DL, Qin R, Steen PD, Aaronson NK, Loprinzi CL. Assessing patient-reported peripheral neuropathy: The reliability and validity of the European Organization for Research and Treatment of Cancer QLQ-CIPN20 Questionnaire. Qual Life Res (in press)
Petersen MA, Aaronson NK, Arraras JI, Chie W-C, Conroy T, Costantini A, Giesinger JM, Holzner B, King MT, Singer S, Velikova G, Verdonck-de Leeuw IM, Young T, Groenvold M. The EORTC computer-adaptive tests (CATs) measuring physical functioning and fatigue exhibited high levels of measurement precision and efficiency J Clin Epidemiol (in press) Victorson DE, Brucker PS, Bode RK, Eton DT, Talcott JA, Clark JA, Knight SJ, Litwin MS, Moinpour CM, Reeve BB, Aaronson NK, Bennett CL, Herr HW, McGuire M, Shevrin D, McVary K, Cella D. Ensuring Comprehensive Assessment of Urinary Problems in Prostate Cancer through PatientPhysician Concordance. Urol Oncol (in press) Chinapaw MJM, Buffart LM, van Mechelen W, Schep G, Aaronson NK, van Harten WH, Stuiver MM, Kersten MJ, Nollet F, Kaspers GJK, van Dulmen-den Broeder E, Huisman J, Takken T, van Tulder M, Brug J. Alpe d’HuZes Cancer Rehabilitation (A-CaRe) research: Four randomized controlled exercise trials and economic evaluations in cancer patients and survivors. Int J Behav Med 2012;19:143-56 Reeve BB, Wyrwich KW, Wu AW, Velikova G, Terwee CB, Snyder CF, Schwartz C, Revicki DA, Moinpour CM, McLeod LD, Lyons JC, Lenderking WR, Hinds Ps, Hays RD, Greenhalgh J, Gershon R, Feeny D, Fayers PM, Cella D, Brundage M, Ahmed S, Aaronson NK, Butt Z on behalf of the International Society for Quality of Life Research (ISOQOL). ISOQOL recommends minimum standards for patientreported Outcome Measures Used in Patient-Centered Outcomes Research. Qual Life Res 2013 Kuijpers W, Groen WG, Aaronson NK, van Harten WH. A systematic review of web-based interventions for patient empowerment and physical activity in chronic diseases: Relevance for cancer survivors. J Med Internet Res (in press)
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A Jenal M, Elkon R, Loayza-Puch F, van Haaften G, Kuhn U, Menzies FM, Oude Vrielink JA, Bos AJ, Drost J, Rooijers, K, Rubinsztein DC, Agami R. The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites. Cell 2012;149:538-553
Division head, group leader Reuven Agami Reuven Agami Group leader Rani Elkon PhD Post-doc Nicolas Léveillé PhD Post-doc Mathias Jenal PhD Post-doc Fabricio Loayza Puch PhD Post-doc Pieter Van Breugel PhD Post-doc Boris Slobodin PhD Post-doc Jarno Drost MSc PhD student Marieke Van Kouwenhove MSc PhD student Arnold Bos MSc PhD student Carlos Melo MSc PhD student Koos Rooijers MSc PhD student Rui Lopes MSc PhD student Mariëtte Schrier PhD Technical staff Joachim Oude Vrielink Technical staff
Publications
Melo CA, Drost J, Wijchers, PJ, van de Werken H, de Wit E, Oude Vrielink JA, Elkon R, Melo SA, Leveille N, Kalluri R, de Laat W and Agami R. eRNAs Are Required for p53-Dependent Enhancer Activity and Gene Transcription. Mol Cell. 2012 Elkon R, Drost J, van Haaften G, Jenal M, Schrier M, Oude Vrielink JA, and Agami R. E2F mediates enhanced alternative polyadenylation in proliferation. Genome Biol 2012;13:R59 Morris AR, Bos A, Diosdado B, Rooijers K, Elkon R, Bolijn AS, Carvalho B, Meijer GA and Agami R. Alternative Cleavage and Polyadenylation during Colorectal Cancer Development. Clin Cancer Res. 2012;18:5256-66 Korkmaz G, le Sage C, Tekirdag KA, Agami R and Gozuacik D. miR376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1. Autophagy 2012;8:165-176
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Wilting SM, Snijders PJ, Verlaat W, Jaspers A, van de Wiel MA, van Wieringen WN, Meijer GA, Kenter GG, Yi Y, le Sage C, Agami R, Meijer CJ, Steenbergen RD. Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis. Oncogene 2012;3;32:106-16 Wilting SM, Verlaat W, Jaspers A, Makazaji NA, Agami R, Meijer CJ, Snijders PJ, Steenbergen RD. Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis. Epigenetics. 2013;8 Tekirdag KA, Korkmaz G, Ozturk DG, Agami R, Gozuacik D. MIR181A regulates starvation- and rapamycininduced autophagy through targeting of ATG5. Autophagy. 2013;9:374-85
Identifying and characterizing novel cancerous processes Our main research objective is to understand the cancerous process in humans – with the focus on RNA-directed gene regulation. We either use RNA as a tool to interfere with cellular gene expression or study various cellular RNA types to uncover novel cancer causing or inhibiting genetic programs. The knowledge we gather is used to design novel therapeutic approaches.
Research line 1: Functional screens using RNA interference (RNAi) All 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. We developed and successfully used an RNA interference (RNAi) approach to inactivate genes in mammalian cells to identify and characterize cancerous genes. As an example, we lately identified and characterized the role of the gene BRD7 (bromodomaincontaining 7) as a protein whose inhibition allows full neoplastic transformation in the presence of the tumor suppressor p53. Our results proposed a tumor suppressive role in breast cancer. Now, we generate a mouse model system for BRD7 to study in detail its in vivo role as a tumor suppressor, and to assess novel therapeutic strategies.
Research line 2: Functional screens to identify cancerous miRNAs In the past years we initiated studies to identify cancerous microRNAs (miRNAs), a newly emerging gene family encoding for endogenous small RNAs. We developed novel and unique genetic approaches to screen for cancer-causing and cancerpreventing miRNAs. With these tools we discovered and characterized the role of the microRNAs in autophagy, tumor growth, cancer and metastasis.
Research line 3: Interplay between miRNAs and RNA binding proteins in cancer We recently noticed that the regions surrounding some functional miRNA targets are highly conserved throughout evolution. We hypothesized that these regions recruit RNA binding proteins (RBPs) to control miRNA function during induction of various stress responses. We performed genetic screens and identified and characterized RBPs that can inhibit or potentiate the accessibility of miRNAs to their target mRNAs. We suggest that the genetic interaction between miRNAs and RBPs influence developmental processes, cellular proliferation, and cancer.
Research line 4: Regulation of alternative cleavage and polyadenylation of mRNAs The 3’-end of most message RNAs (mRNAs) is cleaved and polyadenylated, a process that is required for mRNA function. Recent discoveries revealed that a large proportion of human genes contain more than one polyadenylation site. Therefore, alternative cleavage and polyadenylation (APA) is a widespread phenomenon that generates mRNAs with alternative 3’ends. Potentially, APA generates isoforms differing in their coding and non-coding regions, thereby affecting gene function and its regulation by miRNAs and RBPs. Thus, APA provides an important regulatory layer of gene expression. Interestingly, very strong genome-wide switches in APA are observed when cells are stimulated to proliferate, differentiate, and during cancer progression. However, how APA is regulated and what is its function, are largely unknown. To identify regulators of APA we developed a reporter-based RNAi screen and devised a genome-wide approach to detect 3’ends of mRNAs (named 3’Seq). With these tools we identified the gene PABPN1 as a regulator of APA. Loss-of PABPN1 resulted in extensive 3’UTR shortening and a compromised miRNA-mediated repression. Interestingly mutations in PABPN1 causes the autosomal dominant oculopharyngeal muscular dystrophy (OPMD). Intriguingly, the expression of mutant PABPN1 in both a mouse model of OPMD and human cells elicited 3’UTR shortening, linking for the first time APA with a genetic disease. We conclude that PABPN1 is a suppressor of APA (figure 1). Additionally, we explored, on a transcriptome-wide scale, APA events that are associated with cancer. First, we comprehensively mapped APA events associated with cellular proliferation and transformation, and cancer. Second, we demonstrated that E2F-mediated transcriptional regulation of 3’-end processing genes is one of the key mechanisms that links APA to proliferation. Increase in E2F activity enhances 3’-end processing expression that results in more potent usage of alternative polyadenylation sites (figure 2).
Research line 5: Role of enhancer RNAs in controlling gene expression and in cancer It is well known that the p53 tumor suppressor gene regulates transcription and cell cycle progression by binding within or nearby target genes controlling cell proliferation and survival. We found that p53 binds genomic regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound regions are indeed enhancers. Moreover, these p53-binding sites produce enhancer RNAs (eRNAs) that are required for enhancer activity. The production of eRNAs is interesting, as they can be used to control enhancer activity and phenotype (figure 3).
Figure 1: A model for PABPN1’s Mode of Action in Regultion of APA. Schematic model for PABPN1’s role in suppressing APA (upper and middele panels). Normally, PABPN1 binds APA sites and competes with the 3’-end cleavage and polyadenylation machinery (CPSF). Expression of an OPMD mutant PABPN1 (trePABPN1) sequesters wild type PABPN1 in aggregates causing 3’UTR shortening.
Figure 2: E2F controls alternative cleavage and polyadenylation of mRNAs. Many 3’-end enzymes are under the control of the E2F transcription factor. When cell enter proliferation, E2F level and activity increase, resulting in more 3’-end cleavage activity and induction of broad mRNA shortening.
Figure 3: p53-induced eRNAs are required for p53 function. The p53 tumor suppressor gene binds enhancers to generate eRNAs and activate distal genes. Knocking down eRNAs results in reduced enhancer activity.
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Unraveling signalling networks in cancer
Group leader Roderick Beijersbergen Roderick Beijersbergen PhD Group leader Pasi Halonen PhD Post-doc Kathy Jastrzebski PhD Post-doc Jos Poell PhD Post-doc Jordi Vidal-Rodriguez PhD Post-doc Johan Kuiken MSc PhD student Klaas De Lint MSc PhD student Cor Lieftink MSc Bioinformatician Ben Morris Technical staff Wouter Nijkamp Technical staff
Publications
Kuiken HJ, Egan DA, Laman H, Bernards R, Beijersbergen RL, Dirac AM. Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling. J Cell Mol Med. 2012;16:2140-9 Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, Beijersbergen RL, Bardelli A, Bernards R. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100-3 Huang S, Holzel M, Knijnenburg T, Schlicker A, Roepman P, McDermott U, Garnett M, Grernrum W, Sun C, Prahallad A, Groenendijk FH, Mittempergher L, Nijkamp W, Neefjes J, Salazar R, Ten Dijke P, Uramoto H, Tanaka F, Beijersbergen RL, Wessels LF, Bernards R. MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-beta Receptor Signalling. Cell. 2012;151:937-50
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The complexity and heterogeneity of cancer poses an enormous challenge for the identification and selection of effective cancer therapies. Genomic alterations occurring in human tumours frequently affect components of signalling networks, thereby contributing to cancerous phenotypes. As a result, components of these signalling networks represent potential targets for cancer therapy. However, the complex structure of these networks, the extensive crosstalk between pathways and unanticipated feedback control pose a major challenge in the selection of the right targets for individual patients. Our research evolves around the development and application of RNAi technologies in largescale cell based screening to identify critical components of signalling networks deregulated in cancer that can be explored as drug targets in cancer therapy. Furthermore, we use these RNAi technologies to search for potential mechanisms of resistance and thereby potential combination treatments. We use dynamic phosphoproteomic profiling of signalling pathways in the context of specific tumour associated mutations with the goal to generate predictive models for therapy response to pathway targeted therapeutics in cancer.
Generation of computational models for predicting response to therapy in breast cancer The understanding of the complex dynamic circuitry of signalling pathways in the context of targeted inhibition is highly valuable for identifying biomarkers to stratify patients, and to enable the identification of more effective combination therapies. In this project our focus is on the PI3-Kinase and MAP-kinase signalling pathways, both of which are strongly implicated in breast cancer. Although genetic alterations in these pathways are frequently observed, drugs targeting specific components display limited success in the clinic. The understanding and modelling of these pathways and the complex regulatory feedback regulation will allow for the generation of predictive models for therapy response in individual cancers. We have characterized a set of more than 30 human breast cancer lines for their sensitivity to more than 15 different pathway targeted drugs. We have analysed genomic alterations present in these tumour cell lines together with gene expression analysis. We have used reverse phosphoprotein arrays to analyse the activation state of a large number of individual components in the PI3K and MAPK signalling networks. In a parallel approach we are generating a panel of cell lines, derived from normal mammary epithelial cells, carrying specific genetic alterations in the PI3K and MAPK signalling pathways e.g. PIK3CA mutation or PTEN loss. We use RNAi for perturbation experiments and Luminex technology for the quantification of the activation status of individual pathway components in a dynamic fashion. The data obtained from these different experimental model systems is used to create a dynamic and quantitative computational model of pathway status and the response to perturbation using clinically relevant pathway-targeted drugs.
Enhancers for response to targeted therapeutics Examples of successes with pathway-targeted drugs have fuelled the expectation that this type of drugs holds great promise in cancer treatment. At present, many of these drugs are in clinical development, but their successes are limited. Complicated interactions with other signalling pathways and unexpected feedback loops underlie the limited effectiveness and, in some instances, even accelerate the tumourigenic process. In this project we apply large-scale RNAi screens to identify genes that upon knockdown increase the response to specific inhibitors in the PI3K and MAPK pathways. To this end, we use the pooled shRNA screening technology in combination with next generation sequencing in different model systems including breast, lung and colon cancer. We have screened the colon cancer cell line HCT116, carrying activating mutations in both the PI3K and MAPK pathway, for genes that upon knockdown enhance the response to either PI3-kinase or MEK kinase inhibition or both simultaneously. As expected, we identify mTORC1 as a crucial intersection of these 2 pathways controlling cell survival. However, apart from this crucial node, we also find a number of receptor tyrosine kinase receptors that are selectively required for the survival of cells only in the presence of either or both inhibitors. We are currently exploring these interactions in several cancer cell lines carrying similar alterations in these pathways.
Synthetic lethal interactions with oncogenic RAS For the effective treatment of cancer, there is a great need for drugs that specifically target tumour cells without affecting normal cells. With the use of RNA interference, we explore synthetic lethal phenotypes in mammalian cells. By using our isogenic cell line pair of primary human BJ fibroblasts harbouring an oncogenic RASV12 allele we identified, among others, kinesin family member 18A (KIF18A) as synthetic lethal interaction with oncogenic RAS. We have shown that silencing of KIF18A has a detrimental effect on human fibroblasts expressing a HRAS, KRAS or NRAS oncogene. Time-lapse imaging of mitotic events showed that silencing of KIF18A resulted in a more pronounced mitotic arrest associated with an increase in apoptosis in cells expressing oncogenic RASV12 (see figure). Furthermore, we observe a strong correlation between the effects of KIF18A silencing and the RAS mutation status in a panel of Non-Small-Cell Lung Cancer cell lines. Together these results suggest that KIF18A synthetic lethality is context independent and could be explored as a therapeutic target in tumours carrying an activated RAS oncogene.
Time-lapse microscopy of cells and analysis of duration of mitosis and subsequent cellular faith. Comparison between RAS wild-type cells and cells expression oncogenic RAS in the absence or presence of KIF18A knockdown.
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Investigating the role of the microenvironment in prostate cancer development Epidemiological, histopathological and molecular studies suggest that the microenvironment plays a crucial role in the initiation and progression of multiple cancers including prostate cancer. There is emerging evidence that specifically the immune cells recruited to the microenvironment are an important factor in prostate cancer etiology and development of Castration Resistant Prostate Cancer (CRPC). CRPC reprensents late stage disease with disabling symptoms and a high mortality. The aim of our research is to address the role of the microenvironment in prostate cancer development, development of CRPC and drug and radiotherapy sensitivity with a focus on the adoptive and innate immune system.
Group leader André Bergman
André Bergman MD PhD Group leader Monique Melis PhD Post-doc Johan van Burgsteden MSc Technical staff
Role of lymphocytes in prostate carcinogenesis
Figure 1: Prostates of 12 weeks old mice; wild-type with normal epithelium (A), HiMYC mouse with marked PIN lesions (B), PB-Cre;PTENF/F mouse with invasive adenocarcinoma (C).
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Both overexpression of myc and loss of PTEN are frequent genetic lesions in human prostate cancer. Two mouse models are employed for our studies; one with a prostate specific expression of human myc (HiMYC mice) and one with a prostate specific loss of PTEN (PB-Cre;PTENF/F mice). The HiMYC mice develop preneoplastic Prostatic Intraepithelial Neoplasia (PIN) lesions at the age of two monts and invasive adenocarcinoma after six months (figure 1). The PB-Cre;PTENF/F mice develop invasive lesions much faster, with invasive carcinoma from the age of 12 weeks on. Immune cells are found in low numbers in the prostates of HiMYC and PB-Cre;PTENF/F mice, including lymphocytes, macrophages and mast cells. The role of the lymphocytes in prostate cancer development is addressed by genetic and pharmacological elimination or inhibition of specific subsets of lymphocytes and their soluble mediators. Therefore, HiMYC mice and PB-Cre;PTENF/F mice are crossed to B- and T-cell deficient RAG1 knock out mice (RAG-/-). In our studies, development of adenocarcinoma of the prostate is delayed in HiMYC;RAG1-/- mice compared to HiMYC mice. Preliminary data suggests also a delayed adenocarcinoma development in PB-Cre;PTENF/F;RAG1-/- mice compared with PB-Cre;PTENF/F mice at 24 weeks of age. These differences in latency of the development of prostate cancer between wild-type and immune deficient mice suggest a pivotal role of these immune cells in tumorigenesis. FACS analysis of immune cell populations in prostates of HiMYC and HiMYC;RAG1-/- mice shows absence of CD19 and B220 positive B-lymphocytes and CD3 positive T-lymphocytes populations (figure 2) in HiMYC;RAG1-/- mice but low numbers in HiMYC mice, however, no differences in total immune cell count (CD45), macrophage (F4/80) and total myelocyte (CD11b) populations are found. Mechanistic studies concentrate on temporal and spatial analysis of immune cell populations by immuno histochemistry and expression of proinflammatory soluble mediators in the tumor microenvironment by Luminex assay. The exact interaction between epithelium, tumor and lymphocytes and their soluble mediators is addressed in vitro. Primary epithelial cells of wild-type, HiMYC and PBCre;PTENF/F mice and cell lines of myc (MYCCaP) and PTEN-loss
(PTEN-CaP) driven murine prostate cancer are exposed to lymphocyte subsets and soluble mediators and assessed for proliferation, migration and invasion.
Role of the inflammatory tumor-microenvironment in castration resistant prostate cancer development Bulky prostate tumors of PB-Cre;PTENF/F mice expressing Luciferase (PB-Cre;PTENF/F;L-stop-L-Luciferase mice) in their prostates (figure 3) are harvested and small fragments are ortotopically transplanted into the anterior prostate lobe of mice with a genetically or pharmacological impaired immune system. After outgrowth of the tumors, mice are castrated which results in regression of the tumors. However, tumors will regrow and become castration resistant. Tumor regression and subsequent growth can be assessed by bioluminescence techniques. Differences in the latency of development of castration resistance between wild-type and immune deficient mice are indicative for a role of specific subsets of the immune system in the development of CRPC.
Figure 2: FACS analysis of CD3 positive cells (T-lymphocytes) in prostates of 30 weeks old HiMYC (left panel) and HiMYC;RAG1-/- mice (right panel).
Exploring the role of the microenvironment in human prostate cancer progression In this study we aim to evaluate the contribution of immune cells, attracted to the human prostate cancer associated stroma, in progression of this disease. Based on Gleason score, T stage and lymph node involvement, cohorts of formalin fixed paraffin imbedded whole mount prostatectomies are formed. The frontier between the tumor and the adjacent stroma is indicated by a pathologist (figure 4). A Tissue Micro Array (TMA) is made of two cores at the frontier at the tumor side and two at the adjacent stroma side per level. Per level 10 μm sections are prepared. After deparraffinization, tumor tissue is separated from its adjacent stroma and DNA and RNA are isolated. Purity of the tumor and stroma compartments is assessed by differential expression of epithelial and stromal markers. Markers of immune cells and soluble mediators are evaluated in the tissue compartments by quantitative PCR and immunohistochemistry. In this study we aim to define an immunological signature with predictive properties for metastatic disease. Expression of markers of immune cells, soluble mediators and their receptors are correlated with clinical determinants including local lymph node metastases and biochemical and distant recurrences.
Figure 3: IVIS Bioluminescence imaging of wild-type (left) and PB-Cre;PTENF/F;Lstop-L-Luciferase (right) mice at the age of 14 weeks.
Figure 4: H&E stained whole mount of a human prostate. The tumor/adjacent stroma frontier is indicated by a line (A). Higher magnification of the frontier between the tumor and adjacent stroma (tumor on the left, stroma on the right) (B).
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B Dummer R, Beyer M, Hymes K, Epping MT, Bernards R, Steinhoff M, Sterry W, Kerl H, Heath K, Ahern JD, Hardwick JS, Garcia-Vargas J, Baumann K, Rizvi S, Frankel SR, Whittaker SJ, Assaf C. Vorinostat combined with bexarotene for treatment of cutaneous T-cell lymphoma: in vitro and phase I clinical evidence supporting augmentation of retinoic acid receptor/retinoid X receptor activation by histone deacetylase inhibition. Leuk Lymphoma. 2012;53:1501-8
Division head, group leader René Bernards René Bernards PhD Group leader Katrien Berns PhD Academic staff Valentina Gambino PhD Post-doc Hugo Horlings MD PhD Post-doc Sidong Huang PhD Senior post-doc Jackie Johnson PhD Post-doc Prasanth Kumar PhD Post-doc Ian Majewski PhD Post-doc Lorenza Mittempergher PhD Post-doc Zheng Xue PhD Post-doc Diede Brunen MSc PhD student Floris Groenendijk MD PhD student Guus Heynen MSc PhD student Anirudh Prahallad MSc PhD student Chong Sun MSc PhD student Liqin Wang MSc PhD student Annemiek Bes-Gennissen Technical staff Astrid Bosma Technical staff Marielle Hijmans MSc Technical staff Wipawadee Grernrum Technical staff
Publications
Bernards R. A missing link in genotype-directed cancer therapy. Cell. 2012;151:465-8 Berns K, Bernards R. Understanding resistance to targeted cancer drugs through loss of function genetic screens. Drug Resist Updat. 2012;15:268-75 Brennan DJ, O’Connor DP, Laursen H, McGee SF, McCarthy S, Zagozdzon R, Rexhepaj E, Culhane AC, Martin FM, Duffy MJ, Landberg G, Ryden L, Hewitt SM, Kuhar MJ, Bernards R, Millikan RC, Crown JP, Jirstrom K, Gallagher WM. The cocaine- and amphetamineregulated transcript mediates ligandindependent activation of ERalpha, and is an independent prognostic factor in node-negative breast cancer. Oncogene. 2012;31:3483-94
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Eichhorn PJ, Rodon L, Gonzalez-Junca A, Dirac A, Gili M, Martinez-Saez E, Aura C, Barba I, Peg V, Prat A, Cuartas I, Jimenez J, Garcia-Dorado D, Sahuquillo J, Bernards R, Baselga J, Seoane J. USP15 stabilizes TGF-beta receptor I and promotes oncogenesis through the activation of TGF-beta signalling in glioblastoma. Nat Med. 2012;18:429-35 Geutjes EJ, Bajpe PK, Bernards R. Targeting the epigenome for treatment of cancer. Oncogene. 2012;31:3827-44 Geutjes EJ, Tian S, Roepman P, Bernards R. Deoxycytidine kinase is overexpressed in poor outcome breast cancer and determines responsiveness to nucleoside analogs. Breast Cancer Res Treat. 2012;131:809-18 Huang S, Holzel M, Knijnenburg T, Schlicker A, Roepman P, McDermott U, Garnett M, Grernrum W, Sun C, Prahallad A, Groenendijk FH, Mittempergher L, Nijkamp W, Neefjes J, Salazar R, Ten Dijke P, Uramoto H, Tanaka F, Beijersbergen RL, Wessels LF, Bernards R. MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-beta Receptor Signalling. Cell. 2012;151:937-50 Krijgsman O, Roepman P, Zwart W, Carroll JS, Tian S, de Snoo FA, Bender RA, Bernards R, Glas AM. A diagnostic gene profile for molecular subtyping of breast cancer associated with treatment response. Breast Cancer Res Treat. 2012;133:37-47 Kuiken HJ, Egan DA, Laman H, Bernards R, Beijersbergen RL, Dirac AM. Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling. J Cell Mol Med. 2012;16:2140-9 Majewski IJ, Kluijt I, Cats A, Scerri TS, de Jong D, Kluin RJ, Hansford S, Hogervorst FB, Bosma AJ, Hofland I, Winter M, Huntsman D, Jonkers J, Bahlo M, Bernards R. An alphaE-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol. 2012
Functional Genomics
My group interrogates the genome to identify biomarkers and mechanisms of response to cancer drugs. We use both functional genetic screens and next generation sequencing technologies to achieve these goals.
Identification of mechanisms and biomarkers of drug response Both intrinsic and acquired unresponsiveness to therapy is a recurring problem in the treatment of cancer. It is therefore important to identify the molecular pathways that contribute to unresponsiveness to cancer therapeutics. We use loss-offunction genetic screens with large sets of shRNA vectors to identify genes that contribute to drug resistance, in particular to the new classes of targeted therapeutics. In the past year, we have focused on the identification of genes whose suppression contributes to resistance to receptor tyrosine kinase (RTK) inhibitory drugs and their downstream signalling pathways. Using a large-scale RNAi screen, we identified MED12, a component of the transcriptional MEDIATOR complex, as a determinant of response to ALK and EGFR inhibitors. MED12 is in part cytoplasmic where it negatively regulates TGFβR2 signalling through physical interaction. MED12 suppression therefore results in TGFβ activation, which is both necessary and sufficient for drug resistance. TGFβ signalling causes ERK activation and consequently MED12 suppression also confers resistance to MEK and BRAF inhibitors in other cancers. MED12 loss induces an EMT-like phenotype, which is associated with poor outcome in primary colon cancer and resistance to MEK inhibitors in a large and heterogeneous panel of cancer cell lines. Inhibition of TGFβR signalling restores drug responsiveness in MED12KD cells, suggesting a strategy to treat drug-resistant tumours that have undergone a TGFβ-induced EMT (see figure). Importantly, we also find that the activation of TGFβ that results from MED12KD also induces resistance to a number of chemotherapies. Consistent with this, we find that an EMT-like gene signature that is induced upon MED12KD is associated with resistance to 5-FU based chemotherapy in colon cancer. We are currently studying how EMT induces chemotherapy resistance. Inhibition of the BRAFV600E oncoprotein by the small molecule drug PLX4032 (vemurafenib) is highly effective in the treatment of melanoma. However, colon cancer patients harbouring the same BRAFV600E oncogenic lesion have poor prognosis and show only a very limited response to this drug. Early this year we published that that blockade of the epidermal growth factor receptor (EGFR) shows strong synergy with BRAFV600E inhibition. In multiple BRAFV600E mutant colon cancers inhibition of EGFR by the antibody drug cetuximab or the small molecule drugs gefitinib or erlotinib is strongly synergistic with BRAFV600E inhibition, both in vitro and in vivo. Late in 2012, the first patient with BRAF mutant colon cancer was enrolled at our hospital in
a clinical trial based on the preclinical insights gained earlier the same year. This achievement underscores how effective we are as a comprehensive cancer centre in binging new basic research findings to cancer patients. Melanoma cells express low levels of EGFR and are therefore not subject to this feedback activation. Consistent with this, we find that ectopic expression of EGFR in melanoma cells is sufficient to cause resistance to PLX4032. In collaboration with the Institut Gustave Roussy in Paris, we have shown that some melanoma patients that acquire resistance to MEK or BRAF inhibitors have upregulated EGFR expression, suggesting that such patients too may benefit from a combination of BRAF/MEK and EGFR inhibitor drugs. Following our success in finding kinases whose inhibition is synthetic lethal with BRAF inhibition in colon cancer, we have initiated new projects to find additional synthetic lethal drug pairs. In first instance we have focussed on KRAS mutant colon and lung cancers treated with small molecule MEK inhibitors as these drugs are relatively ineffective in these cancers. To date, we have validated two druggable kinases that are synthetic lethal with MEK inhibition in KRAS mutant colon and lung cancers. Additional studies, including validation in animal models, are ongoing.
Next generation sequencing We have developed a capture reagent to selectively sequence a set of some 600 cancer-related genes, including all 518 kinases. We have used this tool to selectively sequence these genes at considerable depth in two difficult to treat subtypes of breast cancer: 150 invasive lobular carcinomas and an equal number of triple negative breast cancers. For all tumours, long-term outcome data are available. This work was carried out in the context of an EU project named “RATHER”, in which other partners have performed gene expression analysis, Reverse Phase Protein Lysate Array (RPPA) analysis and SNP genotyping. In 2012, we have completed the sequencing of these tumours and we are making progress toward integrating the multiple omics platform data. In a related project with Astra Zeneca and Agendia NV, we have performed kinome sequencing in some 150 colon cancers, for which RPPA data (performed at MDAnderson Cancer Center) and gene expression data (Agendia) have been collected as well. Through this project, we aim to elucidate which signalling pathways are activated in the three “molecular subtypes” of colon cancer identified at Agendia. In a third sequencing project, we aim to identify novel translocations involving kinases through sequencing of the RNA transcripts (RNAseq) of the same 600 kinase-related genes mentioned above. Using this approach on RNA from 100 non small cell lung cancers, we have already identified two novel translocations that are clinically actionable in that small molecule kinase inhibitors are available for these translocation products. In the next year, we will expand this analysis to additional cancer types. Finally, in collaboration with the family cancer clinic of our hospital, we have identified a novel germ line mutation in a large family suffering from hereditary diffuse gastric cancer. We identified a germ line truncating allele of alpha-E-catenin (CTNNA1) in members of the family affected by disease.
Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, Beijersbergen RL, Bardelli A, Bernards R. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012;483:100-3 Rigaill GJ, Cadot S, Kluin RJ, Xue Z, Bernards R, Majewski IJ, Wessels LF. A regression model for estimating DNA copy number applied to capture sequencing data. Bioinformatics. 2012;28:2357-65 Sun T, Simon I, Moreno V, Roepman P, Tabernero J, Snel M, Van’t Veer L, Salazar R, Bernards R, Capella G. A combined oncogenic pathway signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction. Gut. 2012 Tian S, Roepman P, Popovici V, Michaut M, Majewski I, Salazar R, Santos C, Rosenberg R, Nitsche U, Mesker WE, Bruin S, Tejpar S, Delorenzi M, Bernards R, Simon I. A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency. J Pathol. 2012
Drug resistance caused by activated TGFb signalling. MED12 was identified in a RNAi screen for genes whose suppression causes resistance to both RTK inhibitors and chemotherapy in lung cancer. Loss of MED12 expression leads to activated TGFb signalling, which is responsible for drug resistance. A gene signature that results from loss of MED12 expression was found to predict response to chemotherapy in colon cancer.
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Mouse models for cancer
Group leader Anton Berns
Anton Berns PhD Group leader Paul Krimpenfort PhD Academic staff Margriet Snoek PhD Academic staff Hilda De Vries PhD Post-doc Jitendra Badhai PhD Post-doc Ivo Huijbers PhD Post-doc Min-chul Kwon PhD Post-doc Kate Sutherland PhD Post-doc Lorenzo Bombardelli PhD Post-doc Andrej Alendar MSc PhD student Colin Pritchard MSc Research assistant Rahmen Bin Ali Technical staff Miranda Cozijnsen Technical staff Jan Paul Lambooij Technical staff Natalie Proost Technical staff Maaike Hanegraaf Technical staff Fina Van de Ahé Technical staff John Zevenhoven Technical staff
Publications
Krimpenfort P, Song J-Y, Proost N, Zevenhoven J, Jonkers J and Berns A. Deleted in colorectal carcinoma suppresses metastasis in p53deficient mammary tumours. Nature 2012;482:538-541 Walf-Vorderwülbecke V, De Boer J, Horton SJ, van Amerongen R, Proost N, Berns A, and Williams O. Frat2 mediates the oncogenic activation of Rac by MLL fusions. Blood 2012;120:4819-28 Horn KE, Glasgow SD, Gobert D, Bull SJ, Luk T, Girgis J, Tremblay ME, McEachern D, Bouchard JF, Haber M, Hamel E, Krimpenfort P, Murai KK, Berns A, Doucet G, Chapman CA, Ruthazer ES, Kennedy TE. DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain. Cell Reports (in press)
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The mouse is used as a model organism for establishing the role of oncogenes and tumor suppressor genes in tumor development. By utilizing Cre/Lox mediated switching and taking advantage of somatic gene transfer methods, expression of multiple oncogenes and tumor suppressor genes are regulated in a tissue-specific and spatial-temporal fashion permitting accurate modeling of tumorigenesis as it is observed in man. It provides the opportunity to carefully define relevant genotypephenotype correlations and permit us to identify new genes involved in tumor progression using a variety of techniques, such as array CGH, DNA and RNA sequencing and insertional mutagenesis. These models also constitute an excellent experimental system to test new intervention strategies.
Functional analysis of oncogenes and tumor suppressor genes Our emphasis is on lung cancer and mesotheliomas. To achieve (sporadic) activation of oncogenes and inactivation of tumor suppressor genes we use Adeno-Cre or Lentivirus-mediated somatic gene transfer to sporadically switch the conditional oncogenes and tumor suppressor gene alleles in the desired tissues. Subsequently, tumor initiation and progression over time is monitored using a variety of techniques. When Rb and p53 are inactivated specifically in lung, small cell lung cancer (SCLC) develops in nearly 100% of the mice. These tumors closely resemble human SCLC. The tumors often consist of two different cell types, one showing neuroendocrine markers whereas the other has mesenchymal features. Interestingly, these different cells appear often derived from a common progenitor. Subcutaneous grafting of each of the cell types independently gives results in localized tumor growth. However, grafting mixtures resulted in local growth as well as metastasis of the neuroendocrine cells to liver indicating that the nonneuroendocrine cells in the graft endowed the neuroendocrine cells with metastatic potential. This effect can also be studied in culture with invasiveness as readout. In this way we have identified a number of candidate proteins that might be responsible for this paracrine signaling. We have shown that this requires activation of the MAPK pathway in neuroendocrine cells and that one of the critical genes induced in these cells is PEA3, a gene previously found to be involved in invasion and metastasis. We are currently identifying the upstream and downstream components of this signaling cascade. We also wanted to gain insight into the cell-of-origin of SCLC and NSCLC and have developed a series of Adeno-Cre viruses with cell-type specific promoters driving Cre expression. These vectors enable us to switch oncogenes and tumor suppressor genes specifically in Clara cells, Alveolar type II cells, neuroendocrine cells and basal epithelial cells. Last year we showed that neuroendocrine cells are the predominant cell-
Figure 2: Luciferase signals in lung of Rb/F/F;p53F/F chimeras carrying luciferase by itself or in combination with L-Myc and Nfib. The presence of L-myc and Nfib transgenes accelerate to tumorigenesis.
Figure 1: Schematic representation of the effects observed in KrasG12D and KrasG12D;p53F/F lungs of mice treated with the different adenoviruses targeting alveolar type II cells, Clara cells, and neuro-endocrine cells.
of-origin of SCLC. We have performed similar experiments in conditional mutant KrasG12D mice with or without concomitant depletion of p53. Monitoring of tumor development in these mice shows that both the cell-of-origin and the introduced genetic lesions determine the phenotypic characteristics of the resulting adenocarcinomas (figure 1). Both CC10- and SPC-expressing cells can act as the origin for K-rasG12D only tumors whereas tumors arise from all three cell types when p53 is concomitantly inactivated. We propose that p53 loss enhances the plasticity enabling differentiated cells, such as neuroendocrine cells to initiate adenocarcinoma formation.
Mesotheliomas Previously we have generated mouse models for mesotheliomas by the inactivation of Nf2 and Ink4a/Arf or Nf2/p53/Ink4a in conditional knockout mice by intrathoracic Adeno-Cre injection. We have now also generated conditional Bap1 knockout mice as BAP1 appears very frequently inactivated in human mesotheliomas. The various compound mutants are currently being produced to assess the cooperating capacity of the different lesions. Furthermore we have crossed a PiggyBac transposon/transposase into the Ink4ab-p19Arf knockout to identify additional genes that can drive mesothelioma initiation and progression. Parallel to these experiments we have tested protocols to establish cultures from human mesotheliomas. Although the results look promising further characterization of the cells propagated in vitro is necessary. Preliminary data also indicate that we can propagate small pieces of at least some of the primary human mesothelioma in mice.
Role of DCC in tumor progression Since its discovery in the early 1990s the DCC gene, located on
chromosome 18q21, is suggested as tumor suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of Netrin-1 receptors, which play a major role in the development of the nervous system by guiding axonal outgrowth. They also function as dependence receptors and deliver survival signals in the presence of their ligand. However, the role of DCC as a tumor suppressor has remained controversial. We have shown that in a mouse model for mammary carcinoma based on somatic inactivation of p53 additional loss of Dcc promotes metastasis formation without affecting the phenotype of the primary tumor. Furthermore, in cell cultures derived from p53-deficient mouse mammary tumors Dcc expression controls Netrin-1 dependent cell survival providing a mechanistic basis for the enhanced metastatic capacity of tumor cells lacking Dcc. Our data support DCC ‘s function as a context-dependent tumor suppressor that limits survival of disseminated tumor cells.
GEMM-ES cells as a tool We have now established robust methods to substantially accelerate the generation of complex compound mutant mice. The concept is to re-derive embryonic stem (ES) cells from available compound mutant strains and use these ES cells to introduce additional genetic changes by gene targeting or cassette exchange. Using this approach we have successfully derived a series of ES cell lines from the complex strains we routinely use. We have introduced exchange cassettes in these ES cells and have shuttled reporters and genes believed to accelerate tumor development. Chimeras produced from these ES cells showed the tumor acceleration we hoped to see (figure 2). This will greatly accelerate the testing of candidate genes, identified by human cancer genome sequencing or functional screens in cell lines in a realistic in vivo setting. 23
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B Hooijkaas A, Gadiot J, Morrow M, Stewart R, Schumacher T, Blank CU. Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma. Oncoimmunology. 2012;1:609-17 Hooijkaas AI, Gadiot J, van der Valk M, Mooi WJ, Blank CU. Targeting BRAF(V600E) in an inducible murine model of melanoma. Am J Pathol. 2012;181:785-94
Group leader Christian Blank
Christian Blank MD PhD Group leader Andrew Kaiser PhD Post-doc Anna Hooijkaas MSc PhD student Marcel Deken MSc PhD student Aurelie Guislain Technical staff Jules Gadiot Technical staff
Publications
Vogel WV, Guislain A, Kvistborg P, Schumacher TN, Haanen JB, Blank CU. Ipilimumab-induced sarcoidosis in a patient with metastatic melanoma undergoing complete remission. J Clin Oncol. 2012;30:e7-e10 Kaiser AD, Schuster K, Gadiot J, Borkner L, Daebritz H, Schmitt C, Andreesen R, Blank C. Reduced tumor-antigen density leads to PD-1/ PD-L1 mediated impairment of partially exhausted CD8(+) T cells. Eur J Immunol. 2012;42:662-71 Bex A, Etto T, Vyth-Dreese F, Blank C, Griffioen AW. Immunological heterogeneity of the RCC micro environment: do targeted therapies influence immune response? Curr Oncol Rep. 2012;14:230-9 Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, Rutkowski P, Blank CU, Miller WH Jr, Kaempgen E, Martín-Algarra S, Karaszewska B, Mauch C, ChiarionSileni V, Martin AM, Swann S, Haney P, Mirakhur B, Guckert ME, Goodman V, Chapman PB. Dabrafenib in BRAFmutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380:358-65 Mougiakakos D, Okita R, Ando T, Dürr C, Gadiot J, Ichikawa J, Zeiser R, Blank C, Johansson C, Kiessling R. High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis. J Mol Med. 2012;90:935-44
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Kaiser AD, Gadiot J, Guislain A, Blank CU. Mimicking homeostatic proliferation in vitro generates T cells with high anti-tumor function in non-lymphopenic hosts. Cancer Immunol Immunother. 2012 (in press) Haanen J, Blank C. Immunomodulatory Drugs and Cytokines. ESMO handbook Clinical Pharmacology of Anti-cancer Agents. 2012
Combining targeted therapy and immunotherapy in melanoma and renal cell carcinoma We aim to identify mechanisms of tumor immune escape and to develop therapeutic protocols to combine such checkpoint blockade with targeted and other therapies (figure 1). Tumor immune escape mechanisms include inhibitory molecules on tumor cells or on antigen presenting cells and immune regulatory cells in the tumor environment. The functional characterization of inhibitory molecules, exploration of their inhibition and the examination of possible synergy with small molecule-based targeted and other therapies may help in designing novel approaches to improve anticancer immunotherapy.
Development of inducible spontaneous murine melanoma models Targeted and immunotherapy do not only alter tumor cells but also the tumor environment. Thus it is crucial to simulate combination therapies in spontaneous melanoma models that simulate the human cancer reality. We have crossed mice that inducibly express in melanocytes BRAFV600E and loose PTEN, and tested in these mice combinations of targeted and immunotherapy. While combination of BRAF and MEK inhibition improved tumor control, BRAF inhibitors impaired tumor immune infiltration and did not synergize with T-cell checkpoint blockade by anti-PD-1, -PD-L1 and -CTLA-4 mAb (figure 2). Current experiments aim at understanding the mechanism of BRAF inhibitor-mediated decrease of tumor immune infiltration. Additional spontaneous melanoma models harboring second mutations other than PTEN loss and alteration of the model to harbor more (bystander) mutations and immunogenic antigens are currently developed.
Homeostatically proliferating T cells for the treatment of cancer One approach in immunotherapy is the adoptive transfer of tumor-reactive T cells. For effective tumor growth control, tumor-reactive T cells should sufficiently expand and survive, without exhaustion. Transfer of naïve peripheral T cells into lymphopenic murine recipients results in a slow cytokinedriven proliferation of these T cells. During this homeostatic proliferation (HP), T cells acquire effector functions (IFNproduction, lytic activity), while keeping characteristics of naïve T cells. This results in better CD62L-mediated lymph node homing, less anergy induction and better tumor growth control compared to naïve or effector T cells. As induction of lymphopenia by chemo- or chemoradiotherapy is accompanied by serious adverse events, we aim HP-like T cell expansion in vitro. Recently, we could show that these in vitro generated HP T cells have superior tumor growth control capabilities in vivo (in mice) and characterize them in comparison to naïve, primed and memory T cells.
Generation of RCC TIL after tyrosine kinase inhibitor pretreatment TIL therapy is a promising immunotherapeutic approach in melanoma inducing long lasting clinical responses currently tested at several institutes, including the NKI-AVL. Culture of TIL generated from RCC has been described before, but failed to induce clinical responses. Reason for this is that former protocols lacked preconditioning of the patients to induce lymphopenia and combination with small molecules and antibody mediated checkpoint blockade. We found that TIL from sunitinib pretreated RCC patients expanded better due to alteration of the RCC inhibitory environment. Currently, expansion protocols that prevent exhaustion and negative selection of sunitinib pretreated TIL are being developed.
Figure 1: Combination options of targeted and immunotherapy
Role of co-inhibitory molecules during tumor immune escape Appropriate T cell activation depends on T cell receptor (TCR) ligation and a positive secondary signal. Recent work revealed that this secondary signal is not an on-off phenomenon, but a signal of modulated intensity, which is orchestrated by several co-stimulatory and co-inhibitory molecules. We and others have shown that one of the ligands (PD-L1) of one such a co-inhibitory molecule (PD-1) is highly expressed on tumor cells and leads to impaired immune responses. We found an increased PD-L1 expression on metastases compared to primary melanoma in human, but no influence on overall survival, raising the question in which situations PD-L1 inhibits tumor specific T cells. In experiments using murine TCR transgenic T cells, we identified low tumor antigen expression combined with PD-L1 expression to predispose for the strongest inhibition of tumor-specific T cells.
Figure 2: Combination of BRAF inhibition (BRAFi) and CTLA-4 blockade fails, while BRAFi + MEKi act synergistic. BRAF inhibitor treatment (PLX4720) reduces tumor inflammation (A) represented by decreased tumor immune infiltration (B). Combination of BRAFi and CTLA-4 blockade did not improve melanoma control (C), while combination of BRAFi and MEKi (GSK212) improved long term outcome of animals, completely shutting off pERK as shown by histologic staining (E, upper image) and by western blotting (E, lower image).
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B Hahn D, Bleiker E, Woerdeman L, Emmel R. Tepel-transplantatie bij directe borstreconstructie. Eerste ervaringen met nipple banking. Psychosociale Oncologie 2012;20:18
Psychosocial issues in cancer genetics
Long-term psychosocial and medical impact of breast cancer genetic counselling offered soon after diagnosis
Douma KFL, Vasen HFA, Aaronson NK, Bleiker EMA. Reproductive decisionmaking in hereditary colorectal cancer. The role of the gastroenterologist. Colorectal Cancer (in press)
Group leader Eveline Bleiker
Eveline Bleiker PhD Group leader Jessica Baars PhD Post-doc Willem Eijzenga MSc PhD student Marijke Wevers MD PhD student Femme Harinck MD PhD student Maurits De Boer MSc Research assistant Marianne Kuenen Research assistant Grace Sidharta MSc Research assistant Jacobien Kieffer PhD Technical staff
Publications
Mai PL, Malkin, D, Garber J, Schiffman JD, Witzel JN, Strong L, Wyss O, Locke L, Means V, Achatz MI, Hainaut P, Frebourg T, Evans DG, Bleiker E, Patenaude A, Schneider K, Wilfond B, Peters J, Hwang PM, Ford J, Tabori U, Ognjanovic S, Dennis P, Wentzensen IM, Greene MH, Fraumeni JF jr, Savage SA. Li-Fraumeni syndrome: Report of a clinical research workshop and creation of a research consortium. Cancer Genetics 2012;205:479-487 Wevers MR, Hahn DEE, Verhoef, S, Bolhaar DK, Ausems, MGEM, Aaronson, NK, Bleiker EMA. Breast cancer genetic counseling after diagnosis but before treatment: A pilot study on treatment consequences and psychological impact”. Patient Education and Counseling, 2012;89:89-95 Kluijt I, Sijmons RH, Hoogerbrugge N, Plukker JT, Jong D de, Krieken JH van, Hillgersberg R van, Ligtenberg M, Bleiker EMA, Cats A. Familial gastric cancer: guidelines for diagnosis, treatment and periodic surveillance. Familial Cancer 2012;11:363-369 Nieuwenhuis MH, Douma KFL, Bleiker EMA, Aaronson NK, Clevers H, Vasen HFA. Clinical evidence for an association between familial adenomatoes polyposis (FAP) and type II diabetes. International Journal of Cancer, 2012;131:1488-1489
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This research line is being conducted in close collaboration with the Antoni van Leeuwenhoek family cancer clinic. It comprises a number of studies which are focused on two psychosocial themes in genetic counseling for cancer: 1) the uptake and longterm psychosocial impact of genetic counseling and testing and risk-reducing behavior; and 2) early detection of psychosocial problems and the development of psycho-educational interventions.
Screening for psychosocial problems at the family cancer clinic The aim of this KWF-study is to develop a screening checklist and evaluate the efficacy of this screening instrument as implemented in daily practice as an aid in identifying individuals experiencing significant psychosocial problems associated with cancer genetic counseling. In 2009, this screening checklist was developed adopting the EORTC guidelines for questionnaire module development. Twenty-six issues were identified and operationalized into items covering six problemdomains: family, children, genetics, cancer, practical issues, and emotions. In 2010 and 2011, this so called Psycho-OncoGenetics (POG) checklist has been evaluated for its screening properties for detecting psychosocial problems experienced by individuals attending the family cancer clinic of the Antoni van Leeuwenhoek. For this validation study, 127 counselees completed both the POG checklist and a clinical interview with a psychosocial worker (‘gold standard’). The new screening checklist proved to have sufficient screening properties. Therefore, in 2011, a randomized controlled clinical trial was started to evaluate the efficacy of this newly developed POG checklist, as an aid in 1) facilitating communication on psychosocial issues during the genetic counseling session, 2) increasing counselors’ awareness of psychosocial problems of the counselee, and 3) improving the management of these psychosocial problems during and after the process of genetic counseling. In total, 257 counselees from the Netherlands Canver Institute and UMCU are currently participating in this trial. Results will be available in 2013.
Preventive total gastrectomy The aim of this cross-sectional, multi-center study is to investigate the experiences with, and consequences of gastroscopy screening and prophylactic total gastrectomy in CDH1 mutation carriers. Mutations in the CDH1 gene are associated with a 70% lifetime risk for diffuse gastric cancer and an additional 40% risk for lobular breast cancer in women. Six families in the Netherlands were identified with presence of the CDH1-gene mutation. All proven CDH1-gene mutation carriers were invited to complete a self-report questionnaire and to participate in an interview. In total, 25 of the 31 CDH1 mutation carriers returned the questionnaires (81%). Of these, 20 individuals had undergone prophylactic total gastrectomy.
Respondents reported a wide variety of complaints (e.g., early satiety, extreme weight loss, fatigue, a negative impact on daily functioning) which were three years after gastrectomy less frequently reported but still apparent. ‘The level of energy’ was the most important factor determining functioning and quality of life after prophylactic gastrectomy: 55% of the participants experienced increased to severe fatigue. About 40% reported moderate to severe impairment in daily activities. Furthermore, 44% was not satisfied with the dietary care, and the follow-up after surgery was by half of the respondents experienced as suboptimal. A guideline has been developed (Kluijt et al. 2012) and new steps are undertaken to optimize health care for CDH1carriers undergoing total gastrectomy.
The aim of this study is to assess the long-term psychosocial and medical impact of genetic counselling offered soon after diagnosis of breast cancer. The study, which is funded by Pink Ribbon, is performed in collaboration with the department of Medical Genetics of the UMCU (M. Ausems) and has started in February 2011. In total, 112 breast cancer patients who had been actively offered genetic counseling and testing during radio therapy between 1997 and 2004 participate in our study. Their experiences are compared with those of a group of patients who were diagnosed with breast cancer between 2002 and 2004 and received radiotherapy, but who did not fulfill the criteria for referral for cancer genetic counseling and DNA testing (n=127). In total 72% (n=237) returned the self-report questionnaire. The majority of the women who had received DNA-testing remember their DNA-test results correctly (80/109=73%). Alike the comparative group, a relatively small part of patients who received genetic counseling currently experience high levels of depression (5%, n=6). Nearly 75% of the patients had received two or more mammograms in the past three years. In the long term, we do not find indications that a directive offer of genetic counseling and testing for breast cancer patients undergoing radiotherapy are currently experiencing additional distress. Furthermore, most counselees (93%, n=103) perceived themselves as the person who should be responsible to communicate genetic counseling results to their family members.
Surveillance of the pancreas in high risk individuals The aim of this study is to investigate the psychological burden of participating in a pancreatic cancer (PC-) surveillance program. Since 2006, a multi-center prospective study, funded by ZONMw, is investigating the effectiveness of PC-surveillance (EUS and MRI) in high-risk individuals. High-risk individuals are defined as (1) first degree relatives (FDR) of patients with familial pancreatic cancer (FPC) and (2) carriers of a PC-prone gene mutation. In 2009, a retrospective psychosocial study was added to this multicenter surveillance study (including 69 high-risk individuals), followed by. a prospective psychosocial study in 2010. The aim of the prospective study is to investigate the uptake of PC surveillance and possible changes over time in the physical and psychological burden of participating in this surveillance program. Individuals are invited to complete questionnaires two weeks before the first PC surveillance and four weeks after receiving the result of the annual surveillance. Currently 111 patients completed a questionnaire before their initial PC surveillance. Furthermore, respectively 66, 53 and 25 patients have completed a questionnaire after their first, second and third PC annual surveillance. From the retrospective study we may conclude that, from a psychosocial point of view, PC surveillance in high-risk individuals is feasible. Data of the prospective study are being collected on an ongoing basis. 27
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B Van de Kooij B*, Verbrugge I*, de Vries E, Gijsen M, Monserrat V, Maas Ch, Neefjes J, Borst, J. Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) ligase controls cell surface expression of TNF-related apoptosis inducing ligand (TRAIL) receptor 1. J Biol Chem. 2013 (in press)
Division head, group leader Jannie Borst Jannie Borst PhD Group leader Yanling Xiao MD PhD Senior post-doc Ulf Geumann PhD Post-doc Tomasz Ahrends MSc PhD student Bert Van de Kooij MSc PhD student Rogier Rooswinkel MSc PhD student Elise Veraar MSc PhD student Nikolina Babala MSc Technical staff Evert de Vries BSc Technical staff
Publications
Coquet JM, Middendorp S*, van der Horst G*, Kind J, Veraar EAM, Xiao Y, Jacobs H, Borst J. The CD27 and CD70 costimulatory pathway inhibits effector function of T helper 17 cells and attenuates associated autoimmunity. Immunity 2013;38:53-65 Coquet JM*, Ribot JC*, B bała N, Middendorp S, Van der Horst G, Xiao Y, Neves JF, Pereira D, Pennington DP, Jacobs H, Silva-Santos B**, Borst J**. Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27/CD70 pathway. J Exp Med 2013 (in press) Feau S, Garcia Z, Arens R, Yagita H, Borst J, Schoenberger SP. The CD4+ T-cell help signal is transmitted form APC to CD8+ T-cells via CD27-CD70 interactions. Nat Comm. 2012;3:948 Rooswinkel RW, Van de Kooij B, Verheij M, Borst J. Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B. Cell Death Dis. 2012;3:e366
* and **Equal contribution 28
Van de Kooij B*, Rooswinkel RW*, Kok F, Herrebout M, de Vries E, Paauwe M, Janssen GMC, Van Veelen PA, Borst J. Poly-ubiquitination and proteasomal turnover controls the anti-apoptotic activity of Bcl-B. Oncogene. 2013 (in press)
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 "on the edge" between life and death. Our work is inspired by the desire to improve cancer immunotherapy. The second aim of our work is to contribute to the design of novel therapies aimed at killing cancer cells by activating apoptotic pathways.
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 receptor/ligand pair promotes the generation and maintenance of cytotoxic T cells, CTL), the formation of memory CTL cells and their secondary expansion. We have identified CD27 target genes and followed them up by functional experiments in vitro and in vivo. In this way, we have found that CD27 promotes the survival of effector CD8 T cells at tissue sites via autocrine IL-2 signaling and promotes clonal expansion of CD8 T cells at the priming site via Bcl-xL and the Pim-1 serine/threonine kinase. CD27 also directs the expression of specific chemokines in primed CD8 T cells, which promote generation of effector CTL during priming. CD27/CD70 interactions not only promote survival and clonal expansion of CD8 T cells, but also have qualitative effects on CD4 and CD8 T cells. In CD4 T cells, CD27 target genes are diagnostic for a T helper-1 effector function. CD27 signaling inhibits the function of T helper-17 cells, a pro-inflammatory CD4 T cell subset that can cause auto-immune disease. Through our work, the Jun kinase pathway emerged as an important suppressor pathway for T helper-17 cell function, which may have clinical implications. CD4 T-cell help for the CD8 memory T cell response is highly dependent on the CD27-CD70 pathway, which has implications for vaccine design and diagnosis of vaccine efficacy. Memory programming of CD8 T cells relies on the CD27/ CD70 pathway and proceeds in part via a novel molecule that facilitates costimulation. With the aid of our new CD70-deficient mice, we have discovered that CD27-CD70 interactions in the thymus contribute to the generation of regulatory CD4 T cells (Treg). Both epithelial and dendritic cells in the thymic medulla contribute to positive selection of Treg by preventing their apoptosis via the CD27/ CD70 pathway. Current work explores the role of the CD27/CD70 pathway in Treg expansion and exhaustion of conventional T cells in chronic infection. Constitutive expression of CD70 is a hallmark of a (chronically) activated immune system, such as in human lupus and rheumatoid arthritis. By analysis of CD70 transgenic mice that mimic this situation, we have discovered a novel link between
CD27-CD70 and bone homeostasis. CD27 is expressed on a newly defined osteoclast progenitor in the mouse and its sustained engagement by CD70 on activated immune cells inhibits osteoclast development. Gene expression profiling has confirmed the identification of novel osteoclasts precursors and revealed that our data are relevant for the human situation, where these precursors are undefined. Our gene set forms the starting point to identify human osteoclast precursors and novel molecules involved in osteoclast and dendritic cell development. CD70 is also a target for novel therapeutic antibodies in hematopoietic and solid tumors, such as renal cell carcinoma. Novel targeting antibodies to CD70 are entering clinical trials at present. We have discovered that cell surface expression of CD70 and thus the possibility to target this molecule is under specific molecular control. In dendritic cells, the MHC Class II chaperone Invariant chain brings CD70 to late endosomal MHC Class II compartments, from which it can be mobilized to the cell surface, together with MHC Class II. We have developed technology to study this process in real time and are extrapolating this work to renal cell carcinoma.
To better delineate the selectivity of Bcl-2 family interactions, we have used a novel technique that reveals interactions as they occur in the mitochondrial membrane. The comprehensive interaction profile that we have obtained extends and corrects current knowledge. We have studied the anti-apoptotic capacity of all six inhibitory Bcl-2 proteins side-by-side in the same cellular model systems and found that the differential antiapoptotic capacity of these proteins cannot be explained by their BH3 domain selectivity. Rather, ubiquitin- and proteasome dependent turnover of inhibitory Bcl-2 proteins appears to be the decisive parameter for their anti-apoptotic activity. This finding offers a novel viewpoint for the design of targeted drugs.
Conclusion Our findings indicate that targeting the CD27-CD70 costimulatory axis is of interest not only in the context of anti-tumor immunity, but also in auto-immune or inflammatory disease. Follow up in the human system should further validate this. In collaboration with researchers from the novel biotech company BioNovion, we study reagents that target the human molecules with the aim to apply them clinically.
Apoptosis signaling and cancer therapy
The CD27/CD70 receptor-ligand system is an important target for clinical intervention with monoclonal antibodies in cancer (immuno)therapy and autoimmune disease. BioNovion and J. Borst hold a patent on one such agent and are developing it for clinical application.
Pro-apoptotic agents are of great interest for cancer therapy, provided that they can act in a tumor-specific fashion. The TRAIL death receptors conform to this condition; they are not toxic on normal tissue and induce apoptotic cell death in many different tumor types. We have identified a ubiquitin ligase that determines the steady-state cell surface expression of TRAIL receptor-1, but not TRAIL receptor-2. This ligase targets TRAIL receptor-1 for degradation, by promoting its endocytosis. We have identified the mechanistic details of this process, including the definition of a lysine residue in the receptor tail that is essential for interaction with and ubiquitination by the ligase. We have identified another molecule that impacts on intracellular transport of TRAIL receptor-1 in the biosynthetic route. Both mechanisms attenuate apoptosis sensitivity of the tumor cells and may therefore be of clinical relevance. Another important target for tumor therapy are the inhibitory Bcl-2 family members that block apoptosis at the level of the mitochondrial membrane. Many tumor cell types rely on overexpression of these Bcl-2 proteins that enable tumor cells to survive their stressful growth conditions. In response to apoptotic stimuli, BH3 domain-only Bcl-2 proteins induce mitochondrial membrane permeabilization, by activating the effector molecules Bax and Bak. The inhibitory Bcl-2 family members block this, by physical interaction with their proapoptotic relatives. Interaction between pro- and anti-apoptotic Bcl-2 proteins is intensely studied, since novel targeted reagents are based upon it. BH3 domain mimetic drugs act tumor-specifically according to the principle of “oncogene” addiction.
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B Jaspers JE, Kersbergen A, Boon U, et al. Loss of 53BP1 causes PARP inhibitor resistance in BRCA1-mutated mouse mammary tumors. Cancer Discovery 2012
DNA base J
Drug resistance in “spontaneous” mouse tumors
Krumpochova P, Sapthu S, Brouwers JF, et al. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. FASEB J 2012;26:738-747
Group leader Piet Borst
Piet Borst MD PhD Group leader Henri Van Luenen PhD Academic staff Sven Rottenberg DVM PhD Dipl ECVP Research associate Koen Van de Wetering DVM PhD Senior post-doc Charlotte Guyader PhD Post-doc Robert Jansen PhD Post-doc Nikola Banishki MSc PhD student Guotai Xu MSc PhD student Serge Zander DVM MSc PhD student Ewa Gogola MSc PhD student Liesbeth Van Deemter Technical staff Ariena Kersbergen Technical staff Sunny Sapthu Technical staff Wendy Sol Technical staff Asli Küçükosmano lu Technical staff
Rottenberg S, Vollebergh MA, de Hoon B, et al. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1deficient mammary tumors. Cancer Res 2012;72:2350-2361 Van Luenen H, Farris C, Jan S, et al. Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania. Cell 2012;150:909-921 Zander SAL, Beijnen HJ, Van Tellingen O. Sensitive method for plasma and tumor Ko143 quantification using reversed-phase high-performance liquid chromatography and fluore scence detection. J Chromatogr B Analyt Technol Biomed Life Sci 2012 Van de Wetering JK, Sapthu S. ABCG2 functions as a general phytoestrogensulfate transporter in vivo. FASEB 2012;26:4014-4024
Publications
Zander SAL, Sol W, Zhang Y, et al. EZN2208 overcomes ABCG2-mediated topotecan resistance in BRCA1deficient mouse mammary tumors. PLoS One 2012;7:e45248 Heidebrecht T, Fish A, Von Castelmur E, et al. Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or hmC; evidence for a rapid conformational change upon DNA binding. Journal of the American Chemical Society 2012;134:1335713365 Borst P. Cancer drug pan-resistance: pumps, cancer stem cells, quiescence, epithelial to mesenchymal transition, blocked cell death pathways, persisters or what? Open Biol 2012;2:120066 Zander SA, Kersbergen A, Sol W, et al. Lack of ABCG2 Shortens Latency of BRCA1-Deficient Mammary Tumors and This Is Not Affected by Genistein or Resveratrol. Cancer Prev Res (Phila) 2012;5:1053-1060 Rottenberg S, Borst P. Drug resistance in the mouse cancer clinic. Drug Resist Updat 2012;15:81-89
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Beekman CA, Buckle T, van Leeuwen AC, et al. Questioning the value of (99m)Tc-HYNIC-annexin V based response monitoring after docetaxel treatment in a mouse model for hereditary breast cancer. Appl Radiat Isot 2011;69:656-662
vesicles prepared from cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We are using this method now to find the physiological substrates of MRP4, 5 and 6.
Base J (β-glucosyl-hydroxymethyluracil), which we discovered in African trypanosomes in 1993 (Gommers-Ampt et al., Cell 1993;75:1129-1136), is a base present in kinetoplastid flagellates and in Euglena. It replaces 1% of thymine in nuclear DNA and is predominantly located in repetitive sequences, such as telomeric repeats. We have characterized a J-binding protein (JBP1) that binds to J-containing duplex DNA (Cross et al. EMBO J 1999;18:6573-6581). We have shown that JBP1 is a thymidine hydroxylase that catalyses the first step of J biosynthesis, the conversion of specific T-residues in DNA into hydroxymethyluracil. JBP1 belongs to the family of Fe2+- and 2-oxoglutarate-requiring dioxygenases, as does a second putative hydroxylase, JBP2. A JBP1 KO is lethal. In contrast, JBP2 is dispensable in Leishmania, but JBP2 KO strains are hypersensitive to bromodeoxyuridine (BrdU). The J level goes down to 30% of WT in the Leishmania JBP2 KO strains and during growth in BrdU, Leishmania JBP2 KO strains lose even more J (down to 13% of WT). Using immuno-precipitation of J-DNA and deep sequencing, we have found J in Leishmania at transcriptional stops (collaboration with Antoni van Leeuwenhoek deep sequencing unit and Peter Myler, Seattle). Loss of this internal J leads to massive read-through of RNA Polymerase II transcriptional stops, providing a plausible explanation for J-less death (figure 1). With Jonas Korlach (Pacific Biosciences, USA) we are using SMRT sequencing to locate the exact positions of J in DNA. With Anastassis Perrakis (Division of Biochemistry) we are trying to determine the structure of JBP1-J-DNA complexes by crystallography. Experiments in this project were terminated in Amsterdam in 2012 and will be continued by Peter Myler (Seattle, USA).
Drug transporters We are interested in mechanisms of drug resistance in cancer cells and focus on resistance caused by increased ATPdependent transport of drug out of the cell, mediated by ATPbinding cassette (ABC) transporters. We have isolated genes for these transporters and are characterizing their substrate specificity and sensitivity to inhibitors in transfected cells. To study the physiological function of these transporters, we have inactivated genes for several drug transporters by targeted gene disruption in mice. We are mainly studying the Multidrug Resistance Protein (ABCC) family members MRP2, 3, 4, 5 and 6. We have initiated a systematic search for compounds conjugated to glucuronide or sulphate that are transported by MRPs by comparing the derivatives in plasma/urine of WT and KO mice using Mass Spectrometry (MS). We have identified several glucuronidated and sulphated phyto-estrogens, derived from food, as novel MRP2 and MRP3 substrates by this approach, as well as novel substrates of BCRP, MRP4 and MRP5. To speed up and simplify our search we developed “Transportomics”: we incubate extracts of mouse urine or plasma with membrane
The work on drug resistance in mouse tumor models is taken over and continued by Sven Rottenberg, a former postdoc in the Borst group who became an independent investigator in December 2012. Results obtained in 2012 are reported by Rottenberg in this Annual Report.
Figure 2: Transportomics experiments using BCRP-containing Sf9 vesicles. BCRP-containing (A) or control (B) inside-out membrane vesicles were incubated in the presence of 4 mM ATP for 10 min at 37 ˚C in a diluted urine sample of an Bcrp1-/- mouse. The vesicle content was subsequently analysed by an LC/MS based targetedmetabolomics screen, specifically detecting sulphate conjugates. In subsequent experiments we identified the sulphate-conjugates transported by BCRP into the vesicles as phytoestrogen-sulphates.
Figure 1: Base J is required for RNA polymerase II transcription termination in L. tarentolae. A. Genomic organization of L. tarentolae chromosome 4 with the bars representing the protein coding regions organized in long polycistronic transcription units encoded by the top (transcription to the right) or bottom strand (transcription to the left). The arrows indicate the transcriptional orientation of the polycistronic transcription units. B. Base J is specifically located at RNA pol II transcription termination sites and is lost upon knock-out of JBP2. DNA fragments containing J were immunoprecipitated using an anti-J antibody, deep sequenced and mapped to the genome in bins of 100 bp for WT and for JBP2 knock-out cells grown in BrdU-containing medium. C. Loss of J at transcription termination sites results in transcription read-through. The fraction of small RNA derived from the top strand (values between 0 and 1) and bottom-strand (values between 0 and -1) are plotted in bins of 100 bp across the chromosome.
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Experimental Biomedical Genetics
Group leader Thijn Brummelkamp
Our research focuses on the following topics: the development and application of an entirely novel genetic model system to identify genes that play a role in human disease and the role of the Hippo signalling pathway in mammals.
Haploid Genetic Screens Thijn Brummelkamp PhD Group leader Vincent Blomen MSc PhD student Jacqueline Staring MSc PhD student Lucas Jae MSc PhD student Joppe Nieuwenhuis PhD student Ferdy Van Diemen Technical staff Lisa Van den Hengel Technical staff
Publications
Miller EM, Obernosterer G, Raaben M, Herbert AS, Deffieu MS, Krishnan A, Ndungo E, Sandesara RG, Carette JE, Kuehne AI, Ruthel G, Pfeffer SR, Dye JM, Whelan SP, Brummelkamp TR, Chandran K. Ebola virus entry requires the host-programmed recognition of an intracellular receptor. EMBO J. 2012; 31:1946-60 Rosmarin DM, Carette JE, Olive AJ, Starnbach MN, Brummelkamp TR, Ploegh HL. Attachment of Chlamydia trachomatis L2 to host cells requires sulfation. Proc Natl Acad U S A 2012;25,10059-64 Birsoy K, Wang T, Possemato, R, Yilmaz OH, Koch CE, Chen WW, Hutchins AW, Gultekin Y, Peterson TR, Carette JE, Brummelkamp TR, Clish CB, Sabatini DM. MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors. Nat Genetics 2012
The most powerful and direct way to gain insight into complex biological systems is to remove individual components and observe the consequences. By doing so genetics has revolutionized almost all aspects of biology. Although human cell cultures are often used as experimental tool, their genome is largely refractory to efficient mutagenesis due to its diploid state. To address this we have developed an entirely novel genetic model system based on insertional mutagenesis in haploid or near-haploid human cells. We have shown that this platform enables the generation of null alleles for most human genes and can be used to pinpoint genes that are involved in phenotypes of interest. Combined with parallel sequencing approaches, this method generates high-density genetic overviews of genes required for nearly any selectable cell trait. Importantly, we can carry out and analyze a genetic screen in which we interrogate millions of mutant alleles in a period of weeks in a cost-effective manner. In the last years, our lab has completed more than 50 different systematic haploid screens in which we identified gene products required for the induction of apoptosis, numerous critical host factors used by viruses and bacteria, chemical-genetic interactions that provide insight in compound modes of action and novel components of signalling pathways.
manner from its function as cellular cholesterol transporter. We have shown that a single loop of this 13-pass transmembrane protein interacts with the cathepsin-cleaved Ebola glycoprotein. Moreover, Human NPC1 fulfills a cardinal property of viral receptors: it confers susceptibility to filovirus infection when expressed in non-permissive reptilian cells. Thus, we propose a role for the cholesterol transporter NPC1 as the long-sought intracellular entry receptor for Ebola virus.
A host factor Atlas for Viruses The haploid genetic screening approach that we have developed could in principle be used to study any pathogen that affects haploid or near-haploid cells. Conceptually, this approach reverses the conventional genetic route towards host factors. The conventional route encompasses identification of nonpermissive cells followed by complementation experiments using cDNAs. In our haploid screens, we start out with permissive cells and use deep mutagenesis to search for resistant mutants. As a benefit a multitude of host factors can be identified in a single screen. This approach greatly benefits from modern sequencing technologies allowing millions of mutant alleles to be evaluated in a single experiment. Currently, we apply our haploid screening platform to generate a ‘Host Factors Atlas’ for 50 diverse viruses that infect the human population.
The Hippo Signalling Pathway How tissues stop growing upon reaching their correct size remains a mystery in biology. The barriers that tissues encounter when they reach their final size may bear relevance to neoplastic cells during the early stages of tumorigenesis. Drosophila genetics increased our understanding of the biology of organ size control. The Hippo pathway has emerged as a key pathway that controls tissue expansion through the regulation of cell proliferation and death. Interestingly, all components of the Hippo pathway are conserved in mammals and some have been implicated in cancer. We use genetic mouse models and biochemical experiments to address how this signalling pathway regulates tissue size in mammals and how it contributes to tumorigenesis.
Figure 1: Outline of haploid genetic screens. Haploid or near-haploid human cells are mutagenized with a retroviral gene-trap to inactivate genes that are present in a single copy. Mutagenized cells are selected for phenotypes of interest and gene-trap insertion sites are sequenced to link genes to the selected phenotype.
The Entry Route of Ebola Virus Ebola and Marburg filoviruses cause deadly outbreaks of haemorrhagic fever. The entry of Ebola virus is mediated through the viral spike glycoprotein, which attaches viral particles to cells and delivers them to endosomes. It was known that additional factors are required to allow escape of the viral genome from the vesicular compartment but those factors defied identification using conventional methods. We have used haploid genetic screens in order to identify host factors required for Ebola virus entry. This screen pointed out 15 proteins affecting trafficking and lysosome function as key host factors. This includes the HOPS complex, which is involved in the fusion of endosomes with lysososmes, and the lysosomal cholesterol transporter NPC1. NPC1 is mutated in patients with Niemann-Pick disease type I. Cells isolated from NiemannPick patients (harbouring mutations in NPC1) are completely resistant to infection by Ebola or Marburg virus.
Figure 2: Schematic outline of the proposed entry route for Ebola virus. Inside the host cell the viral glycoprotein is cleaved by a protease (Cathepsin B). In the lysosome the cleaved glycoprotein recognizes NPC1 as intracellular entry receptor.
Last year we have addressed in detail how NPC1 contributes to virus entry. We showed that viral attachment and the earliest steps of entry occurred normally in these cells but viral escape from lysosomes required the NPC1 protein, in an independent 32
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The role of class I HDACs in transcription, development and tumorigenesis
Group leader Jan-Hermen Dannenberg Jan-Hermen Dannenberg PhD Group leader Richard Heideman PhD Post-doc Cesare Lancini PhD Post-doc Roel Wilting MSc PhD student
Publications
Heideman MR, Wilting RH, Yanover E, Velds A, Kerkhoven RM, de Jong J, Wessels LF, Jacobs H, Dannenberg JH. Dosage dependent tumor suppression by histone deacetylases 1 and 2 through regulation of c-Myc collaborating genes and p53 function. Blood 2012 (in press) Gallagher SJ, Kofman AE, Huszar JM, Dannenberg JH, Depinho RA, Braun RE, Payne CJ. Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia. Dev Biol. 2012 Wilting RH, Dannenberg JH. Epigenetic mechanisms in tumorigenesis, tumor cell heterogeneity and drug resistance. Drug Resist Updat. 2012;15:21-38
Cancer is a genetic disease in which epigenetic aberrations contribute to tumor initiation and progression. In contrast to genetic mutations, epigenetic changes are reversible and therefore an attractive target for anti-cancer therapy. Histone deacetylases (HDACs), enzymes involved in removing acetyl groups from lysine residues, are important drug targets in the treatment of cancer and several other diseases such as neurological disorders. Small molecule inhibitors of HDACs (HDACi) have entered the clinic in the treatment of hematological malignancies and are being tested in clinical trials involving other malignancies. Despite proven clinical efficacy the use of HDACi is limited due to a lack of knowledge about the mechanism of action and the role of individual HDACs in normal development and tumorigenesis. To tackle these issues we have generated and exploited the experimental merits of mice carrying HDAC conditional knockout mice. By deleting these genes in normal cells as well as tumor cells we wish to discover antitumor relevant targets of HDACs and study their specific and redundant functions in transcription regulation, development and tumorigenesis.
Tumor suppression by HDAC1 and HDAC2 Our recent findings uncovered a dosage dependent tumor suppressor function for HDAC1 and HDAC2, in contrast to a presumed oncogenic function. Mice carrying an allelic series of Hdac1 and Hdac2 knockout alleles in the thymus showed progressive reduction of global HDAC-activity in Hdac1+/Δ;Hdac2Δ/Δ, Hdac1Δ/Δ;Hdac2+/+ and Hdac1Δ/Δ;Hdac2+/Δ thymocytes, which correlated with histone H3 hyperacetylation, deregulated gene transcription and an increase of immature CD8 single positive (ISP) thymocytes. Surprisingly, a reduction in Hdac1/2 levels resulted in tumorigenesis as mice harboring Hdac1+/Δ;Hdac2+/Δ, Hdac1+/Δ;Hdac2Δ/Δ, Hdac1Δ/Δ;Hdac2+/+ or Hdac1Δ/Δ;Hdac2+/Δ thymocytes developed thymic lymphomas with an latency and incidence that inversely correlated with HDAC-activity. Interestingly, complete loss of HDAC1 and HDAC2 (Hdac1Δ/Δ;Hdac2Δ/Δ) resulted in a profound block in early thymocyte development thereby preventing tumor development. These data suggest a model in which HDAC1 and HDAC2 dosage is critical for tumor suppression; complete loss blocks differentiation while reduced levels induce hyper-proliferation.
HDAC1 and HDAC2 collectively regulate p53 Lymphomas appearing in mice harboring reduced HDAC1/2 levels were monoclonal of origin and invariably displayed chromosome 15 trisomy associated with elevated Myc oncogene expression. The tumorigenic potential of Myc-overexpression requires the inactivation of the Cdkn2a-Mdm2-p53 tumor suppressor pathway. Interestingly, Cdkn2a deletions nor Mdm2 amplifications nor p53 mutations were observed in tumors with the lowest HDAC-activity (Hdac1Δ/Δ;Hdac2+/Δ) suggesting that the p53 tumor suppressive pathway was inactivated by other 34
means. Indeed, Hdac1Δ/Δ;Hdac2+/Δ pre-leukemic thymocytes revealed reduced p53 levels and increased viability upon γ-irradition (Figure XX.X). Gene expression analysis of preleukemic thymocytes revealed a HDAC1/2 dosage dependent regulation of various Myc-collaborating, p53-inactivating genes, providing insight into the tumor suppressive function of histone deacetylases.
Jun dimerization partner 2 (Jdp2) is a Hdac1/2 target and essential for lymphoma survival in a p53-dependent manner Combined gene expression analysis of Hdac1+/Δ;Hdac2Δ/Δ, Hdac1Δ/Δ;Hdac2+/+ and Hdac1Δ/Δ;Hdac2+/Δ pre-leukemic thymocytes and lymphomas revealed that expression of Jun dimerization partner 2 (Jdp2), a known p53 suppressing gene, was regulated in a Hdac1/2 dosage dependent manner. Moreover, chromatin immunoprecipitation (ChIP) experiments identified Jdp2 as direct target of Hdac1 and Hdac2 suggesting that these proteins directly regulate Jdp2. Consequently, loss of Hdac1 and Hdac2 would result in elevated Jdp2 and subsequently suppress p53, thereby relieving the need to inactivate the p53 pathway upon Myc overexpression. Inhibition of Jdp2 using shRNA mediated knockdown resulted in cell death of Hdac1Δ/Δ;Hdac2+/Δ lymphomas while this did not occur in p53null lymphomas. These results suggest that inhibition of Jdp2 selectively eliminates lymphomas possibly through activation of wild-type p53 (see figure)
Relevance of HDAC1 mutations in human cancers Recent studies involving full sequencing of cancer genomes identified somatic mutations in HDAC1. Our tumor model indicates that HDAC1 loss-of-function can act as a tumor driving genetic event. Functional characterization of tumor-associated somatic HDAC1 mutations is important to distinguish inactivating mutations from non-relevant mutations as well as the functional impact of such mutations. To this end we have developed a cellular system, which allows us to determine the functional consequences of HDAC1 carrying tumor-associated mutations. Using this assay we have identified inactivating HDAC1 mutations in particular tumor types, which will be studied in more detail.
A. Western blot analysis (left) of p53 protein levels and percentage of viable cells (right) in one week old WT and Hdac1Δ/Δ;Hdac2Δ/+ thymocytes, after mock or γ-irradiation.
B. Cell viability assay (left) and representative pictures (right) of Hdac1Δ/Δ;Hdac2Δ/+ and p53-/- lymphoma cell lines either mock infected or infected with two independent lentiviral Jdp2 shRNA constructs or a nontargeting shRNA (NT) construct.
Reduced HDAC-activity sensitizes cells to HDACi While reduced HDAC-activity resulted in tumorigenesis, these tumors were still dependent on residual HDAC-activity. As such HDAC-activity levels may put the cell on the brink of being viable, we hypothesized that low HDAC-activity results in an increased sensitivity towards HDACi. Indeed, Hdac1Δ/Δ;Hdac2+/Δ lymphomas were highly sensitive to HDACi treatment compared to lymphomas harboring relative high HDAC-activity. These findings have important implications for the treatment of cancer patients with HDACi, since it indicates that full inhibition of Hdac1/2-activity is critical to prevent de novo tumorigenesis. More importantly, these data suggest that tumors harboring inactivating HDAC1 mutations might be sensitive to HDACi.
C. Dose response curves of 4 independent Eμ−Myc and 4 independent Hdac1Δ/Δ;Hdac2Δ/+ tumor cell lines treated with increasing concentrations of suberoylanilide hydroxamic acid (SAHA).
Future work will address the mechanisms underlying the tumor suppressive function of Hdac1 and Hdac2 and will provide us with new drug targets that may synergize with HDAC1/2 inhibition in cancer treatment.
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Impact of the inflammatory tumor microenvironment on metastatic breast cancer and therapy Group leader Karin De Visser
Karin De Visser PhD Group leader Seth Coffelt PhD Post-doc Metamia Ciampricotti MSc PhD student Chris Doornebal MD PhD student Kelly Kersten MSc PhD student Tisee Hau Technical staff
Publications
Ciampricotti M, Hau CS, Doornebal CW, Jonkers J, de Visser KE. Chemotherapy response of spontaneous mammary tumors is independent of the adaptive immune system. Nat Med. 2012;18:344-46 De Visser KE, Ciampricotti M, Michalak EM, Tan DWM, Speksnijder EN, Hau CS, Clevers H, Barker N, Jonkers J. Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland. J Pathol. 2012;228:300-9 Doornebal CW, Klarenbeek S, Braumuller TM, Klijn CN, Ciampricotti M, Hau CS, Hollmann MW, Jonkers J, de Visser KE. A preclinical mouse model of invasive lobular breast cancer metastasis. Cancer Res. 2012 (in press)
Cancer cells cannot develop into invasive cancers without reciprocal interactions with their microenvironment, of which the immune cells are a major component. The focus of our group is on dissecting the mechanisms by which the inflammatory tumor microenvironment influences breast cancer progression, metastasis formation and therapy responsiveness. We aim to utilize this knowledge to design novel immunomodulatory strategies to fight metastatic breast cancer and to increase efficacy of conventional anti-cancer therapies.
Role of the immune system during breast cancer formation and dissemination Immune cells are abundant in the microenvironment of many tumors. Their role during tumorigenesis is, however, controversial, as both tumor-protective and tumor-promoting properties have been reported. To investigate the impact of the immune system on cancer growth and metastasis, we utilize two transgenic mouse models that develop spontaneous mammary carcinomas, i.e. MMTV-NeuT mice and K14cre;EcadF/F; p53F/F mice. These mice develop metastatic mammary carcinomas that resemble human HER2+ breast cancer and human invasive lobular carcinomas, respectively. We found that genetic elimination of the adaptive immune system did not alter latency and outgrowth of primary breast cancers, indicating that immunosurveillance does not play a role during sporadic breast cancer development. Absence of T- and B cells did not affect metastasis formation in the MMTV-NeuT mouse model. Importantly, however, metastasis formation in the K14cre;EcadF/F; p53F/F mouse model was almost completely abrogated. These findings indicate that adaptive immune cells differently modulate these distinct metastatic breast cancer subtypes. We are currently assessing the mechanisms by which the adaptive immune system promotes metastasis formation. We are also assessing the importance of the innate immune system and pro-inflammatory mediators in metastatic breast cancer. We have identified an important role for CCL2, a chemokine involved in myeloid cell recruitment and activation, in lung metastasis formation. We pursue underlying mechanisms and assess the tumor-modulating effects of other inflammatory mediators and myeloid cells. Ultimately, the outcome of these studies may contribute to the rational design of novel agents that target inflammatory pathways to fight cancer progression.
Impact of the immune system on chemotherapy responsiveness of breast cancer
the question whether the altered inflammatory response in chemotherapy-exposed tumors is just a bystander effect, or whether it actually influences the efficacy of chemotherapy. Using genetic and pharmacological approaches, we examine the role of immune cells and inflammatory mediators in responsiveness and resistance to chemotherapy. Against the prevailing dogma, we have demonstrated that adaptive immune cells do not affect the outcome of chemotherapy against established spontaneous mammary tumors. However, we have recently found that components of the innate immune system can counteract the anti-cancer efficacy of chemotherapy. We observed that the intra-tumoral macrophage population changes considerably in a dynamic manner during chemotherapy treatment. Suppression of the macrophage influx in mammary tumors by targeting the CSF1R signalling pathway in K14cre;EcadF/F;p53F/F mice resulted in an enhanced anti-cancer efficacy of cisplatin (figure 1). We are currently pursuing the underlying mechanisms by which macrophages counteract the efficacy of cisplatin. In addition, we are extending these studies to other chemotherapeutics and other immune cell populations. These studies may shift therapeutic focus towards a combined cancer cell-intrinsic and -extrinsic viewpoint.
Figure 1: Kaplan-Meier survival curves of tumor-bearing K14cre;Cdh1f/ f;Trp53f/f mice treated with cisplatin, a CSF1R inhibitor or both. Suppression of macrophage influx by the CSF1R inhibitor resulted in improved anticancer efficacy of cisplatin (p=0.001).
Cancer cell-intrinsic and -extrinsic regulation of organ-specific metastasis formation Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastasis formation has been hampered by the limited availability of clinically relevant mouse models of metastatic disease. We have recently developed a mouse model of spontaneous breast cancer metastasis that mimics the clinical course of metastatic disease in humans. Using the K14cre;Cdh1F/F;Trp53F/F model of de novo mammary tumor formation, we have orthotopically transplanted invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once primary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice developed widespread clinically overt metastatic disease in lymph nodes, lungs, liver and other distant organs (figure 2a, b). Genomic profiling of paired mammary tumors and distant metastases showed that our model provides a unique tool to further explore the biology of metastatic disease. Neoadjuvant and adjuvant intervention studies with standard-of-care chemotherapeutics demonstrated the value of this model in determining therapeutic agents that can target early and late-stage metastatic disease (figure 2c). We are currently using this model to compare sensitivity of primary tumors and metastases to conventional and novel anti-cancer therapies, and to study cancer cell-intrinsic and -extrinsic mechanisms underlying organ-specific metastasis formation. These studies are likely to provide new insights that might support the development of more effective treatment strategies targeting metastatic disease.
Figure 2: (A) Kaplan-Meier metastasisspecific survival curves of recipient mice orthotopically transplanted with tumor fragments from 3 independent K14cre;Cdh1F/F;Trp53F/F-derived tumors. An event is defined as an animal that had developed clinical signs of metastatic disease. (B) Mice spontaneously develop metastases in various organs. Each mouse is depicted as a circle. (C) Metastasis-related survival of mice receiving neo-adjuvant or adjuvant treatment with PBS or docetaxel.
One of the major impediments to effective cancer therapy is acquisition of unresponsiveness to chemotherapeutics. Using genetically engineered mouse models for breast cancer, we are studying the ability of the immune system to modulate chemotherapy response and resistance. We have observed that different types of chemotherapeutic drugs have a differential effect on intra-tumoral influx of immune cells. This raises 36
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H Henken FE, Oosterhuis K, Öhlschläger P, Bosch L, Hooijberg E, Haanen JB, Steenbergen RD. Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7. Vaccine. 2012;30:4259-66
Immunotherapy and immunomonitoring
Ariotti S, Haanen JB, Schumacher TN. Behavior and function of tissueresident memory T cells. Adv Immunol. 2012;114:203-16
Group leader John Haanen
John Haanen MD PhD Group leader Joost Van den Berg PhD Post-doc Bianca Heemskerk PhD Post-doc Pia Kvistborg PhD Post-doc Koen Oosterhuis MSc PhD student Trees De Jong Technical staff Willeke Van de Kasteele Technical staff Martin Van der Maas Technical staff Samira Michels Technical staff
Publications
Ariotti S, Beltman JB, Chodaczek G, Hoekstra ME, van Beek AE, GomezEerland R, Ritsma L, van Rheenen J, Marée AF, Zal T, de Boer RJ, Haanen JB, Schumacher TN. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proc Natl Acad Sci USA. 2012;109:19739-44 Aarntzen EH, de Vries IJ, Lesterhuis WJ, Schuurhuis D, Jacobs JF, Bol K, Schreibelt G, Mus R, De Wilt JH, Haanen JB, Schadendorf D, Croockewit S, Blokx WA, van Rossum MM, Kwok WW, Adema GJ, Punt CJ, Figdor CG. Targeting CD4+ T helper cells improves the induction of anti-tumor responses in dendritic cell based vaccination. Cancer Res. 2012 (in press) Oosterhuis K, Aleyd E, Vrijland K, Schumacher TN, Haanen JB. Rational design of DNA vaccines for the induction of human papillomavirus type 16 e6- and e7-specific cytotoxic T-cell responses. Hum Gene Ther. 2012;23:1301-12 Kvistborg P, Shu CJ, Heemskerk B, Fankhauser M, Thrue CA, Toebes M, van Rooij N, Linnemann C, van Buuren MM, Urbanus JH, Beltman JB, Thor Straten P, Li YF, Robbins PF, Besser MJ, Schachter J, Kenter GG, Dudley ME, Rosenberg SA, Haanen JB, Hadrup SR, Schumacher TN. TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients. Oncoimmunology. 2012;1:409-18 38
Moulin V, Morgan ME, EleveldTrancikova D, Haanen JB, Wielders E, Looman MW, Janssen RA, Figdor CG, Jansen BJ, Adema GJ. Targeting dendritic cells with antigen via dendritic cell-associated promoters. Cancer Gene Ther. 2012;19:303-11 Vogel WV, Guislain A, Kvistborg P, Schumacher TN, Haanen JB, Blank CU. Ipilimumab-induced sarcoidosis in a patient with metastatic melanoma undergoing complete remission. J Clin Oncol. 2012;30:e7-e10 Oosterhuis K, van den Berg JH, Schumacher TN, Haanen JB. DNA vaccines and intradermal vaccination by DNA tattooing. Curr Top Microbiol Immunol. 2012;351:221-50 Haanen J, Blank C. Immunomodulatory Drugs and Cytokines. ESMO handbook Clinical Pharmacology of Anti-cancer Agents. 2012
The main objective of this line of research is to develop novel T-cell immunity based strategies and to apply them in patients with solid tumors, especially melanoma and renal cell carcinoma. We furthermore develop and apply advanced immunomonitoring strategies to evaluate specific immunotherapies. These studies are conducted together with the Schumacher lab at the Netherlands Cancer Institute and with national and international collaborators.
DNA vaccination for the treatment of cancer Induction of immunity following DNA vaccination is generally considered a slow process. We have developed a novel, rapid and potent intradermal DNA vaccination method that results in a highly transient pulse of antigen expression. This generates robust T cell responses, of a magnitude sufficient to reject established subcutaneous tumors in mice, within 14 days. A clinical grade DNA vaccine has been produced in the GMP DNA plasmid production unit (Amsterdam-Biotherapeutics Unit) at the Antoni van Leeuwenhoek/Slotervaart Hospital. Phase I study in melanoma patients
From 2009 till 2012, nine stage IV melanoma patients have been enrolled in a first phase I clinical trial with DNA tattoo vaccination. The study has been closed. DNA vaccination against high risk human papilloma virus (HPV) associated cancers
HPV infection (serotypes 16 and 18) is strongly associated with the development of squamous cell cancer of the cervix, but also penis, vulva, anus en oropharynx. Since persistence of oncogenic high-risk 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 promise in women suffering from cervical cancer. In the upcoming years (in collaboration with Profs G Kenter, M van den Brekel and S Horenblas (Division of Surgical Oncology), we will perform a phase I/II study in patients with HPV 16-positive squamous cell cancer of the head and neck area, penis and cervix, using the intradermal DNA vaccination strategy. For this trial we have developed highly immunogenic and safe HPV 16 E6 and E7 containing DNA vaccine platform. Recently we have produced the first clinical grade E7 DNA vaccine for clinical testing.
Adoptive immunotherapy program TIL therapy
Adoptive therapy with Tumor-infiltrating Lymphocytes (TIL) is based on results from the Surgery Branch, NIH (Bethesda, MD, USA) and the Sheba Medical Center (Tel Aviv, Israel), showing a 50% objective response rate in pretreated stage IV melanoma patients. This treatment uses ex vivo cultured melanomareactive T cells from resected metastases, combined with non-
myeloablative chemotherapy and high dose bolus IL-2. Our goal is to demonstrate efficacy of this treatment in a randomized controlled phase III trial. We will assess overall survival compared to standard treatment and perform a comprehensive analysis of the specificities of the melanoma-reactive TIL prior and after adoptive transfer. We have finished a pilot study and enrolled 5 patients in 2011. Three patients had an objective response (1 CR, 2 PR), 1 patient had SD for 3 months and 1 patient had PD. The safety of the TIL treatment was as expected and side effects could be attributed to chemotherapy and high dose IL-2. We aim to perform an international, randomized controlled phase III trial in stage IV melanoma patients, comparing TIL with standard of care for 2nd line treatment, starting in 2013.
in close collaboration with the lab of Prof Schumacher setup analysis of T cell responses by combinatorial coding flow cytometry, using MHC peptide exchange technology. Dr. Kvistborg and her team have dissected melanoma-specific T cell immunity in peripheral blood and melanoma metastases from patients treated with ipilimumab or TIL. She has elegantly identified T cell responses against several shared tumor antigens and mutated self-antigens (see section Prof. Schumacher).
Together with biotech company Miltenyi, we investigate a novel process of TIL production, using an integrated cell-processing device. We will compare the final infusion product obtained with this device to the one obtained by standard TIL culturing methods, assessing cell viability, total cell yield, phenotype and functional activity. This device may in the future (partially) replace large GMP facilities for TIL production. TCR gene therapy
We have selected a highly avid TCR specific for melanocyte differentiation antigen MART-126-35, called 1D3, for clinical application using a retroviral vector (MP-71) produced by a German GMP manufacturer. This TCR has been equipped with extra safe guards to prevent mispairing with endogenous TCR chains in transduced T cells to prevent potential side effects. The process of clinical grade culturing and peripheral T cell transduction with the 1D3-MP-71 retrovirus has been validated step-by-step in our GMP facility, in order to start a phase I clinical study in melanoma patients in 2012. One patient has recently been included in this trial. We are preparing for a FP7 Health application granted TCR gene therapy trial in HLA-A2- and NY-eso-1 positive melanoma and gastric cancer patients using a NY-eso-1-specific TCR. This trial will be performed in collaboration with several European partners.
Tumor grafting and resistance to targeted agents In close collaboration with Prof D Peeper (Division of Molecular Oncology), we have developed a mouse model for studying resistance mechanisms of human melanomas against small molecules such as BRAF inhibitor vemurafenib and others. Biopsies or resected tumor tissue derived from melanoma metastases are subcutaneously grafted in immunodeficient mice. The tumor take in these animals is highly successful and hence these animals can subsequently be treated with targeted agents to study resistance. Tumor tissues from these animals are subsequently subjected to RNAseq and other molecular diagnostics (see section Prof Peeper)
Immunomonitoring of melanoma patients treated with immunotherapy In recent years, we have obtained tumor tissues and peripheral blood samples from stage IV melanoma patients treated with Ipilimumab (anti-CTLA4), TIL, or TCR gene therapy. We expect to add to this material from patients treated with anti-PD1 and other immune checkpoint inhibitors in the near future. Dr. P Kvistborg is heading the immunomonitoring laboratory and has 39
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H Hauptmann M, Meulepas JM. CT scans in childhood and risk of leukaemia and brain tumours. Lancet. 2012;380:1736; author reply 1736-7 Devriese LA, Bosma AJ, Van de Heuvel MM, Heemsbergen W, Voest EE, Schellens JH. Circulating tumor cell detection in advanced non-small cell lung cancer patients by multimarker QPCR analysis. Lung Cancer. 2012;75:242-7
Group leader Michael Hauptmann
Michael Hauptmann PhD Group leader Marta Lopez PhD Academic staff Wilma Heemsbergen PhD Academic staff José Meulepas MSc PhD student Mariette Kieft Data manager
Key publications
Pijpe A, Andrieu N, Easton DF, Kesminiene A, Cardis E, Noguès C, Gauthier-Villars M, Lasset C, Fricker JP, Peock S, Frost D, Evans DG, Eeles RA, Paterson J, Manders P, Van Asperen CJ, Ausems MG, MeijersHeijboer H, Thierry-Chef I, Hauptmann M, Goldgar D, Rookus MA, Van Leeuwen FE; GENEPSO; EMBRACE; HEBON. Exposure to diagnostic radiation and risk of breast cancer among carriers of BRCA1/2 mutations: retrospective cohort study (GENE-RAD-RISK). BMJ. 2012;345:e5660 Kok M, Koornstra RH, Mook S, Hauptmann M, Fles R, Jansen MP, Berns EM, Linn SC, Van ‘t Veer LJ. Additional value of the 70-gene signature and levels of ER and PR for the prediction of outcome in tamoxifentreated ER-positive breast cancer. Breast. 2012;21:769-78 Van Monsjou HS, Lopez-Yurda MI, Hauptmann M, Van den Brekel MW, Balm AJ, Wreesmann VB. Oral and oropharyngeal squamous cell carcinoma in young patients: The Netherlands Cancer Institute experience. Head Neck. 2013; 35:94-102 Beane Freeman LE, Blair A, Lubin JH, Stewart PA, Hayes RB, Hoover RN, Hauptmann M. Mortality from solid tumors among workers in formaldehyde industries: an update of the NCI cohort. Am J Ind Med. (in press)
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Kreeft AM, Smeele LE, Rasch CR, Hauptmann M, Rietveld DH, Leemans CR, Balm AJ. Preoperative imaging and surgical margins in maxillectomy patients. Head Neck. 2012;34:1652-6 Ackerstaff AH, Rasch CR, Balm AJ, De Boer JP, Wiggenraad R, Rietveld DH, Gregor RT, Kröger R, Hauptmann M, Vincent A, Hilgers FJ. Five-year quality of life results of the randomized clinical phase III (RADPLAT) trial, comparing concomitant intra-arterial versus intravenous chemoradiotherapy in locally advanced head and neck cancer. Head Neck. 2012;34:974-80 Hamming-Vrieze O, Van Kranen SR, Van Beek S, Heemsbergen W, Van Herk M, Van den Brekel MW, Sonke JJ, Rasch CR. Evaluation of tumor shape variability in head-and-neck cancer patients over the course of radiation therapy using implanted gold markers. Int J Radiat Oncol Biol Phys. 2012;84:e201-7 Defraene G, Van den Bergh L, Al-Mamgani A, Haustermans K, Heemsbergen W, Van den Heuvel F, Lebesque JV. The benefits of including clinical factors in rectal normal tissue complication probability modeling after radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys. 2012;82:1233-42
Biostatistics
The Biostatistics group in the Division of Psychosocial Research and Epidemiology concentrates on methodological and statistical research and is closely involved with several international epidemiological studies on cancer incidence and exposure to chemicals and radiation. The group also provides statistical expertise to investigators in the research departments as well as in the hospital on diverse topics from all areas of biomedical cancer research.
Exposure to ionizing radiation from pediatric computerized tomography scans and subsequent cancer risk – The Dutch CT study Radiation doses from diagnostic procedures are usually much lower than therapeutic doses. However, computed tomography (CT) delivers substantially higher radiation doses than most other diagnostic imaging techniques, and its use has increased dramatically during the past 10-15 years. Therefore, radiation protection is a concern, particularly among children, as they are sensitive to radiation-induced cancer and have a long lifespan to express late effects. We are conducting a retrospective recordlinkage cohort study including an anticipated 100,000 children who received one or more electronically archived CT scans in one of the participating Dutch hospitals. Information on all CT scans among those children will be obtained, including body part scanned, indication and radiologist report, as well as technical parameters for organ dose estimation. So far, about 40 of the eligible 50 hospitals in the Netherlands have been contacted. Almost all agreed to participate. Data collection has been completed in several hospitals and is ongoing in others. Once all radiology records have been obtained, they will be linked with the Netherlands Cancer Registry in order to determine cancer incidence and with other registries to collect information on potential confounders (Perinatal Registry of the Netherlands for congenital and other inborn disorders, European Bone Marrow Transplant registry for use of leukemogenic drugs, Statistics Netherlands for socio-economic status). The data will then be used to evaluate the association between radiation exposure from pediatric CT scans and risk of childhood and young adult leukemia. The data will also be contributed to the EPI-CT consortium for a pooled analysis of data from several European countries, in order to investigate risk for rarer forms of cancer, e.g., brain tumors. Our group is leading the statistical analysis of the combined European consortium data, which is expected to include about one million children. Besides providing safety information for this popular and invaluable imaging procedure, the study will uniquely contribute to etiologic knowledge on childhood/young adult leukemia as well as low-dose radiation risks and associated mechanisms.
Radiation dose-response and second primary cancers of stomach, esophagus and pancreas: an international study of cancer survivors Radio- and chemotherapy have become efficient treatment modalities for cancer at a large number of sites. However, the late effects of such treatments, which emerge only years after treatment, may substantially impact the survivors’ quality of life. Late effects in the gastrointestinal tract have so far not been evaluated in great detail. Our group collaborates on a large international study in which detailed treatment data are collected within a cohort of over 700,000 cancer patients from Denmark, Finland, Sweden, Norway, USA and the Netherlands, who were followed for second primary cancers of the stomach, the esophagus or the pancreas, based on a series of nested case-control studies. We are leading the analysis of cancer of the stomach and the pancreas following testicular cancer. Analyses on 92 stomach cancer cases and 180 matched controls have been completed in 2012 and reveal a strong association between the radiation dose to the stomach and subsequent risk of cancer in this organ. Patients who received more than 25 Gy to the stomach were at a strongly and significantly increased threefold risk compared with those who received 25 Gy or less, with the excess relative risk increasing by 0.3 per Gy. The distal sites of the stomach appeared to be more radiosensitive compared with the proximal parts (homogeneity p=.005).
from breast cancer treatment with tamoxifen, circulating tumor cells predictive of advanced non-small cell lung cancer, and ototoxicity after cisplatin-based chemoradiation in patients with locally advanced head-and-neck cancer.
Health effects of exposure to formaldehyde The group has been closely involved in two major epidemiological studies on formaldehyde and cancer, namely the US National Cancer Institute cohort of industrial workers and the US embalmers study. Both studies have contributed in a major way to reconsiderations of the carcinogenicity of formaldehyde by the International Agency for Research on Cancer (IARC), US regulatory bodies such as the Environmental Protection Agency (EPA) and the National Toxicology Program (NTP) and the European Commission. In 2012, we have completed the analysis of solid cancer mortality in an update of the cohort of industrial workers. The results confirm the previously observed association between exposure to formaldehyde and nasopharyngeal cancer and show an 11-fold significantly increased relative risk in the highest category of average intensity (>=1 part per million) compared with intensities between 0.1 and 0.5 parts per million, based on 10 cases observed in a cohort of over 25,619 workers followed for a median of 42 years. Other formaldehyde-related research includes a meta-analysis of cytogenetic studies and the evaluation of effects of the timing of exposure to formaldehyde. The analyses are done in close collaboration with the US National Cancer Institute.
Biostatistics We provide state-of-the-art statistical expertise to researchers and doctors in the Institute and the hospital. This involves developing and implementing diverse statistical approaches to cover a wide range of topics including the design and analysis of epidemiologic studies and clinical trials, the identification of prognostic and predictive biomarkers, sample size calculations, risk prediction, as well as animal and in vitro experiments. In 2012, we were involved in, among others, analyses of diagnostic radiation exposure and breast cancer in an international study of carriers of BRCA mutations, biomarkers to predict benefit 41
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Genes involved in breast cancer
Group leader John Hilkens
John Hilkens PhD Group leader Gerjon Ikink MSc PhD student Mandy Boer Technical staff
The aim of my group is to identify novel genes involved in breast cancer by using insertional mutagenesis in various mouse models for human breast cancer and subsequently characterize these genes and the oncogenic pathways they control. A series of candidate cancer causing genes has been identified and several of these have been validated as genuine oncogenes. Presently, we are uncovering the function and molecular pathways of a selection of these genes, including Eras, Irs4 and R-spondin genes. These studies can lead to novel drug targets and treatment options, and to more personalized therapy for patients with breast cancer.
R-spondin genes R-spondins (RSPO’s) are secreted proteins that act in the Wntsignaling pathway. WNT and RSPO proteins bind to different coreceptors but strongly synergize in reporter assays for canonical Wnt signaling. Indeed, we found Rspo and Wnt genes frequently MMTV-tagged in the same tumors. The Rspo gene family consists of four members of which we found Rspo2 and Rspo3 tagged at high frequency, Rspo1 only at low frequency while Rspo4 was never targeted by MMTV. We have investigated Rspo3 in more detail by generating transgenic mice that express this gene in the mammary gland. Except for frequent squamous metaplasias, no major effects on mammary gland histology or function were observed. However, these mice develop mammary tumors with a mean latency of 320 days. Tumors in Wnt1 and Rspo3 compound transgenic mice appeared with a slightly, but significant, shorter latency. The Rspo3 induced mammary tumors were predominantly adeno-squamous or squamous carcinomas, whereas Wnt1 induced tumors were generally poorly to well differentiated adenocarcinomas with only infrequent squamous differentiation (in collaboration with M van der Valk). These results indicate that the mammary tumors induced by these genes may originate from different progenitor cells. The MMTV-LTR controlled Rspo3 transgene expression also affects the skin of tail and ears. The effected skin contained highly abnormally branched hair follicles with extensive growth of the isthmus and/or infundibular epithelium leading to cyst-like structures surrounded by squamous epithelium. In addition, the hair bulbs were highly hyperplastic (figure 1). We also found Rspo3 activated in the small intestine, in particular the duodenum, leading to intraepithelial hyperplasia but not malignancies even in old mice. As Rspo genes are known to affect the stem cell compartment in various tissues, we have investigated the affected hair follicles using stem cell and differentiation markers (in collaboration with Pablo Socades, Sonnenberg group). The number of Sox9 positive cells was largely increased and overlapped with the PCNA positive cells. This suggest that Rspo3 increases the Sox9 positive progenitor cells that then differentiate to squamous epithelial cells in the 42
upper part of the hair follicle and to hyperplastic hair bulbs in the lower part of the hair follicle.
Eras Eras was significantly more frequently tagged in tumors from MMTV infected ErbB2 trangenic mice than in tumors from MMTV infected wild-type mice indicating that Eras and ErbB2 collaborate in tumorigenesis. Ectopic expression of Eras in immortalized mouse and human mammary epithelial cell lines rendered these cells tumorigenic in nude mice, validating Eras as an oncogene. Transduction of Eras in ErbB2 positive cells caused a dramatic increase in anchorage independent growth and strongly accelerated tumor growth in mice. We showed that ERAS strongly activates the PI3-kinase pathway, whereas it does not activate the MAPK pathway. It is well established that ERBB2 preferentially forms a heterodimeric complex with ERBB3 wherein ERBB2 predominantly activates the MAPK pathway and ERBB3 the PI3K pathway. Thus activation of the PI3K pathway via ERAS may replace activation of this pathway through ERBB3 in cells overexpressing ErbB2. Indeed, we found ErbB3 highly expressed in all tumors that arose in ErbB2 transgenic mice but only in 57% of the tumors that developed in MMTV infected ErbB2 transgenic mice, suggesting that induction of ErbB3 expression in ErbB2 induced tumors can be replaced by retroviral activation of a PI3K pathway activating gene. Indeed, Eras expression is negatively correlated with ErbB3 expression in MMTV induced tumors. ERAS mRNA is expressed to a variable degree in more than 15% of human breast cancers suggesting involvement of this, in adult tissues silent gene, in human breast carcinogenesis.
Irs4 Irs4 was also found frequently tagged in our MMTV screen. Although in contrast to Eras, it was found with approximately the same frequency in tumors that arose in MMTV infected wild-type and ErbB2 transgenic mice. Interestingly, the closely related Irs1 and Irs2 genes have not been found as target neither in retroviral nor in transposon insertional mutagenesis screens. NMuMG cells overexpressing Irs4 are tumorigenic in nude mice, while vector control cells do not form tumors. Like Eras, Irs4 also activated the PI3K pathway in mammary epithelial cells, whereas it did not activate the MAPK pathway in these cells. Cells overexpressing Irs4 were no longer dependent on insulin when growing at low serum concentrations. Coexpression of Irs4 and ErbB2 synergistically stimulated growth rate in vitro, anchorage independent growth and tumor growth in nude mice. However, we did not see any a collaborative effect on growth rate and soft agar colony formation when Irs1 or Irs2 were co-expressed with ErbB2.
Figure 1: Tail skin of Rspo3 transgenic mouse. Hair follicles exhibit extensive growth of the isthmus and/or infundibular epithelium leading to cyst-like structures surrounded by squamous epithelium and hyperplastic hair bulbs.
Figure 2: Eras expression strongly reduces the sensitivity for Lapatinib. The graph shows proliferation of ERBB2 positive MCF10A cells transfected with Eras or empty control vector in the presence or absence of lapatinib. Cell proliferation was determined in the Incucyte cell imager. Lapatinib concentration: 10µM.
Eras and Irs4 affect the sensitivity ErbB2 positive cells against Lapatinb Patients with ERBB2 positive breast cancers are treated with monoclonal antibodies against ERBB2 (Herceptin/Trastuzomab) or small molecule inhibitors such as Lapatinib usually combined with chemotherapy. Although the treatment is initially effective, the disease usually recurs due to mutations activating the PI3K pathway. Activation of genes that stimulate PI3K activity may similarly cause resistance. We have investigated whether Irs4 and Eras can affect the sensitivity against Lapatinib. Our results indicate that both genes can dramatically affect the sensitivity of ErbB2 transduced MCF10A cells Lapatinib (figure 2). 43
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Cancer cell migration
Group leader Metello Innocenti
Metello Innocenti PhD Group leader Andrew Campbell PhD Post-doc Tadamoto Isogai MSc PhD student Rob Van der Kammen Technical staff
Cells move during the development of our body and to maintain it healthy. However, if uncontrolled, cell motility can also lead to disease and cancer metastasis. We aim at understanding how cells move to combat the metastatic dissemination of cancer. Since movement requires force generated by the polymerization of the protein actin into filaments, we study the molecular mechanisms dictating actin polymerization within normal and malignant cells. We deploy a multi-disciplinary approach based on biochemistry, structural and cell biology, genetics and advanced imaging and exploit cellular and animal models.
Actin nucleators and actin-based protrusions Cellular actin cannot self-assemble into filaments without actin nucleators, proteins catalysing the production of very short actin polymers. The growth of these filaments pushes forward the plasma membrane, thereby resulting in the formation of cell protrusions key to cell migration. Therefore, the function and the regulation of actin nucleators take centre stage in our research. Two types of actin-based protrusions have been observed in mammalian cells: lamellipodia/ruffles and filopodia. Lamellipodia and ruffles are veil-shaped or upward-curled protrusions of the plasma membrane, respectively, consisting of a dense meshwork of branched actin filaments. The Arp2/3 complex generates these actin-based protrusions upon activation by the WAVE family of nucleation-promoting factors (NPFs). Filopodia are finger-like and highly dynamic extensions of the cell surface composed of a bundle of linear actin filaments. The nucleator involved in the making of filopoda is still debated: both the Formin mDia2 and the Arp2/3 complex have been suggested to control filopodium initiation. Furthermore, the role of filopodia in cell migration remains unclear.
Function and regulation of mDia2 The Diaphanous-related formin mDia2 nucleates linear actin filaments regulating the life cycle of invasive finger-like and bleb-like protrusions. We demonstrated that mDia2 plays a crucial role in the making of filopodia, the prime example of finger-like cell protrusions. Furthermore, increasing evidence links mDia2 to non-apoptotic blebs involved in the amoeboidtype of cell motility. Not surprisingly, dysregulation of mDia2 is emerging as a key event in cancer and other pathological conditions. How mDia2 controls the formation of different types of cell protrusions and what specifies its action remain unclear. To answer these questions, we have identified the mDia2 interactome and systematically test how these interacting proteins affect mDia2-depedent actin polymerization, filopodium and bleb formation. We aim to assess how blockade of either type of cell protrusions impacts on cell migration and invasion.
Mechanisms of formation and roles of filopodia in cell migration The mechanisms whereby filopodia are generated and the function of filopodia in cell migration remain mysterious. To tackle these issues, we have generated a genetically modified cell line that allows us to induce filopodium formation in tissue culture (see figure). Loss-of-function genetic and proteomic screens will be performed to inventory filopodium-regulatory proteins and to reveal the filopodium protein signature, respectively.
mDia1 KD cells form filopodia upon EGF stimulation. Control (Ctr) or mDia1 knockdown cells were serum-starved and stimulated with EGF for 7 minutes. Filamentous actin was visualized with TRITC-conjugated phalloidin. Maximal projections are shown.
Function and regulation of the Arp2/3 complex Arp2/3 complex nucleates branched actin filaments powering the formation of lamellipodia, which are required for the mesenchymal-type of migration. In the past, we identified WAVE proteins as key Arp2/3-complex activators regulating cell migration. Nevertheless, the role of the Arp2/3 complex and WAVE proteins in invasion remains controversial and tumor-type-dependent regulatory proteins may determine either their pro- or antimetastatic functions. We investigate this possibility by focusing on the WAVE interacome and tissue-specific Arp2/3-complex knockout mice.
Regulation of WAVE activity WAVE proteins play an essential role in cell migration and in a host of actin-based processes, including different forms of endocytosis. However, a mechanistic explanation for the high versatility of WAVE proteins is still lacking. We have identified a new WAVE-binding protein that confers functional specificity on WAVE and dictates its contribution to macropinocytosis. 44
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Programmed mutagenesis
Group leader Heinz Jacobs
Heinz Jacobs PhD Group leader Marc Hogenbirk MSc PhD student Niek Wit MSc PhD student Paul Van den Berk Technical staff
Publications
Krijger PHL, Tsaalbi-Shtylik A, Wit N, Van den Berk PCM, De Wind N, and Jacobs H. Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2/Ung2 hybrid pathway. Eur. J. Immunol. (in press) Heideman RM, Wilting RH, Yanover E, Velds A, De Jong J, Kerkhoven RM, Jacobs H, Wessels LF, Dannenberg J-H. Dosage dependent tumor suppression by histone deacetylases 1 and 2 through regulation of c-Myc collaborating genes and p53 function. Blood. (in press) Coquet JM, Middendorp S, Van der Horst G, Veraar EAM, Kind J, Xiao Y, Jacobs H, and Borst J. The CD27/ CD70 pathway inhibits Th-17 effector function and attenuates associated autoimmunity. Immunity 2012. (in press) Hogenbirk MA, Velds A, Kerkhoven RM, Jacobs H. Reassessing genomic targeting of the activation induced cytidine deaminase. Nat Immunol. 2012;13:797-8 Temviriyanukul P, Van Hees-Stuiven berg S, Delbos F, Jacobs H, De Wind N, and Jansen JG. Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome. DNA Repair 2012;11:550-8
Lymphocytes are licensed to transiently activate specific mutator pathways that enable efficient remodelling of genes coding antigen-receptor chains. To generate the enormous diversity of clonotypic antigen receptors, specific DNA lesions have to be generated and resolved at defined developmental stages. This hallmark of lymphocytes provides an ideal model system to study the role of specific DNA modifiers as well as generic DNA repair and DNA damage tolerance (DDT) pathways in shaping the immunoglobulin (Ig) repertoire and their impact on genome stability. Our research is focused on three subjects: (i) Non-coding function of stable IgH transcripts in establishing allelic exclusion (ii) Targeting specificity of AID and its role in malignant transformation (iii) The contribution of DDT to secondary Ig gene diversification
Non-coding function of stable IgH transcripts in establishing allelic exclusion Though potentially each B cell can express two different IgH and six different IgL chains (2k and 4λ) and hence twelve different antigen specificities, Ig allelic exclusion ensures that B cells are mono-specific and antibody responses antigen-specific. How this critical ‘one B cell – one antibody rule’ is established at the molecular level is still enigmatic, but is controlled at three main levels: lineage specificity (e.g., completed rearrangements of B cell loci occur only in B cells and not T cells), coordinated rearrangement within a given lineage (e.g., the heavy chain locus in B cells rearranges before the light chain loci), and allelic exclusion (cell surface expression of only one functionally rearranged allele). Present data are best compatible with the regulated model of allelic exclusion, where the product of the first productively rearranged allele prohibits further rearrangement of the second. However, while for decades this feedback loop appeared to be controlled at the level of the protein product, our recent findings made in specific IgH knock in mice indicate that a progenitor B cell is capable of sensing and distinguishing a productive from a non-productive rearrangement on the basis of differential mRNA stability. These data argue for a crirtcal regulatory, non-coding function of stable, coding Igμ transcripts in establishing IgH chain gene allelic exclusion. These insights provided a new research area: Unraveling the mechanism of transcript mediated IgH allelic exclusion.
Targeting specificity of AID and its role in malignant transformation
Both SHM and CSR require the activity of the activation induced cytidine deaminase (AID). AID binds single stranded DNA and deaminates cytosine (C) into uracil (U). AID is expressed in dark zone B cells of the GC and proposed to mutate genome wide through direct DNA interactions. Besides SHM and CSR, AID has been implicated in active DNA demethylation in non-B cells. To access the role of AID in promoting genome instability and its potential in epigenetic programming of B cells we applied RNASeq, MethylCap, and DamID. Our data contrast those published by Yamane et al. in Nature Immunology 2011. Applying ChIP-Seq to switch-induced B cells, it was argued that AID binds to >5900 genes. This controversy led us to reanalyse their ChIP-Seq data. We conclude that their experimental design and data do not allow any conclusion on genome wide AID binding (Hogenbirk et al. Nature Immunology, 2012).
The contribution of DDT to secondary Ig gene diversification Besides these genome-wide aspects on the biology of AID, a large proportion of our research activities have been devoted to the characterization of SHM pathways, specifically the contribution and regulation of error prone translesion synthesis (TLS) polymerases during the process of immunoglobulin gene SHM. Over the past years we provided seminal contribution to the regulation and generation of defined point mutations during the process of SHM. Given the defined mutation signatures of individual TLS polymerases, the analysis of SHM provides an effective read-out system for the activity of diverse TLS polymerases and their regulation in mammals. While the generation of A/T mutations downstream of MutSα strongly depends on PCNA-Ub, the generation of G/C transversions is independent of PCNA-Ub. In this context we were the first to reveal the critical and selective role of the Y-family TLS polymerase Rev1 in generating selectively G/C to C/G transversions. Given the central role of PCNA-ubiquitination (PCNA-Ub) in the regulation of TLS polymerases in yeast, we generated mice with a non-modifiable K146R mutation in PCNA (PCNAK164R). Using these mice and SHM as a read out for mammalian TLS activity, we were the first to demonstrate a selective relevance of PCNA-Ub in recruiting the Y-family polymerase η (Polη) to generate mutations at template A/T during SHM. Unlike Polη, Rev1 generates G/C to C/G transversions independent of PCNA-Ub, indicating a differential relevance of PCNA-Ub in activating these TLS polymerases. Furthermore, a quantitative analysis on the dependence of nucleotide substitutions on the glycosylase Ung2, the mismatch repair complex MutSα and PCNA-Ub during somatic hypermutation revealed the existence of a fourth SHM pathway where mismatch repair (MMR) and base excision repair (BER) activities are intertwined to generate a large proportion of GC transversions. Our most recent studies suggest that Rev1 generates G/C to C/G transversions within the Ung2 pathway and the MutSα/Ung2 hybrid pathway. However, in the absence of Rev1 or its catalytic activity, Polη can replace Rev1 in the MutSα/Ung2 hybrid pathway but not the Ung2 pathway (P. Krijger et al.; Eur. J. Immunol., in press).
Besides V(D)J recombination taking place in progenitor B and T cells, B cells of the germinal center (GC) can activate two pathways of secondary Ig gene diversification: somatic hypermutation (SHM) and class switch recombination (CSR). 46
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Telomere-dependent control of cancer development and aging
Group leader Jacqueline Jacobs
Jacqueline Jacobs PhD Group leader Vera Boersma MSc PhD student Inge De Krijger MSc PhD student Marieke Peuscher MSc PhD student Jaco Van der Torre MSc PhD student Zeliha Yalçin MSc PhD student
Publications
Peuscher MH and Jacobs JJL. Posttranslational control of telomere maintenance and the telomere damage response. Cell Cycle 2012;11:15241534 Jacobs JJL. Fusing telomeres with RNF8. Nucleus 2012;3:143-149
Telomeres are specialized nucleoprotein structures that protect natural chromosome ends from being recognized as broken DNA ends. Telomeres in human somatic cells progressively shorten with every cell division, eventually causing telomere dysfunction. Loss of telomere protection leads to activation of a DNA damage-like response that causes cells to die or stop proliferating indefinitely (senesce). By limiting the replicative life span of cells this response contributes to organismal aging. In addition, this response represents an important tumor suppressor mechanism as it prevents unlimited outgrowth of potentially cancerous cells. However, deprotected chromosome ends are also subject to processing by DNA repair factors that lead to chromosome end-to-end fusions. If cells with fused telomeres manage to divide, this results in breakage-fusionbridge cycles generating complex, unbalanced chromosome rearrangements that promote the development of cancer. On a mechanistic level, the cellular responses to telomere dysfunction are poorly understood. Therefore, our laboratory undertakes both candidate-driven and unbiased approaches to identify the genes and activities that play important roles in the telomere damage response and telomere-driven genomic instability. We mechanistically study these genes and their associated biological processes to uncover their precise role in the cellular response to telomere dysfunction. By doing so we aim to significantly increase our understanding of how dysfunctional telomeres affect genome stability, cancer development and aging.
Identification of critical factors controlling DNA damage response signaling and repair activities at telomeres To identify novel factors that contribute to telomere-driven genomic instability we developed an RNAi loss-of-function genetic screen that we call TIGIR screen for telomere-induced genomic instability regulators. The TIGIR screen relies on telomere uncapping via well-controlled reversible inactivation of the telomeric protein TRF2, using a temperature-sensitive mutant TRF2 allele in a p53-deficient background. The basis of our approach is that in this system telomere fusions and genomic instability eventually become so severe that cells die. However, interference with the efficiency of telomere fusion, such as upon DNA-ligaseIV-deficiency, reads out as enhanced survival. Indeed, small-scale TIGIR screens already identified multiple factors that, when inhibited with RNAi, allow cells to survive the lethal consequences of severe telomere uncapping. Among the screen hits are several factors with a known activity in the telomere damage response, such as NBS1 and ATM. Importantly, TIGIR screens also identified thus far a dozen factors that were not previously known to act at uncapped telomeres. These latter factors are at different stages of validation or mechanistic follow-up. Together these screen hits indicate that the TIGIR screen approach identifies factors 48
across the entire telomere damage response, from factors acting upstream in the sensing of uncapped telomeres, to signal transduction and recruitment of repair factors, until the final processing of uncapped telomeres that creates chromosome end-to-end fusions. In addition to the TIGIR screen approach, we have successfully set up a different, but complimentary screen approach that uses the well-controllable properties of the TRF2ts system to specifically identify activities that contribute to the initiation of a DNA damage response at telomeres, or to its shutdown.
Functional characterization of novel factors controlling DNA damage responses at telomeres The genes and their associated biological activities, identified though our screens or selected via candidate-driven approaches, are investigated in-depth for their role in the cellular response to telomere uncapping. As a significant portion of our TIGIR screen hits are factors that have been linked to ubiquitylation, so-called ‘ubiquitin-TIGIRs’, we became especially interested in how posttranslational modification by ubiquitylation would affect telomere-driven genomic instability. We are addressing this by studying the role of ubiquitin-TIGIR screen hits in the telomere damage response, as well as by investigating the contribution of potential candidate TIGIRs involved in ubiquitylation. For instance, we previously studied the potential telomere damage response activity of RNF8, a ubiquitin E3 ligase that we initially selected as a candidate factor based on its known role at DNA double-strand breaks, but that also emerged as a prominent screen hit in parallel TIGIR screens. We found that RNF8 promotes processing of uncapped telomeres and telomere-induced genomic instability by controlling the ubiquitylation of telomeric histone H2A and H2AX, and by facilitating the accumulation of both 53BP1 and p-ATM at uncapped telomeres. Most recently we concentrated our functional studies on three factors each identified in TIGIR screens performed with different RNAi libraries. These are factors with diverse biological activities and without a previously identified role at telomeres. They include factors directly involved in posttranslational modification by ubiquitylation or by methylation. All three factors contribute to telomere-driven genomic instability, but each of these factors appears to control the processing of uncapped telomeres through a distinct mechanism. Our current focus is to further uncover the mechanistic details of their role at telomeres. In addition, we are also investigating the activity of these factors in the response to DNA lesions such as DNA double-strand breaks. By doing so our work not only leads to significantly increased mechanistic understanding of the consequences of telomere uncapping but also has the potential to lead to new insights in the responses to damaged DNA. Proper processing of DNA lesions is of crucial importance for the maintenance of genome integrity and defects in DNA repair can lead to cancer and aging and affect treatment responses to anti-cancer therapies. Together, our research yields new mechanistic insights that facilitate the development of novel therapeutic and diagnostic strategies for cancer and (premature) aging-related disease.
Figure 1: Schematic representation of the main consequences of loss of telomere protection at chromosome ends that play important roles in aging and cancer development.
Figure 2: Telomere fusions upon inactivation of TRF2 in p53-deficient mouse fibroblasts (top). These fusions are markedly reduced upon shRNAmediated knockdown of a ubiquitinconjugating enzyme identified as a new telomere-induced genomic instability regulator (TIGIR) (bottom).
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J Kedziora K, Jalink K. Fluorescence resonance energy transfer microscopy (FRET). In: Advanced Fluorescence Microscopy: Methods and Protocols (PJ Vergeer, Editor) Humana Press. (in press)
Group leader Kees Jalink
Kees Jalink PhD Group leader Bram Van den Broek PhD Staff physicist Marcel Raspe PhD Post-doc Daan Visser MSc PhD student Kasia Kedziora MSc PhD student Daniela Leyton Puig MSc PhD student Leila Nahidi Msc PhD student Jeffrey Klarenbeek MSc Technical staff
Publications
Frijns E, Kuikman I, Litjens S, Raspe M, Jalink K, Ports M, Wilhelmsen K, Sonnenberg A. Phosphorylation of threonine 1736 in the C-terminal tail of integrin b4 contributes to hemidesmosome disassembly. Mol Biol Cell 2012;23:1475-85 Middelbeek J, Kuipers AJ, Henneman L, Visser D, Eidhof I, van Horssen R, Wieringa B, Canisius SV, Zwart W, Wessels LF, Sweep FC, Bult P, Span PN, van Leeuwen FN, Jalink K. TRPM7 is required for breast tumor cell meta stasis. Cancer Res 2012;72:4250-61 Zhao Q, Schelen B, Schouten R, Oever R, Leenen R, Kuijk H, Peters I, Polderdijk F, Bosiers J, Raspe M, Jalink K, Geert J, Jong S, Geest B, Stoop K, Young T. MEM-FLIM: all-solid-state camera for fluorescence lifetime imaging. Journal of Biomedical Optics (in press) Klarenbeek J, Jalink K. Detecting cAMP with an Epac-based FRET sensor in single living cells. Methods In Molecular Biology (R. Newman and J. Zhang, editors). (in press) Goedhart J, Hink M, Jalink K. An introduction to fluorescence imaging techniques geared towards biosensor applications. Methods in Molecular Biology (R. Newman and J. Zhang, editors). (in press)
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Biophysics of cell signaling
Employing advanced imaging and other biophysical techniques, we study cell signaling events and cytoskeletal dynamics with high spatial and temporal resolution. Electrophysiological (e.g. patch clamping) and advanced imaging (e.g. Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), Fluorescence Cross Correlation Spectroscopy (FCCS) and photorelease of caged compounds) are used in research projects in our group as well as in collaborations within and outside our institute. We also develop FRET hard- and software and biosensors for various intracellular messengers. A recent focus in the lab is on the development and application of ‘super-resolution’ light microscopy.
T-Epac-VV, a superior FRET sensor for the second messenger cAMP We are finalizing further development of a new generation of FRET-based cAMP sensors in the lab. We have made a library of construction vectors, consisting of a FRET donor, polylinker and FRET acceptor that are ideal ‘one-step’ construction blocks to build FRET sensors by simply inserting the protein of choice into the polylinker. A large choice of donors and acceptors are available. New additions to this library, notably the incorporation of the excellent donors mTurquoise and mTurquoise2, have yielded far superior FRET probes, in terms of signal-tonoise, photostability and mono-exponential decay. The latter is important for Fluorescence Lifetime imaging and it allows simultaneous read-out of several FRET sensors in a single living cell. We further prepared a set of cAMP sensors with higher affinity to be used in neuronal cells, which often display very moderate changes in this second messenger. The Epac-based cAMP sensors have been employed to study cAMP-sensitivity of TRPM7 channels in our laboratory and in a number of (inter) national collaborations with other laboratories.
Super resolution imaging The cation channel TRPM7 in the control of invadosomes and invasive migration In a continuing collaboration with Dr F van Leeuwen (Nijmegen) we studied the role of the atypical ‘channel-kinase’ TRPM7 as novel component of invadosomes and as determinant of cell adhesion and migration. TRPM7 is thought to function as a mechanosensor and regulator of local Ca2+ entry. Phospholipase C signaling triggers TRPM7-mediated Ca2+ influx and enhances invadosome formation. TRPM7 overexpressing neuroblastoma cells display a highly invasive phenotype, both in vitro (time-lapse imaging, Transwell assays) and in vivo (tail-vein injection in nude mice). Conversely, RNAi-mediated knockdown of TRPM7 strongly suppresses migration in MDA-MB-231 breast carcinoma cells, a model for invasive breast cancer. Moreover, mRNA expression profiling of human breast carcinoma specimens reveals that high expression of TRPM7 at the time of diagnosis predicts a poor prognosis and is correlated with distant metastasis. This result has now been confirmed in large published databases and, by QPCR, in an independent set of tumor biopsies in Nijmegen. These findings provide strong support for a role of TRPM7 in tumor cell dissemination. Furthermore, knockdown of TRPM7 strongly decreased metastasis formation of MDA-MB-231 cells when injected in the tail vein of nude mice. We have started analyzing the role of some 40 proteins in the TRPM7 interactome, many of which are implicated in cytoskeletal regulation, cell adhesion and migration. Ca2+ and PIP2-binding proteins present in this set suggest an important role for these intracellular messengers. However, and at variance with recently published reports, our detailed analysis of local Ca2+ signaling events (“Ca2+ sparks”) did not reveal evidence for localized Ca2+ signaling in the control of invadosome formation or turnover. The exact role of TRPM7 in these processes therefore remains enigmatic. Our current investigations address, by combining biophysical readout techniques with mutational analysis of the TRPM7 C terminus, the details of TRPM7 sensitivity to Ca2+ and phosphoinositides, and the exact role of PLCd1 which appears to mediate a Ca2+-influx dependent feedback loop in the activation of TRPM7.
This year we started the STW-funded super-resolution project, in collaboration with Prof. Dr. E. Manders (UvA). In light microscopy, it has been known for over a century that the laws of physics limit the achievable resolution to ~ 250 nm. At this resolution, many details of the cell architecture are out of reach, yielding blurry, so-called defraction-limited images. Super-resolution light microscopy employs a set of ‘tricks’ to circumvent this limitation to achieve resolution down to ~10 nm (see figure). We have opted to implement the GSDIM (Ground-State Depletion IMaging) method, using Leica hardware. In this implementation, super-resolution imaging of fluorescently labeled specimen is achieved by rapidly converting all fluorophores to a transient dark-state using a very powerful laser. When most fluorophores are in the dark state, the remaining few fluorophores yield individual diffraction-limited fluorescence spots that can be localized with very high precision by pin-pointing their mathematical centre in a high-resolution constructed image. Subsequently, the fluorophores revert to the dark state and other fluorophores may convert to the bright state in a stochastic manner, yielding a new set of coordinates to be added to the high-resolution image. This process is typically repeated several thousand times to achieve SR images with astonishing detail. GSDIM is a very powerful and well-established technique but it is also still in its infancy. In this project, we aim to optimize preparation techniques and image acquisition for SR microscopy and employ the equipment in running research lines, both within our group as well as in collaborations with other groups at the NKI. Over the past year, we have succeeded to develop very stable preparations that allow image acquisition for many hours, and we have obtained the first dual-color SR images. Obtaining accuracy to ~7 nm, we have constructed SR images of invadopodia and the cytoskeleton, and in collaboration with Dr. Metello Innocenti, Dr. Wouter Moolenaar, Dr. Arnoud Sonnenberg and others, we have started applying our techniques to resolve scientific questions. Nevertheless, before GSDIM may be considered a routine technique, many technical shortcomings need to be addressed over the next years.
Super-resolution Imaging of the intermediate filement Vimentin in cultured HUVEC cell. The images demonstrate clearly the increased detail visible in the reconstructed image (right panel), when compared to the normal fluorescence micrograph (left). Image size is ~15 x 15 micrometer.
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J Ciampricotti M, Hau CS, Doornebal CW, Jonkers J, de Visser KE. Chemotherapy response of spontaneous mammary tumors is independent of the adaptive immune system. Nat Med. 2012;18:344-6
Division head, group leader Jos Jonkers Jos Jonkers PhD Group leader Peter Bouwman PhD Post-doc Linda Henneman PhD Post-doc Ewa Michalak PhD Post-doc Petra Ter Brugge PhD Post-doc Marieke Van de Ven PhD Post-doc Martine Van Miltenburg PhD Post-doc Lisette Cornelissen MSc PhD student Julian De Ruiter MSc PhD student Rinske Drost MSc PhD student Janneke Jaspers MSc PhD student Sjors Kas MSc PhD student Sjoerd Klarenbeek MSc PhD student Hanneke Van der Gulden Technical staff Ingrid Van der Heijden Technical staff Ellen Wientjens Technical staff Ute Boon Research assistant Tanya Braumuller Research assistant Eva Kregel Research assistant Eline Van der Burg Research assistant
Publications
Vollebergh MA, Jonkers J*, Linn SC*. Genomic instability in breast and ovarian cancers: translation into clinical predictive biomarkers. Cell Mol Life Sci. 2012;69: 223-45 (* joint corresponding authors) Krimpenfort K, Song JY, Proost N, Zevenhoven J, Jonkers J, Berns A. Deleted in Colorectal Carcinoma (DCC) suppresses metastasis formation in p53 deficient mammary tumors. Nature 2012;482:538-41 Shimada S, Mimata A, Sekine M, Mogushi K, Akiyama Y, Fukamachi H, Jonkers J, Tanaka H, Eishi Y, Yuasa Y. Synergistic tumor suppressor activity of E-cadherin and p53 in a conditional mouse model for metastatic diffusetype gastric cancer. Gut 2012;61:344-53 Van Miltenburg MH, Jonkers J. Using genetically engineered mouse models to validate candidate cancer genes and test new therapeutic approaches. Curr Opin Genet Dev. 2012;22:21-7
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Rottenberg S, Vollebergh MA, de Hoorn B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Lodén M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res. 2012; 72: 2350-61 Giske CG, Jonkers J. Editorial. Drug Resist Updat. 2012;15:1 Warmoes M, Jaspes JE, Pham TV, Piersma SR, Oudgenoeg G, Massink MP, Waisfisz Q, Rottenberg S, Jonkers J, Jimenez C. Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with diagnostic and prognostic value in human breast cancer. Mol Cell Proteomics 2012;11:M111.013334 Jonkers J. Tracking evolution of BRCA1-associated breast cancer. Cancer Discov. 2012;2:486-8 Zander SA, Kersbergen A, Sol W, Gonggrijp M, van de Wetering K, Jonkers J, Borst P, Rottenberg S. Lack of ABCG2 shortens latency of BRCA1-deficient mammary tumors and this is not affected by genistein or resveratrol. Cancer Prev Res. 2012;5:1053-60 Bouwman P, Jonkers J. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat Rev Cancer 2012; 12: 587-98 Zander SA, Sol W, Greenberger L, Zhang Y, van Tellingen O, Jonkers J, Borst P, Rottenberg S. EZN-2208 (PEGSN38) overcomes ABCG2-mediated topotecan resistance in BRCA1deficient mouse mammary tumors. PLoS One 2012; 7: e45248 De Visser KE*, Ciampricotti M, Michalak E, Wei-Min Tan D, Speksnijder EN, Hau CS, Clevers H, Barker N, Jonkers J*. Developmental stagespecific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland. J Pathol. 2012;228:300-9 (* joint corresponding authors)
Mouse models of breast cancer
The focus of our group is on the genetic dissection of human breast cancer through the use of genetically engineered mouse models (GEMMs). For this, we have developed models for p53-induced breast cancer, BRCA1- and BRCA2- associated hereditary breast cancer, and E-cadherin-mutated lobular breast cancer. We are using these models to (1) investigate genotype-phenotype relations in mammary tumorigenesis; (2) study therapy response and resistance of primary tumors and metastases; (3) identify genetic changes underlying breast tumorigenesis.
Functional assays in BRCA1-deficient cells We have developed a functional complementation assay for testing human BRCA1 variants of unknown clinical significance (VUS). For this, we have engineered conditional Brca1 knockout mouse embryonic stem cells (ESCs) containing a docking site for introduction of human BRCA1 cDNAs by recombinase mediated cassette exchange (RMCE). Introduction of wildtype hBRCA1 – but not pathogenic hBRCA1 mutants – rescues the growth defect of ES cells lacking endogenous BRCA1. We have so far tested 100 defined hBRCA1 VUSs in our functional complementation assay.
Mouse models for BRCA-associated breast cancer We have previously generated GEMMs for BRCA1- and BRCA2associated hereditary breast cancer. The BRCA1-deficient mouse mammary tumors are highly proliferative, poorly differentiated adenocarcinomas that lack expression of hormone receptors (ER, PR) and ERBB2 and thus mimic BRCA1mutated triple-negative breast cancers (TNBCs) in women. Interestingly, we have found that mammary tumor formation in our BRCA1 models is still estrogen-dependent, suggesting that SERMs or aromatase inhibitors may be effective agents for chemoprevention of breast cancer in BRCA1-mutation carriers. The central role of BRCA1 and BRCA2 in the DNA damage response (DDR) implies that BRCA-deficient tumors are especially sensitive to DNA-damaging agents. In collaboration with Sven Rottenberg and Piet Borst we have used our BRCA1/2 models to test the anti-tumoral efficacy of the PARP inhibitor AZD2281 (olaparib), which displays selective toxicity to BRCAdeficient cells. While administration of olaparib to mice with BRCA1- or BRCA2-deficient mammary tumors caused tumor shrinkage without signs of toxicity, long-term treatment induced acquired drug resistance caused by upregulation of the P-glycoprotein (Pgp) drug efflux transporter. To study Pgp-independent mechanisms of olaparib resistance, we have crossed the BRCA1 mammary tumor model onto a Pgp-deficient background and transplanted the resulting BRCA1- and Pgpdeficient mammary tumors into wildtype recipients. Repeated treatment of these mice with olaparib resulted in induction of drug resistance without loss of target (PARP) inhibition. In 25%
of the cases, olaparib resistance was caused by inactivating mutations in Trp53bp1, highlighting the role of this DDR factor in therapy resistance. To study the effects of specific BRCA1 mutations on tumorigenesis and therapy response, we have generated mouse mutants mimicking the human BRCA1-185delAG, BRCA15382insC and BRCA1-C61G founder mutations and crossed these 3 mouse strains into our BRCA1 mammary tumor model. All 3 mutants fail to suppress mammary tumor formation, but show different activities in the DNA damage response following treatment of tumors with platinum drugs or PARP inhibitors. Whereas BRCA1-null and BRCA1-5382insC tumors never develop resistance to cisplatin, the BRCA1-185delA and BRCA1-C61G tumors readily become resistant while retaining the Brca1 founder mutation, suggesting that BRCA1 RING function is required for tumor suppression but dispensable for therapy resistance. In collaboration with Jelle Wesseling, we have established a panel of patient-derived tumor xenograft (PDX) models of BRCA1deficient and –proficient TNBC. We have also used these models to investigate response and acquired resistance to cisplatin and olaparib. Interestingly, BRCA1-methylated PDX tumors develop resistance via loss of BRCA1 promoter hypermethylation as well as via intrachromosomal rearrangements that lead to transcription of BRCA1 from heterologous promoters.
Mouse models for E-cadherin-mutated invasive lobular breast cancer Invasive lobular carcinomas (ILC), which account for 10-15% of all breast cancers, show frequent mutations in E-cadherin and components of the PI3K pathway. To study the role of E-cadherin loss and PI3K pathway activation in lobular breast cancer formation, we have generated mice with mammaryspecific loss of E-cadherin and PTEN. Ecad–/–;Pten–/– mammary tumors resemble human classical ILC and develop significantly faster than Ecad–/– or Pten–/– mammary tumors, demonstrating synergism between and E-cadherin mutation and PTEN loss. To study the role of PI3K pathway activation in maintenance of established ILCs, we are using the Ecad–/–;p53–/– and Ecad–/–; Pten–/– mammary tumor models for intervention studies with mTOR inhibitors and other PI3K pathway inhibitors. To identify additional cancer genes that collaborate with E-cadherin loss in ILC development, we have performed Sleeping Beauty (SB) transposon mutagenesis screens in our mammary-specific E-cadherin knockout mice. We identified recurrent inactivating SB insertions in several candidate tumor suppressor genes and activating mutations in Fgfr2. The constitutive activity of the SB transposon system allowed us to use our panel of SB-induced mouse ILCs to screen for genes conferring resistance to FGFR inhibitors. To this end, we performed orthotopic transplantations with SB-induced tumors with Fgfr2 overexpression and treated the tumor-bearing mice with the FGFR inhibitor AZD4547. All tumors regressed completely but eventually acquired resistance to AZD4547. We are currently analyzing therapy-resistant tumors for the presence of additional SB insertions at or near candidate resistance genes.
Identification and validation of novel cancer genes To facilitate functional in vivo validation of candidate cancer genes, we have developed together with the Berns group a rapid procedure for RMCE-mediated introduction of additional mutations in ESCs derived from established GEMMs of human cancer. The resulting GEMM-ESCs can be used to generate experimental cohorts of chimeric mice via blastocyst injections. We are currently using this procedure to introduce a number of different candidate cancer genes in our BRCA1 and E-cadherin mammary tumor models.
Buckle T, Kuil J, van den Berg NS, Bunschoten A, Lamb HJ, Yuan H, Josephson L, Jonkers J, Borowsky AD, van Leeuwen FW. Integrating target validation with in vivo and ex vivo imaging of mouse tumor lesions resembling human DCIS using a single hybrid imaging agent. PLoS One 2012 (In press) Rottenberg S*, Jonkers J*. MEK inhibition as a strategy for targeting residual breast cancer cells with low DUSP4 expression. Breast Cancer Res. 2012;14: 324 (* joint corresponding authors) Jaspers J, Kersbergen A, Boon U, Sol W, van Deemter L, Zander SA, Drost R, Wientjens E, Ji J, Aly A, Doroshow JH, Cranston A, Martin NM, Lau A, O’Connor MJ, Ganesan S, Borst P, Jonkers J*, Rottenberg S*. Loss of 53BP1 causes PARP inhibitor resistance in BRCA1-mutated mouse mammary tumors. Cancer Discov. 2012 (* joint corresponding authors) Doornebal CW, Klarenbeek S, Braumuller TM, Klijn CN, Ciampricotti M, Hau CS, Hollmann MW, Jonkers J*, de Visser KE*. A preclinical mouse model of invasive lobular breast cancer metastasis. Cancer Res. 2012 (* joint corresponding authors) Majewski IJ, Kluijt I, Cats A, Scerri TS, de Jong D, Kluin RJ, Hansford S, Hogervorst FB, Bosma AJ, Hofland I, Winter M, Huntsman D, Jonkers J, Bahlo M, Bernards R. An alphaE-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol. 2012 Sotiropoulou PA, Karambelas AE, Debaugnies M, Candi A, Bouwman P, Moers V, Revenco T, Rocha AS, Sekiguchi K, Jonkers J, Blanpain C. BRCA1 deficiency in skin epidermis leads to selective loss of hair follicle stem cells and their progeny. Genes Dev. 2012
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L Keszthelyi D, Dackus GH, Masclee GM, Kruimel JW, Masclee AA. Increased proton pump inhibitor and NSAID exposure in irritable bowel syndrome: results from a case-control study. BMC Gastroenterol. 2012;12:121
Group leader Sabine Linn
Kok M, Koornstra RH, Mook S, Hauptmann M, Fles R, Jansen MP, Berns EM, Linn SC, Van ‘t Veer LJ. Additional value of the 70-gene signature and levels of ER and PR for the prediction of outcome in tamoxifentreated ER-positive breast cancer. Breast. 2012;21:769-778
Sabine Linn MD PhD Group leader Karin Beelen MD PhD student Gwen Dackus MD PhD student Rutger Koornstra MD PhD student Izhar Salomon MSc PhD student Philip Schouten MSc PhD student Tesa Severson MSc PhD student Mark Opdam Technical staff
Loibl S, Han SN, von Minckwitz G, Bontenbal M, Ring A, Giermek J, Fehm T, Van Calsteren K, Linn SC, Schlehe B, Gziri MM, Westenend PJ, Muller V, Heyns L, Rack B, Van Calster B, Harbeck N, Lenhard M, Halaska MJ, Kaufmann M, Nekljudova V, Amant F. Treatment of breast cancer during pregnancy: an observational study. Lancet Oncol. 2012;13:887-896
Publications
Beelen K, Zwart W, Linn SC. Can predictive biomarkers in breast cancer guide adjuvant endocrine therapy? Nat Rev Clin Oncol. 2012;9:529-541 Criscitiello C, Azim HA, Jr., Schouten PC, Linn SC, Sotiriou C. Understanding the biology of triple-negative breast cancer. Ann Oncol. 2012;23 Suppl 6:vi13-18 Dackus GH, Loffeld SM, Loffeld RJ. Use of acid suppressive therapy more than 10 years after the endoscopic diagnosis of reflux esophagitis with specific emphasis to trademark and generic proton pump inhibitors. J Gastroenterol Hepatol. 2012;27:368371 Griffith M, Mwenifumbo JC, Cheung PY, Paul JE, Pugh TJ, Tang MJ, Chittaranjan S, Morin RD, Asano JK, Ally AA, Miao L, Lee A, Chan SY, Taylor G, Severson T, Hou YC, Griffith OL, Cheng GS, Novik K, Moore R, Luk M, Owen D, Brown CJ, Morin GB, Gill S, Tai IT, Marra MA. Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer. Pharmacogenomics J. 2012 Jager NG, Rosing H, Linn SC, Schellens JH, Beijnen JH. Importance of highly selective LC-MS/MS analysis for the accurate quantification of tamoxifen and its metabolites: focus on endoxifen and 4-hydroxytamoxifen. Breast Cancer Res Treat. 2012;133:793-798
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Retel VP, Joore MA, Linn SC, Rutgers EJ, van Harten WH. Scenario drafting to anticipate future developments in technology assessment. BMC research notes. 2012;5:442 Rottenberg S, Vollebergh MA, de Hoon B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Loden M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res. 2012;72:2350-2361
Molecular dissection of cancer by differential drug sensitivity In the clinic, we mainly use anticancer drugs based on outcomes of clinical trials that have been carried out in the general breast cancer population, whereas little is known about the molecular mechanisms underlying differential drug sensitivity. The same holds true for other cancer types, including gastrointestinal malignancies, and ovarian cancer. The focus of our research line is to unravel these molecular 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, gastrointestinal, and ovarian cancer patients into resistant and sensitive to a particular drug. We have a close collaboration with the groups of Jos Jonkers, Piet Borst and Wilbert Zwart, who use conditional mouse models for breast cancer and human breast cancer cell lines to study differential drug sensitivity in a controlled fashion. A second research line focuses on the impact of prognostic molecular classifiers on adjuvant systemic treatment advice in breast cancer.
Development of a predictive test for tamoxifen resistance in breast cancer Activation of the PI3K/MAPK pathways results in anti-estrogen resistance in vitro, however a biomarker with clinical validity has not been identified. We tested the predictive value of PI3K/MAPK pathway drivers as well as downstream activated proteins in postmenopausal breast cancer patients. We collected primary tumor tissue from 563 estrogen receptorpositive breast cancer patients, randomized between adjuvant tamoxifen (1-3 years) versus no systemic treatment. PIK3CA mutations were assessed by Sequenom Mass Spectometry. Immunohistochemistry was performed for PTEN, IGF-1R and the downstream markers p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interaction between these markers and treatment. No interaction between the PI3K/MAPK pathway drivers and tamoxifen was found. However, interactions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. After correcting for multiple testing, p-p70S6K remained significantly associated with tamoxifen resistance. Patients whose tumor did not express p-p70S6K derived significant benefit from tamoxifen (HR 0.24, 95 % CI= 0.12-0.47, p< 0.0001), while patients whose tumor did express p-p70S6K did not (HR=1.02, 95% CI=0.48-2.21, p=0.95), p for interaction 0.003. We conclude that the downstream marker of PI3K/ MAPK activation p-p70S6K predicts tamoxifen resistance in postmenopausal breast cancer patients.
Molecular mechanisms underlying sensitivity to high dose alkylating agents Collaboration with Petra Nederlof, Division of Molecular Pathology, and Sjoerd Rodenhuis, Division of Medical Oncology The inability of breast cancer cells deficient in homologous recombination (HR), such as BRCA1/-2-mutated cells, to repair DNA double strand breaks (DSBs) offers a target for DSB-inducing therapies, such as intensified alkylating therapy. Our group previously employed array Comparative Genomic Hybridization (aCGH) to assess the genomic profiles of BRCA1and BRCA2-mutated breast cancers. We called these aCGH profiles BRCA-likeCGH profiles. We demonstrated that high-risk, stage III, HER2-negative breast cancer patients with BRCAlikeCGH tumors had five times less risk to die from breast cancer when they were treated with adjuvant high-dose (HD) carboplatin-thiotepa-cyclophosphamide (CTC) with autologous stem cell rescue, instead of standard fluorouracil-epirubicincyclophosphamide (FEC) chemotherapy. In collaboration with the University of Heidelberg, Germany, we have analyzed a case-control series of 117 high-risk German breast cancer patients. The majority had been treated with HD alkylating chemotherapy with autologous stem cell rescue. Similar results were obtained for BRCA1-likeCGH patients, with a positive test for interaction between BRCA1-like status and increased benefit from HD alkylating therapy (p<0.05). Since male breast cancer has been associated with BRCA2 germline mutations, we have initiated a pilot study to analyze the prevalence of BRCA-likeCGH tumors among male breast cancers. Of 24 cases, 15 cases scored BRCA2-like (63%), while none scored BRCA1-like. This series will now be enlarged. We have generated aCGH profiles of 86 BRCA-mutated ovarian cancers and compared these with BRCA-mutated breast cancer aCGH profiles. Overlapping aberrant genomic regions are further characterized in order to identify potential druggable targets.
Figure 1: Kaplan Meier survival analysis according to tamoxifen treatment in patients whose tumor does not express p-p70S6K (A) and patients whose tumor does express p-p70S6K (B)
Figure 2: Comparative KCsmart of 20 BRCA1 mutated breast cancers compared to 43 BRCA1 mutated ovarian cancers. Smoothing was done with a 5 megabase kernel. On the x-axis genomic position and smoothed log2 ratio of breast (dark grey) and ovarian (light grey) on the y-axis. Vertical lines represent chromosomes, dashed vertical lines centromeres.
Netherlands Breast Cancer Project (NBCP) In collaboration with the Dutch Cancer Registry we have initiated a project to find answers for clinical and translational research questions that will never be answered anymore through prospective clinical trials. For this, we make use of the Dutch Cancer Registry, where data of over 150,000 breast cancer patients is stored with clinical follow-up. The ultimate aim is to combine clinical data with molecular data of tumor material that has been traced back through PALGA, the Dutch nationwide surgical pathology registry.
Identification of druggable targets in lobular breast cancer and triple negative breast cancer As participant of the FP7 RATHER consortium, together with the Bernards group (Division of Molecular Carcinogenesis), we have molecularly characterized 150 lobular and 150 triple negative breast cancers. We have used next generation sequencing, together with exon capture technology, to sequence genomic regions of interest. The final goal is to deliver diagnostic tests along with the right targeted therapy – chemotherapy combination to improve treatment options and outcome for these difficult to treat breast cancer subtypes.
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M Janssen A and Medema RH. Genetic instability: tipping the balance. Oncogene (in press) Macurek L, Benada J, Mullers E, Halim VA, Krejcikova K, Burdova K, Pechackova S, Hodny Z, Lindqvist A, Medema RH and Bartek J. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis. Cell Cycle (in press)
Group leader René Medema
René Medema PhD Group leader Benjamin Rowland PhD Senior postdoc Anja Duursma PhD Post-doc Vincentius Halim PhD Post-doc Andre Koch PhD Post-doc Rita Maia PhD Post-doc Daniel Warmerdam PhD Post-doc Yan Juan Xu PhD Post-doc Wytse Bruinsma MSc PhD student Ahmed Elbatsh MSc PhD student Femke Feringa MSc PhD student Judith Haarhuis MSc PhD student Roy Van Heesbeen MSc PhD student Aniek Janssen MSc PhD student Lenno Krenning MSc PhD student Jonne Raaijmakers MSc PhD student Indra Shaltiel MSc PhD student Mihoko Tame MSc PhD student Melinda Aprelia Technical staff Rob Klompmaker Technical staff
Publications
Medema RH and Macurek L. Checkpoint control and cancer. Oncogene 2012;31:2601-13 Monteiro LJ, Khongkow P, Kongsema M, Morris JR, Man C, Weekes D, Koo CY, Gomes AR, Pinto PH, Varghese V, Kenny LM, Charles Coombes R, Freire R, Medema RH, Lam EW. The Forkhead Box M1 protein regulates BRIP1 expression and DNA damage repair in epirubicin treatment Oncogene (in press) Raaijmakers JA, van Heesbeen RG, Meaders JL, Geers EF, FernandezGarcia B, Medema RH and Tanenbaum ME. Nuclear envelope-associated dynein drives prophase centrosome separation and enables Eg5independent bipolar spindle formation. EMBO J. 2012;31:4179-90 van der Waal MS, Saurin AT, Vromans MJ, Vleugel M, Wurzenberger C, Gerlich DW, Medema RH, Kops GJ, Lens SM. Mps1 promotes rapid centromere accumulation of Aurora B. EMBO Rep. 2012;13:847-54 Janssen A. Chromosome segregation errors: a double-edged sword. PhD thesis. Utrecht: University Utrecht
Bruinsma W, Raaijmakers JA, Medema RH. Switching Polo-like kinase-1 on and off in time and space. Trends Biochem Sci. 2012;37:534-42 Fairley JA, Mitchell LE, Berg T, Kenneth NS, von Schubert C, Sillje HH, Medema RH, Nigg EA, White RJ. Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase-1. Mol Cell 2012;45:541-52 Hopker K, Hagmann H, Khurshid S, Chen S, Hasskamp P, SeegerNukpezah T, Schilberg K, Heukamp L, Lamkemeyer T, Sos ML, Thomas RK, Lowery D, Roels F, Fischer M, Liebau MC, Resch U, Kisner T, Rother F, Bartram MP, Muller RU, Fabretti F, Kurschat P, Schumacher B, Gaestel M, Medema RH, Yaffe MB, Schermer B, Reinhardt HC, Benzing T. AATF/ Che-1 acts as a phosphorylationdependent molecular modulator to repress p53-driven apoptosis. EMBO J. 2012;31:3961-75
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Cell cycle checkpoints and chromosome segregation
The Medema group moved to the Netherlands Cancer Institute at the beginning of 2012. The research in the Medema group aims to gain a better understanding of the cellular response to classic anti-cancer drugs that damage the DNA or perturb mitotic spindle assembly. His group uses the knowledge that is generated to define and experimentally test new anti-cancer strategies. The Medema group studies 3 topics:
Checkpoint recovery; how do cells recover from a DNA damaging insult? Our primary aim in this project is to unravel the mechanism that promotes cell cycle re-entry once the checkpoint is turned off. We would like to understand how activation of the pathway responsible for cell cycle re-entry is coordinated with repair of the damage. In 2012, we have completed a large phosphoproteomic analysis comparing damage-arrested and recovering cultures to identify novel regulators of checkpoint recovery. This has resulted in the identification of several new regulators, the function of which was validated using siRNAmediated depletion. Their exact role in the recovery process is currently evaluated. In addition, in collaboration with Libor Mac rek and Zden k Kleibl in Prague, we have identified heterozygous mutations in the Wip1 phosphatase (also known as PPM1D). Wip1 is a negative regulator of p53 for which we previously demonstrated a role in recovery. The respective mutations lead to truncation and stabilization of the Wip1 protein, resulting in overexpression of active Wip1 and a consequent partial inactivation of the p53 pathway in cells containing wild-type p53. Importantly, these mutations can be found in the germ-line of cancer patients, suggesting that individuals heterozygous for these mutations could have an increased risk of cancer. In addition, we have established a number of new biosensors that allow us to monitor the appearance and repair of double-stranded DNA breaks, as well as inactivation and reactivation of the cell cycle machinery in a single living cell. Moreover, we have established a cellular system to study recovery from a G1 arrest that we have subsequently used to identify several kinases and phosphatases that control G1 recovery. We are currently completing our studies that address how these factors control the recovery process.
of dynein in spindle assembly does not require dynactin. The secondary aim in our studies on chromosome segregation has been to exploit chromosome segregation errors as a means to selectively target the fitness of cancer cells. In collaboration with researchers at the Netherlands Center for Translational Research we successfully developed orally available inhibitors for Mps1 that we are currently using in various preclinical models to validate Mps1 as a useful target for anti-cancer therapies.
Schematic overview of the different functions of dynein, dynactin and adaptor proteins during cell division.
Chromosome cohesion; How is chromosome cohesion established and removed? Research on this topic is supervised by Benjamin Rowland, who joined the group about in 2012. As a postdoc in the group of Kim Nasmyth he demonstrated that acetylation the Smc3 subunit in cohesion relieves an anti-establishment activity. His group now aims to further characterize this anti-establishment activity, both in yeast as well as in human cells.
Chromosome segregation; how to assemble a bipolar spindle and how do segregation errors affect tumor fitness? In our work on chromosome segregation, our emphasis has recently shifted towards the mechanisms underlying bipolar spindle assembly, particularly to understand how a correct balance of forces is established in the spindle. For this we monitor spindle assembly and chromosome segregation in living cells. In 2012, we have uncovered a novel role for nuclear envelope-associated dynein in the separation of centrosomes in prophase. In addition, we were able to show that the function 57
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M Stortelers C, Moolenaar WH. The global gene expression program of LPA-stimulated fibroblasts. In: Lysophospholipid Receptors: Signaling and Biochemistry. Eds.: Chun J, Hla T, Moolenaar WH, Spiegel S. Publisher: John Wiley (in press)
Lipid growth factor signaling
Houben, A. Autotaxin: biochemical and functional studies. PhD thesis. Leiden: Leiden University 2012
Group leader Wouter Moolenaar
Wouter Moolenaar PhD Group leader Elisabetta Argenzio PhD Post-doc Maikel Jongsma PhD Post-doc Elisa Matas-Rico PhD Post-doc Dalila Elouarrat MSc PhD student Anna Houben MSc PhD student Michiel Van Veen MSc PhD student
Publications
Moolenaar WH, Hla T. SnapShot: Bioactive lysophospolipids. Cell 2012;148:378-378.e2 Lai SL, Yao WL, Tsao KC, Houben AJ, Albers HM, Ovaa H, Moolenaar WH, Lee SJ. Autotaxin/Lpar3 signaling regulates Kupffer’s vesicle formation and left-right asymmetry in zebrafish. Development 2012;139:4439-48 Jansen S, Perrakis A, Ulens C, Winkler C, Andries M, Joosten RP, Van Acker M, Luyten FP, Moolenaar WH, Bollen M. Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification. Structure. 2012;20:1948-59 Houben AJ, Van Wijk XM, Van Meeteren LA, Van Zeijl L, Van de Westerlo EM, Hausmann J, Fish A, Perrakis A, Van Kuppevelt TH, Moolenaar WH. The polybasic insertion in autotaxin-alpha confers specific binding to heparin and cell-surface heparan sulfate proteoglycans. J Biol Chem. (in press) Hausmann J, Perrakis A, Moolenaar WH. Structure-function relationships of autotaxin, a secreted lysophospholipase D. Adv Biol Regul. (in press) Moolenaar WH, Houben AJ, Lee SJ, van Meeteren LA. Autotaxin in embryonic development. Biochim Biophys Acta (in press)
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The major focus of our research is on bioactive lysophos pholipids, most notably the lipid growth factor lysophosphatidic acid (LPA), its signaling properties, biosynthesis and role in health and disease. LPA signals through distinct G proteincoupled receptors, showing both overlapping and distinct signaling properties. LPA signaling influences proliferation, migration and other functions in numerous cell types. LPA is produced by a secreted lysophospholipase D, named autotaxin (ATX), originally identified as an autocrine motility factor for tumor cells. The ATX-LPA receptor system axis is vital for embryonic development, as shown by studies in mice and zebrafish, and when hyperactive promotes tumor formation and metastasis in mice. As such, the ATX-LPA signalling axis qualifies as an attractive target for therapy. Our current research focuses on ATX structure-function relation ships as well as the characterization of novel LPA receptor signaling pathways relevant to cancer. These studies should lead to novel ways of interfering with ATX-LPA receptor signalling and with undue LPA production in the tumor-stroma microenvironment. A new line of research focuses on the function of glycero phosphodiester phosphodiesterases, notably GDE2 and GDE3, enigmatic ecto-enzymes implicated in the regulation of cell differentiation.
Autotaxin structure-function analysis While the LPA-generating activity of ATX has been well characterized, the molecular basis of substrate recognition and catalysis by ATX, and how it interacts with target cells has been elusive. Close collaboration with the groups of Anastassis Perrakis (Division of Biochemistry), Andrew Morris (University of Kentucky, USA), Mathieu Bollen (KU Leuven, Belgium) and Huib Ovaa (Division of Cell Biology) has led to the elucidation of the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. The ATX structure reveals a unique hydrophobic lipidbinding pocket and a nearby open tunnel, features not found in the closest relative of ATX, termed NPP1. Whether the ATX tunnel may have a role in lipid substrate entrance or LPA product release is currently under investigation. We have mapped key residues required for catalysis and selection between nucleotide and phospholipid substrates. Furthermore, ATX was found to interact with cell-surface integrins via its the N-terminal somatomedin-Blike domains through an atypical mechanism.
ATX isoforms The intricacy of the ATX-LPA signaling axis is further enhanced by alternative splicing of ATX mRNA, giving rise to distinct isoforms. However, isoform-specific functions of ATX remain elusive. Most ATX research has focused on the β isoform, but three other isoforms are known to be produced. The ATXα isoform includes a polybasic insertion, thought to confer proteolytic instability to the protein. We find that although the insertion is cleaved at a furin consensus site, this cleavage neither has an impact on
protein stability nor affects enzyme activity. We also find that ATXα has similar kinetics to ATXβ, raising the question of why this domain is present. We find that ATXα binds specifically to heparin with nanomolar affinity, whereas the canonical isoform (ATXβ) does not. This effect was mirrored by the ability of ATXα, but not ATXβ, to bind to cell-surface heparan sulfate proteoglycans on carcinoma cells (collaboration Toin van Kuppevelt, Radboud University Nijmegen). Intriguingly, heparin also increased ATXα activity by up to two-fold. Although ATX can be targeted to the cell surface by integrin binding, these results point to a second, isoform-specific targeting mechanism that both localizes and activates ATXα to optimize LPA production and signaling. Our studies raise the possibility that ATXα and ATXβ may act on distinct LPA receptors, depending on their membrane localization, a scenario that warrants further investigation.
Figure 2: A polybasic loop in ATX-α mediates binding to cell-surface heparan sulfate proteoglycans, thereby targeting LPA production and release to specific LPA receptors at the plasma membrane.
LPA receptor signalling: CLIC4, a new signaling intermediate LPA receptors strongly couple to the G(13)-RhoA pathway to regulate the actin cytoskeleton and promote cell migration. We discovered that LPA-induced RhoA activation is accompanied by rapid recruitment of “Chloride Intracellular Channel” protein 4 (CLIC4) to the plasma membrane. CLIC4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain elusive. Intriguingly, CLIC4 translocation depends on conserved residues, including a reactive cysteine, the equivalents of which are critical for the enzymatic function of GSTs. This suggests that membrane-targeted CLIC4 may catalyse a yet unknown chemical reaction. Through yeast two-hybrid screening and biochemical studies, we have identified and validated novel binding partners of CLIC4 that may shed new light on its possible function(s) as a signaling intermediate. In particular, we find that CLIC4 is important for the recycling of integrin subunits to the plasma membrane and thereby affects cell migration in response to LPA and epidermal growth factor.
Figure 1: The autotaxin-LPA receptor signaling axis. (A) Autotaxin (ATX) converts carrier-bound lysophosphatidylcholine (LPC) into bioactive LPA, which in turn acts on specific G protein-coupled receptors to activate multiple signaling cascades and cellular responses. (B) Structures of LPA and LPC.
Transmembrane glycerophosphodiester phosphodiesterases The mammalian glycerophosphodiester phosphodiesterases (GDEs or GDPDs) are enigmatic ecto-enzymes with multiple transmembrane domains implicated in the metabolism of deacylated phospholipids. However, their biochemical and biological functions remain poorly understood. GDE2 and GDE3 are of particular interest because of their reported role in cell differentiation. We have begun to characterize the possible role of GDE2 in neurite formation in neuroblastoma cells. Forced overexpression of GDE2 promotes morphological changes in a manner dependent on its ecto-GDPD activity, reminiscent of an altered Rho/Rac activity balance. Furthermore, we find that GDE2 localizes to the plasma membrane and endocytic vesicles, suggesting that GDE2 undergoes internalization from the plasma membrane in response to an as yet unknown serum factor. We are currently exploring the biochemical activity of GDE2 and how it may translate into altered Rho/Rac signalling. We are also examining how GDE2 expression correlates with clinical outcome in neuroblastoma patients (collaboration R. Versteeg, Academic Medical Center, Amsterdam). 59
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Statistics and machine learning
Group leader Sach Mukherjee
Sach Mukherjee DPhil Group leader Nicolas Städler PhD Post-doc Steven Hill PhD Post-doc Frank Dondelinger BSc MSc Post-doc Chris Oates BA MSc PhD student Anas Rana MSci MSc PhD student
Publications
Oates CJ, Mukherjee S. Network inference and biological dynamics. Ann Appl Stat. 2012;6:1209-35 Hill SM, Neve RM, Bayani N, Kuo W-L, Ziyad S, Spellman PT, Gray JW, Mukherjee S. Integrating biological knowledge into variable selection: an empirical Bayes approach with an application in cancer biology. BMC Bioinformatics. 2012;13:94 Oates CJ, Hennessy BT, Lu Y, Mills GB, Mukherjee S. Network Inference Using Steady-State Data and GoldbeterKoshland Kinetics. Bioinformatics. 2012;28:2342-48 Hill SM, Lu Y, Molina J, Heiser LM, Spellman PT, Speed TP, Gray JW, Mills GB, Mukherjee S. Bayesian Inference of Signaling Network Topology in a Cancer Cell Line. Bioinformatics. 2012;28:2804-10 Nam C, Mukherjee S, Schilk M, Mukherjee J. Statistical Analysis of Varieties of English. J Roy Stat Soc A (in press)
The Mukherjee lab focuses on statistical and machine learning approaches in systems biology and personalized medicine. Our long-term vision is to develop computational approaches that can aid clinicians in matching therapies to those patients most likely to benefit. Realizing this vision requires not only predictive machine learning tools but also scalable approaches by which to explore the biological heterogeneity that underpins variation in therapeutic response. Accordingly, our current efforts encompass relevant aspects of basic biology, notably signaling downstream of receptor tyrosine kinases, as well as prediction of therapeutic response. We work on both specific biological questions, addressed in collaboration with experimental groups, and methodological research in statistics and machine learning motivated by such questions. The potential of quantitative approaches in cancer is clear, but the challenges posed by noisy and incomplete data, inherent biological variability and complex underlying processes and dynamics remain substantial. Our work is aimed at developing and exploiting statistical and machine learning methods that can help to surmount some of these challenges. Networks and graphical models, high-dimensional problems, combinatorial influences and inference for dynamical systems are key methodological themes in much of our work.
Ongoing projects include: Data-driven characterization of signaling networks How is the genomic heterogeneity of cancer manifested at the level of signaling networks? Do cancers show altered “wiring” due to genomic aberrations? And if so, how? Addressing these questions requires proteomic assays that can interrogate multiple signaling proteins through time, under a range of conditions and perturbations, and computational approaches by which to model the data. We are working on both theoretical and applied aspects of these questions, in collaboration with the Beijersbergen, Wessels, Bernards and Jonkers laboratories at the NKI, and the laboratories of Paul Spellman and Joe Gray (OHSU Knight Cancer Institute, USA) and Gordon Mills and Prahlad Ram (MD Anderson Cancer Center, USA). We model signaling using network inference approaches rooted in probabilistic graphical models and dynamical models based on differential equations. We are also developing approaches that bring together network inference and dynamical models. Furthermore, we are exploring whether tumor subtypes show evidence of shared signaling topology and whether network analyses can help uncover novel subtypes.
Statistical methods for personalized medicine We are developing computational approaches by which to predict drug response from multiple high-throughput data types. Several recent drug response studies have started to clarify some of the statistical challenges that arise in developing such predictive approaches. These include the large number of potential predictors (high-dimensionality), limited number of 60
samples (especially within any given lineage or cancer subtype) and challenges in integrating in vitro and clinical datasets. Within the broad area of personalized approaches to cancer therapy, our efforts focus on two specific aspects. First, we are investigating the use of network models to aid in prediction of therapeutic response. Second, we are exploiting recent advances in machine learning and high-dimensional statistics to improve predictive ability within lineage or subtype.
High-dimensional genome analysis
Data-driven characterization of signaling networks. Reverse-phase protein arrays interrogate signaling dynamics in samples of interest. Sample-specific network topology is inferred using probabilistic models known as dynamic Bayesian networks. These models integrate phosphoproteomic time-course data with existing biochemical knowledge via a principled approach rooted in Bayesian statistics.
Proteins bind to DNA in a manner that depends on chromatin organization. At the same time, DNA binding data for pairs of proteins can shed light on interplay between those proteins. In collaboration with the Van Steensel laboratory, we are combining both views, by developing models that simultaneously describe dependence of binding scores across the genome and between proteins. The aim of this work is twofold. First, to understand whether a high-dimensional view, with models that describe binding for more than one hundred proteins at once, can shed light on genome organisation. Second, to develop an understanding of protein-protein interplay that is specific to genomic region.
Stochastic models for transition processes Many physiological, pathological and experimental processes involve cell transitions through biologically distinct states. During such transition processes cell populations become heterogeneous, since at a given time individual cells within the population may be in different states. However, at present, most genome-wide assays yield only population-average data. This has limited our ability to dissect mechanisms underlying cellular transitions. At the same time, the prospect to carry out genomewide assays with single or small numbers of cells remains limited. We are developing stochastic models by which to shed light on transition processes of this kind. In collaboration with the van Steensel laboratory we are using these approaches to shed light on cell transitions in oncogenic transformation. Furthermore, with the Jaenisch laboratory (Whitehead Institute, USA) and Saha laboratory (University of Wisconsin-Madison, USA) we are exploring these ideas in the context of cellular reprogramming.
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N Huang S, Hölzel M, Knijnenburg T, Schlicker A, Roepman P, McDermott U, Garnett M, Grernrum W, Sun C, Prahallad A, Groenendijk FH, Mittempergher L, Nijkamp W, Neefjes J, Salazar R, Ten Dijke P, Uramoto H, Tanaka F, Beijersbergen RL, Wessels LF, Bernards R. MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling. Cell. 2012;151:937-50
Division head, group leader Jacques Neefjes Jacques Neefjes PhD Group leader Ilana Berlin PhD Post-doc Gosia Garska PhD Post-doc Petra Paul PhD Post-doc Xiaohang Qiao PhD Post-doc Tiziana Scanu PhD Post-doc Robbert Spaapen PhD Post-doc Inge Verbrugge PhD Post-doc Amy Dohmen MD PhD Post-doc Josine Quispel-Janssen MD PhD Post-doc Jeroen Bakker MSc PhD student Sjoerd Van Deventer MSc PhD student Marlieke Jongsma MSc PhD student Rik Van der Kant MSc PhD student Baoxu Pang MSc PhD student Laurel Schunselaar MSc PhD student Ruud Wijdeven MSc PhD student Li-en Wu MSc PhD student Lennert Janssen Technical staff
Publications
Kuijl C, Pilli M, Alahari SK, Janssen H, Khoo P-S, Ervin KE, Calero M, Jonnalagadda S, Scheller RH, Neefjes J and Junutula JR. Rac and Rab GTPases Dual effector Nischarin facilitates vesicle maturation and survival of intracellular Salmonella typhimurium. EMBO J. 2013;110:175-180 Menendez-Benito V, van Deventer SJ, Jimenez-Garcia V, Roy-Luzarraga M, van Leeuwen F, Neefjes J. Spatiotemporal analysis of organelle and macromolecular complex inheritance. Proc Natl Acad Sci U S A 2013;110:175-180
Wijdeven RH, Bakker JM, Paul P, Neefjes J. Exploring genome-wide datasets of MHC class II antigen presentation. Mol Immunol. (in press) de Jong A, Merkx R, Berlin I, Rodenko B, Wijdeven RH, El Atmioui D, Yalçin Z, Robson CN, Neefjes JJ, Ovaa H. Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. Chembiochem. 2012;13:2251-8 de Leeuw R, Flach K, Bentin Toaldo C, Alexi X, Canisius S, Neefjes J, Michalides R, Zwart W. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance. Oncogene. 2012;1-9 Neefjes J, Sadaka C. Into the intracellular logistics of crosspresentation. Front Immunol. 2012;3:31 Majera D, Kristan K , Neefjes J, Turk D, Miheli M. Expression, purification and assembly of soluble multimeric MHC class II-invariant chain complexes. FEBS Lett. 2012;586:1:318-24 Houtman R, de Leeuw R, Rondaij M, Melchers D, Verwoerd D, Ruijtenbeek R, Martens JW, Neefjes J, Michalides R. Serine-305 phosphorylation modulates estrogen receptor alpha binding to a coregulator peptide array, with potential application in predicting responses to tamoxifen. Mol Cancer Ther. 2012;11:805-16 Hoorn Tv, Paul P, Janssen L, Janssen H, Neefjes J. Dynamics within tetraspanin pairs affect MHC class II expression. J Cell Sci. 2012;125:328-39 Spaapen RM, Neefjes J. Immuno-waste exposure and further management. Nat Immunol. 2012;13:109-11
Souwer Y, Griekspoor A, de Wit J, Martinoli C, Zagato E, Janssen H, Jorritsma T, Bar-Ephraïm YE, Rescigno M, Neefjes J, van Ham SM. Selective infection of antigen-specific B lymphocytes by salmonella mediates bacterial survival and systemic spreading of infection. PLoS One. 2012;7:e50667
Uusi-Rauva K, Kyttälä A, van der Kant R, Vesa J, Tanhuanpää K, Neefjes J, Olkkonen VM, Jalanko A. Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments. Cell Mol Life Sci. 2012;69:2075-89
Garstka MA, Neefjes J. How to target MHC class II into the MIIC compartment. Mol Immunol. (in press)
Petra Paul. The Systems Biology of MHC class II antigen presentation. Thesis. Leiden 2012 (cum laude)
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Chemical biology of antigen presentation and anticancer drugs The Neefjes lab is studying the cell biology of antigen presentation and introducing chemical biology into the field of immunology in order to define small molecules manipulating immune responses. This work is performed in close collaboration with the Ovaa lab in the Division and integrates immunology, cell biology and chemistry. These activities are also translated to drug screening programs on human primary mesothelioma and head and neck tumor cells in collaboration with the clinic. We use modern technologies to reevaluate the activities of anti-cancer drugs such as doxorubicin, again integrating chemistry, cell biology and molecular biology to define new mechanisms for an old but effective anticancer drug. Finally, we have started a program aimed at defining new ways to induce cell transformation and combined the cell biology of bacterial infections with transformation assays and mouse studies. These data suggest an active contribution of Salmonella infection in the induction phase of cell transformation.
MHC class I antigen presentation MHC class I molecules present fragments of intracellular antigens including tumor proteins to the immune system that than respond by eliminating the cells expressing these. The process is defined in detail. We have performed a haploid cell screen (in collaboration with Dr Thijn Brummelkamp) to define new proteins required for successful antigen presentation and are currently characterizing these. In addition, we have studied the process of peptide binding to MHC class I molecules using a combination of chemistry, biophysics and crystallography. We show that MHC class I first binds many different peptides followed by a conformational change to select only peptides with a low off rate. This is important for the design of MHC class I molecules with peptides at wish and explains how MHC class I selects peptides for presentation.
MHC class II antigen presentation MHC class II molecules present peptides encountered in the endocytic pathway in a process understood in detail. This is important to drive many immune responses but also in most auto-immune diseases where this process is uncontrolled. We have performed a genome-wide siRNA screen to identify new molecules in control of MHC class II antigen presentation. We have deconvoluted the hits to solve a new pathway in control of MHC class II transport in dendritic cells. We have integrated chemical inactivation and genetic silencing to search for compounds and corresponding targets controlling MHC class II expression. We have used the siRNA screen data base to study other processes in detail. These include the control of MHC class II transport by a new RING containing protein, DUBs in the control of MHC class II transport and transcription and GTPases controlling bidirectional transport of MHC class II compartments. Our aim is to identify new cell biology in the MHC class II pathway and compounds to manipulate MHC class II controlled immune responses.
Chemical Biology of old anticancer drugs: Doxorubicin Doxorubicin, like Etoposide, is a topo-isomerase II inhibitor that generates DNA double stranded breaks to induce apoptosis. Yet, Doxorubicin is considerably more potent as an anticancer drug than Etoposide with also more side effects. We have used new technologies to visualize the effects of these drugs on cells and their genome. We have shown that Doxorubicin has an additional effect, histone eviction. As a result DNA repair is stalled, epigenetics are altered and an additional signal for apoptosis is provided. Using combinations of the Doxorubicin structure, we have explained the molecular mechanism and defined various of the consequences in cells, mice and human cancer patients. This unique effect of Doxorubicin is now further explored to define some of its effects in combination trials. In addition, our data rationalize the use of Doxorubicin in anticancer treatment.
Model of the structure of the anticancer drug doxorubicin (yellow) in the DNA (green) and nucleosome (blue) structure. Doxorubicin destabilizes these structures resulting in histone eviction, attenuation of DNA repair and epigenetic changes.
Finding new drug combinations for mesothelioma and head&neck cancer We have started to establish cultures of primary cancer cells isolated from mesothelioma and head&neck cancer patients. These cells are characterized and exposed to a series of (combinations of) anti-cancer drugs. The effect of drug exposure on cell viability is used to select drug combinations that can be effective against these tumor cells. For mesothelioma, we use mesothelial cells as a control that should not be affected by the various drug combinations. In collaboration with the clinic, we are now establishing a pipeline for obtaining patient mesothelioma cells to test whether chemical profiling will indicate patient responses and whether there is any patient to patient variation. New combinations of anticancer drugs will be further tested for safety before the information can be translated into patient treatment.
The contribution of bacterial infections in cell transformation and cancer formation It is believed that bacteria may by inducing a chronic inflammation contribute to cancer. Chronic typhoid carriers in India have an increased risk in gallbladder cancer. We have used primary cells with one or more pre-transforming mutations to show that infection with Salmonella suffices to induce transformation. In fact, only a short infection suffices to imprint the transformed state in these cells and we are currently studying the molecular basis of this new phenomenon. We have established a mouse model system to test a contribution of chronic Salmonella infection in colon tumor formation and the results are expected early 2013. Our data indicate that Salmonella infection contributes to cancer formation and suggest a new mechanism of cancer formation.
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O De Jong A, Merkx R. et al. Ubiquitinbased probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. Chembiochem 2012:13:2251-2258
Biological chemistry
De Jong A, Schuurman KG,, et al. Fluorescence-based proteasome activityprofiling. Methods Mol Biol 2012:803:183-204
Group leader Huib Ovaa
Huib Ovaa PhD Group leader Harald Albers PhD Post-doc Paul Geurink PhD Post-doc Remco Merkx PhD Post-doc Alessia Amore PhD Post-doc Aysegul Sapmaz PhD Post-doc Monique Mulder PhD Post-doc Farid El Oualid PhD Post-doc Boris Rodenko PhD Post-doc Sander Van Kasteren PhD Post-doc Katharina Witting MSc PhD student Annemieke De Jong MSc PhD student Dharjath Hameed MSc PhD student Rieuwert Hoppes MSc PhD student Jolien Luimstra MSc PhD student Reggy Ekkebus MSc PhD student Gabrielle Van Tilburg MSc Technical staff Kim Wals ing Technical staff Henk Hilkmann ing Technical staff Raymond Kooy ing Technical staff Dris El Atmioui ing Technical staff
Publications
Amore A, Wals K, Koekoek E, Hoppes R, Toebes M, Schumacher TN, Rodenko B, Ovaa H. Development of a Hypersensitive Periodate-Cleavable Amino Acid that is Methionine- and Disulfide-Compatible and its Application in MHC Exchange Reagents for T Cell Characterisation. Chembiochem 2013:14:123-131 Albers HM, Ovaa H. Chemical evolution of autotaxin inhibitors. Chem Rev 2012:112:2593-260 Berkers CR, Leestemaker Y, et al. Probing the specificity and activity profiles of the proteasome inhibitors bortezomib and delanzomib. Mol Pharm 2012:9:1126-1135 Burns KE, McAllister FE,, et al. Mycobacterium tuberculosis prokaryotic ubiquitin-like proteindeconjugating enzyme is an unusual aspartate amidase. J Biol Chem 2012:287:37522-37529
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El Oualid F, Hameed DS, et al. Synthesis of atypical diubiquitin chains. Methods Mol Biol 2012:832:597-609 Federico L, Ren H, et al. Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice. Mol Endocrinol 2012:26:786-797 Geurink PP, El Oualid F, et al. A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents. Chembiochem 2012:13:293-297 Lai SL, Yao WL, et al. Autotaxin/ Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish. Development 2012:139:4439-4448 Merkx R, Burns KE, et al. Synthesis and evaluation of a selective fluorogenic Pup derived assay reagent for Dop, a potential drug target in Mycobacterium tuberculosis. Chembiochem 2012:13:2056-2060 Schulz S, Chachami G,, et al. Ubiquitinspecific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions. EMBO Rep 2012:13:930-938
Cellular protein degradation and many other cellular processes are controlled by post-translational modification of proteins with ubiquitin. Ubiquitin itself is a small protein that consists of 76 amino acids and ubiquitin modification is tightly controlled. Approximately 1,000 ubiquitin ligases and 100 DUBs are encoded in the human genome. Malfunction of several components that control ubiquitination frequently are associated with cancer. When specific ubiquitin modifications target for protein destruction, the proteasome can recognize these signals and destroy the ubiquitintagged protein. In this manner misfolded or redundant proteins are removed. Accumulation of such proteins can lead to diseases associated with protein aggregation. Viral and tumor proteins are also destroyed by the proteasome and protein fragments can be presented to the immune system to indicate cancer or infection. These protein fragments are loaded into major histocompatibility class I complexes that present these antigens to cytotoxic T-cells that can subsequently eliminate infected and tumor cells. The Ovaa lab studies the biochemistry of ubiquitin modification, proteasomal proteolysis and antigen presentation using chemical approaches. It is our aim to develop novel methods and reagents that allow us to study these processes in unprecedented detail and to develop - where possible - novel small molecules to intervene with these processes to study potential new drug targets. Our research is carried out in close collaboration with various NKI investigators. Three topics are central in the lab: 1. Ubiquitin chemistry, biology & proteomics. 2. Proteasome action. 3. MHC class I antigen presentation.
2. Proteasome action Over the last years we have developed reagents that allow monitoring of proteasomal activity. These reagents have been very instrumental in studying the specificity of inhibitors that are currently in use in the clinic or that are currently being evaluated in clinical trials. Such reagents were needed as the proteasome has six distinct active sites with different specificities. With our proteasome probes we are able to distinguish all these activities while overall proteasome activity can also be monitored in live cells. We are currently investigating how proteasome activity is regulated as this is obviously a tightly regulated process. The tight regulation of proteasome activity is an understudied topic. The proteasome is one of the most abundant and stable complexes and its activity is likely tightly controlled.
3. MHC class I antigen presentation
Figure 1: Peptide exchange by means of in situ generation of empty class I complexes. MHC molecules are folded with a UV-senstive ligand that disintegrates upon UV-irradiation. Complex degradation can be prevented by addition of another peptide.
Over the last few years we have developed technologies for the parallel loading of MHC complexes with either physical or chemical triggers in collaboration with the Schumacher lab. As a result high throughput measurements of MHC affinity are now routine, allowing high throughput searches for novel antigens or vaccine candidates. With this technology we have synthesized and tested over 10,000 peptides and over 50,000 other molecules for MHC affinity with the goal of identifying novel epitopes and to find molecules that may act as immunomodulators.
Drug testing on short term tumor cultures This new project, in collaboration with Jacques Neefjes and AVL clinicians Paul Baas, Michiel van den Brekel, Lotje Zuur, Josine Quispel-Janssen and Amy Dohmen, aims at the development of personalized therapies, as well as the development of novel agents with action against mesothelioma and head & neck cancer. The role of our lab in this project concerns small molecule screening on primary material and the further development of active “chemical hits” towards molecules with therapeutic potential with the ultimate goal to develop personalized therapies.
Figure 2: Chemically synthesized atypical ubiquitin chains.
1. Ubiquitin chemistry, biology and ubiquitin proteomics We study the ubiquitin system with chemical approaches. In doing so, we take advantage of organic synthesis. We have recently developed methods to synthesize ubiquitin and virtually any ubiquitin conjugate. With these chemical techniques we can synthesize virtually any reagent that is needed for our research. For example, we recently synthesized all isopeptide-linked ubiquitin topoisomers. We currently use these conjugates to study how different ubiquitin linkage topologies interact with the proteasome. All ubiquitin chain topoisomers can be found in cells, but the role of only few chains is relatively well understood. This is understandable since they cannot currently be generated in isolation by biochemical means. With our chemical methods we are now well positioned to study the role of different ubiquitin chains in cellular signal transduction events and we can now generate the reagents required to detect interacting proteins with specific linkage-specificities using proteomics approaches. 65
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Dissecting cancer cell signaling networks & Identifying therapeutic cancer targets Division head, group leader Daniel Peeper Daniel Peeper PhD Group leader Renee Van Amerongen PhD Research associate Yann Neuzillet MD Clinical fellow Patricia Abrao Possik PhD Post-doc Mirjam Epping PhD Post-doc Tristan Gallenne PhD Post-doc Kylie Greig PhD Post-doc Kristel Kemper PhD Post-doc Christelle Lenain MD PhD Post-doc Katrin Meissl PhD Post-doc Judith Mueller PhD Post-doc Marjon Smit PhD Post-doc Celia Vogel PhD Post-doc Sedef Iskit MSc PhD student Joanna Kaplon MSc PhD student Sirith Douma MSc Technical staff Nils Visser MSc Technical staff
Figure 1: Adora2B depletion or targeted inhibition suppresses filopodia formation. Representative immunofluorescence imaging of filopodia in LM2 human breast cancer cells depleted for Adora2B or treated with the Adora2B inhibitor theophylline (100 μM). Merged images of the staining of DNA, F-actin and a-tubulin are shown.
To investigate these questions, we make use of advanced approaches, including function-based screens with 100.000-vector RNAi libraries and next-generation sequencing, but also classical biochemical and genetic approaches. While we are studying a diverse array of tumor types, there is a particular focus on melanoma (a highly aggressive skin cancer) and metastatic breast cancer. At the cellular level, we have a special interest in several cancer-relevant events: (i) override of Oncogene-Induced cellular Senescence (‘OIS’, a potent tumor suppressor mechanism limiting the proliferative capacity of incipient cancer cells); (ii) mechanisms driving metastasis; (iii) (non-) oncogene addiction and drug enhancer and resistance screens.
Publication
Vredeveld LCW, Possik PA, Smit MA, Meissl K, Michaloglou C, Horlings HM, Ajouaou A, Kortman PC, Dankort D, McMahon M, Mooi WJ, Peeper DS. Abrogation of BRAFV600E-induced senescence by PI3K pathway activation contributes to melanomagenesis.
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The development of cancer relies on several steps that are all in principle rate-limiting for completion of the transformation process. My laboratory aims to tackle both basic and translational questions in this context. Are there any intrinsic fail-safe mechanisms protecting us against cancer? How can we identify and dissect tumor-driving genetic networks? How can we best use recently available techniques to uncover novel drug targets and make a difference in the clinic? And along these lines, can we find ways to enhance the activity, or avoid resistance, of currently used targeted drugs?
Our results over the past decade demonstrate that these approaches, together, lead to the identification of signaling networks, deregulation of which allows for tumorigenesis and metastasis. Our more recent results indicate that among these, several factors exist that can be exploited for therapeutic intervention of cancer. Figure 2: Reactivation of senescence features in melanoma upon PI(3)K inhibition. (A) Samples were kept in medium containing a dose range of Pi-103 for 7 d and fixed and stained with crystal violet. (B) Melanoma cell lines were treated with 0.5 μM Pi-103 and analyzed by Western blotting for the senescence-associated tumor suppressor p15INK4B.
Ongoing
I. Breast cancer metastasis: mechanism and drug target identification Metastasis commonly underlies the malignancy of cancers, representing the principal cause of cancer-treatment failure. We have recently discovered a novel critical mediator of breast cancer metastasis, the Fra-1 transcription factor. Fra-1 depletion by shRNA from human breast cancer cells reduced their metastatic potential by >3 orders of magnitude. Further, by analyzing the expression of a set of Fra-1 target genes we could make accurate predictions on the clinical outcome of breast cancer. And using a synthetic lethal drug screen we identified inhibitors of the adenosine receptor Adora2B, which are cytotoxic to metastasizing, Fra-1-expressing, cells. Concordantly, Adora2B inhibitors suppress the outgrowth of metastases. We found that either silencing, or inhibition of ADORA2B with specific inhibitors led to a reduction in formation of filopodia, cellular protrusions associated with the ability of cells to migrate and invade (figure 1). Mining the prognostic Fra1 signature, we have found several additional factors that may
be amenable to targeted inhibition. Furthermore, our preliminary results suggest that some of those factors engage in a large network driving breast cancer metastasis. We are currently validating this hypothesis, which should eventually lead to the development of companion diagnostics for breast cancer.
II. Oncogene-Induced cellular Senescence (OIS): mechanism and drug target identification We are also screening for novel OIS pathways whose deregulation contributes to cancer. In this setting too, we are combining function-based, genome-wide screens with classical molecular biological approaches. For example, we study the genetic basis for malignant progression of benign moles (nevi) to malignant melanoma. We, in a longstanding collaboration with prof. Wolter Mooi (VUmc), were the first to discover that nevi display several OIS hallmarks in vivo. Using a combination of genetic profiling and laser capture microdissection of compound clinical nevus-melanoma specimens, we found that activation of the PI(3)Kinase pathway represents a frequent event by which nevi escape from OIS, thereby allowing their progression to melanomas. Targeted inhibition of PI(3)Kinase restores senescence features in melanoma cells, raising the possibility that these results can be applied clinically (figure 2). Furthermore, we found that combined inhibition of BRAF and PI(3)Kinase acts synergistically toxic to melanoma cells, providing a rationale the combined treatment of melanomas with these inhibitors. We have also recently discovered a functional connection between cellular metabolism and OIS. By comprehensive metabolic flux profiling and functional analysis we unmasked the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) as an enzyme critically contributing to OIS. OIS was accompanied by the simultaneous downregulation of the inhibitory PDH kinase PDK1 and induction of the PDH-activating phosphatase PDP2, leading to the concerted activation of PDH. This caused a major shift in metabolite flux from glycolysis to the tricarboxylic acid (TCA) cycle, increasing pyruvate oxidation. Conversely, OIS abrogation coincided with PDH inhibition. Enforced normalization of PDK1 or PDP2 levels inhibited PDH and abrogated OIS, causing continued cell proliferation. These results establish a functional link between OIS and a key mitochondrial signaling axis balancing glycolysis and respiration.
The BRAF kinase inhibitor vemurafenib has generated tremendous excitement recently, following unprecedented responses observed in clinical trials of patients with metastatic melanoma carrying the BRAFV600E mutation. As already eluded to, despite such early promise, patients commonly relapse with drug-resistant tumours. There is an urgent need to find drugs that can be used in combination with vemurafenib to reduce the risk of resistance. This is best done in a physiologically relevant setting. Therefore, we have established, in collaboration with our clinical colleagues prof. John Haanen and dr. Christian Blank and with prof. Ton Schumacher, a xenograft platform for human melanoma. Matched patient samples, taken prior to therapy and after the patient has relapsed are taken and placed into immunocompromised mice, which engraft melanomas highly efficiently. The first results show that the cellular and genetic profiles are similar among the original melanomas and derived xenografts, while drug-sensitive and resistant traits were largely retained. These xenografts will be used for a variety of studies, aiming to find novel treatment modalities for melanoma. Finally, we have set up several screens to identify novel therapeutic targets for melanoma. Exploiting the phenomenon of synthetic lethality (SL) as a mean of therapeutic intervention is gaining increasing interest. For example, set out to screen for synthetic lethal partners of BRAFV600E. To date, this has led to the identification of a gene involved in signal transduction, which we are currently characterizing. We have begun to do this also in vivo, using large-scale RNAi libraries and massive parallel sequencing. Together, these approaches should lead to the identification of novel therapeutic targets in melanoma and possibly other cancer types.
III. Screening for novel therapeutic targets in melanoma In an attempt to translate the findings of this metabolic study to a preclinical setting, we depleted PDK1 from normal and melanoma cells. In a panel of human melanoma cell lines, shRNA-mediated PDK1 depletion induced massive cell death. Furthermore, depletion of PDK1 sensitized BRAF mutant melanoma cell lines to the clinical BRAF inhibitor vemurafenib. This is particularly relevant because melanoma patients treated with this drug invariably show a relapse at seven months on average after initial strong responses. We found that in vivo, stably depleted melanoma cells failed to produce tumors, while induction of PDK1 shRNA in already established melanomas induced strong tumor regression. We aim to collaborate with pharma to use pharmacologic PDK1 inhibitors to recapitulate the effect of PDK1 RNAi, exploring the possibility to use PDK1 inhibition as a new treatment modality in melanoma. 67
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P Heidebrecht T, Fish A, von Castelmur E, Johnson KA, Zaccai G, Borst P, Perrakis A. Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding. J Am Chem Soc. 2012;134:13357-65
Group leader Anastassis Perrakis
Hausmann J, Perrakis A, Moolenaar WH. Structure-function relationships of autotaxin, a secreted lysophospholipase D. Advances in biological regulation. 2012 Caillat C, Perrakis A. Cdt1 and geminin in DNA replication initiation. Subcellular biochemistry. 2012;62:71-87
Anastassis Perrakis PhD Group leader Christophe Caillat PhD Post-doc Eleonora von Castelmur PhD Post-doc Krista Joosten PhD Post-doc Robbie Joosten PhD Post-doc Leonie van Zeijl PhD Post-doc Jens Haussman MSc PhD student Tatjana Heidebrecht Technical staff
Publications
Van Luenen HG, Farris C, Jan S, Genest PA, Tripathi P, Velds A, Kerkhoven RM, Nieuwland M, Haydock A, Ramasamy G, Vainio S, Heidebrecht T, Perrakis A, Pagie L, van Steensel B, Myler PJ, Borst P. Glucosylated hydroxymethyluracil, DNA base j, prevents transcriptional readthrough in leishmania. Cell. 2012;150:909-21 Suijkerbuijk SJ, van Dam TJ, Karagoz GE, von Castelmur E, Hubner NC, Duarte AM, Vleugel M, Perrakis A, Rudiger SG, Snel B, Kops GJ. The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase. Developmental cell. 2012;22:1321-9 Joosten RP, Joosten K, Murshudov GN, Perrakis A. PDB_REDO: constructive validation, more than just looking for errors. Acta Crystallogr D Biol Crystallogr. 2012;68:484-96 Jansen S, Perrakis A, Ulens C, Winkler C, Andries M, Joosten RP, Van Acker M, Luyten FP, Moolenaar WH, Bollen M. Structure of NPP1, an Ectonucleotide Pyrophosphatase/Phosphodiesterase Involved in Tissue Calcification. Structure. 2012 Houben AJ, van Wijk XM, van Meeteren LA, van Zeijl L, van de Westerlo EM, Hausmann J, Fish A, Perrakis A, van Kuppevelt TH, Moolenaar WH. The Polybasic Insertion in Autotaxin-alpha Confers Specific Binding to Heparin and Cell-Surface Heparan Sulfate Proteoglycans. J Biol Chem. 2012
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Structural biology
Our major projects remain the in-house collaborations about the function of Autotaxin (with Wouter Moolenaar), JBP1 (with Piet Borst) and mitotic kinases (with René Medema and with Geert Kops at UMC Utrecht), while we keep our long-term interest in proteins that regulate DNA replication licensing. Our efforts in structural bioinformatics are focusing on the PDB_REDO project, a tool and a resource for better structures by X-ray crystallography.
Structural studies of Autotaxin ATX is an ecto-nucleotide phosphodiesterase 2 (ENPP2), capable of both lysoPLD and nucleotide pyrophosphatase activities. We determined the structure of ATX last year. ATX produces the signaling phospholipid lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA and ATX have been shown by numerous studies to be involved in cancer metastasis and other pathogenic situations, such as chronic inflammation. Our current focus is to understand the interaction of ATX with cell-surface integrins and heparin in the cell surface. Our data on the interaction of ATX with platelet integrins and more recent data on binding of the ATXα isoform to heparan sulphate proteoglycans, suggest a mechanism for the delivery of LPA close to the cell surface. We are proceeding to study the molecular mechanism and the importance of these bindings to ATX and LPA mediated signalling.
Structural studies of JBP1 The JBP1 protein, discovered by Piet Borst and colleagues, binds to DNA that contains base J (β-D-glucosyl-hydroxymethyluracil) and is also a thymidine hydroxylase. Almost twenty years following the discovery of base J, Piet Borst and colleagues found a function of base J in non-telomeric regions, being responsible for terminating transcription. We had shown that JBP1 recognizes J-containing DNA through a small DNA Binding Domain (DBD), with ten thousand-fold preference over normal DNA, and that a single aspartate residue is responsible for this remarkable discrimination capability. We obtained pre-steady state DNA-binding kinetics data that suggested that while DB-JBP1 (a protein containing only the DBD domain) binds to DNA in a single step, full-length JBP1 binding to DNA takes place in two distinct steps. Analysing the apparent binding rates and combining this with small angle neutron scattering data that allowed us to look at the shape of the protein before and after binding to J-DNA, we demonstrated that the two-step mechanism represents a conformational change. We suggest that JBP1 binding to J-DNA through the DB-domain triggers a conformational change that brings the hydroxylase domain towards the DNA, into position to hydroxylate thymidine.
Structural studies of proteins involved in mitotic progression The Spindle Assembly Checkpoint (SAC) is a protein network that ensures that the cell does not proceed with separating the sister chromatids in mitosis before all chromosomes have been aligned and attached to the spindle machinery. Our focus has been on understanding the role of the regulatory domain of Mps1 kinase, one of the SAC kinases. The MPS (MonoPolar Spindle) family of kinases is a dual specificity protein kinase in vitro, capable of autophosphorylation on serine, threonine and tyrosine residues. Mps1 allows resolution of merotelic attachments and ensures that single kinetochores achieve the strength of checkpoint signalling sufficient to prevent premature anaphase onset and chromosomal instability. We have previously determined the structure of the N-terminal domain of Mps1, which adopts the TPR fold, also found in the Bub1 and BubR1 family of kinases. In collaboration with the group of Geert Kops (UMC Utrecht), we have shown with site directed mutagenesis and domain deletion and swapping studies, that a module in the N-terminus of Mps1, comprised by an N-Terminal Extension (NTE) and the TPR domain of Mps1, is important for localization of Mps1 to the kinetochores. In addition we showed that this Mps1 module interacts with the HEC1 protein of the NDC80 complex in the outer kinetochores, establishing for the first time a physical interaction between the spindle assembly checkpoint and the microtubule-binding end of kinetochores.
correct errors of existing models. The first results of the new PDB_REDO, incorporating a decade of knowledge and algorithms that were used to build the ARP/wARP software, show marked improvement of the quality of both old but also new models in the PDB. PDB_REDO is developing to a decision-making system making rational decisions for the best protocols on a case-bycase basis, based on a decision-making framework we described this year and we continue to develop.
Geminin and its homologues in DNA replication licensing A new twist to our research on the role of Geminin in replication licensing was provided by the discovery of two homologue of Geminin: Idas and Lygeas (in Greek mythology, Ίδας and Λυγέας are the cousin of the two Gemini). Lygeas (who unfortunately made its first appearance in scientific literature under the more mundane name GemC1) directly mediates replication initiation through TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins. We want to investigate the hypothesis if the assigned functions of Geminin in proliferation and development are possibly through the combined action of Geminin with its two homologue proteins. We have determined the structure of a complex between Geminin and Idas, that form a parallel coiled coil heterodimer. Biophysical analysis of this complex showed that Geminin prefers to make complexes with Idas than to itself, and Idas prefers Geminin to itself as well. These data combined with functional studies in Xenopus oocyte cell extracts and mammalian cell cycle analysis, show that the formation of this heterodimer is likely to regulate the function of Geminin in tissues where Idas is expressed, like the forebrain. We have also determined the structure of GemC1, which is an unusual homodimer where the first heptad of the coiled coil adopts an unusual conformation, hinting as to why GemC1 has very different functionality compared to its relatives.
Methods for X-ray crystallography We are focusing on bringing up-to-date the crystallographic models in the Protein Data Bank (PDB), that were deposited there over several decades, and were created using the methods and software available at the time. The state of the art software of nowadays is used in a fully automated procedure, PDB_REDO, which we have designed to improve the accuracy and 69
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P Houben D, Demangel C, van Ingen J, Perez J, Baldeón L, Abdallah AM, Caleechurn L, Bottai D, van Zon M, de Punder K, van der Laan T, Kant A, Bossers-de Vries R, Willemsen P, Bitter W, van Soolingen D, Brosch R, van der Wel N, Peters PJ. ESX-1mediated translocation to the cytosol controls virulence of mycobacteria. Cell Microbiol. 2012;14:1287-98
Group leader Peter Peters
Peter Peters PhD Group leader Nicole Van der Wel PhD Associate staff scientist Mary Morphey Visiting staff scientist Sue Godsave PhD Post-doc Pekka Kujala PhD Post-doc Alicia Lammerts van Buren PhD Post-doc Massimiliano Maletta PhD Post-doc Musa Sani PhD Post-doc Matthijn Vos PhD Post-doc Pavel Afanasyev MSc PhD student Karin De Punder MSc Technical staff Hans Jansen BSc Technical staff Maaike Van Zon BSc Technical staff Nico Ong Research assistant
Publications
Godsave S, Wille H, Pierson J, Prusiner S. Plasma membrane invaginations containing clusters of full length PrPSc are an early form of prion-associated neuropathology in vivo. Neurobiology of Aging (in press) Barker N, Rookmaaker MB, Kujala P, Ng A, Leushacke M, Snippert H, van de Wetering M, Tan S, Van Es JH, Huch M, Poulsom R, Verhaar MC, Peters PJ, Clevers H. Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep. 2012;2:540-52 De Lau W, Kujala P, Schneeberger K, Middendorp S, Li VS, Barker N, Martens A, Hofhuis F, DeKoter RP, Peters PJ, Nieuwenhuis E, Clevers H. Peyer's patch M cells derived from Lgr5(+) stem cells require SpiB and are induced by RankL in cultured "miniguts". Mol Cell Biol. 2012;32:3639-47
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The first EM image of purified ESX complexes, isolated from M. marinum.
Nanobiology
Our team is working hard to reach a deeper understanding of the intricacies of a biological cell down to the nanoscale. We are imaging biological components of functional entities, and ultimately to understand the building blocks of a cell under normal and pathogenic conditions. This cellular nanocosmos comprises an amazing wealth of macromolecular complexes and biological nanomachines and we are developing methods to study them. Imaging of biological complexes in a native state and under experimental conditions is our focus with the ultimate goal of reaching an understanding of their functions in a cell. In order to identify such macromolecular complexes and determine their structures in situ we obtained a grant this year for state-ofthe-art correlative high-resolution light and electron microscopy (CLEM). While NKI researchers exploit live-cell imaging using GFP-tagged proteins (available for almost all gene products in the genome), rapid immobilization and cryo-electron tomography (ET) is now also an established and available technique. Cryo-ET together with image averaging can provide the extreme resolution needed to construct 3D images at the 3-5 nm scale. Our team worked over the last year at providing such an infrastructure. An amazing development of imaging methods over the last few years has revolutionized our ability to directly visualize protein complexes. We participated in creating the Netherlands Centre for Electron Nanoscopy (NeCEN), a facility with two top-end Titan Krios cryo-TEMs that came into use a year ago. These expensive instruments can only be fully exploited when the proper samples have been produced and we have been pre-screening many in our lab. Recent advances by the group of Kees Jalink in optical super resolution fluorescence microscopy (SR-LM) resulted in a new ambitious endeavor to integrate the strengths of cryo-TEM and SR-LM in a unique setting. The majority of cells are too thick to be imaged in an intact state and therefore methods such as cryo-sectioning of unperturbed vitreous, fully hydrated samples are needed. Our lab has been improving this technique over the years. As an alternative to vitreous cryo-sectioning, we have recently used live-cell light microscopy of migrating white blood cells to show that they can naturally migrate into the 1000 nm high space of a “nanochamber”. We are developing this chamber with siliconbased MEMS technology in collaboration with colleagues at the Kavli Institute in Delft. The development of the device that we propose will allow us to study the behavior of intact migrating malignant cells and their response to drug-induced changes at the molecular level. The project is high risk but when successful it will open up entirely new avenues towards nanoscale studies of intact mammalian cells. Together with FEI, we are further developing technology to vitrify the cells in this state. They will then be sufficiently thin to use a cryo-FIB/SEM making them 200nm, thus useful for electron cryo-ET without further need for cryo-sectioning.
Mycobacteria Why do we study mycobacteria? Pathogenic mycobacteria are devastating pathogens, but controlled use of certain strains of mycobacteria activates macrophages in ways that can be harnessed for therapy. The most common treatment of superficial/noninvasive bladder cancer is to inject BCG mycobacteria into the bladder. This mediates the regression of transitional cell carcinomas by stimulating a vigorous local immune response, bathing tumors in cytokines and activated immune cells. It is unclear how this provides a beneficial effect in eliminating bladder cancer, and this needs to be better understood to improve the therapy and perhaps use it for other noninvasive carcinomas. Nearly one-third of the population worldwide has latent tuberculosis (TB). Migration from high-incidence countries has increased the number of cases of multi-drug resistant (MDR) TB. More youngsters visit those high incidence countries for an extended period and become exposed. It is now well known that anti-TNF (tumor necrosis factor) activates latent TB. The cytokine and immunostimulant, TNF-alpha, is able to prevent breast cancer tumor formation in vivo under experimental conditions (Al-zoubi M, et al., Cell Cycle, 2012;12). Inhibition of TNF using antibodies has revolutionized the treatment of rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and Crohn disease. Anti-TNF, also promotes reactivation of intracellular pathogens, including MDR TB, resulting in potentially life-threatening infections. Therefore, TB screening and or preventive antibiotic treatment recently became routine prior to starting a patient on an anti-TNF antibody therapy. The use of other biological agents that target specific immune molecules is becoming more widespread for cancer treatment. The soluble CTLA4 molecule that is used to treat melanoma can cause symptoms consistent with autoimmunity, but since disseminated TB can be confused with an autoimmune disease this problem might dramatically increase with MDR TB. Humans can carry the pathogen for decades and when the immune system is compromised the bacilli become virulent. Treatment of a wide variety of cancers, especially lymphoma, is associated with increased risk of TB. Since we are treating patients from other countries, we are bound to have patients that develop TB when their cancer is treated, and they can transmit MDR TB to other patients in the hospital. Our discovery of cytosolic mycobacteria published earlier in Cell (2007) challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. Until recently the biological relevance of mycobacterial translocation to the cytosol remained unclear. In a study published this year we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patientderived mycobacteria strains were found to translocate to the cytosol, while non-pathogenic strains did not. We completed this study linking cytosolic translocation with pathogenicity, by introducing the ESX-1 (type VII) secretion system into the nonvirulent, exclusively phagolysosomal M. bovis BCG. Furthermore, we show that translocation is dependent on the 11-amino acid C-terminus of the early-secreted antigen ESAT-6, a 6kDa secreted ESX-1 protein. Together these data demonstrated that the ability to translocate from the phagolysosome to the cytosol determines mycobacterial virulence. ESX-5 is a homologous type VII secretion system (T7SS) of
pathogenic mycobacteria that plays a role in pathogenesis. We showed that a functional ESX-5 T7SS is required for inducing caspase-independent cell death in macrophages. Host cell death induction was dependent on mycobacterial internalization and translocation to the cytosol, but independent of TNF- and TLR2 signaling. Addition of cathepsin B inhibitors abrogated the induction of host cell death by both M. tuberculosis wild-type and their ESX-5 mutants. Our study established a role for an ESX-5 T7SS in cathepsin B-dependent cell death by pathogenic mycobacteria. The structure of this T7SS is not known. This year we had a breakthrough in the biochemical isolation of this 1.5 mDa macromolecular complex. The first EM picture of the intact complex is revealed above. In the coming years we hope to disclose the biochemical isolation and to resolve its 3D structure at a resolution below 1nm.
Prions The cellular prion protein (PrPC) is a glycoprotein and is abundantly expressed in various tissues. PrPC has been implicated in cell signaling, survival and cancer progression. PrPC expression correlates with more aggressive colorectal carcinomas. It is associated with ER-negative breast cancer and with a lower sensitivity to adjuvant chemotherapy. During prion disease cellular prion protein (PrPC) is refolded into a pathogenic isoform (PrPSc) that accumulates in the central nervous system and causes neurodegeneration and death. This year we used immunofluorescence, quantitative cryoimmunogold EM and tomography to detect nascent, full-length PrPSc in the hippocampus of prion-infected mice (in collaboration with Stanley Prusiner) from early pre-clinical disease stages onwards. Comparison of uninfected and infected brains showed that sites containing full-length PrPSc could be recognized in the neuropil by bright spots and streaks of immunofluorescence, and by clusters of cryo-immunogold EM labeling. PrPSc was found mainly on neuronal plasma membranes, most strikingly on membrane invaginations and sites of cell-to-cell contact, and was evident by 65 days postinoculation, or 54% of the incubation period to terminal disease. Both axons and dendrites in the neuropil were affected. We hypothesize that closely apposed plasma membranes provide a favorable environment for prion conversion and intercellular prion transfer. Only a small proportion of clustered PrP immunogold labeling was found at synapses, indicating that synapses are not targeted specifically in prion disease.
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R Rottenberg S, Vollebergh MA, de Hoon B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Lodén M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res. 2012;72:2350-61
Group leader Sven Rottenberg
Sven Rottenberg DVM PhD Dipl ECVP Group leader Piet Borst MD PhD Adviser Charlotte Guyader PhD Post-doc Nikola Banishki MSc PhD student Ewa Gogola MSc PhD student Janneke Jaspers Msc PhD student Guotai Xu MSc PhD student Serge Zander DVM MSc PhD student Ariena Kersbergen Technical staff Asli Küçükosmano lu Technical staff Wendy Sol Technical staff
Publications
Rottenberg S, Jonkers J. MEK inhibition as a strategy for targeting residual breast cancer cells with low DUSP4 expression. Breast Cancer Res. 2012;14:324 Jaspers JE, Kersbergen A, Boon U, Sol W, van Deemter L, Zander SA, Drost R, Wientjens E, Ji J, Aly A, Doroshow JH, Cranston A, Martin NM, Lau A, O’Connor MJ, Ganesan S, Borst P, Jonkers J, Rottenberg S. Loss of 53BP1 Causes PARP Inhibitor Resistance in Brca1-Mutated Mouse Mammary Tumors. Cancer Discov. 2012 Zander SA, Sol W, Greenberger L, Zhang Y, van Tellingen O, Jonkers J, Borst P, Rottenberg S. EZN-2208 (PEGSN38) overcomes ABCG2-mediated topotecan resistance in BRCA1deficient mouse mammary tumors. PLoS One. 2012;7:e45248 Zander SA, Kersbergen A, Sol W, Gonggrijp M, van de Wetering K, Jonkers J, Borst P, Rottenberg S. Lack of ABCG2 shortens latency of BRCA1-deficient mammary tumors and this is not affected by genistein or resveratrol. Cancer Prev Res (Phila). 2012;5:1053-60
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Warmoes M, Jaspers JE, Pham TV, Piersma SR, Oudgenoeg G, Massink MP, Waisfisz Q, Rottenberg S, Boven E, Jonkers J, Jimenez CR. Proteomics of mouse BRCA1-deficient mammary tumors identifies DNA repair proteins with potential diagnostic and prognostic value in human breast cancer. Mol Cell Proteomics. 2012;11:M111.013334 Rottenberg S, Borst P. Drug resistance in the mouse cancer clinic. Drug Resist Updat. 2012;15:81-9
Drug resistance in “spontaneous” mouse tumors In collaboration with Piet Borst and Jos Jonkers, we are studying drug resistance mechanisms in “spontaneous” mammary tumors arising in genetically engineered mice. In particular, we are using mammary tumors with conditional defects of the Brca1 and p53 genes. In this model we are focusing on (1) the identification of markers that are useful to predict therapy response, (2) mechanisms of acquired drug resistance, and (3) the characterization of drug tolerant tumor cells.
Identification of markers to predict therapy response We searched for gene expression patterns that correlate with docetaxel or cisplatin response in our mouse model for BRCA1deficient breast cancer. Array-based expression profiling did not identify a single marker gene predicting docetaxel response, despite an increase in Abcb1 (P-glycoprotein) expression that was sufficient to explain resistance in several poor responders. Intertumoral heterogeneity explained the inability to identify a predictive gene expression signature for docetaxel. To address this problem, we used the Detection of Imbalanced Differential Signal (DIDS) algorithm designed by Jorma de Ronde (group of Lodewyk Wessels). This approach detects differential gene expression in a subgroup of the poor responders, and we could identify tumors with increased Abcb1 transcript levels. In contrast, standard analytical tools, such as significance analysis of microarrays, detected a marker only if it correlated with response in a substantial fraction of tumors. For example, low expression of the Xist gene correlated with cisplatin hypersensitivity in most tumors, and it also predicted long recurrence-free survival of HER2-negative, stage III breast cancer patients treated with intensive platinum-based chemotherapy. Our findings may prove useful for selecting patients with high-risk breast cancer who could benefit from platinum-based therapy. In collaboration with Connie Jimenez (VU Amsterdam) we also investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes that show a differential cisplatin response. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared to that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma (ILC). Major discriminating markers that were upregulated in the resistant model were predominantly involved in (fatty acid) metabolism, such as fatty acid synthase (FASN). In this context we studied the role of platinum-induced fatty acids (PIFAs) for cisplatin resistance in collaboration with Laura Daenen and Emile Voest (Utrecht Medical Center). PIFAs appear to protect tumor cells against DNA damaging chemotherapeutic agents through a mechanism involving early DNA damage repair. Non-functional homologous recombination due to the BRCA1 defect abrogated such PIFA-induced resistance. Only after restoration of homologous recombination, PIFA-induced cisplatin resistance was observed.
Mechanisms of acquired drug resistance When treated with the maximum tolerable dose (MTD) of doxorubicin, docetaxel, topotecan, or the Poly-ADP-ribose polymerase (PARP) inhibitor olaparib, the BRCA1-deficient mammary tumors initially respond but eventually develop resistance. Resistance is often associated with upregulation of the Abcb1a/b and Abcg2 genes, which encode the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We have crossed disrupted alleles for the Abcb1 or Abcg2 genes into our mouse model to further test the importance of these transporters in drug resistance and to uncover other forms of resistance. After orthotopic grafting of P-gp-deficient tumors into wild-type mice we found that tumor-specific inactivation of P-gp increases the longterm response of BRCA1-deficient mouse mammary tumors to olaparib, but these tumors eventually developed PARPi resistance. In a fraction of cases, this resistance is caused by partial restoration of homologous recombination due to somatic loss of 53BP1. Importantly, we minimized PARPi resistance by long-term treatment with the novel PARP inhibitor AZD2461, which is a poor P-gp substrate. To identify what other mechanisms may explain olaparib resistance, we performed loss-of-function shRNA screens using Brca1-/-;p53-/- cell lines derived from our model. Among other hits, we found that loss of Rev7 (Mad2l2) induced olaparib resistance and restored the formation of ionizing radiation-induced RAD51 foci (figure 1). When we applied the DIDS algorithm on the genome wide expression profiles of the olaparib resistant Abcb1a/b-/-;Brca1-/-;p53-/- mammary tumors, reduced Rev7 gene expression was also a top hit.
Figure 1: Validation of 2 hairpins targeting Rev7/Mad2l2 that were picked up in a shRNA screen for olaparib resistance using the Brca1-/-; p53-/- mouse mammary tumor cell line B11. (A) Relative Rev7 gene expression of cells transduced with the empty vector or 2 Rev7-targeting hairpins. (B) Clonogenic assay of the B11 cells shown in (A) using 500nM olaparib. (C) RAD51 foci formation of the B11 cells 5 h after irradiation (10 Gy).
Drug tolerance Despite their high sensitivity to platinum drugs, we have found that mammary tumors arising in our mouse model for BRCA1deficient breast cancer are usually not eradicated, not even by a frequent dosing schedule. Although relapse-free survival is tumor size-dependent, tumors of only 2-3mm in diameter still contain enough cisplatin-tolerant tumor cells to escape elimination. We found that the resistance of “remnants” is not due to specific biochemical defense mechanisms of putative tumor-initiating cells, but due to the ability of a sub-fraction of the cells to stall in their cell cycle progression until the drug is gone and the DNA damage has been repaired. After treatment of tumors with cisplatin, most tumor cells initially became giant multi-nuclear cells; relapse comes, however, from cells avoiding entry into S phase. Slowly cycling cells are present within the drug-naïve tumor population and are enriched in tumor remnants. High dose platinum therapy was not tolerated in mice, but we found that the nimustine MTD eradicates all tumors. Complete eradication is dose-dependent and lowering the nimustine dose by 50% results in relapse of all tumors (figure 2). In contrast to platinum drugs, nimustine greatly reduces the number of G0/G1 cells, which appears to cause disease relapse in our model.
Figure 2: Time until relapse of BRCA1deficient mouse mammary tumors after treatment with the nimustine MTD, or 75%, 50% and 25% of the MTD.
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S Koppelmans V, de Ruiter MB, van de Lijn F, Boogerd W, Seynaeve C, van der Lugt A. Vrooman H, Niessen WJ, Schagen, SB Breteler MM. Global and Focal White Matter Integrity in Breast Cancer Survivors 20 Years After Adjuvant Chemotherapy. Human Brain Mapping. (in press) M Klein, SB Schagen. Klinische Neuropsychologie. Intracraniale en extracraniale tumoren bij volwassenen. 2012, Uitgeverij Boom
Group leader Sanne Schagen
Sanne Schagen PhD Group leader Michiel De Ruiter PhD Post-doc Riejanne Seigers PhD Post-doc Vincent Koppelmans MSc PhD student Sanne Menning MSc PhD student Myrle Kemperman MSc PhD student Heleen Feenstra MSc PhD student Wendy Jakobs MSc PhD student Jacobien Kieffer MSc Statistical analyst Marianne Kuenen Research assistant Kim Kersten Research assistant Agnetha Fruijtier Research assistant Monica Kobus Research assistent
Key publications
Koppelmans V, Breteler MM, Boogerd W, Seynaeve C, Gundy C, Schagen SB. Neuropsychological performance in survivors of breast cancer more than 20 years after adjuvant chemotherapy. J Clin Oncol. 2012;1;30:1080-6 Koppelmans V, de Ruiter MB, van der Lijn F, Boogerd W, Seynaeve C, van der Lugt A, Vrooman H, Niessen WJ, Breteler MM, Schagen SB. Global and focal brain volume in long-term breast cancer survivors exposed to adjuvant chemotherapy. Breast Cancer Res Treat. 2012;132:1099-106 De Ruiter MB, Reneman L, Boogerd W, Veltman DJ, Caan M, Douaud G, Lavini C, Linn SC, Boven E, van Dam FS, Schagen SB. Late effects of high-dose adjuvant chemotherapy on white and gray matter in breast cancer survivors: Converging results from multimodal magnetic resonance imaging. Hum Brain Mapp. 2012;33:2971-83 Wefel JS, Schagen SB. Chemotherapyrelated cognitive dysfunction. Curr Neurol Neurosci Rep. 2012;12:267-75 Bulwalda B, Schagen SB. Is basic research providing answers if adjuvant anti-estrogen treatment of breast cancer can induce cognitive impairment? Life Sciences, (in press) 74
Ahles TA, Schagen SB, Vardy J. Clinical Psycho-oncology: An International Perspective. Neurocognitive effects of Anticancer treatments. 2012, WileyBlackwell
Cognitive function in cancer patients
RT patients and 18 HC showed a reduction in N-acetyl aspartate in parietal white matter in the CTC-FEC patients, which suggests axonal injury.
Cognitive and brain MRI study in non-small cell lung cancer patients after prophylactic cranial irradiation
Many cancer patients report cognitive changes during their disease course and following treatment that interfere with their daily life activities. The projects constituting this research line center around the investigation of the incidence, nature and cause of cognitive problems associated with cancer and its treatments, and at the development of strategies to diminish these symptoms.
White matter integrity in breast cancer survivors 20 years after adjuvant chemotherapy In a collaborative study with the Erasmus MC the long-term effect of chemotherapy on white matter microstructural integrity was investigated. Diffusion tensor imaging was performed in 187 CMF chemotherapy exposed breast cancer survivors, mean age 64 years, who had been diagnosed with cancer on average 21 years before, and in 374 age-matched cancer-free reference subjects from a population- based cohort study. No significant group differences were observed in white matter integrity. Within the breast cancer survivors, time since treatment was inversely associated with lower global and focal white matter integrity.
Prospective multimodality MRI study into cognitive dysfunction in breast cancer patients In a collaborative study with the Academic Medical Center we prospectively investigate structural and functional properties of the brain with MRI in relation to cognition. Currently, 37 patients who will receive chemotherapy, 39 patients who will not undergo chemotherapy and 44 healthy controls completed baseline assessments. Baseline performance data obtained in the scanner (memory and executive function) showed no significant differences between the groups. No significant differences in brain activation during these tasks were found. To date, 22 patients who received chemotherapy, 34 patients who did not receive chemotherapy, and 19 healthy controls completed follow-up assessments.
Late effects of different dosages of chemotherapy in breast cancer survivors on white matter integrity and brain function In a collaborative study with the Academic Medical Center we investigate the late effects of chemotherapy on cognition, brain structure and brain function in breast cancer (BC) survivors. Data of patients who received standard-dose chemotherapy (FEC) and healthy controls (HC) were collected and added to previously obtained and reported data in BC treated with highdose adjuvant CT (FEC-CTC) and BC patients who only received radiotherapy (RT) following surgery. For each participant an overall cognitive impairment score (Mahalanobis distance) was calculated. On average 10 years after completion of chemotherapy, CTC-FEC and FEC patients had a significantly higher MD than the HC. Analysis of 17 CTC-FEC, 19 FEC and 14
In collaboration with the Radiotherapy Department of the Antoni van Leeuwenhoek and the University Medical Center Maastricht/ MAASTRO Clinic we prospectively investigate neurotoxic side effects of prophylactic cranial irradiation (PCI) in non-small cell lung cancer patients (nSCLC). Patients are evaluated with cognitive tests and MRI in the context of a phase III randomized trial on the efficacy of PCI in decreasing brain metastasis, and the impact of PCI on neurological symptoms and health-related quality of life in nSCLC. Patients undergo two MRI scans (baseline assessment before PCI/no PCI and one year after PCI/no PCI) and six cognitive examinations (at baseline and three months to two years after PCI/no PCI). Currently, twenty patients have been enrolled in the study.
stereotype threat and priming. In a collaborative study with the Radboud University the influence of informing patients about the association between cognitive problems and chemotherapy will be studied in breast cancer patients receiving standard dose (neo)adjuvant chemotherapy before, 6 months after and 12 months after chemotherapy. Conditions under which ‘stereotype priming’-effects occur, mediating processes and the severity and duration of these effects will be investigated, as well as interventions to diminish these effects.
Molecular and cellular mechanisms of cognitive impairment following chemotherapy Together with the VU University the potential cognitive impact of six chemotherapeutic agents was explored. Approximately 300 mice were treated with saline, cyclophosphamide, docetaxel, doxorubicin, 5-fluorouracil, methotrexate or topotecan, Cognitive examination started two and 15 weeks after treatment with the novel location recognition test (NLR), the novel object recognition test (NOR), Barnes maze (BM), and a simple choice reaction time task (SCRTT). Cyclophosphamide induced cognitive impairment on NLR, NOR, and SCRTT. Docetaxel affected general behavior and cognition on NLR and SCRTT. Doxorubicin influenced general behavior and behavior on NLR, NOR, and BM. 5-fluorouracil induced cognitive deficits on NLR, NOR, and BM. Methotrexate influenced general behavior and NLR. Topotecan influenced general behavior and cognition on NLR, NOR, BM, and SCRTT. The results show that several cytotoxic agents causes impairment in various cognitive domains in mice. The neurobiological effects will at present be explored using immunohistochemistry and by analyzing the proteome of the synapse.
An online testing approach to assess cognitive problems Neuropsychological tests are the gold standard criterion for measuring cognitive function. However, traditional face-to-face assessments can be time-consuming and labor intensive, which restrains the conduction of large studies. In order to advance the research in the field of cancer and cognition it is important to obtain data in large samples. Online testing provides the opportunity to do so in a relatively quick and cost efficient way. Together with the University of Amsterdam and the VU University an online neuropsychological test battery is developed, and we are currently performing usability studies. Next steps include a validation study and the collection of normative data.
Downsides of Being Well-Informed Informing patients about the association between chemotherapy and cognitive problems may contribute to the occurrence of cognitive problems. An explanation for this mere information effect is derived from social psychological research on 75
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S Devriese LA, Witteveen PO, Marchetti S, Mergui-Roelvink M, Reyderman L, Wanders J, Jenner A, Edwards G, Beijnen JH, Voest EE, Schellens JH. Pharmacokinetics of eribulin mesylate in patients with solid tumors and hepatic impairment. Cancer Chemother Pharmacol. 2012;70: 823-32
Group leader Jan Schellens
Jan Schellens MD PhD Group leader Jos Beijnen PhD Academic staff Serena Marchetti MD PhD Academic staff Neeltje Steeghs MD PhD Academic staff Suzanne Leijen MD PhD student Ruud Van der Noll MSc PhD student Geert Frederix MSc PhD student Bart Jacobs MSc PhD student Robin Van Geel MSc PhD student Didier Meulendijks MSc PhD student Bojana Milojkovic MD PhD student Jeroen Hendrikx MSc PhD student Rik Stuurman MSc PhD student Dick Pluim Technical staff Artur Burylo Technical staff
Publications
Pluim D, Jacobs BA, Beijnen JH, Schellens JHM. ABC-01765-2012. R1 - Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination. Analytical and Bioanalytical Chemistry 2012 (in press) Van Hasselt JGC, van Eijkelenburg NKA, Beijnen JH, Schellens JHM, Huitema ADR. Optimizing drug development of anti-cancer drugs in children using modelling and simulation. British Journal of Clinical Pharmacology. 2012 (in press) Helgason HH; Kruijtzer CMF; Koolen SLW; van Werkhoven E; Malingre MM; Huitema ADR; Schot M; Smit W; Beijnen JH; Schellens JHM. Phase II and pharmacological study of oral docetaxel plus cyclosporin A in anthracycline pre-treated metastatic breast cancer. Current Clinical pharmacology. 2012 (in press) Goey AKL, Mooiman KD, Beijnen JH, Schellens JHM, Meijerman I. Relevance of in vitro and clinical data for predicting CYP3A4-mediated herb-drug interactions in cancer patients. Cancer Treatment Reviews. 2012 (in press) 76
Nijenhuis CM, Haanen JB, Schellens JH, Beijnen JH. Is combination therapy the next step to overcome resistance and reduce toxicities in melanoma? Cancer Treat Rev. 2012 (in press) Van Hasselt JG, Schellens JH, Mac Gillavry MR, Beijnen JH, Huitema AD. Model-based evaluation and optimization of cardiac monitoring protocols for adjuvant treatment of breast cancer with trastuzumab. Pharm Res. 2012;29:3499-511 Lankheet N, Schaake E, Rosing H, Burgers J, Schellens J, Beijnen J, Huitema A. Quantitative determination of erlotinib and O-desmethyl erlotinib in human EDTA plasma and lung tumor tissue. Bioanalysis. 2012;4:2563-77 Dubbelman AC, Upthagrove A, Beijnen JH, Marchetti S, Tan E, Krone K, Anand S, Schellens JH. Disposition and metabolism of (14)C-dovitinib (TKI258), an inhibitor of FGFR and VEGFR, after oral administration in patients with advanced solid tumors. Cancer Chemother Pharmacol. 2012;70:653-63 Hendrikx JJ, Lagas JS, Rosing H, Schellens JH, Beijnen JH, Schinkel AH. P-glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel. Int J Cancer. 2012 (in press) Moes J, Koolen S, Huitema A, Schellens J, Beijnen J, Nuijen B. Development of an oral solid dispersion formulation for use in low-dose metronomic chemotherapy of paclitaxel. Eur J Pharm Biopharm. 2012 (in press) Vermaat JS, Gerritse FL, van der Veldt AA, Roessingh WM, Niers TM, Oosting SF, Sleijfer S, Roodhart JM, Beijnen JH, Schellens JH, Gietema JA, Boven E, Richel DJ, Haanen JB, Voest EE. Validation of serum amyloid α as an independent biomarker for progression-free and overall survival in metastatic renal cell cancer patients. Eur Urol. 2012;62:685-95
Pharmacodynamics of anti-cancer drugs
Diagnostic and therapeutic studies of blood brain barrier (BBB) 1. A glutathione (GSH) pegylated liposomal doxorubicin hydrochloride formulation (2B3-101), expected to penetrate the BBB by the process of endocytosis, is recruiting patients with solid tumors and brain metastases or malignant glioma currently in cohort 7 (60 mg/m2). 2. A diagnostic study in patients with leptomeningeal meta stases aims to determine the sensitivity and specificity of circulating tumor cells and standard cytology diagnostics in cerebrospinal fluid. Till now two patients have been recruited.
Detection and enumeration of mature circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) Biomarkers of angiogenesis are being developed as a clinical tool. Endothelial cells and their progenitors defined as: CD45-/ CD34+/CD146+/CD133-(CEC) and CD45-/CD34+/CD146+/ CD133+(EPC) are currently being studied for this purpose. However, their detection and enumeration is problematic because these markers are shared by other cells in human peripheral blood. Magnetic CD34 and CD146-conjugated beads are used to isolate the CEC and EPC fraction from human peripheral blood, followed by staining with anti-CD45 to distinguish from hematopoietic blood cells, endothelial markers CD34, CD146 and progenitor marker CD133. The numbers of CECs and EPCs in human blood are low; hence, the type of tube for blood withdrawal might be of influence. Different tubes and methods of sample processing are being tested for the highest yield of cells. CECs and EPCs are being detected and enumerated by flow cytometry.
Impact of structural uncertainty on costeffectiveness outcomes: Towards a standardized cost-effectiveness model for adjuvant breast cancer therapies In a review of published cost-effectiveness analyses (CEAs) of tamoxifen and anastrazole, we identified several structural differences in model components and differences in parameterization. Implementing these differences individually and combined resulted in substantial differences in estimated patient life expectancy. We therefore recommend standardized model structures and parameterization for early breast cancer CEAs.
Determination of dihydropyrimidine dehydrogenase and thymidylate synthase activity in human peripheral blood mononuclear cells in healthy volunteers The circadian rhythm of the fluoropyrimidine catabolizing enzyme dihydropyrimidine dehydrogenase (DPD), and of its drug target thymidylate synthase (TS) were studied in peripheral blood mononuclear cells (PBMCs) from 12 healthy volunteers
to explore opportunities for chronotherapy. Mean trough (1 pm) and peak (5 am) DPD activity were 8.09 and 12.88 nmol/ mg/h, suggesting highly increased fluoropyrimidine clearance during the night (figure 1). Activity of TS also showed circadian variation with average trough (1 am) and peak (5 pm) activities of 0.068 and 0.110 nmol/mg/h respectively, indicating circadian sensitivity to fluoropyrimidine therapy. These observational results support the rationale for efficacy and tolerability studies of chronomodulated fluoropyrimidine therapy.
BRAF inhibitor plus anti-EGFR based combination therapies in patients with BRAF mutant metastatic colorectal cancer Pre-clinical studies have demonstrated very promising results with BRAF inhibitor plus anti-EGFR agent based regimens in BRAF mutated colorectal cancer (CRC) cells and xenograft models (Prahallad, A. et al. Nature 483, 100–3 (2012). On the basis of this concept, we developed a phase I/II clinical trial to determine the safety, tolerability and efficacy of LGX818 (BRAF inhibitor) in combination with cetuximab, or in combination with both cetuximab and the PI3K-inhibitor BYL719 in patients with BRAF mutant CRC. This study has started recently with the first patient on study worldwide being treated in the Netherlands Cancer Institute – Antoni van Leeuwenhoek hospital.
Docetaxel intestinal toxicity is related to systemic exposure of docetaxel In preclinical experiments with knock-out mouse models, we showed that intestinal toxicity is similar after oral and intraperitoneal administration of docetaxel. Intestinal toxicity is characterized as depletion and mitotic arrest of the deep crypts in the intestinal tract combined with infiltrating inflammation. The intestinal toxicity is dose related and the absolute docetaxel load in the lumen does not influence intestinal toxicity.
Phase II study of docetaxel, oxaliplatin, capecitabine, bevacizumab, and in case of HER2-positivity trastuzumab for advanced gastric cancer This is a phase II study in advanced inoperable or metastatic disease of the stomach or gastro-esophageal junction. Patients are treated based on tumor HER2 expression. HER2 analysis is performed at a central lab prior to initiation of treatment. After enrolling the first five patients at the NKI-AVL in 2011, we have now started up the study in nine other centers in the Netherlands.
Average dihydropyrimidine dehydrogenase (DPD) enzyme activity curve during a 24 hour cycle (n=12 volunteers). DPD trough (1 pm) and peak (5 am) activities were 8.09 and 12.88 nmol/h/mg (*** P < 0.001 for repeated measures ANOVA and Bonferroni post hoc tests). Error bars represent 95% CI.
Phase II, Pharmacological Study with Wee1 Inhibitor MK-1775 in Combination with Carboplatin in Patients with p53 Mutated Epithelial Ovarian Cancer that show Relapse or Progression after Standard First Line Treatment with Carboplatin – Paclitaxel Combination Therapy Epithelial ovarian cancers often harbor p53 abnormalities. First line therapy of ovarian cancer consists of carboplatin or platinum containing therapy. Re-exposing patients with early relapse (during or within 3 month after 1st line therapy) to carboplatin in combination with Wee1 inhibitor MK-1775 is an ultimate proof of principal study. Ca-125 marker responses and responses according to (Response Evaluation Criteria In Solid Tumors) RECIST have already been observed in a significant number of patients.
Improved high throughput pharmacodynamic assay for the determination of poly(ADP-ribose) polymerase activity after ex vivo radiation of peripheral blood mononuclear cells (PBMC) at low temperature Poly(ADP-ribose) polymerase is an enzyme that forms PAR at the site of single strand DNA damage for the recruitment of repair proteins. PARP inhibitors like olaparib are a new and promising class of anti cancer agents. PARP activity in PBMC is too low for accurate and precise quantification of the effect of PARP inhibitors. Therefore, we induced PARP activity 50-fold by ex vivo gamma radiation at 8 Gy followed by 1 h of incubation at 0°C. The method was validated and implemented into a Phase I olaparib dose escalating trial, combining olaparib and irradiation.
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S Kerklaan BM, Diéras V, Le Tourneau C, Mergui-Roelvink M, Huitema AD, Rosing H, Beijnen JH, Marreaud S, Govaerts AS, Piccart-Gebhart MJ, Schellens JH, Awada A. Phase I study of lonafarnib (SCH66336) in combination with trastuzumab plus paclitaxel in Her2/ neu overexpressing breast cancer: EORTC study 16023. Cancer Chemother Pharmacol. 2012 (in press) Ploquin A, Olmos D, Lacombe D, A'Hern R, Duhamel A, Twelves C, Marsoni S, Morales-Barrera R, Soria JC, Verweij J, Voest EE, Schöffski P, Schellens JH, Kramar A, Kristeleit RS, Arkenau HT, Kaye SB, Penel N. Prediction of early death among patients enrolled in phase I trials: development and validation of a new model based on platelet count and albumin. Br J Cancer. 2012;107:1025-30
S Joerger M, Kraff S, Huitema AD, Feiss G, Moritz B, Schellens JH, Beijnen JH, Jaehde U. Evaluation of a pharmacology-driven dosing algorithm of 3-weekly paclitaxel using therapeutic drug monitoring: a pharmacokineticpharmacodynamic simulation study. Clin Pharmacokinet. 2012;51:607-17
Boss DS, Glen H, Beijnen JH, Keesen M, Morrison R, Tait B, Copalu W, Mazur A, Wanders J, O'Brien JP, Schellens JH, Evans TR. A phase I study of E7080, a multitargeted tyrosine kinase inhibitor, in patients with advanced solid tumours. Br J Cancer. 2012;106:1598604
Keizer RJ, Gupta A, Shumaker R, Beijnen JH, Schellens JH, Huitema AD. Model-based treatment optimization of a novel VEGFR inhibitor. Br J Clin Pharmacol. 2012;74:315-26
Devriese LA, Mergui-Roelvink M, Wanders J, Jenner A, Edwards G, Reyderman L, Copalu W, Peng F, Marchetti S, Beijnen JH, Schellens JH. Eribulin mesylate pharmacokinetics in patients with solid tumors receiving repeated oral ketoconazole. Invest New Drugs. 2012 (in press)
Pluim D, Devriese LA, Beijnen JH, Schellens JH. Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular-signalregulated kinase and DNA in circulating tumor cells. Cytometry A. 2012;81:664-71
Lankheet NA, Hillebrand MJ, Rosing H, Schellens JH, Beijnen JH, Huitema AD. Method development and validation for the quantification of dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma by liquid chromatography coupled with tandem mass spectrometry. Biomed Chromatogr. 2012 (in press)
Deenen MJ, Klümpen HJ, Richel DJ, Sparidans RW, Weterman MJ, Beijnen JH, Schellens JH, Wilmink JW. Phase I and pharmacokinetic study of capecitabine and the oral mTOR inhibitor everolimus in patients with advanced solid malignancies. Invest New Drugs. 2012;30:1557-65
Sparidans RW, Tang SC, Nguyen LN, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;905:150-4
Keizer RJ, Zandvliet AS, Beijnen JH, Schellens JH, Huitema AD. Two-stage model-based design of cancer phase I dose escalation trials: evaluation using the phase I program of barasertib (AZD1152). Invest New Drugs. 2012;30:1519-30
Sparidans RW, Ahmed TT, Muilwijk EW, Welzen ME, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for therapeutic drug monitoring of the tyrosine kinase inhibitor pazopanib in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;905:137-40
Devriese LA, Witteveen PO, Wanders J, Law K, Edwards G, Reyderman L, Copalu W, Peng F, Marchetti S, Beijnen JH, Huitema AD, Voest EE, Schellens JH. Pharmacokinetics of eribulin mesylate in patients with solid tumors receiving repeated oral rifampicin. Br J Clin Pharmacol. 2012 (in press)
Keizer RJ, Zandvliet AS, Beijnen JH, Schellens JH, Huitema AD. Performance of methods for handling missing categorical covariate data in population pharmacokinetic analyses. AAPS J. 2012;14:601-11
Dubbelman AC, Jansen RS, Rosing H, Darwish M, Hellriegel E, Robertson P Jr, Schellens JH, Beijnen JH. Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine. Drug Metab Dispos. 2012;40:1297-307
Leijen S, Middleton MR, Tresca P, Kraeber-Bodéré F, Dieras V, Scheulen ME, Gupta A, Lopez-Valverde V, Xu ZX, Rueger R, Tessier JJ, Shochat E, Blotner S, Naegelen VM, Schellens JH, Eberhardt WE. Phase I dose-escalation study of the safety, pharmacokinetics, and pharmacodynamics of the MEK inhibitor RO4987655 (CH4987655) in patients with advanced solid tumors. Clin Cancer Res. 2012;18:4794-805
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Jager NG, Rosing H, Linn SC, Schellens JH, Beijnen JH. Importance of highly selective LC-MS/MS analysis for the accurate quantification of tamoxifen and its metabolites: focus on endoxifen and 4-hydroxytamoxifen. Breast Cancer Res Treat. 2012;133:793-8 Jansen RS, Rosing H, Wijermans PW, Keizer RJ, Schellens JH, Beijnen JH. Decitabine triphosphate levels in peripheral blood mononuclear cells from patients receiving prolonged lowdose decitabine administration: a pilot study. Cancer Chemother Pharmacol. 2012;69:1457-66
Joerger M, Burgers SA, Baas P, Smit EF, Haitjema TJ, Bard MP, Doodeman VD, Smits PH, Vincent A, Huitema AD, Beijnen JH, Schellens JH. Germline polymorphisms in patients with advanced nonsmall cell lung cancer receiving first-line platinum-gemcitabine chemotherapy: a prospective clinical study. Cancer. 2012;118:2466-75 Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the VEGFR inhibitor cediranib and its primary human metabolite cediranibN+-glucuronide in plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;895-896:169-73 Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;895-896:174-7 Dubbelman AC, Tibben M, Rosing H, Gebretensae A, Nan L, Gorman SH, Robertson P Jr, Schellens JH, Beijnen JH. Development and validation of LCMS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2012; 893-894:92-100
De Vries NA, Buckle T, Zhao J, Beijnen JH, Schellens JH, van Tellingen O. Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug transporters P-gp and BCRP. Invest New Drugs. 2012;30:443-9 Van Hasselt JG, van den Heuvel MM, Schellens JH, Beijnen JH, Brandsma D, Huitema AD. Severe cannabinoid intoxication in a patient with nonsmall-cell lung cancer. J Palliat Care. 2012;28:60-1
Olmos D, A'hern RP, Marsoni S, Morales R, Gomez-Roca C, Verweij J, Voest EE, Schöffski P, Ang JE, Penel N, Schellens JH, Del Conte G, Brunetto AT, Evans TR, Wilson R, Gallerani E, Plummer R, Tabernero J, Soria JC, Kaye SB. Patient selection for oncology phase I trials: a multi-institutional study of prognostic factors. J Clin Oncol. 2012;30:996-1004 Sparidans RW, Durmus S, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;889-890:144-7 Dubbelman AC, Rosing H, Thijssen B, Gebretensae A, Lucas L, Chen H, Shumaker R, Schellens JH, Beijnen JH. Development and validation of LC-MS/ MS assays for the quantification of E7080 and metabolites in various human biological matrices. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;887-888:25-34 Koolen SL, van Waterschoot RA, van Tellingen O, Schinkel AH, Beijnen JH, Schellens JH, Huitema AD. From mouse to man: predictions of human pharmacokinetics of orally administered docetaxel from preclinical studies. J Clin Pharmacol. 2012;52:37080 Joerger M, Ferreri AJ, Krähenbühl S, Schellens JH, Cerny T, Zucca E, Huitema AD. Dosing algorithm to target a predefined AUC in patients with primary central nervous system lymphoma receiving high dose methotrexate. Br J Clin Pharmacol. 2012;73:240-7
Bergamo A, Gaiddon C, Schellens JH, Beijnen JH, Sava G. Approaching tumour therapy beyond platinum drugs: status of the art and perspectives of ruthenium drug candidates. J Inorg Biochem. 2012;106:90-9 Deenen MJ, Cats A, Mandigers CM, Soesan M, Terpstra WE, Beijnen JH, Schellens JH. Prevention of severe toxicity from capecitabine, 5-fluorouracil and tegafur by screening for DPD-deficiency. Ned Tijdschr Geneeskd. 2012;156:A4934 Manson ML, Derissen EJ, Wijermans PW, Schellens JH, Beijnen JH. Demethylating medication in myelodysplastic syndrome. Ned Tijdschr Geneeskd. 2012;156:A3167 Van Geel RM, Beijnen JH, Schellens JH. Concise drug review: pazopanib and axitinib. Oncologist. 2012;17:1081-9
Kontny NE, Würthwein G, Boos J, Boddy AV, Krischke M, Fuhr U, Thompson PA, Jörger M. Schellens JHM, Hempel G. Population pharmacokinetics of doxorubicin: establishment of a NONMEM model for adults and children older than three years. Cancer Chemotherapy and Pharmacology 2012 (in press) Meulenaar J, Beijnen JH, Schellens JH, Nuijen B. Slow dissolution behaviour of amorphous capecitabine. Int J Pharm 2012 (in press) Deenen MN, Rosing H, Hillebrand MJ, Schellens JHM, Beijnen JH. Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. Journal of Chromatography B 2012 (in press)
Mooiman KD, Goey AK, Meijerman I, Beijnen JH, Schellens JH. Letter to the editor regarding "A prospective, controlled study of the botanical compound mixture LCS101 for chemotherapy-induced hematological complications in breast cancer" by Yaal-Hahoshen et al. (The Oncologist 2011;16:1197-1202). Oncologist. 2012;17:740-1; author reply 742-3 Opdam FL, Guchelaar HJ, Beijnen JH, Schellens JH. Lapatinib for advanced or metastatic breast cancer. Oncologist. 2012;17:536-42 Van Leeuwen WA, Schellens JH, Becker HE, de Haan L, van Westrhenen R. The subclinical course of a paracetamol intoxication: pitfall for patient and clinician. Tijdschr Psychiatr. 2012;54:555-9
Dubbelman AC, Rosing H, Jansen RS, Mergui-Roelvink M, Huitema AD, Koetz B, Lymboura M, Reyderman L, Lopez-Anaya A, Schellens JH, Beijnen JH. Mass balance study of [¹4C]eribulin in patients with advanced solid tumors. Drug Metab Dispos. 2012;40:313-21
Frederix GW, Severens JL, Hövels AM, Raaijmakers JA, Schellens JH. Reviewing the cost-effectiveness of endocrine early breast cancer therapies: influence of differences in modeling methods on outcomes. Value Health. 2012;15:94-105
Devriese LA, Bosma AJ, van de Heuvel MM, Heemsbergen W, Voest EE, Schellens JH. Circulating tumor cell detection in advanced non-small cell lung cancer patients by multimarker QPCR analysis. Lung Cancer. 2012;75:242-7
Harmsen S, Meijerman I, MaasBakker RFM, Schellens JHM; Beijnen JH. PXR-mediated P-glycoprotein induction by small molecule tyrosine kinase inhibitors. European Journal of Pharmaceutical Sciences 2012 (in press)
Joerger M, Burgers JA, Baas P, Doodeman VD, Smits PH, Jansen RS, Vainchtein LD, Rosing H, Huitema AD, Beijnen JH, Schellens JH. Gene polymorphisms, pharmacokinetics, and hematological toxicity in advanced non-small-cell lung cancer patients receiving cisplatin/gemcitabine. Cancer Chemother Pharmacol. 2012;69:25-33
Goey AKL, Mooiman KD, Meijerman I, Schellens JHM, Beijnen JH. Relevance of in vitro and clinical data for predicting CYP3A4-mediated herb-drug interactions in cancer patients. Cancer Treatment Reviews 2012 (in press)
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S Tang SC, Lankheet NA, Poller B, Wagenaar E, Beijnen JH, Schinkel AH. P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict brain accumulation of the active sunitinib metabolite N-desethyl sunitinib. J Pharmacol Exp Ther. 2012;341:164-73
Group leader Alfred Schinkel
Alfred Schinkel PhD Group leader Dilek Iusuf MSc PhD student Seng Chuan Tang MSc PhD student Selvi Durmus MSc PhD student Jeroen Hendrikx MSc PhD student Ning Xu MSc PhD student Anita Van Esch Technical staff Els Wagenaar Technical staff
Publications
Koolen SL, van Waterschoot RA, van Tellingen O, Schinkel AH, Beijnen JH, Schellens JH, Huitema AD. From mouse to man: predictions of human pharmacokinetics of orally administered docetaxel from preclinical studies. J Clin Pharmacol. 2012;52:370-80 Tang SC, Lagas JS, Lankheet NA, Poller B, Hillebrand MJ, Rosing H, Beijnen JH, Schinkel AH. Brain accumulation of sunitinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by oral elacridar and sunitinib coadministration. Int J Cancer. 2012;130:223-33 Iusuf D, van de Steeg E, Schinkel AH. Functions of OATP1A and 1B transporters in vivo: insights from mouse models. Trends Pharmacol Sci. 2012;33:100-8 Van de Steeg E, Stránecký V, Hartmannová H, Nosková L, H ebí ek M, Wagenaar E, van Esch A, de Waart DR, Oude Elferink RP, Kenworthy KE, Sticová E, al-Edreesi M, Knisely AS, Kmoch S, Jirsa M, Schinkel AH. Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J Clin Invest. 2012;122:519-28
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Sparidans RW, Durmus S, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;889-890:144-7 Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;895-896:174-7 Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the VEGFR inhibitor cediranib and its primary human metabolite cediranib-N+glucuronide in plasma. J Chromatogr B Analyt Technol Biomed Life Sci.2012;895-896:169-73 Zhu P, Hata R, Ogasawara M, Cao F, Kameda K, Yamauchi K, Schinkel AH, Maeyama K, Sakanaka M. Targeted disruption of organic cation transporter 3 (Oct3) ameliorates ischemic brain damage through modulating histamine and regulatory T cells. J Cereb Blood Flow Metab. 2012;32:1897-908 Lagas JS, Damen CW, van Waterschoot RA, Iusuf D, Beijnen JH, Schinkel AH. P-gp, Mrp2, Cyp3a, and carboxylesterase affect the oral availability and metabolism of vinorelbine. Mol Pharmacol. 2012;82:636-44 Iusuf D, Sparidans RW, van Esch A, Hobbs M, Kenworthy K, van de Steeg E, Wagenaar E, Beijnen JH, Schinkel AH. Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice. Mol Pharm. 2012;9:2497-504 André P, Saubaméa B, CochoisGuégan V, Marie-Claire C, Cattelotte J, Smirnova M, Schinkel AH, Scherrmann JM, Cisternino S. Transport of biogenic amine neurotransmitters at the mouse blood-retina and blood-brain barriers by uptake1 and uptake2. J Cereb Blood Flow Metab. 2012;32:1989-2001
Genes and proteins involved in anticancer drug resistance and pharmacokinetics Our research focuses on genes and proteins that cause drug resistance or drug susceptibility 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 and more generally pharmacotherapy approaches for cancer and other diseases; ii) increase insights into 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. Below we describe a few recent studies that illustrate our approach.
Vemurafenib oral availability and brain accumulation is restricted by Abcb1 and Abcg2 Vemurafenib is a novel tyrosine kinase inhibitor that has clinical efficacy against metastatic melanoma. We aimed to establish whether oral availability and brain penetration of vemurafenib could be restricted by the multidrug efflux transporters P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), as these might limit therapeutic efficacy. In vitro, vemurafenib was efficiently transported by both human ABCB1 and ABCG2, and very efficiently by mouse Abcg2. Upon oral administration of vemurafenib Abcb1a/1b-/- mice had a 1.6-fold increased, Abcg2-/- mice a 2.3-fold increased, and Abcb1a/1b-/-;Abcg2-/- mice a 6.6-fold increased plasma AUC, respectively, compared to wild-type mice, indicating a marked and additive role of these transporters in limiting vemurafenib oral availability. Brain-to-plasma ratios of vemurafenib were not increased in Abcg2-/- mice, only 1.7-fold in Abcb1a/1b-/- mice, but 21.4-fold in Abcb1a/1b-/-;Abcg2-/- mice, indicating pronounced overlapping functions of these transporters in reducing vemurafenib brain accumulation. Oral coadministration of the dual ABCB1 and ABCG2 inhibitor elacridar almost completely eliminated the roles of Abcb1 and Abcg2 in restricting oral availability and brain accumulation of vemurafenib. Our data suggest that elacridar coadministration may be considered to improve the therapeutic efficacy of vemurafenib, especially for brain metastases located behind a functional blood-brain barrier.
Abcb1, Abcc2, Cyp3a, and carboxylesterase affect the oral availability and metabolism of vinorelbine We investigated the interactions of the anticancer drug vinorelbine with drug efflux transporters and cytochrome P450 3A drug-metabolizing enzymes. Vinorelbine was transported by human multidrug-resistance associated protein (ABCC) 2, and Abcc2 knockout mice displayed increased vinorelbine plasma exposure after oral administration, suggesting that Abcc2 limits the intestinal uptake of vinorelbine. Using Abcb1, Cyp3a-, and Abcb1/Cyp3a knockout mice, we found that the absence
of Abcb1 or Cyp3a resulted in increased vinorelbine plasma exposure, both after oral and intravenous administration. Surprisingly, Abcb1/Cyp3a knockout mice displayed markedly lower vinorelbine plasma concentrations than wild-type mice upon intravenous administration but higher concentrations upon oral administration. This could be explained by highly increased formation of 4’-O-deacetylvinorelbine, an active vinorelbine metabolite, especially in Abcb1/Cyp3a knockout plasma. Using wild-type and Cyp3a knockout liver microsomes, we found that 4’-O-deacetylvinorelbine formation was 4-fold increased in Cyp3a knockout liver and was not mediated by Cyp3a or other cytochrome P450 enzymes. In vitro incubation of vinorelbine with plasma revealed that vinorelbine deacetylation in Cyp3a and especially in Abcb1/Cyp3a knockout mice was strongly upregulated. Metabolite formation in microsomes and plasma could be completely inhibited with the nonspecific carboxylesterase (CES) inhibitor bis(4-nitrophenyl) phosphate and partly with the CES2-specific inhibitor loperamide, indicating that carboxylesterase Ces2a, which was appropriately up-regulated in Cyp3a and Abcb1/Cyp3a knockout liver was responsible for the 4’O-deacetylvinorelbine formation. Abcb1, Abcc2, Cyp3a, and Ces2a thus clearly restricted vinorelbine availability in mice. Variation in activity of their human homologs may also affect vinorelbine pharmacokinetics in patients.
Sparidans RW, Tang SC, Nguyen LN, Schinkel AH, Schellens JH, Beijnen JH. (2012) Liquid chromatography-tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma. J. Chromatogr B Analyt Technol Biomed Life Sci. 2012;905:150-4 Iusuf D, van de Steeg E, Schinkel AH. Hepatocyte hopping of OATP1B substrates contributes to efficient hepatic detoxification. Clin Pharmacol Ther. 2012;92:559-62 Durmus S, Sparidans RW, Wagenaar E, Beijnen JH, Schinkel AH. Oral availability and brain penetration of the B-RAFV600E inhibitor vemurafenib can be enhanced by the P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) inhibitor elacridar. Mol Pharm. 2012;9:3236-45
Putative structure of a prototypic ABC drug transporter.
Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. We analyzed 8 Rotorsyndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotortype hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.
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S Kirchhoff T, Gaudet MM, Antoniou AC et al. Breast Cancer Risk and 6q22.33: Combined Results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2. PLoS One. 2012;7:e35706
Group leader Marjanka Schmidt
Marjanka Schmidt PhD Group leader Annegien Broeks PhD Academic staff Susanne Rebers PhD Post-doc Eric Vermeulen PhD Post-doc Heleen Bronsveld MSc PhD student Caroline Drukker MD PhD student Sandra Van den Broek MSc PhD student Alexander Brandenburg MSc Research assistant Sten Cornelissen Technical staff Renske Keeman MSc Data manager
Rebers S, van der Valk T, Meijer GA, van Leeuwen FE and Schmidt MK. De toekomst van zeggenschap bij nader gebruik van lichaamsmateriaal: in het belang van de patiënt? [Consent for the secondary use of human residual tissue: the patient is best served by an ‘opting-out’ procedure]. Ned Tijdschr Geneeskd. 2012;156:A4485 Fasching PA, Pharoah PD, Cox A et al, and Schmidt MK. The role of genetic breast cancer susceptibility variants as prognostic factors. Hum Mol Genet. 2012;21:3926-39 Lambrechts D, Truong T, Justenhoven C et al. 11q13 is a Susceptibility Locus for Hormone Receptor Positive Breast Cancer. Hum Mutat. 2012;33:1123-32 Stevens KN, Fredericksen Z, Vachon CM et al. 19p13.1 is a triple negativespecific breast cancer susceptibility locus. Cancer Res. 2012;72:1795-803
Publications
Weischer M, Nordestgaard BG, Pharoah P et al, Schmidt MK and Bojesen S. CHEK2*1100delC Heterozygosity in Women With Breast Cancer Associated With Early Death, Breast Cancer-Specific Death, and Increased Risk of a Second Breast Cancer. J Clin Oncol. 2012;30:4308-16 Jamshidi M, Schmidt MK, Dörk T et al. Germline variation in TP53 regulatory network genes associates with breast cancer survival and treatment outcome. Int J Cancer. 2012 Hein R, Maranian M, Hopper JL, Kapuscinski MK et al. Comparison of 6q25 Breast Cancer Hits from Asian and European Genome Wide Association Studies in the Breast Cancer Association Consortium (BCAC). PLoS One. 2012;7:e42380 Warren H, Dudbridge F, Fletcher O et al. 9q31.2-rs865686 as a susceptibility locus for estrogen receptor-positive breast cancer: evidence from the Breast Cancer Association Consortium. Cancer Epidemiol Biomarkers Prev. 2012;21:1783-91 Wishart GC, Bajdik CD, Dicks E et al. PREDICT Plus: development and validation of a prognostic model for early breast cancer that includes HER2. Br J Cancer. 2012;107:800-7
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Ghoussaini M, Fletcher O, Michailidou K et al. Genome-wide association analysis identifies three new breast cancer susceptibility loci. Nat Genet. 2012;44:312-8 Hilbers FS, Boekel NB, van den Broek AJ, van Hien R, Cornelissen S, Aleman BM, van ‘t Veer LJ, van Leeuwen FE, Schmidt MK. Genetic variants in TGFβ-1 and PAI-1 as possible risk factors for cardiovascular disease after radiotherapy for breast cancer. Radiother Oncol. 2012;102:115-21 Stephens PJ, Tarpey PS, Davies H et al. The landscape of cancer genes and mutational processes in breast cancer. Nature. 2012;486:400-4
Molecular and genetic breast cancer epidemiology Adjuvant therapy recommendations for breast cancer patients are based on the estimated average risk of tumor relapse and death, which is mostly based on tumor characteristics but not on patient genotype. We study the impact of germline variants on the development of (contralateral) breast cancer, treatment response and long-term survival. This information may eventually be included in guidelines or prediction tools for improved disease management, or in the pre-selection of women for breast cancer screening programs. A second research line focuses on patient information and consent procedures, and return of results from research using human materials.
Genetic determinants of breast cancer risk and outcome The genetic determinants of breast cancer susceptibility and prognosis are still largely unexplained. We are continuously identifying and validating new single nucleotide polymorphisms (SNPs) within the Breast Cancer Association Consortium (BCAC; Coordinator: D. Easton, Cambridge, UK). Our group maintains the BCAC database table including tumor characteristics and clinical information from 65 studies comprising ~110,000 patients. We identified a number of additional breast cancer risk variants. All validated variants now explain approximately 9% of familial clustering of breast cancer. Identification of SNPs related to survival turned out to be more challenging. Using the BCAC database, we analyzed 11 breast cancer susceptibility SNPs and 62 additional variants which had been suggested as breast cancer risk SNPs by a GWAS or as candidate SNPs from individual studies. Analyses of all SNPs in 25,853 patients with available follow up showed that only one SNP in TOX3 (rs3803662) was associated with breast cancer-specific survival in homozygous carriers at P<0.01 (HR: 1.3, 95%CI: 1.1–1.5, P=0.0003), suggesting a recessive mode of action. We concluded that breast cancer survival and susceptibility may be influenced by distinct sets of germline variants. This is however not true for some moderate risk variants. Analyzing 25,571 patients from 22 studies participating in the BCAC, we found that estrogen receptor positive breast cancer patients carrying the CHEK2 1100delC variant had a significantly worse breast cancer survival (P=8x10-9). These results are in line with our earlier findings in Dutch patients (Schmidt et al., J Clin Oncol. 2007;25:64-9) In collaboration with other cancer consortia, the BCAC has designed a 200k cancer SNP chip (‘iCOGS’ chip) for SNP analysis of 45,290 breast cancer cases and 41,880 controls of European ancestry from 41 studies. Results from analyses using these data are awaiting publication. Also breast cancer risk analyses investigating interactions between SNPs, lifestyle and environmental factors are ongoing. In collaboration with L. Van ‘t Veer and E. Rutgers, we have collected ~1,500 risk
factor questionnaires and screening data for ~1,400 women >50 years of the ~2,000 Dutch breast cancer patients included in the MINDACT cohort. For a subset of patients, also blood was collected and DNA is being isolated for SNP analysis on the iCOGS chip. We are also analyzing the effect of the iCOGS SNPs on the development of contralateral breast cancer in the BCAC series.
governance of biobanks and the optimal way to inform donors about research results. Current practices of BBMRI.NLregistered biobanks are explored by means of a questionnaire sent to 143 biobank representatives, interviews with selected representatives, and expert meetings. Donor experiences and preferences will be investigated in interviews and results will be circulated through a secure web portal.
In collaboration with the Division of Psychosocial Research and Epidemiology (PSOE) and clinical groups, we are investigating prognosis and long-term outcome of BRCA1/2 mutation carriers among young (<50 years of age) breast cancer patients in our nationwide, consecutive breast cancer cohort (‘BOSOM’). We have expanded this cohort and finished BRCA1 and BRCA2 genotyping for 5,356 patients, detecting 3.4% and 1.2% mutation carriers, respectively. We have performed preliminary analyses on the development of contralateral breast cancer and survival. Moreover we systematically reviewed all breast cancer outcome studies in BRCA1/2 carriers. For the HEBON-Resource study (a nationwide cohort of breast cancer patients from families that have been screened for BRCA1/2 mutations; coordinator: M Rookus), we are collecting clinico-pathological and treatment information, which will allow us to study prognosis and treatment of familial breast cancer patients, and acceptance of prophylactic measures.
Prognosis and mortality prediction models We shared data with the Cambridge group to support the evaluation of the PREDICT tool for breast cancer survival. We also prepared and submitted other datasets to receive Adjuvant Online! and PREDICT outcome predictions in order to assist finetuning of these prediction models for more specified groups of breast cancer patients.
Biobanking: consent procedures and return of results In collaboration with the Division of PSOE (Flora van Leeuwen, Neil Aaronson), we are conducting research on consent procedures for secondary use of tissue for scientific research. In the Netherlands and many other countries, the use of residual tissues for research is regulated by an ‘opt-out’ procedure, in which patients can decide to disagree with the use of their tissue on the basis of written information. At present, the amount and quality of information differs widely across Dutch hospitals. Our previous research showed that cancer patients are unsatisfied with the current information procedure: they are mostly unaware of residual tissue storage for their own clinical benefit and possible use in research, although only a minority wishes to refuse this. We are currently surveying 1,800 patients in 5 Dutch hospitals to investigate different consent procedures for the secondary use of human materials in clinical research. To date, 450 patients have received an informed consent procedure or an opt-out procedure, either with or without active information provision. Using questionnaires and interviews, we will examine patient satisfaction and their preference for the different procedures. We will also examine the impact of the different consent procedures on the quality of clinical research and on the medical staff. Biobank donors are interested in receiving research results from biobank research. In a BBMRI funded project we aim to identify strategies for the involvement of donors in the 83
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S Ariotti S, Haanen JB, Schumacher TN. Behavior and function of tissueresident memory T cells. Adv Immunol 2012;114:203-16 Heemskerk B, Kvistborg P, Schumacher TN. The cancer antigenome. EMBO J. 2012 (in press)
Group leader Ton Schumacher
Ton Schumacher PhD Group leader Gavin Bendle PhD Post-doc Joost Beltman PhD Post-doc Andreas Hollenstein PhD Post-doc Shalin Naik PhD Post-doc Leila Perie PhD Post-doc Jan Rohr MD Post-doc Ferenc Scheeren PhD Post-doc Silvia Ariotti MSc PhD student Marit Van Buuren MSc PhD student Feline Dijkgraaf MSc PhD student Lorenzo Fanchi MSc PhD student Sander Kelderman MSc PhD student Carsten Linnemann MSc PhD student Riccardo Mezzadra MSc PhD student Laura Bies Technical staff Laura Van Dijk Technical staff Raquel Gomez Technical staff Mels Lodder Technical staff Daisy Philips Technical staff Nienke Van Rooij Technical staff Mireille Toebes Technical staff Jos Urbanus Technical staff
Publications
Andersen RS, Kvistborg P, Frosig TM, Pedersen NW, Lyngaa R, Bakker AH, Shu CJ, Straten P, Schumacher TN, Hadrup SR. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers. Nat Prot. 2012;7:891-902 Andersen RS, Thrue CA, Junker N, Lyngaa R, Donia M, Ellebaek E, Svane IM, Schumacher TN, Thor Straten P, Hadrup SR. Dissection of T-cell antigen specificity in human melanoma. Cancer Res. 2012;72:1642-50 Ariotti S, Beltman JB, Chodaczek G, Hoekstra ME, van Beek AE, GomezEerland R, Ritsma L, van Rheenen J, Maree AF, Zal T, de Boer RJ, Haanen, JB, Schumacher TN. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proc Natl Acad Sci USA. 2012;109:19739-44
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Kvistborg P, Shu CJ, Heemskerk B, Fankhauser M, Thrue CA, Toebes M, van Rooij N, Linnemann C, van Buuren MM, Urbanus JH, Beltman JB, Thor Straten P, Li YF, Robbins PF, Besser MJ, Schachter J, Kenter GG, Dudley ME, Rosenberg SA, Haanen JB, Hadrup SR, Schumacher TN. TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients. Oncoimmunol. 2012;1:409-18 Oosterhuis K, Aleyd E, Vrijland K, Schumacher TN, Haanen JB. Rational design of DNA vaccines for the induction of Human Papillomavirus type 16 E6- and E7-specific Cytotoxic T-cell responses. Human Gene Ther. 2012 Oosterhuis K, van den Berg JH, Schumacher TN, Haanen JB. DNA vaccines and intradermal vaccination by DNA tattooing. Curr Top Microbiol Immunol. 2012;351:221-250 Ritsma L, Steller EJ, Beerling E, Loomans CJ, Zomer A, Gerlach C, Vrisekoop N, Seinstra D, van Gurp L, Schafer R, Raats DA, de Graaff A, Schumacher TN, de Koning EJ, Rinkes IH, Kranenburg O, Rheenen J. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci Transl Med. 2012;4: 158ra145 Singh SK, Tummers B, Schumacher TN, Gomez R, Franken KL, Verdegaal EM, Laske K, Gouttefangeas C, Ottensmeier C, Welters MJ, Britten CM, van der Burg SH. The development of standard samples with a defined number of antigen-specific T cells to harmonize T cell assays: a proofof-principle study. Cancer Immunol Immunther. 2012 (in press) van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJ, Behjati S, Hilkmann H, el Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM, Kesmir C, Haanen JB, Kvistborg P, Schumacher TN. Tumor exome analysis reveals neoantigen-specific T cell reactivity in an Ipilimumab-responsive melanoma. J Clin Oncol. 2012 (in press)
Dissecting and manipulating antigenspecific T cell immunity The aim of our research is straightforward 1). To design novel technologies to examine and modify antigen-specific T cell immunity 2). To use these technologies 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 of cancer.
Dissecting antigen-specific T cell immunity in human cancer There is now substantial evidence that therapeutic manipulation of immune reactivity can result in clinically meaningful effects on human cancer. For example, antibodies to CTLA4 or PD1 show substantial activity in metastatic melanoma. Likewise, infusion of ex vivo expanded tumor-infiltrating lymphocytes (TIL) can lead to marked tumor regressions. However, it is largely unknown which cytotoxic T cell (CTL) reactivities mediate such cancer regressions. Knowledge of such reactivities would be of value, both to monitor current therapies and to further design targeted strategies that specifically increase immune reactivity against these antigens. In the past years, we have developed a broadly applicable “MHCbased toolkit” for the parallel detection of dozens of different T cell populations within a single sample. We have used this technological framework to determine the effects of both TIL therapy and anti-CTLA4 treatment on the CTL repertoire in melanoma patients. These analyses have demonstrated that CTL responses against non-mutated (‘shared’) melanomaassociated epitopes occur frequently, and that both therapies lead to a broadening of the CTL response against these antigens. Strikingly however, the combined magnitude of these CTL responses is very low, suggesting a potentially important role of T cells that recognize patient-specific neo-antigens that arise as a consequence of mutations. To address this possibility, we have developed an approach to identify potential neo-antigens in human cancer, on the basis of cancer exome sequencing data. Importantly, the first evidence for the feasibility of this approach to identify human neoantigens has recently been obtained. In the coming years, this approach should offer the possibility to determine – presumably also for cancers other than melanoma – to what extent the mutational landscape in human tumors is recognized by CTL.
Dissection of cellular immunity through single cell tracing The above work measures immune responses at the level of cell populations. To also allow the tracing of immune cell fate at the level of single cells, we have developed technology that enables tagging of individual cells with genetic barcodes. This technology relies on the use of oncoretroviral and lentiviral libraries containing thousands of different DNA ‘barcodes’. Introduction of these genetic barcodes in progenitor cells and
subsequent analysis of the barcodes present within the cell populations that arise from them reveals lineage relationships. In recent work, we have used this barcode labeling strategy, together with second generation sequencing, to measure the output of individual antigen-specific T cells. The data obtained demonstrate that T cell responses are dominated by the output of only a very small number of naïve antigen-specific T cells. Furthermore, T cell families vary markedly with respect their phenotypic markers of memory potential and the families that remained small during the primary response display a disproportional potential for renewed expansion. Collectively, these data indicate that the reproducibility of T cell responses that has for long been observed at the population level is achieved by the ‘averaging’ of individually remarkably divergent cellular behaviors. In a parallel project we have addressed which (types of) progeny are generated by individual multipotent hematopoietic progenitors (LMPP) in vivo. These data demonstrate that LMPPs, previously defined as multipotent, only produce a restricted set of output cell types in vivo. Importantly, ‘sibling analyses’ that assess the output of sister cells transferred into different mice, demonstrate that at least part of these specific output patterns are due to a pre-existing imprinting; ‘sisters behave alike’. Collectively, these data suggest a graded commitment model of hematopoiesis, in which the imprinting of possible cell fates occurs at a much earlier developmental phase than previously appreciated. The vast majority of studies that analyze antigen-specific T cell responses primarily assess the development of antigen-specific T cell responses within the blood compartment. To also allow the analysis of T cell reactivity at the sites of action, the Haanen group and our group have developed an intravital imaging system in which virus-specific T cell responses can be followed in skin. In recent work we have demonstrated the existence of a specialized antigen-specific T cell population of “skin patrolling memory T cells” that migrate through former sites of infection. In ongoing work, we are evaluating how this T cell population can form a possible link between renewed antigen production and the development of a state of ‘pathogen alert’ at that site.
Adoptive T cell therapy (collaboration with Haanen lab) The cornerstone of our translational work is the development and evaluation of adoptive T cell therapies for human cancer. In addition to the TIL trial that is led by the Haanen group, within the past year we have also started a first TCR gene therapy trial in which MART-I - one of the melanoma antigens - is targeted. In parallel, we are preparing for a substantially larger clinical program in which we aim to test the clinical value of a series of different TCRs that target various tumor-associated antigens. Towards this goal, we have developed a technology termed TCR capture that allows rapid TCR gene identification from many samples, and have utilized it to generate a library of TCR genes to target a series of around 10 different cancer/germline antigens. In subsequent work, we will evaluate this large panel of TCRs with respect to safety and efficacy, in order to select those TCR genes that will be used for future TCR gene therapy trials. Furthermore, in parallel work, we have made substantial progress in the development of optimized transposon-based gene transfer strategies to create the TCR-modified T cells that will be used in such clinical studies.
Vogel WV, Guislain A, Kvistborg P, Schumacher TN, Haanen JB, Blank CU. Ipilimumab-induced sarcoidosis in a patient with metastatic melanoma undergoing complete remission. J Clin Oncol 2012;30:e7-e10 Wolkers MC, Gerlach C, Arens R, Janssen EM, Fitzgerald P, Schumacher TN, Medema JP, Green DR, Schoenberger SP. Nab2 regulates secondary CD8+ T-cell responses through control of TRAIL expression. Blood. 2012;119:798-804
Cancer exome-based identification of neo-antigen specific T cell reactivity. 1). The repertoire of tumor-specific mutations is identified by exome sequencing. 2). T cell epitopes are predicted 3). Autologous T cell activity against these epitopes is evaluated.
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S Kanga S, Bernard D, Mager-Heckel AM, Erpapazoglou Z, Mattiroli F, Sixma TK, Leon S, Urban-Grimal D, Tarassov I, Haguenauer-Tsapis R. A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit. PLoS One. 2012;7
Division head, group leader Titia Sixma Titia Sixma PhD Group leader Marcello Clerici PhD Post-doc Alex Fish PhD Post-doc Rick Hibbert PhD Post-doc Prakash Rucktooa PhD Post-doc Mariano Stornaiuolo PhD Post-doc Francesca Mattiroli MSc PhD student Judith Smit MSc PhD student Flora Groothuizen MSc PhD student Danny Sahtoe MSc PhD student Michael Uckelman MSc PhD student Robbert Kim Msc PhD student Pim Van Dijk Technical staff Herrie Winterwerp Technical staff
Publications
Hibbert RG, Sixma TK. Intrinsic Flexibility of Ubiquitin on Proliferating Cell Nuclear Antigen (PCNA) in Translesion Synthesis. J Biol Chem. 2012;287:39216-23 Mattiroli F, Vissers JH, van Dijk WJ, Ikpa P, Citterio E, Vermeulen W, Marteijn JA, Sixma TK. RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 2012;150:1182-95 Smit JJ, Monteferrario D, Noordermeer SM, van Dijk WJ, van der Reijden BA, Sixma TK. The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension. EMBO J. 2012;31:3833-44
Radner S, Celie PH, Fuchs K, Sieghart W, Sixma TK, Stornaiuolo M. Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells. J Struct Biol. 2012;179:46-55 Mallette FA, Mattiroli F, Cui G, Young LC, Hendzel MJ, Mer G, Sixma TK, Richard S. RNF8- and RNF168dependent degradation of KDM4A/ JMJD2A triggers 53BP1 recruitment to DNA damage sites. EMBO J. 2012;31:1865-78 Faesen AC, Luna-Vargas MP, Sixma TK. The role of UBL domains in ubiquitinspecific proteases. Biochem Soc Trans. 2012;40:539-45 Nacerddine K, Beaudry JB, Ginjala V, Westerman B, Mattiroli F, Song JY, van der Poel H, Ponz OB, Pritchard C, Cornelissen-Steijger P, Zevenhoven J, Tanger E, Sixma TK, Ganesan S, van Lohuizen M. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer. J Clin Invest. 2012;122:1920-32 Osipov AV, Rucktooa P, Kasheverov IE, Filkin SY, Starkov VG, Andreeva TV, Sixma TK, Bertrand D, Utkin YN, Tsetlin VI. Dimeric alpha-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors. J Biol Chem. 2012;287:6725-34
Structural biology
Development of cancer is generally due to errors that occur in cellular pathways. Understanding the mechanisms of underlying processes will help to determine where the errors occur and how they can be treated. We study proteins using a combination of biochemical and biophysical methods, in particular X-ray crystallography to provide three-dimensional structures. This leads to insights in molecular mechanisms that we validate in cells. In addition our structures provide targets for drug design studies. In this work we focus primarily on proteins involved in ubiquitin conjugation, particularly in stress response and DNA repair pathways. In addition we study DNA mismatch repair pathways and nicotinic receptor signaling.
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 non-fully complementary DNA strands. We make use of variants of MutS that specifically maintain the dimer or tetramer state of these proteins in order to be able to provide quantitative data on the mismatch recognition cycle. In collaboration with the groups of Terence Strick (Paris), Peter Friedhoff (Giessen) and Joyce Lebbink (Rotterdam) this allows a precise analysis of the steps involved in mismatch recognition and the initiation of repair. Also these mutants allowed novel crystallographic analysis of the proteins with and without DNA, leading to new insight into the state of the protein prior to DNA binding. We use crystallography and SAXs to define the states that are observed and we use it to create novel tools to study the initiation of DNA mismatch repair. Quantitative analysis of the obligate dimer and tetramer variants of the MutS has allowed novel insights in the ATP dependent release of the mismatched DNA.
ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. We are particularly interested in the factors that determine the rate, the specificity and the selectivity of the E2/E3 dependent reaction especially in DNA dependent processes. Recently we found that UbcH5c is sufficient to provide specificity for K164 in PCNA. This reaction is very efficient and after optimization allowed a characterization of ubiquitinated PCNA biophysically. In a combination of SAXS, MALLS and NMR we could show that ubiquitin is flexible when it is coupled to PCNA. We have studied the interesting process of linear ubiquitin chain formation by the E3-ligase complex LUBAC. We could show that the specificity for the amino terminal modification is entirely within the HOIP RBR-LDD domain (figure 1). We could validate that this protein functions via a RING/HECT mechanism, similar to Parkin. Then we could show that the C-terminal LDD region of this protein is required for correct positioning of the target ubiquitin for linear fusion. Much of our attention is focused on the modification of H2A in nucleosomes, both by the polycomb repressive complex and by the DNA damage response proteins, RNF8 and RNF168. We were able to show that RNF168 is able to modify the nucleosome upon DNA damage at K13/15, whereas RNF8 does not. This surprising insight predicts the presence of a different target for RNF8 and uncouples the effects of the polycomb PRC1 complex at K119 and that of the DNA damage response in H2A ubiquitination (figure 2). Interestingly, this newly identified site on H2A is spatially close to a different DNA damage dependent site on H2B on lysine 120 (Mattiroli et al, 2012).
Nicotinic Acetylcholine receptor homolog AChBP The acetylcholine binding protein (AChBP) is a homolog of the extracellular domain of the nicotinic acetylcholine receptor, and a representative of the cys-loop receptor family. It remains the best model for atomic resolution structures of ligand binding to this pharmaceutically important family of ion channels. In collaboration with the group of Iwan de Esch (VU) novel interactors for AChBP we identified novel scaffolds for interaction with this protein, with unexpected binding properties. Currently we are studying variants of these compounds in order to understand the mode of interaction.
Figure 1: Linear ubiquitin chain formation is promoted by the LUBAC complex, but the RBR-LDD region are sufficient to generate the specificity for the N-terminus of ubiquitin. The LDD region seems to act as a platform for this ubiquitination (Smit et al, EMBO J, 2012)
Figure 2: RNF168 can modify H2A in nucleosomes during the DNA damage response. This modification is required to drive DNA damage signalling. It takes place at Lysine 13 and 15, and hence is spatially separate from the ubiquitination performed by Ring1b in the polycomb PRC1 complex (Mattiroli et al, Cell 2012)
Ubiquitin conjugation Rucktooa P, Haseler CA, van Elk R, Smit AB, Gallagher T, Sixma TK. Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein. J Biol Chem. 2012;287:23283-93
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Ubiquitin conjugation is critical for signaling in almost all cellular processes, including DNA repair, apoptosis, cell cycle, chromatin regulation and endocytosis. Since these processes are so important for cellular stability, deregulation of ubiquitindependent processes often leads to cancer. Structure analysis of the proteins involved in ubiquitin conjugation could help to develop novel drugs inhibiting critical pathways in ubiquitin conjugation. 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 87
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S Margadant C, van den Bout I, van Boxtel AL, Thijssen VL, Sonnenberg A. Epigenetic regulation of galectin-3 expression by integrin a5b1 promotes cell adhesion and migration. J Biol Chem. 2012;287:44684-93
Division head, group leader Arnoud Sonnenberg Arnoud Sonnenberg PhD Group leader Pablo Secades PhD Post-doc Mirjam Ketema MSc PhD student Coert Margadant MSc PhD student Maaike Kreft Technical staff
Sachs N, Secades P, van Hulst L, Kreft M, Song J-Y, Sonnenberg A. Loss of integrin a3 prevents skin tumor formation by promoting epidermal turnover and depletion of slowcycling cells. Proc Natl Acad Sci USA 2012;109:21468-73 Margadant C, Cremers L, Sonnenberg A, Boonstra J. MAPK uncouples cell cycle progression from cell spreading and cytoskeletal organization in cycling cells. Cell Mol Life Sci. (in press) Sachs N, Sonnenberg A. Cell-matrix adhesion of podocytes in physiology and disease. Nature Reviews Nephrology (in press)
Publications
Sachs N, Claessen N, Aten J, Kreft M, Teske GJ, Koeman A, Zuurbier CJ, Janssen H, Sonnenberg A. Blood pressure influences end-stage renal disease of Cd151 knockout mice. J Clin Invest. 2012;122:348-58 Frijns E, Kuikman I, Litjens S, Raspe, M., Jalink K, Ports M, Wilhelmsen K, Sonnenberg A. Phosphorylation of T1736 in the C-terminal tail of integrin b4 contributes to hemidesmosome disassembly. Mol Biol Cell 2012;23:1475-85 Postel R, Sonnenberg A. Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish. PLoS One 2012;7:e39881 Deng X, Li Q, Hoff J, Novak M, Yang H, Jin H, Erfani SF, Sharma C, Zhou P, Rabinovitz I, Sonnenberg A, Yi Y, Zhou P, Stipp CS, Kaetzel DM, Hemler ME, Yang XH. Integrin-associated CD151 drives ErbB2-evoked mammary tumor onset and metastasis. Neoplasia 2012;14:678-89 Margadant C, Kreft M, de Groot DJ, Norman JC, Sonnenberg, A. Distinct roles of talin and kindlin in regulating integrin a5b1 function and trafficking. Curr Biol. 2012;22:1554-63 Nicolaou N, Margadant C, Kevelam SH, Lilien MR, Oosterveld MJ, Kreft M, van Eerde, AM, Pfundt R, Terhal PA, van der Zwaag B, Nikkels PG, Sachs N, Goldschemding R, Knoers NVAM, Renkema KY, Sonnenberg A. Gain-ofglycosylation in integrin a3 causes lung disease and nephrotic syndrome. J Clin Invest. 2012;122:4375-87 88
Frijns E. Controling hemidesmosome dynamics by phosphorylation of the integrin b4 subunit. PhD thesis. Amsterdam: University of Amsterdam, 2012 Ketema M. Nesprin-3 as a LINC between the nucleus and intermediate filaments. PhD thesis. Amsterdam: University of Amsterdam, 2012.
Receptors for matrix adhesion
The main objective of our group is to study the mechanisms involved in cell adhesion. We are specifically interested in understanding the significance of and characterizing the interactions that take place between cells and the extracellular matrix component laminin in both the epidermis and the glomerulus of the kidney. A second line of research involves the outer nuclear membrane protein nesprin-3, which binds to the cytoskeletal linker protein plectin, thereby establishing a link between the intermediate filament system and the nucleus. The physiological and pathological roles of these proteins and their interaction are studied using knockout mice for the various proteins.
Regulation of a5b1 trafficking Integrin function depends on conformational rearrangements that increase affinity for ligand (“activation”), and by intracellular trafficking. We investigate how activation and trafficking of the fibronectin receptor, integrin a5b1, are regulated by two NPxY motifs in the b1-cytoplasmic tail. We therefore generated disrupting mutations in the first NPxY (b1Y783A), the second NPxY (b1Y795A), or both (b1Y783/795A), and stably expressed these mutants in b1-deficient cells. Wild-type a5b1 induces epithelial-to-mesenchymal transition (EMT)-like events characterized by disruption of cell-cell contacts, cell scattering, increased motility, organization of a fibronectin network, and a contractile, fibroblast-like morphology with multiple protrusions. This phenotype is recapitulated by expression of b1Y795A, but not by b1Y783A or b1Y783/795A, indicating that the first but not the second NPxY motif is absolutely required for a5b1 function. However, cell surface levels and total-protein levels of b1Y795A and b1Y783/795A are low (50% and 70% of wild-type, respectively), and they accumulate in late endosomes/lysosomes, where they co-localize with internalized fibronectin, suggesting that the second NPxY motif regulates integrin trafficking. Indeed, whereas internalization rates for all mutants are similar to that of wild-type a5b1, recycling to the plasma membrane of b1Y795A and b1Y783/795A, but not b1Y783A, is considerably impaired. Instead, a large part of the internal pool of these mutants is degraded. Together, these data show that the first NPxY motif regulates activation of a5b1, whereas the second NPxY regulates recycling of ligand-bound a5b1 to the plasma membrane.
Regulation of hemidesmosome disassembly by growth factors receptors During wound healing, hemidesmome disassembly enables keratinocyte migration and proliferation. Hemidesmosome dynamics can be altered downstream of EGFR activation following the phosphorylation of integrin b4 residues, S1356 and S1364, which reduces the interaction with plectin. This event, however, is insufficient to drive complete hemidesmosome disassembly. We have used a FRET-based assay to demonstrate
that the connecting segment (CS) and carboxy-terminal tail (C-tail) of the b4 cytoplasmic domain come into close proximity and facilitate the formation of a tertiary binding platform for plectin. Additionally, analysis of b4 mutants containing either phospho-mimicking or non-phosphorylatable residues at threonine 1736 (T1736) in the C-tail show that this residue regulates the interaction with the plectin-plakin domain, since binding of b4 to the plectin-plakin domain was prevented when T1736 was replaced by aspartic acid but not by alanine. Furthermore, the aspartic acid mutation of b4 T1736 impaired hemidesmosome formation in PA-JEB/b4 keratinocytes. Phosphorylation of integrin b4 at T1736 was shown to be regulated by PKD1, which requires translocation to the plasma membrane and subsequent activation. In conclusion, we identified PKD1 as a possible novel player in the regulation of hemidesmosome disassembly, a dynamically regulated process involving phosphorylation of b4 on S1356 and S1364, and T1736.
ITGA3 mutation causes interstitial lung disease and congenital nephrotic syndrome The laminin-binding integrin a3b1 is highly expressed in lung epithelium and kidney podocytes, and loss of a3b1 expression in mice causes severe lung and kidney defects. In collaboration with the groups of Dr. Kirsten Renkema and Prof. Roel Goldschmeding (UMC, Utrecht), we identified a patient carrying a homozygous recessive missense mutation in the human ITGA3 gene, causing a fatal multi-organ condition comprising interstitial lung disease and congenital nephrotic syndrome. The mutation causes an alanine to serine substitution, thereby introducing an N-glycosylation motif. As a consequence, the mutant a3-subunit is hyperglycosylated, leading to degradation and impaired a3b1 expression at the cell surface. Thus, these findings uncover for the first time a human ITGA3 mutation as the cause of a complex multi-organ disorder, suggesting that mutations in ITGA3 underlie certain congenital lung and kidney diseases of unknown etiology.
Immunofluorescence staining for the integrin a3 subunit on a kidney biopsy from a control (unaffected) individual and patient carrying an ITGA3 missense mutation.
Loss of integrin a3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling bulge cells Progression of skin tumors is correlated with altered expression of the epidermal integrin a3b1 suggesting a causal role during tumorigenesis. Using epidermis-specific Itga3 conditional knockout mice subjected to the DMBA/TPA twostage skin carcinogenesis protocol, we showed that efficient tumor development is critically dependent on the presence of a3b1. In the absence of a3b1, tumor initiation is dramatically decreased due to increased epidermal turnover leading to a loss of DMBA-initiated slow-cycling keratinocytes. Lineage tracing revealed emigration of a3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, proliferation and growth of the few tumors that do arise in the mice with an epidermis-specific deletion of Itga3 was strongly reduced. We also show that progression of a3b1 null squamous cell carcinomas to undifferentiated, invasive carcinomas occurs at an increased rate. In conclusion, we identify a3b1 as a critical modulator of skin carcinogenesis with contrasting effects in early and late tumor stages.
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S Seemann I, Gabriels K, Visser NL, Hoving S, te Poele JA, Pol JF, Gijbels MJ, Janssen BJ, van Leeuwen FW, Daemen MJ, Heeneman S, Stewart FA. Irradiation induces modest changes in murine cardiac function despite progressive structural damage to the myocardium and microvasculature. Radiother Oncol. 2012;103:143-50
Group leader Fiona Stewart
Fiona Stewart PhD Group leader Nicola Russell MD PhD Academic staff Saske Hoving PhD Post-doc Marion Scharpfenecker PhD Post-doc Ingar Seemann MSc PhD student Karin De Cortie Technical staff Ben Floot Technical staff Johannes Te Poele Technical staff
Publications
Gabriels K, Hoving S, Seemann I, Visser NL, Gijbels MJ, Pol JF, Daemen MJ, Stewart FA, Heeneman S. Local heart irradiation of ApoE-/- mice increases inflammation and causes endocardial damage with modest changes in cardiac function. Radiother Oncol. 2012;105:358-364 Hoving S, Heeneman S, Gijbels MJJ, Te Poele JAM, Visser N, Cleutjens J, Russell NS, Daemen MJAP, Stewart FA. Irradiation induces different inflammatory and thrombotic responses in carotid arteries of wildtype C57BL/6J and atherosclerosis-prone ApoE-/- mice. Radiother Oncol. 2012;105:365-370 Nyst HJ, Wildeman MA, Indrasari SR, Karakullukcu B, van Veen RLP, Adham M, Stewart FA, Levendag PC, Sterenborg HJCM, Tan IB. Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent nasopharyngeal carcinoma after curative radiotherapy: A feasibility study. Photodiagnosis and Photodynamic Therapy 2012;9:274-281 Scharpfenecker M, Floot B, Russell NS, Stewart FA. The TGF-β co-receptor endoglin regulates macrophage infiltration and cytokine production in the irradiated mouse kidney. Radiother Oncol. 2012;105:313-320
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Stewart FA. Mechanisms and doseresponse relationships for radiationinduced cardiovascular disease. Ann ICRP, 2012;41:72-79 Russell NS, Scharpfenecker M, Hoving S, Woerdeman LAE. Consequences of radiotherapy for breast reconstruction” In: “Reconstructive Surgery” Ed; Danilla S. Intech open access publishing. 2012 Stewart FA et al. ICRP Publication 118: ICRP Statement on Tissue Reactions and Early and Late Effects of Radiation in Normal Tissues and Organs Threshold Doses for Tissue Reactions in a Radiation Protection Context. Ann ICRP. 2012;41:1-322
Radiation-induced vascular damage
Advances in cancer diagnosis and treatment, as well as better supportive care, have lead to improvements in patient survival for most tumour types. However, this has also resulted in an increased number of patients at risk for developing treatmentrelated side effects. At least half of all long-term cancer survivors receive radiotherapy and vascular damage is one of the main underlying causes of late toxicity and morbidity in these people. In our lab we are studying the cellular and molecular mechanisms involved in development of radiation-induced vascular damage. A greater understanding of this process is required to optimize treatment and management of irradiated cancer patients.
Radiation-induced microvascular damage and fibrosis development Collaboration with Rob Coppes, UMCG, Groningen Radiation-induced vascular damage mainly affects capillary blood vessels, which become dilated and thin-walled (telangiectasia). This compromises the functional integrity of tissues and precipitates other events such as fibrosis, inadequate tissue perfusion and necrosis. Transforming growth factor-beta (TGF-β) is an important regulator of vascular homeostasis and neovessel formation. In endothelial cells (EC), TGF-β signals through receptors ALK1 and ALK5, with modulation by co-receptor endoglin. TGF-β is upregulated and activated by irradiation, which subsequently leads to excessive connective tissue formation (fibrosis). We used mouse kidneys (a microvascular rich organ) as a model to study the underlying mechanisms of radiation-induced telangiectasia and fibrosis. These studies identified endoglin as being critically involved in both processes. Mice expressing reduced levels of endoglin (Eng+/- mice) developed less radiationinduced vascular damage, less inflammatory cell infiltration (mainly macrophages) and less fibrosis. Irradiated Eng+/- mice also expressed reduced levels of the pro-inflammatory/ angiogenic/fibrotic cytokines interleukin 6 (Il6) and interleukin 1 beta (Il1β); all of which are produced by macrophages. We subsequently investigated whether the reduction in proinflammatory cytokine expression in irradiated Eng+/- kidneys could be explained by shift in macrophage subpopulations. Macrophages comprise a heterogeneous population of cells with phenotypic plasticity. Depending on the tissue environment, they may either polarize toward a “classically activated, M1like” phenotype that promotes inflammation or toward an “alternatively-activated, M2-like’’ phenotype that plays a role in angiogenesis, wound healing, and tissue remodelling. We showed that all except one “M2-like” markers were reduced in irradiated Eng+/- compared to Eng+/+ kidneys, suggesting that Eng+/- mice do not display a shift in macrophage subpopulations after irradiation. In summary, these results suggest that pro-
inflammatory cytokine production by macrophages contributes to the vascular and fibrotic phenotype after irradiation. Endoglin is expressed on inflammatory cells, as well as EC. Therefore we are currently investigating the role of endoglin on bone-marrow-derived cell (BMDC) infiltration and cytokine production, versus its role in resident EC in irradiated tissues. To this end, we treated Eng+/+ and Eng+/- mice with total body irradiation followed by local kidney irradiation and grafted these animals with bone marrow from Eng+/+ or Eng+/- mice. We are now monitoring kidney function and the development of vascular damage and fibrosis. In addition, we will investigate whether infiltrating macrophage populations in the different treatment groups are differentially skewed. We are also investigating how endoglin regulates the immune response in other irradiated tissues and whether depleting macrophages after irradiation leads to a reduction in the development of normal tissue damage after irradiation. In addition, we aim to modulate TGF-β/endoglin-mediated BMDC recruitment to irradiated tissues by anti-vascular treatments used in cancer therapy, to investigate whether development of late vascular damage after irradiation can be prevented.
Radiation induced cardiac damage Collaboration with Sylvia Heeneman, Cardiovascular Research Institute, Maastricht and Mat Daemen, Department of Pathology, AMC, Amsterdam Radiotherapy of thoracic and chest-wall tumors increases the long-term risk of cardiovascular damage. This risk is further increased by anthracycline chemotherapy or drugs used to block ErbB2 signaling in HER2-positive breast cancers, but the mechanisms are unknown. We have studied the effects of cardiac irradiation in both wild type C57BL/6J mice, which do not develop atherosclerosis, and in ApoE-/- mice that have elevated serum cholesterol levels and develop atherosclerosis with age. Microvascular density in the heart decreased progressively after irradiation, with functional damage to remaining microvasculature, as well as progressive development of myocardial inflammation and fibrosis. These changes were more severe in ApoE-/- mice. Hearts of irradiated ApoE-/- mice also developed atherosclerotic lesions in midsized coronary arteries. Microarray pathway analysis indicated a stimulated inflammatory response and highly upregulated collagen pathway in ApoE-/- mice only. In vitro studies showed that incubation of human cardiomyocytes with the ErbB2 inhibitor trastuzimab did not enhance cell killing by doxorubicin or irradiation. Lapatinib (ErbB1/2 inhibitor) fed to mice for 20 weeks after irradiation or doxorubicin did not exacerbate cardiac functional damage and actually inhibited radiation-induced inflammation and doxorubicin-induced fibrosis. However, lapatinib did increase the mitochondrial damage and degeneration of cardiomyocytes after doxorubicin treatment. Endoglin is highly expressed in damaged EC and is crucial for proliferation and vascular repair. We therefore examined the role of endoglin in vascular damage and repair in the irradiated heart. Eng+/- mice had a reduced early inflammatory response to irradiation and inflammatory, fibrotic and immune-regulatory genes were less prominently regulated in Eng +/- mice at 4 weeks after irradiation. At later times these differences were less apparent and results suggest that endoglin insufficiency only limits inflammation and fibrosis in the early phase of damage.
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Genomic instability and carcinogenesis
Division head, group leader Hein Te Riele Hein Te Riele PhD Group leader Erika Cantelli PhD Post-doc Henri Van de Vrugt PhD Post-doc Camiel Wielders PhD Post-doc Kamila Wojciechowicz-Grzadka PhD Post-doc Tim Harmsen MSc PhD student Tanja Van Harn MSc PhD student Hellen Houlleberghs MSc PhD student Marleen Dekker Technical staff Elly Delzenne-Goette Technical staff Anja Van der Wal Technical staff
Publications
Bakker ST, Van de Vrugt HJ, Visser JA, Delzenne-Goette A, Van der Wal A, Berns MAD, Van de Ven M, Oostra AB, De Vries S, Kramer P, Arwert F, Van der Valk M, De Winter JP, and Te Riele H. Fancf-deficient mice are prone to develop ovarian tumors. J Pathology 2012;226:28-39 Ezzatizadeh V, Pinto RM, Sandi C, Sandi M, Al-Mahdawi S, Te Riele H, Pook MA. The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model. Neurobiol Dis. 2012;46:165-71 Tomosugi M, Sowa Y, Yasuda S, Tanaka R, Te Riele H, Ikawa H, Koyama M, Sakai T. RB independent G1-phase arrest by flavone, phosphatidylinositol 3-kinase inhibitor and histone deacetylase inhibitor. Cancer Sci. 2012;103:2139-43 Tomé S, Simard JP, Slean MM, Holt I, Morris GE, Wojciechowicz K, Te Riele H, Pearson CE. Tissue-specific mismatch repair protein expression: MSH3 is higher than MSH6 in multiple mouse tissues. DNA Repair 2012 Bakker ST, De Winter JP, te Riele H. Learning from a paradox: recent insights into Fanconi anemia through studying mouse models. Disease Models Mech. 2012 (in press) 92
A new mouse model for Lynch syndrome. MSH2-deficient crypts, assessed by immunohistochemistry using MSH2 antibodies in (A) an LS patient with a heterozygous MSH2 mutation (taken from Kloor et al. Lancet Oncol 2012;13:598) and (B) in an Msh2-Lynch mouse. MSH2-deficient cells (light, indicated by arrowheads) are present amidst an excess of MSH2-positive cells (dark).
Genomic instability and deregulated cell cycle control are hallmarks of human cancer. Our research involves both aspects focusing on (1) the role of the DNA mismatch repair and Fanconi anemia genome maintenance pathways in mutation avoidance and (2) the role of cell cycle checkpoints in tumor suppression. The principle tools include gene modification in murine embryonic stem cells (ESC) and analyses of the phenotypic consequences in ESC, mutant mice and cell lines derived thereof.
DNA Mismatch Repair Lynch syndrome/hereditary non-polyposis colorectal cancer (LS/HNPCC) is caused by inherited defects in DNA mismatch repair (MMR). The primary function of MMR is correction of DNA replication errors, which are recognized by MSH2/MSH6 or MSH2/MSH3 protein complexes. Subsequent recruitment of another dimer, MLH1/PMS2, activates exonucleolytic activity to remove the error-containing DNA strand allowing resynthesis of a new error-free strand. Mismatches can also arise by replication of damaged bases such as O6-methylguanine. This lesion elicits futile cycles of repair, leading to double-strand break formation and cell death. Thus, DNA MMR acts anti-mutagenic and mediates the toxicity of methylating agents.
Oligonucleotide-directed gene modification Mismatches can also arise artificially, e.g., during oligonucleotide-directed gene modification, when a singlestranded oligodeoxyribonucleotide (ssODN) of ~35 nucleotides hybridizes to its chromosomal complement. We found this to happen during DNA replication (Aarts and Te Riele, NAR 2010;38:6956), however, the incomplete base paring elicits a MMR reaction that aborts the gene modification reaction (Dekker et al., NAR 2003;31:e27). ESCs can be made permissive for ‘oligo targeting’ by shRNAmediated transient suppression of either MSH2 (Aarts et al., NAR 2006;34:e147) or MLH1 (Dekker et al., Mutation Res 2011;715:52-60), allowing effective substitution of 3-4 adjacent nucleotides or even a single nucleotide, respectively. To identify cells carrying a single base-pair alteration, we developed a real-time PCR protocol that makes use of so-called mismatchamplification-mutation-assay (MAMA) primers. However, transient suppression of MMR is not entirely innocent. Particularly, MLH1 knockdown strongly increased the frequency of slippage errors at mononucleotide repeats (Dekker et al., Mutation Res 2011;715:52-60). The accumulation of undesired mutations may interfere with the phenotype of cells and mice. We have recently found ways to circumvent the MMR problem. Furthermore, we are developing protocols for oligo targeting in murine and human induced pluripotent stem cells.
Unclassified variants of MMR genes Single codon variants of MMR genes are widespread in the
human population and in (suspected) LS patients but their phenotypic consequences are often difficult to predict. We use oligo targeting to recreate suspected variants of MMR genes in ESCs in order to assess their capacity to support MMR. We have thus far analyzed nine of such ‘Variants of uncertain significance’ (VUS) of MSH2 and found four to fully abrogate MMR activity (Wielders et al., Human Mutation 2011;32:389-96). Four MSH6 variants carrying codon substitutions at conserved positions showed wild-type activity. We are extending this type of analysis to MLH1 VUS.
A new mouse model for Lynch syndrome We have recently generated a novel mouse model in which, similar to LS patients, MMR-defective crypts arise amidst an excess of MMR-proficient crypts (Fig. 1). We use this model to identify internal and external modifiers of tumor development with the aim to reduce cancer risk in LS patients.
Fanconi anemia Another cancer predisposition syndrome caused by defects in DNA repair is Fanconi anemia (FA), a recessive disorder characterized by skeletal malformations, progressive anemia and high incidence of AML and HNSCC. FA is caused by bi-allelic defects in either one of 15 genes, FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P. The products of these genes are essential for the repair of DNA interstrand crosslinks (ICL). In collaboration with the VU University FA research group (Hans Joenje/Johan de Winter), we have generated Fancfand Fancm-deficient mice to study the significance of the FA pathway in suppression of cancer. FANCF and FANCM are part of the FA core complex that is essential for monoubiquitylation of FANCD2 and FANCI. Both, Fancf and Fancm deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. Fancf-deficient female mice were highly prone to development of granulosa cell tumors (Bakker et al., J Pathology 2012;226:28-39). Also Fancm deficiency caused decreased tumor-free survival (Bakker et al., Hum Mol Genet 2009;18:3484), however, no specific tumor type was seen. At the cellular level, FANCM was found to suppress spontaneous (but not induced) sister chromatid exchanges, a role that seems independent of its function in the FA core complex. Crossing the Fancf and Fancm knockout alleles into cancer prone Apc+/- and Eµ-Pim transgenic mice revealed tissueand gene-specific acceleration of tumorigenesis.
kinase inhibitors p27KIP1 and p21CIP1, the latter pointing to activation of the DNA Damage Response (DDR) (Foijer et al., Cancer Cell 2005;8:455). Indeed, G2-arrested cells accumulated DNA double-strand breaks (DSBs) that resulted from severe replication stress. Upon mitogen re-addition, G2 arrested cells repaired part of the DSBs and re-entered the cell cycle. Cell cultures derived from TKO cells that had been transiently mitogen deprived showed aneuploidy indicating that cells devoid of G1/S control are sensitive to genomic instability (Van Harn et al., Genes Dev 2010;24:1377).
Anchorage independence TKO MEFs were also dependent on a solid support (anchor), even if they ectopically expressed RASV12 (Vormer et al., MCB 2008;28:7263). Apparently, a cell cycle mechanism still operates to restrict proliferation of TKO cells in the absence of anchorage. To identify this mechanism, we have used both gain- and loss-of-function screening. We found that ectopic expression of the immortalizing oncogene TBX2 allowed anchorage-independent growth of RASV12-expressing TKO MEFs. For loss-of-function screening, we have developed a new technique that allows enzymatic production of shRNA libraries from cDNAs extracted from arrested cells. Screening of these libraries uncovered several novel suppressors of anchorageindependent growth.
Cell cycle checkpoints Loss of G1/S control is a frequent if not mandatory event in tumor development. G1/S control relies on the pocket proteins pRB, p107 and p130, which collectively regulate the activity of E2F transcription factors. We use MEFs devoid of pocket proteins (TKO MEFs) to study residual cell cycle control mechanisms and to identify events that promote oncogenic transformation.
Growth-factor independence TKO MEFs still rely on mitogens for survival and proliferation. In the absence of mitogens, many TKO MEFs die whereas surviving cells arrested in the G2 phase of the cell cycle. Suppression of apoptosis by ectopic expression of Bcl2 revealed that G2 arrest was effectuated through induction of the cyclin-dependent93
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V Retèl VP, van der Molen L, Hilgers FJ, Rasch CR, L’Ortye AA, Steuten LMR, Van Harten WH A Cost-effectiveness analysis of a preventive exercise program for patients with advanced head and neck cancer treated with concomitant chemo-radiotherapy. BMC Cancer.2011;11:475
Group leader Wim Van Harten
Early stage technology assessment, operations research and cancer rehabilitation In this group three research topics are covered: Early Stage Technology Assessment, Improving Oncology Services and Cancer Rehabilitation.
Early Stage Technology Assessment Wim Van Harten MD PhD Group leader Lotte Steuten PhD Academic staff Peter Van Berkel Msc Research staff Wineke Van Lent Msc Research staff Arturo Perez Research staff Valesca Retèl MSc Research staff Wim Groen PhD Post-doc Wilma Kuijpers MSc PhD student Janne Mewes PhD student Abinaya Rajan PhD student Anna Miquel Cases PhD student
Publications
Rajan A, Sullivan R, Bakker S, Van Harten WH Critical appraisal of translational research models for suitability in performance assessment of cancer centers. The Oncologist 2012;17:e48-57 Mewes JC, Steuten LMG, IJzerman MJ, Van Harten WH. Effectiveness of multidimensional cancer survivor rehabilitation and cost effectiveness of cancer rehabilitation in general: a systematic review. The Oncologist, 2012;17:1581-93 Van Lent WAM, Deetman JW, Teertstra HJ, Muller SH, Hans EW, Van Harten WH. Reducing the throughput time of the diagnostic track involving CT scanning with computer simulation. Eur J Radiol. 2012;81:3131-40 Retèl VP, Joore MA, Van Harten WH. Head-to-head comparison of the 70-gene signature versus the 21-gene assay: Cost-effectiveness and the effect of compliance. Breast Cancer Research and Treatment, 2012;131:627-36 Koelewijn WT, Ehrenhard ML, Groen AJ, Van Harten WH Intraorganizational dynamics as drivers of entrepreneurschip among physicians and managers in hospitals of western countries. Soc Sci Med. 2012;75:795800
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As healthcare costs are continuously increasing and demographics and technologic developments in oncology cause especially high service – and financial burdens on health systems, sustainability of future oncology services will inevitably become an issue. Gradually we can expect Health Technology Assessment (HTA) not only to be involved in policy- and coverage decisions, but also in an earlier stage in the translational research process. From 2003 through 2006, a technology assessment study was conducted on the introduction of a 70-gene micro array test as a prognostic tool in the treatment of node negative breast cancer (the RASTER-study) and as a side study of the European MINDACT-study. As the diffusion of this technology was in an early stage and the course of development not easy to predict, we chose an early stage evaluation approach that takes the technology dynamics into account, constructive technology assessment (CTA). Most papers from this study have been published or submitted and we hope to end this series by a Cost Effectiveness Analysis on a prospective series. Additionally, in cooperation with the University of Twente, we have initiated an early stage technology assessment of TIL-transfer technology in advanced melanoma. In 2011 Anna Miquel Cases started to work as PhD student on early stage technology assessment in the application of diagnostic/prognostic markers in neoadjuvant breast cancer treatment. This is part of the CTMM program BREASTCARE. We foresee to be increasingly involved in translational research projects.
of this project. This projects aims to draft a pilot Excellence Designation System that is to be piloted in the coming years. Benchmarking is a possibly powerful tool to inform management on improvement options and patients on the quality of services. In 2013 the EU-subsidized project BENCH-CAN will start to develop and pilot a European benchmarking system on benchmarking comprehensive cancer care for which a PhD student will be localised in the NKI.
Rehabilitation, Physical Activity and Cancer Survivorship care and rehabilitation are important elements of a cancer centre’s program. In 2009, a multidisciplinary rehabilitation program was started for breast cancer survivors receiving adjuvant treatment. In addition, a rehabilitation program for head-and-neck cancer patients has been approved by health insurers and was rolled out beginning of 2010. Finally, a major Alpe d’Huzes KWF project was started early 2011, focusing on patient empowerment, return to work, tele-monitoring and implementation of relevant findings and programs related to physical exercise and supported by innovative IT. This program totalling up to 2,8 million euro, will run for 5 years. The IT supported patient empowerment part is staffed with a PhD student and a postdoc and is lead by the NKI. Furthermore, PhD student Janne Mewes is working on the cost effectiveness and budget impact of multidisciplinary/multi faceted rehabilitation inter-ventions and is supervised by WH Van Harten. She is located at the University of Twente.
Operations Improvement in Oncology In cooperation with the University of Twente, PhD student Peter van Berkel worked on a mathematical analysis and scheduling of care pathways within the oncologic hospital setting, and the effect of increased focus on efficient capacity use. Recently a mathematical model was developed using Industrial Engineering techniques for Radiotherapy planning and this will be used to prepare an elaborate project proposal. A study is ongoing on the comparison of characteristics of colorectal surgery pathways using structured efficiency measures and the national colorectal quality registry (DSCA). Together with the University of Twente and the Integraal Kanker Centrum Noord-Oost, a PhD student works on a project to evaluate the added value of accrediting oncology departments in General Hospitals. As part of the Eurocan Platform project, work package 12 is directed towards the development of scientometrics and a designation system for excellent Comprehensive Cancer Centers. PhD student Abinaya Rajan works on the latter part 95
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Division head, group leader Flora Van Leeuwen
Alexander Brandenburg Research assistant Sandra Fase MSc Research assistant Miranda Gerritsma Research assistant Esther Janssen Research assistant Kim Kersten MSc Research assistant Karen Kooijman MSc Research assistant Gabey Ouwens Research assistant Saskia Pelders MSc Research assistant Merian Van Wouwe MD Research assistant
Publications
Cancer epidemiology
The cancer epidemiology group is currently concentrating on three principal research lines: 1) Long-term adverse health outcomes after cancer treatment; 2) Long-term cancer risk after subfertility treatment; 3) The etiology of hormone related cancers.
Late effects of cancer treatment De Haas EC, Altena R, Boezen HM, Zwart N, Smit AJ, Bakker SJ, van Roon AM, Postma A, Wolffenbuttel BH, Hoekstra HJ, van Leeuwen FE, Sleijfer DT, Gietema JA. Early development of the metabolic syndrome after chemotherapy for testicular cancer. Ann Oncol 2012
Research associate Matti Rookus
Flora Van Leeuwen PhD Group leader Matti Rookus PhD Research associate Michael Schaapveld PhD Research associate Mars Van ‘t Veer PhD Hematooncologist Nicky Decker PhD Post-doc Annemieke Opstal-van Winden PhD Post-doc Carmen Paalman PhD Post-doc Anouk Pijpe PhD Post-doc Susanne Rebers PhD Post-doc Sandra Van den Belt-Dusebout PhD Post-doc Frederieke Van der Baan PhD Post-doc Naomi Boekel MSc PhD student Richard Brohet MSc PhD student Harmke Groot MSc PhD student Inge Krul MSc PhD student Lieske Schrijver MSc PhD student Mandy Spaan MSc PhD student Sandra Van den Broek MSc PhD student Anja Van Eggermond MSc PhD student Rianne Van Nimwegen MSc PhD student Janneke Verloop MSc PhD student Cherita Sombroek MSc Junior scientific researcher Thea Mooij MSc Statistical analyst Marie-José Blom MSc Data manager Judith De Lange MSc Data manager Cora Knol MSc Data manager Rosemarie Wijnands MSc Data manager
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Dommering C, Marees T, Hout A, Imhof S, Meijers-Heijboer H, Ringens P, Leeuwen F, Moll A. RB1 mutations and second primary malignancies after hereditary retinoblastoma. Familial Cancer 2012;11:225-233 Elias S, Grooters HG, BauschGoldbohm RA, van den Brandt PA, Kampman E, van Leeuwen FE, Peeters PH, de Vries E, Wigger S, Kiemeney LA. [The Dutch Cancer Society Cancer Risk Test]. Ned Tijdschr Geneeskd 2012;156:A4888 Groeneveld E, Lambers MJ, Stakelbeek MEF, Mooij TM, Belt-Dusebout AW, Heymans MW, Schats R, Hompes PGA, Hoek A, Burger CW, van Leeuwen FE, Lambalk CB. Factors associated with dizygotic twinning after IVF treatment with double embryo transfer. Human Reproduction 2012;27:2966-2970 Hilbers F, Boekel N, van den Broek A, van Hien R, Cornelissen S, Aleman B, van ‘t Veer L, van Leeuwen F, Schmidt M. Genetic variants in TGFβ1 and PAI-1 as possible risk factors for cardiovascular disease after radiotherapy for breast cancer. Radiotherapy and Oncology 2012;102:115-121
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 radiotherapy (RT) and chemotherapy (CT) for Hodgkin’s lymphoma (HL) testicular cancer and breast cancer up to 40 yrs after treatment. As part of a program grant awarded in 2011 a comprehensive assessment is made of the late effects of treatment for HL. In 2012 we assessed the risk of colorectal cancer (CRC), focusing on the risks for colon subsites and rectum, in a cohort of 2,820 5-year HL survivors, treated 1965-1995 (median follow-up, 22 yrs). So far only few studies examined the risk of CRC after HL. The overall standardized incidence ratio (SIR) for CRC was 2.7, with 6.3 excess cases per 10,000 patient-years. The cumulative incidence of CRC risk was 2.1% at 30 years of follow-up. The highest SIR was observed for cancer of the transverse colon (SIR 7.5). Especially patients treated with para-aortic and spleen fields had a strongly increased risk of cancer of the transverse colon (SIR 17.1). In multivariable analyses, patients treated with high-dose procarbazine (>4.2g/m2) had a significantly increased risk of CRC (Hazard ratio (HR) 2.4) compared with patients treated without procarbazine. The risk of CRC was especially high (HR 3.9) for patients who received procarbazine-containing CT and infradiaphragmatic RT, compared with patients receiving none of these treatments. Our previous studies showed strongly increased risk of breast cancer after chest RT for HL, prompting us to investigate whether specific genes affect the magnitude of the risk of RT-induced breast cancer. In collaboration with the Childhood Cancer Survivor Study (US) and the Institute for Cancer Research UK (Prof.dr. A. Swerdlow) we determined 200,000 selected SNPs in 418 HL survivors who developed breast cancer and 520 matched HL survivors without breast cancer. Analyses are ongoing. To study long-term risk of CVD following HL treatment, we are performing a set of case-control studies that aims to quantify separate and joint effects of radiation dose to the heart, anthracycline dose, other CT, life style and established CVD risk factors. So far we included 101 cases with valvular disease, 73 with heart failure, 155 with myocardial infarction and 75 with angina pectoris, each with at least one control. RT dosimetry is being performed in collaboration with Prof. S. Darby and Dr. D.
Cutter, Oxford (UK). Preliminary results on the risk of heart failure after HL show an increased odds ratio (OR) of 3.7 for patients treated with 36 Gy or more at the mediastinum, compared to patients who did not receive mediastinal RT, and an OR of 4.3 for patients treated with a high dose of anthracyclines (>250mg/m2), compared to patients who did not. The Dutch BETTER consortium (Better care after HL: Evaluation of long-Term Treatment Effects and screening Recommendations) develops screening guidelines for HL survivors at risk of late effects of treatment. In 2012 a large grant was obtained from KWF-Alpe d’HuZes to implement the guidelines and to establish an infrastructure for survivorship clinics. This project is coordinated by the NKI-AvL and the UMCN. The national screening guidelines were further detailed. Important steps were made in the organisation of the BETTER outpatient clinics, the database and survivorship care plan that will support these clinics. Additional RT centres were invited to join, resulting in a total of 21 participating centres. The identification of eligible HL survivors has continued and been expanded to include non-Hodgkin lymphoma survivors as well. In 2012, the informative BETTER website about treatment-related adverse effects was used by 9437 visitors. We recently evaluated leukemia risk in our nationwide cohort of 3,800 testicular germ cell tumor (TGCT) patients treated 1965-1995. Several reports have linked receipt of etoposide and cisplatin with increased risk of leukemia. Seventeen patients developed leukemia or the myelodysplastic syndrome (MDS) after a mean interval of 10 yrs, translating into a 10-yr cumulative incidence of 0.24%. Leukemia risk was 1.8-fold increased compared with the general population. Platinumcontaining CT was associated with a 4.8-fold increased risk of acute myeloid leukemia/ MDS. We also evaluated risk of a late recurrence (LR) ≥2 yrs after completion of initial CT in non-seminoma patients. The median time until LR was 6 yrs and the 10-yr cumulative incidence was 2.6% for LR comprising only mature teratoma (MT) and 1.8% for LR with vital germ cell tumor (VGCT). LRs were most frequently detected after patient complaints (43%). The 10-yr cancer specific survival rates after LR were 84% and 64% for patients with MT and VGCT, respectively (p=0.043). It is not yet clear whether platinum-based CT may induce long-term effects. In 2012 we received a KWF-grant to assess the long-term effects of platinum-containing CT on solid second cancer and CVD risk in a large hospital-based cohort, which will include over 10,000 testicular cancer patients treated 1976-2006. In a previous study among breast cancer patients we showed increased risks of cardiac morbidity and mortality in patients treated 1970-1986. To evaluate long-term CVD risk in survivors of breast cancer treated with more contemporary regimens we are conducting two large cohort studies and two nested case-control studies. The first cohort is a population-based cohort of patients with invasive breast cancer (n=97,747) and ductal carcinoma in situ of the breast (DCIS; n=13,545) diagnosed 1989-2004. In 2012, analyses of CVD risk after DCIS were completed. Patients treated with RT to the left breast, who received a higher RT dose to the heart, are not at increased risk for CVD mortality (HR=0.89) or hospital admission for CVD (HR=0.95) compared to patients treated with RT to the right breast.
Jakubowska A, Rozkrut D, Antoniou A, et al. Rookus M, Aalfs CM, Kluijt I, Boessenkool-Pape JL, MeijersHeijboer HE, Oosterwijk JC, van Asperen CJ, Blok MJ, Nelen MR, van den Ouweland AM, Seynaeve C, van der Luijt RB, Devilee P, et al. Mai PL, Loud JT, Lubinski J. Association of PHB 1630 C>T and MTHFR 677 C>T polymorphisms with breast and ovarian cancer risk in BRCA1/2 mutation carriers: results from a multicenter study. Br J Cancer 2012;106:20162024 Jones ME, van Leeuwen FE, Hoogendoorn WE, Mourits MJ, Hollema H, van Boven H, Press MF, Bernstein L, Swerdlow AJ. Endometrial cancer survival after breast cancer in relation to tamoxifen treatment: pooled results from three countries. Breast Cancer Res 2012;14:R91 Kirchhoff T, Gaudet M, Antoniou A, McGuffog L, et al. HEBON, Hogervorst F, Verheus M, Rookus M, Seynaeve C, Oldenburg R, Ligtenberg M, Ausems M, Aalfs C, Gille H, Wijnen J, et al. Chenevix-Trench G, Easton D, GENICA N, on behalf of BCAC/CIMBA. Breast Cancer Risk and 6q22.33: Combined Results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1. PLoS ONE 2012;7:e35706 Knijnenburg S, Jaspers M, van der Pal H, Schouten-van Meeteren A, Bouts A, Lieverst J, Bökenkamp A, Koning C, Oldenburger F, Wilde J, van Leeuwen F, Caron H, Kremer L. Renal Dysfunction and Elevated Blood Pressure in LongTerm Childhood Cancer Survivors. Clinical Journal of the American Society of Nephrology 2012;7:1416-1427 Ma Y, van Leeuwen F, Cooke R, Broeks A, Enciso-Mora V, Olver B, Lloyd A, Broderick P, Russell N, Janus C, Ashworth A, Houlston R, Swerdlow A. FGFR2 genotype and risk of radiationassociated breast cancer in Hodgkin lymphoma. Blood 2012;119:1029-1031 Moll A, Dommering C, Bosscha M, de Graaf P, Kors WA, van Leeuwen F. Risk Factors for the Incidence of Second Cancers in Survivors of Retinoblastoma With a Family History. Journal of Clinical Oncology 2012;30:3028
Morton LM, Gilbert ES, Hall P, Andersson M, Joensuu H, Vaalavirta L, Dores GM, Stovall M, Holowaty EJ, Lynch CF, Curtis RE, Smith SA, Kleinerman RA, Kaijser M, Storm HH, Pukkala E, Weathers RE, Linet MS, Rajaraman P, Fraumeni JF, Brown LM, van Leeuwen FE, Fossa SD, Johannesen TB, Langmark F, Lamart S, Travis LB, Aleman BMP. Risk of treatment-related esophageal cancer among breast cancer survivors. Annals of Oncology 2012;23:3081-3091 Nes JGH, Fontein DBY, Hille ETM, Voskuil DW, Leeuwen FE, Haes JCJM, Putter H, Seynaeve C, Nortier JWR, Velde CJH. Quality of life in relation to tamoxifen or exemestane treatment in postmenopausal breast cancer patients: a Tamoxifen Exemestane Adjuvant Multinational (TEAM) Trial side study. Breast Cancer Res Treat 2012;134:267-276 Overbeek A, van den Berg MH, Kremer LC, van den Heuvel-Eibrink MM, Tissing WJ, Loonen JJ, Versluys B, Bresters D, Kaspers GJ, Lambalk CB, van Leeuwen FE, Dulmen-den Broeder E. A nationwide study on reproductive function, ovarian reserve, and risk of premature menopause in female survivors of childhood cancer: design and methodological challenges. BMC Cancer 2012;12:363 Overbeek A, van den Berg MH, Hukkelhoven CWPM, Kremer LC, Heuvel-Eibrink MM, Tissing WJE, Loonen JJ, Versluys AB, Bresters D, Kaspers GJL, Lambalk CB, van Leeuwen FE, Dulmen-den Broeder E, On Behalf of the DCOG LATER/VEVO Study Group. Validity of self-reported data on pregnancies for childhood cancer survivors: a comparison with data from a nationwide populationbased registry. Human Reproduction 2012 Pijpe A, Andrieu N, Easton DF, Kesminiene A, Cardis E, Nogues C, Gauthier-Villars M, Lasset C, Fricker JP, Peock S, Frost D, Evans DG, Eeles RA, Paterson J, Manders P, van Asperen CJ, Ausems MG, MeijersHeijboer H, Thierry-Chef I, Hauptmann M, Goldgar D, Rookus MA, van Leeuwen FE. Exposure to diagnostic radiation and risk of breast cancer among carriers of BRCA1/2 mutations: retrospective cohort study (GENE-RADRISK). BMJ 2012;345:e5660
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V Ramus S, Antoniou A, Kuchenbaecker K, et al. Rookus M, van Leeuwen F, Meijers-Heijboer H, van Asperen C, van Roozendaal KEP, Hoogerbrugge N, et al. Easton D, Couch F, ChenevixTrench G, on behalf of the Consortium of Investigators of Modifiers of BRCA. Ovarian cancer susceptibility alleles and risk of ovarian cancer in BRCA1 and BRCA2 mutation carriers. Hum Mutat 2012;33:690-702 Rebers S, van der Valk T, Meijer GA, van Leeuwen FE, Schmidt MK. [Consent for the secondary use of human residual tissue: the patient is best served by an 'opting-out' procedure]. Ned Tijdschr Geneeskd 2012;156:A4485 Schaapveld M, van den Belt-Dusebout AW, Gietema JA, de Wit R, Horenblas S, Witjes JA, Hoekstra HJ, Kiemeney LA, Louwman WJ, Ouwens GM, Aleman BM, van Leeuwen FE. Risk and prognostic significance of metachronous contralateral testicular germ cell tumours. Br J Cancer 2012;107:16371643 Sieswerda E, Postma A, van Dalen EC, van der Pal HJH, Tissing WJE, Rammeloo LAJ, Kok WEM, van Leeuwen FE, Caron HN, Kremer LCM. The Dutch Childhood Oncology Group guideline for follow-up of asymptomatic cardiac dysfunction in childhood cancer survivors. Annals of Oncology 2012;23:2191-2198 Van der Pal H, van Dalen E, van Delden E, van Dijk I, Kok W, Geskus R, Sieswerda E, Oldenburger F, Koning C, van Leeuwen F, Caron H, Kremer L. High Risk of Symptomatic Cardiac Events in Childhood Cancer Survivors. Journal of Clinical Oncology 2012;30:1429-1437 Van Dijk I, Cardous-Ubbink M, van der Pal H, Heinen R, van Leeuwen F, Oldenburger F, van Os R, Ronckers CM, Schouten-van Meeteren A, Caron H, Koning C, Kremer L. Dose-Effect Relationships for Adverse Events After Cranial Radiation Therapy in Long-term Childhood Cancer Survivors. International Journal of Radiation Oncology*Biology*Physics
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V For the case-control studies we have collected detailed treatment data for 139 cases with myocardial infarction, 79 congestive heart failure cases and matched controls. From copies of RT charts individual radiation dose to the heart will be estimated (S. Darby, Oxford).
Etiology of hormone-related cancers We investigate the etiology of hormone related cancers in four national cohort studies 1. A cohort of BRCA1/2 and nonBRCA1/2 families (HEreditary Breast and Ovarian cancer study, Netherlands, HEBON Study); 2. A cohort of women treated for subfertility (OMEGA Study); 3. A cohort of women exposed to diethylstilbestrol in utero (DES Netherlands, (DESnet Study); 4. A cohort of nurses (the Nightingale Study) and two international studies on BRCA1/2 mutation carriers (International BRCA1/2 mutation Carrier Cohort Study (IBCCS) and Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA)). In our nationwide cohort study among families with a BRCA1/2 mutation and tested in a clinical genetic center (HEBON study, 7473 family members; www.hebon.nl), we are studying whether 1) hormonal/life-style factors modify cancer risk in BRCA1/2 families, and 2) common genetic alterations are associated with the risk of breast cancer among BRCA1/2 mutation carriers, and 3) treatment effects are different for BRCA1/2 mutation carriers (M. Schmidt, Division of Molecular Pathology). The Hebon collaboration was formalized by a Steering Committee and a collaboration agreement, aiming to involve more centers in the research program and to support future continuation. For a pilot study on a new mixed-mode (webbased/paper-based) enrolment procedure we invited 900 BRCA1/2 mutation carriers. After two reminders the response was 59%. With an improved procedure we invited 27,000 women tested for BRCA1/2 to the study in December 2012, involving the media to increase response. As early life risk factors have been found to be associated with risks of breast and ovarian cancer in the general population, we investigated the role of these risk factors in 1021 Dutch BRCA1/2 mutation carriers. A higher birth order (≥2) compared with being first child was associated with a decreasing risk of postmenopausal breast cancer among both BRCA1 (Hazard Ratio (HR): 0.54) and BRCA2 carriers (HR: 0.41). Having been taller than peers during childhood was associated with a 2.13-fold increased risk of postmenopausal breast cancer. Thus, early life risk factors seem to be involved in the etiology of breast cancer among BRCA1/2 mutation carriers. We continued the coordination of the IBCCS study, and increased the number of BRCA1/2 mutation carriers in this follow-up study to 7514 women of whom 3800 were still unaffected with breast cancer. During follow-up 322 women opted for prophylactic bilateral mastectomy and 231 women were diagnosed with breast cancer. First prospective analyses will start soon. In 1,993 BRCA1/2 mutation carriers we examined the association between diagnostic radiation exposure and breast cancer risk. Exposure to diagnostic radiation before age 30 was associated with an increased risk of breast cancer at dose levels considerably lower than those at which increases have been found in other cohorts exposed to radiation. Any exposure to diagnostic radiation before the age of 30 was associated with a 1.9-fold increased risk of breast cancer, with a dose-response
pattern. The HRs by quarter of estimated cumulative dose <0.0020 Gy, ≥0.0020-0.0065 Gy, ≥0.0066-0.0173 Gy, and ≥0.0174 Gy were 1.63, 1.78, 1.75, and 3.84, respectively. The results of this study support the use of non-ionising radiation imaging techniques (such as magnetic resonance imaging) as the main tool for surveillance in young women with BRCA1/2 mutations. We harmonized the epidemiologic risk factor data for the CIMBA study, 12916 BRCA1/2 carriers (7977 BRCA1 and 4917 BRCA2). Within this group 6464 BRCA1/2 carriers had a history of breast cancer and 1457 a history of ovarian cancer. The iCOGS chip (200k SNPs, selected on associations with hormone-related cancers) became available for analyses on more than 23,000 BRCA1/2 carriers (database in Cambridge). Using pre-iCOGS data 14 independent genetic variants were found to be associated with risk of breast or ovarian cancer in BRCA1 or BRCA2 carriers. The aim of the nation-wide OMEGA study is to assess the risk of hormone-related cancers after fertility treatment, such as ovarian stimulation for in-vitro fertilization (IVF). Preliminary analyses in the OMEGA I cohort (19,146 IVF-treated women (1983-1994) and 6,006 subfertile controls) showed no increased risk of breast cancer or melanoma in IVF-treated women when compared to subfertile women not treated with IVF (HRs 0.99 and 0.79 respectively). Recently, we completed a large mailing of invitations to 17,000 women treated 1995-2000, (OMEGA II project, www.omega-onderzoek.nl). Women were invited to complete an internet-based or paper questionnaire to collect data regarding lifestyle, reproductive factors and health outcomes, including health of offspring. Three invitations resulted in an overall response rate of 54%; 58% in the IVF group and 44% in the subfertile control group. Of the participants, 80% also sent toenail clippings for future extraction of DNA. Three-quarters of the women chose for the internet questionnaire and 25% for a paper version. About 7% of the women refused to participate. The most important reasons for non-participation were emotional reasons or lack of time. Because of their increased risk of clear cell adenocarcinoma of the vagina and cervix women exposed to diethylstilbestrol (DES) in utero have been intensively screened. We evaluated the effectiveness of cytological screening in a case-control study (39 cases with vaginal/cervical cancer and 186 age-matched controls) nested in the DESnet cohort. Women with a Pap smear within 5.5 to 0.5 years preceding (pseudo) diagnosis had no reduced risk of vaginal and cervical cancer (Odds Ratio 1.48) compared to non-screened women. The stage distribution for cervical cancer was slightly more favorable in DES-daughters compared to the general population (stage I in 90% and 77%). We concluded that cytological screening is not effective in preventing cervical/vaginal cancer in DES daughters, although stage at diagnosis might be more favorable. As DES daughters often present with an enlarged transfor mation zone we investigated whether the risk of cervical intra-epithelial neoplasia (CIN) and cervical cancer was increased. In a prospective cohort study of 12,182 DES daughters no increased risk was observed for CIN2+ (CIN grade 2, 3 or cancer) compared to the screened general population (Standardized incidence ratio (SIR) 0.96). The incidence of CIN
grade 1 was borderline statistically significantly increased (SIR=1.5). The incidence rate of CIN (all grades) was largely determined by the number of screening episodes. Thus, DES daughters do not seem to have an increased risk of squamous cell cervical cancer, while the risk of clear cell adenocarcinoma continued to be increased at higher ages. In 2012, we finished recruitment for the Nightingale Study on shift work and breast cancer risk among Dutch nurses. We started processing and cleaning the data collected through the online questionnaire. In total, we recruited 59,945 nurses (31% of invited group), of whom 2011 women reported a history of breast cancer and 39% returned a sample of toenail clippings for future DNA analyses on clock genes. The mean age of the responders was 46.9 years. Eighty percent (N=48,072) indicated to have ever worked in night shifts regularly (i.e. at least 1 night per month for at least 6 months) between 1960 and 2011. The average number of night shifts per month decreased over the past 50 years from 7 night shifts per month to 4 night shifts per month. We are currently writing the first manuscript on the rationale, design, and baseline characteristics of the study population.
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V Van Leeuwen F, Frederiks F, Terweij M, De Vos D, Bakker BM. News About Old Histones - A Role for Histone Age in Controlling the Epigenome. Cell Cycle 2012;11:11-12
Group leader Fred Van Leeuwen
Fred Van Leeuwen PhD Group leader Iris Stulemeijer PhD Post-doc Marit Terweij MSc PhD student Hanneke Vlaming MSc PhD student Tibor Van Welsem Technical staff
Publications
Chromatin Dynamics
Role and regulation of histone methylation
Fred van Leeuwen’s group is interested in understanding the basic principles of epigenetics, the process by which genes are stably switched on or off. Although an identical set of genes is found in every cell in the body they are not all in the same on or off state, which allows each cell to function differently. These patterns of gene activity are also passed on to future cell generations. Thereby, non-genetic changes can have long-term consequences for cellular function. The Van Leeuwen lab is interested in how cells maintain their identity and pass on this epigenetic memory of gene activity to daughter cells through cell division, which can lead to insights into the development of cancer.
Epigenetic regulation of gene expression
Van Bemmel JG, Filion GJ, Rosado A, Talhout W, de Haas M, van Welsem T, van Leeuwen F, van Steensel B. A network model of the molecular organization of chromatin in Drosophila. Mol Cell.2013;49:759-771 Ontoso D, Acosta I, van Leeuwen F, Freire R, San-Segundo PA. Dot1Dependent Histone H3K79 Methylation Promotes Activation of the Mek1 Meiotic Checkpoint Effector Kinase by Regulating the Hop1 Adaptor. PLoS Genetics. 2013;9:e1003262 Menendez-Benito V, van Deventer SJ, Jimenez-Garcia V, Roy-Luzarraga M, van Leeuwen F, Neefjes J. Spatiotemporal analysis of organelle and macromolecular complex inheritance. Proc Natl Acad Sci USA. 2013;110:175-80 Hegnauer AM, Hustedt N, Shimada K, Pike BL, Vogel M, Amsler P, Rubin SM, van Leeuwen F, Guenole A, van Attikum H, Thoma NH, Gasser SM. An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks. EMBO Journal 2012;31:3768-83 Radman-Livaja M, Quan TK, Valenzuela L, Armstrong JA, Van Welsem T, Kim TS, Lee KJ, Buratowski S, van Leeuwen F, Rando OJ, Harzorg, GA A Key Role for Chd1 in Histone H3 Dynamics at the 3’ Ends of Long Genes in Yeast. PLoS Genetics 2012;8:e1002811 Vlaming H, van Leeuwen F. Cross-talk between aging and the epigenome. Epigenomics 2012;4:5-7 100
of RITE to study the spatiotemporal inheritance of organelles in dividing cells.
Switching genes on or off and keeping them in that state involves ‘packaging’ the DNA in the nucleus to form chromatin by wrapping it around proteins called histones. Histones carry different chemical modifications that affect the packaging of chromatin, which in turn affects the ease with which the cellular machinery can access the DNA sequence and regulate gene expression. One of the goals of the van Leeuwen lab is to investigate if and how these accessibility instructions are involved in epigenetic memory. By taking advantage of budding yeast as a model system the van Leeuwen lab is developing new tools to investigate the basic principles of chromatin-based gene regulation.
Histone dynamics and inheritance It is widely believed that the long-lived histone proteins that package eukaryotic genomes can carry non-genetic or epigenetic information and thus transmit information about genome activity from one cell generation to the next. However, inheritance of genomic packaging status is hard to study and still poorly understood. Studies on the behavior of long-lived proteins in dividing cells have been hampered by the lack of biochemical and genetic technologies to analyze them. To solve this problem, we take advantage of budding yeast genetics and develop tools to simultaneously monitor old and new proteins in living cells. One of the tools we developed is RecombinationInduced Tag Exchange (RITE). Using RITE in combination with genomics and proteomics methods, we unexpectedly found that histone protein inheritance can vary along the genome, that histone proteins may walk along active regions, and that histone proteins carrying epigenetic information can be replaced by new naïve histone proteins, potentially erasing information. Since the inheritance and dynamics of histones are expected to influence the epigenetic landscape and epigenetic memory, we are searching for the mechanisms responsible for these processes. We recently developed a barcode-sequencing method (Epi-ID) to systematically identify histone dynamics factors and other chromatin regulators. In collaboration with the Neefjes lab (Division of Cell Biology II) we have applied fluorescent versions
Errors in the chemical modifications of histones, such as acetylation and methylation, can lead to changes in gene expression and cause cancer. We are particularly interested in histone methylation, which plays a critical role in maintaining cell identity and in tumor development. Here we use budding yeast as a discovery platform, and translate our knowledge to mouse models and human cells. We previously discovered a novel histone methyltransferase Dot1, which does not have the SET domain commonly found in histone methyltransferases. Dot1 can add one, two, or three methyl groups to lysine 79 of histone H3 (H3K79), which is located on the top and bottom of the nucleosome core. Dot1 influences heterochromatin structure and the DNA damage response, and has been implicated in oncogenic transformation in mammals. A major goal of our research is to understand the regulation of H3K79 methylation and its function in gene regulation and DNA damage response. Our studies have shown that Dot1 is a non-processive enzyme in vivo. This uncommon mechanism affects the function of the methylated lysine and determines how methylation can be regulated, for example by trans-histone crosstalk to ubiquitination of histone H2B. In yeast, in agreement with the methylation mechanism and the apparent absence of histone H3K79 demethylase activity, the degree of methylation of H3K79 increases progressively on histones and depends on the growth rate, suggesting that this methyl mark constitutes a timer mechanism. Using the RITE tool, we found that genomic locations that show high turnover of histones show lower degrees of H3K79 methylation, indicating that histone turnover and inheritance fine tune the levels of H3K79 methylation.
Figure 1: Model for chromatin-based epigenetic inheritance.
Figure 2: Recombination-induced tag exchange (RITE) distinguishes Old from New chromatin proteins in replicating cells.
Together, our studies are aimed at providing a deeper understanding of the inheritance of protein-based information in dividing cells, a fundamental process not only relevant for cancer, but also for other diseases, evolution, and aging. Figure 3: Epigenetic memory in budding yeast.
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V Westerman BA, Blom M, Tanger E, van der Valk M, Song JY, van Santen M, Gadiot J, Cornelissen-Steijger P, Zevenhoven J, Prosser HM, Uren A, Aronica E, van Lohuizen M. GFAP-CreMediated Transgenic Activation of Bmi1 Results in Pituitary Tumors. PLoS One. 2012;7:e35943
Division head, group leader Maarten Van Lohuizen Maarten Van Lohuizen PhD Group leader Elisabetta Citterio PhD Senior post-doc Karim Nacerdine PhD Post-doc Inka Pawlitzky PhD Post-doc Gaetano Gargiulo PhD Post-doc Anke Sparmann PhD Post-doc Bas Tolhuis PhD Post-doc Nienke De Vries PhD Post-doc Cesare Lancini PhD Post-doc Waseem Akthar PhD Post-doc Michela Serresi PhD Post-doc Joep Vissers MSc PhD student Martijn Koppens MSc PhD student Santiago Gisler MSc PhD student Marleen Blom Technical staff Paulien Cornelissen Technical staff Danielle Hulsman Technical staff Ellen Tanger Technical staff Huub Van Vugt Technical staff
Publications
Posfai E, Kunzmann R, Brochard V, Salvaing J, Cabuy E, Roloff TC, Liu Z, Tardat M, van Lohuizen M, Vidal M, Beaujean N, Peters AH. Polycomb function during oogenesis is required for mouse embryonic development. Genes Dev. 2012;1:26:920 Nacerddine K, Beaudry JB, Ginjala V, Westerman B, Mattiroli F, Song JY, van der Poel H, Ponz OB, Pritchard C, Cornelissen-Steijger P, Zevenhoven J, Tanger E, Sixma TK, Ganesan S, van Lohuizen M. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer. J Clin Invest. 2012;1:122:1920-32 Tolhuis B, Blom M, van Lohuizen M. Chromosome conformation capture on chip in single Drosophila melanogaster tissues. Methods. 2012;58:231-42
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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 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 cancerpredisposed mice to identify cancer-relevant networks of oncogenes and tumor-suppressor genes. Model organisms comprise Mouse and Drosophila.
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 complex (PRC1) is represented by several subcomplexes all carrying the central Bmi1/Ring1B or Mel18/Ring1B ubiquitin E3 ligase that can monoubiquitinate H2A or H2Ax at K119. We focused on key PRC1 and PRC2 enzymes in gain- and loss-offunction studies in mice and Drosophila. Conditional Ring1b and Bmi1 loss-of-function experiments indicate an essential role for maintenance of Pc-G repression in development and stem cell maintenance. An outstanding question is how the activity of PcG enzymes is regulated; we recently obtained evidence that phosphorylation of Bmi1 is required for E3 ligase activity of PRC1 and by mutating the essential phosphorylation sites demonstrated that these are important for Bmi1’s oncogenic capacity (figure 1) and also for its role in double strand break repair. In collaborative experiments with Dr S. Ganesan, New Jersey Cancer Institute, we demonstrated rapid recruitment of a specific subclass of PRC1 to sites of induced DNA damage and using functional assays showed that this contributes to optimal DNA DS break repair. In addition we showed that the deubequitinating enzyme Usp3 can remove the K119ub mark and conditional loss of USP3 in mice leads to increased DNA damage and chromosomal instability and leads to increased cancer incidence. A major question is where and how Pc-G complexes bind to chromatin. After establishing genome wide binding profiles for PRC1 and PRC2 in Drosophila we recently showed that the ± 400 Polycomb domains encompass conserved developmental regulatory genes controlling differentiation. With In vivo 4C (chromatin conformation capture on Chip) we demonstrated that these domains interact in vivo in 3D nuclear space in Drosophila neural tissues.
Connections between Pc-G gene repression, control of stem cell fate and cancer We originally identified Bmi1 as a cMyc-cooperating lymphomainducing oncogene in mice. We have recently exploited new conditional knockout and transgenic mouse models to extend the observed wide-spread Pc-G deregulation to models of solid cancer. This indicated important roles for overexpression of both Bmi1 and Ezh2 in breast- prostate- and non-small cell lung cancer as well as in glioblastoma (figure 2). In contrast, using tissue specific loss of function for Bmi1, Ezh2 or the essential PRC2 component Eed in mice we recently showed profound effects on stem cell/progenitor compartments in the breast, small intestine and brain, often associated with premature induction of proliferation arrest and aberrant differentiation (in several cases at least in part due to induction of the prominent Pc-G target and tumor suppressor Ink4a/Arf), highlighting a dual role for Pc-G in controlling both proliferation and differentiation. To select the critical cancer-inducing Bmi1 target genes among the many Pc-G targets in the genome we performed highthroughput ChIP to define a conserved set of Bmi1 targets in mouse and human glioblastoma cells. We subsequently used these to generate a custom shRNAi library that was used in in vivo glioma-inducing shRNAi screens to select shRNAs that are specifically gained or lost. This robust screening method yielded novel tumor suppressors and candidate oncogenes several of which could be independently validated and which form the basis for investigating possible new intervention strategies exploiting glioma-subtype specific synthetic lethal interactions. In light of the wide spread deregulation of Pc-G in diverse cancers we are currently investigating using both small molecule inhibitors and in parallel inducible shRNAi for Bmi1 and Ezh2 their possible use in preclinical intervention studies in mouse models for basal breast cancer, colon cancer, Glioblastoma and lung cancer.
In vivo genetic screens to identify new groups of collaborating oncogenes or tumor suppressors In close collaboration with Anton Berns, Jos Jonkers, Lodewyk Wessels and D Adams/The Sanger Centre, Hinxton, UK, we have developed high-throughput insertional mutagenesis techniques. The power of this approach as a cancer gene discovery platform is highlighted by screens accelerating hemapoietic tumors induced in wild type, p53 or p19Arf deficient mice. We recently extended these screens to p15Ink4b, p21 and p27 defcient mice and to Pten-deficient mice prone to MMTV-induced mammary tumorigenesis. These screens yielded over 10.000 insertion sites implicating over 300 loci in tumorigenesis and uncovered new pathway-specific oncogenes and candidate tumor-suppressors. Cross species comparative analysis with a large array-CGH dataset of human cancer cell lines revealed both new and novel candidate oncogenes and tumor-suppressor genes. In addition, this screen allowed the identification of both mutual exclusive as well as cooperative interactions between these genes. The role and mechanism of action of several of these new putative oncogenes or tumor suppressors, is under investigation hemapoietic- and mammary fat pad celltransplantation systems.
Figure 1: PTEN-deficient human prostate cancer cells (LNCaP) in which Bmi1 is ablated using inducible shRNAi (+dox) are ‘addicted’ to Bmi1 overexpression as shown with complementation assays using shRNAi insensitive cDNA (Bmi1-WT). Mutation of 3 critical phosphorylation sites (Bmi1-3A) abrogates the proliferation rescue. Illustrating the importance of posttranslational regulation of the Bmi1/Ring1b E3 ligase in cancer.
Figure 2: 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 orthotopically transplanted in the forebrain of recipient mice.
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V Meuleman W, Peric-Hupkes D, Kind J, Beaudry JB, Pagie L, Kellis M, Reinders M, Wessels L, van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence. Genome Res. (in press)
Group leader Bas Van Steensel
Bas Van Steensel PhD Group leader Mario Amendola PhD Post-doc Carolyn De Graaf PhD Post-doc Jop Kind PhD Senior post-doc Alexey Pindyurin PhD Post-doc Joris Van Arensbergen PhD Post-doc Eva Brinkman MSc PhD student Tao Chen MSc PhD student Wouter Meuleman MSc PhD student Joke Van Bemmel MSc PhD student Ludo Pagie PhD Bioinformatician Marcel De Haas Technical staff Sandra De Vries MSc Technical staff Arantxa Rosado BSc Technical staff
Publications
Van Bemmel JG, Filion G, Rosado A, Talhout W, de Haas M, van Welsem T, van Leeuwen F, van Steensel B. A network model of the molecular organization of chromatin in Drosophila. Mol Cell. (in press)
Chromatin Genomics
Chromatin is probably the most complex molecular ensemble in the cell. It consists of genomic DNA together with all directly or indirectly associated protein and RNA molecules. This includes histones, DNA-binding factors (DBFs), the basal transcription machinery and its nascent transcripts, replication and repair machineries that copy and maintain DNA, and many other molecules that interact with any of these components. All of these components work in concert, and cannot be fully understood unless they are studied in their complete context. In addition, the spatial organization of interphase chromosomes is thought to be of key importance for genome expression and maintenance. Yet, this three-dimensional chromosome organization and its impact on gene regulation and other functions are still poorly understood. In order to gain insight into these fundamental processes, we take a broad integrative genomics approach, using both fruit fly and mammalian cells as model systems. We conduct our studies in the living cell, in the context of the entire genome. We develop and apply new genomics techniques to reveal the interplay among many chromatin proteins, and to visualize the architecture of chromosomes inside the nucleus.
Chromatin Protein Discovery Project Pindyurin AV, van Steensel B. Hox in space: gene cluster regulation linked to folding of chromatin. Nucleus 2012;3:118-22 Steglich B, Filion GJ, van Steensel B, Ekwall K. The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe. Nucleus 2012;3:77-87 Kubben N, Adriaens M, Meuleman W, Voncken JW, van Steensel B, Misteli T. Mapping of lamin A- and progerin-interacting genome regions. Chromosoma 2012;121:447-64 Van Luenen HG, Farris C, Jan S, Genest PA, Tripathi P, Velds A, Kerkhoven RM, Nieuwland M, Haydock A, Ramasamy G, Vainio S, Heidebrecht T, Perrakis A, Pagie L, van Steensel B, Myler PJ, Borst P. Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell 2012;150:909-21 De Graaf CA, van Steensel B. Chromatin organization: form to function. Curr Opin Genet Dev. (in press)
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In 2012 we completed the Chromatin Protein Discovery Project, aimed to identify and functionally characterize hitherto unknown chromatin proteins in Drosophila. We selected more than 100 candidate proteins by computational predictions that take into account protein domain structure and interactions with known chromatin proteins. For each of these we generated fullgenome, high-resolution DamID maps, which reveal whether each protein interacts with specific parts of the genome. This yielded informative binding maps of 42 novel proteins. By extensive computational analysis of these binding maps we predicted the functions and molecular interactions for each protein. Several of these predictions were further evaluated by wet-lab experiments. For example, we identified a novel regulator of DNA replication. Thus, this project broadens our view of chromatin by identifying and annotating dozens of novel components.
The network of interactions in chromatin Our previous work has led to the insight that chromatin in Drosophila consists of five principal types, which are defined by distinct combinations of proteins and histone marks. These chromatin types are distributed along the genome in domains that can be >100kb long. We identified a repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and regulate distinct classes of genes (Filion et al, 2010, Cell 143:212-224). What are the mechanisms that drive the formation of these different chromatin types at distinct locations along the
genome? And how does each chromatin type contribute to the regulation of genes? In order to elucidate these mechanisms, we developed several new strategies. First, we use computational methods such as Bayesian Network Inference to construct models of the interaction network, based on genome-wide DamID maps of more than 100 chromatin proteins (Figure 1). Second, we are developing a high-throughput DamID assay to monitor in parallel the changes in binding patterns of dozens of proteins after perturbation of one or more other proteins. Third, together with the Van Lohuizen and Wessels labs, we have developed a new parallel reporter gene assay which enables us to dissect the impact of specific chromatin environments on gene expression for thousands of locations in the genome. This assay is now being used to unravel the interplay of chromatin and local sequence signals in the regulation of gene expression.
Mechanisms and dynamics of genome – nuclear lamina interactions The nuclear lamina (NL) is a fibrous protein sheet at the nucleoplasmic surface of the nuclear envelope. By DamID mapping, we previously found that the genome of mammalian cells is associated with the NL through ~1,300 LaminaAssociated Domains (LADs). These LADs tend to be large (median size 0.5Mb) and harbor thousands of genes that are transcriptionally repressed (Guelen et al 2008, Nature 453:948951). We hypothesize that the NL provides an anchoring scaffold that helps to organize the genome in nuclear space. Some LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. Together with the Wessels lab we have subjected these different LAD types to an extensive analysis. By comparison of mouse and human lamina interaction maps, we found that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this “A/T rule” in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition. Finally, we visualized the fate of LADs in single cells using a novel ‘molecular contact memory’ approach (Figure 2). This revealed that in each nucleus, only ~30% of LADs is positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL is linked to transcriptional repression and H3K9 dimethylation in single cells. We identified the H3K9 methyltransferase G9a as a regulator of NL contacts. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes in relation to gene regulation.
Figure 1: Network model of the interplay among 112 chromatin components in Drosophila cells, obtained by Bayesian network inference using genome-wide maps generated in our lab.
Figure 2: Confocal “molecular contact memory” image of DNA that has recently touched the nuclear lamina.
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V Van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta 2012 (in press) Vens C, Sobol RW. Targeting DNA repair pathways for Cancer Therapy. Cell Death Signaling in Cancer Biology and Treatment; Daniel E. Johnson Editor, Humana Press; 2013:137-180
Group leader Marcel Verheij
Yang TJ, Haimovitz-Friedman A, Verheij M. Anticancer therapy and apoptosis imaging. Exp Oncol 2012;34:269-76
Marcel Verheij MD PhD Group leader Conchita Vens PhD Senior scientist Baukelien Van Triest MD PhD Academic staff Michiel Van den Brekel MD PhD Academic staff Michel Van den Heuvel MD PhD Academic staff Albert Van Hell PhD Post-doc Saurabh Dayal PhD Post-doc Damien Van Berlo PhD Post-doc Burcu Inanc PhD Post-doc Lília Cordeiro Pedrosa PhD student Caroline Verhagen MSc PhD student David Vossen MSc Bioinformatician Monique De Jong MD Resident - PhD student Rosemarie de Haan MD Resident - PhD student Shuraila Zerp MSc Technical staff
Publications
Borst GR, McLaughlin M, Kyula JN, Neijenhuis S, Khan A, Good J, Zaidi S, Powell NG, Meier P, Collins I, Garrett MD, Verheij M, Harrington. Targeted radiosensitization by the Chk1 inhibitor SAR-020106. Int J Radiat Oncol Biol Phys. 2012 (in press) Haimovitz-Friedman A, Yang TI, Thin TH, Verheij M. Imaging radiotherapyinduced apoptosis. Radiat Res. 2012;177:467-82 Rooswinkel RW, van der Kooij B, Verheij M, Borst J. Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1 or Bcl-B. Cell Death Dis. 2012;9:e366 Svilar D, Vens C, Sobol RW. Quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons. J Vis Exp. 2012;66:e4168
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Image-guided Radiotherapy for small animals
Strategies to enhance chemosensitivity and radiosensitivity Targeted radiosensitization Novel Radiotherapy (RT) and chemotherapy (CT) combination strategies aim at achieving tumor-specific sensitization while sparing normal tissue from additional damage. Candidate agents that could enable such “biological” targeting should result in dose intensification in the tumor cells only. We explore and test such novel combination strategies with a focus on DNA repair and response modulation. DNA damage repair inhibitors: We explore the interaction between radiation and inhibitors of DNA repair. A first strategy targets NAD+ biosynthesis to enhance radiation-induced cell death. APO866 is a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the regulation of NAD+ biosynthesis. Inhibition of NAMPT reduces cellular NAD+ levels which impacts on multiple biochemical processes, including regulation of DNA repair, replication, transcription and apoptosis. We investigated the cytotoxic effect of APO866 alone and in combination with radiation in vitro and in vivo. Nanomolar APO866 induces a timeand dose-dependent depletion of cellular NAD+ levels in a panel of human prostate tumor cell lines. In vitro, APO866-mediated reduction of NAD+ levels resulted in enhanced cytotoxicity and, in combination with ionizing radiation, decreased clonogenic survival. Addition of NAD+ rescued the cells from APO866 mediated cell death, demonstrating the essential role of NAD+ depletion in the induction of tumor cell death. In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD+ levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Collectively, our in vitro and in vivo studies have shown that APO866-induced NAD+ depletion enhances radiation response in prostate cancer. Direct PARP inhibitors are also good candidates for combined use with DNA damaging agents. The main mechanism by which both radiation and cisplatin kill tumor cells is by an accumulation of un- or misrepaired DNA damage. PARP inhibitors increase radiation and chemotherapy (Cisplatin) responses in preclinical studies. We have designed 3 phase I-II studies evaluating the safety and tolerability of Olaparib, an effective PARP inhibitor, in combination with (cisplatin-based chemo-) radiotherapy in locally advanced breast cancer, NSCLC and head and neck cancer. The lung trial is recruiting and no dose-limiting toxicities (DLT) have been encountered in the first Olaparib dose level. In support of these trials, biomarkers for the radiosensitization efficacy of PARP inhibitors have been developed and are evaluated.
repair defects, in particular crosslink repair, are common in Head and Neck cancers. We analyzed a panel of 30 HNSCC cell lines and determined crosslinker MMC sensitivity as a functional read out of defects in the Fanconi anemia (FA) pathway or in homologous recombination (HR) driven double strand break repair. A considerable proportion of HNSCC cell lines were MMC hypersensitive. MMC induced G2 blocks and FANCD2ubiquitination defects confirmed that FA gene defects may underlie crosslink sensitivity. Sensitivity to the PARP inhibitor Olaparib further revealed DNA repair defects in this panel. Expression analysis and next generation sequencing revealed mutations and alterations in the FA pathway genes that are likely to be the cause of the observed hypersensitivity. Such defects can be exploited by the use of PARP inhibitors that specifically target FA/HR defected cells. Our data reveal such an opportunity for HNSCC in particular when combined with chemoradiation. Exploitation of tumor specific DNA repair defects for tumortargeted radiosensitization: A proportion of tumors exhibit alterations in the cellular base excision repair/single strand break repair (BER/SSBR) pathway. These are often based on the expression of mutant DNA polymerase beta (polβ) or the overexpression of crucial BER proteins such as AP-endonuclease or polβ. In collaboration with A. Begg we have previously shown that the expression of truncated polymerase dead polβ inhibits BER/SSBR upon radiation. We demonstrated that this inhibition caused the formation of replication associated double strand breaks and exposed the involvement of HR in the repair of these secondarily induced damages. As a consequence these polβmutated cells heavily rely on HR for survival upon radiation. This led us to propose a novel tumor-targeted radio-sensitization strategy by targeting the backup HR pathway in these cells. Within the BER pathway, balanced protein levels are crucial to avoid the accumulation of BER intermediates that could be converted into potentially lethal double strand breaks. Polβ overexpression has been demonstrated to occur frequently throughout different cancer types. We observed an increased radiosensitivity and dependence on HR driven repair pathways in replicating tumor cell lines when overexpressing polβ. Evaluation of tumor-specific radiosensitization strategies and their therapeutic gain: With our recently acquired image-guided mouse irradiator (µIGRT) (Figure 1) we will be able to design new combination strategies that mimic clinical practice, in terms of treatment set-up, scheduling and expected normal tissue toxicity. To assess the therapeutic benefit of any novel RT combination strategy the effects of the combination treatment on healthy tissue need to be evaluated. We are therefore establishing a sensitive mouse RT lung toxicity model to determine changes in radiation-induced toxicity in the lung. Molecular, cellular and functional markers are monitored to allow the assessment of the benefit or risks of novel combination strategies and to test scheduling options in the preclinical setting.
Response prediction Identification of exploitable DNA repair defects: In order to improve radiotherapy, combination strategies should be developed that exploit defects or alterations in the tumors while sparing normal tissue. In a collaborative project (financed by the Verwelius fund) with M. van den Brekel, we found that DNA
In a recent study of early stage larynx tumors from patients treated with radiotherapy alone, Begg et al. found that expression of the putative stem cell marker CD44 was the most significant predictor of local control. This was validated in a separate series of larynx tumors in which CD44 staining
intensity and frequency on a tissue microarray made from pretreatment biopsies correlated with outcome after radiotherapy. We will therefore pursue the relationship of CD44 expression with factors known to influence radiotherapeutic response. In addition to the mRNA profiling studies, Begg et al. have studied both global mRNA and miR expression in a unique panel of 33 head and neck cancer cell lines with known radiosensitivities. Around 200 mRNAs and 7 miRs were found to correlate with radiosensitivity. Many of the 200 mRNAs and the most strongly associated miRs are known to be involved in epithelial to mesenchymal transition (EMT). Interestingly, CD44 has also been linked to EMT. We have carried out miR and mRNA sequencing on formalin fixed paraffin embedded material from patients with T2-3 larynx carcinomas, treated with single modality radiotherapy in the NKI over the last 10 years. In addition, we plan to modify the EMT-inducing miRs in HNSCC cell lines to study the effect on gene expression and radiosensitivity.
Improved drug delivery The cellular plasma membrane is an effective barrier for exogenous compounds to accumulate in the tumor cell. We identified a well-defined class of sphingolipid analogs that effectively overcomes this barrier, catalyzing drug-membrane traversal preferential for tumor cell membranes. A liposomal coformulation of the short-chain lipid N-octanoyl-glucosylceramide (GC) and doxorubicin was applied in a genetically engineered mouse (GEM) breast tumor model (Wcre;Cdh1F/F;Trp53F/F). These tumors, which are resistant to a variety of conventional and biological antitumor therapies, responded to doxorubicin treatment when combined with the membrane modulation strategy. Co-administration of GC generated a sustained anti-tumor response and significantly improved overall survival. Using a nuclear isolation procedure, we showed that the presence of GC enhanced the intracellular tumor accumulation in vivo by a factor 1.9 (p < 0.05). In contrast, the accumulation within normal heart tissue was not elevated. When compared to either clinically available formulations of doxorubicin (free or PEGylated liposomal doxorubicin) a favorable efficacy, pharmacokinetic and toxicity profile was obtained by co-formulation of doxorubicin with GC. In order to elucidate the mechanism of action of the sphingolipid analogues, in vitro and model membrane studies and in silico molecular dynamics simulation experiments were designed. In artificial lipid membranes of well-defined compositions the short-chain sphingolipids enhanced doxorubicin-membrane traversal similar to cell membranes; N-octanoyl-glucosylceramide elevated the translocation rate of doxorubicin by a factor 1.93 (p < 0.05). Molecular dynamics simulations, in collaboration with the University of Groningen, revealed that the energetic barrier for the hydrophilic part of the doxorubicin to translocate to the opposite side of the membrane/inner membrane leaflet reduced by two-fold (p < 0.05) due to the presence of shortchain lipids. This phenomenon of facilitated traversal is explained by a transient hydrophilic gateway (lipidic pore) through which amphiphilic drugs can traverse. Our data suggest that concomitant targeting of the cellular plasma membrane can widen the therapeutic window of classical or newly developed cancer therapeutics. Currently, extensive toxicology studies are carried out to prepare the translation of this concept in a clinical setting. 107
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W Donker M, Straver ME, Rutgers EJ, Valdés Olmos RA, Loo CE, Sonke GS, Wesseling J, Vrancken Peeters MJ. Radioguided occult lesion localisation (ROLL) in breast-conserving surgery after neoadjuvant chemotherapy. European Journal of Surgical Oncology. 2012;38:1218-24
Group leader Jelle Wesseling
Jelle Wesseling MD PhD Group leader Sjoerd Rodenhuis MD PhD Academic staff Petra Nederlof PhD Academic staff Esther Lips PhD Post-doc Petra Ter Brugge PhD Post-doc Jorma De Ronde MSc PhD student Lotte Elshof MD PhD student Petra Kristel Technical staff Lennart Mulder Technical staff
Publications
Bijker N, Donker M, Wesseling J, Heeten den GJ, Rutgers EJ. Is DCIS Breast Cancer, and How Do I Treat it? Curr Treat Options Oncol. 2012 (in press) De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LF. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer. Breast Cancer Res Treat. 2012 (in press) Lips EH, Mukhtar RA, Yau C, de Ronde JJ, Livasy C, Carey LA, Loo CE, Vrancken-Peeters MJ, Sonke GS, Berry DA, Van't Veer LJ, Esserman LJ, Wesseling J, Rodenhuis S, Shelley Hwang E; I-SPY TRIAL Investigators. Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer. Breast Cancer Res Treat. 2012;136:35-43 Koolen BB, Vrancken Peeters MJ, Wesseling J, Lips EH, Vogel WV, Aukema TS, van Werkhoven E, Gilhuijs KG, Rodenhuis S, Rutgers EJ, Valdés Olmos RA. Association of primary tumour FDG uptake with clinical, histopathological and molecular characteristics in breast cancer patients scheduled for neoadjuvant chemotherapy. Eur J Nucl Med Mol Imaging. 2012;39:1830-8
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Rottenberg S, Vollebergh MA, de Hoon B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Lodén M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Research. 2012;72:2350-61 Oonk AM, van Rijn C, Smits MM, Mulder L, Laddach N, Savola SP, Wesseling J, Rodenhuis S, Imholz AL, Lips EH. Clinical correlates of “BRCAness” in triple-negative breast cancer of patients receiving adjuvant chemotherapy. Ann Oncol. 2012;23:2301-5 Jones S, Li M, Parsons DW, Zhang X, Wesseling J, Kristel P, Schmidt MK, Markowitz S, Yan H, Bigner D, Hruban RH, Eshleman JR, Iacobuzio-Donahue CA, Goggins M, Maitra A, Malek SN, Powell S, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N. Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types. Hum Mutat. 2012;33:100-3 Lips EH, Mulder L, de Ronde JJ, Mandjes IA, Vincent A, Vrancken Peeters MJ, Nederlof PM, Wesseling J, Rodenhuis S. Neoadjuvant chemotherapy in ER+ HER2- breast cancer: response prediction based on immunohistochemical and molecular characteristics. Breast Cancer Res Treat. 2012;131:827-36
Molecular pathology of breast cancer
Mechanisms of therapy response and resistance in patient-derived xenograft (PDX) models for BRCA1-deficient triple negative breast cancer We have generated patient-derived xenograft (PDX) models for BRCA1-deficient triple negative breast cancer (TNBC) and used these to test response to alkylating agents and PARP inhibitors. Initially, these models respond well to such treatments, but eventually resistance develops frequently. For the hypermethylated BRCA1-deficient models, we found treatment-induced demethylation of the BRCA1 promoter or complex genetic rearrangements resulting in fusion transcripts with the BRCA1 mRNA downstream of exons of an unrelated gene. Both mechanisms restore full length BRCA1 expression. For a BRCA1-mutated model, resistant tumors harbor additional small deletions restoring the BRCA1 reading frame, similar to the mechanism found in patients. Strikingly, response was not directly correlated to BRCA1 expression in a series of 24 TNBC PDX models treated with cisplatin (collaboration with the Curie Institute, Paris, France). Additional analyses of candidate predictive factors are ongoing.
Development of clinically useful tests to predict chemotherapy response in breast cancer In the CTMM BreastCare project, we aim to develop tests to predict response to preoperative chemotherapy. For this purpose, we have established a large collection of pre- and post-treatment tumor samples from breast cancer patients who received neoadjuvant anthracyline-based chemotherapy. First, we used a previously established MLPA assay (Lips et al., Breast Cancer Res 2011;13:R107) to show that the majority (67%) of triple negative breast cancers from our cohort have a BRCA1like phenotype. Interestingly, we found that BRCA1 methylation and mutation are mutually exclusive events. Second, we identified the SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B genes as chemotherapy resistance markers (collaboration with Lodewyk Wessels). Third, we confirmed that invasive lobular carcinoma is less sensitive than invasive ductal carcinoma to neoadjuvant chemotherapy; however, this difference is dependent on differences in tumor characteristics that are known to determine sensitivity to chemotherapy (collaboration with the USA I-Spy trial). Finally, we compared various definitions of breast cancer molecular subtypes in our set (n=560) of neoadjuvantly treated patients. We found that histological grade, ER, PR, and HER2 were the best predictive factors for chemotherapy response in breast cancer and propose the conventional use of these markers.
lesions appropriately. Therefore, we need to discover features predicting recurrent disease. Using a unique nationwide DCIS cohort with actively collected data of 11,524 patients diagnosed with a DCIS as first cancer between 1989 and 2005, we will assess the risk of developing a subsequent ipsilateral or contralateral invasive BC and BC mortality. We will compare immunohistochemical tumor markers and copy number variation profiles for 500 DCIS and their subsequent invasive BC. Also, we will conduct a nested case-control study in which we will review histopathological characteristics of the same 500 DCIS patients (cases), who developed an ipsilateral invasive BC, and 1000 agematched DCIS patients (controls) who did not develop ipsilateral invasive BC. Subsequently, we will evaluate the association of clinical and histopathological characteristics of the DCIS with risk of developing invasive ipsilateral BC.
Figure 1: Pie chart showing the frequency of the combination of BRCAness characteristics and BRCA1 mutation status in 86 triple-negative breast cancers (TNBCs) from the neoadjuvant series. 76% of all TNBCs were BRCA1-like, which can be explained by a mutation, promoter methylation, or a currently unknown cause. 24% of TNBCs were non-BRCA1like.
Figure 2: Recurrence-free survival for the surrogate intrinsic subtypes based on grade (Log-rank p = 0.003). Luminal A is low or medium grade, Luminal B is high grade.
Finding the balance between overdiagnosis and undertreatment of Ductal Carcinoma in Situ (DCIS) We aim to identify subsets of DCIS patients with a very low risk of developing invasive ipsilateral breast cancer (BC), to refrain from overtreatment, but treat more aggressive 109
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W Huang S, Holzel M, Knijnenburg T, Schlicker A, Roepman P, McDermott U, Garnett M, Grernrum W, Sun C, Prahallad A, Groenendijk FH, Mittempergher L, Nijkamp W, Neefjes J, Salazar R, Ten Dijke P, Uramoto H, Tanaka F, Beijersbergen RL, Wessels LFA, Bernards R. MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-beta Receptor Signalling. Cell. 2012;151:937-50
Group leader Lodewyk Wessels
Lodewyk Wessels PhD Group leader Nicos Angelopoulos PhD Post-doc Nicola Bonzanni PhD Post-doc Sander Canisius PhD Post-doc Theo Knijnenburg PhD Post-doc Magali Michaut PhD Post-doc Andreas Schlicker PhD Post-doc Hayssam Soueidan PhD Post-doc Ewald Van Dyk MSc PhD student Johann De Jong MSc PhD student Jorma De Ronde MSc PhD student Julian De Ruiter MSc PhD student Christine Staiger MSc PhD student Jelle Ten Hoeve MSc Bioinformatician David Vossen MSc Bioinformatician
Publications
Schlicker A, Beran G, Chresta M C, McWalter G, Pritchard A, Weston S, Runswick S, Davenport S, Heathcote K, Alferez Castro D, Orphanides G, French T, Wessels LFA. Subtypes of primary colorectal tumours correlate with response to targeted treatment in colorectal cell lines. BMC Medical Genomics. 2012 (in press) De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LFA. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer. Breast Cancer Res Treat. 2013;137:213-23 Michailidou et al. Large-scale genotyping identifies 38 new breast cancer susceptibility loci. Nature Genetics 2012 (in press)
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Lips EH, Mukhtar RA, Yau C, de Ronde JJ, Livasy C, Carey LA, Loo CE, Vrancken-Peeters MJ, Sonke GS, Berry DA, Van’t Veer LJ, Esserman LJ, Wesseling J, Rodenhuis S, Shelley Hwang E; I-SPY TRIAL Investigators. Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer. Breast Cancer Res Treat. 2012;136:35-43 Biankin AV et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012;491:399-405 Meuleman W, Peric-Hupkes D, Kind J, Beaudry JB, Pagie L, Kellis M, Reinders M, Wessels LFA, van Steensel B. Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence. Genome Res. 2012 De Leeuw R, Flach K, Bentin Toaldo C, Alexi X, Canisius S, Neefjes J, Michalides R, Zwart W. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance. Oncogene. 2012 Middelbeek J, Kuipers AJ, Henneman L, Visser D, Eidhof I, van Horssen R, Wieringa B, Canisius SV, Zwart W, Wessels LFA, Sweep FC, Bult P, Span PN, van Leeuwen FN, Jalink K. TRPM7 is required for breast tumour cell metastasis. Cancer Res. 2012;72:425061 Stiekema J, Boot H, Aleman BM, Wessels LFA, van Sandick JW. Prognostication and prediction using gene expression profiling in oesophageal cancer. Eur J Surg Oncol. 2013;39:17-23 Farazi TA, Brown M, Morozov P, Ten Hoeve JJ, Ben-Dov IZ, Hovestadt V, Hafner M, Renwick N, Mihailovi A, Wessels LFA, Tuschl T. Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next-generation sequencing. Methods. 2012;58:171-87
Bioinformatics and Statistics
The Bioinformatics and Statistics group conducts research in computational biology and performs state-of-the-art analyses of a wide array of data types to further our basic understanding of cancer and to translate these findings to the clinic. Research topics include stratifying tumours into groups with distinct and homogeneous outcome and therapy response; the characterization of genes and pathways involved in tumourigenesis and understanding molecular regulatory and signalling mechanisms. A number of exemplary projects are presented below in more detail.
Predicting drug sensitivity as a logic combination of mutations All cancers arise due to alterations in their genomes. Although insight into the genetic lesions in tumours by genome sequencing does already assist in selecting some drug regimens, it rarely results in disease eradication due to the emergence of drugresistance. More sophisticated combination therapies in which several pathways are targeted simultaneously could alleviate this problem. However, at present we are unable to extract and interpret the necessary information from tumours to predict which drug regimen will be most adequate. In this project we collaborate with the Welcome Trust Sanger Institute and employ the 1000 cancer cell line data set to tackle this problem. This data set comprises large-scale molecular characterization and cancer drug screening of the 1000 cell lines for 400 anti-cancer drugs. The molecular data currently consists of gene expression, copy number, and whole exome sequencing data. We have developed a novel computational approach based on integer programming that infers logic combinations of mutations that predict the observed drug response based on the mutation status of the genes. The use of a logic formalism enables the formulation of intelligible models, allowing rapid hypotheses generation. Our models show that for most drugs, combinations of mutations explain the drug response better than single mutations. Thus far we have identified tissue types where current biomarker-drug combinations are not effective and potential drug combinations that overcome resistance mechanisms.
A scale-space method for detecting recurrent DNA copy number changes Tumour formation is partially driven by DNA copy number changes, which are typically measured using array comparative genomic hybridization (aCGH), SNP arrays and DNA sequencing platforms. Many techniques are available for detecting recurring aberrations across multiple tumour samples including CMAR, STAC, GISTIC and KC-SMART. GISTIC is widely used and detects both broad and focal (potentially overlapping) recurring events. However GISTIC performs FDR control on probes instead of events. We have developed a novel approach, ADMIRE (Analytical Multi-scale Identification of Recurrent Events), a multi-scale Gaussian smoothing approach, for the detection of both broad
and focal (potentially overlapping) recurring copy number alterations. Importantly, FDR control is performed analytically (no need for permutations) on events rather than probes. The method does not require segmentation or calling on the input dataset and therefore reduces the potential loss of information due to discretization. An important characteristic of the approach is that the error rate is controlled across all scales and that the algorithm outputs a single profile of significant events selected from the appropriate scales. We performed extensive simulations on artificial datasets and showcase its utility on the TCGA Glioblastoma SNP-array dataset. Importantly, ADMIRE detects focal events that are missed by GISTIC, including two events involving known glioma tumour-suppressor genes: CDKN2C and NF1.
Pathway construction from the literature and protein-protein interaction networks Many key cellular processes, like cell proliferation or differentiation, are responses to changes in environment. Signals from the cell surface are transmitted downstream by sequential protein interactions, represented as an interaction network. These networks contain signalling pathways, representing a consensus, expert description of the function of a subset of the interactions. The interactions describing these pathways are typically documented in the literature. Reverse engineering signalling pathways from experimental data is very hard, as there are many possible interactions and typically insufficient data to fully support this process. In addition, a large number of published studies need to be surveyed to find support for interactions detected in the data. For that reason, reliable (semi-) automated pathway reconstruction from literature evidence is indispensable. We propose a novel approach, called LINK (Latent Inference of NetworKs), to characterize, compare and build signalling pathways by combining the protein interactome with literature evidence. Our approach is based on Latent Semantic Indexing that identifies key concepts in published abstracts. Latent Semantic Indexing produces a space of manageable dimensionality, in which we can represent individual articles and signalling pathways. We used this factor space to identify pathways by measuring textual similarities between articles describing individual interactions. More specifically, we showed that 1) we are able to distinguish, with high precision, expert-curated signalling pathways from randomly connected components; 2) in this space, articles can be clustered by pathways, and that 3) complete pathways can be recovered from the interactome by expanding a small set of articles describing a subset of the interactions constituting a pathway. LINK offers vastly improved performance over state-of-the-art approaches that do not use literature information. For example, complete pathways can be recovered from an interactome by expanding small seed sets of articles or proteins involved in a pathway. Figure 1 shows how LINK reconstructs the IL-2 signalling pathway from a small starting set of 10 proteins and successfully identifies true pathway interactions between these proteins from all possible interactions known in the interactome. For comparison, Figure 2 depicts a network involving the same set of 34 proteins as in Figure 1 but now including all possible interactions. Our approach is a very useful tool for experimentalists, since it allows reliable checking and expansion of existing pathway hypotheses and also adds value to experimental results by providing a knowledge-based context.
Rigaill GJ, Cadot S, Kluin RJ, Xue Z, Bernards R, Majewski IJ, Wessels LFA. A regression model for estimating DNA copy number data applied to capture sequencing data. 2012;28:2357-65 Van Vliet MH, Horlings HM, van de Vijver MJ, Reinders MJ, Wessels LFA. Integration of clinical and gene expression data has a synergetic effect on predicting breast cancer outcome. PLoS One. 2012;7:e40358 Rottenberg S, Vollebergh MA, de Hoon B, de Ronde JJ, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Loden M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels LFA, Linn SC, Jonkers JJ, Borst P. Impact of intertumoural heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumours. Cancer Res. 2012
Figure 1: LINK reconstruction of the IL-2 signalling pathway. LINK reconstruction starting from 10 proteins (grey symbols) when allowed to grow to 70 proteins. Black (white) nodes are true (false) positives.
Staiger C, Cadot S, Kooter R, Dittrich M, Müller T, Klau GW, Wessels LFA. A critical evaluation of network and pathway based classifiers for outcome prediction in breast cancer, PLoS One 2012;7:e34796 Pérez-Mancera PA, Rust AG, van der Weyden L, Kristiansen G, Li A, Sarver AL, Silverstein KAT, Grützmann R, Aust D, Rümmele P, Knösel T, Herd C, Stemple DL, Kettleborough R, Brosnan JA, Li A, Morgan R, Knight S, Yu J, Stegeman S, Collier LS, ten Hoeve J, de Ridder J, Goggins M, Hruban RH, Biankin AV, Grimmond SM, APGI, Wessels LFA, Wood SA, IacobuzioDonahue CA, Pilarsky C, Largaespada DA, Adams DA, Tuveson DA. The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma, Nature 2012;486:266-70
Figure 2: LINK reconstruction of the IL-2 signalling pathway. A network involving the same set of 34 proteins as in Figure 1 but now including all possible interactions in the interactome. Solid (dashed) edges are true (false) positives.
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Mechanisms of cell cycle control in cancer
Cell duplication in cancer Group leader Rob Wolthuis
Rob Wolthuis PhD Group leader Cornelia Rumpf PhD Post-doc Michiel Boekhout MSc PhD student Linda Clijsters MSc PhD student Erik Voets MSc PhD student Janneke Ogink Technical staff
Publication
Voets E, Wolthuis RMF. Stable government of mitosis by Greatwall: the emperor’s best servant. Mol Cell Biol. 2012;32:1334-6
Cell division forms the deadly essence of cancer and should therefore be a key target of directed therapy. My group aims to understand the mechanisms that govern major transitions in the cell cycle, such as mitotic entry and initiation of DNA replication. Such mechanisms are clearly sensitive to therapeutic intervention, a principle often used in existing cancer therapy. However, genetic variability in cancer cells, combined with redundancies in signal transduction pathways or the involvement of feed-back loops, make drug responses hard to predict. It is our goal to extract the molecular principles of the most critical hurdles in the cancer cell division process. Subsequently, we aim to reveal the level of control of these pathways in specific tumors and design strategies that lethally interfere with cancer growth.
Mechanisms that control cell cycle transitions Cells pause in G2 phase when the conditions are unfavourable for mitosis, such as after exposure to ionizing radiation. The ability to delay cell division is controlled by different converging mechanisms that inhibit the principal mitotic kinase, cyclin B1-CDK1. We try to unravel the parameters that control cyclin B1-CDK1 activity and its effects, i.e. the decision of cells to stay in G2 phase or enter mitosis. For example, we found that regulated inhibition of the PP2A phosphatase, which counteracts the effects of cyclin B1-CDK1, strongly facilitates mitotic entry. In G2 phase, PP2A is inhibited by the human Greatwall kinase, MASTL (Voets and Wolthuis, 2012). By a genome-wide genetic screen for factors that make mitosis partially independent of CDK1, we found a new regulator of CDK1 and PP2A (Voets et al., manuscript in preparation). Once cells are in mitosis, they need to ensure that all the duplicated chromosomes are attached to the mitotic spindle. This is safe-guarded by the mitotic spindle checkpoint, a regulatory pathway that inhibits another key enzyme of our interest, the Anaphase-Promoting Complex (APC/C) ubiquitin ligase. The success of mitosis and chromosome segregation strongly depends on accurate regulation of APC/C and its activator CDC20. APC/C-CDC20 drives the degradation of cyclin B1 and securin, a process that can be followed in live cells as they divide (figure 1). Recently, we also found a new role for the spindle checkpoint in coordinating the transition from mitosis to S-phase, as APC/C-CDC20 directs geminin degradation and the functionality of the DNA replication licensing factor Cdt1 (figure 2). Furthermore, we found that the levels of the DNA replication licensing factor CDC6 are down-regulated by activation of APC/C-CDH1, followed by a novel replication-coupled mechanism that is independent of the APC/C. In return, CDC6 is up-regulated again at the G0/G1 transition by inhibition of the E2F7/E2F8 transcriptional repressors (collaboration with Bart Westendorp and Alain De Bruin, Utrecht).
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Revealing the cell cycle control state of cancer cells Exactly how growth is de-regulated in cancer cells is very hard to monitor. In a HFSP project, we collaborate with a bioinformatics group and an organic chemistry lab to improve identification, definition, and classification of consecutive cell cycle transitions by accounting for a larger spectrum of molecular and intracellular changes. Therefore, we use livecell time-lapse imaging of non-transformed human cell lines, expressing distinct markers of unique features that, either directly or indirectly, define the cell cycle. Acquired image data are processed by new computer-based data extraction methods for automated image recognition and dynamic description of a spectrum of sub-cellular changes. In addition, we develop flexible cell permeable compounds to assign key cell-cycle landmarks without the strict need for genetic engineering. This should enable more advanced ‘cell cycle profiling’ in any given cell type. Next, by analyzing cell cycle markers in tumors, we hope our work will lead to a better understanding of specific prognostic factors of proliferation capacity. Next, we intend to design ways to intervene with newly identified cancer-relevant steps in the cell cycle and predict which pathways will determine the eventual responses to therapy.
Figure 1: Measuring APC/C activity by time-lapse fluorescence microscopy of Cyclin B1 destruction. The top panel shows a cell undergoing mitotic division, followed in time. The bottom panel shows the localization and quantitative levels of fluorescent cyclin B1 in the same cells.
Interfering with cancer cell growth and responses to cell cycle blockage When cells are forced to arrest in the cell cycle, for instance in response to drugs or radiation, they gradually lose fitness. For example, cells that delay in mitosis, e.g. in response to the cancer drug paclitaxel, cannot initiate gene expression to generate a stress response because transcription is actively silenced in mitosis. How rapidly cell cycle-arrested cells deteriorate, could be crucial for the efficacy of cancer treatment. We found that a specific change in the translation initiation machinery resulted in a unique mitotic protein expression program, involving cell cycle genes, chaperones and regulators of apoptosis. This shows that regulated de novo protein synthesis forms a crucial adaptation mechanism when cells are forced to stall in the cell cycle. For this project, which will be continued and further completed in 2013, we are collaborating with the different groups inside and outside the Netherlands Cancer Institute.
Figure 2: Regulation of DNA-replication licensing by spindle checkpointdependent control of geminin levels. Dividing cells stably expressing both geminin-Cherry and Cdt1-Venus were imaged by fluorescence microscopy. Geminin is degraded when the spindle checkpoint is switched off. Cdt1-Venus localization acts as a marker for DNA replication licensing (Clijsters et al, submitted).
Future goals In 2012, thanks to support by the Nefkens Foundation and the Netherlands Cancer Institute, three Applied Precision DeltaVision deconvolution cell imaging systems have been taylor-designed by our group, to address emerging questions in cell division research posed at the NKI. These have been implemented in a new microscopy lab, shared with the group of René Medema, at the Division of Cell Biology. To translate the growing fundamental knowledge of the cell cycle into clinical applications, we will now aim at i) developing markers that rapidly display unique features of cancer cell cycles; ii) identifying the most relevant drugtargets in cancer cell cycles, and iii) understanding feed-back mechanisms and drug-resistance pathways invoked by candidate therapeutic drugs. Altogether, the projects described here build a deeper understanding of cell division control in normal and cancer cells. The obtained insights are applicable in tumor marker development for prognostic and predictive analyses, and may help to translate cell cycle science into cancer therapeutic strategies. 113
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Z De Leeuw R, Flach K, Bentin Toaldo C, Alexi X, Canisius S, Neefjes J, Michalides R, Zwart W. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance. Oncogene. 2012
Translational endocrinology in breast, prostate and endometrial cancer
Magnani L, Carroll J, Zwart W, Palmieri C. ChIPing away at breast cancer. Lancet Oncol. 2012;13:1185-7
Group leader Wilbert Zwart
Wilbert Zwart PhD Group leader Xanthippi Alexi PhD Post-doc Marjolein Droog MSc PhD student Koen Flach MSc PhD student Suzan Stelloo MSc PhD student
Publications
Zwart W, Koornstra R, Wesseling J, Rutgers E, Linn S, Carroll JS. A carrier-assisted ChIP-seq method to enable Estrogen Receptor/chromatin interaction assessment from core needle biopsies of breast tumors. BMC Genomics. 2012 (in press) Desmet CJ, Gallenne T, Alexandre Prieur A, Reyal F, Visser NL, Wittnerg BS, Smit MA, Geiger TR, Laoukili J,Iskit S, Rodenko B, Zwart W, Evers B, Horlings H, Ajouaou A, Zevenhoven J, van Vliet M, Ramaswamy S, Wessels LFA, Peeper DS. Identification of a pharmacologically tractable Fra-1/ ADORA2B axis promoting breast cancer metastasis. Proc Natl Acad Sci U S A 2012 (in press) Mohammed H, D’Santos C, Serandour AA, Ali HR, Brown GD, Atkins A, Rueda OM, Holmes KA, Theodorou V, Zwart W, Robinson JLL, Saadi A, Ross-Innes CS, Chin S, Menon S, Stingl J, Palmieri C, Caldas CS, Carroll JS. Endogenous purification from primary material reveals GREB1 as a key Estrogen Receptor regulatory factor. Cell reports. 2012 (in press) Nautiyal J, Steel JH, Rosell Mane M, Oduwole O, Poliandri A, Alexi X, Wood N, Poutanen M, Zwart W, Stingl J, Parker MG. The transcriptional co-factor RIP140 regulates mammary gland development by promoting the generation of key mitogenic signals. Development. 2012 (in press)
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Krijgsman O, Roepman P, Zwart W, Carroll JS, Tian S, de Snoo FA, Bender RA, Bernards R, Glas AM. A diagnostic gene profile for molecular subtyping of breast cancer associated with treatment response. Breast Cancer Res Treat. 2012;133:37-47 Beelen K, Zwart W, Linn SC. Can predictive biomarkers in breast cancer guide adjuvant endocrine therapy? Nat Rev Clin Oncol. 2012;9:529-41 Middelbeek J, Kuipers AJ, Henneman L, Visser D, Eidhof I, van Horssen R, Wieringa B, Canisius SV, Zwart W, Wessels LF, Sweep FC, Bult P, Span PN, van Leeuwen FN, Jalink K. TRPM7 is required for breast tumor cell metastasis. Cancer Res. 2012;72:4250-61
Our group is interested in hormonal regulation of breast, endometrial and prostate cancer, and how this links with patient response to treatment in the clinic. Drug treatment is often directed at inhibiting hormonal action, but resistance is common. Our group combines cell biology, genomics, endocrinology and clinical information to determine which patients would benefit the most from a specific hormonal treatment.
Primary breast tumor genomics that enables personalized endocrine treatment 75% of all breast tumors are of the luminal subtype and their proliferation is thought to depend on activity of the estrogen receptor α (ERa). Inhibition of ERa by anti-estrogens (e.g. tamoxifen) or aromatase inhibitors is therefore a major treatment modality for these tumors. Still, relatively little is known about the molecular determinants of aromatase inhibitor response. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we mapped in primary breast tumor tissue the genome-wide chromatin binding profiles of the estrogen receptor alpha (ERa) and epigenetic modifications H3K4me3 and H3K27me3 that dictate gene (in)activation. Differential binding patterns were found between patients with a good and poor disease outcome after treatment with aromatase inhibitors. Integrating these findings with gene expression and progressionfree survival data enabled us to identify distinct epigenetic and ERa/chromatin binding profiles that hallmark outcome in breast cancer patients who received first-line treatment with aromatase inhibitors. These data could be validated in a second cohort of aromatase inhibitor-treated breast cancer patients. Classifications based on mRNA expression levels of genes proximal to altered ERa and H3K27me3 binding events showed significant correlation with progression-free survival, while this was not observed for H3K4me3. Expression of proximal genes did not correlate with survival after receiving tamoxifen, illustrating a treatment-selective classification.
Estrogen Receptor genomics and tamoxifen resistance Using ChIP-seq, we found a selective subgroup of 111 ERαresponsive genes under the control of the coregulator SRC3, which could be used as a classifier for tamoxifen resistance (Zwart et al., EMBO J 2011;30:4764-76). Prediction-analysis-ofmicroarrays (PAM) enabled us to refine this gene list to only 4 genes, which (also after multivariate corrections) still showed significant correlations with progression-free survival using two independent tamoxifen-treated cohorts of breast cancer patients. Each of these 4 genes directly influenced ERα activity and siRNA targeting resulted in an inhibition of ERα-mediated cell proliferation, gene expression and transcription complex formation. We are currently setting up immunohistochemical stainings to assess correlations with survival after tamoxifen treatment using standard pathological assays.
5-10% of ERα positive tumors are also positive for the human epidermal growth factor 2 (HER2). HER2 signaling is frequently implicated in endocrine therapy resistance. Most reports focus on HER2-mediated kinase signaling, neglecting its possible influence on hormonal signaling and the transcriptional program of ERα. ChIP-seq analysis of MCF-7 cells with doxycyclineinducible HER2 expression revealed a substantial increase of ERα binding sites in HER2 overexpressing MCF-7 cells, specifically upon tamoxifen treatment. We are currently correlating differential ERα chromatin binding with HER2mediated changes in mRNA synthesis. Our hypothesis is that HER2 alters the transcriptional repertoire of ERα by reprogramming ERα binding patterns, thereby activating pathways that drive endocrine resistance.
Tamoxifen-induced endometrial cancer Tamoxifen is the most frequently prescribed drug for ERαpositive breast cancer, but it does have side effects. The drug inhibits ERα in luminal breast cancer cells, but it stimulates the receptor in endometrial cells, resulting in a 7-fold increased risk for endometrial cancer (Bergman et al., Lancet 2000;356:8817). We are studying the mechanisms underlying the tissuespecific effects of tamoxifen, with the aim to identify biomarkers that predict risk for tamoxifen-induced endometrial cancer development. In breast cancer cells, the transcriptional regulator FOXA1 is essential for ERa function. FOXA1 enables ERa/chromatin interactions, ERa responsive gene activation and subsequent estradiol-driven cell proliferation (Hurtado et al., Nat Genet 2011;43:27-33). Importantly, ERa apparently uses an alternative mechanism for its chromatin interactions and functionality in endometrial cells, which do not express FOXA1. Using ChIPseq on endometrial cancer cell lines and primary tumor tissue, we found that FOXA2 plays a central role in ERa biology in endometrial cells. Analogous to FOXA1 in breast cancer cells, FOXA2 binds to ERa/chromatin interaction sites in the absence of hormone. Targeting FOXA2 by siRNA in endometrial cancer cells greatly diminished ERa/chromatin interactions, leading to inhibition of tamoxifen-induced gene activation and proliferation. Next, we analyzed the clinical implications of FOXA2 in tamoxifeninduced endometrial cancer. Gene expression data was analyzed from endometrial tumors, from breast cancer patients who were either or not treated with tamoxifen. Tumors were stratified over high versus low levels of FOXA2, where patients with high endometrial FOXA2 levels had a significantly shorter interval between breast surgery and endometrial cancer development (p<0.0001; HR=3.0, 95% CI: 1.7-5.3). Importantly, this correlation with FOXA2 levels was not observed for patients who did not receive tamoxifen treatment. Thus, we found that the tamoxifenstimulatory effects on ERa and endometrial tissue is dictated by FOXA2 and that its expression levels correlate with an increased risk for tamoxifen-induced endometrial cancer development.
Figure 1: Estrogen Receptor/chromatin binding patterns hallmark aromatase inhibitor resistance. (A) Example ERα binding sites in tumor tissue, enriched between good (left panel) and poor (right panel) outcome. (B) Clustering analyses on enriched binding sites shows correlation between Good and Poor outcome patients. (C) Genes with altered proximal ERα binding sites enable the identification of breast cancer patients with a poor survival after aromatase inhibitor treatment.
Figure 2: High FOXA2 mRNA expression levels correlate with an increased development of endometrial cancer after receiving tamoxifen for breast cancer treatment. 115
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Division head Marcel Stokkel
DEPARTMENT OF CLINICAL CHEMISTRY Willem Nooijen PhD Academic staff Daan Van den Broek PhD Academic staff Olaf Van Tellingen PhD Academic staff Tiny Korse PhD Research associate Levi Buil Technical staff Marian Buning-Kager Technical staff Dorothé Linders Technical staff Lin Fan PhD student DEPARTMENT OF NUCLEAR MEDICINE Else Aalbersberg MSc Research fellow Bernies Van der Hiel MD Academic staff Cornelis Hoefnagel MD PhD Academic staff Saar Muller PhD Academic staff Michiel Sinaasappel PhD Academic staff Ferida Sivro-Prndelj MD Academic staff Marcel Stokkel MD PhD Academic staff Renato Valdés Olmos MD PhD Academic staff Erik Vegt MD PhD Academic staff Linda de Wit-van der Veen MSC Academic staff Wouter Vogel MD PhD Academic staff Oscar Brouwer MD Clinical Research fellow Bas Koolen MD Clinical Research fellow Jacob Kist MD Clinical Research fellow Saskia Baank Technical staff Martine Bakker Technical staff Natascha Bruin Technical staff Christel Feenstra Technical staff Danielle Gardeniers Technical staff Bert Pool Technical staff Yvonne Pluister Technical staff Chelvi Mylvaganan Technical staff Kirsten Peen Technical staff Lyandra Rooze Technical staff Mariska Sonneborn Technical staff Colinda Vroonland Technical staff
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DEPARTMENT OF RADIOLOGY Peter Besnard MD Academic staff Annemarie Bruining MD Academic staff Kenneth Gilhuijs PhD Academic staff Wim Koops MD Academic staff Charlotte Lange MD Academic staff Robert Kröger MD Academic staff Claudette Loo MD Academic staff Saar Muller PhD Academic staff Warner Prevoo MD Academic staff Michiel Sinaasappel PhD Academic staff Jelle Teertstra MD Academic staff Stijn Heijmink MD Academic staff Philip Pevenage MD Academic staff Amir Yazdi MD Academic staff Gonneke Winter-Warnars MD PhD Academic staff Fijs Van Leeuwen PhD Academic staff Ingrid Westerveld-de Zwart MD PhD Academic staff Patrick Chin PhD Post-doc Saske Hoving PhD Post-doc Joeri Kuil PhD Post-doc Anton Bunschoten PhD Post-doc Edoardo Pasca PhD Post-doc Wen Qi PhD Post-doc Christian Siedschlag PhD Post-doc Ingar Seemann PhD student Lukas Batteau Technical staff Tessa Buckle Technical staff Anita Paape Technical staff Maddalena Rossi Technical staff Chantal Beekman Technical staff DEPARTMENT OF PATHOLOGY Hester Van Boven MD PhD Head Mijke Bol PhD Academic staff Olga Balague Ponz MD PhD Academic staff Annegien Broeks PhD Academic staff Frans Hogervorst PhD Academic staff Jeroen De Jong MD PhD Academic staff Jettie Muris MD PhD Academic staff Petra Nederlof PhD Academic staff Renée Van Pel MD Academic staff Efraim Rosenberg PhD Academic staff Loes Van Velthuysen MD PhD Academic staff Jelle Wesseling MD PhD Academic staff Bart Van de Wiel MD Academic staff Joyce Sanders MD Academic staff Lucie Boerrigter-Barendsen Technical staff Aafke Wieringa-Ariaens Technical staff Henrique Ruijter-Schippers Technical staff Esther Scheerman Technical staff Ivon Tielen Technical staff Miranda Van Dongen Technical staff Els Verhoeven Technical staff Cristiane Bentin Toaldo Technical staff Tjitte Rijpkema Technical staff Jan-Nico Ridderbos Technical staff Daoin Hunneman Technical staff Candy Van Riel Technical staff Pilar Salinas Technical staff Donné Majoor Technical staff Linde Braaf Technical staff Ingrid Hofland Technical staff Renate De Groot Technical staff Marcel Winter Technical staff Dennis Peters Technical staff Huib Vroege Medical Informatician
Division of Diagnostic Oncology
responsive when ABT-888 is given in combination with elacridar. We also investigated the in vivo relevance of the finding that sildenafil was reported to be an inhibitor of Abcb1 and Abcg2. Sildenafil was not able to enhance the brain penetration of two substrate drugs (docetaxel and topotecan) and did not enhance the efficacy of doxorubicin against Abcb1 expressing CT-26 tumor cells in vivo. Sildenafil did enhance the plasma levels of the cytotoxic drugs. Our results do not support further clinical testing of sildenafil.
Department of clinical chemistry Pharmacological studies in mice Lin Fan, Levi Buil, Nishita Thoti, Eloy Moreno Roig, Lisette Hoogendijk, Diana Hanekamp and Olaf van Tellingen
We have developed/available a series of preclinical models that will allow us to test the usefulness of agent targeting aberrant pathways in high-grade gliomas/glioblastoma multiforme (HGG/ GBM). PARP inhibitors, such as ABT-888 interfere with the base excision repair pathway (BER) and may therefore synergize with temozolomide chemoradiation therapies as used for treatment of HGG/GBM. With these models we have identified two factors that may be important with respect to the clinical use of this agent. The first factor is the expression of Abcb1 (P-glycoprotein) and Abcg2 (BCRP). These ABC transporters are present at the BBB and also in a subpopulation of GBM tumor cells. ABT-888 is a good substrate of both transporters. We now found that the dual inhibitor elacridar is increasing the brain penetration to the level in Abcg2;Abcb1 KO mice without altering the systemic exposure. The efficacy of ABT-888 and temozolomide was better against intracranial tumors growing in Abcb1/Abcg2 knockout mice than in WT mice. Importantly, concomitant use of elacridar further enhanced the efficacy. BCRP was not only expressed in tumor vessels, but also in a subset of tumor cells. Most likely, elacridar also inhibits these drug efflux transporters. Because TMZ is also to some extent a substrate of both transporters, elacridar may also help this drug to reach into the tumor cells. The second factor is the PTEN status of the tumor cells. Previous in vitro studies demonstrated that PTEN deficient astrocytes have a compromised homologous recombination repair (HRR) pathway. Simultaneous abrogation of both HRR and BER pathways creates a synthetic lethality when cells are exposed to DNA damaging therapies. Accordingly, our PTEN deficient GBM-derived cell lines are much more vulnerable to TMZ + ABT-888 in vitro than PTEN proficient cells. We took advantage of the fact that we could further investigate the relevance of these findings using our in vivo models. PTEN deficient GBM696677 cells were injected in WT and KO mice and treated with TMZ and ABT-888 (without elacridar). PTEN deficient tumor cells are also more responsive to TMZ/ABT-888 in vivo than PTEN proficient cells. Notably, the effect-size was not so dramatic as seen in vitro, reminding us that the tumor environment of GBM cells proliferating in vivo may help these cells to escape from the lethal effects of drugs.
We developed new bio-analytical assays in order to support preclinical studies. One assay involves the PI3K-mTOR inhibitor NVP-BEZ 235. We discovered that this compound had excellent fluorescence properties, which allowed us to design a highly sensitive method. Similarly, the ABCG2 inhibitor Ko143 also had good fluorescence properties that allowed sensitive detection. Notably, the stability of Ko143 was adversely affected by plasma esterases, but this could be tackled by use of NaF and cold storage. We also developed an HPLC-fluorescence assay for compound Alfa, an agent that was selected from a screen to target Brca1 deficient tumor. This compound was found to have a remarkable brain penetration. Since a few months we have available an API3000 mass spectrometer, which will greatly facilitate setting up bioanalytical assays in the future. We already used this system to setup a method for the MPS-1 inhibitor NMS-5, which was accomplished in less than two weeks.
THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC Frans Hogervorst PhD Academic staff, head Diagnostic Laboratory PFT Efraim Rosenberg Academic staff Laura Van ’t Veer PhD Academic staff Irma Kluijt MD Academic staff Marielle Ruijs MD PhD Academic staff Lizet Van der Kolk MD PhD Academic staff Senno Verhoef MD PhD Academic staff Eveline Bleiker PhD Academic staff Daniela Hahn Academic staff Petra Nederlof PhD Academic staff Merel Maiburg MD Academic staff Emma Van der Riet MD Academic staff Libertje Bosma MD Academic staff Sophie Van der Velden Genetic associate Anja Van Rens Genetic associate Daoud Ait Moha Research assistant Kiki Jeanson Research assistant Mohamed Achachah Technical staff Roelof Pruntel Technical staff Majella Boutmy-de Lange Technical staff Esther Scheerman Technical staff Mobien Kasiem Technical staff Abderrahim Ajouaou Technical staff
Publications
Neuroendocrine tumours In collaboration with the Comprehensive Cancer Center (Utrecht, Otto Visser) and the Pathology (Loes van Velthuysen) Many epidemiological studies on neuroendocrine tumors (NET) show a major increase in incidence. To investigate this increase, epidemiological data from the Netherlands were evaluated according to histological grade. 47,800 patients with NET (diagnosed 1990-2010) from the population-based Netherlands Cancer Registry were stratified according to the latest WHO classification: well-differentiated NET grade 1 and 2 (G1NET and G2NET), and poorly differentiated (grade 3) neuroendocrine carcinoma, subdivided in large cell (G3-LCNEC) and small cell (G3-SCNEC). The age-standardized incidence rate (excluding G3-SCNEC) increased from 2.1/100,000 in 1990 to 4.9/100,000 in 2010. The incidence of G1NET increased from 2.0 to 3.0; there was a large increase in G2NET from 0.01 in 1990 to 0.20 in 2010, and of the G3-LCNEC from 0.01 to 1.8. In G3-SCNEC incidence in men decreased from 21.3 to 10.1, whereas in women it increased from 4.5 to 7.7. The 5-year survival improved in G1NET, particularly in metastatic disease, from 30% in 19902000 to 47% in 2001-2010. We concluded that the increasing incidence of NET (without G3-SCNEC) was mainly due to the increase of G3-LCNEC. This increase is related to improved diagnostic procedures and to shifting in pathology from other entities, such as undifferentiated carcinoma, to NET. Improved survival was seen in all sites and stages, especially in patients with metastatic G1NET.
Alvarez Paez AM, Brouwer OR, Veenstra HJ, van der Hage JA, Wouters M, Nieweg OE, Valdés-Olmos RA. Decisive role of SPECT/CT in localization of unusual periscapular sentinel nodes in patients with posterior trunk melanoma: three illustrative cases and a review of the literature. Melanoma Res 2012;22:278283 Antoniou AC, [… ]; CIMBA, SWEBRCA; HEBON; EMBRACE; GEMO Collaborators Study; kConFab Investigators. Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. Breast Cancer Res 2012;14:R33 Van den Berg NS, Brouwer OR, Klop WM, Karakullukcu B, Zuur CL, Tan IB, Balm AJ, van den Brekel MW, Valdés Olmos RA, van Leeuwen FW. Concomitant radio- and fluorescenceguided sentinel lymph node biopsy in squamous cell carcinoma of the oral cavity using ICG-(99m)Tcnanocolloid. Eur J Nucl Med Mol Imaging 2012;39:1128-36
Betsalel OT, Pop A, Rosenberg EH, Fernandez-Ojeda M. Creatine Transporter Research, Group, Jakobs C, Salomons GS. Detection of variants in SLC6A8 and functional analysis of unclassified missense variants. Mol Genet Metab 2012;105:596-601 Bex A, Brouwer OR, Valdés Olmos RA. Preoperative and intraoperative lymphatic mapping for radioguided sentinel node biopsy in kidney and bladder cancers. In: Mariani G, Manca G, Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: Springer-Verlag, 2012:285-293 Bijker N, Donker M, Wesseling J, den Heeten GJ, Rutgers EJ. Is DCIS Breast Cancer, and How Do I Treat it? Curr Treat Options Oncol. 2012 de Bonilla-Damiá A, Roberto Brouwer O, Meinhardt W, ValdésOlmos RA. Lymphatic drainage in prostate carcinoma assessed by lymphoscintigraphy and SPECT/CT: its importance for the sentinel node procedure. Rev Esp Med Nucl Imagen Mol 2012;31:66-70 Borovski T, Beke P, van Tellingen O, Rodermond HM, Verhoeff JJ, Lascano V, Daalhuisen JB, Medema JP, and Sprick MR. Therapy-resistant tumor microvascular endothelial cells contribute to treatment failure in glioblastoma multiforme. Oncogene 2012 Brouwer van den Berg NS, Brouwer OR, Klop WM, Karakullukcu B, Zuur CL, Tan IB, Balm AJ, van den Brekel MW, Valdés Olmos RA, van Leeuwen FW. Concomitant radio- and fluorescenceguided sentinel lymph node biopsy in squamous cell carcinoma of the oral cavity using ICG-(99m)Tc-nanocolloid. Eur J Nucl Med Mol Imaging. 2012;39:1128-1136 Brouwer OR, Donker M, Woerdeman LA, Vrancken Peeters MJ. [Local recurrence after skin-sparing mastectomy]. Ned Tijdschr Geneeskd 2012;156:A4692 Brouwer OR, Buckle T, Vermeeren L, Klop WM, Balm AJ, van der Poel HG, van Rhijn BW, Horenblas S, Nieweg OE, van Leeuwen FW, Valdés Olmos RA. Comparing the hybrid fluorescentradioactive tracer indocyanine green-99mTc-nanocolloid with 99mTc-nanocolloid for sentinel node identification: a validation study using lymphoscintigraphy and SPECT/CT. J Nucl Med 2012;53:1034-1040
Together, our results suggest that patients whose tumor is deficient in PTEN (40% of all GBMs) may be most responsive to temozolomide treatment. Other tumors may become more 119
Brouwer OR, Buckle T, Bunschoten A, Kuil J, Vahrmeijer AL, Wendler T, Valdés-Olmos RA, van der Poel HG, van Leeuwen FW. Image navigation as a means to expand the boundaries of fluorescence-guided surgery. Phys Med Biol 2012;57:3123-3136 Brouwer OR, van Leeuwen FWB, van der Poel HG, Horenblas S, Valdés Olmos RA, Meinhardt W. De schildwachtklierprocedure bij prostaatkanker. Tijdschrift voor Urologie 2012;2:84-91 Brouwer OR, Vermeeren L, van der Ploeg IM, Valdés Olmos RA, Loo CE, Pereira-Bouda LM, Smit F, Neijenhuis P, Vrouenraets BC, Sivro-Prndelj F, Jap-a-Joe SM, Borgstein PJ, Rutgers EJ, Oldenburg HS. Lymphoscintigraphy and SPECT/CT in multicentric and multifocal breast cancer: does each tumour have a separate drainage pattern? Results of a Dutch multicentre study (MULTISENT). Eur J Nucl Med Mol Imaging 2012;39:1137-1143 Brouwer OR, Klop WM, Buckle T, Vermeeren L, van den Brekel MW, Balm AJ, Nieweg OE, Valdés Olmos RA, van Leeuwen FW. Feasibility of sentinel node biopsy in head and neck melanoma using a hybrid radioactive and fluorescent tracer. Ann Surg Oncol 2012;19:1988-1994 Brouwer OR, Meinhardt W, Horenblas S, Valdés Olmos RA. Preoperative and intraoperative lymphatic mapping for radioguided sentinel node biopsy in cancers of the male reproductive system. In: Mariani G, Manca G, Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: SpringerVerlag, 2012:269-283 Buckle T, Brouwer OR, Valdés Olmos RA, van der Poel HG, van Leeuwen FW. Relationship between intraprostatic tracer deposits and sentinel lymph node mapping in prostate cancer patients. J Nucl Med 2012;53:1026-1033 Buckle T, van Berg NS, Kuil J, Bunschoten A, Oldenburg J, Borowsky AD, Wesseling J, Masada R, Oishi S, Fujii N, van Leeuwen FW. Noninvasive longitudinal imaging of tumor progression using an (111)indium labeled CXCR4 peptide antagonist. Am J Nucl Med Mol Imaging 2012;2:99-109 Chen W, Stroom J, Sonke JJ, Bartelink H, Schmitz AC, Gilhuijs KG. Impact of negative margin width on local recurrence in breast conserving therapy. Radiother Oncol 2012;104:148-154
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Couch FJ et al. Consortium of Investigators of Modifiers of BRCA1/2. Common variants at the 19p13.1 and ZNF365 loci are associated with ER subtypes of breast cancer and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev. 2012 Apr;21(4):645-57 Dekker TJ, Borg ST, Hooijer GK, Meijer SL, Wesseling J, Boers JE, Schuuring E, Bart J, van Gorp J, Mesker WE, Kroep JR, Smit VT, van de Vijver MJ. Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach. Breast Cancer Res 2012;14:R93 Deurloo EE, Sriram JD, Teertstra HJ, Loo CE, Wesseling J, Rutgers EJ, Gilhuijs KG. MRI of the breast in patients with DCIS to exclude the presence of invasive disease. Eur Radiol. 2012 Donker M, Straver ME, Rutgers EJ, Valdés Olmos RA, Loo CE, Sonke GS, Wesseling J, Vrancken Peeters MJ. Radioguided occult lesion localisation (ROLL) in breast-conserving surgery after neoadjuvant chemotherapy. Eur J Surg Oncol 2012;38:1218-1224 Emmering J, Vogel WV, Stokkel MP. Intramuscular metastases on FDG PETCT: a review of the literature. Nucl Med Commun 2012;33:117-20 Evers DJ, Nachabé R, Hompes D, van Coevorden F, Lucassen GW, Hendriks BH, van Velthuysen ML, Wesseling J, Ruers TJ. Optical sensing for tumor detection in the liver. Eur J Surg Oncol 2013;39:68-75 Evers DJ, Nachabé R, Klomp HM, van Sandick JW, Wouters MW, Lucassen GW, Hendriks BH, Wesseling J, Ruers TJ. Diffuse reflectance spectroscopy: a new guidance tool for improvement of biopsy procedures in lung malignancies. Clin Lung Cancer 2012;13:424-431 Geyer FC, Lacroix-Triki M, Colombo PE, Patani N, Gauthier A, Natrajan R, Lambros MB, Khalifeh I, Albarracin C, Orru S, Marchiò C, Sapino A, Mackay A, Weigelt B, Schmitt FC, Wesseling J, Sneige N, Reis-Filho JS. Molecular evidence in support of the neoplastic and precursor nature of microglandular adenosis. Histopathology 2012;60:E115-30 Ghoussaini M, […] Easton DF. Genomewide association analysis identifies three new breast cancer susceptibility loci. Nat Genet 2012;44:312-318
Neuroendocrine tumours In collaboration with the Division of Medical Oncology (Margot Tesselaar, Lisette Saveur) and medical student (Anouk Keizer) The diagnosis of neuroendocrine tumors (NET) is often delayed due to the vague symptoms and rareness of the disease. We investigate the association of delay in diagnosis and stage, grade, symptoms, primary site of the tumor and survival in patients with low-grade NET. We collected data of 216 patients who were referred to the NKI-AVL between 2003 and 2009. The start date and nature of the first symptoms, the first referral to a specialist and the date of NET diagnosis were reported in order to calculate two types of delay: patient (first symptoms to first referral to the specialist) and total delay (first symptoms to diagnosis). The median patient and total delay were 7 (0-296) and 17 (0-296) months, respectively. There was no association between survival and the delays. A NET of the small bowel showed longer delay compared to other sites. Patients with a longer delay were more likely to have psychological problems.
Non-small cell lung cancers In collaboration with the Division of Medical Oncology (Michel vd Heuvel, Wilma Uyterlinde), Department Biometrics (Andrew Vincent) and MAASTRO Clinic (Maastricht, Cary Oberije) In all patients with cytological or histological proven locally advanced NSCLC treated with concurrent chemoradiation between 2008 and 2010, we investigated the relevance of tumor markers in addition to clinical parameters and tumor characteristics. We measured Cyfra 21.1, CEA, NSE, SCC, cytokeratine fragments (CK8, CK18 and CK19) and IL-6 before, during and after treatment. Prediction models as developed in our group will be compared with the patient chort from Maastricht en vice versa.
Mesothelioma In collaboration with the Division of Medical Oncology (Paul Baas, Wieneke Buikhuisen) and Department of Biometrics (Andrew Vincent) As secondary endpoint in the randomized phase III study testing thalidomide as new second-line treatment, we measured VEGF, bFGF, IL6, mesothelin and Cyfra21.1 in patients with malignant pleural mesothelioma. We investigated their prognostic and predictive value in 73 patients. IL6 exhibited the best association with survival (p<0.0001), followed by VEGF and Cyfra 21.1 (both p=0.01). None of the five biomarkers tested were predictive for the use of thalidomide.
Pro-Gastrin releasing peptide Gastrin releasing peptide (GRP) is a molecule that has been implicated in a number of (patho)physiological processes. Its preprohormone is after cleavage further processed into GRP and the more stable molecule pro-Gastrin releasing peptide (ProGRP). ProGRP can be an aid in the differential diagnosis in lung cancer and the management of patients with small cell lung cancer. In collaboration with other centers, we are evaluating the new developed proGRP assay on our routine automated instrument, the Cobas 6000 analyzer. Therefore, we evaluate the technical performance and the clinical relevance of proGRP.
DEPARTMENT OF NUCLEAR MEDICINE Marie-Jeanne Vrancken Peeters, Axel Bex, Paula Elkhuizen, Kenneth Gilhuijs, Jos van der Hage, Simon Horenblas, Claudette Loo, Wim Meinhardt, Omgo Nieweg, Hester Oldenburg, Kenneth Pengel, Henk van der Poel, B van Rhijn, Sjoerd Rodenhuis, Emiel Rutgers, Jelle Teertstra, Michel Wouters, NS van den Berg, Oscar Brouwer, Bernies van der Hiel, Kees Hoefnagel, Gijs Kleinjan, Bas Koolen, Fijs van Leeuwen, Hanna Matheron, Saar Muller, Ferida Sivro, Michiel Sinaasappel, Marcel Stokkel, Suzana Teixeira, Renato Valdés Olmos, Erik Vegt, Wouter Vogel, Linda de Wit-van der Veen
Advances in radioguided surgical navigation and network cooperation Two important investigational lines were further delineated for radioguided surgical navigation. The hybrid approach, which started a few years ago following the introduction of ICG-99mTc-nanocolloid, has been based until now upon the use of two portable camera devices for the separate detection of the radioactive and fluorescence signals; it has been applied in different malignancies such as cutaneous melanoma, head and neck tumors, penile cancer, and vulvar cancer. In further steps the integration of the fluorescence signal to the portable gamma camera will be explored firstly based on the display of both signals in one screen, and subsequently on the development of an integrated device. For this purpose cooperation with the Polytechnic University of Valencia (Instrumentation Institute for Molecular Imaging and Oncovision) has been started. A second model of surgical navigation includes both augmented reality and mixed reality. In the protocol of augmented reality, generated freehand SPECT probe images obtained using a tracking device are incorporated in real time to the surgical area enabling 3D information of the radioactivity in the tumor or lymph node to be excised. In the protocol of mixed reality, 3D generated images of a preoperative acquired SPECT/CT using a tracking device are incorporated in the sentinel node procedure enabling surgical navigation in complex anatomical areas. This work is implemented in the context of a Eurostars project (E!7103 Real-time fhSPECT) in cooperation with the Technical University of Munich and Surgic Eye. Following participation in advanced training activities coordinated by the International Atomic Energy Agency (IAEA) and the European Association of Nuclear Medicine (EANM) a first step was taken to develop an educational and research network on radioguided surgery and interventional molecular imaging research; this initiative includes both clinical institutions (NKIAVL, LUMC, University Hospital Pisa, University Hospital Clinic Barcelona, and Claude Bernard University Hospital Lyon) and private sector partners (Oncovision Valencia, Surgic Eye Munich, and Eurorad Strasbourg) with a leading role and recognized experience in the field.
Grootendorst DJ, Jose J, Wouters MW, van Boven H, Van der Hage J, Van Leeuwen TG, Steenbergen W, Manohar S, Ruers TJ. First experiences of photoacoustic imaging for detection of melanoma metastases in resected human lymph nodes. Lasers Surg Med 2012;44:541-549 Harinck F, Kluijt I, van Mil SE, Waisfisz Q, van Os TA, Aalfs CM, Wagner A, Olderode-Berends M, Sijmons RH, Kuipers EJ, Poley JW, Fockens P, Bruno MJ. Routine testing for PALB2 mutations in familial pancreatic cancer families and breast cancer families with pancreatic cancer is not indicated. Eur J Hum Genet 2012;20:577-579 Hartog H, Van Der Graaf WT, Boezen HM, Wesseling J. Treatment of breast cancer cells by IGF1R tyrosine kinase inhibitor combined with conventional systemic drugs. Anticancer Res 2012;32:1309-1318 Hein R, ... Dunning AM. Comparison of 6q25 breast cancer hits from Asian and European Genome Wide Association Studies in the Breast Cancer Association Consortium (BCAC). PLoS One 2012;7 Van Herk-Sukel MP, Shantakumar S, Overbeek LI, van Boven H, Penning-van Beest FJ, Herings RM. Occurrence of Comorbidities before and after Soft Tissue Sarcoma Diagnosis. Sarcoma 2012;2012 Hoefnagel LD, Moelans CB, Meijer SL, van Slooten HJ, Wesseling P, Wesseling J, Westenend PJ, Bart J, Seldenrijk CA, Nagtegaal ID, Oudejans J, van der Valk P, van Gils CH, van der Wall E, van Diest PJ. Prognostic value of estrogen receptor α and progesterone receptor conversion in distant breast cancer metastases. Cancer 2012;118:4929-4935 Van Hooff SR, Leusink FK, Roepman P, Baatenburg de Jong RJ, Speel EJ, van den Brekel MW, van Velthuysen ML, van Diest PJ, van Es RJ, Merkx MA, Kummer JA, Leemans CR, Schuuring E, Langendijk JA, Lacko M, De Herdt MJ, Jansen JC, Brakenhoff RH, Slootweg PJ, Takes RP, Holstege FC. Validation of a gene expression signature for assessment of lymph node metastasis in oral squamous cell carcinoma. J Clin Oncol 2012;30:4104-4110 Van Hulsteijn LT, Corssmit EP, van der Hiel B, Smit JW, Stokkel MP. Is there a role for radioguided surgery with iodine-labeled metaiodobenzylguanidine in resection of neuroendocrine tumors? Clin Nucl Med 2012;37:1083-1088
Van Hulsteijn LT, van der Hiel B, Smit JW, Stokkel MP, Corssmit EP. Intraoperative detection of ganglioneuromas with 123I-MIBG. Clin Nucl Med 2012;37:768-771 Jakubowska A, Rozkrut D, Antoniou A, Hamann U, Scott RJ, McGuffog L, Healy S, Sinilnikova OM, Rennert G, Lejbkowicz F, Flugelman A, Andrulis IL, Glendon G, Ozcelik H; OCGN, Thomassen M, Paligo M, Aretini P; SWE-BRCA, Kantala J, Aroer B, von Wachenfeldt A, Liljegren A, Loman N, Herbst K, Kristoffersson U, Rosenquist R, Karlsson P, StenmarkAskmalm M, Melin B, Nathanson KL, Domchek SM, Byrski T, Huzarski T, Gronwald J, Menkiszak J, Cybulski C, Serrano P, Osorio A, Cajal TR, Tsitlaidou M, Benítez J, Gilbert M; HEBON, Rookus M, Aalfs CM, Kluijt I, Boessenkool-Pape JL, Meijers-Heijboer HE, Oosterwijk JC, van Asperen CJ, Blok MJ, Nelen MR, van den Ouweland AM, Seynaeve C, van der Luijt RB, Devilee P; EMBRACE, Easton DF, Peock S, Frost D, Platte R, Ellis SD, Fineberg E, Evans DG, Lalloo F, Eeles R, Jacobs C, Adlard J, Davidson R, Eccles D, Cole T, Cook J, Godwin A, Bove B; GEMO Study Collaborators, Stoppa-Lyonnet D, Caux-Moncoutier V, Belotti M, Tirapo C, Mazoyer S, Barjhoux L, Boutry-Kryza N, Pujol P, Coupier I, Peyrat JP, Vennin P, Muller D, Fricker JP, Venat-Bouvet L, Johannsson OT, Isaacs C, Schmutzler R, Wappenschmidt B, Meindl A, Arnold N, Varon-Mateeva R, Niederacher D, Sutter C, Deissler H, Preisler-Adams S, Simard J, Soucy P, Durocher F, Chenevix-Trench G, Beesley J, Chen X; KConFab, Rebbeck T, Couch F, Wang X, Lindor N, Fredericksen Z, Pankratz VS, Peterlongo P, Bonanni B, Fortuzzi S, Peissel B, Szabo C, Mai PL, Loud JT, Lubinski J; CIMBA, the Consortium of Investigators of Modifiers of BRCA1/2-Related Cancer. Association of PHB 1630 C>T and MTHFR 677C>T polymorphisms with breast and ovarian cancer risk in BRCA1/2 mutation carriers: results from a multicenter study. Br J Cancer 2012;106:20162024 Jones ME, van Leeuwen FE, Hoogendoorn WE, Mourits MJ, Hollema H, van Boven H, Press MF, Bernstein L, Swerdlow AJ. Endometrial cancer survival after breast cancer in relation to tamoxifen treatment: pooled results from three countries. Breast Cancer Res 2012;14:R91
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Jones S, Li M, Parsons DW, Zhang X, Wesseling J, Kristel P, Schmidt MK, Markowitz S, Yan H, Bigner D, Hruban RH, Eshleman JR, Iacobuzio-Donahue CA, Goggins M, Maitra A, Malek SN, Powell S, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N. Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types. Hum Mutat 2012;33:100-103
Koolen BB, Vrancken Peeters MJ, Wesseling J, Lips EH, Vogel WV, Aukema TS, van Werkhoven E, Gilhuijs KG, Rodenhuis S, Rutgers EJ, Valdés Olmos RA. Association of primary tumour FDG uptake with clinical, histopathological and molecular characteristics in breast cancer patients scheduled for neoadjuvant chemotherapy. Eur J Nucl Med Mol Imaging 2012;39:1830-1838
Joosse SA, Brandwijk KI, Devilee P, Wesseling J, Hogervorst FB, Verhoef S, Nederlof PM. Prediction of BRCA2association in hereditary breast carcinomas using array-CGH. Breast Cancer Res Treat 2012;132:379-389
Koolen BB, Valdés Olmos RA, Elkhuizen PH, Vogel WV, Vrancken Peeters MJ, Rodenhuis S, Rutgers EJ. Locoregional lymph node involvement on 18F-FDG PET/CT in breast cancer patients scheduled for neoadjuvant chemotherapy. Breast Cancer Res Treat 2012;135:231-240
Kirchhoff T, Gaudet MM, Antoniou AC, McGuffog L, Humphreys MK, Dunning AM, Bojesen SE, Nordestgaard BG, Flyger H, Kang D, Yoo KY, Noh DY, Ahn SH, Dork T, Schürmann P, Karstens JH, Hillemanns P, Couch FJ, Olson J, Vachon C, Wang X, Cox A, Brock I, Elliott G, Reed MW, Burwinkel B, Meindl A, Brauch H, Hamann U, Ko YD; GENICA Network, Broeks A, Schmidt MK, Van ‘t Veer LJ, Braaf LM, Johnson N, Fletcher O, Gibson L, Peto J, Turnbull C, Seal S, Renwick A, Rahman N, Wu PE, Yu JC, Hsiung CN, Shen CY, Southey MC, Hopper JL, Hammet F, Van Dorpe T, Dieudonne AS, Hatse S, Lambrechts D, Andrulis IL, Bogdanova N, Antonenkova N, Rogov JI, Prokofieva D, Bermisheva M, Khusnutdinova E, van Asperen CJ, Tollenaar RA, Hooning MJ, Devilee P, Margolin S, Lindblom A, Milne RL, Arias JI, Zamora MP, Benítez J, Severi G, Baglietto L, Giles GG; kConFab; AOCS Study Group, Spurdle AB, Beesley J, Chen X, Holland H, Healey S, Wang-Gohrke S, Chang-Claude J, Mannermaa A, Kosma VM, Kauppinen J, Kataja V, Agnarsson BA, Caligo MA, Godwin AK, Nevanlinna H, Heikkinen T, Fredericksen Z, Lindor N, Nathanson KL, Domchek SM; SWE-BRCA, Loman N, Karlsson P, Stenmark Askmalm M, Melin B, von Wachenfeldt A; HEBON, Hogervorst FB, Verheus M, Rookus MA, Seynaeve C, Oldenburg RA, Ligtenberg MJ, Ausems MG, Aalfs CM, Gille HJ, Wijnen JT, Gómez García EB; EMBRACE, Peock S, Cook M, Oliver CT, Frost D, Luccarini C, Pichert G, Davidson R, Chu C, Eccles D, Ong KR, Cook J, Douglas F, Hodgson S, Evans DG, Eeles R, Gold B, Pharoah PD, Offit K, Chenevix-Trench G, Easton DF; BCAC/CIMBA. Breast cancer risk and 6q22.33:combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2. PLoS One. 2012;7:
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Koolen BB, Vogel WV, Vrancken Peeters MJ, Loo CE, Rutgers EJ, Valdés Olmos RA. Molecular Imaging in Breast Cancer: From Whole-Body PET/ CT to Dedicated Breast PET. J Oncol Epub 2012 Koolen BB, Vegt E, Rutgers EJ, Vogel WV, Stokkel MP, Hoefnagel CA, FiooleBruining A, Vrancken Peeters MJ, Valdés Olmos RA. FDG-avid sclerotic bone metastases in breast cancer patients: a PET/CT case series. Ann Nucl Med 2012;26:86-91 Koolen BB, Vrancken Peeters MJ, Aukema TS, Vogel WV, Oldenburg HS, van der Hage JA, Hoefnagel CA, Stokkel MP, Loo CE, Rodenhuis S, Rutgers EJ, Valdés Olmos RA. 18F-FDG PET/CT as a staging procedure in primary stage II and III breast cancer: comparison with conventional imaging techniques. Breast Cancer Res Treat 2012;131:117-126 Korse CM, Taal BG, Vincent A, van Velthuysen ML, Baas P, Buning-Kager JC, Linders TC, Bonfrer JM. Choice of tumour markers in patients with neuroendocrine tumours is dependent on the histological grade. A marker study of Chromogranin A, Neuron specific enolase, Progastrin-releasing peptide and cytokeratin fragments. Eur J Cancer 2012;48:662-671 Kluijt I, Siemerink EJ, Ausems MG, van Os TA, de Jong D, Simões-Correia J, van Krieken JH, Ligtenberg MJ, Figueiredo J, van Riel E, Sijmons RH, Plukker JT, van Hillegersberg R, Dekker E, Oliveira C, Cats A, Hoogerbrugge N; Dutch Working Group on Hereditary Gastric Cancer. CDH1-related hereditary diffuse gastric cancer syndrome: clinical variations and implications for counseling. Int J Cancer 2012;131:367-76
Sentinel node detection with combined fluorescent and radioactive surgical guidance Following a reproducibility study with ICG-99mTc-nanocolloid, which demonstrated a 100% correlation with the standard 99m Tc-nanocolloid, in cooperation with the group Interventional Molecular Imaging of the LUMC the application of this novel tracer for the sentinel node procedure was further extended to various malignancies. The use of ICG-99mTc-nanocolloid enables preoperative lymphatic mapping using lymphoscintigraphy and SPECT/CT followed by intraoperative sentinel node localisation using separate camera devices for radioactive and fluorescence signal detection. In a feasibility study including 14 patients with oral cavity squamous cell carcinoma 43 sentinel nodes were removed using combined radio- and fluorescence guidance; in four patients sentinel nodes were localized close to the primary injection site, including three cases with localization in level I of the neck. In another evaluation involving 48 patients with penile squamous cell carcinoma and cN0 groins, preoperative lymphatic mapping identified a total of 138 sentinel nodes. All these nodes could be localized in the operation room using combined radio- and fluorescence guidance. Visualization using a fluorescence camera was possible in 98% of the sentinel nodes whereas only 53% was stained by the blue dye. In a study including 76 cN0 melanoma patients (39 head/neck, 27 trunk and 10 extremities) lymphoscintigraphy in combination with SPECT/CT identified 205 sentinel nodes. Intraoperatively, 94% of these nodes could be localized by detecting the radioactive signal. The 6% remaining sentinel nodes concerned head/neck melanoma patients and were localized in the vicinity of the injection site only by detection using a fluorescence camera. In the patients in whom blue dye was used, merely 63% of the sentinel nodes stained blue (45% head/neck, 77% trunk/extremities). Besides hybrid sentinel node detection, the fluorescent component of the tracer ICG-99mTc-nanocolloid can be used for tumor demarcation. In a pilot study including five patients with T1-2 tongue carcinoma scheduled for sentinel node biopsy, all fluorescent tracer deposits could be detected surrounding the primary tumor. A second study using ultrasound guided tracer microinjections for intraoperative fluorescence guided tumor delineation and resection has recently been started.
Augmented and mixed reality models in surgical navigation The feasibility of 3D intraoperative imaging with a freehand SPECT probe to guide tumor excision was evaluated in a study including 11 patients with non-palpable breast cancer. The procedure was performed following a protocol of augmented reality and in two patients was combined with sentinel node biopsy. Radioguidance was based in 8 patients on an intratumoral injection of 99mTcnanocolloid (ROLL) and in 3 on the localization of a seed of 125I (RSL). Freehand SPECT image acquisition took 131.5 seconds for ROLL, 155.5 for ROLL + sentinel node, and 69.2 seconds for RSL on average. Ex-vivo real time display of the radioactivity in the excised tissue specimen in relation to the margins of the specimen was performed in all patients. Histopathologic margins were tumor negative in all 11 cases in accordance with the ex-vivo images. This experience will be further extended in the context of the EU-Eurostars project “E!7103 Real-time fhSPECT” which has recently been started.
Following a protocol of mixed reality a study was initiated to provide a proof of concept of how intraoperative navigation based on preoperative SPECT/CT images may help to improve hybrid radio-and fluorescence guided surgery. Based on phantom experiences, preoperative SPECT/CT images of 10 patients undergoing an inguinal sentinel node biopsy were acquired with a fixed reference target placed on a rigid anatomical structure. By also attaching a reference target to the fluorescence endoscopic/gamma probe, preoperative SPECT/CT were transferred to the navigation system providing a virtual 3D view of SPECT/CT in the operation room. Optical tracking could be used to position the tip of the fluorescence endoscope/gamma probe in relation to the preoperative 3D imaging. This navigation approach allowed the localization of preoperatively defined lesions with an error of < 1 cm in the inguinal area. The same approach was used to navigate towards the prostate in a patient undergoing robot-assisted prostatectomy.
Dedicated PET imaging in breast cancer F-FDG PET/CT using a dedicated protocol for breast imaging based on prone patient position (hanging breast technique) and 2 mm slices was evaluated for locoregional staging in breast cancer patients scheduled for neoadjuvant chemotherapy. In 311 patients PET/CT reached a sensitivity of 82% and a specificity of 92% with an accuracy of 84%. The positive predictive value was 98% and the negative predictive value 53%. Occult lymph node metastases were detected in 26 patients (8%) in the internal mammary chain and in 32 (10%) in the periclavicular area. Due to its high predictive value a positive 18F-FDG PET/CT may render pre-chemotherapy sentinel node biopsy. Risk for locoregional recurrence in breast cancer depends on tumor size and the number and location of tumor-positive locoregional lymphnodes. Postoperative irradiation of chest wall and/or locregional nodes is advised in patients at high risk for locoregional recurrence but remains controversial at intermediate risk. In this context detection of occult N3-disease or ≥ 4 FDG-avid axillary nodes may lead to changes in clinical management. In a study involving 278 patients scheduled for neoadjuvant chemotherapy PET/CT upstaged 5 of 47 initially considered low risk patients and 38 of 144 intermediate risk patients to the high risk group; this implicated postoperative chest wall and/or locoregional irradiation. In another study, 18F-FDG PET/CT was able to detect the primary tumor in 54 of 62 patients (87%) with invasive T1 breast cancer. PET/CT sensitivity varied from 59% (10/17) in tumors ≤ 10 mm to 98% in tumors > 10 mm. All triple negative and HER2-positive tumors and 40/48 (88%) ER-positive/HER2-negative tumors were visualized. In 203 patients with FDG-avid breast tumors scheduled for neoadjuvant chemotherapy, the distance and relative difference in FDG uptake between the core biopsy area (indicated by a marker) and the most metabolic part of the lesion were compared by calculating SUVmax. A distance ≥ 2 cm and a relative difference in SUVmax ≥ 25% were considered clinically relevant and a combination of both was defined as noncorrespondence. Non-correspondence, found in 28 of 203 tumors (14%), was significantly associated with T-stage, lesion morphology on MRI and PET/CT, tumor diameter, and histologic type. It was more often seen in tumors with a higher T-stage, diffuse and multifocal tumors on MRI and PET/CT, tumors
Kraeber-Bodéré F, Carlier T, Naegelen VM, Shochat E, Lumbroso J, Trampal C, Nagarajah J, Chua S, Hugonnet F, Stokkel M, Gleeson F, Tessier J. Differences in the Biologic Activity of 2 Novel MEK Inhibitors Revealed by 18F-FDG PET: Analysis of Imaging Data from 2 Phase I Trials. J Nucl Med 2012;53:1836-1846 Laitman Y, Kuchenbaecker KB, Rantala J, Hogervorst F, Peock S, Godwin AK, Arason A, Kirchhoff T, Offit K, Isaacs C, Schmutzler RK, Wappenschmidt B, Nevanlinna H, Chen X, Chenevix-Trench G, Healey S, Couch F, Peterlongo P, Radice P, Nathanson KL, Caligo MA, Neuhausen SL, Ganz P, Sinilnikova OM, McGuffog L, Easton DF, Antoniou AC, Wolf I, Friedman E. The KL-VS sequence variant of Klotho and cancer risk in BRCA1 and BRCA2 mutation carriers. Breast Cancer Res Treat 2012;132:1119-1126
Majewski IJ, Kluijt I, Cats A, Scerri TS, de Jong D, Kluin RJ, Hansford S, Hogervorst FB, Bosma AJ, Hofland I, Winter M, Huntsman D, Jonkers J, Bahlo M, Bernards R. An alphaE-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol. 2012 Mavaddat N, […]; Consortium of Investigators of Modifiers of BRCA1/2. Pathology of breast and ovarian cancers among BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Cancer Epidemiol Biomarkers Prev 2012;21:134-147 Maxwell CA, …, Pujana MA. Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer. PLoS Biol 2011;9
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Van Lent W, Deetman JW, Teertstra HJ, Muller SH, Hans EW, van Harten WH. Reducing the throughput time of the diagnostic track involving CT scanning with computer simulation. Eur J Radiol. 2012 Lin F, Chandrasekaran G, de Gooijer MC, Beijnen JH, van Tellingen O. Determination of NVP-BEZ235, a dual PI3K and mTOR inhibitor, in human and mouse plasma and in mouse tissue homogenates by reversed-phase highperformance liquid chromatography with fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci 2012;901:9-17 Lips EH, Mukhtar RA, Yau C, de Ronde JJ, Livasy C, Carey LA, Loo CE, Vrancken-Peeters MJ, Sonke GS, Berry DA, Van ‘t Veer LJ, Esserman LJ, Wesseling J, Rodenhuis S, Shelley Hwang E; I-SPY TRIAL Investigators. Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer. Breast Cancer Res Treat 2012;136:35-43 Lips EH, Mulder L, de Ronde JJ, Mandjes IA, Vincent A, Vrancken Peeters MT, Nederlof PM, Wesseling J, Rodenhuis S. Neoadjuvant chemotherapy in ER+ HER2-breast cancer: response prediction based on immunohistochemical and molecular characteristics. Breast Cancer Res Treat 2012;131:827-836 Lodder WL, Gilhuijs KG, Lange CA, Pameijer FA, Balm AJ, van den Brekel MW. Semi-automated primary tumor volume measurements by dynamic contrast-enhanced MRI in patients with head and neck cancer. Head Neck 2012;9
Meinhardt W, van der Poel HG, Valdés Olmos RA, Bex A, Brouwer OR, Horenblas S. Laparoscopic sentinel lymph node biopsy for prostate cancer: the relevance of locations outside the extended dissection area. Prostate Cancer. 2011 Mertens LS, Bruin NM, Vegt E, de Blok WM, Fioole-Bruining A, van Rhijn BW, Horenblas S, Vogel WV. Catheterassisted 18F-FDG-PET/CT imaging of primary bladder cancer: a prospective study. Nucl Med Commun 2012;33:11951201 Mertens LS, Fioole-Bruining A, van Rhijn BW, Kerst JM, Bergman AM, Vogel WV, Vegt E, Horenblas S. FDG-PET/CT in Monitoring Response of Pelvic Lymph Node Metastases to Neoadjuvant Chemotherapy in Bladder Cancer. J Urol 2012 Mingels MJ, Roelofsen T, van der Laak JA, de Hullu JA, van Ham MA, Massuger LF, Bulten J, Bol M. Tubal epithelial lesions in salpingooophorectomy specimens of BRCAmutation carriers and controls. Gynecol Oncol 2012;127:88-93 Moghadasi S, Hofland N, Wouts JN, Hogervorst FB, Wijnen JT, Vreeswijk MP, van Asperen CJ. Variants of Uncertain Significance in BRCA1 and BRCA2 assessment of in silico analysis and a proposal for communication in genetic counselling. J Med Genet 2012 Van Monsjou HS, van Velthuysen ML, van den Brekel MW, Jordanova ES, Melief CJ, Balm AJ. Human papillomavirus status in young patients with head and neck squamous cell carcinoma. Int J Cancer 2012;130:1806-1812
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Oonk AM, van Rijn C, Smits MM, Mulder L, Laddach N, Savola SP, Wesseling J, Rodenhuis S, Imholz AL, Lips EH. Clinical correlates of ‘BRCAness’ in triple-negative breast cancer of patients receiving adjuvant chemotherapy. Ann Oncol 2012;23:2301-2305 Orsini F, Guidoccio F, Vidal Sicart S, Valdés Olmos RA, Mariani G. General concepts on radioguided sentinel lymph node biopsy: preoperative imaging, intraoperative gamma-probe guidance, intraoperative imaging, and multimodality imaging. In: Mariani G, Manca G,Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: Springer-Verlag, 2012:95-110 Van der Poel HG, Balm AJ, Nieweg OE, Valdés Olmos RA. Comment on Heuveling et al.: Nanocolloidal albumin-IRDye 800CW: a nearinfrared fluorescent tracer with optimal retention in the sentinel lymph node. Eur J Nucl Med Mol Imaging 2012;39:1510-1511 Roelofsen T, van Ham MA, Wiersma van Tilburg JM, Zomer SF, Bol M, Massuger LF, Bulten J. Pure compared with mixed serous endometrial carcinoma: two different entities? Obstet Gynecol. 2012;120:1371-1381 De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LF. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer. Breast Cancer Res Treat 2012 Rottenberg S, Vollebergh MA, de Hoon B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Lodén M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res 2012;72:2350-2361 Ruibal A, Benlloch JM, Valdés Olmos RA, Langstrom B. Molecular imaging in breast cancer. J Oncol 2012 (in press) Salm LP, Van der Hiel B, Stokkel MP. Neurolymphomatosis Diagnosed by 18F-FDG PET-CT. Clin Nucl Med. 2012 Salm LP, Van der Hiel B, Stokkel MP. Increasing importance of 18F-FDG PET in the diagnosis of neurolymphomatosis. Nucl Med Commun 2012;33:907-916
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Stokkel MP, van der Hiel B, Olmos RV, Hoefnagel K. Fluorodeoxyglucose PET/CT in subcutaneous sarcoidosis mimicking cutaneous lymphoma. Eur J Nucl Med Mol Imaging 2012;39:919-920 Schaake EE, Kappers I, Codrington HE, Valdés Olmos RA, Teertstra HJ, van Pel R, Burgers JA, van Tinteren H, Klomp HM. Tumor response and toxicity of neoadjuvant erlotinib in patients with early-stage non-small-cell lung cancer. J Clin Oncol 2012;30:2731-2738 Schmitz AC, Pengel KE, Loo CE, van den Bosch MA, Wesseling J, Gertenbach M, Alderliesten T, Mali WP, Rutgers EJ, Bartelink H, Gilhuijs KG. Pre-treatment imaging and pathology characteristics of invasive breast cancers of limited extent: potential relevance for MRI-guided localized therapy. Radiother Oncol 2012;104:11-18 Spurdle AB, Healey S, Devereau A, Hogervorst FB, Monteiro AN, Nathanson KL, Radice P, StoppaLyonnet D, Tavtigian S, Wappenschmidt B, Couch FJ, Goldgar DE; ENIGMA. ENIGMA--evidence-based network for the interpretation of germline mutant alleles: an international initiative to evaluate risk and clinical significance associated with sequence variation in BRCA1 and BRCA2 genes. Hum Mutat 2012;33:2-7 Stevens KN, […]; kConFab Investigators; AOCS Group, Beesley J, Chen X, Mannermaa A, Kosma VM, Hartikainen JM, Soini Y, Easton DF, Couch FJ. 19p13.1 is a triple-negativespecific breast cancer susceptibility locus. Cancer Res 2012;72:1795-1803 Stokkel MP, van der Hiel B, Olmos RV, Hoefnagel K. Fluorodeoxyglucose PET/CT in subcutaneous sarcoidosis mimicking cutaneous lymphoma. Eur J Nucl Med Mol Imaging 2012;39:919-920 Stokkel MP, de Wit-van der Veen LJ, Boekhout A. I-123-MIBG myocardial imaging in trastuzumab-based cardiotoxicity: the first experience. Nucl Med Commun 2013;34:19-24 Stokkel MPM. Tratiamiento de tumores neuroendocrinos en medicina nuclear. In: Castrejon AS, Martin-Comin J, Garcia AM (eds). Medicina Nuclear en la practica clinica 2° edicion. Madrid: Aula Medica 2012:545-56 Thorwarth D, Beyer T, Boellaard R, de Ruysscher D, Grgic A, Lee JA, Pietrzyk U, Sattler B, Schaefer A, van Elmpt W, Vogel W, Oyen WJ, Nestle U. Integration of FDG-PET/CT into external beam radiation therapy planning: technical aspects and recommendations on methodological approaches. Nuklearmedizin 2012;51:140-153
>3cm, and lobular carcinomas. In these patient categories FDG information may lead to improved pre-treatment tumor sampling. Following the installation of the definitive prototype of the dedicated PET ring camera MAMMI, the heterogeneity of primary tumor FDG uptake was evaluated in patients with breast cancer scheduled for neoadjuvant chemotherapy. Due to its superior resolution MAMMI was able to show higher heterogeneity scores in 11 of 35 patients (31%) when compared with prone PET/CT imaging. Significantly higher heterogeneity scores were seen in large tumors and tumors with high FDG uptake. These findings appear to provide a rational for FDG-guided tumor sampling using MAMMI and a specially designed biopsy device.
Developments radiopharmaceuticals In 2012, further diversification of PET techniques available for diagnostic use was achieved. Besides the regularly applied 18F-FDG, 18F-choline, 68Ga-octreotate and 124I-iodine, new tracers were introduced for clinical and pre-clinical imaging. Within the scope of the ARTFORCE research initiative, the antibody cetuximab was radiolabeled with 89Zirconium. This tracer can be applied to visualise and quantify the distribution of the pharmaceutical cetuximab in vivo, with potential for treatment outcome prediction and patient selection. The clinical PET/CT scanner was calibrated and validated for this new radionuclide, and inclusion of patients has started. Furthermore, the acquisition and reconstruction of 4D motion-compensated PET/CT was optimized, with a main focus on voxel-based response monitoring during chemotherapy and radiotherapy. To accommodate all these new clinical and research PET initiatives in the coming decade, a second PET/CT scanner was installed, featuring a big bore to allow radiotherapy positioning. In addition, the development of a new radiopharmaceutical for gamma camera imaging was initiated. The radionuclide 195mPlatinum was produced in collaboration with NRG Petten. Clinical and preclinical SPECT/CT were successfully calibrated and applied for imaging of this tracer. In subsequent projects, the radiopharmaceutical 195mPt-cisplatin will be applied to visualize and quantify the biodistribution of the chemotherapy cisplatin in vivo, in mice and humans.
DEPARTMENT OF PATHOLOGY PATHOLOGY Pathology is the key for tumor tissue related research by facilitating basal, translational, and clinical research to optimize precision (personalized) medicine for cancer patients. Most research questions relate to finding, validating, and implementing prognostic and predictive biomarkers, combined with tumor classification issues. For this, molecular analyses are becoming increasingly important. To ensure adequate and prudent tissue banking and use of these samples, the Core Facility Molecular Pathology was established. Below, most research topics, in which our department is involved, are briefly described.
Discovery, evaluation and validation of prognostic and predictive factors to tailor diagnosis and treatment of breast cancer patients Theo Ruers, Danny Evers, Claudette Loo, Sabine Linn, Emiel Rutgers, Mila Donker, Bas Koolen, Joyce Sanders, Petra Nederlof, Jelle Wesseling
Cancer is a heterogeneous disease requiring an individualized approach for optimal diagnosis and treatment. Therefore, we collaborate with a variety of research groups, mainly within the institute, but also worldwide, to ensure that our concerted action ultimately will benefit breast cancer patient survival. As such, we team up on the development of new imaging techniques (e.g. collaboration with the Theo Ruers group), improvement of existing biomarker assessment (e.g. HER2 testing), as well as development of new biomarker tests to determine for example the tamoxifen sensitivity in breast cancer positive for the estrogen receptor (see for a detailed description the contribution of Sabine Linn). Note: Jelle Wesseling’s main breast cancer research is carried out at the Section of Molecular Pathology (Head: Jos Jonkers).
EGFR mutation in lung carcinomas and response to Iressa therapy Petra Nederlof, Jeroen de Jong, Erik Thunnissen (VUMC), Michel van den Heuvel
In a nation-wide study, clinical data and EGFR/KRAS mutation data has been collected for all lung carcinomas from 2005-2011 for which molecular diagnostics has been performed. Mutation frequency, tumor characteristics and clinical response to tyrosine kinase inhibitors will be evaluated.
PTEN mutations and deletions and response in melanoma Petra Nederlof, Bart van de Wiel, Christian Blank
We will determine the PTEN mutation status (including deletions) in 120 melanomas from the NKI who were treated with (BRAF mutated cases were also treated with a BRAF inhibitor) and 30 melanoma patients treated with BRAF inhibitor only. Several publications indicate that possibly patients with inactivated PTEN are less sensitive for BRAF inhibitors or show a shorter PFS. PI3K pathway activation has been implicates as mediator in escape to BRAF inhibitors. (Villanueva et al Cancer Cell 18:683695, Nazarian, R.et al, 2010 Nature 468:973-977. Peruzzi, F.et al 1999 Mol Cell Biol 19:7203-7215). We will further study the correlation between response to ipilimumab en BRAF/NRAS/ PTEN status. (Han SJ et al Neuroreport. 2009;20:1597-602, Parsa AT et al, Nat Med. 2007;13:84-8.
Tran L, Huitema A, Vogel W, Beijnen J, Baars J. Lack of tumor uptake of 131-I labeled rituximab in a patient with a CD20 positive lymphoma lesion. J Oncol Pharm Pract 2012;18:417-420
Vidal-Sicart S, Valdés Olmos RA. Sentinel node for breast cancer: current situation. J Oncol. 2012
Valdés Olmos RA, Brouwer OR, Feenstra C. De schildwachtklier procedure en SPECT-CT. Gamma Professional 2012;62:13-18
Van der Veen BJ, Al Younis I, de Roos A, Stokkel MP. Assessment of global cardiac I-123 MIBG uptake and washout using volumetric quantification of SPECT acquisitions. J Nucl Cardiol 2012;19:752-62
Valdés Olmos RA, Vidal Sicart S. SPECT/CT image generation and criteria for sentinel node mapping. In: Mariani G, Manca G, Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: Springer-Verlag, 2012:111-119
Van der Veen BJ, Al Younis I, AjmoneMarsan N, Westenberg JJ, Bax JJ, Stokkel MP, de Roos A. Ventricular dyssynchrony assessed by gated myocardial perfusion SPECT using a geometrical approach: a feasibility study. Eur J Nucl Med Mol Imaging. 2012;39:421-429
Valdés Olmos RA, Klop WMC, Brouwer OR. Preoperative and intraoperative lymphatic mapping for radioguided sentinel node biopsy in head and neck cancers. In: Mariani G, Manca G, Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: SpringerVerlag, 2012:199-208
Veenstra HJ, Brouwer OR, van der Ploeg IM, Kroon BB, Nieweg OE. Five-year follow-up of 16 melanoma patients with a Starz I-involved sentinel node in whom completion lymph node dissection was omitted. Melanoma Res 2012;22:436-439
Veenstra HJ, Vermeeren L, Valdés Olmos RA, Nieweg OE. The additional value of lymphatic mapping with routine SPECT/CT in unselected patients with clinically localized melanoma. Ann Surg Oncol. 2012;19:1018-1023 Verhagen MM, Last JI, Hogervorst FB, Smeets DF, Roeleveld N, Verheijen F, Catsman-Berrevoets CE, Wulffraat NM, Cobben JM, Hiel J, Brunt ER, Peeters EA, Gómez Garcia EB, van der Knaap MS, Lincke CR, Laan LA, Tijssen MA, van Rijn MA, Majoor-Krakauer D, Visser M, van ‘t Veer LJ, Kleijer WJ, van de Warrenburg BP, Warris A, de Groot IJ, de Groot R, Broeks A, Preijers F, Kremer BH, Weemaes CM, Taylor MA, van Deuren M, Willemsen MA. Presence of ATM protein and residual kinase activity correlates with the phenotype in ataxia-telangiectasia: a genotype-phenotype study. Hum Mutat 2012;33:561-571 Vidal-Sicart S, Brouwer OR, Mathéron HM, Bing Tan I, Valdés-Olmos RA. Sentinel node identification with a portable gamma camera in a case without visualization on conventional lymphoscintigraphy and SPECT/CT. Rev Esp Med Nucl Imagen Mol 2012p Vidal Sicart S, Valdés Olmos RA. Preoperative and intraoperative lymphatic mapping for radioguided sentinel node biopsy in cutaneous melanoma. In: Mariani G, Manca G, Orisini F, Vidal Sicart S, Valdés Olmos RA (eds). Atlas of lymphoscintigraphy and sentinel node mapping. Milan: Springer-Verlag, 2012:169-180
Verburg FA, Dietlein M, Freudenberg L, Leboulleux S, Pitoia F, Reiners C, Stokkel M, Luster M. Recombinant Human TSH Versus Thyroid Hormone Withdrawal. J Nucl Med 2012;53:18151816 Vogel WV, Valdés Olmos RA, Tijs TJ, Gillies MF, van Elswijk G, Vogt J. Intervention to lower anxiety of 18F-FDG PET/CT patients by use of audiovisual imagery during the uptake phase before imaging. J Nucl Med Technol 2012;40:92-98 Vogel WV, Guislain A, Kvistborg P, Schumacher TN, Haanen JB, Blank CU. Ipilimumab-induced sarcoidosis in a patient with metastatic melanoma undergoing complete remission. J Clin Oncol 2012;30: e7-e10 Wessels R, de Bruin DM, Faber DJ, van Boven HH, Vincent AD, van Leeuwen TG, van Beurden M, Ruersa TJ. Optical coherence tomography in vulvar intraepithelial neoplasia. J Biomed Opt 2012;17 Zaknum JJ, Gianmarile F, Valdés Olmos RA, Vidal Sicart S, Mariani G. Changing paradigms in radioguided surgery and intraoperative imaging: the GOSTT concept. Eur J Nucl Med Mol Imaging 2012;39:1-3 Zander SA, Sol W, Greenberger L, Zhang Y, van Tellingen O, Jonkers J, Borst P, and Rottenberg S. EZN-2208 (PEG-SN38) overcomes ABCG2mediated topotecan resistance in BRCA1-deficient mouse mammary tumors. PLoS One 2012;7
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ALK mutations in lung carcinomas
Core facility molecular pathology and biobank
Petra Nederlof, Jeroen de Jong, Michel van den Heuvel, Paul Baas
Annegien Broeks, Jose Overwater, Linde Braaf, Ingrid Hofland, Renate de Groot, Marcel Winter, Huib Vroege, Donne Majoor, Dennis Peters
As part of a European project, NKI-AVL archival FFPE material of resection specimens of non-small cell long carcinomas of a group of approximately 70 patients will be analysed for a panel of molecular characteristics. To start with, EML4-ALK translocation will be analysed. The 70 cases will be screened by IHC and positive cases will be analysed by FISH. Lung cancer is a heterogeneous disease in which small subgroups are currently rapidly defined by the discovery of new genetic aberrations with major therapeutic implications. This project is the first step in molecular characterization of a large European cohort (2000-2400) of stage I-IIIB resected NSCLC. Lungscape is a translational research project designed by European Thoracic Oncology Platform (ETOP)
Deletion of p16 as a biomarker in cytological specimen to identify mesothelioma Petra Nederlof, Jeroen de Jong, Josine Quispel
We will investigate if 9p21 deletion detection by FISH is valuable biomarkers for mesothelioma. Approximately 70-85% of the malignant mesothelioma (epithelial type) show deletion of CDKN2A. Using this marker the diagnosis mesothelioma in cytological specimen could possibly be improved. Illei et al, Clin Cancer Res. 2003;9:2108-13, CT-S Chung, J Clin Pathol. 2010;63:630-634.
The CF-MPB registers, coordinates, assists and facilitates research involving archived human/patient material (biospecimens). This concerns all research using Biobank material both form the department of Pathology (the formalin fixed paraffin embedded (FFPE) -block archive, and fresh frozen (FF) tissue bank with) and the department of Clinical Chemistry (the serum and blood Biobank (protocols under approval). The facility provides professional expertise, appropriate samples and tissue based experimentation in the context of optimally controlled medical-ethical issues according to ‘the code of conduct’. To register all requests in a central database we have developed a, tailor made, online system ‘the Application and request tool ‘ART’’. From the 176 studies that have been registered from the start in 2010, 80 have been registered in 2012. These studies requested the use of e.g FFPE and FF tissue for Immunohistochemistry and DNA/ RNA isolation for e.g. Sequenom mutation analysis and Next Gen Sequencing.
THE NETHERLANDS CANCER INSTITUTE FAMILY CANCER CLINIC
Subtyping of second tumors after breast cancer Jelle Wesseling, Loes van Velthuysen, Sabine Linn, Neeltje Steeghs, Petra Nederlof, Marjanka Schmidt
With the increased incidence of breast cancer and the improved survival, studies on breast cancer outcome have become exceedingly important. Survivors of breast cancer are at an increased risk for developing second cancers, but the risk of these cancers may vary based on the type of the first breast cancer, age at first cancer diagnosis, and therapy for the first breast cancer. It is still largely unknown which are the predominant morphological and molecular subtypes of these second primary cancers that occur after breast cancer, and whether the development of these subtypes is affected by patient characteristics and treatment of the primary breast cancer. This is an important question because of the worse prognosis for certain subtypes. The aim of this pilot study is to characterize second primary breast and gastro-intestinal tumors which developed after a primary breast cancer diagnosis by immunohistochemical markers and Comparative Genomic Hybridization. Protein expression and genomic profiles of the primary breast tumors will be compared with those of the second primary breast and gastrointestinal tumors. Information from the clinical genetic center and molecular pathology about known germline or tumor mutations will be taken into account. Patient characteristics, clinicopathological factors and treatment will be investigated to explore the driving factors for the similarities or differences of these first and second tumors.
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Mohamed Achachah, Abderrahim Ajouaou, Libertje Bosma, Majella Boutmy-de Lange, Daniela Hahn, Frans Hogervorst, Mobien Kasiem, Irma Kluijt, Lizet van der Kolk, Merel Maiburg, Petra Nederlof, Rob Plug, Roelof Pruntel, Anja van Rens, Emma van der Riet, Efraim Rosenberg, Marielle Ruijs, Esther Scheerman, Laura van ’t Veer, Sophie van der Velden, Senno Verhoef
Like in previous years, also in this year the number of families who have sought genetic advice through our hospital has increased substantially, amounting to over 1000 patients for the year 2012. Most families seen at our family cancer clinic present with a possible genetic predisposition for breast and/or ovarian cancer. Other indications for referral are a suspicion of non polyposis colorectal cancer (Lynch syndrome), familial renal cancer, pancreatic cancer, stomach cancer and melanoma. The DNA-diagnostic laboratory has screened more than 3000 families for germ line mutations in the BRCA1/2 genes since the start in 1995. These families are obtained through our Family Cancer Clinic and the Oncogenetic section of the Department of Clinical Genetics of the Academic Medical Center. The laboratory has been testing new techniques in order to shorten the through put time of samples. Last year we tested the high resolution melting curve analysis of BRCA1 which proves to be less complex in handling and this year we implemented the technique in routine analysis for BRCA1. Furthermore, we introduced an 8 channel pipetting robot to facilitate rapid and controlled pipetting. Using Array CGH for hereditary breast/ovarian cancer we study whether the genomic aberrations in breast tumors are similar to the specific
aberrations found in BRCA1 and BRCA2 tumors (in collaboration with Petra Nederlof, Division of Experimental Therapy and Diagnostic Oncology). More than 250 a-CGH analyses have been completed, for high risk families and unclassified variant analysis mostly. A BRCA2 classifier has been validated and implemented in the routine diagnostics together with the assessment of methylation of the BRCA1 promotor. For families with hereditary non polyposis colorectal cancer we have mutation scanning tests for mismatch repair genes hMLH1, hMSH2 and hMSH6 and the MutY (MYH) gene. Microsatellite instability tests and immunohistochemistry for the mismatch repair genes is carried out in collaboration with the pathologist Daphne de Jong. Furthermore methylation of the hMLH1 promoter and the presence of a specific somatic mutation in the BRAF gene (V600E) are being assessed. About 50% of the micro satellite instable(MSI-high) tumors with absent staining of hMLH1 have a methylated promoter. This result has direct consequences for the clinical interpretation of the family history data. In a multi-centre study, the ‘TIME- study’ in collaboration with the Division of Medical Genetics of the Utrecht Medical Centre, In 2008 a PhD student started a project on the implementation of genetic counseling and where necessary rapid DNA-testing in recently diagnosed breast cancer patients. Patients from 14 different hospitals have been enrolled. The analysis of the data from the 271 included patients from a potential of 351 (ie 77% recruitment) is in progress. The Family Cancer Clinic contributes data to several multicenter national and international research projects, e.g. GEO-HEBON (gene environment interactions in hereditary breast and ovarian cancer, see Division Psychosocial Research and Epidemiology), DNA-profiling by classic cGH and array cGH of breast cancer patients (see Division of Experimental Therapy), the BCAC and CIMBA consortiums which focus on the contribution of SNPs to cancer risk (HEBON resource, Matti Rookus, dept of Psychosocial Research and Epidemiology), studies into the biological significance of so called unclassified variants (DNA changes of which it is uncertain whether they be pathogenic mutations or polymorphisms) in collaboration with the dept Molecular Biology groups of Jos Jonkers (BRCA1 UVs) and Hein te Riele (MMR UVs), in national and international collaborations with other DNAdiagnostic and research labs, eg ENIGMA for BRA1/2 UVs (Frans Hogervorst), psychosocial studies, in collaboration with the department of Psychosocial Research and Epidemiology and clinical and genetic research in families with gastrointestinal cancer, including stomach cancer and pancreatic cancer (Annemieke Cats, Division of Medical Oncology). New initiatives for collaboration are in a study on premature aging in BRCA1/2 carriers (BRAVA study), and on the effect of tailored internet information in preparation for genetic counsellling (e-infogene, in collaboration with Dr. M Ausems, UMC Utrecht).
DEPARTMENT OF RADIOLOGY 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, pre-treatment assessment of tumor extent, imageguided therapy and response monitoring. The focus of the department was on prostate cancer imaging and follow-up and breast cancer imaging and follow-up using MRI and on image guided treatment procedures of liver tumors, lung tumors and renal tumors. Finally, there is a close clinical collaboration with the department of nuclear medicine and radiotherapy in the development of new imaging tools and therapeutic options.
Impact of negative margin width on local recurrence in breast conserving therapy Wei Chen, Joep Stroom, Jan-Jakob Sonke, Harry Bartelink, Sander Schmitz, Kenneth Gilhuijs
Background and purpose: This study aims to explain the unexpected weak association between the width of the negative surgical margin and the risk of local recurrence in breast conserving therapy. Materials and methods: We utilized a classical tumor-control probability (TCP) model to estimate the risk of local recurrence, considering the heterogeneity of microscopic disease spread observed around the invasive index tumor in a pathology dataset (N=60). The estimated result was compared with the true risk observed in the EORTC boost-versus-no-boost trial (N=1616). Results: The disease volume beyond any given distance from the edge of the index tumor varied considerably among patients. Adopting this disease volume variation in the TCP model accurately reproduced the local recurrence rate as function of surgical margin width in the boost-versus-no-boost trial (Pearson’s correlation coefficients are 0.652 and 0.862, and significant at the 0.05 and 0.01 level for absence and presence of a radiation boost, respectively). Conclusions: The impact of a negative margin width on local recurrence is limited due to the large variation of microscopic disease that can reach large quantities beyond any given distance from the edge of the index tumor across the patient population of breast-conserving therapy.
Pre-treatment imaging and pathology characteristics of invasive breast cancers of limited extent: potential relevance for MRI-guided localized therapy Sander Schmitz, Kenneth Pengel, Claudette Loo, MA van den Bosch, Jelle Wesseling, M Gertenbach, Tanja Alderliesten, WP Mali, Emiel Rutgers, Harry Bartelink, Kenneth Gilhuijs
Background and purpose: Identifying breast cancers of limited extent (BCLE) is becoming increasingly important, especially for (image guided) minimally invasive therapy and partial breast irradiation. The purpose of this study is to establish characteristics at functional imaging and Materials and Methods: Seventy-five patients (77 breasts) with invasive breast cancer were prospectively included. Excision specimens were processed using complete embedding. Microscopic findings were reconstructed and correlated with contrast-enhanced MRI. Tumors were stratified by absence or presence of occult disease ≥10 mm from the MRI-visible lesion: BCLE and non127
BCLE, respectively. Imaging and pathology characteristics were evaluated for their ability to discriminate between BCLE and non-BCLE. Multivariate binary logistic regression was employed to create a prediction model for BCLE. Results: At univariate analysis, imaging as well as pathology characteristics were indicative for BCLE (39/77=51%). At multivariate analysis, a mass on mammography, the absence of tumor washout, positive ER and low quantity of DCIS in the index tumor retained significance (area under ROC curve=0.87). Conclusions: Pre-treatment assessment of mammography findings, MRI washout kinetics, ER status and quantity of DCIS in the index tumor has the potential to accurately identify BCLE.
Semi-automated primary tumor volume measurements by dynamic contrast-enhanced MRI in patients with head and neck cancer
(n=19, 26.0%) or type 3 curve, respectively (n=42, 57.5%). At multivariate analysis, the most predictive features for excluding presence of invasive disease were absence of enhancement or a type 1 curve on MRI (negative predictive value 98.5%; A(Z) 0.80, P=0.00006). Conclusions: Contrast medium uptake kinetics at MRI provide high negative predictive value to exclude presence of invasion and may be useful in primary surgical planning in patients with a preoperative diagnosis of pure DCIS. Key points: It is important to determine invasion in breast DCIS. MRI contrast medium uptake kinetics can help exclude the presence of invasion. However, the positive predictive value for the presence of invasion is limited. MRI features were more accurate at predicting invasion than mammographic features alone.
WL Lodder, Kenneth Gilhuijs, Charlotte Lange, Frank Pameijer, Fons Balm, Michiel van den Brekel
Background and purpose: Tumor volume is a significant prognostic factor in the treatment of malignant head and neck tumors. Unfortunately, it is not routinely measured because of the workload involved. Methods: Twenty-one patients, between 2009 and 2010, were studied. Dynamic contrast-enhanced MRI (DCE-MRI) at 3.0T was performed. A workstation previously developed for semiautomated segmentation of breast cancers on DCE-MRI was used to segment the head and neck cancers. The Pearson correlation analysis was used to assess the agreement between volumetric measurements and the manually derived gross tumor volume (GTV). Results: In 90.5% of the patients (19 of 21) correlation could be made between DCE-MRI and the manually derived GTV. The Pearson correlation coefficient between the automatically derived tumor volume at DCE-MRI and the manually derived GTVs was R(2) = 0.95 (p < .001). Conclusions: Semi-automated tumor volumes on DCE-MRI were representative of those derived from the manually derived GTV (R(2) = 0.95; p <.001).
MRI of the breast in patients with DCIS to exclude the presence of invasive disease E Deurloo, Jincey Sriram, Jelle Teertstra, Claudette Loo, Jelle Wesseling, Emiel Rutgers, Kenneth Gilhuijs
Objectives: Core biopsy underestimates invasion in more than 20% of patients with preoperatively diagnosed ductal carcinoma in situ (DCIS) without evidence of invasion (pure DCIS). The aim of the current study was to evaluate the efficacy of preoperative magnetic resonance imaging (MRI) to discriminate between patients with DCIS who are at high risk of invasive breast cancer and patients at low risk. Methods: One hundred and twenty-five patients, preoperatively diagnosed with pure DCIS (128 lesions; 3 bilateral) by core-needle biopsy, were prospectively included. Clinical, mammographic, histological (core biopsy) and MRI features were assessed. All patients underwent breast surgery. Analyses were performed to identify features associated with presence of invasion. Results: Eighteen lesions (14.1%) showed invasion on final histology. Seventy-three lesions (57%) showed suspicious enhancement on MRI with a type 1 (n=12, 16.4%), type 2 128
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Division head John Haanen
John Haanen MD PhD Head Joke Baars MD PhD Academic staff Paul Baas MD PhD Academic staff André Bergman MD PhD Academic staff Jos Beijnen PhD Academic staff Christian Blank MD PhD Academic staff Willem Boogerd MD PhD Academic staff Henk Boot MD PhD Academic staff Wieneke Buikhuisen MD PhD Academic staff Sjaak Burgers MD PhD Academic staff Annemieke Cats MD PhD Academic staff Jan Paul De Boer MD PhD Academic staff Dieta Brandsma MD PhD Academic staff Cecile Grootscholten MD PhD Academic staff Alwin Huitema PhD Academic staff Martijn Kerst MD PhD Academic staff Monique Van Leerdam MD PhD Academic staff Sabine Linn MD PhD Academic staff Anne Lukas MD PhD Academic staff Serena Marchetti MD Academic staff Bastiaan Nuijen PhD Academic staff Frans Opdam Academic staff Sjoerd Rodenhuis MD PhD Head Jan Schellens MD PhD Academic staff Gabe Sonke MD Academic staff Neeltje Steeghs MD PhD Academic staff Jacqueline Stouthard MD PhD Academic staff Babs Taal MD PhD Academic staff Hans Van Thienen Academic staff Margot Tesselaar MD Academic staff Michel Van den Heuvel MD PhD Academic staff Marchien Van der Weide MD PhD Academic staff Roel Van Gijn PhD Academic staff Yasser Alderlieste Temporary staff Adriaan Bins Temporary staff Rogier Boshuizen Temporary staff Annette Coumou Temporary staff Foke Van Delft Temporary staff Michael Van Geer Temporary staff Annebeth Haringhuizen Temporary staff Marieke Van Harskamp Temporary staff Michiel Van der Heijden Temporary staff Jan Van Meerbeeck MD PhD Temporary academic staff David Van Nieuwenhuizen Temporary staff 130
Diederik Ten Oever Temporary staff Noortje Schoenmakers Temporary staff Wim Van der Steeg Temporary staff Sander Wagemakers Temporary staff Hilde Wolzak Temporary staff Josine Quispel Temporary staff Dominic Snijder Temporary staff Suzan Vrijaldenhoven Temporary staff Karin Beelen PhD student and temporary staff Ellen Derissen PhD student Thomas Dorlo PhD student Anne Charlotte Dubbelman PhD student Geert Frederikx PhD student Robin Van Geel PhD student Andrew Goey PhD student Coen Van Hasselt PhD student Iris Van der Heijden PhD student Helgi Helgason PhD student Jeroen Hendrikx PhD student Bart Jacobs PhD student Nynke Jager PhD student Tine Janssens PhD student Aarti Jibodh PhD student Anita Kort PhD student Heinz Josef Klümpen PhD student Wiete Kromdijk PhD student Nienke Lankheet PhD student Suzanne Leijen PhD student Jelte Meulenaar PhD student Didier Meulendijks PhD student Bojana Milojkovic PhD student Johannes Moes PhD student Ruud Van de Noll PhD student Nalini Radhakishun PhD student Emilia Sawicki PhD student Philip Schouten PhD student Rik Stuurman PhD student Bas Teunissen PhD student Linda Tulner PhD student Jolanda Van den Hoven PhD student Mariska Van Vliet PhD student Annemieke Van Winden PhD student Michel Hillebrand Technical staff Joke Schol Technical staff Matthijs Tibben Technical staff Marja Merqui-Roelvink Clinical trial manager Sandra Adriaansz Nurse practitioner Annelies Boekhout Nurse practitioner Karina Bucholtz Nurse practitioner i.o. Ria Dubbelman Nurse practitioner Joke Foekema Nurse practitioner Emmy Harms Nurse practitioner Marjo Holtkamp Nurse practitioner Marianne Keessen Nurse practitioner Annemieke Koenen Nurse practitioner i.o. Maria Kuiper Nurse practitioner Elsbeth Van der Laan Nurse practitioner i.o. Lisette Saveur Nurse practitioner Henk Mallo Nurse practitioner Annemarie Nol Nurse practitioner Suzanne Onderwater Nurse practitioner Margaret Schot Nurse practitioner Wilma Uyterlinde Nurse practitioner Jana Van der Sar Nurse practitioner Dick Visser Nurse practitioner i.o. Marion Zimmerman Nurse practitioner i.o.
Division of Medical Oncology
Research performed in the year 2012 I Pharmaceutical formulation II Bioanalytical method development + implementation in PK studies III Early clinical phase I/II studies I
The Division of Medical Oncology comprises a growing number of research activities with focus on translational research, early drug development and clinical research. In this report only research from the largest groups is presented. Much of the work is multidisciplinary and involves groups from different divisions or different institutions both in the Netherlands and abroad. One of the major common themes between the different research groups within this division is personalized medicine, in order to offer patients the optimal treatment for their specific type of cancer.
CLINICAL PHARMACOLOGY OF ANTICANCER DRUGS Jan Schellens, Henk Boot, Annemieke Cats, Bastiaan Nuijen, Hilde Rosing, Serena Marchetti, Frans Opdam, Dieta Brandsma, Neeltje Steeghs, Alwin Huitema, Jos Beijnen
Background and Objectives Over the last year the division of clinical pharmacology has further invested in clinical and translational research involving “personalized medicine” by expanding the number of trials in which patient selection on the basis of a tumor derived genotype proceeds entry into the trial. Besides this, the department remains involved in research in different phases of anticancer drug development. This concerns: I) Pharmaceutical formulation, II) Bioanalytical method development and implementation in clinical pharmacological studies, III) Early clinical phase I/II studies and IV) supportive care studies in patients with breast cancer. Research activities of the division of Clinical Pharmacology of the department of Medical Oncology, the department of Pharmacy & Pharmacology and the department of Molecular Pathology (group Schellens) are closely integrated. In 2012 we continued clinical research fully compliant with ICH-GCP guidelines, previously certified by the Dutch Ministry of Health. The department of Pharmacy & Pharmacology successfully continued its official governmental GLP (in the field of analytical chemistry), GMP (formulation and manufacturing of investigational cytotoxic drugs) and GDP (ISO9002 for worldwide distribution of clinical supplies) licenses.
Pharmaceutical formulation
Three pharmaceutical formulation projects aiming at the development of oral dosage forms of the anti-cancer agents docetaxel, paclitaxel, and capecitabine are still ongoing. A phase I study was initiated to evaluate the release characteristics of extended release (ER) formulations of capecitabine (ModraCape). As in vitro tests appeared not to be predictive for the in vivo pharmacokinetics (PK), a matrix of different ER formulations of capecitabine was prepared. Furthermore, several projects, including pharmaceutical development and/or clinical manufacturing of cyclodextrin and liposomal formulations of various (anticancer) compounds are ongoing. During 2012, several patients were treated in phase I clinical studies with cellular therapy products (TIL, TCR) prepared in the manufacturing facilities of the Department of Pharmacy & Pharmacology. The Department takes part in several EU programs focused on the further development of cellular products (T-lymphocytes) and pDNA vaccines. A research program in collaboration with Utrecht University (UU) and University of Twente (UT) aiming at the pharmaceutical development of non-viral vectors (e.g. lipoplexes and polyplexes) for improvement of DNA-plasmid transfection is ongoing. The Department of Pharmacy & Pharmacology holds a Manufacturer’s licence issued by the Dutch Health Inspectorate (IGZ). II Bioanalytical method development + implementation in PK studies
To improve our research on pharmacokinetics and metabolite profiling, we purchased an Ultra Performance Liquid Chromatograph (UPLC, Waters) coupled to a highly sensitive mass spectrometer (API5500 trap, AB Sciex). In bioanalysis we are faced with requests to develop highly sensitive methods, for example to support clinical studies where metronomic dosing is applied, for the analysis of high potent (low dose) cytotoxic drugs, to determine drug concentrations in tumor tissue and to quantify low abundant metabolites. Using this new combination, we have succeeded to develop and validate an ultra sensitive bioanalytical method to support a phase I study with MSC2015103, an orally bio-available, selective, and highly potent small molecule inhibitor of MEK1/2. The lower limit of quantitation of the method in plasma is 2.5 pg/mL. Another advantage of the new UPLC-API5500 combination is the possibility to use lower sample volumes. This will facilitate the support of microsampling studies such as pre-clinical studies, studies using dried blood spots (DBS) sampling and studies where fine needle aspirates of tumor tissue are collected. The trap facility of the third quadrupole of the mass spectrometer enables us to execute MS3, and thus to introduce extra selectivity in our bioanalytical methods. This feature is being tested in the development of assays for the determination of nucleotides in peripheral blood mononuclear cells (PBMCs).
Publications
Aarntzen EH, de Vries IJ, Lesterhuis WJ, Schuurhuis D, Jacobs JF, Bol K, Schreibelt G, Mus R, De Wilt JH, Haanen JB, Schadendorf D, Croockewit S, Blokx WA, van Rossum MM, Kwok WW, Adema GJ, Punt CJ, Figdor CG. Targeting CD4+ T helper cells improves the induction of antitumor responses in dendritic cell based vaccination. Cancer Res. 2012 Ariotti S, Beltman JB, Chodaczek G, Hoekstra ME, van Beek AE, GomezEerland R, Ritsma L, van Rheenen J, Marée AF, Zal T, de Boer RJ, Haanen JB, Schumacher TN. Tissue-resident memory CD8+ T cells continuously patrol skin epithelia to quickly recognize local antigen. Proc Natl Acad Sci U S A. 2012;109:19739-44 Ariotti S, Haanen JB, Schumacher TN. Behavior and function of tissueresident memory T cells. Adv Immunol. 2012;114:203-16 Baas P. Primum non nocere? Does this also apply to mesothelioma? Lung Cancer. 2012;77:1 Beelen K, Zwart W, Linn SC. Can predictive biomarkers in breast cancer guide adjuvant endocrine therapy? Nat Rev Clin Oncol. 2012;9:529-541 Bergamo A, Gaiddon C, Schellens JH, Beijnen JH, Sava G. Approaching tumour therapy beyond platinum drugs: status of the art and perspectives of ruthenium drug candidates. J Inorg Biochem. 2012;106:90-9 Bergman AM. Cabazitaxel: de grote stap voorwaarts bij chemotherapie voor castratieresistent prostaatcarcinoom (CRPC)? Tijdschrift voor Urologie. 2012;2:45-52 Bex A, Etto T, Vyth-Dreese F, Blank C, Griffioen AW. Immunological heterogeneity of the RCC microenvironment: do targeted therapies influence immune response? Curr Oncol Rep. 2012;14:230-9 Blank CU, Kapiteijn E. AEB071 en SECIRA-UM, 2 nieuwe studies voor gemetastaseerde oogmelanoompatiënten, met doelgerichte therapie en immuuntherapie, Nederlands Tijdschrift voor Oncologie 2012
Blayney JK, Ceresoli GL, Castagneto B, O’Brien ME, Hasan B, Sylvester R, Rudd R, Steele J, Busacca S, Porta C, Mutti L, O’Byrne KJ, Scullin P, Gaafar R, Baas P, Van Meerbeeck J, Fennell DA. Response to chemotherapy is predictive in relation to longer overall survival in an individual patient combined-analysis with pleural mesothelioma. Eur J Cancer. 2012;48:2983-92 Boogerd W, Brandsma D, Sanders EACM. Neuro-oncologie in beeld. Springer, Houten. 2012 Boogerd W, Groeneveld F, Linn S, Baars JW, Brandsma D, van Tinteren H. Chemotherapy as primary treatment for brain metastases from breast cancer: analysis of 115 one year survivors. J Cancer Res Clin Oncol. 2012:138:1395-1403 Boshuizen R, Kuhn P, van den Heuvel M. Circulating tumor cells in non-small cell lung carcinoma. J Thorac Dis. 2012;4:456-8 Boshuizen RC, Besnard APE, Boot H, van den Heuvel MM. Kortademigheid en hoestklachten na radiofrequency ablation of liver metastases- Dyspnea and coughing after radiofrequency ablation of liver metastases. Ned Tijdschr Oncol. 2012;9:77-80 Boshuizen RC, Vincent AD, Kunst PW, Burgers JA, van den Heuvel MM. Talc Instillation Consensus Aids Differentiating Successful from Unsuccessful Pleurodesis: A Survey on the Interpretation of Pleural Approximation after Chest Tube Placement. Respiration. 2012 Boss DS, Glen H, Beijnen JH, Keesen M, Morrison R, Tait B, Copalu W, Mazur A, Wanders J, O’Brien JP, Schellens JH, Evans TR. A phase I study of E7080, a multitargeted tyrosine kinase inhibitor, in patients with advanced solid tumours. Br J Cancer. 2012;106:1598604 Bot I, Blank CU, Brandsma D. Clinical and radiological response of leptomeningeal melanoma after whole brain radiotherapy and ipilimumab. J Neurol. 2012;259:1976-8 Bromberg JE, Doorduijn JK, Baars JW, van Imhoff GW, Enting R, van den Bent MJ. Acute painful lumbosacral paresthesia after intrathecal rituximab. J Neurol 2012;259:559-561
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Cavaletti G, Cornblath DR, Merkies IS, Postma TJ, Rossi E, Frigeni B, Alberti P, Bruna J, Velasco R, Argyriou AA, Kalofonos HP, Psimaras D, Ricard D, Pace A, Galiè E, Briani C, Dalla Torre C, Faber CG, Lalisang RI, Boogerd W, Brandsma D, Koeppen S, Hense J, Storey D, Kerrigan S, Schenone A, Fabbri S, Valsecchi MG; the CIPeriNomS Group. The chemotherapyinduced peripheral neuropathy outcome measures standardization study from consensus to the first validity and reliability findings. Ann Oncol 2012 Courrech Staal EFW, Bloemendal K, Bloemer MC, Aleman BMP, Cats A, van Sandick JW. Oesophageal cancer treatment in a tertiary referral hospital evaluated by indicators for quality of care. Eur J Surg Oncol, 2012;38:150156 Criscitiello C, Azim HA, Jr., Schouten PC, Linn SC, Sotiriou C. Understanding the biology of triple-negative breast cancer. Ann Oncol. 2012;23 Suppl 6:vi13-18 Damhuis RA, Schroten C, Burgers JA. Population-based survival for malignant mesothelioma after introduction of novel chemotherapy. Eur Respir J. 2012;40:185-9 De Jonge V, Sint Nicolaas J, Cahen DL, Moolenaar W, Ouwendijk RJ, Tang TJ, van Tilburg AJ, Kuipers EJ, van Leerdam ME; SCoPE Consortium. Quality evaluation of colonoscopy reporting and colonoscopy performance in daily clinical practice. Gastrointest Endosc. 2012;75:98-106 De Miguel Luken MJ, Beijnen JH, Schellens JHM. Crizotinib. Concise drug review. The Oncologist. 2012 (in press) De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LF. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer. Breast Cancer Res Treat. 2012 De Ruiter MB, Reneman L, Boogerd W, Veltman DJ, Caan M, Douad G, Lavini C, Linn SC, Boven E, van Dam FS, Schagen SB. Late effects of high-dose adjuvant chemotherapy on white and gray matter in breast cancer survivors: converging results from multimodal magnetic resonance imaging. Hum Brain Mapp 2012;33:2971-2983 De Vries NA, Buckle T, Zhao J, Beijnen JH, Schellens JH, van Tellingen O. Restricted brain penetration of the tyrosine kinase inhibitor erlotinib due to the drug transporters P-gp and BCRP. Invest New Drugs. 2012;30:443-9 132
De Wijkerslooth TR, de Haan MC, Stoop EM, Bossuyt PM, Thomeer M, EssinkBot ML, van Leerdam ME, Fockens P, Kuipers EJ, Stoker J, Dekker E. Burden of colonoscopy compared to non-cathartic CT-colonography in a colorectal cancer screening programme: randomised controlled trial. Gut. 2012;61:1552-9 De Wijkerslooth TR, Stoop EM, Bossuyt PM, Mathus-Vliegen EM, Dees J, Tytgat KM, van Leerdam ME, Fockens P, Kuipers EJ, Dekker E. Adenoma detection with capassisted colonoscopy versus regular colonoscopy: a randomised controlled trial. Gut. 2012;61:1426-34 De Wijkerslooth TR, Stoop EM, Bossuyt PM, Meijer GA, van Ballegooijen M, van Roon AH, Stegeman I, Kraaijenhagen RA, Fockens P, van Leerdam ME, Dekker E, Kuipers EJ. Immunochemical fecal occult blood testing is equally sensitive for proximal and distal advanced neoplasia. Am J Gastroenterol. 2012;107:1570-8 Deenen MJ, Cats A, Mandigers CM, Soesan M, Terpstra WE, Beijnen JH, Schellens JH. Prevention of severe toxicity from capecitabine, 5-fluorouracil and tegafur by screening for DPD-deficiency. Ned Tijdschr Geneeskd. 2012;156:A4934 Deenen MJ, Cats A, Mandigers CM, Soesan M, Terpstra WE, Beijnen JH, Schellens JH. Het nut van testen op dihydropyrimidine dehydrogenase (DPD) deficiëntie in de behandeling met capecitabine, 5-fluorouracil en UFT. [Prevention of severe toxicity from capecitabine, 5-fluorouracil and tegafur by screening for DPD-deficiency] Ned Tijdschr Geneeskd. 2012;156:A4934 Deenen MJ, Dewit L, Boot H, Beijnen JH, Schellens JHM, Cats A. A phase I-II study of simultaneous integrated boost – intensity modulated radiation therapy with concomitant capecitabine and mitomycin-C for locally advanced anal carcinoma. Int J Rad Biol Physiol (in press) Deenen MJ, Klümpen HJ, Richel DJ, Sparidans RW, Weterman MJ, Beijnen JH, Schellens JH, Wilmink JW. Phase I and pharmacokinetic study of capecitabine and the oral mTOR inhibitor everolimus in patients with advanced solid malignancies. Invest New Drugs. 2012;30:1557-65
A DBS method was developed for the determination of tamoxifen and its active metabolite endoxifen. We are in the process of validating the method. For the therapeutic drug monitoring (TDM) of “NIB-compounds” a combination assay for the determination of gefitinib, erlotinib, dasatinib, imatinib, sunitinib, sorafenib, lapatinib and nilotinib has been validated, which is currently being offered for TDM service. For the support of a mass balance study with docetaxel and ritonavir, assays were validated in human plasma, urine and feaces. Furthermore an assay for the determination of olaparib has been developed and validated to support upcoming studies with the drug. The following sample analysis were performed for clinical and pre-clinical trials within and outside the Institute concerning docetaxel, cabacitaxel, topotecan, irrinotican and its metabolite SN-38, capecitabine and its metabolites, trabectedin, pimasertib (an inhibitor of MEK 1 and MEK2), a prodrug of gemcitabine (CO-101), gemcitabine and dFdU, gemcitabine triphosphate, cyclophosphamide (in combination with fludarabine) and its metabolite 4-hydroxy cyclophosphamide, eribulin, gancyclovir, and platinum (originating from cisplatin, carboplatin and oxaliplatin). III Early Clinical Phase I/II studies III-1 Novel approaches to improve oral bioavailability
The ‘boosting’ concept of oral docetaxel plus ritonavir, an efficacious inhibitor of gut wall and hepatic CYP3A4, has been further developed this year. The major step forward is that the maximum tolerated dose (MTD) of the oral capsule formulation of docetaxel, denoted ModraDoc001, has been established at 60 mg plus 100 mg ritonavir taken together QD. Main toxicities are diarrhea and general malaise. The MTD has also been reached in the BID schedule of docetaxel-ritonavir and was 20/100 mg. The novel oral paclitaxel capsule formulation (ModraPac001) showed excellent systemic exposure to paclitaxel the phase I study with daily low-dose or metronomic ModraPac001 plus booster drug ritonavir has reached levels of 20 mg plus 100 mg ritonavir BID. The concentration-time profiles of paclitaxel, administered as ModraPac001, at the tested dose-levels administered together with ritonavir are shown in figure 1. III-2 Pharmacology (PK/PD, ADME, mass balance) of novel anticancer drugs
Currently, we perform fourty phase I/II, pharmacological and proof of concept studies, which number has slightly increased compared with last year. We recruited more than 250 new patients for these studies this year. Twenty-nine of these studies are two- or multicenter studies. Representative examples are described. a. Studies with the poly-ADP-ribose polymerase (PARP1) inhibitor olaparib (AZD2281)
The ongoing three-center phase I trial to test the safety, optimal dose and schedule of AZD2281 when given daily BID in combination with three-weekly carboplatin and paclitaxel, or in combination with weekly paclitaxel has entered its 27th dose-level. We demonstrated promising anticancer activity in a range of tumor types, but especially patients with tumors harboring BRCA1/2 mutations benefitted most. Prolonged myelosuppression is the main toxicity of the combination.
Deenen MN, Rosing H, Hillebrand MJ, Schellens JHM, Beijnen JH. Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. Journal of Chromatography B 2012 (in press)
b. Phase I studies with oral fibroblast growth factor receptor (FGFR) inhibitors
The two trials with BGJ398 and AZD4547 respectively are ongoing. The study with AZD4547 has reached its MTD, which is hyperphosphatemia, skin and nail toxicity and general malaise. Standard phosphate lowering medication is given to prevent FGFR mediated hyperphosphatemia. Trials continue with patient prescreening for FGFR1,2 mutation or FGFR3 amplification. c.
Studies with the monoclonal antibody (MoAb) TWEAK
The first in man trial with the MoAb TWEAK directed against the ligand of the Fn14 transmembrane protein has delivered pharmacological proof of principle. Prescreening on the basis of Fn14 expression determined by IHC proceeds patient inclusion. No relevant safety concerns were documented up to 7200 mg per two weeks. The trial continues with combinations of TWEAK and erlotinib or cetuximab. d.
Other phase I studies
This year we started a number of first-in-man phase I studies, including the novel anti-Her3 MoAb, the novel combination of the anti-Her3 MoAb LJM716 plus trastuzumab, and the oral HDM2 inhibitor SAR405838. The first-in-man study with the combination of erlotinib and the PI3K inhibitor GDC0941 progressed. We continued the phase I study with the Wee1 inhibitor in combination with or gemcitabine, cisplatin or carboplatin. Main toxicities are general malaise, nausea and vomiting, which can be controlled well with intensive antiemetic medication. This concept is currently been tested in tumors with p53 pathway mutation, especially refractory ovarian cancer and advanced melanoma. Execution of the phase II study of MK1775 plus carboplatin in p53 mutant advanced platinum refractory or resistant ovarian cancer progressed. Among 14 evaluable patients 5 partial responders were documented. We started the unique concept of combining a BRAF and an mEGFR inhibitor in BRAF V600 mutant colorectal cancer to proof the principle that combined inhibition would overcome tumor unresponsiveness as has been documented preclinically (Prahallad et al., Nature 2012). We started the phase I study with the novel GSH-coated liposomal formulation of doxorubicin (2B-3-101) targeting CNS metastases and primary brain tumors. Safety of threeweekly dosing up to 50 mg/m2 was demonstrated. We also started a study with 2B3-101 in patients with leptomeningeal carcinomatosis supported by quantitative monitoring of circulating tumor cells in liquor. III-3 Pharmacokinetic and pharmacodynamic (PK/PD) modelling and simulation
Two previously developed PK/PD models describing the relationship between drug exposure and major dose limiting toxicity of two anticancer agents have been used to develop optimal clinical management protocols for toxicity in order to maintain the highest achievable dose intensity. For trastuzumab, optimal timing of left ventricular ejection fraction (LVEF) measurement and a algorithm for dosing based on LVEF was developed. For the investigational drug E7080 a protocol for adequate management of hypertension was developed which has been implemented in further clinical studies. For the oral targeted anticancer agents imatinib, sunitinib and erlotinib is was shown that a major part of the patients did not reach adequate trough levels after the standard dose.
Figure 1: Plasma concentration-time profiles of patients who were treated with oral paclitaxel formulated as capsules ModraPac001 on all occasions taken together with one single dose of 100 mg of the boosting agent ritonavir.
De Jonge V, Sint Nicolaas J, van Baalen O, Brouwer JT, Stolk MF, Tang TJ, van Tilburg AJ, van Leerdam ME, Kuipers EJ; SCoPE consortium. The incidence of 30-day adverse events after colonoscopy among outpatients in the Netherlands. Am J Gastroenterol. 2012;107:878-84 Devriese LA, Bosma AJ, van de Heuvel MM, Heemsbergen W, Voest EE, Schellens JH. Circulating tumor cell detection in advanced non-small cell lung cancer patients by multimarker QPCR analysis. Lung Cancer. 2012;75:242-7 Devriese LA, Mergui-Roelvink M, Wanders J, Jenner A, Edwards G, Reyderman L, Copalu W, Peng F, Marchetti S, Beijnen JH, Schellens JH. Eribulin mesylate pharmacokinetics in patients with solid tumors receiving repeated oral ketoconazole. Invest New Drugs. 2012 (in press) Devriese LA, Witteveen PO, Marchetti S, Mergui-Roelvink M, Reyderman L, Wanders J, Jenner A, Edwards G, Beijnen JH, Voest EE, Schellens JH. Pharmacokinetics of eribulin mesylate in patients with solid tumors and hepatic impairment. Cancer Chemother Pharmacol. 2012;70:823-32 Devriese LA, Witteveen PO, Wanders J, Law K, Edwards G, Reyderman L, Copalu W, Peng F, Marchetti S, Beijnen JH, Huitema AD, Voest EE, Schellens JH. Pharmacokinetics of eribulin mesylate in patients with solid tumors receiving repeated oral rifampicin. Br J Clin Pharmacol. 2012 (in press) Dikken JL, Cats A, Verheij M, van de Velde CJ. Randomized trials and quality assurance in gastric cancer surgery. J Surg Oncol. 2012 Dikken JL, Lemmens VE, Wouters MW, Wijnhoven BP, Siersema PD, Nieuwenhuijzen GA, van Sandick JW, Cats A, Verheij M, Coebergh JW, van de Velde CJ. Increased incidence and survival for oesophageal cancer but not for gastric cardia cancer in the Netherlands. Eur J Cancer. 2012;48:1624-32
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Dikken JL, Stiekema J, van de Velde CJ, Verheij M, Cats A, Wouters MW, van Sandick JW. Quality of Care Indicators for the Surgical Treatment of Gastric Cancer: A Systematic Review. Ann Surg Oncol. 2012 Dikken JL, van de Velde CJ, Coit DG, Shah MA, Verheij M, Cats A Treatment of resectable gastric cancer. Therap Adv Gastroenterol. 2012;5:49-69 Dikken JL, van de Velde CJH, Verheij M, Jansen EPM, Cats A. Resectabel maagcarcinoom: introductie en chirurgische behandeling. Ned Tijdsch Oncol, 2012;9:15-24 Dikken JL, van de Velde CJH, Verheij M, Jansen EPM, Cats A Resectabel maagcarcinoom: neoadjuvante en adjuvante behandeling. Ned Tijdsch Oncol, 2012;9:26-34 Dikken JL, van Grieken NC, P Krijnen, Gönen M, Tang LH, Cats A, Verheij M, Brennan MF, van de Velde CJ, Coit DG. Preoperative chemotherapy does not influence the number of evaluable lymph nodes in resected gastric cancer. Eur J Surg Oncol. 2012;38:319-25 Dikken JL, van Sandick JW, Allum WH, Johansson J, Jensen LS, Putter H, Coupland VH, Wouters MW, Lemmens VE, van de Velde CJ, van der Geest LG, Larsson HJ, Cats A, Verheij M. Differences in outcomes of oesophageal and gastric cancer surgery across Europe. Br J Surg. 2013;100:83-94 Dikken JL, Wouters MW, Lemmens VE, Putter H, van der Geest LG, Verheij M, Cats A, van Sandick JW, van de Velde CJ. Influence of hospital type on outcomes after oesophageal and gastric cancer surgery. Br J Surg. 2012;99:954-63 Donker M, Hage JJ, Woerdeman LA, Rutgers EJ, Sonke GS, Vrancken Peeters MJ. Surgical complications of skin sparing mastectomy and immediate prosthetic reconstruction after neoadjuvant chemotherapy for invasive breast cancer. Eur J Surg Oncol. 2012;38:25-30
Dubbelman AC, Rosing H, Jansen RS, Mergui-Roelvink M, Huitema AD, Koetz B, Lymboura M, Reyderman L, Lopez-Anaya A, Schellens JH, Beijnen JH. Mass balance study of [¹4C]eribulin in patients with advanced solid tumors. Drug Metab Dispos. 2012;40:313-21 Dubbelman AC, Rosing H, Thijssen B, Gebretensae A, Lucas L, Chen H, Shumaker R, Schellens JH, Beijnen JH. Development and validation of LC-MS/ MS assays for the quantification of E7080 and metabolites in various human biological matrices. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;887-888:25-34 Dubbelman AC, Tibben M, Rosing H, Gebretensae A, Nan L, Gorman SH, Robertson P Jr, Schellens JH, Beijnen JH. Development and validation of LCMS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;893-894:92-100 Dubbelman AC, Upthagrove A, Beijnen JH, Marchetti S, Tan E, Krone K, Anand S, Schellens JH. Disposition and metabolism of (14)C-dovitinib (TKI258), an inhibitor of FGFR and VEGFR, after oral administration in patients with advanced solid tumors. Cancer Chemother Pharmacol. 2012;70:653-63 Eechoute K, van der Veldt AA, Oosting S, Kappers MH, Wessels JA, Gelderblom H, Guchelaar HJ, Reyners AK, van Herpen CM, Haanen JB, Mathijssen RH, Boven E. Polymorphisms in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) predict sunitinib-induced hypertension. Clin Pharmacol Ther. 2012;92:503-10 Fennell DA, McDowell C, Busacca S, Webb G, Moulton B, Cakana A, O’Byrne KJ, Meerbeeck JV, Donnellan P, McCaffrey J, Baas P. Phase II Clinical Trial of First or Second-Line Treatment with Bortezomib in Patients with Malignant Pleural Mesothelioma. J Thorac Oncol. 2012;7:1466-1670
Donker M, Straver ME, Rutgers EJ, Valdés Olmos RA, Loo CE, Sonke GS, Wesseling J, Vrancken Peeters MJ. Radioguided occult lesion localisation (ROLL) in breast-conserving surgery after neoadjuvant chemotherapy. Eur J Surg Oncol. 2012;38:1218-24
Fink C, Hasan B, Deleu S, Pallis AG, Baas P, Brien MO. High prevalence of osteoblastic bone reaction in computed tomography scans of an European Organisation for Research and Treatment of Cancer prospective randomised phase II trial in extensive stage small cell lung cancer. Eur J Cancer. 2012;48:3157-60
Dubbelman AC, Jansen RS, Rosing H, Darwish M, Hellriegel E, Robertson P Jr, Schellens JH, Beijnen JH. Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine. Drug Metab Dispos. 2012;40:1297-307
Frederix GW, Severens JL, Hövels AM, Raaijmakers JA, Schellens JH. Reviewing the cost-effectiveness of endocrine early breast cancer therapies: influence of differences in modeling methods on outcomes. Value Health. 2012;15:94-105
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Therefore, therapeutic drug monitoring strategies were developed based on published and/or developed population PK models and Bayesian predictions. III-4 Pharmacogenomics to identify patients at risk for toxicity or undertreatment
Pharmacogenetic screening of patients treated with 5-FU/Capecitabine
DYPD*2 genotyping in routine clinical practice was continued. To date a population of more than 2000 patients has been genotyped prior to start of capecitabine therapy, amongst which seventeen heterozygotes have been identified. Dose-adaptation could be performed in twelve of these patients. Safety was excellent. No major toxicities were reported and no toxic deaths occurred. The cost analysis of the genotyping approach testing demonstrated that the adaptive dosing in DPYD*2A patients is cost-effective. IV S upportive care studies in patients with breast cancer
We continued the multicenter cardiac protection trial with the angiotensine II (ATI) receptor antagonist candesartan in patients with Her 2 positive breast cancer who received adjuvant treatment with trastuzumab. This is a double blind randomized trial versus placebo. Recruitment of the planned 200 patients has finished in the 19 centers. The last patient out will be in May 2013. Collaboration with the Dutch Medicines Evaluation Board
We performed the following dossiers for the CBG/EMA this year: Rapporteur report about regorafenib (Stivarga) for treatment of patients with metastatic colorectal cancer experiencing disease progression after all standard treatment options available; Rapporteur report about cabozantinib (Cometriq) for the treatment of patients with medullary thyroid cancer; Rapporteur report about paclitaxel albumin (Abraxane) (type II variation) for weekly treatment of patients with breast cancer. In addition Scientific advices were given twice about paclitaxel albumin (Abraxane), one in melanoma and one in breast cancer, about vemurafenib (BRAF inhibitor) in melanoma, about AZD2281 (PARP inhibitor) in ovarian cancer, about Radium-223 dichloride (Xofigo) in prostate cancer, about GDC0973 in melanoma, about Apo-Rituximab in chronic lymphocytic leukemia, and about rucaparib (PARP inhibitor) in ovarian cancer. We continued the scientific advices to the minister of Health and the Dutch Health Insurance Authority (NZA) as chairperson of the “Committee for Pharmaceutical Aid”.
CLINICAL IMMUNOTHERAPY AND TARGETED THERAPY John Haanen, Christian Blank, Hans van Thienen, Sandra Adriaansz, Henk Mallo, Elsbeth van der Laan, Loes Pronk, Marieke Groot-Obbink, Willem Boogerd, Dieta Brandsma, André Bergman
Background and Objectives The clinical immuno- and targeted therapy group is primarily involved in the treatment of melanoma and renal cell carcinoma patients. Translational immunotherapy research focuses on adoptive cellular therapies, such as T-cell receptor gene therapy and treatment with tumor-infiltrating lymphocytes (TIL) for melanoma and DNA vaccination studies for HPV-related squamous cell cancers. For renal cell cancer our group is leading in the development of investigator-initiated phase II/ III trials to improve the treatment with small molecule receptor tyrosine kinase inhibitors (RTKI), mTOR kinase inhibitors and combinations of cytokines and anti-angiogenesis drugs. Melanoma Translational research with DNA vaccination in melanoma patients
In 2012 we finished a first in man study with DNA tattoo vaccination. In this dose-escalating phase I clinical study advanced-stage HLA-A*0201-positive melanoma patients were treated with an in-house manufactured DNA vaccine, pDERMATT, encoding Tetanus Toxin Fragment C (TTFC) fused to an immunodominant epitope of the melanocyte differentiation antigen MART-1. The DNA vaccine (5 mg/ml) has been tattoed into the epidermis of patients. In 2012 the 9th and last patient was included in this study. Only local vaccination site toxicity (grade 1 and 2) was observed. In biopsies taken one week after the last tattoo (both priming and booster) from the vaccination site showed the presence of CD8+ MART-1-specific T cells, indicating that a cellular immune response had been induced.
Figure 2: Complete response 20 weeks after TIL therapy in TIL treated patient NKI3
Translational research with tumor-infiltrating lymphocytes in melanoma patients:
We finished a pilot study using tumor-infiltrating lymphocytes for patients with metastatic melanoma. This is based on impressive and consistent results from single arm, single institution trials performed USA and Israel, showing a 50% or more objective response rate in heavily pre-treated stage IV melanoma patients (see also Clinical Pharmacology Section). After receiving approval from the Central Committee on Research involving Human Subjects (CCMO) in 2011 for a pilot study (n=5), we finished this study in 2012. Toxicity was as expected and objective clinical responses were observed (figure 2). Our next step is to start a RCT phase III trial comparing TIL to standard of care. Anti-CTLA4 treatment:
From June 2010 – August 2011, Ipilimumab was available at our institute in a Named Patient Program for treatment of patients with metastatic melanoma, who have failed 1st line treatment. Over 100 patients have been enrolled in this program. Patient characteristics, objective response rate, survival and blood samples have being analysed in order to find predictive or prognostic markers. In addition, blood samples have been studied for changes in cellular immune responses upon ipilimumab treatment (see section IV). 135
Goede SL, van Roon AH, Reijerink JC, van Vuuren AJ, Lansdorp-Vogelaar I, Habbema JD, Kuipers EJ, van Leerdam ME, van BallegooijenCost-effectiveness of one versus two sample faecal immunochemical testing for colorectal cancer screening. M. Gut. 2012 (in press) Goey AKL, Mooiman KD, Beijnen JH, Schellens JHM, Meijerman I. Relevance of in vitro and clinical data for predicting CYP3A4-mediated herb-drug interactions in cancer patients. Cancer Treatment Reviews. 2012 (in press) Groenewegen G, Walraven M, Vermaat J, de Gast B, Witteveen E, Giles R, Haanen J, Voest E. Targeting the endothelin axis with atrasentan, in combination with IFN-alpha, in metastatic renal cell carcinoma. Br J Cancer. 2012;106:284-9 Grünwald V, Karakiewicz PI, Bavbek SE, Miller K, Machiels JP, Lee SH, Larkin J, Bono P, Rha SY, Castellano D, Blank CU, Knox JJ, Hawkins R, Anak O, Rosamilia M, Booth J, Pirotta N, Bodrogi I; REACT Study Group. An international expanded-access programme of everolimus: addressing safety and efficacy in patients with metastatic renal cell carcinoma who progress after initial vascular endothelial growth factor receptortyrosine kinase inhibitor therapy. Eur J Cancer. 2012;48:324-32 Haanen H, Blank CU. Immunomodulatory Drugs and Cytokines. ESMO handboek Clinical Pharmacology of Anti-cancer Agents, 2012 Haanstra JF, de Vos Tot Nederveen Cappel WH, Gopie JP, Vecht J, Vanhoutvin SA, Cats A, van der Zaag-Loonen HJ, Langers AM, Bergmann JH, van de Meeberg PC, Dekker E, Kleibeuker JH, Vasen HF, Nagengast FM, van Duijvendijk P Harmsen S, Meijerman I, MaasBakker RFM, Schellens JHM; Beijnen JH. PXR-mediated P-glycoprotein induction by small molecule tyrosine kinase inhibitors. European Journal of Pharmaceutical Sciences 2012 (in press) Hart A, Baars JW, Brandsma D. Primair centraal zenuwstelsel lymfoom. Foliolum 2012;25:7-9
Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, Rutkowski P, Blank CU, Miller WH Jr, Kaempgen E, Martín-Algarra S, Karaszewska B, Mauch C, ChiarionSileni V, Martin AM, Swann S, Haney P, Mirakhur B, Guckert ME, Goodman V, Chapman PB. Dabrafenib in BRAFmutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380(9839):358-65 Helgason HH; Kruijtzer CMF; Koolen SLW; van Werkhoven E; Malingre MM; Huitema ADR; Schot M; Smit W; Beijnen JH; Schellens JHM. Phase II and pharmacological study of oral docetaxel plus cyclosporin A in anthracycline pre-treated metastatic breast cancer. Current Clinical pharmacology. 2012 (in press) Hendrikx JJ, Lagas JS, Rosing H, Schellens JH, Beijnen JH, Schinkel AH. P-glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel. Int J Cancer. 2012 (in press) Henken FE, Oosterhuis K, Öhlschläger P, Bosch L, Hooijberg E, Haanen JB, Steenbergen RD. Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7. Vaccine. 2012;30:4259-66 Hol L, Kuipers EJ, van Ballegooijen M, van Vuuren AJ, Reijerink JC, Habbema DJ, van Leerdam ME. Uptake of faecal immunochemical test screening among nonparticipants in a flexible sigmoidoscopy screening programme. Int J Cancer. 2012;130:2096-102 Hollevoet K, Reitsma JB, Creaney J, Grigoriu BD, Robinson BW, Scherpereel A, Cristaudo A, Pass HI, Nackaerts K, Rodríguez Portal JA, Schneider J, Muley T, Di Serio F, Baas P, Tomasetti M, Rai AJ, van Meerbeeck JP. Serum mesothelin for diagnosing malignant pleural mesothelioma: an individual patient data meta-analysis. J Clin Oncol. 2012;30:1541-9 Hooijkaas A, Gadiot J, Morrow M, Stewart R, Schumacher T, Blank CU. Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma. Oncoimmunology. 2012;1:609-617 Hooijkaas AI, Gadiot J, van der Valk M, Mooi WJ, Blank CU. Targeting BRAF(V600E) in an Inducible Murine Model of Melanoma. Am J Pathol. 2012;181:785-94
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BRAF V600E mutated melanoma
In 2011 we participated in the vemurafenib Expanded Access Program (EAP) for patients with BRAF mutated stage IV melanoma. We screened more than 100 patients in the EAP and more than 50 patients were enrolled. Tumor and blood samples have been collected for translational research. In addition, we participated in phase II trial with MEK1/2 inhibitor MEK162 and a phase III trial with BRAF V600E/K inhibitor Dabrafenib. Trials in which combination of BRAF and MEK inhibitors are being compared to BRAF inhibitors alone are ongoing.
BREAST CANCER THERAPY AND RESPONSE PREDICTION Sabine Linn, Sjoerd Rodenhuis, Gabe Sonke, Jacqueline Stouthard, Karin Beelen, Jos Beijnen, Jorma de Ronde, Marjo Holtkamp, Alwin Huitema, Inge Kemper, Rutger Koornstra, Esther Lips, Ingrid Mandjes, Lennart Mulder, Jettie Muris, Petra Nederlof, Margaret Schot, Philip Schouten, Marieke Vollebergh, Jelle Wesseling, Lodewyk Wessels
Background and objectives Renal cell carcinoma In the renal cell carcinoma program we closely collaborate with Dr Axel Bex from the urology-oncology group. The idea of defining the optimal timing for nephrectomy in primary metastatic RCC was adopted by the EORTC GU group and has been approved as a randomized controlled multicenter phase III trial (NCT01099423) that has started enrolment in 2010. Patients are being randomized to receive either 3 cycles of sunitinib treatment prior to tumor nephrectomy or immediate nephrectomy. Post surgery both groups will (re)start sunitinib treatment until disease progression. Study endpoint: Progression-free and overall survival. In addition, we recently started participation in two Dutch RCC consortium (WIN-O) trials: the ROPETAR trial which studies the effect of alternating pazopanib and everolimus on the resistance against TKI. The Cyclo/Everolimus study investigates the effect of eliminating Tregs on the outcome of everolimus treatment in mRCC.
The objective of this program is to develop and improve potentially curative therapy for patients with locoregional or oligometastatic breast cancer. For all studies, close collaborations are maintained with many other clinical departments and research divisions. In 2012, over 170 patients were entered in sixteen medical studies in breast cancer, either focusing on the treatment of early breast cancer or on advanced disease.
Preoperative chemotherapy The preoperative chemotherapy program continues to accrue over 100 patients per year and now includes data and tissues of more than 500 patients. The program is the core of a multidisciplinary research effort to optimize response prediction and to improve response monitoring. It consists of separate studies for ER+/HER2- breast cancer (single institution), Triple Negative tumors (neo-TN randomized multicenter trial) and HER2+ tumors (multicenter studies). The effort to identify mRNA gene expression patterns associated with chemotherapy resistance continues, and the validation of lists of candidate genes for each breast cancer subtype is ongoing. Next generation sequencing has been employed in sensitive and resistant triple-negative tumors and the analysis of the results is in progress. An aCGH signature predictive for a response to bifunctional alkylating agents has been defined previously. A newly developed MLPA assay for the same purpose was validated in independent series of samples and is now undergoing prospective testing in the Neo-TN study (NCT01057069). Since the addition of participating hospitals, patient accrual per unit of time of this multicenter randomized trial now meets its objective. The TRAIN-1 study (NCT00768859) completed its accrual and analysis of prognostic biomarkers is underway in over 120 trastuzumab treated patients. The TRAIN-2 study will be a multicenter phase II study employing dual HER2 targeting as well as combined endocrine and anti-HER2 targeted agents. The study is expected to start accruing patients in early 2013. The large number of available primary breast cancer samples with complete clinical data has allowed the detailed evaluation of a range of biomarkers for their ability to predict the response to neoadjuvant chemotherapy. In luminal tumors, standard immunohistochemistry and pathological grade were superior to both intrinsic subtypes (PAM 50) and ‘surrogate intrinsic subtypes’. Lobular histology had no independent predictive value when pathological grade and hormone receptor expression were taken into account.
Hubers AJ, Heideman DA, Herder GJ, Burgers SA, Sterk PJ, Kunst PW, Smit HJ, Postmus PE, Witte BI, Duin S, Snijders PJ, Smit EF, Thunnissen E. Prolonged sampling of spontaneous sputum improves sensitivity of hypermethylation analysis for lung cancer. J Clin Pathol. 2012;65:541-5 Jager NG, Rosing H, Linn SC, Schellens JH, Beijnen JH. Importance of highly selective LC-MS/MS analysis for the accurate quantification of tamoxifen and its metabolites: focus on endoxifen and 4-hydroxytamoxifen. Breast Cancer Res Treat. 2012;133:793-798 Jansen EP, Boot H, van de Velde CJ, van Sandick J, Cats A, Verheij M. Can adjuvant chemoradiotherapy replace extended lymph node dissection in gastric cancer? Recent Results Cancer Res. 2012;196:229-40 Jansen RS, Rosing H, Wijermans PW, Keizer RJ, Schellens JH, Beijnen JH. Decitabine triphosphate levels in peripheral blood mononuclear cells from patients receiving prolonged lowdose decitabine administration: a pilot study. Cancer Chemother Pharmacol. 2012;69:1457-66 Joerger M, Burgers JA, Baas P, Doodeman VD, Smits PH, Jansen RS, Vainchtein LD, Rosing H, Huitema AD, Beijnen JH, Schellens JH. Gene polymorphisms, pharmacokinetics, and hematological toxicity in advanced non-small-cell lung cancer patients receiving cisplatin/gemcitabine. Cancer Chemother Pharmacol. 2012;69:25-33 Joerger M, Burgers SA, Baas P, Smit EF, Haitjema TJ, Bard MP, Doodeman VD, Smits PH, Vincent A, Huitema AD, Beijnen JH, Schellens JH. Germline polymorphisms in patients with advanced nonsmall cell lung cancer receiving first-line platinum-gemcitabine chemotherapy: a prospective clinical study. Cancer. 2012;118:2466-75 Joerger M, Ferreri AJ, Krähenbühl S, Schellens JH, Cerny T, Zucca E, Huitema AD. Dosing algorithm to target a predefined AUC in patients with primary central nervous system lymphoma receiving high dose methotrexate. Br J Clin Pharmacol. 2012;73:240-7 Joerger M, Kraff S, Huitema AD, Feiss G, Moritz B, Schellens JH, Beijnen JH, Jaehde U. Evaluation of a pharmacology-driven dosing algorithm of 3-weekly paclitaxel using therapeutic drug monitoring: a pharmacokineticpharmacodynamic simulation study. Clin Pharmacokinet. 2012;51:607-17
Kaiser AD, Gadiot J, Guislain A, Blank CU. Mimicking homeostatic proliferation in vitro generates T cells with high anti-tumor function in non-lymphopenic hosts. Cancer Immunol Immunother. 2012 Kaiser AD, Schuster K, Gadiot J, Borkner L, Daebritz H, Schmitt C, Andreesen R, Blank C. Reduced tumor-antigen density leads to PD-1/ PD-L1 mediated impairment of partially exhausted CD8(+) T cells. Eur J Immunol. 2012;42:662-71 Kaminski MF, Bretthauer M, Zauber AG, Kuipers EJ, Adami HO, van Ballegooijen M, Regula J, van Leerdam M, Stefansson T, Påhlman L, Dekker E, Hernán MA, Garborg K, Hoff G. The NordICC Study: rationale and design of a randomized trial on colonoscopy screening for colorectal cancer. Endoscopy. 2012;44:695-702 Kapidzic A, Korfage IJ, van Dam L, van Roon AH, Reijerink JC, Zauber AG, van Ballegooijen M, Kuipers EJ, van Leerdam ME. Quality of life in participants of a CRC screening program. Br J Cancer. 2012;107:1295301 Keizer RJ, Gupta A, Shumaker R, Beijnen JH, Schellens JH, Huitema AD. Model-based treatment optimization of a novel VEGFR inhibitor. Br J Clin Pharmacol. 2012;74:315-26 Keizer RJ, Zandvliet AS, Beijnen JH, Schellens JH, Huitema AD. Performance of methods for handling missing categorical covariate data in population pharmacokinetic analyses. AAPS J. 2012;14:601-11 Keizer RJ, Zandvliet AS, Beijnen JH, Schellens JH, Huitema AD. Two-stage model-based design of cancer phase I dose escalation trials: evaluation using the phase I program of barasertib (AZD1152). Invest New Drugs. 2012;30:1519-30 Kerklaan BM, Diéras V, Le Tourneau C, Mergui-Roelvink M, Huitema AD, Rosing H, Beijnen JH, Marreaud S, Govaerts AS, Piccart-Gebhart MJ, Schellens JH, Awada A. Phase I study of lonafarnib (SCH66336) in combination with trastuzumab plus paclitaxel in Her2/ neu overexpressing breast cancer: EORTC study 16023. Cancer Chemother Pharmacol. 2012 (in press) Kersseboom R, Dubbink HJ, Corver WE, van Tilburg AJ, Poley JW, van Leerdam ME, Atmodimedjo PN, van de Laar IM, Collée JM, Dinjens WN, Morreau H, Wagner A. PTEN in colorectal cancer: a report on two Cowden syndrome patients. Clin Genet. 2012;81:555-62
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Kerst JM, Moonen L, Graafland NM, Bergman AM, Pos J, Horenblas S. Textbook o penile cancer (Eds Muneer A, Arya M, Horenblas S). Chapter 12: The role of chemotherapy and radiotherapy in the treatment of Penile Cancer (Springer Verlag) 2012 Kluijt I, Sijmons RH, Hoogerbrugge N, Plukker JT, de Jong D, van Krieken JH, van Hillegersberg R, Ligtenberg M, Bleiker E, Cats A. Familial gastric cancer: guidelines for diagnosis, treatment and periodic surveillance. Fam Cancer. 2012;11:363-9 Kok M, Koornstra RH, Mook S, Hauptmann M, Fles R, Jansen MP, Berns EM, Linn SC, Van ‘t Veer LJ. Additional value of the 70-gene signature and levels of ER and PR for the prediction of outcome in tamoxifentreated ER-positive breast cancer. Breast. 2012;21:769-778 Kontny NE, Würthwein G, Boos J, Boddy AV, Krischke M, Fuhr U, Thompson PA, Jörger M. Schellens JHM, Hempel G. Population pharmacokinetics of doxorubicin: establishment of a NONMEM model for adults and children older than three years. Cancer Chemotherapy and Pharmacology 2012 (in press) Koolen BB, Valdés Olmos RA, Elkhuizen PH, Vogel WV, Vrancken Peeters MJ, Rodenhuis S, Rutgers EJ. Locoregional lymph node involvement on 18F-FDG PET/CT in breast cancer patients scheduled for neoadjuvant chemotherapy. Breast Cancer Res Treat. 2012;135:231-40 Koolen BB, Vrancken Peeters MJ, Aukema TS, Vogel WV, Oldenburg HS, Van der Hage JA, Hoefnagel CA, Stokkel MP, Loo CE, Rodenhuis S, Rutgers EJ, Valdés Olmos RA. 18F-FDG PET/CT as a staging procedure in primary stage II and III breast cancer: comparison with conventional imaging techniques. Breast Cancer Res Treat. 2012;31:117-26 Koolen BB, Vrancken Peeters MJ, Wesseling J, Lips EH, Vogel WV, Aukema TS, Van Werkhoven E, Gilhuijs KG, Rodenhuis S, Rutgers EJ, Valdés Olmos RA. Association of primary tumour FDG uptake with clinical, histopathological and molecular characteristics in breast cancer patients scheduled for neoadjuvant chemotherapy. Eur J Nucl Med Imaging. 2012;39:1830-8 Koolen SL, van Waterschoot RA, van Tellingen O, Schinkel AH, Beijnen JH, Schellens JH, Huitema AD. From mouse to man: predictions of human pharmacokinetics of orally administered docetaxel from preclinical studies. J Clin Pharmacol. 2012;52:370-80 138
Koppelmans V, Breteler MM, Boogerd W, Seynave C, Gundy C, Schagen SB. Neuropsychological performance in survivors of breast cancer more than 20 years after adjuvant chemotherapy. J Clin Oncol 2012;30:1080-1086 Koppelmans V, de Ruiter MB, van der Lijn F, Boogerd W, Seynave C, van der Lugt A, Vrooman H, Niessen WJ, Breteler MM, Schagen SB. Global and focal brain volume in long-term breast cancer survivors exposed to adjuvant chemotherapy. Breast Cancer Res Treat 2012;132:1099-1106 Korsse SE, Dewint P, Kuipers EJ, van Leerdam ME. Small bowel endoscopy and Peutz-Jeghers syndrome. Best Pract Res Clin Gastroenterol. 2012;26:263-78 Koster GT, Brandsma D, Boot H, Kruyt ND. Multiple cerebral air emboli during upper gastrointestinal endoscopy. J Neurol Neurosurg Psychiatry 2012;83:1110-1 Kubal TE, Spiess PhE, Bergman AM. Essentials and Updates in Urologic Oncology (Eds Ph. E. Spiess). Chapter 31: Management of advanced penile cancer Kvistborg P, Shu CJ, Heemskerk B, Fankhauser M, Thrue CA, Toebes M, van Rooij N, Linnemann C, van Buuren MM, Urbanus JH, Beltman JB, Thor Straten P, Li YF, Robbins PF, Besser MJ, Schachter J, Kenter GG, Dudley ME, Rosenberg SA, Haanen JB, Hadrup SR, Schumacher TN. TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients. Oncoimmunology. 2012;1:409-418 Kwint M, Uyterlinde W, Nijkamp J, Chen C, de Bois J, Sonke JJ, van den Heuvel M, Knegjens J, van Herk M, Belderbos J. Acute esophagus toxicity in lung cancer patients after intensity modulated radiation therapy and concurrent chemotherapy. Int J Radiat Oncol Biol Phys. 2012;84:e223-8 Lankheet N, Schaake E, Rosing H, Burgers J, Schellens J, Beijnen J, Huitema A. Quantitative determination of erlotinib and O-desmethyl erlotinib in human EDTA plasma and lung tumor tissue. Bioanalysis. 2012;4:2563-77 Lankheet NA, Hillebrand MJ, Rosing H, Schellens JH, Beijnen JH, Huitema AD. Method development and validation for the quantification of dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib and sunitinib in human plasma by liquid chromatography coupled with tandem mass spectrometry. Biomed Chromatogr. 2012 (in press)
Adjuvant systemic therapy, prognosis and prediction The primary objective of the Matador study is to identify predictive gene expression profiles for a disease-free survival benefit of either a docetaxel-containing regimen (docetaxel-doxorubicin-cyclophosphamide (TAC)) or an accelerated doxorubicin-cyclophosphamide (ACdd) regimen (ISRCTN61893718). As such, frozen tissue of the primary tumor is mandatory to be eligible. Accrual of 660 patients has been completed. Thirty-eight of the 50 events in the first 200 patients, needed to perform first analyses, have occurred. In an exploratory sub-study, GSTP1 14Ala/114Val polymorphism was associated with an increased risk of peripheral polyneuropathy. The translational preoperative window trial to study responsiveness of hormone-receptor positive breast cancers to tamoxifen, anastrozole or fulvestrant in postmenopausal patients has been extended to other centers (NCT00738777), and 48 patients have been accrued. Changes in Ki67 expression are used as a read-out for hormonal therapy responsiveness. In collaboration with Leiden University Medical Center and the TEAM study group, the TEAM IIb trial has been initiated to study the benefit of three years adjuvant oral ibandronate in addition to standard adjuvant systemic therapy in postmenopausal patients with hormone-receptor positive early breast cancer (currently ~840 patients included) (ISRCTN17633610).
Oligometastatic breast cancer The OLIGO study (NCT01646034) randomizes patients with limited metastatic disease (up to three distant metastases amenable for radical local treatment) between conventional chemotherapy and high dose alkylating treatment. Only patients whose tumor harbor homologous recombination deficiency as determined by the aCGH profile or by BRCA1/2 mutation are eligible for randomization. The aim of the study is to provide randomized evidence to support an aggressive oligometastatic approach and to validate the aCGH signature in this population.
THORACIC ONCOLOGY Paul Baas, Sjaak Burgers, Wieneke Buikhuisen, Michel van den Heuvel, Josine Quispel, Jose Belderbos, Houke Klomp, Petra Nederlof, Sjaak Neefjes, Annegien Broeks, Jan Schellens
The department of Thoracic Oncology focuses on studies of chemo-radiotherapy, malignant mesothelioma and immunomodulating studies.
Non-small cell lung cancer (NSCLC) In early stage disease the results of a neoadjuvant trial using erlotinib were recently presented supporting the potential of neo-adjuvant targeted therapy. In locally advanced disease detailed follow up of toxicity and other relevant clinical data since 2007 has started to led to publications on toxicity and its management. Results on the trial combining concurrent chemoradiotherapy with or without Cetuximab (NTR2230) were presented at ESTRO and ASCO. A phase I trial combining the PARP inhibitor Olaparib with radiotherapy with or without cisplatin has started (NCT1562210). In advanced stage disease our main attention focuses on molecular profiling. Standard of care involves profiling a set of 20 genes for actionable alterations (Oncocarta). Next Generation Sequencing is being implemented at present in an oncology-wide initiative that involves the establishment of a tumor sequencing board. Clinical trials that specifically use this molecular profiling on for instance ALK-translocations, KRASmutations, and PD1 ligand expression are ongoing. Furthermore, we participate in several trials such as first line treatment with carboplatin, taxol, bevacizumab plus or minus nitroglycerine patches. Other initiatives in non-small lung cancer are in conjunction with the European Thoracic Oncology Platform for maintenance therapy or the so-called Lungscape study in which retrospective analysis of resection material is analysed for different activating mutations.
Small cell lung cancer For patient with limited stage disease we participate in the phase III European CONVERT study, which investigates the effects of concurrent twice-daily radiotherapy and chemotherapy versus sequential chemotherapy and radiotherapy. For extensive stage disease the impact of local chest irradiation is examined (CREST study). Currently no other studies are ongoing.
Pleural diseases A national study was initiated comparing indwelling catheters vs. standard thorax drainage with pleurodesis in patients with malignant effusions (NTR2518) and the first results have been published. The group continues the development of a biobank on pleural effusions for the development of innovative diagnostic and treatment algorithms on malignant pleural effusions. Over 600 patients have given consent to biomarker research applying both frozen serum and pleural fluid samples.
Lankheet NAG, Steeghs N, Rosing H, Schellens JHM, Beijnen JH, Huitema ADR Quantification of sunitinib and N-desethyl sunitinib in human EDTA plasm by liquid chromatography coupled with electrospray ionization tandem mass spectrometry: validation and application in routine therapeutic drug monitoring. Ther Drug Monit 2012 (in press) Leenen CH, van Lier MG, van Doorn HC, van Leerdam ME, Kooi SG, de Waard J, Hoedemaeker RF, van den Ouweland AM, Hulspas SM, Dubbink HJ, Kuipers EJ, Wagner A, Dinjens WN, Steyerberg EW. Prospective evaluation of molecular screening for Lynch syndrome in patients with endometrial cancer ≤ 70 years. Gynecol Oncol. 2012;125:414-20 Leijen S, Middleton MR, Tresca P, Kraeber-Bodéré F, Dieras V, Scheulen ME, Gupta A, Lopez-Valverde V, Xu ZX, Rueger R, Tessier JJ, Shochat E, Blotner S, Naegelen VM, Schellens JH, Eberhardt WE. Phase I dose-escalation study of the safety, pharmacokinetics, and pharmacodynamics of the MEK inhibitor RO4987655 (CH4987655) in patients with advanced solid tumors. Clin Cancer Res. 2012;18:4794-805 Lips EH, Mukhtar RA, Yau C, de Ronde JJ, Livasy C, Carey LA, Loo CE, Vrancken-Peeters MJ, Sonke GS, Berry DA, Van’t Veer LJ, Esserman LJ, Wesseling J, Rodenhuis S, Shelley Hwang E; I-SPY TRIAL Investigators. Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer. Breast Cancer Res Treat. 2012;136:35-43
Majewski IJ, Kluijt I, Cats A, Scerri TS, de Jong D, Kluin RJ, Hansford S, Hogervorst FB, Bosma AJ, Hofland I, Winter M, Huntsman D, Jonkers J, Bahlo M, Bernards R. An alpha-E-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer. J Pathol. 2012 Manson ML, Derissen EJ, Wijermans PW, Schellens JH, Beijnen JH. Demethylating medication in myelodysplastic syndrome. Ned Tijdschr Geneeskd. 2012;156:A3167 Mertens LS, Fioole-Bruining A, van Rhijn BW, Kerst JM, Bergman AM, Vogel WV, Vegt E, Horenblas S. FDG-PET/CT in Monitoring Response of Pelvic Lymph Node Metastases to Neoadjuvant Chemotherapy in Bladder Cancer. J Urol. 2012 Mertens LS, Meijer RP, Kerst JM, Bergman AM, van Tinteren H, van Rhijn BW, Horenblas S. Carboplatin based induction chemotherapy for nonorgan confined bladder cancer--a reasonable alternative for cisplatin unfit patients? J Urol. 2012;188:1108-1113 Meulenaar J, Beijnen JH, Schellens JH, Nuijen B. Slow dissolution behaviour of amorphous capecitabine. Int J Pharm 2012 (in press) Moes J, Koolen S, Huitema A, Schellens J, Beijnen J, Nuijen B. Development of an oral solid dispersion formulation for use in low-dose metronomic chemotherapy of paclitaxel. Eur J Pharm Biopharm. 2012 (in press)
Lips EH, Mulder L, De Ronde, JJ, Mandjes IAM, Vincent A, Vrancken Peeters, MTFD, Nederlof PM, Wesseling J, Rodenhuis S. Neoadjuvant chemotherapy in ER+ HER2− breast cancer: response prediction based on immunohistochemical and molecular characteristics. Breast Cancer Res Treat. 2012;131:827-36
Mooiman KD, Goey AK, Meijerman I, Beijnen JH, Schellens JH. Letter to the editor regarding “A prospective, controlled study of the botanical compound mixture LCS101 for chemotherapy-induced hematological complications in breast cancer” by Yaal-Hahoshen et al. (The Oncologist 2011;16:1197-1202). Oncologist. 2012;17:740-1; author reply 742-3
Lips IM, van Gils CH, Kotte AN, van Leerdam ME, Franken SP, van der Heide UA, van Vulpen M. A doubleblind placebo-controlled randomized clinical trial with magnesium oxide to reduce intrafraction prostate motion for prostate cancer radiotherapy. Int J Radiat Oncol Biol Phys. 2012;83:653-60
Mougiakakos D, Okita R, Ando T, Dürr C, Gadiot J, Ichikawa J, Zeiser R, Blank C, Johansson C, Kiessling R. High Expression of GCLC is Associated with Malignant Melanoma of Low Oxidative Phenotype and Predicts a Better Prognosis. Journal of Molecular Medicine. 2012;90:935-44
Loibl S, Han SN, von Minckwitz G, Bontenbal M, Ring A, Giermek J, Fehm T, Van Calsteren K, Linn SC, Schlehe B, Gziri MM, Westenend PJ, Muller V, Heyns L, Rack B, Van Calster B, Harbeck N, Lenhard M, Halaska MJ, Kaufmann M, Nekljudova V, Amant F. Treatment of breast cancer during pregnancy: an observational study. Lancet Oncol. 2012;13:887-896
Moulin V, Morgan ME, EleveldTrancikova D, Haanen JB, Wielders E, Looman MW, Janssen RA, Figdor CG, Jansen BJ, Adema GJ. Targeting dendritic cells with antigen via dendritic cell-associated promoters. Cancer Gene Ther. 2012;19:303-11
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Mulder SA, Kranse R, Damhuis RA, Ouwendijk RJ, Kuipers EJ, van Leerdam ME. The incidence and risk factors of metachronous colorectal cancer: an indication for follow-up. 2012;55:522-31 Nijenhuis CM, Haanen JB, Schellens JH, Beijnen JH. Is combination therapy the next step to overcome resistance and reduce toxicities in melanoma? Cancer Treat Rev. 2012 Nijkamp J, Rossi M, Lebesque J, Belderbos J, van den Heuvel M, Kwint M, Uyterlinde W, Vogel W, Sonke JJ. Relating acute esophagitis to radiotherapy dose using FDG-PET in concurrent chemo-radiotherapy for locally advanced non-small cell lung cancer. Radiother Oncol. 2012 Numan RC, Klomp HM, Li W, Buitelaar DR, Burgers JA, Van Sandick JW, Wouters MW. A clinical audit in a multidisciplinary care path for thoracic surgery: An instrument for continuous quality improvement. Lung Cancer. 2012;78:270-5 Olmos D, A’hern RP, Marsoni S, Morales R, Gomez-Roca C, Verweij J, Voest EE, Schöffski P, Ang JE, Penel N, Schellens JH, Del Conte G, Brunetto AT, Evans TR, Wilson R, Gallerani E, Plummer R, Tabernero J, Soria JC, Kaye SB. Patient selection for oncology phase I trials: a multi-institutional study of prognostic factors. J Clin Oncol. 2012;30:996-1004 Oonk AM, Van Rijn C, Smits MM, Mulder L, Laddach N, Savola SP, Wesseling J, Rodenhuis S, Imholz AL, Lips EH. Clinical correlates of ‘BRCAness’ in triple-negative breast cancer of patients receiving adjuvant chemotherapy. Ann Oncol. 2012;23:2301-5 Oosterhuis K, Aleyd E, Vrijland K, Schumacher TN, Haanen JB. Rational design of DNA vaccines for the induction of human papillomavirus type 16 e6- and e7-specific cytotoxic T-cell responses. Hum Gene Ther. 2012;23:1301-12 Oosterhuis K, van den Berg JH, Schumacher TN, Haanen JB. DNA vaccines and intradermal vaccination by DNA tattooing. Curr Top Microbiol Immunol. 2012;351:221-50 Opdam FL, Guchelaar HJ, Beijnen JH, Schellens JH. Lapatinib for advanced or metastatic breast cancer. Oncologist. 2012;17:536-42
Penel N, Demetri GD, Blay JY, Cousin S, Maki RG, Chawla SP, Judson I, Van Mehren M, Schöffski P, Verweij J, Casali P, Rodenhuis S, Schütte HJ, Cassar A, Gomez J, Nieto A, Zintl P, Pontes MJ, Le Cesne A. Growth modulation index as metric of clinical benefit assessment among advanced soft tissue sarcoma patients receiving trabectedin as a salvage therapy. Ann Oncol. 2012 Ploquin A, Olmos D, Lacombe D, A’Hern R, Duhamel A, Twelves C, Marsoni S, Morales-Barrera R, Soria JC, Verweij J, Voest EE, Schöffski P, Schellens JH, Kramar A, Kristeleit RS, Arkenau HT, Kaye SB, Penel N. Prediction of early death among patients enrolled in phase I trials: development and validation of a new model based on platelet count and albumin. Br J Cancer. 2012;107:1025-30 Pluim D, Devriese LA, Beijnen JH, Schellens JH. Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular-signal-regulated kinase and DNA in circulating tumor cells. Cytometry A. 2012;81:664-71 Pluim D, Jacobs BA, Beijnen JH, Schellens JHM. ABC-01765-2012. R1 - Correction of peripheral blood mononuclear cell cytosolic protein for hemoglobin contamination. Analytical and Bioanalytical Chemistry 2012 (in press) Quality of Life After Surgery for Colon Cancer in Patients With Lynch Syndrome: Partial Versus Subtotal Colectomy. Dis Colon Rectum. 2012;55:653-659 Retel VP, Joore MA, Linn SC, Rutgers EJ, van Harten WH. Scenario drafting to anticipate future developments in technology assessment. BMC research notes. 2012;5:442 Rottenberg S, Vollebergh MA, de Hoon B, de Ronde J, Schouten PC, Kersbergen A, Zander SA, Pajic M, Jaspers JE, Jonkers M, Loden M, Sol W, van der Burg E, Wesseling J, Gillet JP, Gottesman MM, Gribnau J, Wessels L, Linn SC, Jonkers J, Borst P. Impact of intertumoral heterogeneity on predicting chemotherapy response of BRCA1-deficient mammary tumors. Cancer Res. 2012;72:2350-2361 Schaake EE, Belderbos JS, Buikhuisen WA, Rossi MM, Burgers JA, Sonke JJ. Mediastinal lymph node position variability in non-small cell lung cancer patients treated with radical irradiation. Radiother Oncol. 2012
Malignant pleural mesothelioma (MPM) The results of the randomised phase III switch maintenance study of Thalidomide after standard chemotherapy has been submitted (negative outcome). For second and third line therapy a randomized phase III study of Vorinostat versus placebo has been evaluated with negative outcome (submitted). The randomized phase IB-II study of chemotherapy plus or minus axitinib with translational research has been finalised and results are expected in 2013. In the laboratory of Jaques Neefjes, tumor cells obtained form pleural fluid of patients with malignant mesothelioma are cultured. In these short time cell cultures a library of DUBS and drugs are tested. Together with the Sanger Institute (Cambridge) full genome and exone sequencing is performed to identify targets for treatment. Promising new drugs will then be tested in animal models. In addition, in the lab of Anton Berns patient material is implanted in SCID mice for these purposes.
GASTROENTEROLOGY Henk Boot, Annemieke Cats, Cecile Grootscholten, Monique van Leerdam, Babs Taal, Margot Tesselaar, Rene Bernards, Maarten Deenen, Luc Dewit, Johan Dikken, Edwin Jansen, Daphne de Jong, Irma Kluijt, Tiny Korse, Ian Majewski, Corrie Marijnen, Didier Meulendijks, Johanna van Sandick, Lisette Saveur, Jan Schellens, Baukelien van Triest, Anouk Trip, Steven Vanhoutvin, Marcel Verheij
Background and Objectives
Figure 3: 1a: LN per Tcat MSK
The division of Gastroenterology and Hepatology is involved in different phases of clinical research, with emphasis on innovative multimodality treatments for gastric, anal and rectal cancer, NET and hereditary tumors.
In patients with primary irresectable gastric cancer (M0) the NARCIS-study investigated the role for neoadjuvant chemoradiotherapy with carboplatin and paclitaxel. This study showed demonstrated its feasibility, and promising (pathological) response and R0 resection rate in collaboration with 2 other Dutch institutes to determine. For patients with metastatic gastric cancer we initiated a multicenter study with this regimen in combination with bevacizumab and trastuzumab for Her2-positive tumors. More than 20 patients have started treatment. In a nationwide study, we investigated reasons to opt for (or against) DNA-testing, experience with surveillance and prophylactic gastrectomy, decisional conflict, and the effect of gastrectomy on cancer worries in 31 patients with hereditary diffuse-type gastric cancer with a CDH1-mutation. We also investigated the impact of a prophylactic total gastrectomy on daily life and compared this with patients undergoing a gastrectomy for gastric cancer. We are planning to study this further prospectively.
Schilder CM, Seynave C, Linn SC, Boogerd W, Beex LV, Gundy CM, Nortier JW, van de Velde CJ, van Dam FS, Schagen SB. Self-reported cognitive functioning inostmenopausal breast cancer patients before and during endocrine treatment: findings from the neuropsychological TEAM side-study. Psychooncology 2012;21:479-487 Sigmond J, Leon LG, Kamphuis JAE, Bergman AM, Peters GJ. Effect of staurosporine and ucn-01 on gemcitabine cytotoxicity in relation to cell cycle effects and p53 status. Cancer Therapeutics Research. 2012
Gastric cancer The international, randomized, controlled, CRITICS-study continues to accrue patients with primary resectable gastric cancer and preoperative chemotherapy and investigates the role of postoperative chemotherapy versus chemoradiotherapy. The study has now included more than 500 of the 788 required patients, of which most have been entered in the NKI-AVL. A quality control program has shown to improve lymph node yield in participating institutes and will be further prolonged. Several international and nation-wide studies evaluated the role of treatment factors, such as hospital type and hospital volume, that play an important role on primary gastric cancer outcomes. In collaboration with researchers from the Memorial Sloan Kettering Cancer Center in New York and the Comprehensive Cancer Center Leiden it was shown that neoadjuvant chemotherapy does not affect lymph node yield in gastric cancer resection specimens. (figure 3 and figure 4)
Between 30-40% of hereditary diffuse gastric cancer (HDGC) can be explained by defective germline alleles of E-cadherin (CDH1), but for the remaining families the factors driving susceptibility remain unknown. We had access to a large HDGC 140
Schaake EE, Kappers I, Codrington HE, Valdés Olmos RA, Teertstra HJ, van Pel R, Burgers JA, van Tinteren H, Klomp HM. Tumor response and toxicity of neoadjuvant erlotinib in patients with early-stage non-small-cell lung cancer. J Clin Oncol. 2012;30:2731-8
Figure 4: 1b: LN per Tcat NCR The difference of absent in LNN yield after chemotherapy
Sint Nicolaas J, de Jonge V, Cahen DL, Ouwendijk RJ, Tang TJ, van Tilburg AJ, van Leerdam ME, Kuipers EJ; SCoPE Consortium. Awareness of surveillance recommendations among patients with colorectal adenomas. Clin Gastroenterol Hepatol. 2012;10:405-11 Sint Nicolaas J, de Jonge V, de Man RA, Ter Borg F, Cahen DL, Moolenaar W, Stolk MF, van Tilburg AJ, Valori RM, van Leerdam ME, Kuipers EJ; for the SCoPE consortium. The Global Rating Scale in clinical practice: A comprehensive quality assurance programme for endoscopy departments. Dig Liver Dis. 2012;44:919-924 Sint Nicolaas J, de Jonge V, Korfage IJ, Ter Borg F, Brouwer JT, Cahen DL, Lesterhuis W, Ouwendijk RJ, Kuipers EJ, van Leerdam ME. Benchmarking patient experiences in colonoscopy using the Global Rating Scale. Endoscopy. 2012;44:462-72 Sparidans RW, Ahmed TT, Muilwijk EW, Welzen ME, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for therapeutic drug monitoring of the tyrosine kinase inhibitor pazopanib in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;905:137-40 Sparidans RW, Durmus S, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;889-890:144-7
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Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the VEGFR inhibitor cediranib and its primary human metabolite cediranibN+-glucuronide in plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;895-896:169-73 Sparidans RW, Durmus S, Xu N, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;895-896:174-7 Sparidans RW, Tang SC, Nguyen LN, Schinkel AH, Schellens JH, Beijnen JH. Liquid chromatography-tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;905:150-4 Stoop EM, de Haan MC, de Wijkerslooth TR, Bossuyt PM, van Ballegooijen M, Nio CY, van de Vijver MJ, Biermann K, Thomeer M, van Leerdam ME, Fockens P, Stoker J, Kuipers EJ, Dekker E. Participation and yield of colonoscopy versus non-cathartic CT colonography in population-based screening for colorectal cancer: a randomised controlled trial. Lancet Oncol. 2012;13:55-64 Stoop EM, de Wijkerslooth TR, Bossuyt PM, Stoker J, Fockens P, Kuipers EJ, Dekker E, van Leerdam ME. Face-toface vs telephone pre-colonoscopy consultation in colorectal cancer screening; a randomised trial. Br J Cancer. 2012;107:1051-8 Tiseo M, Giovannetti E, Tibaldi C, Camerini A, Di Costanzo F, Barbieri F, Burgers JA, Vincent A, Peters GJ, Smit EF, Ardizzoni A. Pharmacogenetic study of patients with advanced non-small cell lung cancer (NSCLC) treated with second-line pemetrexed or pemetrexed-carboplatin. Lung Cancer. 2012;78:92-9 Tran L, Huitema A, Vogel W, Baars J. Lack of tumor uptake of 131-I labeled rituximab in a patient with a CD20 positive lymphoma lesion. J Oncol Pharm Pract. 2012;18:417-420 Uyterlinde W, Chen C, de Bois J, Kwint M, Belderbos J, Sonke J.J, van den Heuvel M. Clinical and dosimetric parameters for acute esophagus toxicity in concurrent chemotherapy and Intensity Modulated radiotherapy for locally advanced non-small cell lung cancer. International Journal of Radiation Oncology *Biology* Physics. 2012
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Uyterlinde W, Zimmerman M, Belderbos J, Kwint M, Burgers S, van den Heuvel M. Bij lokaal gevorderd niet-kleincellig longcarcinoom Gelijktijdige chemoradiatie Oncologica. 2012;29:30-3 Van Cutsem, Tejpar S, Vanbeckevoort D, Peeters M, Humblet Y, Gelderblom H, Vermorken JB, Viret F, Glimelius B, Gallerani E, Hendlisz A, Cats A, Moehler M, Sagaert X, Vlassak S, Schlichting M, Ciardiello F. Intrapatient cetuximab dose-escalation in metastatic colorectal cancer according to the grade of early skin reactions: the randomized EVEREST study. JCO 2012;30:2861-8 Van de Schans SA, Aben KK, Mulders PF, Haanen JB, van Herpen C, Verhoeven RH, Karim-Kos HE, Oosterwijk E, Kiemeney LA. Modest improvement in 20 years of kidney cancer care in the Netherlands. Eur J Cancer. 2012;48:1822-30 Van der Bij S, Koffijberg H, Burgers JA, Baas P, van de Vijver MJ, de Mol BA, Moons KG. Prognosis and prognostic factors of patients with mesothelioma: a population-based study. Br J Cancer. 2012;107:161-4 Van der Veldt AA, Vroling L, de Haas RR, Koolwijk P, van den Eertwegh AJ, Haanen JB, van Hinsbergh VW, Broxterman HJ, Boven E. Sunitinibinduced changes in circulating endothelial cell-related proteins in patients with metastatic renal cell cancer. Int J Cancer. 2012;131:E484-93 Van Geel RM, Beijnen JH, Schellens JH. Concise drug review: pazopanib and axitinib. Oncologist. 2012;17:1081-9 Van Hasselt JG, Schellens JH, Mac Gillavry MR, Beijnen JH, Huitema AD. Model-based evaluation and optimization of cardiac monitoring protocols for adjuvant treatment of breast cancer with trastuzumab. Pharm Res. 2012;29:3499-511 Van Hasselt JG, van den Heuvel MM, Schellens JH, Beijnen JH, Brandsma D, Huitema AD. Severe cannabinoid intoxication in a patient with nonsmall-cell lung cancer. J Palliat Care. 2012;28:60-1 Van Hasselt JGC, van Eijkelenburg NKA, Beijnen JH, Schellens JHM, Huitema ADR. Optimizing drug development of anti-cancer drugs in children using modelling and simulation. British Journal of Clinical Pharmacology. 2012 (in press)
pedigree with no obvious mutation in CDH1, and applied exome sequencing to identify new genes involved in gastric cancer. We identified a germline truncating allele of alpha-E-catenin (CTNNA1) that was present in two family members with invasive diffuse gastric cancer and four in which intramucosal signet ring cells were detected as part of endoscopic surveillance. The remaining CTNNA1 allele was silenced in the two diffuse gastric cancers from the family that were available for screening, and this was also true for signet ring cells identified in endoscopic biopsies. Since alpha-Ecatenin functions in the same complex as E-cadherin, our results call attention to the broader signaling network surrounding these proteins in HDGC. We also detected somatic mutations in one tumour and found substantial overlap with genes mutated in sporadic gastric cancer, including PIK3CA, ARID1A, MED12 and MED23.
Rectal cancer In inoperable rectal cancer patients, we test the feasibility of EBRT and increasing doses of HDRBT. Currently, 19 patients are included in the study and as dose-limiting toxicity was not met yet, the study was amended to higher radiotherapy dose levels.
Anal cancer A phase I, dose-finding study assessed the feasibility and efficacy of simultaneous integrated boost – intensity modulated radiation therapy (SIB-IMRT) with concomitant capecitabine and mitomycin-C in locally advanced anal cancer. Patients with the GSTP1 313A>G variant allele experienced more frequently more severe toxicity, but showed a significantly higher complete response rate of 100% compared to 50% in wild type patients. This treatment regimen has currently been applied to an extended cohort and will be evaluated for its efficacy, and compared to a historical cohort.
UROLOGIC ONCOLOGY André Bergman, Martijn Kerst, Elsbeth van der Laan, John Haanen, Christian Blank, Sandra Adriaansz, Eric van Muilekom
Background and objectives The urologic oncology group is involved in the treatment of prostate, bladder and testicular cancer. This new subdivision of the division of Medical Oncology aims to contribute significantly to international trials and play a leading role in initiation of national trials and translational research.
Prostate cancer Exploring the role of the microenvironment in prostate cancer progression
In this study we aim to evaluate the contribution of immune cells, attracted to the prostate cancer associated stroma, in progression of this disease. Based on Gleason score, T stage and lymph node involvement, cohorts of paraffin imbedded prostatectomies are formed. After deparraffinization, tumor tissue is separated from its adjacent stroma and markers of immune cells and soluble mediators are evaluated in the tissue compartments by quantitative PCR and immunohistochemistry.
In this study we aim to define an immunological signature with predictive properties for metastatic disease Anti-CTLA4 treatment
In 2010 we initiated participation in a randomized double-blind Phase III trial comparing Ipilimumab (Yervoy™) with placebo after a single radiation of a bone metastases in prostate cancer patients previously treated with docetaxel. Our site included a significant number of patients, rated number 12 worldwide. The first results are expected in 2013. Docetaxel in combination with custirsen
This randomized open-label Phase III study compares patients treated with first line docetaxel therapy with or without the addition of custirsen (OGX-011). Custirsen is an antisense RNA blocking the synthesis of the cell-survival protein clusterin, which might enhance docetaxel efficacy. Starting in 2011, our side included 17 patients.
Bladder cancer Phase I study evaluating combined radiotherapy with panitumumab
Despite intensive treatment of muscle invasive bladder cancer, the average recurrence rate is about 50%. These recurrences are most frequently at distant sides, which holds promise to organ spearing approaches. In an investigator initiated Phase I study, patients are treated with platinum based neoadjuvant chemotherapy after a pelvic lymph node dissection, followed by radiotherapy of the bladder in combination with panitumumab. We aim to include 31 patients. To date 17 patients are enrolled in this study and no local recurrences are observed. Testicular cancer
Our department continued its role as reference center for testicular cancer, covering the treatment of all subtypes and all clinical stages, including relapse treatment using high dose chemotherapy and autologous stem cell transplantation. In 2012 initiatives for future trials have been developed, including evaluation of a carboplatin containing regimen in the treatment of metastatic seminoma testis.
Van Heel NC, Haringsma J, Boot H, Cats A, Vanhoutvin SA, Kuipers EJ. Comparison of 2 expandable stents for malignant esophageal disease: a randomized controlled trial. Gastrointest Endosc. 2012;76:52-8 Van Kooten H, de Jonge V, Schreuders E, Sint Nicolaas J, van Leerdam ME, Kuipers EJ, Veldhuyzen van Zanten SJ. Awareness of postpolypectomy surveillance guidelines: a nationwide survey of colonoscopists in Canada. Can J Gastroenterol. 2012;26:79-84 Van Leeuwen WA, Schellens JH, Becker HE, de Haan L, van Westrhenen R. The subclinical course of a paracetamol intoxication: pitfall for patient and clinician. Tijdschr Psychiatr. 2012;54:555-9 Van Lier MG, Korsse SE, MathusVliegen EM, Kuipers EJ, van den Ouweland AM, Vanheusden K, van Leerdam ME, Wagner A. Peutz-Jeghers syndrome and family planning: the attitude towards prenatal diagnosis and pre-implantation genetic diagnosis. Eur J Hum Genet. 2012;20:236-9 Van Loon J, Siedschlag C, Stroom J, Blauwgeers H, van Suylen RJ, Knegjens J, Rossi M, van Baardwijk A, Boersma L, Klomp H, Vogel W, Burgers S, Gilhuijs K. Microscopic Disease Extension in Three Dimensions for NonSmall-Cell Lung Cancer: Development of a Prediction Model Using Pathology-Validated Positron Emission Tomography and Computed Tomography Features. Int J Radiat Oncol Biol Phys. 2012;82:448-56 Van Putten PG, Ter Borg F, Adang RP, Koornstra JJ, Romberg-Camps MJ, Timmer R, Poen AC, Kuipers EJ, Van Leerdam ME. Nurse endoscopists perform colonoscopies according to the international standard and with high patient satisfaction. Endoscopy. 2012;44:1127-32
Vermaat JS, Gerritse FL, van der Veldt AA, Roessingh WM, Niers TM, Oosting SF, Sleijfer S, Roodhart JM, Beijnen JH, Schellens JH, Gietema JA, Boven E, Richel DJ, Haanen JB, Voest EE. Validation of serum amyloid α as an independent biomarker for progression-free and overall survival in metastatic renal cell cancer patients. Eur Urol. 2012;62:685-95 Vogel WV, Guislain A, Kvistborg P, Schumacher TN, Haanen JB, Blank CU. Ipilimumab-induced sarcoidosis in a patient with metastatic melanoma undergoing complete remission. J Clin Oncol. 2012;30:e7-e10 Von Meyenfeldt EM, Wouters MW, Fat NL, Prevoo W, Burgers SA, van Sandick JW, Klomp HM. Local treatment of pulmonary metastases: from open resection to minimally invasive approach? Less morbidity, comparable local control. Surg Endosc. 2012;26:2312-21 Wendel M, Bazhenova L, Boshuizen R, Kolatkar A, Honnatti M, Cho EH, Marrinucci D, Sandhu A, Perricone A, Thistlethwaite P, Bethel K, Nieva J, Heuvel M, Kuhn P. Fluid biopsy for circulating tumor cell identification in patients with early-and late-stage non-small cell lung cancer: a glimpse into lung cancer biology. Phys Biol. 2012;9:016005 Yield van Lier MG, Leenen CH, Wagner A, Ramsoekh D, Dubbink HJ, van den Ouweland AM, Westenend PJ, de Graaf EJ, Wolters LM, Vrijland WW, Kuipers EJ, van Leerdam ME, Steyerberg EW, Dinjens WN; LIMO Study Group. of routine molecular analyses in colorectal cancer patients ≤70 years to detect underlying Lynch syndrome. J Pathol. 2012;226:764-74
Van Roon AH, Goede SL, van Ballegooijen M, van Vuuren AJ, Looman CW, Biermann K, Reijerink JC, Mannetje HT, van der Togt AC, Habbema JD, van Leerdam ME, Kuipers EJ. Random comparison of repeated faecal immunochemical testing at different intervals for population-based colorectal cancer screening. Gut. 2012 Van Roon AH, Hol L, van Vuuren AJ, Francke J, Ouwendijk M, Heijens A, Nagtzaam N, Reijerink JC, van der Togt AC, van Ballegooijen M, Kuipers EJ, van Leerdam ME. Are fecal immunochemical test characteristics influenced by sample return time? A population-based colorectal cancer screening trial. Am J Gastroenterol. 2012;107:99-107
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Division head Theo Reurs
Board Theo Ruers MD PhD Division head Marc Van Beurden MD PhD Academic staff Charlotte Zuur MD PhD Academic staff General Surgical Oncology Emiel Rutgers MD PhD Head Akash Mehta MD Academic staff Arend Aalbers MD Academic staff Frits Van Coevorden MD PhD Academic staff Hester Oldenburg MD PhD Academic staff Houke Klomp MD PhD Academic staff Jeroen Hagendoorn MD PhD Academic staff Johanna Van Sandick MD PhD Academic staff Jos Van der Hage MD PhD Academic staff Koert Kuhlmann MD Academic staff Marianne Piek-den Hartog MD Academic staff Marie-Jeanne Baas-Vrancken Peeters MD PhD Academic staff Michel Wouters MD Academic staff Omgo Nieweg MD PhD Academic staff Theo Ruers MD PhD Academic staff Vic Verwaal MD PhD Academic staff Martijn Stuiver Physiotherapist Ronnie Wessels Research assistant Mila Donker MD Research assistant Rachel Numan PhD student Eva Schaake Research physician Jarich Spliethoff PhD student Niels Langhout PhD student Head and Neck Oncology and Surgery Michiel Van den Brekel MD PhD Head Alfons Balm MD PhD FRCS FACS Academic staff Frans Hilgers MD PhD Academic staff Baris Karakullukcu MD Academic staff Martin Klop MD PhD Academic staff Menno Krap DDS Academic staff Michiel Lieshout DDS Academic staff Peter Lohuis MD PhD Academic staff Ludi Smeele MD DDS PhD Academic staff Bing Tan MD PhD Academic staff Corina Van As-Brooks PhD Academic staff Charlotte Zuur MD PhD Academic staff 144
David Vossen MSc Biostatistician Kahairi Abdullah MD Fellow Bart Knockaert MD Fellow Jos Straetmans MD Fellow Hannah Tefsen Nurse practitioner i.o. Oscar Brouwer MD PhD student Renee Clapham MA PhD student/SLP Elin Derks MA PhD student Amy Dohmen MD PhD student Tihana Ibrahim MD PhD student Anne Marijn Kreeft MD PhD student Wouter Lodder MD PhD student Iain Nixon MD PhD student Heike Nyst MD PhD student Jimmy Pramana MD PhD student Sharon Stoker MD PhD student Noortje Theunissen MD PhD student Jacqueline Timmermans MD PhD student Maarten Van Alphen MSc PhD student Cindy Van den Boer MD PhD student Nynke Van den Berg MD PhD student Paul Van der Eerden MD PhD student Hester Van Monsjou MD PhD student Caroline Verhagen MD PhD student Hidde Veenstra MD PhD student Maarten Wildeman MD PhD student Irene Jacobi PhD Post-doc Lisette Van der Molen PhD Post-doc Rob Van Son PhD Post-doc Liset Lansaat MSc Research coordinator Renske Fles MSc Researcher Lisa Caesar MD Resident Mischa De Ridder MD Resident Dion Eijkenboom MD Resident Roel Henneman MD Resident Casper Jolink MD Resident Cuna Knegt MD Resident Madeline Ravesloot MD Resident Andrea Remmelts MD Resident Pim Schreuder MD Resident Fokko Smits MD Resident Matthijs Valstar MD Resident Urologic Oncology Simon Horenblas MD PhD FEBU Head Axel Bex MD PhD Academic staff Wim Meinhardt MD PhD Academic staff Henk Van de Poel MD PhD Academic staff Richard Meijer MD Academic staff Bas Van Rhijn MD PhD Academic staff Garry Pigot Fellow Rosa Djajdiningrat MD Research physician Laura Mertens MD Research physician Gynaecology Gemma Kenter MD PhD Head Marc Van Beurden MD PhD Academic staff Monique Brood MD Academic staff Petra Biewenga MD Academic staff Willemien Van Driel MD PhD Academic staff Jan Lange MD Academic staff Christianne Lok MD PhD Academic staff Henry Zijlmans MD Academic staff
Division of Surgical Oncology
Image guided surgery Theo Ruers, Marc van Beurden, Henk van der Poel, Michel Wouters, Germaine Relyveld, Jelle Wesseling, Sven Rottenberg, Danny Evers, Jarich Spliethoff, Ronni Wessels, Diederik Grootendorst, Niels Langhout, Breast Surgery group, Lung Surgery Group, Urology group, Head and Neck Oncology group
This research line aims to optimize surgical procedures by better surgical guidance during operative procedures. To this end new imaging technologies are developed and tested to improve tumor mapping and staging pre and intra-operative. These imaging and surgical guidance procedures should lead to more radical resections while sparing normal tissue and organ function. The research line is a strong collaboration between the Antoni van Leeuwenhoek, Technical University Twente and industrial partners. For the moment three projects are running. In the first project we are developing a tool for optical biopsies by means of spectroscopy and fluorescence techniques. An optical needle was developed together with industry. An extensive series of ex vivo tissue specimens was analyzed and compared with histopathological analysis with an accuracy of over 90%.These results were confirmed by in vivo testing in a controlled setting in breast cancer, liver metastases and lung tumors. This study in over 100 patients was finished in December 2012. The next steps will concentrate to incorporate the developed technology into surgical and radiological tools and in the work flow of clinical practice. In a second project, in cooperation with the group Biomedical Physics and Biomedical Photonics of AMC, optical coherence tomography is tested for improved tissue diagnosis in vulvar neoplasia (VIN), penis cancer and skin malignancies. For VIN lesions we were able to show that OCT images match well with definite histology, meaning that OCT can have a significant role in the clinical workflow of this disease. A third project concentrates on the use of photo acoustic imaging for diagnosis of lymph node metastases and is running in close collaboration with the Biomedical Photonic Imaging group of the University of Twente. We recently showed that photo acoustic imaging is able to detect microscopic tumor foci within lymph nodes of melanoma patients. In addition, in a rat prostate tumor model we were able to visualize small tumor deposits in lymph nodes by using new iron and carbon nanoparticles as contrast agents for photo acoustic imaging. Future work will focus on the introduction of a hand held clinical system. The research performed in the present projects leads to strong synergy with the new innovative minimal invasive operating theatre complex planned to be build in 2014 and will result in a technology platform that can be used for further clinical studies.
GYNAECOLOGY Gemma Kenter, Marc van Beurden, Willemien van Driel, Petra Biewenga, Monique Brood, Pia Kvistborg, Luc van Lonkhuizen and Henry Zijlmans
The department focuses on innovative treatment for ovarian cancer, on immunotherapy for HPV related (pre)malignant neoplasms and on interventions to improve quality of life for premature (iatrogenic) menopausal symptoms Research takes place in close cooperation with the other centers from the Center for Gynaecologic Oncology Amsterdam (CGOA) i.e. AMC and VUmc.
Ovary In a randomized multicenter phase III clinical trial for stage III ovarian carcinoma coordinated by the Antoni van Leeuwenhoek (Willemien van Driel) the effect of secondary debulking surgery with or without hyperthermic intraperitoneal cisplatinum is being studied. Inclusion started March 2007 and currently runs in 6 centers in The Netherlands and 1 center in Belgium. Primary endpoint of this study is progression free survival. Up until now a total of 156 patients have been randomized. The beneficial effect of a laparoscopy in order to predict the operability in high stage ovarian carcinoma is being studied in a multicenter randomized trial coordinated by the CGOA-AMC. The effect of hormonal replacement therapy on menopausal complaints related to biochemical changes in surgically and naturally postmenopausal women is investigated in a prospective observational comparative study. (Vermeulen, Marc van Beurden, Tiny Korse)
Vulva Participation in the GROINSS-V II study, an international multicenter observational study on patients with vulvar cancer. Patients with positive lymph nodes, diagnosed by sentinal node technique, undergo radiation without full lymphadenectomy. Endpoints of this study are recurrence free survival and quality of life. For patients with locally advanced carcinoma of the vulva an efficacy study is ongoing during which patients are treated with induction chemoradiation and if necessary followed by surgery in order to reduce postoperative morbidity. (AMC locally advanced vulvar cancer efficacy study). Patients with VIN III take part in a multicenter randomized trial to study the effect of vaccination with synthetic long HPV 16 E6/ E7 peptides with or without imiquimod on the vaccination site. Inclusion has been closed and data are being analysed.
Plastic and Reconstructive surgery J Joris Hage MD PhD Head Leonie Woerdeman MD PhD Academic staff Marieke Van der Berg MD Academic staff Martine Van Huizum MD Academic staff Marije Hoornweg MD PhD Academic staff Fientje Van der Veen MD Academic staff Brigitte Drost MD Academic staff Anesthesiology Peter Schutte MD Head Karin Ariese-Beldman Academic staff Sannine Buma Academic staff Dick Buitelaar Academic staff Julia ten Cate Academic staff Katina Efthymiou Academic staff Cristoph Hahn Academic staff Sandra Huissoon Academic staff Lenie Hulshoff Academic staff Anne Lukas MD PhD Academic staff Michael Srámek MD PhD Academic staff Ingeborg Vergouwe Academic staff Marchien van der Weide MD PhD Academic staff Dermatology Marianne Crijns MD PhD Head Germaine Relyveld MD PhD Academic staff Biljana Zupan-Kajcovski MD Academic staff Wietze Van der Veen MD PhD Academic staff Soe Janssens MD PhD Academic staff Inka Nieuweboer-Krobotova MD Academic staff
Publications
Ackerstaff AH, Rasch CRN, Balm AJM, de Boer JP, Wiggenraad R, Rietveld DHF, Gregor RT, Kröger R, Hauptmann M, Vincent A, Hilgers FJM. Five-years Quality of Life results of the randomized clinical phase III (RADPLAT) trial, comparing concomitant intra-arterial versus intravenous chemoradiotherapy in locally advanced head and neck cancer. Head Neck. 2012;34:974-80
Cervix A multicenter trial is running to study the safety and immunogenicity of combined chemo-immunotherapy in high stage or recurrent carcinoma of the cervix. Furthermore, preparations for a phase 1 trial with DNA HPV 16 E7 vaccination in patients with HPV related disorders of the genital region or head and neck region are being made. This trial is part of a European project to study the molecular pathways and potentials for innovative drugs in advanced cervical carcinoma. (RAID, EU7). The role of the lymph nodes in the immune surveillance in cancer of the cervix is being studied by gathering lymph node scrapings during surgery.
Adham M, Kurniawan AN, Muhtadi AI, Roezin A, Hermani B, Gondhowiardjo S, Tan IB, Middeldorp JM. Nasopharyngeal carcinoma in Indonesia: epidemiology, incidence, signs, and symptoms at presentation. Chin J Cancer. 2012;31:185-96
Alvarez Paez AM, Brouwer OR, Veenstra HJ, van der Hage JA, Wouters M, Nieweg OE, Valdés-Olmos RA. Decisive role of SPECT/CT in localization of unusual periscapular sentinel nodes in patients with posterior trunk melanoma: three illustrative cases and a review of the literature. Melanoma Res. 2012;22:278-83 Arnold M, Wildeman MA, Visser O, Karim-Kos HE, Middeldorp JM, Fles R, Tan IB, Coebergh JW. Lower mortality from nasopharyngeal cancer in The Netherlands since 1970 with differential incidence trends in histopathology. Oral Oncol (in press) Balm AJM, de Boer JP, Rasch CRN. Recurrent salivary gland carcinoma and pleomorphic adenoma. In: Head and Neck Cancer Recurrence. Evidencebased, Multidisciplinary Management. Editors: HM Mehanna, KK Ang, Thieme. 2012:211-21 Bex A, Etto T, Vyth-Dreese F, Blank C, Griffioen AW. Immunological heterogeneity of the RCC microenvironment: do targeted therapies influence immune response? Curr Oncol Rep. 2012;14:230-9 Bex A, Gore M, Mulders P, Sternberg CN. Recent advances in the treatment of advanced renal cell carcinoma: towards multidisciplinary personalized care. BJU Int. 2012;110:1289-300 Bex A, Kroon BK, de Bruijn R. Is there a role for neoadjuvant targeted therapy to downsize primary tumors for organ sparing strategies in renal cell carcinoma? International journal of surgical oncology. 2012;2012:250479 Bex A, Powles T. Selecting patients for cytoreductive nephrectomy in advanced renal cell carcinoma: who and when. Expert review of anticancer therapy. 2012;12:787-97 Bex A, Sonke GS. Low incidence of brain metastases from small cell carcinoma of the urinary bladder. European journal of Clinical and Medical Oncology. 2012;4:59-63 Bex A, Valdés Olmos RA. GOSTT in kidney cancer. In: International Atomic Energy A, editor. Guided intraoperative scintigraphic tumour targeting (GOSTT): IAEA; 2012 (in press) Bex A. Choosing the right option for renal-cell carcinoma. The lancet oncology. 2012;13:970-1 Bex A. Interferon-alpha in Combination with Sorafenib: RAPSODY or Requiem? European Urology. 2012;63:262-4
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Bex A. Type Ii Papillary Histology Predicts Poor Outcome in Patients with Renal Cell Carcinoma and Vena Cava Thrombus. BJU Int. 2012 Bijker N, Donker M, Wesseling J, den Heeten GJ, Rutgers EJ. Is DCIS Breast Cancer, and How Do I Treat it? Curr Treat Options Oncol 2012 Bleeker MCG, Heideman DAM, Snijders PJF, Horenblas S, Meijer CJLM. Epidemiology and Etiology of Penile Cancer. In: Muneer A, Arya M, Horenblas S, editors. Textbook of Penile Cancer: Springer; 2012. p.1-12 Boone J, Bex A, Prevoo W. Percutaneous radiofrequency ablation of a small renal mass complicated by appendiceal perforation. Cardiovasc Intervent Radiol. 2012;35:695-9 Bostrom PJ, Alkhateeb S, Trottier G, Athanasopoulos PZ, Mirtti T, Kortekangas H, et al. Sex differences in bladder cancer outcomes among smokers with advanced bladder cancer. BJU Int. 2012;109:70-6 Brölmann FE, Groenewold MD, Spijker R, van der Hage JA, Ubbink DT, Vermeulen H. Does evidence permeate all surgical areas equally? Publication trends in wound care compared to breast cancer care: a longitudinal trend analysis. World J Surg. 2012;36:2021-7 Brouwer OR, Buckle T, Bunschoten A, Kuil J, Vahrmeijer AL, Wendler T, et al. Image navigation as a means to expand the boundaries of fluorescence-guided surgery. Phys Med Biol. 2012;57:3123-36 Brouwer OR, Buckle T, Vermeeren L, Klop WMC, Balm AJM, van der Poel HG, van Rhijn BW, Horenblas S, Nieweg OE, van Leeuwen FW, Valdés Olmos RA. Comparing the hybrid fluorescent-radioactive tracer Indocyanine green-99mTc-nanocolloid with 99mTc-nanocolloid for sentinel node identification: a validation study using lymphoscintigraphy and SPECT/ CT. J Nucl Med. 2012;53:1034-40 Brouwer OR, Donker M, Woerdeman LA, Vrancken Peeters MJ. Local recurrence after skin-sparing mastectomy. Ned Tijdschr Geneeskd 2012;156:A4692 Brouwer OR, Donker M, Woerdeman LA, Vrancken Peeters MJ. Lokaal recidief na huid-sparende mastectomie. Ned Tijdschr Geneeskd. 2012;156:A4692.
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Brouwer OR, Horenblas S, Nieweg OE, Valdés Olmos RA. GOSTT in testicular cancer. In: International Atomic Energy A, editor. Guided intraoperative scintigraphic tumour targeting (GOSTT): IAEA; 2012 (in press) Brouwer OR, Klop WMC, Buckle T, Vermeeren L, Van den Brekel MWM, Balm AJM, Nieweg OE, Valdés Olmos RA, Van Leeuwen FWB. Feasibility of sentinel node biopsy in head and neck melanoma using a hybrid radioactive and fluorescent tracer. Ann Surg Oncol 2012;19:1988-1994 Brouwer OR, Vermeeren L, van der Ploeg IM, Valdés Olmos RA, Loo CE, Pereira-Bouda LM, Smit F, Neijenhuis P, Vrouenraets BC, Sivro-Prndelj F, Jap-a-Joe SM, Borgstein PJ, Rutgers EJ, Oldenburg HS. Lymphoscintigraphy and SPECT/CT in multicentric and multifocal breast cancer: does each tumour have a separate drainage pattern? Results of a Dutch multicentre study (MULTISENT). Eur J Nucl Med Mol Imaging. 2012;39:1137-43 Bruggink-Hoornweg MJ. Thesis; On infantile hemangiomas University of Amsterdam, September 12, 2012 Buckle T, Brouwer OR, Valdes Olmos RA, van der Poel HG, van Leeuwen FWB. Relationship between intraprostatic tracer deposits and sentinel lymph node mapping in prostate cancer patients. J Nucl Med. 2012;53:1026-33 Bunschoten A, Buckle T, Kuil J, Nieweg OE, van Leeuwen FWB. Targeted noncovalent self-assembled nanoparticles based on human serum albumin. Biomaterials 2012;33:867-875 Cibula D, Abu-Rustum NR, Dusek L, Slama J, Zikán M, Zaal A, Sevcik L, Kenter G, Querleu D, Jach R, Bats AS, Dyduch G, Graf P, Klat J, Meijer CJ, Mery E, Verheijen R, Zweemer RP. Bilateral ultrastaging of sentinel lymph node in cervical cancer: Lowering the false-negative rate and improving the detection of micrometastasis. Gynecol Oncol. 2012;127:462-6 Cibula D, Abu-Rustum NR, Dusek L, Zikán M, Zaal A, Sevcik L, Kenter GG, Querleu D, Jach R, Bats AS, Dyduch G, Graf P, Klat J, Lacheta J, Meijer CJ, Mery E, Verheijen R, Zweemer RP. Prognostic significance of low volume sentinel lymph node disease in earlystage cervical cancer. Gynecol Oncol. 2012;124:496-501
Making use of the large database from AVL, AMC and VUmc a prognostic model study is ongoing in order to individualize prognosis for survival in patients with early stage cervical cancer. (Biewenga et al). Together with the LUMC we study the needs for assessment in patients and professionals on sexual complaints after treatment for cervical cancer.
BREAST CANCER Emiel Rutgers, Hester Oldenburg, Marie-Jeanne Vrancken Peeters, Jos Van der Hage, Mila Donker, Caroline Drukker, Bas Koolen
After the introduction of population-based screening breast cancer incidence increased while breast cancer mortality declined. Our previous analyses suggested screen detection might be associated with a higher likelihood of a biologically low risk tumor assessed by the 70-gene signature (MammaPrint™). Drukker et al confirmed this by analyzing all Dutch breast cancer patients enrolled in the MINDACT trial, who were invited for the Dutch screening program (n=1409). A significant difference was seen between the screen-detected carcinomas and interval carcinomas (pX2= 0.002) in all 70-gene signature risk groups. 51% of the tumors detected in the first screening round had an ultralow risk 70-gene signature result. Further analysis, including a multinominal logistic regression model with covariants, such as age, tumor size, histological type, ER, PgR and HER2, are planned for the beginning of 2013. New diagnostic tools were investigated: The first experience with a dedicated, high-resolution breast PET for hanging breast molecular imaging was described. The MAMMI PET, developed to improve primary tumor detection and characterization, visualized 31 (97%) of 32 tumors. SUVmax on MAMMI PET was consistently higher (average ratio 2.7) than on whole body PET/CT. A review by our group compared all currently available dedicated breast PETs. The additional value of dedicated breast PETs should mainly be sought in dense breast imaging, screening in high-risk patients, tumor visualization in case of an occult or inconclusive lesion on conventional imaging, accurate FDG uptake determination, response monitoring, or FDG-guided biopsies. Evers et al investigated the diffuse reflectance spectroscopy (DRS) as a new technique for breast cancer diagnosis. Ex vivo analysis was performed of the breast in which, in an individual patient, normal versus malignant breast tissue could be accurately discriminated. Further in vivo analysis in needed to confirm the presented results as a next step towards the clinical application of smart biopsy tools and surgical instruments. Many of these screen detected tumors (invasive and DCIS) are non-palpable tumors. We started to use radioactive iodine seeds to localize these tumors in order to facilitate the operation. We first showed that the 125I seed and Tc-99 nanocolloid (needed for the Sentinel node procedure) can be used in one procedure without interference in radiation energy. We feel that the usage of the 125I seed is a next step within fine-tuning breastconserving surgery that should lead to further investigation to
confirm its value. We therefore set up a database and currently data of 138 patients that were localized with an Iodine seed are compared with 288 patients that were localized with the conventional ROLL technique using a non-radioactive marker followed by injection of radioactive Technetium. As opposed to patients with early-stage breast cancer who are in general treated with primary surgery, patients with locally advanced tumors are usually treated with neoadjuvant chemotherapy (NAC). Adequate diagnostic workup and response motoring is essential to patient tailor this treatment. Koolen et al investigated the role of PET/CT in this setting. We found that primary tumor FDG uptake was sufficient for response monitoring in 203 (95%) of 214 stage II-III breast cancer patients, a significantly higher SUVmax was seen in patients with unfavourable tumor characteristics. Sensitivity and negative predictive value for the detection of axillary metastases were suboptimal (82 and 53%, respectively), but positive predictive value was excellent (98%). The proportion of missed metastases with PET/CT was significantly lower in patients with unfavourable tumor characteristics: higher N-stage, triple negative tumors, high Ki-67, and high grade. Occult N3-disease in the internal mammary chain (IMC) and periclavicular area was detected in 26 (8%) and 32 (10%) patients, respectively. This resulted in changed radiotherapy planning in 50 (16%) patients. Furthermore, we showed the superiority of the PET/CT over conventional diagnostic workup in detecting distant metastases PET/CT response monitoring during NAC was also useful, but heavily depended on the breast cancer subtype. It seemed accurate in ER-positive/HER2-negative and triple negative tumors, but appeared inaccurate in HER2-positive patients.
Melanoma Anton Bunschoten, Jos Van der Hage, Bin Kroon, Omgo Nieweg, Hidde Veenstra, Michel Wouters
The research interest of the melanoma surgeons comprises the anatomy and physiology of the lymphatic system, implications for dissemination, implementation of new forms of imaging techniques and other new forms of diagnostic and staging methods, sentinel node biopsy, aspects of inguinal node dissection, in transit metastases and aspects of regional isolation perfusion. Lymphoscintigraphy can visualize sentinel nodes before the operation. One issue hampering lymphatic mapping is the observation that a sentinel lymph node often does not accumulate all of the 99mTc-nanocolloid that is administered at the tumor site. Some of it passes through downstream to lodge in second- or higher-echelon nodes. The multitude of radioactive nodes hampers the identification of the sentinel node on the lymphoscintigrams and particularly in the operating room, since the gamma-ray detection probe cannot discern one from the other.
Courrech Staal EFW, Bloemendal KM, Bloemer MC, Aleman BMP, Cats A, van Sandick JW. Oesophageal cancertreatment in a tertiary referral hospital evaluated by indicators for quality of care. Eur J Surg Oncol. 2012;38:150-6 De Bonilla-Damia A, Brouwer OR, Meinhardt W, Valdes-Olmos RA. Lymphatic Drainage in Prostate Carcinoma assessed by Lymphoscintigraphy and SPECT/CT: Its importance for the Sentinel Node Procedure. Rev Esp Med Nucl Imagen Mol. 2012;31:66-70 De Bruin-Visser JC, Ackerstaff AH, Rehorst H, Hilgers FJM. Integration of a smoking cessation program in the treatment protocol for patients with head and neck and lung cancer. Eur Arch ORL. 2012;269:659–65 De Melker H, Kenter G, van Rossum T, Conyn-van Spaendonck M. Developments in HPV vaccination. Ned Tijdschr Geneeskd. 2012;156:A5410 De Ridder M, Smeele LE, Balm AJM. Klinische les: Het pleiomorf adenoom van de glandula parotis; regels voor resectie. Ned Tijdschr Geneeskd. 2012;156:773-8 De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LF. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer.Breast Cancer Res Treat. 2013;137:213-23 De Visscher SA, Dijkstra PU, Tan IB, Roodenburg JL, Witjes MJ. mTHPC mediated photodynamic therapy (PDT) of squamous cell carcinoma in the head and neck: A systematic review. Oral Oncol (in press) De Vos van Steenwijk PJ, Ramwadhdoebe TH, Löwik MJ, van der Minne CE, Berends-van der Meer DM, Fathers LM, Valentijn AR, Oostendorp J, Fleuren GJ, Hellebrekers BW, Welters MJ, van Poelgeest MI, Melief CJ, Kenter GG, van der Burg SH. A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions. Cancer Immunol Immunother. 2012;61:1485-92 De Vries RR, Kauer P, van Tinteren H, van der Poel HG, Bex A, Meinhardt W, et al. Short-term outcome after cystectomy: comparison of two different perioperative protocols. Urol Int. 2012;88:383-9
De Vries RR. The surgical management of invasive bladder cancer [urol NKI]. Amsterdam: Free University; 2012 Den Brok MH, Nierkens S, Wagenaars JA, Ruers TJ, Schrier CC, Rijke EO, Adema GJ. Saponin-based adjuvants create a highly effective anti-tumor vaccine when combined with in situ tumor destruction. Vaccine. 2012;30:737-44 Deurloo EE, Sriram JD, Teertstra HJ, Loo CE, Wesseling J, Rutgers EJTh, Gilhuijs KG. MRI of the breast in patients with DCIS to exclude the presence of invasive disease. Eur Radiol. 2012;22:1504-11 Dikken JL, Lemmens VE, Wouters MW, Wijnhoven BP, Siersema PD, Nieuwenhuijzen GA, van Sandick JW, Cats A, Verheij M, Coebergh JW, van de Velde CJ. Increased incidence and survival for oesphageal cancer but not for gastric cardia cancer in the Netherlands. Eur J Cancer. 2012;48:1624-32 Dikken JL, Stiekema J, van de Velde CJ, Verheij M, Cats A, Wouters MW, van Sandick JW. Quality of care indicators for the surgical treatment of gastric cancer: a systematic review. Ann Surg Oncol. 2012 Dikken JL, van Sandick JW, Allum WH, Johansson J, Jensen LS, Putter H, Coupland VH, Wouters MW, Lemmens VE, van de Velde CJ, van der Geest LG, Larsson HJ, Cats A, Verheij M. Differences in outcomes of oesophageal and gastric cancer surgery across Europe. Br J Surg. 2012 Dikken JL, Wouters MW, Lemmens VE, Putter H, van der Geest LG, Verheij M, Cats A, van Sandick JW, van de Velde CJ. Influence of hospital type on outcomes after oesophageal and gastric cancer surgery. Br J Surg. 2012;99:954-63 Djajadiningrat R, Horenblas S. Competing-Risks Analysis in Patients with T1 Squamous Cell Carcinoma of The Penis. BJU Int. 2012 Donker M, Hage JJ, Woerdeman LA, Rutgers EJTh, Sonke GS, Vrancken Peeters MJTFD. Surgical complications of skin sparing mastectomy and immediate prosthetic reconstruction after neoadjuvant chemotherapy for invasive breast cancer. Eur J Surg Oncol. 2012;38:25-30 Donker M, Straver ME, Rutgers EJ, Vrancken Peeters MJ. Radioguided occult lesion localisation (ROLL) in breast-conserving surgery after neoadjuvant chemotherapy. Eur J Surg Oncol 2012
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Duijts SF, van Beurden M, Oldenburg HS, Hunter MS, Kieffer JM, Stuiver MM, Gerritsma MA, Menke-Pluymers MB, Plaisier PW, Rijna H, Lopes Cardozo AM, Timmers G, van der Meij S, van der Veen H, Bijker N, de Widt-Levert LM, Geenen MM, Heuff G, van Dulken EJ, Boven E, Aaronson NK. Efficacy of cognitive behavoiral therapy and physical exercise in alleviating treatment-induced menopausal symptoms in patients with breast cancer: results of a randomized, controlled, multicenter trial. J Clin Oncol, 2012;30:4124-33 Escudier B, Osanto S, Ljungberg B, Porta C, Wagstaff J, Mulders P, et al. Multidisciplinary management of metastatic renal cell carcinoma in the era of targeted therapies. Cancer Treat Rev. 2012;38:127-32 Evers DJ, Hendriks B, Lucassen G, Ruers T. Optical spectroscopy: current advances and future applications in cancer diagnostics and therapy. Future Oncol. 2012;8:307-20 Evers DJ, Nachabé R, Klomp HM, van Sandick JW, Wouters MW, Lucassen GW, Hendriks BH, Wesseling J, Ruers TJ. Diffuse reflectance spectroscopy: a new guidance tool for improvement of biopsy procedures in lung malignancies. Clin Lung Cancer. 2012;13:424-31
Ficarra V, Sooriakumaran P, Novara G, Schatloff O, Briganti A, Van der Poel H, et al. Systematic review of methods for reporting combined outcomes after radical prostatectomy and proposal of a novel system: the survival, continence, and potency (SCP) classification. Eur Urol. 2012;61:541-8 Fontein DB, Nortier JW, Liefers GJ, Putter H, Meershoek-Klein Kranenbarg E, van den Bosch J, Maartense E, Rutgers EJTh, van de Velde CJ. High non-compliance in the use of letrozole after 2.5 years of extended adjuvant endocrine therapy. Results from the IDEAL randomized trial. Eur J Surg Oncol. 2012;38:110-7 Godefroy WP, Klop WM, Smeele LE, Lohuis PJ. Free-flap reconstruction of large full-thickness lip and chin defects. Ann Otol Rhinol Laryngol. 2012;121:594-603 Govaert KM, van Kessel CS, Lolkema M, Ruers TJ, Borel Rinkes IH. Does Radiofrequency Ablation Add to Chemotherapy for Unresectable Liver Metastases? Curr Colorectal Cancer Rep. 2012;8:130-137 Graafland NM, Horenblas S. Follow-Up of Patients with Penile Cancer. In: Muneer A, Arya M, Horenblas S, editors. Textbook of Penile Cancer: Springer; 2012. p. 283-90
Evers DJ, Nachabe R, Vranken Peeters MJTFD, van der Hage J.A., Oldenburg H.S., Rutgers EJTh, et al. Diffuse reflectance spectroscopy: towards clinical application in breast cancer. Breast Cancer Res Treat 2013;137:155-165
Graafland NM, Valdés Olmos RA, Horenblas S. GOSTT in penile cancer. In: International Atomic Energy A, editor. Guided intraoperative scintigraphic tumour targeting (GOSTT): IAEA; 2012 (in press)
Evers DJ, Nachabé R, Wesseling J, Klomp HM, van Sandick JW, Wouters MW, Lucassen GW, Hendriks BHW, Ruers TJ. Diffuse reflectance spectroscopy: a new guidance tool for improvement of biopsy procedures in lung malignancies. Clin Lung Cancer. 2012;13:424-31
Graveland AP, Braakhuis BH, Eerenstein SE, de Bree R, Bloemena E, de Maaker M, Van den Brekel MW, Dijk F, Mesker WE, Tanke HJ, Leemans CR, Brakenhoff RH. Molecular diagnosis of minimal residual disease in head and neck cancer patients. Cell Oncol. 2012;35:367-75
Ficarra V, Novara G, Ahlering TE, Costello A, Eastham JA, Graefen M, et al. Systematic review and metaanalysis of studies reporting potency rates after robot-assisted radical prostatectomy. Eur Urol. 2012;62:418-30
Griffioen AW, Mans LA, de Graaf AM, Nowak-Sliwinska P, de Hoog CL, de Jong TA, et al. Rapid angiogenesis onset after discontinuation of sunitinib treatment of renal cell carcinoma patients. Clinical cancer research : an official journal of the American Association for Cancer Research. 2012;18:3961-71
Ficarra V, Novara G, Rosen RC, Artibani W, Carroll PR, Costello A, et al. Systematic review and metaanalysis of studies reporting urinary continence recovery after robotassisted radical prostatectomy. Eur Urol. 2012;62:405-17
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Grootendorst DJ, Jose J, Wouters MW, van Boven H, Van der Hage J, Van Leeuwen TG, Steenbergen W, Manohar S, Ruers TJ. First experiences of photoacoustic imaging for detection of melanoma metastases in resected human lymph nodes. Lasers Surg Med. 2012;44:541-9
To optimize sentinel node surgery, multiple studies concerning image guided surgery of the sentinel node are currently being employed. These studies range from the experimental use of new tracers like immunofluoarescent labeled particles, to studies addressing the efficacy of new types op real-time perioperative SPECT/CT scanning.
Colorectal liver metastases Theo Ruers, Frits van Coevorden, Koert Kuhlmann, Erik Tanis
Research is focused on imaging and local tumour destruction. The results of an international practice changing randomized study on the use of RFA for unresectable colorectal liver metastases was recently published by our group. In 2012 a follow up study was started in collaboration with the EORTC to compare RFA for resectable colorectal liver metastases with resection of the metastases. Further research is directed at immunological cell death and immunostimulation after local tumour destruction (HIFU) within a CTMM consortium. In addition, a European multicentre study was initiated to evaluate anti MUC-1 vaccination in patients treated pre-operatively with radiotherapy. Moreover, together with the Technical University Twente gold nanoshells, in combination with NIR light, are used for tumour detection and ablation of resection margins. In collaboration with industrial partners the use spectroscopy for tumour diagnosis and guidance of minimal invasive procedures is investigated. The research line is funded by KWF, the UTwente, EORTC and industrial partners.
Oesophageal and gastric cancer Johanna van Sandick, Frits van Coevorden, Marie-Jeanne Vrancken Peeters, Jurriën Stiekema (research physician), Sera de Leeuw (student), Sabrine Kol (student)
A database of all patients who underwent surgery for a gastric malignancy in the Antoni van Leeuwenhoek since 1995 has been completed. In one study, the outcome of patients who were treated with potentially curative surgery for a gastric carcinoma was analysed (N=132). Striking differences were found between patients with intestinal type tumours and those with diffuse type tumours, especially with regard to the rate of R1 resections (2% versus 24%, respectively) and - irrespective of R status – median overall survival (129 versus 17 months, respectively). In 33% of patients with recurrent disease from diffuse type gastric cancer, the site of recurrence was in the peritoneal cavity (paper submitted). Based on these results, for patients with an irradical resection, the role of adjuvant chemoradiotherapy is being explored, and for patients with peritoneal dissemination from gastric cancer, the role of HIPEC will be investigated in a feasibility study (protocol in preparation). In another study, the surgical treatment of gastrointestinal stromal tumours located in the stomach has been analysed (N=47). Irradical resection, tumour spill and progressive disease on neo-adjuvant imatinib at
the time of surgery were associated with poor prognosis (paper submitted). For translational research, pre-treatment biopsy samples of oesophageal cancer patients have been collected. Samples from patients with oesophageal adenocarcinoma who were treated with neo-adjuvant chemoradiotherapy followed by surgical resection (N=66), have been prepared for DNA and RNA sequencing.
Lower GI and HIPEC Vic Verwaal, Arend Aalbers, Theo Ruers, Koert Kuhlmann
In 2012 a change of the research focus of the HIPEC and Lower GI has taken place. In the pervious year the focus was mainly on the HIPEC procedure it self and on the clinical outcome. Now the scope is focus on translational research on the biology of metastasizing patterns and on clinical studies of rectal cancers. To enable this two PhD projects are started. The first study-line aims at finding differences in pathway activation between tumors with a small number large metastasis and those who with a large number of small metastasis. The first step in this study is to grow patient’s tumor cells in cultures. This part of the study is now successful. The analysis of the pathways has recently been started. The second study line contains clinical studies of rectal tumors treatment. A number of import findings are on the way to be published. The major outcomes are; large rectal tumors have similar survival to small rectum tumors, low rectal tumors have equal outcome to high rectal tumors when treated in a cancer center, most failures after rectal tumor treatment occur systemically and not locally. The treatment of recurrent rectal cancer has reasonable good survival. Beside of these studies a larger study is performed on the outcome of all Dutch HIPEC centers including the evaluation of the learning curve. The last study shows that new centers start off at a higher level on the curve and have a steeper learning curve, they end off at the seem level as the most experienced center.
THORACIC CANCER SURGERY Houke Klomp, Michel Wouters, Eva Schaake, Rachel Numan, Matthijs van Gool, Tjeerd Aukema
The development of targeted therapy has introduced new options to improve treatment outcome in selected patients with non-small cell lung cancer (NSCLC). Response measurements in patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have proven to be difficult. The M06NEL-study provided data from a group of patients that received neoadjuvant erlotinib. Patients received erlotinib 150mg once daily for 3 weeks before surgery. Response to treatment was evaluated using [18F] fluorodeoxyglucose positron emission tomography (PET) and computed tomography (CT) scans during treatment and histological examination of the resection specimen. Primary end points were toxicity and pathologic response.
Hage JJ, van der Heeden JF, Lankhorst KM, Romviel SMG, Vluttters ME, Woerdeman LAE, Bart Visser B, Veeger HEJ. Impact of combined skin sparing mastectomy and immediate subpectoral prosthetic reconstruction on the pectoralis major muscle function: A preoperative and postoperative comparative study. Ann Plast Surg. (in press) Hamming-Vrieze O, van Kranen SR, van Beek S, Heemsbergen W, van Herk M, Van den Brekel MW, Sonke JJ, Rasch CR. Evaluation of tumor shape variability in head-and-neck cancer patients over the course of radiation therapy using implanted gold markers. Int J Radiat Oncol Biol Phys. 2012;84:e201-7 Heideman DAM, Bleeker MCG, Ahmed HU, Arya M, Horenblas S, Snijders PJF, et al. Molecular Biology of Penile Cancer. In: Muneer A, Arya M, Horenblas S, editors. Textbook of Penile Cancer: Springer; 2012. p. 13-26 Heijnsdijk EA, Warner E, Gilbert FJ, Tilanus-Linthorst MM, Evans G, Causer PA, Eeles RA, Kaas R, Draisma G, Ramsay EA, Warren RM, Hill KA, Hoogerbrugge N, Wasser MN, Bergers E, Oosterwijk JC, Hooning MJ, Rutgers EJTh, Klijn JG, Plewes DB, Leach MO, de Koning HJ. Differences in natural history between breast cancers in BRCA1 and BRCA2 mutation carriers and effects of MRI screening-MRISC, MARIBS, and Canadian studies combined. Cancer Epidemiol Biomarkers Prev. 2012;21:1458-68 Hilgers FJM, Ackerstaff AH, van den Brekel MWM. Bedeutung von HME für die ganzheitliche Rehabilitation nach LE. In: Pulmonale Rehabilitation nach Laryngektomie. Editor: K. Lorenz, Unimed Verlag (in press) Hilgers FJM, Dirven R, Wouters Y, Jacobi I, Marres HAM, van den Brekel MWM. A multicenter prospective clinical trial evaluating a novel adhesive baseplate (Provox StabiliBase) for enhanced peristomal attachment of postlaryngectomy pulmonary and voice rehabilitation devices. Laryngoscope. 2012;122:2447-53 Hilgers FJM, Lorenz KJ, Maier H, Meeuwis CA, Kerrebijn JD, Vander Poorten V, Vinck AS, Quer M, van den Brekel MWM. Development and (pre-) clinical assessment of a novel surgical tool for primary and secondary tracheoesophageal puncture with immediate voice prosthesis insertion, the Provox Vega Puncture Set. Eur Arch Otorhinolaryngol (in press)
Hilgers FJM, Scheenstra RJ, Muller SH, van den Brekel MWM. Aktuelle Forschung zur Verbesserung der Wärme und Feuchtigkeitskapazität von HME for Laryngektomierte patienten. In: Pulmonale Rehabilitation nach Laryngektomie. Editor: K. Lorenz, Unimed Verlag (in press) Hilgers FJM, van den Brekel MWM. Practical tips for voice rehabilitation after pharyngolaryngectomy. In: Pearls and Pitfalls in Head and Neck Surgery; practical tips to minimize complications. Editor: CR Cernea; associate editors: FL Dias, D Fliss, RA Lima, EN Myers, WI Wei, Karger Publishers. 2012:94-5 Hoornweg MJ, Smeulders MJ, Ubbink DT, van der Horst CM. The prevalence and risk factors of infantile hemangiomas: A case-control study in the Dutch population. Paediatr Perinat Epidemiol. 2012;26:156-62 Hoornweg MJ, Theunissen CIJM, Hage JJ, van der Horst CMAM. Malignant differential diagnosis in children referred for infantile hemangioma. Ann Plast Surg (in press) Horenblas S. Sentinel lymph node biopsy in penile carcinoma. Seminars in diagnostic pathology. 2012;29:90-5 Howard JH, Thompson JF, Mozzillo N, Nieweg OE, Hoekstra HJ, Roses DF, Sondak VK, Reintgen DS, KashaniSabet M, Karakousis CP, Coventry BJ, Kraybill WG, Smithers BM, Elashoff R, Stern SL, Cochran AJ, Faries MB, Morton DL. Metastasectomy for distant metastatic melanoma: analysis of data from the first Multicenter Selective Lymphadenectomy Trial (MSLT-I). Ann Surg Oncol 2012;19:2547-2555 Indrasari SR, Timmermans AJ, Wildeman MA, Karakullukcu MB, Herdini C, Hariwiyanto B, Tan IB. Remarkable response to photodynamic therapy in residual T4N0M0 nasopharyngeal carcinoma: A case report. Photodiagnosis Photodyn Ther. 2012;9:319-20 Jansen EPM, Boot H, van de Velde CJH, van Sandick JW, Cats A, Verheij M. Can adjuvant chemoradiotherapy replace extended lymph node dissection in gastric cancer? Recent Results Cancer Res. 2012;196:229-40 Karakullukcu B, Nyst HJ, van Veen RL, Hoebers FJ, Hamming-Vrieze O, Witjes MJ, de Visscher SA, Burlage FR, Levendag PC, Sterenborg HJ, Tan IB. mTHPC mediated interstitial photodynamic therapy of recurrent nonmetastatic base of tongue cancers: Development of a new method. Head Neck. 2012;34:1597-606
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Karakullukcu B, Stoker SD, Wildeman AP, Copper MP, Wildeman MA, Tan IB. A matched cohort comparison of mTHPCmediated photodynamic therapy and trans-oral surgery of early stage oral cavity squamous cell cancer. Eur Arch Otorhinolaryngol (in press) Karakullukçu B, van der Vlies D, Hoebers FJ. A rare late complication after concomitant chemoradiation of an oropharyngeal tumor: cervical necrotizing fasciitis. Kulak Burun Bogaz Ihtis Derg. 2012;22:105-8 Kenter G, Wijma S. NVOG celebrates 125 years. Ned Tijdschr Geneeskd. 2012;155:A5178 Kenter GG. Premaligne aandoeningen. Obstetrie en gynaecologie. De voortplanting van de mens. Heineman M.J. Reed Business.Amsterdam 2012 Kerst JM, Moonen L, Graafland NM, Bergman AM, Pos FJ, Horenblas S. The role of chemotherapy and radiotherapy in the treatment of penile cancer. In: Muneer A, Arya M, Horenblas S, editors. Textbook of Penile Cancer: Springer; 2012 Kocken M, Berkhof J, van Kemenade FJ, Louwers JA, Zaal A, Nobbenhuis MA, Kenter G, Snijders PJ, Meijer CJ, Helmerhorst TJ. Long-term CIN3+ risk in women with abnormal cytology; role of hrHPV testing. Br J Cancer. 2012;106:817-25 Koers K, Francken AB, Haanen J; Woerdeman LA, Van Der Hage J. Vemurafenib as neoadjuvant treatment for irresectable regional metastatic melanoma. JCO. (in press) Koning CC, Blank LE, Koedooder C, van Os RM, van de Kar M, Jansen E, et al. Brachytherapy after external beam radiotherapy and limited surgery preserves bladders for patients with solitary pT1-pT3 bladder tumors. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2012;23:2948-53 Koolen BB, Aukema TS, González Martínez AJ, Vogel WV, Caballero Ontanaya L, Vrancken Peeters MJTFD, Vroonland CJJ, Rutgers EJTh, Benlloch Baviera JM, Valdés Olmos RA, First clinical experience with a dedicated PET for hanging breast molecular imaging. Q J Nucl Med Mol Imaging 2012 (in press)
Koolen BB, Pengel KE, Wesseling J, Vogel WV, Vrancken Peeters MJTFD, Vincent AD, Gilhuijs KGA, Rodenhuis S, Rutgers EJTh, Valdés Olmos RA, FDG PET/CT during neoadjuvant chemotherapy may predict response in ER-positive/HER2-negative and triple negative, but not in HER2-positive breast cancer. Breast 2012 (in press) Koolen BB, Valdés Olmos RA, Elkhuizen PHM, Vogel WV, Vrancken Peeters MJTFD, Rodenhuis S, Rutgers EJTh, Locoregional lymph node involvement on 18F-FDG PET/CT in breast cancer patients scheduled for neoadjuvant chemotherapy. Breast Cancer Res Treat 2012;135:231-40 Koolen BB, Vegt E, Rutgers EJTh, Vogel WV, Stokkel MPM, Hoefnagel CA, Fioole-Bruining A, Vrancken Peeters MJTFD, Valdés Olmos RA, FDG-avid sclerotic bone metastases in breast cancer patients: a PET/CT case series. Ann Nucl Med 2012;26:86-91 Koolen BB, Vogel WV, Vrancken Peeters MJTFD, Loo CE, Rutgers EJTh, Valdés Olmos RA Molecular imaging in breast cancer: from whole body PET/ CT to dedicated breast PET J Oncol 2012;2012:438647 Koolen BB, Vrancken Peeters MJTFD, Aukema TS, Vogel WV, Oldenburg HSA, van der Hage JA, Hoefnagel CA, Stokkel MPM, Loo CE, Rodenhuis S, Rutgers EJTh, Valdés Olmos RA, 18F-FDG PET/CT as a staging procedure in primary stage II and III breast cancer: comparison with conventional imaging techniques. Breast Cancer Res Treat 2012;131:117-26 Koolen BB, Vrancken Peeters MJTFD, Wesseling J, Lips EH, Vogel WV, Aukema TS, van Werkhoven E, Gilhuijs KGA, Rodenhuis S, Rutgers EJTh, Valdés Olmos RA, Association of primary tumour FDG uptake with clinical, histopathological, and molecular characteristics in breast cancer patients scheduled for neoadjuvant chemotherapy. Eur J Nucl Med Mol imaging 2012;37:1830-8 Kreeft AM, Krap M, Wismeijer D, Speksnijder CM, Smeele LE, Bosch SD, Muijen MS, Balm AJM. Oral function after maxillectomy and reconstruction with an obturator. Int J Oral Maxillofac Surg. 2012;41:1387-92 Kreeft AM, Rasch CR, Muller SH, Pameijer FA, Hallo E, Balm AJM. Cine MRI of swallowing in patients with advanced oral or oropharyngeal carcinoma: a feasibility study. Eur Arch Otorhinolaryngol. 2012;269:1703-11
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Sixty patients were included. Seven patients stopped treatment prematurely (12%). Skin toxicity was present in 37 patients (62%), and diarrhea was present in 21 patients (35%). PET evaluation revealed metabolic response (> 25% standardized uptake value decrease) in 16 patients (27%); CT evaluation using RECIST showed response in three patients (5%). At surgery, no unexpected complications occurred. Pathologic examination showed more than 50% necrosis in 14 patients (23%), of whom three (5%) had more than 95% tumor necrosis. The response rate in the enriched population was 34% (10 of 29 patients). The report clearly shows the limitations of CT scanning and provides support for PET as a more reliable biomarker of response. This study reports considerable activity of neoadjuvant erlotinib in an enriched population of operable NSCLC patients, thereby underlining the importance of patient selection for EGFR TKIs.
Head and neck oncologyy Alfons Balm, Michiel van den Brekel, Frans Hilgers, Baris Karakullukcu, Martin Klop, Peter Lohuis, Ludi Smeele, Bing Tan, Charlotte Zuur
The department is a national referral center and one of the larger clinical departments in this field treating about 600 new patients annually. The department is active in clinical and translational research. Currently, 5 full professors are part of the department and the staff members have part-time appointments at the Academic Medical Center (AMC) of the University of Amsterdam. Being a multidisciplinary field, there are many clinical and research connections within the institute and also with the AMC.
Translational research In 2012 the head and neck department was involved in several translational research projects. Together with Adrian Begg and Conchita Vens the Fanconi pathway in oral cancer samples as well as in cell lines was studied. So far, over 150 patient samples have been deep-sequenced. The findings are currently analysed. A clinical trial using PARP inhibitors in combination with radiotherapy is initiated. In a collaborative project with Marcel Verheij and Adrian Begg a study looking at micro-RNA profiles in laryngeal cancer is being conducted. In an international study lead by Olga Hamming (Artforce) Cetuximab will be combined with radiotherapy and several expression profile studies are attached. Together with Jacques Neefjes and Huib Ovaa primary HNSCC short term cultures are explored. These can be used for testing a patients individual chemo-sensitivity prior to the intended treatment and also for testing the chemical compound library. Early detection and monitoring treatment outcome of nasopharyngeal cancer using (anti-) Epstein-Barr Virus (EBV) based tumor markers is studied in the Netherlands, UI-Jakarta, UGM-Yogyakarta together with the VUmc. With the same group a project on lytical EBV induction therapy in recurrent nasopharyngeal cancer has been approved by the KWF. Tumor microenvironment studies have been initiated with Jan-Paul de Boer.
Photodynamic therapy The department is active in research on photodynamic therapy (PDT) and several grants have been obtained (VENI, ZonMw, NWO, KWF). Currently the focus of this research is on interstitial PDT. In several projects treatment planning and dosimetry for interstitial PDT is studied by means of developing pre-treatment planning tools and intraoperative imaging guidance. In a national study with participation of all academic medical centers in the Netherlands, the cost effectiveness of Foscan PDT for incurable recurrent head and neck cancers is being studied. Monitoring of tissue oxygen saturation and Foscan concentration during PDT to optimize treatment parameters is another project.
Rehabilitation Rehabilitation research focuses on several topics. One concerns prevention of swallowing and communication problems in patients treated with chemo-radiation for advanced head and neck cancer. Research on postlaryngectomy vocal and pulmonary rehabilitation, in cooperation with Atos Medical Sweden, has resulted in the development of a new surgical instrument for voice prosthesis implantation, and in new heatand-moist-exchangers. Mucociliary transport in the trachea after laryngectomy is another field of interest. Together with the Amsterdam Centre for Language and Communication (ACLC) of the University of Amsterdam, the possibilities are explored to use automatic speech analyzers for the assessment of intelligibility of pathologic voices, e.g. after laryngectomy or chemoradiation. Also with the ACLC a project on physicianpatient communication is started. A head and neck rehabilitation center implementing an evidence-based rehabilitation program developed in the NKI has been set up in 2011 and within this setting several strategies are studied.
Clinical research Clinical research is quite diverse. Fields of interest are fluorescent imaging of sentinel nodes, impact of sentinel node surgery in melanoma, and clinical and biological aspects (HPV) of head and neck cancer in young patients. Together with the departments of gynaecology (Gemma Kenter), medical oncology (Jan Paul de Boer) and urology (Simon Horenblas) HNSCC microenvironment studies and a phase 1 HPV vaccination study are in preparation. Prediction of inoperability and development of virtual surgery for oral function to predict morbidity after surgery are conducted in cooperation with the Technical University Twente and VUmc.
Kreeft AM, Smeele LE, Rasch CR, Hauptmann M, Rietveld DH, Leemans CR, Balm AJM. Preoperative imaging and surgical margins in maxillectomy patients. Head Neck. 2012;34:1652-6 Kroon BBR. Surgery for distant metastatic melanoma improves survival. Ann Surg Oncol. 2012;19:2426-7 Kvistborg P, Shu CJ, Heemskerk B, Fankhauser M, Thrue CA, Toebes M, van Rooij N, Linnemann C, van Buuren MM, Urbanus JH, Beltman JB, Thor Straten P, Li YF, Robbins PF, Besser MJ, Schachter J, Kenter GG, Dudley ME, Rosenberg SA, Haanen JB, Hadrup SR, Schumacher TN. TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients. Oncoimmunology. 2012;1:409-418 Lips EH, Mukhtar RA, Yau C, de Ronde JJ, Livasy C, Carey LA, Loo CE, Vrancken-Peeters MJTFD, Sonke GS, Berry DA, Van’t Veer LJ, Esserman LJ, Wesseling J, Rodenhuis S, Shelley Hwang E; I-SPY TRIAL. Lobular histology and response to neoadjuvant chemotherapy in invasive breast cancer. Investigators. Breast Cancer Res Treat. 2012;136:35-43 Lips EH, Mulder L, de Ronde JJ, Mandjes IA, Vincent A, Vrancken Peeters MJTFD, Nederlof PM, Wesseling J, Rodenhuis S. Neoadjuvant chemotherapy in ER+ HER2- breast cancer: response prediction based on immunohistochemical and molecular characteristics. Breast Cancer Res Treat. 2012;131:827-36 Litière S, Werutsky G, Fentiman IS, Rutgers EJTh, Christiaens MR, Van Limbergen E, Baaijens MH, Bogaerts J, Bartelink H. Breast conserving therapy versus mastectomy for stage I-II breast cancer: 20 year follow-up of the EORTC 10801 phase 3 randomised trial. Lancet Oncol. 2012;13:412-9 Lodder WL, Gilhuijs KG, Lange CA, Pameijer FA, Balm AJ, van den Brekel MW. Semi-automated primary tumor volume measurements by dynamic contrast-enhanced MRI in patients with head and neck cancer. Head Neck (in press) Lodder WL, Pameijer FA, Rasch CR, van den Brekel MW, Balm AJ. Prognostic significance of radiologically determined neck node volume in head and neck cancer: a systematic review. Oral Oncol. 2012;48:298-302
Lok CA, Snijder KS, Nieuwland R, Van Der Post JA, de Vos P, Faas MM. Microparticles of pregnant women and preeclamptic patients activate endothelial cells in the presence of monocytes. Am J Reprod Immunol. 2012;67:206-15 Lorenz KJ, Hilgers FJM, Maier H. A novel puncture instrument: the Provox-Vega® puncture set: Its use in voice prosthesis insertion following laryngectomy. HNO (in press) Maaskant-Braat AJ, Roumen RM, Voogd AC, Pijpers R, Luiten EJ, Rutgers EJTh, Nieuwenhuijzen GA. Sentinel Node and Recurrent Breast Cancer (SNARB): Results of a Nationwide Registration Study. Ann Surg Oncol. 2012 Meijer RP, Meinhardt W, van der Poel HG, van Rhijn BW, Kerst JM, Pos FJ, et al. Local control rate and prognosis after sequential chemoradiation for small cell carcinoma of the bladder. International journal of urology: official journal of the Japanese Urological Association. 2012 Meijer RP, Nunnink CJ, Wassenaar AE, Bex A, van der Poel HG, van Rhijn BW, et al. Standard lymph node dissection for bladder cancer: significant variability in the number of reported lymph nodes. J Urol. 2012;187:446-50 Meijer RP, van Rhijn BW. A plea for long-term surveillance in bacillus Calmette-Guerin-treated non-muscleinvasive bladder cancer. European Urology. 2012;61:508-9 Meinhardt W, van der Poel HG, Valdes Olmos RA, Bex A, Brouwer OR, Horenblas S. Laparoscopic sentinel lymph node biopsy for prostate cancer: the relevance of locations outside the extended dissection area. Prostate Cancer. 2012;2012:751753 Meinhardt-Wollweber M, Krebs R. A computational model for previtamin D(3) production in skin. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology. 2012;11:731-7 Mérol JC, Charpiot A, Langagne T, Hémar P, Ackerstaff AH, Hilgers FJM. Randomized controlled trial on postoperative pulmonary humidification after total laryngectomy: External Humidifier versus Heat and Moisture Exchanger. Laryngoscope. 2012;122:275-81
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Mertens LS, Bruin NM, Vegt E, de Blok WM, Fioole-Bruining A, van Rhijn BW, et al. Catheter-assisted 18F-FDG-PET/ CT imaging of primary bladder cancer: a prospective study. Nuclear medicine communications. 2012;33:1195-201 Mertens LS, Fioole-Bruining A, van Rhijn BW, Kerst JM, Bergman AM, Vogel WV, et al. FDG-PET/CT in Monitoring Response of Pelvic Lymph Node Metastases to Neoadjuvant Chemotherapy in Bladder Cancer. J Urol. 2012 Mertens LS, Meijer RP, Kerst JM, Bergman AM, van Tinteren H, van Rhijn BW, et al. Carboplatin based induction chemotherapy for nonorgan confined bladder cancer--a reasonable alternative for cisplatin unfit patients? J Urol. 2012;188:1108-13 Mertens LS, Meijer RP, van Werkhoven E, Bex A, van der Poel HG, van Rhijn BW, et al.. Differences in histopathological evaluation of standard lymph node dissections result in differences in nodal count but not in survival. World journal of urology 2012 Mertens LS, Meinhardt W, Rier WB, Nooter RI, Horenblas S. Extravasation of intravesical chemotherapy for non-muscle-invasive bladder cancer. Urologia internationalis. 2012;89:332-6 Mieog JS, Morden JP, Bliss JM, Coombes RC, van de Velde CJ; IES Steering Committee. Carpal tunnel syndrome and musculoskeletal symptoms in postmenopausal women with early breast cancer treated with exemestane or tamoxifen after 2-3 years of tamoxifen: a retrospective analysis of the Intergroup Exemestane Study. Lancet Oncol. 2012;13:420-32 Milstein DM, van Kuijen AM, Copper MP, Karakullukçu B, Tan IB, Lindeboom JA, Fokkens WJ, Ince C. Monitoring microcirculatory alterations in oral squamous cell carcinoma following photodynamic therapy. Photodiagnosis Photodyn Ther. 2012;9:69-75 Montorsi F, Wilson TG, Rosen RC, Ahlering TE, Artibani W, Carroll PR, et al. Best practices in robotassisted radical prostatectomy: recommendations of the Pasadena Consensus Panel. Eur Urol. 2012;62:368-81 Nacerddine K, Beaudry JB, Ginjala V, Westerman B, Mattiroli F, Song JY, et al. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer. The Journal of clinical investigation. 2012;122:1920-32
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Nieweg OE, Hoekstra HJ. Gerandomizeerd onderzoek verschaft meer duidelijkheid over sentinelnodebiopsie bij melanoom. Ned Tijdschr Oncol 2012;3:131-134. Nieweg OE, Hoekstra HJ. Respons op ‘Gerandomiseerd onderzoek leidt to meer vragen over schildwachtklierbiopsie bij het melanoom’, Van Akkooi ACJ, Verhoef C, De Wilt JHW. Ned Tijdschr Oncol 2012;3:134-135 Nieweg OE. Behandeling van hematogene metasasen van melanoom in beweging. Editorial bij de bijdrage van K.P. Wevers, G.A.P. Hospers en H.J. Hoekstra, getiteld ‘Fase IIIstudie: chirurgische of systemische behandeling van resectabele hematogene melanoommetastasen’. Ned Tijdschr Oncol 2012;9:42-43 Nieweg OE. Oncologie en de rol van de chirurg. In: Gooszen HG, Aronson DC, Blankensteijn JD, Gouma DJ, Kroon BBR, Lange JF, van Vugt AB, redacteuren. Leerboek chirurgie. Bohn Stafleu van Loghum, Houten, 2012 Nieweg OE. The sentinel lymph node concept in oncologic surgery. In: Mariani G, Manca G, Vidal-Sicart S, Valdés Olmos RA, redacteuren. Atlas of lymphoscintigraphy and sentinel node mapping. Springer, Heidelberg, 2012 Numan RC, Klomp HM, Li W, Buitelaar DR, Burgers JA, Van Sandick JW, Wouters MW. A clinical audit in a multidisciplinary care path for thoracic surgery: an instrument for continuous quality improvement. Lung Cancer. 2012;78:270-5 Nyst HJ, Wildeman MA, Indrasari SR, Karakullukcu B, Van Veen RL, Adham M, Stewart FA, Levendag PC, Sterenborg HJ, Tan IB. Temoporfin mediated photodynamic therapy in patients with local persistent and recurrent nasopharyngeal carcinoma after curative radiotherapy: A feasibility study. Photodiagnosis Photodyn Ther. 2012;9:274-81 Osanto S, Bex A, Hulsbergen-van de Kaa CA, Soetekouw PM, Stemkens D. [The Dutch guideline ‘Renal cell carcinoma’]. Nederlands tijdschrift voor geneeskunde. 2012;156:A4462 Peters M, Moman MR, van der Poel HG, Vergunst H, de Jong IJ, Vijverberg PL, et al. Patterns of outcome and toxicity after salvage prostatectomy, salvage cryosurgery and salvage brachytherapy for prostate cancer recurrences after radiation therapy: a multi-center experience and literature review. World journal of urology. 2012
UROLOGIC ONCOLOGY Simon Horenblas, Wim Meinhardt, Axel Bex, Henk Van de Poel, Bas Van Rhijn, Rosa Djajadiningrat, Laura Mertens; Departments of Immunology, Radiology, Nuclear medicine, Radiotherapy and Biostatistics, VUMC, Department of Pathology, LUMC, Department of Clinical Imaging
Research in urologic oncology has been centred on the following themes: Improved staging of urologic tumours, organ and function sparing, fundamental research in prostate, kidney and penis cancer.
Improved staging of urologic tumours Research has been focussed on the role of sentinel node biopsy in kidney, prostate, testicular and penile cancer. In all above mentioned tumours, apart from kidney cancer, sentinel node biopsy has proven to be extremely useful in minimizing the morbidity of surgery of the lymph nodes and at the same time maximizing the detection rate of occult lymph node metastases. The collaboration with the department of nuclear physics at the Antoni van Leeuwenhoek and the dept. of clinical imaging in the LUMC, has been very fruitful. Especially the use of SPECT /CT scans and intra-operative imaging with a mobile gamma camera (Sentinella®) has proven to be a clinical useful adjunct. In prostate cancer no false negative findings were observed. The sentinel node strategy together with the Sentinella was effective in localizing sentinel nodes in a variety of anatomical locations, otherwise not removed during lymph node dissection. The scope of research has been widened by collaboration with the department of clinical physics. A comparative trial was started, comparing patent blue, ICG and ICG with technetium (N09IGF). Using a fluorescence camera, ICG is seen after i.v. injection. Integration of the fluorescence camera in the robot camera is being investigated. In testicular cancer also no false negative findings have been observed. Accrual in this clinical study is very slow unfortunately, mainly because of the fact that stage I germ cell tumours are mostly been treated outside of Antoni van Leeuwenhoek. In penile cancer the false negative rate dropped from 20% to an acceptable 5.1% to increase again after the last analysis to 10.8% Reasons for this is analyzed. For renal cancer we continue with the sentinel node study, approved in 2008, with the aim of elucidating the lymphatic pathways of renal cancer and the immunological effect of renal cancer in the sentinel nodes. The first 20 patients were assessed. Main problem remains non visualisation in a large proportion of patients.
Organ and function sparing The role of cytoreductive surgery in metastatic renal cancer is being investigated in a randomized EORTC study comparing immediate versus deferred nephrectomy for patients with synchronous metastatic renal cell carcinoma and the primary tumour in situ. This study (E 30073) coordinated by Axel Bex, followed the experiences of the phase II study with neo-adjuvant Sutent (tyrosine kinase inhibitor). In bladder cancer we continue to expand our experiences with the so called Sexuality Preserving Cystectomy and Neobladder
(SPCN). In a recent publication excellent continence and potency rates were seen without any danger of increased recurrences. Despite international scepsis we continue to advocate this procedure in well selected patients. In line with the wish to decrease the morbidity of the surgery robotic assisted cystectomy was started in 2010. The robot cystectomy was enlarged by intracorporeal formation of the urinary deviation. A difficult operation, with no advantages so far after initial experience. We are involved in the active surveillance study for prostate cancer (Prias, initiated by Rotterdam). We are partners together with Rotterdam and Groningen in the PCMM consortium regarding prostate cancer and prostatectomy. Another modality will be investigated: the use of photodynamic therapy in localized prostate cancer. A randomized study is launched comparing active surveillance and photodynamic therapy. Using a new device measuring endothelial function a study was launched assessing this function after robot prostatectomy, with the aim to better predict sexual rehabilitation.
Translational research in prostate, kidney and penile cancer Prostate cancer
The role of mTOR inhibition in chemotherapy is being assessed in a phase I study in collaboration with Andre Bergman, medical oncologist. In this study the combined use of everolimus (mTOR inhibitor) and cyclophosphamide in CPRC will be tested. Further trials are: Ipilimumab + radiation in M+ prostate cancer (M10IPI), (M11DPC) Docetaxel and Custirsen. In collaboration with Prof. Ton Schumacher the role of T-cell receptors in prostate cancer model vaccination studies was addressed. Penis cancer
The role of HPV in penis cancer was further investigated in close collaboration with the department of pathology of the Free University medical centre. Using molecular and serological analyses for a wide range of HPV types and comparing serological findings with age-matched male controls. Primarily HPV 16 infection is directly involved in penile carcinogenesis. Based on earlier micro-array results a renewed analysis was started comparing HPV+ and HPV- tumors. A research project was started in collaboration with the Department of Head and Neck surgery, Gynaecologic Surgery and Department of Immunotherapy to assess the role of the microenvironment.
Pieters BR, Horenblas S, Koning CC. Does Partial Cystectomy Compromise Oncologic Outcomes for Patients with Bladder Cancer Compared to Radical Cystectomy? A Matched Case-Control Analysis. J Urol. 2012 Retèl VP, Joore MA, Linn SC, Rutgers EJTh, van Harten WH. Scenario drafting to anticipate future developments in technology assessment. BMC Res Notes. 2012;5:442 Rijkaart DC, Berkhof J, Rozendaal L, van Kemenade FJ, Bulkmans NW, Heideman DA, Kenter GG, Cuzick J, Snijders PJ, Meijer CJ. Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial. Lancet Oncol. 2012;13:78-88 Ruers T, Punt C, Van Coevorden F, Pierie JP, Borel-Rinkes I, Ledermann JA, Poston G, Bechstein W, Lentz MA, Mauer M, Van Cutsem E, Lutz MP, Nordlinger B; EORTC Gastro-Intestinal Tract Cancer Group, Arbeitsgruppe Lebermetastasen und—tumoren in der Chirurgischen Arbeitsgemeinschaft Onkologie (ALM-CAO) and the National Cancer Research Institute Colorectal Clinical Study Group (NCRI CCSG. Radiofrequency ablation combined with systemic treatment versus systemic treatment alone in patients with nonresectable colorectal liver metastases: a randomized EORTC Intergroup phase II study (EORTC 40004). Ann Oncol. 2012;23:2619-26 Ruers TJ, Hagendoorn J. Treatment dilemmas in patients with synchronous colorectal liver metastases. Recent Results Cancer Res. 2012;196:37-49 Ruiterkamp J, Voogd AC, Tjan-Heijnen VC, Bosscha K, van der Linden YM, Rutgers EJTh, Boven E, van der Sangen MJ, Ernst MF; Dutch Breast Cancer Trialists’ Group (BOOG). Systemic therapy with or without up front surgery of the primary tumor in breast cancer patients with distant metastases at initial presentation. BMC Surg. 2012;12:5
Kidney cancer
In collaboration with the Department of Immunology, the Department of Angiogenesis of Arjan Griffioen at the Free University medical centre and Eric Jonasch, MD Anderson Cancer Center, Houston, kidney cancer tissue is analysed, specifically looking into neo-vascularization and immune modulation. Also together with the department of immunology analysis of rapid expansion of TIL cells was started.
Russell NS, Scharfenecker M, Hoving S, Woerdeman LA. Consequences of radiotherapy for breast reconstruction. In: Danilla S, Ed. Selected Topics in Plastic Reconstructive Surgery. New York, InTech, 2012 Rutgers EJTh. How can breastconserving surgery be improved even more?. Ned Tijdschr Geneeskd. 2012;156
Rutten MJ, Gaarenstroom KN, Van Gorp T, van Meurs HS, Arts HJ, Bossuyt PM, Ter Brugge HG, Hermans RH, Opmeer BC, Pijnenborg JM, Schreuder HW, Schutter EM, Spijkerboer AM, Wensveen CW, Zusterzeel P, Mol BW, Kenter GG, Buist MR. Laparoscopy to predict the result of primary cytoreductive surgery in advanced ovarian cancer patients (LapOvCa-trial): a multicentre randomized controlled study. BMC Cancer. 2012;12:31 Saadatmand S, Rutgers EJTh, Tollenaar RA, Zonderland HM, Ausems MG, Keymeulen KB, Schlooz-Vries MS, Koppert LB, Heijnsdijk EA, Seynaeve C, Verhoef C, Oosterwijk JC, Obdeijn IM, de Koning HJ, Tilanus-Linthorst MM. Breast density as indicator for the use of mammography or MRI to screen women with familial risk for breast cancer (FaMRIsc): a multicentre randomized controlled trial. BMC Cancer. 2012;12:440 Sadeghi R, Gholami H, Zakavi SR, Kakhki VR, Horenblas S. Accuracy of 18F-FDG PET/CT for diagnosing inguinal lymph node involvement in penile squamous cell carcinoma: systematic review and meta-analysis of the literature. Clinical nuclear medicine. 2012;37:436-41 Samant S, van den Brekel MW, Kies MS, Wan J, Robbins KT, Rosenthal DI, Rasch C, Weber RS. Concurrent chemoradiation for adenoid cystic carcinoma of the head and neck. Head Neck. 2012;34:1263-8 Santegoets LA, van Baars R, Terlou A, Heijmans-Antonissen C, Swagemakers SM, van der Spek PJ, Ewing PC, van Beurden M, van der Meijden WI, Helmerhorst TJ, Blok LJ. Different DNA damage and cell cycle checkpoint control in low- and high-risk human papillomavirus infections of the vulva. Int J Cancer. 2012;130:2874-85 Schaake EE, Kappers I, Codrington HE, Valdés Olmos RA, Teertstra HJ, van Pel R, Burgers JA, van Tinteren H, Klomp HM. Tumor response and toxicity of neoadjuvant erlotinib in patients with early-stage non-small-cell lung cancer. J Clin Oncol. 2012;30:2731-8 Schaapveld M, van den Belt-Dusebout AW, Gietema JA, de Wit R, Horenblas S, Witjes JA, et al. Risk and prognostic significance of metachronous contralateral testicular germ cell tumours. British journal of cancer. 2012;107:1637-43
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Schellekens PPA, Aukema TS, Hage JJ, Prevoo W, Kon M. Can previous diagnostic examinations prevent preoperative angiographic assessment of the internal mammary perforators for (micro)surgical use? Ann Plast Surg. (in press)
Theunissen EAR, Timmermans AJ, Zuur CL, Hamming-Vrieze O, de Boer JP, Hilgers FJM, van den Brekel MWM. Total laryngectomy for a dysfunctional larynx after (chemo)radiotherapy. Arch Otolaryngol Head Neck Surg. 2012;138:548-55
Schmitz AC, Pengel KE, Loo CE, van den Bosch MA, Wesseling J, Gertenbach M, Alderliesten T, Mali WP, Rutgers EJTh, Bartelink H, Gilhuijs KG. Pre-treatment imaging and pathology characteristics of invasive breast cancers of limited extent: potential relevance for MRI-guided localized therapy. Radiother Oncol. 2012;104:11-8
Timmermans AJ, Krap M, Hilgers FJM, van den Brekel MWM. Spraakrevalidatie na totale laryngectomie. Ned Tijdschr Tandheelk. 2012;119:357-61
Schwartz GF, Reis-Fihlo J, Pusztai L, Fentiman IS, Holland R, Bartelink H, Rutgers EJTh, Solin LJ, Palazzo J; Consensus Committee. Adjuvant therapy in stage I carcinoma of the breast: the influence of multigene analyses and molecula phenotyping. Cancer. 2012;118:2031-8 Stiekema J, Boot H, Aleman BM, Wessels LF, van Sandick JW. Prognostication and prediction using gene expression profiling in oesophageal cancer. Eur J Surg Oncol. 2012 Stoker SD, Greijer AE, Wildeman MA, Novalic Z, de Boer JP, Huitema ADR, Fles R, Karakullukcu B, Middeldorp JM, Tan IB. Nieuwe diagnostische middelen en behandeltechnieken voor het nasofarynxcarcinoom. Ned Tijdschr Oncol. 2012;9:262-8 Sylvester R, Collette L, Bex A, Clarke N, Sternberg CL, Tombal B. The EORTC Genito-Urinary Cancers Group: 35 years of achievements and future strategy. EJC Suppl. 2012;10:58-65 Tanis E, van de Velde CJ, Bartelink H, van de Vijver MJ, Putter H, van der Hage JA. Locoregional recurrence after breast-conserving therapy remains an independent prognostic factor even after an event free interval of 10 years in early stage breast cancer. Eur J Cancer. 2012;48:1751-6 Tanis PJ, Nieweg OE. Anatomy and physiology of lymphathic circulation In: Mariani G, Manca G, Vidal-Sicart S, Valdés Olmos RA, redacteuren. Atlas of lymphoscintigraphy and sentinel node mapping Terlou A, Santegoets LA, van der Meijden WI, Heijmans-Antonissen C, Swagemakers SM, van der Spek PJ, Ewing PC, van Beurden M, Helmerhorst TJ, Blok LJ. An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155. J invest Dermatol. 2012;132:658-66
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Timmermans AJ, van den Brekel MWM, van der Molen L, Navran A, Nijssen TF, Hilgers FJM. Nieuwe ontwikkelingen op het gebied van zorg en nazorg van hoofd-halskanker. Ned Tijdschr Geneeskd. 2012;156:1719-24 Truong HT, Ramos AM, Yalcin F, de Ruiter M, van der Poel HJ, Huvenaars KH, et al. Sequence-based genotyping for marker discovery and co-dominant scoring in germplasm and populations. PloS one. 2012;7:e37565 Turker P, Bostrom PJ, Wroclawski ML, van Rhijn B, Kortekangas H, Kuk C, et al. Upstaging of urothelial cancer at the time of radical cystectomy: factors associated with upstaging and its effect on outcome. BJU Int. 2012;110:804-11 Valdés Olmos RA, Klop WMC, Brouwer OR. Preoperative and intraoperative lymphatic mapping for radioguided sentinel node biopsy in head and neck cancers. In: Atlas of Lymphoscintigraphy and Sentinel Lymph Node Mapping. Editors: G. Mariani, G. Manca, S. Vidal-Sicart, RA Valdés Olmos, Springer. 2012:199-208. Valdés Olmos RA, Meinhardt W, Vermeeren L, Brouwer OR. GOSTT in prostate cancer. In: International Atomic Energy A, editor. Guided intraoperative scintigraphic tumour targeting (GOSTT): IAEA; 2012 (in press) Van den Berg NS, Brouwer OR, Klop WMC, Karakullukcu B, Zuur CL, Tan IB, Balm AJM, van den Brekel MWM, Valdés Olmos RA, van Leeuwen FW. Concomitant radio- and fluorescenceguided sentinel lymph node biopsy in squamous cell carcinoma of the oral cavity using ICG-(99m)Tc-nanocolloid. Eur J Nucl Med Mol Imaging. 2012;39:1128-36 Van den Berg NS, van Leeuwen FW, van der Poel HG. Fluorescence guidance in urologic surgery. Current opinion in urology. 2012;22:109-20
Improved quality control of treatment results
Using prospective data collection, almost all uro-surgical treatments at the Antoni van Leeuwenhoek can be analysed now. Extensive use of these databases was made for prostate, bladder and penile cancer. Quality of life analysis was done of in patients with localized prostate cancer managed by brachytherapy or robot assisted laparoscopic prostatectomy (RALP). Prospective data are analysed for risk estimation of treatment or active surveillance for prostate cancer within the framework of the IMPACT-study, funded by ZonMW.
Malignant differential diagnosis in children referred for infantile hemangioma From April, 2003 through December, 2009, 251 girls and 172 boys were referred for a “hemangioma” to the AMC, Amsterdam. This diagnosis was confirmed in 377 children (89%). Seven of the remaining children (2%) were diagnosed with a rhabdomyosarcoma (n = 2), a sarcoma (n = 1), a poorly differentiated round and spool cell sarcoma (n = 1), a nerve sheath sarcoma (n = 1), a dermatofibrosarcoma protuberans (n = 1), or a lymphoma (n = 1). Early age malignant tumors can mimic benign infantile hemangioma. In cases where the diagnosis of infantile hemangioma is equivocal, biopsy has to be performed in a specialized center to prevent delay or omission of proper treatment.
Plastic and Reconstructive Surgery Marieke Van Den Berg, Brigitte Drost, J Joris Hage, Marije Hoornweg, Martine Van Huizum, Fientje Van der Veen, Leonie Woerdeman
Our research is focused on innovative reconstructive techniques after ablative surgery by other specialists. Additionally, ongoing vascular and functional studies are executed in collaboration with the Department of Plastic Surgery at the University Medical Centre Utrecht (prof dr M Kon) and the Faculty of Human Movement Sciences at the Free University, Amsterdam (prof dr HEJ Veeger). This year, Marije Hoornweg took her PhD “On Infantile Hemangiomas”. Even tough infantile hemangiomas are benign neonatal vascular tumours, they may be mimicked by early age malignancies.
Assessment of previous diagnostic examinations can prevent preoperative angiography of the internal mammary perforators to be used for (micro)surgical reconstruction Preoperative assessment of the internal mammary artery perforating (IMAP) branches enhances IMAP-based reconstructive procedures. A radiologist and a plastic surgeon jointly assessed whether such information could sufficiently be obtained from previously performed diagnostic thoracic CT scans of 12 head and neck cancer patients and 12 breast cancer patients, and from the mammary MRI of 12 breast cancer patients. Secondly, we retrospectively assessed in how many of 10 IMAP-patients and 10 DIEP-patients such previous diagnostic examinations were informative regarding the level of the dominant perforator. All 24 CT scans and 11 of the 12 MRI scans sufficiently allowed assessment of the level of the dominant IMAP. Previous information had already been available in all 10 DIEP-patients and 6 of the 10 IMAP-patients. Hence, we concluded that previously performed diagnostic CT scans and MRI scans that included the parasternal region usually allow sufficient preoperative assessment of the internal mammary perforators for reconstructive procedures. We advocate re-assessment of such previous examinations before ordering additional angiography.
DERMATOLOGY Marianne Crijns, Germaine Relyveld, Biljana Zupan-Kajcovski, Wietze Van der Veen, Inka Nieuweboer-Krobotova, Soe Janssens
Optical Coherence Tomography in melanocytic lesions Optical coherence tomography (OCT) is an emerging biomedical optical imaging technique that performs high resolution, cross sectional tomographic imaging generating pictures that resemble histopathological examination. The images are generated by scanning an optical beam across the tissue and measuring the echo time delay and intensity of backscattered light. The image penetration depth of OCT is determined by optical scattering and is up to 2 mm in tissue. We use (in collaboration with the Biomedical Engineering and Physics department of the AMC) the Santec Inner Vision 2000 with a 10 µm axial resolution, 11 µm lateral resolution, and light with a wavelength around 1300 nm. This high resolution OCT functions as a type of ‘optical biopsy’, providing cross sectional images of tissues in analogy to histopathology but without removal or staining tissue. With regard to tissue diagnosis: we are focusing on the use of OCT in melanocytic lesions. We would like to determine the possible value of OCT in distinguishing benign nevi from malignant melanoma in patients with suspicion of malignant melanoma in comparison with histopathology as the gold standard, by viewing OCT images and by determining the attenuation coefficient of the B-scan OCT images. At this moment, we are including and imaging patients with one or more suspicious lesions with OCT.
Local immunotherapy by the synergism of monobenzone and imiquimod cream (MI) for cutaneous metastases in stage III-IV melanoma patients Melanoma is a good candidate for treatment with immunotherapy, during which vitiligo development is a favourable prognostic sign. At the department of Dermatology of the AMC, we have developed a new therapy for melanoma, based on the potent vitiligo-inducing effect of monobenzone combined with the immunostimulatory adjuvants imiquimod (MI therapy). MI therapy both induces strong melanoma-reactive immunity, which effectively eradicated established melanoma in mice (van den Boorn al, PLoS one 2010).
Van den Brekel MW, Lodder WL, Stel HV, Bloemena E, Leemans CR, Van der Waal I. Observer variation in the histopathologic assessment of extranodal tumorspread in lymph node metastases in the neck. Head Neck. 2012;34:840-5 Van den Brekel MWM, Greven AJ. Keel-, neus- en oorafwijkingen. In: Algemene Ziekteleer voor Tandartsen. Editors: HS Brand, DE van Diermen, PC Makkes, Bohn Stafleu van Loghum. 2012:167-76 Van den Brekel MWM, Hilgers FJM. Preoperative workup of the neck in head and neck squamous cell carcinoma. In: Pearls and Pitfalls in Head and Neck Surgery; practical tips to minimize complications. Editor: CR Cernea; associate editors: FL Dias, D Fliss, RA Lima, EN Myers, WI Wei, Karger Publishers. 2012:36-7 Van der Drift MA, Karim-Kos HE, Siesling S, Groen HJ, Wouters MW, Coebergh JW, de Vries E, JanssenHeijnen ML. Progress in standard of care therapy and modest survival benefits in the treatment of nonsmall cell lung cancer patients in the Netherlands in the last 20 years. J Thorac Oncol. 2012;7:291-8 Van der Heiden-van der Loo M, de Munck L, Visser O, Westenend PJ, van Dalen T, Menke MB, Rutgers EJTh, Peeters PH. Variation between hospitals in surgical margins after first breast-conserving surgery in the Netherlands. Breast Cancer Res Treat. 2012;131:691-8 Van der Molen L, van Rossum MA, Jacobi I, van Son RJJH, Smeele LE, Rasch CRN, Hilgers FJM. Pre- and posttreatment voice and speech outcomes in patients with advanced head and neck cancer treated with chemoradiotherapy: expert listeners’ and patient’s perception. J Voice. 2012;26:664 Van der Ploeg APT, Van Akkooi ACJ, Rutkowski P, Cook M, Nieweg OE, Rossi CR, Testori A, Verhoef C, Eggermont AMM; European Organization for Research and Treatment of Cancer Melanoma Group. Prognosis in patients with sentinel node positive without immediate completion lymph node dissection. Br J Surg 2012;99:13961405 Van der Poel HG, Balm AJ, Nieweg OE, Valdes Olmos RA. Comment on Heuveling et al.: Nanocolloidal albumin-IRDye 800CW: a near-infrared fluorescent tracer with optimal retention in the sentinel lymph node. European journal of nuclear medicine and molecular imaging. 2012;39:1510-1
Van der Poel HG, Balm AJM, Nieweg OE, Valdés Olmos RA. Comment on Heuveling et al.: Nanocolloidal albumin-IRDye 800CW: a nearinfrared fluorescent tracer with optimal retention in the sentinel lymph node. Eur J Nucl Med Mol Imaging 2012;39:1510-1511 Van der Poel HG, Brouwer OR, van den Berg NS, Buckle T, van Leeuwen FWB. Reply to Karol Polom, Dawid Murawa, Wojciech polom’s letter to the editor re: Henk G. van der Poel, Tessa Buckle, Oscar R. Brouwer, Renato A. Valdés Olmos, Fijs W.B. van Leeuwen. Intraoperative laparoscopic fluorescence guidance to the sentinel lymph node in prostate cancer patients: Clinical proof of concept of an integrated functional imaging approach using a multimodal tracer. European Urology. 2012;61:e19-e20 Van der Poel HG, de Blok W, Tillier C, van Muilekom E. Robot-assisted laparoscopic prostatectomy: nodal dissection results during the first 440 cases by two surgeons. Journal of endourology / Endourological Society. 2012;26:1618-24 Van der Poel HG, Tillier C, de Blok W, van Muilekom E. Extended nodal dissection reduces sexual function recovery after robot-assisted laparoscopic prostatectomy. Journal of endourology / Endourological Society. 2012;26:1192-8 Van der Poel HG. Words of wisdom: Re: Super extended versus extended pelvic lymph node dissection in patients undergoing radical cystectomy for bladder cancer: a comparative study. Eur Urol. 2012;61:1266-7 Van der Steen LP, Bakema JE, Sesarman A, Florea F, Tuk CW, Kirtschig G, Hage JJ, Sitaru C, van Egmond M. Blocking FCα receptor I on granulocytes prevents tissue damage induced by IgA autoantbodies. J Immunol. 2012;189:1594-601 Van Hooff SR, Leusink FKJ, Roepman P, Baatenburg de Jong RJ, Speel EJM, Van den Brekel MWM, Van Velthuysen MLF, Van Diest PJ, Van Es RJJ, Merkx MAW, Kummer JA, Leemans CR, Schuuring E, Langendijk JA, Lacko M, De Herdt MJ, Jansen JC, Brakenhoff RH, Slootweg PJ, Takes RP, Holstege FCP. Validation of a gene expression signature of assessment of lymph node metastasis in oral squamous cell carcinoma. J Clin Oncol. 2012;30:4104-10
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Van Lonkhuijzen L, Stekelenburg J, van Roosmalen J. Maternity waiting facilities for improving maternal and neonatal outcome in low-resource countries. Cochrane Database Syst Rev. 2012;10:CD006759 Van Monsjou HS, Van Velthuysen ML, Van den Brekel MWM, Jordanova ES, Melief CJ, Balm AJM. Human papillomavirus status in young patients with head and neck squamous cell carcinoma. Int J Cancer. 2012;130:1806-12 Van Rhijn BD, Smout AJ, Bredenoord AJ. [Eosinophilic oesophagitis: a frequently missed cause of dysphagia]. Nederlands tijdschrift voor geneeskunde. 2012;156:A4716 Van Rhijn BW, Liu L, Vis AN, Bostrom PJ, Zuiverloon TC, Fleshner NE, et al. Prognostic value of molecular markers, sub-stage and European Organisation for the Research and Treatment of Cancer risk scores in primary T1 bladder cancer. BJU Int. 2012;110:1169-76 Van Rhijn BW, van der Kwast TH, Alkhateeb SS, Fleshner NE, van Leenders GJ, Bostrom PJ, et al. A new and highly prognostic system to discern T1 bladder cancer substage. Eur Urol. 2012;61:378-84 Van Rhijn BW, van der Kwast TH, Liu L, Fleshner NE, Bostrom PJ, Vis AN, et al. The FGFR3 mutation is related to favorable pT1 bladder cancer. J Urol. 2012;187:310-4 Van Rhijn BW. Combining molecular and pathologic data to prognosticate non-muscle-invasive bladder cancer. Urologic oncology. 2012;3:518-23 Van Rhijn BW. Editorial comment. J Urol. 2012;187:462 Van Rhijn BWG, van der Kwast TH, Jewett MAS, Zlotta AR. Reply to Maximilian Burger, Wolfgang Otto, and Arndt Hartmann’s letter to the editor re: Bas W.G. van Rhijn, Theo H. van der Kwast, Sultan S. Alkhateeb, et al. A new and highly prognostic system to discern T1 bladder cancer substage. Eur Urol 2012;61:378-84 Van Rhijn BWG. Combining molecular and pathologic data to prognosticate non-muscle-invasive bladder cancer. Urologic Oncology: Seminars and Original Investigations. 2012;30:518-23
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Van Rhijn BWG. Reply to Anna Orsola, Ines de Torres and Juan Morote’s Letter to the Editor re: Bas W.G. van Rhijn, Theo H. van der Kwast, Sultan S. Alkhateeb, et al. A New and Highly Prognostic System to Discern T1 Bladder Cancer Substage. Eur Urol 2012:61:378-84. European Urology. 2012;61:E55-E6 Van Vugt HA, Kranse R, Steyerberg EW, van der Poel HG, Busstra M, Kil P, et al. Prospective validation of a risk calculator which calculates the probability of a positive prostate biopsy in a contemporary clinical cohort. European journal of cancer. 2012;48:1809-15 Van Vugt HA, Roobol MJ, van der Poel HG, van Muilekom EH, Busstra M, Kil P, et al. Selecting men diagnosed with prostate cancer for active surveillance using a risk calculator: a prospective impact study. BJU Int. 2012;110:180-7 Van Walsum GA, de Ridder JA, Verhoef C, Bosscha K, van Gulik TM, Hesselink EJ, Ruers TJ, van den Tol MP, Nagtegaal ID, Brouwers M, van Hillegersberg R, Porte RJ, Rijken AM, Strobbe LJ, de Wilt JH; Dutch Liver Surgeons Group. Resection of liver metastases in patients with breast cancer: survival and prognostic factors. Eur J Surg Oncol. 2012;38:910-7 Veenstra HJ, Brouwer OR, Kroon BBR, Van der Ploeg IMC, Kroon BBR, Nieweg OE. Five-year follow up of sixteen melanoma patients with a Starz I-involved sentinel node in whom completion lymph node dissection was omitted. Melanoma Res 2012;22:436439 Veenstra HJ, Brouwer OR, van der Ploeg IM, Kroon BB, Nieweg OE. Five-year follow-up of 16 melanoma patients with a Starz I-involved sentinel node in whom completion lymph node dissection was omitted. Melanoma Res. 2012;22:436-9 Veenstra HJ, Klop WMC, Speijers MJ, Lohuis PJFM, Nieweg OE, Hoekstra HJ, Balm AJM. Lymphatic drainage patterns from melanomas on the shoulder or upper trunk to cervical lymph nodes and implications for the extent of neck dissection. Ann Surg Oncol 2012;19:3906-3912 Veenstra HJ, Vermeeren L, Valdés Olmos RA, Nieweg OE. The additional value of lymphatic mapping with routine SPECT/CT in unselected patients with clinically localized melanoma. Ann Surg Oncol 2012;19:1018-1023
Based on these data, we started a phase II (proof of concept) clinical trial of monobenzone-imiquimod (MI) therapy in stage III-IV melanoma patients at the Antoni van Leeuwenhoek. The trial is open for patient accrual since March 2011. Twenty-one melanoma patients with cutaneous metastases will be treated locally with monobenzone and imiquimod during 12 weeks. Study objectives are: 1) to study the clinical efficacy of local MI treatment on cutaneous metastases, 2) to study the induction of local tumor-specific immunity by MI treatment, as measured by the accumulation of melanoma/melanocyte specific T-cells at the treatment site. In addition, potential systemic immunity will be measured in the peripheral blood. The MI regimen is a low-cost, simple therapy, which is applicable in broad range of patients regardless of HLA-haplotype. It does not require elaborate patient-specific in vitro cultures nor non-myeloablative lymphodepletion, reducing patient treatment burden. The MI compounds have each already been used in humans, making it readily available and easily applicable in the clinic. This trial will provide information on the efficacy of monobenzone and imiquimod to raise melanocyte/melanomaspecific (auto)immunity, which may be effective for the treatment of melanoma.
Screening for physical and psychosocial problems at the Skin and Melanoma Centre The aim of the so called ‘Skinlast Study’ is to investigate the physical and psychosocial impact of a (pre) malignant skin disorder in individuals who visit the ‘Skin and Melanoma center’ of Antoni van Leeuwenhoek with a new (non) pigmented dermatological skin problem. We will investigate which questionnaire in onco-dermatological daily practice is most suitable to identify the physical and psychosocial impact. We also aim to investigate to what extent additional psychosocial support is desired. At the outpatient clinic of the ‘Skin and Melanoma center’ (Huid- en melanoom center) of the Antoni van Leeuwenhoek, approximately 35 patients with a new pigmented skin disorder are diagnosed weekly. During a three months period, all new patients (n=300) will be invited to complete the Lastmeter and the Skindex-29. Respondents are also invited to evaluate both questionnaires for their appropriateness, and their ability to assess relevant psychosocial and skin-related problems. Data collection has started in November 2012, and will continue in 2013.
Verhoeven RH, Janssen-Heijnen ML, Saum KU, Zanetti R, Caldarella A, Holleczek B, et al. Population-based survival of penile cancer patients in Europe and the United States of America: No improvement since 1990. European journal of cancer. 2012 Vestjens JH, Pepels MJ, de Boer M, Borm GF, van Deurzen CH, van Diest PJ, van Dijck JA, Adang EM, Nortier JW, Rutgers EJTh, Seynaeve C, MenkePluymers MB, Bult P, Tjan-Heijnen VC. Relevant impact of central pathology review on nodal classification in individual breast cancer patients. Ann Oncol. 2012;23:2561-6 Vidal-Sicart S, Brouwer OR, Mathéron HM, Tan IB, Valdés-Olmos RA. Sentinel node identification with a portable gamma camera in a case without visualization on conventional lymphoscintigraphy and SPECT/CT. Rev Esp Med Nucl Imagen Mol (in press) Von Meyenfeldt EM, Gooiker GA, van Gijn W, Post PN, van de Velde CJ, Tollenaar RA, Klomp HM, Wouters MW. The relationship between volume or surgeon specialty and outcome in the surgical treatment of lung cancer: a systematic review and meta-analysis. J Thorac Oncol. 2012;7:1170-8 Von Meyenfeldt EM, Wouters MW, Fat NL, Prevoo W, Burgers SA, van Sandick JW, Klomp HM. Local treatment of pulmonary metastases: from open resection to minimally invasive approach? Less morbidity, comparable local control. Surg Endosc. 2012;26:2312-21
Wildeman MA, Fles R, Adham M, Mayangsari ID, Luirink I, Sandberg M, Vincent AD, Fardizza F, Musa Z, Armiyanto, Middeldorp JM, Gerritsen G, Suwanto R, Tan IB. Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia. PLoS One. 2012;7 Wilting SM, Snijders PJ, Verlaat W, Jaspers A, van de Wiel MA, van Wieringen WN, Meijer GA, Kenter GG, Yi Y, le Sage C, Agami R, Meijer CJ, Steenbergen RD. Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis. Oncogene. 2012 Wouters MW, Gooiker GA, van Sandick JW, Tollenaar RA. The volume-outcome relation in the surgical treatment of esophageal cancer: a systematic review and meta-analysis. Cancer. 2012;118:1754-63 Wouters MWJM, Nieweg OE. Melanoom en andere huidtumoren In: Gooszen HG, Aronson DC, Blankensteijn JD, Gouma DJ, Kroon BBR, Lange JF, van Vugt AB, redacteuren. Leerboek chirurgie. Bohn Stafleu van Loghum, Houten, 2012. Zijlmans HJ, Punt S, Fleuren GJ, Trimbos JB, Kenter GG, Gorter A. Role of IL-12p40 in cervical carcinoma. Br J Cancer. 2012;107:1956-62
Wegdam W, Moerland PD, Meijer D, de Jong SM, Hoefsloot HC, Kenter GG, Buist MR, Aerts JM. A critical assessment of SELDI-TOF-MS for biomarker discovery in serum and tissue of patients with an ovarian mass. Proteome Sci. 2012;10:45 Wessels R, de Bruin DM, Faber DJ, van Boven HH, Vincent AD, van Leeuwen TG, van Beurden M, Ruersa TJ. Optical coherence tomography in vulvar intraepithelial neoplasia. J Biomed Opt. 2012;17:116022 Wildeman M A, Novalic Z, Verkuijlen SA, Juwana H, Huitema AD, Tan IB, Middeldorp JM, de Boer JP, Greijer AE. Cytolytic virus activation therapy for epstein-barr virus-driven tumors. Clin Cancer Res. 2012;18:5061-70
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Division head Marcel Verheij
Marcel Verheij MD PhD Head Berthe Aleman MD PhD Academic staff Harry Bartelink MD PhD Academic staff José Belderbos MD PhD Academic staff Monique Bloemers MD Academic staff Eugène Damen PhD Academic staff Roel de Boer PhD Academic staff Gerben Borst MD PhD Academic staff Luc Dewit MD PhD Academic staff Paula Elkhuizen MD PhD Academic staff Marloes Frantzen PhD Academic staff Rick Haas MD PhD Academic staff Olga Hamming-Vrieze MD Academic staff Wilma Heemsbergen PhD Academic staff Edwin Jansen MD PhD Academic staff Joost Knegjens MD Academic staff Joos Lebesque MD PhD Academic staff Ben Mijnheer PhD Academic staff Luc Moonen MD PhD Academic staff Arash Navran MD Academic staff Heike Peulen MD Academic staff Floris Pos 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 Jan-Jakob Sonke PhD Academic staff Ylanga Van der Geld MD Academic staff Judi Van Diessen MD Academic staff Marcel Van Herk PhD Academic staff Baukelien Van Triest MD PhD Academic staff Corine Van Vliet-Vroegindeweij PhD Academic staff Jeroen Van de Kamer PhD Academic staff Uulke Van der Heide PhD Academic staff Thelma Witteveen MD Academic staff Frits Wittkämper PhD Academic staff Rosemarie De Haan MD Temporary staff Jos Elbers MD Temporary staff Jan Groenewegen MD Temporary staff Jolien Heukelom MD Temporary staff Karel Hinnen MD PhD Temporary staff Birgit Hollmann MD Temporary staff Monique De Jong MD Temporary staff Kim De Vries MD Temporary staff Philip Meijnen MD PhD Temporary staff Stella Mook MD PhD Temporary staff Marlies Nowee MD PhD Temporary staff Eva Schaake MD Temporary staff 158
Anouk Trip MD Temporary staff Brenda Tomasoa MD Temporary staff Femke Van der Leij MD Temporary staff Wouter Vogel MD PhD Temporary staff Ben Vanneste MD Temporary staff Francine Voncken MD Temporary staff Tanja Alderliesten PhD Post-doc Glenda Bochove PhD Post-doc Chun Chen PhD Post-doc Laurens Van Buuren PhD Post-doc Dosti Dihalu PhD Post-doc Daniel Polders PhD Post-doc Alessia Gasparini PhD Post-doc Greetje Groenendaal PhD Post-doc Andrea Holt PhD Post-doc Anette Houweling PhD Post-doc Tomas Janssen PhD Post-doc Anton Mans PhD Post-doc Anke Van Mourik PhD Post-doc Jasper Nijkamp PhD Post-doc Edoardo Pasca PhD Post-doc Roel Rozendaal PhD Post-doc Alize Scheenstra PhD Post-doc Hanno Spreeuw PhD Post-doc Javier Salguero PhD Post-doc Barbara Stam PhD Post-doc Rajko Topolnjak PhD Post-doc Marnix Witte PhD Post-doc Anne Lisa Wolf PhD Post-doc Johan De Boer MSc PhD student Roman Bohoslavsky PhD student Wei Chen PhD student Hermine Dees-Ribbers PhD student Folkert Koetsveld MSc PhD student Simon Van Kranen MSc PhD student Anneke van der Reijden MSc PhD student Matthijs Kruis PhD student Angelo Mencarelli PhD student Kiri Nichol PhD Physicist Uros Stankovic PhD student Hua Zhang PhD student Barry Doodeman Physician assistant Marcel Jonker Physician assistant Robin Kalisvaart Physician assistant Margriet Kwint Physician assistant Cherita Sombroek Physician assistant Corine Veenstra Physician assistant Sandra Vreeswijk Physician assistant Annemarie IJpelaar Physician assistant Erdenay Alpay MSc Technical staff Anja Betgen MSc Technical staff Natascha Bruin Technical staff Josien De Bois Technical staff Rianne De Jong MSc Technical staff Joop Duppen Technical staff Joeri Honnef Technical staff Nico Jessurun Technical staff Tim Kaasenbrood Technical staff Agnieszka Olszewska MSc Technical staff Carmen Panneman MSc Technical staff Carolien Peters Technical staff Kenneth Pengel MSc Technical staff Lennert Ploeger PhD Technical staff Maddalena Rossi MSc Technical staff René Tielenburg Technical staff Lambert Zijp MSc Technical staff
Division of Radiotherapy
In 2012, the department has focused its research activities on two central themes: image-guided adaptive radiotherapy (IGART) and targeted radiosensitization. The combination of geometric, biological and functional imaging information acquired before, during and after treatment, allows adaptation of the treatment plan if necessary, thereby contributing to the ultimate ambition of delivering individualized radiotherapy. As one of the few departments in the world, we apply true IGART for multiple indications, both as standard-of-care and within research protocols. As member of the international MRI-Linac Consortium, we conduct tumor site-specific studies on MRI-guided treatment delivery and prepare the introduction of this new imaging/ treatment machine in our clinic. In addition to chemoradiotherapy, the combination of targeted agents and radiation is an attractive strategy to improve treatment outcome and limit toxicity. Our lab research is focused on the identification and preclinical testing of these targeted radiosensitizers. Some of these agents are evaluated in our clinic, including PARP inhibitors (lung, head and neck, breast), EGFR-blockers (head and neck, lung, bladder), angiogenesis inhibitors (soft tissue sarcoma), apoptosis modulators (head and neck). The Radiotherapy department continues to promote the introduction of proton therapy in The Netherlands. In 2012, the Antoni van Leeuwenhoek hospital, VU Medical Center and Academic Medical Center have joined their strength within the Amsterdam Proton Therapy Center (APTC) initiative. This consortium aims at realizing a joint proton facility in Amsterdam (location Antoni van Leeuwenhoek) in collaboration with the National Center for Pediatric Oncology (NKOC) and the University Medical Center Utrecht. In 2012 two co-workers of the division successfully defended their theses: Jasper Nijkamp ((Un-)certainties in radiotherapy of rectal cancer) and in collaboration with Leiden University: Johan Dikken (Gastric cancer: staging, treatment, and surgical quality assurance).
IMAGE ACQUISITION AND PROCESSING Tanja Alderliesten, Suzanne Van Beek, Anja Betgen, Johan De Boer, Roman Bohoslavsky, Hermine Dees-Ribbers, Joop Duppen, Alessia Gasparini, Rutger Heddes,Maarten Hulshof, Rianne De Jong, Matthijs Kruis, Simon Van Kranen, Joos Lebesque, Patricia Lindsay1, Corrie Marijnen2, Angelo Mencarelli, Jasper Nijkamp, Hans van Piggelen, Lennert Ploeger, Floris Pos, Coen Rasch3, Peter Remeijer, Simon Rit4, Peter De Ruiter, Rajko Topolnjak, Jeroen van de Kamer, Corine Van Vliet-Vroegindeweij, Marnix Witte, Lambert Zijp, Jan-Jakob Sonke, Marcel Van Herk
The Radiotherapy Department of the Antoni van Leeuwenhoek hospital has a long history of developments in imaging solutions for image guidance and multi-modal imaging for target volume definition and follow-up. In 2012, the group has been strengthened with a new group leader (50% research) with a very strong background in MRI.
Developments in image guidance In collaboration with Elekta Oncology Systems, a new version of the Synergy image guidance solution is being prepared (XVI 5). This is the third release since 2005. The first major novelty of this release is an improvement is reconstruction towards better Hounsfield unit values, which also paves the way towards correction of other artifacts such as ghosting. The other is the incorporation of cone beam CT imaging during delivery of rotational radiotherapy. In this way, critical treatments such as hypofractionated radiotherapy of lung tumors (SABR) can be verified during delivery. SABR is a successful treatment for small lung tumors where a very high ablative dose is delivered in a few fractions. Image guidance has greatly improved the precision of this treatment, but ensuring patient and tumor stability during actual treatment delivery remains an issue. With intrafraction imaging during rotational therapy (volume modulated arc therapy, VMAT) delivery, patient and tumor stability during SABR can be observed by acquiring conebeam CT (CBCT) simultaneously while VMAT is being delivered. However, CBCT is only available after each delivery arc is completed (we use 2 per fraction). In another investigation, the frequency of stability monitoring is being increased by on-line reconstruction and analysis of multiple overlapping digital tomosynthesis (DTS) reconstructions, or short angle CBCT, during each treatment arc. We found: 1) that including DTS reconstruction in the in-line reconstruction workflow is feasible without increasing computation time as low-resolution DTS reconstruction adds only 12 ms computation per projection image; 2) Registration of 30 degree DTS reconstructions made during a verification scan with corresponding reconstructions from a monitoring scan takes 2 s. It is sensitive enough to detect shifts exceeding 4 mm, providing registration failures are filtered by human inspection; 3) This methodology extents the number of time points at which patient stability can be monitored from 2 (we deliver in 2 arcs)
1 PMH, Radiation Medicine Program, Toronto, Canada 2 LUMC, Department of Radiotherapy, Leiden 3 AMC, Department of Radiotherapy, Amsterdam
Publications
Alderliesten T, Sonke JJ, Betgen A, Honnef J, van Vliet-Vroegindeweij C, Remeijer P. Accuracy evaluation of a 3-Dimensional surface imaging system for guidance in deep-inspiration breathold radiation therapy. Int J Radiat Oncol Biol Phys 2012 (in press) Alderliesten T, Sonke JJ, Betgen A, van Vliet-Vroegindeweij C, Remeijer P. 3D surface imaging for monitoring intrafraction motion in frameless stereotactic body radiotherapy of lung cancer. Radiother Oncol 2012;102:155-60 Bartelink H, Bourgier C, Elkhuizen P. Has partial breast irradiation by IORT or brachytherapy been prematurely introduced into the clinic? Radiother Oncol 2012;104:139-42 Bissonnette JP, Balter PA, Dong L, Langen KM, Lovelock DM, Miften M, Moseley DJ, Pouliot J, Sonke JJ, Yoo S. Quality assurance for image-guided radiation therapy utilizing CT-based technologies: a report of the AAPM TG179. Med Phys 2012;39:1946-63 Bloemers MC, Portelance L, Ruo R, Parker W, Souhami L. A dosimetric evaluation of dose escalation for the radical treatment of locally advanced vulvar cancer by intensity-modulated radiation therapy. Med Dosim 2012;37:310-3 Boersma LJ, Janssen T, Elkhuizen PH, Poortmans P, van der Sangen M, Scholten AN, Hanbeukers B, Duppen JC, Hurkmans C, van Vliet C. Reducing interobserver variation of boostCTV delineation in breast conserving radiation therapy using a pre-operative CT and delineation guidelines. Radiother Oncol 2012;103:178-82 Borst GR, McLaughlin M, Kyula JN, Neijenhuis S, Khan A, Good J, Zaidi S, Powell NG, Meier P, Collins I, Garrett MD, Verheij M, Harrington. Targeted radiosensitization by the Chk1 inhibitor SAR-020106. Int J Radiat Oncol Biol Phys 2012 (in press) Brouwer CL, Steenbakkers RJ, van den Heuvel E, Duppen JC, Navran A, Bijl HP, Chouvalova O, Burlage FR, Meertens H, Langendijk JA, van ‘t Veld AA. 3D variation in delineation of head and neck organs at risk. Radiat Oncol 2012;7:32
Cernohorsky P, de Bruin DM, van Herk M, Bras J, Faber DJ, Strackee SD, van Leeuwen TG. In-situ imaging of articular cartilage of the first carpometacarpal joint using co-registered optical coherence tomography and computed tomography. J Biomed Opt 2012;17:060501-3 Chai X, van Herk M, Hulshof MC, Bel A. A voxel-based finite element model for the prediction of bladder deformation. Med Phys 2012;39:55-65 Chai X, van Herk M, Betgen A, Hulshof M, Bel A. Automatic bladder segmentation on CBCT for multiple plan ART of bladder cancer using a patientspecific bladder model. Phys Med Biol 2012;57:3945-62 Chai X, van Herk M, Betgen A, Hulshof M, Bel A. Semiautomatic bladder segmentation on CBCT using a population-based model for multipleplan ART of bladder cancer. Phys Med Biol 2012;57:525-41 Chen W, Stroom J, Sonke JJ, Bartelink H, Schmitz AC, Gilhuijs KG. Impact of negative margin width on local recurrence in breast conserving therapy. Radiother Oncol 2012;104:148-54 Courrech Staal EF, Bloemendal K, Bloemer MC, Aleman BM, Cats A, van Sandick JW. Oesophageal cancer treatment in a tertiary referral hospital evaluated by indicators for quality of care. Eur J Surg Oncol 2012;38:150-6 De Boer J, de Bois J, van Herk M, Sonke JJ. Influence of the number of elongated fiducial markers on the localization accuracy of the prostate. Phys Med Biol 2012;57:6211-26 De Ruysscher D, Belderbos J, Reymen B, van Elmpt W, van Baardwijk A, Wanders R, Hoebers F, Vooijs M, Ollers M, Lambin P. State of the art radiation therapy for lung cancer 2012: a glimpse of the future. Clin Lung Cancer 2012 (in press) Defraene G, van den Bergh L, Al-Mamgani A, Haustermans K, Heemsbergen W, van den Heuvel F, Lebesque JV. The benefits of including clinical factors in rectal normal tissue complication probability modelling after radiotherapy for prostate cancer. Int J Radiat Oncol Biol Phys 2012;82:1233-42 Devriese LA, Bosma AJ, van den Heuvel MM, Heemsbergen W, Voest EE, Schellens JH. Circulating tumor cell detection in advanced non-small cell lung cancer patients by Multimarker QPCR analysis. Lung Cancer 2012;75:242-7
4 University of Lyon, France
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Dikken JL, van Grieken NC, Krijnen P, Gönen M, Tang LH, Cats A, Verheij M, Brennan MF, van de Velde CJ, Coit DG. Preoperative chemotherapy does not influence the number of evaluable lymph nodes in resected gastric cancer. Eur J Surg Oncol 2012;38:319-25 Dikken JL, van de Velde CJ, Coit DG, Shah MA, Verheij M, Cats A. Treatment of resectable gastric cancer. Therap Adv Gastroenterol 2012;5:49-69 Dikken JL, Lemmens VE, Wouters MW, Wijnhoven BP, Siersema PD, Nieuwenhuijzen GA, van Sandick JW, Cats A, Verheij M, Coebergh JW, van de Velde CJ. Increased incidence and survival for oesophageal cancer but not for gastric cardia cancer in the Netherlands. Eur J Cancer 2012;48:1624-32 Dikken JL, Dassen AE, Lemmens VE, Putter H, Krijnen P, van der Geest L, Bosscha K, Verheij M, van de Velde CJ, Wouters MW. Effect of hospital volume on postoperative mortality and survival after oesophageal and gastric cancer surgery in the Netherlands between 1989 and 2009. Eur J Cancer 2012;48:1004-13 Dikken JL, Cats A, Verheij M, van de Velde CJ. Randomized trials and quality assurance in gastric cancer surgery. J Surg Oncol 2012 (in press) Dikken JL, van de Velde CJ, Gönen M, Verheij M, Brennan MF, Coit DG. The New American Joint Committee on Cancer/International Union against cancer staging system for adenocarcinoma of the stomach: increased complexity without clear improvement in predictive accuracy. Ann Surg Oncol 2012;19:2443-51 Dikken JL, Wouters MW, Lemmens VE, Putter H, van der Geest LG, Verheij M, Cats A, van Sandick JW, van de Velde CJ. Influence of hospital type on outcomes after oesophageal and gastric cancer surgery. Br J Surg 2012;99:954-63 Dikken JL, Baser RE, Gonen M, Kattan MW, Shah MA, Verheij M, van de Velde CJ, Brennan MF, Coit DG. Conditional probability of survival nomogram for 1-, 2-, and 3-year survivors after an R0 resection for gastric cancer. Ann Surg Oncol 2012 (in press) Dikken JL, Stiekema J, van de Velde CJ, Verheij M, Cats A, Wouters MW, van Sandick JW. Quality of care indicators for the surgical treatment of gastric cancer: a systemic review. Ann Surg Oncol 2012 (in press)
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Dikken JL, van Sandick JW, Allum WH, Johansson J, Jensen LS, Putter H, Coupland VH, Wouters MW, Lemmens VE, van de Velde CJ, van der Geest LG, Larsson HJ, Cats A, Verheij M. Differences in outcomes of oesophageal and gastric cancer surgery across Europe. Br J Surg (in press) Grills IS, Hope AJ, Guckenberger M, Kestin LL, Werner-Wasik M, Yan D, Sonke JJ, Bissonnette JP, Wilbert J, Xiao Y, Belderbos J. A collaborative analysis of stereotactic lung radiotherapy outcomes for early-stage non-small-cell lung cancer using daily online cone-beam computed tomography image-guided radiotherapy. J Thorac Oncol 2012;7:1382-93 Groenendaal G, Borren A, Moman MR, Monninkhof E, van Diest PJ, Philippens ME, van Vulpen M, van der Heide UA. Pathologic validation of a model based on diffusion-weighted imaging and dynamic contrast-enhanced magnetic resonance imaging for tumor delineation in the prostate peripheral zone. Int J Radiat Oncol Biol Phys 2012;82:e537-44 Groenendaal G, van Vulpen M, Pereboom SR, Poelma-Tap D, Korporaal JG, Monninkhof E, van der Heide UA. The effect of hormonal treatment on conspicuity of prostate cancer: implications for focal boosting radiotherapy. Radiother Oncol 2012;103:233-8 Guckenberger M, Kestin LL, Hope AJ, Belderbos J, Werner-Wasik M, Yan D, Sonke JJ, Bissonnette JP, Wilbert J, Xiao Y, Grills IS. Is there a lower limit of pretreatment pulmonary function for safe and effective stereotactic body radiotherapy for early-stage nonsmall cell lung cancer? J Thorac Oncol 2012;7:542-51 Guckenberger M, Klement RJ, Kestin LL, Hope AJ, Belderbos J, WernerWasik M, Yan D, Sonke JJ, Bissonnette JP, Xiao Y, Grills IS. Lack of a doseeffect relationship for pulmonary function changes after stereotactic body radiation therapy for early-stage non-small cell lung cancer. Int J Radiat Oncol Biol Phys 2012 (in press) Haas RL, Delaney TF, O’Sullivan B, Keus RB, Le Pechoux C, Olmi P, Poulsen JP, Seddon B, Wang D. Radiotherapy for management of extremity soft tissue sarcomas: Why, when, and where? Int J Radiat Oncol Biol Phys 2012;84:572-80
to 36, using overlapping 30 degree arcs in steps of 10 degrees. This method is a feasible extension of simultaneous CBCT acquisition during VMAT and reduces the time delay between unacceptable patient motion and its detection. It will therefore further improve treatment accuracy, although its largest impact will be for the few ‘instable’ patients. Improvements in image quality are being pursued and based on novel reconstruction techniques. Compressed sensing based cone-beam reconstruction is introduced to reduce streaking artifacts due to undersampling in four-dimensional (4D) cone-beam computed tomography (CBCT). This reconstruction, however, is typically time-consuming, especially for 4D CBCT. We propose a novel approach, which introduces a first-order method, called continuation accelerated Nesterov's descent, to improve convergence rate. We extract the respiratory signal directly from projections, sort them into ten subsets, and reconstruct each subset into a 3D CBCT by compressed sensing based total-variation minimization. The reconstruction iteration is solved by Nesterov's descent algorithm. In each loop, we adapt the step size by sufficient reducing global Lipschitz constant, and update variants by gradient-mapping parameters to proximal points. The proposed method is assessed by a thorax phantom and a patient, which are scanned with a CBCT scanner. The reconstructed data is compared among FDK algorithm, projection onto convex sets (POCS) which is a steepest decent based optimization, and our method. A region of homogeneous voxel values is manually selected, and the root-mean-square-error between CBCT and CT in this region is defined as streaking artifacts. It has reduced 27.2% in phantom data, when our method is compared to traditional FDK methods for 2-minute scan. Correlation ratio between CBCT and CT is increased for 10.2% when our method is compared to FDK for patient data. For the convergence rate, our method only needs 25 iterations to get the same total-variation residual of POCS at 250 iterations. In addition, for other cut-offs, it seems an order of magnitude faster. Our proposed method decreases the streaking artifacts and convergence time in 4D CBCT reconstruction. The image quality assessment suggests that our method has improved image quality and better correlation.
Motion compensation of 4D CT and 4D PET has also been implemented showing improved CT image quality for target volume delineation, in particular for tumors in the mediastinum. Since early 2012, the so-called mid-position CT scan is routinely used for RT planning. Such a scan has a much better image quality than a free-breathing CT (much less image deformations) or a single frame of a 4D CT scan (our previous mid-ventilation approach). In addition, it helps to mitigate the effect of respiration on the geometry of the planning CT, i.e., the mid-position CT minimizes configuration differences between tumor and normal tissues relative to the treatment situation. The application of motion compensation to PET is under active development. It allows better definition of the tumor’s boundary (motion blurring is reduced), and a better quantification of the PET activity for patient selection and follow-up measurements.
Small animal research Novel developments in radiotherapy depend ultimately on clinical trials to demonstrate their efficacy. Due to the improvements in image guidance, however, a new therapeutic window is opening that can only slowly be explored. For instance, the higher precision allows a better sparing of normal tissues, but to translate that into a higher tumor dose to improve control is scary because the effect of the remaining hot spots on the normal tissue are unknown. By translating the human image guidance solution back to small animal research, it now becomes possible to explore high-dose high-precision treatments in preclinical work in combination with all sorts of smart drugs. In addition, the volume effect of irradiation normal tissue can be further explored. To support this work, a commercial small animal irradiation unit produced by Precision X-Ray Inc in the USA has been interfaced with the XVI image guidance software. This year, the system has been characterized, demonstrating a very high accuracy of 0.1 mm and sharp treatments fields that can be collimated down to 1 cm or less. As a result, it is now possible to selectively irradiate very small parts of the animals rather than a ‘half mouse’ as was the state of the art until a few years ago. A start has been made with the first studies, looking into the effect of partial heart and lung irradiation, and quantifying response timelines after re-irradiation of orthotopically implanted breast tumors.
Imaging software and database development Motion compensation Fast motion, e.g., due to respiratory causes a blurring of moving structures in a CT, PET and CBCT. As a result, small lung tumors may become invisible and some larger lung tumors are displayed with so much to motion blur that they cannot be accurately localized. Previously we developed a 4D reconstruction technique for CBCT based on sorting according to the respiration trace to overcome these problems, and this solution has been in years of use and integrated into the commercial image guidance product. A disadvantage of this technique is that, in order to collect enough projection images, acquisition needs to be performed for around 60 respiration cycles, or 4 minutes scan time. This year, a novel motion compensated reconstruction technique was implemented in our clinic. In this solution, a motion model is derived from the 4D planning CT, which is subsequently used to counteract the motion observer during acquisition of CBCT. The motion compensation techniques allow shortening the scan time to 1 minute without loss of tumor localization accuracy.
A fully functional connection has been made between two types of Electronic Case Report Form (ECRF) system and a hospital Picture Archiving and Communication System (PACS). The setup that allows copying of anonymized PACS image data to a central SFTP server while storing the anonymized patient ID's in the ECRF is as follows: 1) TENALEA or OPENCLINICA (central) has added JavaScript code to initiate a HTML connection to a local web server inside the hospitals firewall; 2) Conquest WEB SERVER (local on a dedicated machine) runs OPENCLINICA/ TENALEA specific Lua scripts controlling the Conquest DICOM server and returns the correct anonymized UIDs to OPENCLINICA; 3) Conquest DICOM server (local on a dedicated machine) makes contact with the PACS and collects, anonymizes, and sends the correct identifiers to be stored in the ECRF; 4) The local PACS system responds to requests from the Conquest DICOM server and sends data to the Conquest DICOM server; This data is then forwarded over SFTP to a central image server for the clinical study. 5) The data received on the SFTP server
Hamming-Vrieze O, van Kranen SR, van Beek S, Heemsbergen W, van Herk M, van den Brekel MW, Sonke JJ, Rasch CR. Evaluation of Tumor Shape Variability in Head-and-Neck Cancer Patients Over the Course of Radiation Therapy Using Implanted Gold Markers. Int J Radiat Oncol Biol Phys 2012;84:201-7 Haimovitz-Friedman A, Yang TI, Thin TH, Verheij M. Imaging radiotherapyinduced apoptosis. Radiat Res 2012;177:467-82 Haverkort MA, van de Kamer JB, Pieters BR, Koning CC, van Herk M, Tiehoven G. Adaptive margin radiotherapy for patients with prostate carcinoma: what’s the benefit? Radiother Oncol 2012;105:203-6 Hilbers FS, Boekel NB, van den Broek AJ, van Hien R, Cornelissen S, Aleman BM, van ‘t Veer LJ, van Leeuwen FE, Schmidt MK. et al. Genetic variants in TGFbeta-1 and PAI-1 as possible risk factors for cardiovascular disease after radiotherapy for breast cancer. Radiother Oncol 2012;102:115-21 Holt A, van Gestel D, Arends MP, Korevaar EW, Schuring D, KunzeBusch MC, Louwe RJW, van VlietVroegindeweij C. Multi-institutional comparison of volumetric modulated arc therapy vs intensity-modulated radiation therapy for head-and-neck cancer: a planning study. Radiat Oncol 2012 (in press) Hoving S, Heeneman S, Gijbels MJ, Te Poele JA, Visser N, Cleutjens J, Russell NS, Daemen MJ, Stewart FA. Irradiation induces different inflammatory and thrombotic responses in carotid arteries of wildtype C57BL/6J and atherosclerosis-prone ApoE(-/-) mice. Radiother Oncol 2012;105:365-70 Hurkmans CW, Knegjens JL, Oei BS, Maas AJ, Uiterwaal G, van der Borden AJ, Ploegmakers MM, van Erven L. Management of radiation oncology patients with a pacemaker or ICD: a new comprehensive practical guideline in the Netherlands. Radiat Oncol 2012;7:198 Hurkmans CW, Dijckmans I, Reijnen M, van der Leer J, van Vliet-Vroegindeweij C, van der Sangen M. Adaptive radiation therapy for breast IMRTsimultaneously integrated boost: threeyear clinical experience. Radiother Oncol 2012;103:183-7
Immink JM, Putter H, Bartelink H, Cardoso JS, Cardoso MJ, van der Hulst-Vijgen MH, Noordijk EM, Poortmans PM, Rodenhuis CC, Struikmans H. Long-term cosmetic changes after breast-conserving treatment of patients with stage I-II breast cancer and included in the EORTC ‘boost versus no boost’ trial. Ann Oncol 2012;23:2591-8 Jansen EP, Boot H, van de Velde CJ van Sandick J, Cats A, Verheij M. Can adjuvant chemoradiotherapy replace extended lymph node dissection in gastric cancer? Recent results. Cancer Res 2012;196:229-40 Jia X, Tian Z, Lou Y, Sonke JJ, Jiang SB. Four-dimensional cone beam CT reconstruction and enhancement using a temporal nonlocal means method. Med Phys 2012;39:5592-602 Karakullukcu B, Nyst H, van Veen RL, Hamming-Vrieze O, Witjes MJ, de Visscher SA, Burlage FR, Levendag PC, Sterenborg HJ, Tan IB. mTHPC mediated interstitial photodynamic therapy of recurrent nonmetastatic base of tongue cancers: Development of a new method. Head Neck 2012;34:1597-606 Koning CC, Blank LE, Koedooder C, van Os RM, van de Kar M, Jansen E, Battermann JJ, Beijert M, Gernaat C, van Herpen KA, Hoekstra C, Horenblas S, Jobsen JJ, Krol AD, Lybeert ML, van Onna IE, Pelger RC, Poortmans P, Pos FJ, van der Steen-Banasik E, Slot A, Visser A, Pieters BR. Brachytherapy after external beam radiotherapy and limited surgery preserves bladders for patients with solitary pT1-pT3 bladder tumors. Ann Oncol 2012;23:2948-53 Koolen BB, Valdés Olmos RA, Elkhuizen PH, Vogel WV, Vrancken Peeters MJ, Rodenhuis S, Rutgers EJ. Locoregional lymph node involvement on 18F-FDG PET/CT in breast cancer patients scheduled for neoadjuvant chemotherapy. Breast Cancer Res Treat 2012;135:231-40 Kwint M, Uyterlinde W, Nijkamp J, Chen C, de Bois J, Sonke JJ, van den Heuvel M, Knegjens J, van Herk M, Belderbos J. Acute esophagus toxicity in lung cancer patients after intensity modulated radiation therapy and concurrent chemotherapy. Int J Radiat Oncol Biol Phys 2012;84:e223-8 Litière S, Werutsky G, Fentiman IS, Rutgers E, Christiaens MR, Van Limbergen E, Baaijens MH, Bogaerts J, Bartelink H. Breast conserving therapy versus mastectomy for stage I-II breast cancer: 20 year follow-up of the EORTC 10801 phase 3 randomised trial. Lancet Oncol 2012;13:412-9
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Liu F, Yorke ED, Belderbos JS, Borst GR, Rosenzweig KE, Lebesque JV, Jackson A. Using Generalized Equivalent Uniform Dose Atlases to Combine and Analyze Prospective Dosimetric and Radiation Pneumonitis Data From 2 Non-Small Cell Lung Cancer Dose Escalation Protocols. Int J Radiat Oncol Biol Phys 2012 (in press) Lutz MP, Zalcberg JR, Ducreux M, Ajani JA, Allum W, Aust D, Bang YJ, Cascinu S, Hölscher A, Jankowski J, Jansen EP, Kisslich R, Lordick F, Mariette C, Moehler M, Oyama T, Roth A, Rueschoff J, Ruhstaller T, Seruca R, Stahl M, Sterzing F, van Cutsem E, van der Gaast A, van Lanschot J, Ychou M, Otto F; First St Gallen EORTC Gastrointestinal Cancer Conference 2012 Expert Panel. Highlights of the EORTC St. Gallen International Expert Consensus on the primary therapy of gastric, gastroesophageal and oesophageal cancer – Differential treatment strategies for subtypes of early gastroesophageal cancer. Eur J Cancer 2012;48:2941-53 Ma YP, van Leeuwen FE, Cooke R, Broeks A, Enciso-Mora V, Olver B, Lloyd A, Broderick P, Russell NS, Janus C, Ashworth A, Houlston RS, Swerdlow AJ. FGFR2 genotype and risk of radiation-associated breast cancer in Hodgkin lymphoma. Blood 2012;119:1029-31 Mencarelli A, van Beek S, van Kranen S, Rasch C, van Herk M, Sonke JJ. Validation of deformable registration in head and neck cancer using analysis of variance. Med Phys 2012;39:6879-84 Morton L, Gilbert ES, Hall P, Andersson M, Joensuu H, Vaalavirta L, Dores GM, Stovall M, Holowaty EJ, Lynch CF, Curtis RE, Smith SA, Kleinerman RA, Kaijser M, Storm HH, Pukkala E, Weathers RE, Linet MS, Rajaraman P, Fraumeni JF Jr, Brown LM, van Leeuwen FE, Fossa SD, Johannesen TB, Langmark F, Lamart S, Travis LB, Aleman BM. Risk of treatmentrelated esophageal cancer among breast cancer survivors. Ann Oncol 2012:23:3081-91 Nijkamp J, Rossi M, Lebesque J, Belderbos J, van den Heuvel M, Kwint M, Uyterlinde W, Vogel W, Sonke JJ. Relating acute esophagitis to radiotherapy dose using FDG-PET in concurrent chemo-radiotherapy for locally advanced non-small cell lung cancer. Radiother Oncol 2012 (in press)
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Nijkamp J, de Haas-Kock, Beukema JC, Neelis KJ, Woutersen D, Ceha H, Rozema T, Slot A, Vos-Westerman H, Intven M, Spruit PH, van der Linden Y, Geijsen D, Verschueren K, van Herk MB, Marijnen CA. Target volume delineation variation in radiotherapy for early stage rectal cancer in the Netherlands. Radiother Oncol 2012;102:14-21 Nijkamp J, Doodeman B, Marijnen C, Vincent A, van Vliet-Vroegindeweij C. Bowel exposure in rectal cancer IMRT using prone, supine, or a belly board. Radiother Oncol 2012;102:22-9 Nijkamp J, Marijnen C, van Herk M, van Triest B, Sonke JJ. Adaptive radiotherapy for long course neoadjuvant treatment of rectal cancer. Radiother Oncol 2012;103:353-9 Nijkamp J, Swellengrebel M, Hollmann B, de Jong R, Marijnen C, van VlietVroegindeweij C, van Triest B, van Herk M, Sonke JJ. Repeat CT assessed CTV variation and PTV margins for short- and long-course pre-operative RT of rectal cancer. Radiother Oncol 2012;102:399-405 Nijman JL, Sixma H, van Triest B, Keus R, Hendriks M. The quality of radiation care: The results of focus group interviews and concept mapping to explore the patient’s perspective. Radiother Oncol 2012;102:154-60 Ohri N, Werner-Wasik M, Grills IS, Belderbos J, Hope A, Yan D, Kestin LL, Guckenberger M, Sonke JJ, Bissonnette JP, Xiao Y. Modeling local control after hypofractionated stereotactic body radiation therapy for stage I non-small cell lung cancer: a report from the elekta collaborative lung research group. Int J Radiat Oncol Biol Phys 2012;84:379-84 Overgaard J, Bartelink H. Introduction: towards predicting outcome of radiotherapy-at last. Semin Radiat Oncol 2012;22:87-90 Peulen H, Hanbeukers B, Boersma L, van Baardwijk A van den Ende P, Houben R, Jager J, Murrer L, Borger J. Forward intensity-modulated radiotherapy planning in breast cancer to improve dose homogeneity: feasibility of class solutions. Int J Radiat Oncol Biol Phys 2012;82:394-400 Poortmans P, Aznar M, Bartelink H. Quality indicators for breast cancer: revisiting historical evidence in the context of technology changes. Semin Radiat Oncol 2012;22:29-39
is automatically loaded into a central DICOM server, which is accessed through WADO to allow ECRF users anywhere to view and inspect images submitted. This system is intended to run at each hospital collaborating in specific clinical trials and has to be setup to allow communication with the PACS. A critical point there is that the PACS administrator has access to the anonymization routine and can adjust it to his or her liking before approval of submitting data. To facilitate ‘minor’ software development, e.g. by PhD students or postdocs, a scripting system has been setup in an NKI image registration applications. The scripting system uses the popular scripting language ‘Lua’ developed from 1993 at the Pontifical Catholic University of Rio de Janeiro, in Brazil. A user-friendly and efficient development environment has been achieved by coupling the advanced image processing tools available in NKI’s in-house Worldmatch application, such as deformable image registration, with the easy of interactive coding, debugging and modification of data in Lua through an open source development environment “zerobrane studio”.
Probabilistic planning To deal with geometric uncertainties in radiotherapy, conventionally a PTV is defined. The rationale behind the PTV concept is that if the PTV receives a certain minimum dose, the CTV is expected to receive a certain minimum dose. Probabilistic plan evaluation takes a different approach by directly estimating the expected delivered dose, based on population statistics, describing the geometric uncertainties. Since a year, we use both conventional and probabilistic plan evaluation for all prostate patients. We compared the two approaches of plan evaluation with the actual delivered dose, for prostate patients. Twenty-five patients receiving 39x2 Gy on the prostate plus seminal vesicles +4 mm (group A) and 56 patients, receiving 39x1.85 Gy on the prostate plus seminal vesicles +7 mm, with an additional boost of 39x2 Gy on the prostate +4 mm (group B) were studied. For each patient, the planned dose on the PTV and the dose delivered with 90% confidence to the CTV using probabilistic evaluation have been determined. The probabilistic evaluation simulates both random and systematic geometric uncertainties and takes translations and rotations into account. Each patient has marker implants, and on average 17.3 (range: 13-25) CBCT’s were taken per patient. Using the CBCT setup data, we estimated the actual delivered dose, assuming dose invariance under shifts and rotations and a rigid target volume. For both group A and B we find no significant correlation between planned and delivered dose, while in both cases the probabilistically estimated dose is significantly correlated to the delivered dose. Our results show explicitly that probabilistic plan evaluation, based on population statistics give a significantly better prediction of the actual delivered minimum dose, compared to conventional plan evaluation. We conclude that it is feasible and clinically advantageous to introduce acceptance criteria, based on probabilistic plan evaluation, instead of conventional plan evaluation. Probabilistic dose estimation such as in the previous study assumes invariance of the dose under motion of organs and setup error. This assumption holds well for photon beams in homogeneous parts of the body such as the pelvis. However, in regions such as the lung or the head and neck, this assumption is less valid. To improve the dose estimation without a full recalculation of the dose for each simulated setup error or
organ motion, an interpolation strategy has been implemented where dose is calculated at a few steps of the simulated motion, followed by a non-linear interpolation at in-between steps. Tests with this approach show that dose errors can be reduced to within 1% for many clinical purposes. Deformation of anatomy shows complex patterns. A PCA based model was developed to predict the day-to-day variations in rectum shape. To build the model we used data from 19 prostate cancer patients, who received a planning computed tomography (CT) scan and 9-13 repeat CT scans. We took into account that rectum shapes are related to time and planning CT volume. The model is based on principal component analysis (PCA); this allows summarizing large dimensionality of geometrical data from multiple CT scans to a meaningful few-parametric statistical model of rectum shapes. We verified the model by comparing the accumulated doses of the originally delineated recta and simulated recta for a 5 coplanar beams treatment technique (IMRT) with a prescription dose of 78 Gy. The 9-13 repeat CT scans of each patient are regarded as the golden standard and provide a good estimate of the actual dose during the treatment. Our study shows that, with the EUD, mean dose and the standard deviation (SD) of the dose distribution as criteria, the PCA simulated rectums yield a better prediction of the actual dose than the dose prediction based on the planning CT alone.
EPID DOSIMETRY Anton Mans, Ali Jomehzadeh, Igor Olaciregui-Ruiz, Roel Rozendaal, Jan-Jakob Sonke, Hanno Spreeuw, Marcel van Herk, Ben Mijnheer
In vivo dosimetry in external beam radiotherapy can be used to detect major errors, to assess clinically relevant differences between planned and delivered dose, to record dose received by individual patients, and in some countries to fulfill legal requirements. In our institution, all treatments with curative intent are irradiated with either an IMRT or VMAT technique, and almost all are verified by means of EPID-based in vivo dosimetry during three fractions in the first week of their treatment. The reconstructed 2D or 3D in vivo dose distribution is compared with the planned dose distribution by means of a 3D gamma evaluation method. Although such an approach is capable of detecting deviations in the in vivo dose distribution, the clinical relevance of gamma parameters is far from obvious. It seems therefore worthwhile to correlate the 3D gamma evaluation results with other, clinically more common parameters used to determine the quality of a dose distribution, such as EUDs for specific regions of interest and parameters derived from DVH analyses. A pilot study was started to investigate the usefulness of these parameters for the EPID-based in vivo dose verification of head-and-neck VMAT treatments. The first results indicate that for several regions of interest the gamma evaluation pass-rate criteria do not correlate well with DVH parameters, but the mean gamma values do show a relationship with the D50 values. These results pave the way for moving from gamma evaluation data to clinically more common and insightful DVH-derived parameters.
Rit S, van Herk M, Zijp L, JJ Sonke. Quantifications of the variability of diaphragm motion and implications for treatment margin Construction. Int J Radiat Oncol Biol Phys 2012;82:e399407 Roelofs E, Engelsman M, Rasch C, Persoon L, Qamhiyeh S, de Ruysscher D, Verhaegen F, Pijls-Johannesma M, Lambin P; ROCOCO Consortium. Results of a multicentric in silico clinical trial (ROCOCO): comparing radiotherapy with photons and protons for nonsmall cell lung cancer. J Thorac Oncol 2012;7:165-76 Rooswinkel RW, van der Kooij B, Verheij M, Borst J. Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1 or Bcl-B. Cell Death Dis 2012;9:e366 Santanam L, Hurkmans C, Mutic S, van Vliet-Vroegindeweij C, Brame S, Straube W, Galvin J, Tripuraneni P, Michalski J, Bosch W. Standardizing naming conventions in radiation oncology. Int J Radiat Oncol Biol Phys 2012;83:1344-9 Schaake EE, Kappers I, Codrington HE, Valdés Olmos RA, Teertstra HJ, van Pel R, Burgers JA, van Tinteren H, Klomp HM. Tumor response and toxicity of neoadjuvant erlotinib in patients with early-stage non-small-cell lung cancer. J Clin Oncol 2012;30:2731-8 Schaake EE, Belderbos JS, Buikhuisen WA, Rossi MM, Burgers JA, Sonke JJ. Mediastinal lymph node position variability in non-small cell lung cancer patients treated with radical irradiation. Radiother Oncol 2012;105:150-4 Schaapveld M, van den Belt-Dusebout AW, Gietema JA, de Wit R, Horenblas S, Witjes JA, Hoekstra HJ, Kiemeney LA, Louwman WJ, Ouwens GM, Aleman BM, van Leeuwen FE. Risk and prognostic significance of metachronous contralateral testicular germ cell tumours. Br J Cancer 2012;107:1637-43 Scharpfenecker M, Floot B, Russell NS, Stewart FA. The TGF-beta co-receptor endoglin regulates macrophage infiltration and cytokine production in the irradiated mouse kidney. Radiother Oncol 2012;105:313-20 Schmitz A, Pengel K, Loo CE, van den Bosch MA, Wesseling J, Gertenbach M, Alderliesten T, Mali WP, Rutgers EJ, Bartelink H, Gilhuijs KG. Pre-treatment imaging and pathology characteristics of invasive breast cancers of limited extent: potential relevance for MRIguided localized therapy. Radiother Oncol 2012;104:11-8
Schwartz G, Reis-Fihlo J, Pusztai L, Fentiman IS, Holland R, Bartelink H, Rutgers EJ, Solin LJ, Palazzo J; Consensus Committee. Adjuvant therapy in stage I carcinoma of the breast: the influence of multigene analyses and molecular phenotyping. Cancer 2012;118:2031-8 Schwartz G, Bartelink H, Burstein H, Cady B Cataliotti L, Fentiman IS, Holland R, Hughes KS, Masood S, McCormick B, Palazzo JA, Pressman PI, Reis-Filho J, Pusztai L, Rutgers EJ, Seidman AD, Solin LJ, Sparano JA. Adjuvant therapy in stage I carcinoma of the breast: the influence of multigene analyses and molecular phenotyping. Breast J 2012;18:303-11 Servant N, Bollet MA, Halfwerk H, Bleakley K, Kreike B, Jacob L, Sie D, Kerkhoven RM, Hupé P, Hadhri R, Fourquet A, Bartelink H, Barillot E, Sigal-Zafrani B, van de Vijver MJ. Search for a gene expression signature of breast cancer local recurrence in Young women. Clin Cancer Res 2012;18:1704-15 Stewart F, Akleyev A, Hauer-Jensen M, Hendry JH, Kleiman NJ, Macvittie TJ, Aleman BM, Edgar AB, Mabuchi K, Muirhead CR, Shore RE, Wallace WH. ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs – threshold doses for tissue reactions in a radiation protection context. Ann ICRP 2012;41:1-322 (abstract) Stiekema J, Boot H, Aleman BM, Wessels LF, van Sandick JW. Prognostication and prediction using gene expression profiling in oesophageal cancer. Eur J Surg Oncol 2012 (in press) Tanis E, van de Velde CJ, Bartelink H, van de Vijver MJ, Putter H, van der Hage JA. Locoregional recurrence after breast-conserving therapy remains an independent prognostic factor even after an event free interval of 10years in early stage breast cancer. Eur J Cancer 2012;48:1751-6 Theunissen EA, Timmermans AJ, Zuur CL, Hamming-Vrieze O, Paul de Boer J, Hilgers FJ, van den Brekel MW. Total laryngectomy for a dysfunctional larynx after (chemo) radiotherapy, total laryngectomy for a dysfunctional larynx. Arch Otolaryngol Head Neck Surg 2012;138:548-55 Topolnjak R, Borst GR, Nijkamp J, Sonke JJ. Image-guided Radiotherapy for Left-sided Breast Cancer Patients: Geometrical Uncertainty of the Heart. Int J Radiat Oncol Biol Phys 2012;82:647-55
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Van Blitterswijk WJ, Verheij M. Anticancer mechanisms and clinical application of alkylphospholipids. Biochim Biophys Acta 2012 (in press) Van der Heide UA, Houweling AC, Groenendaal G, Beets-Tan RG, Lambin P. Functional MRI for radiotherapy dose painting. Magn Reson Imaging 2012;30:1216-23 Van der Kaaij M, Heutte N, Meijnders P, Abeilard-Lemoisson E, Spina M, Moser EC, Allgeier A, Meulemans B, Simons AH, Lugtenburg PJ, Aleman BM, Noordijk EM, Fermé C, Thomas J, Stamatoullas A, Fruchart C, Brice P, Gaillard I, Bologna S, Ong F, Eghbali H, Doorduijn JK, Morschhauser F, Sebban C, Roesink JM, Bouteloup M, Van Hoof A, Raemaekers JM, Henry-Amar M, Kluin-Nelemans HC. Premature ovarian failure and fertility in longterm organisation for research and treatment of Cancer Lymphoma Group and Groupe d’Étude des Lymphomes de lÁdulte Cohort Study. J Clin Oncol 2012;30:291-9 Van der Kaaij MA, Heutte N, Meijnders P, Abeilard-Lemoisson E, Spina M, Moser LC, Allgeier A, Meulemans B, Dubois B, Simons AH, Lugtenburg PJ, Aleman BM, Noordijk EM, Fermé C, Thomas J, Stamatoullas A, Fruchart C, Brice P, Gaillard I, Doorduijn JK, Sebban C, Smit WG, Bologna S, Roesink JM, Ong F, André MP, Raemaekers JM, Henry-Amar M, KluinNelemans HC. Parenthood in survivors of Hodgkin lymphoma; an EORTC-GELA general population case-control study. J Clin Oncol 2012;30:3854-63 Van Elmpt W, De Ruysscher D, van der Salm A, Lakeman A, van der Stoep J, Emans D, Damen E, Ollers M, Sonke JJ, Belderbos J.The PET-boost randomised phase II dose-escalation trial in nonsmall cell lung cancer. Radiother Oncol 2012;104:67-71 Van der Leij F, Elkhuizen PH, Bartelink H, van de Vijver MJ. Predictive factors for local recurrence in breast cancer. Semin Radiat Oncol 2012;22:100-7 Van Kesteren Z, Janssen TM, Damen E, van Vliet-Vroegindeweij C. The dosimetric impact of leaf interdigitation and leaf width on VMAT treatment planning in Pinnacle: comparing Pareto fronts. Phys Med Biol 2012;57:2943-52 Van Kesteren Z, Belderbos J, van Herk M, Olszewska A, Lamers E, De Ruysscher D, Damen E, van Vliet-Vroegindeweij C. A practical technique to avoid the hippocampus in prophylactic cranial irradiation for lung cancer. Radiother Oncol 2012;102:225-7
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Van Loon J, Siedschlag C, Stroom J, Blauwgeers H, van Suylen RJ, Knegjens J, Rossi M, van Baardwijk A, Boersma L, Klomp H, Vogel W, Burgers S, Gilhuijs K. Microscopic disease extension in three dimensions for nonsmall-cell-lung cancer: development of a prediction model using pathologyvalidated positron emission tomography and computed tomography features. Int J Radiat Oncol Biol Phys 2012;82:448-56 Vanneste BG, Borst GR, van den Heuvel M, Belderbos JS. Diagnose en behandeling van radiatiepneumonitis. Ned Tijdschr voor Oncol 2012,9:344-9 Wendling M, McDermot LN, Mans A, Olaciregui-Ruiz I, PecharromanGallego R, Sonke JJ, Stroom J, van Herk M, Mijnheer BJ. In acqua vivo EPID dosimetry. Med Phys 2012;39:367-77 Yang TJ, Haimovitz-Friedman A, Verheij M. Anticancer therapy and apoptosis imaging. Exp Oncol 2012;34:269-76
In order to assess the sensitivity of our EPID-based in vivo dosimetry method, patient-related errors were simulated by changing position and dimensions of an anthropomorphic (Alderson) phantom. The phantom was irradiated using 2-arc head-and-neck, prostate and lung VMAT techniques. The errors comprised vertical and horizontal phantom shifts, rotations, and the addition of tissue-equivalent material. Dose distributions reconstructed from EPID images and the original planned dose distributions were compared using 3D gamma evaluation. The preliminary results showed that EPID dose verification of headand-neck VMAT treatments is more sensitive for positional errors than verification of prostate and lung VMAT treatments, and that EPID verification of VMAT delivery is more sensitive to thickness variations than to positional errors. The results will be used to define clinically relevant action levels. EPID-based in vivo dosimetry can only be applied in a routine way in a busy clinic if the software includes fully automated image acquisition and data analysis. Due to the large number of patients, there was an urgent need at NKI-AVL for automatic analysis of in vivo dose verification of IMRT and VMAT. Since August 2011 dose reconstruction and gamma evaluation software runs automatically yielding a dosimetry report for inspection within minutes after treatment delivery without any manual intervention. Currently the reports of all in vivo dose verifications, approximately 7,500 in 2012, are automatically produced and checked directly after delivery without human intervention. With the current set of criteria for errors, 82% of the IMRT and VMAT treatments in 2012 were automatically approved. In this way, more time is available to investigate the reasons for differences between measured and planned dose distributions. It will enable the identification of potential errors in dose calculation, data transfer, dose delivery, patient setup and changes in patient anatomy. Automatic analysis may also allow a more frequent use of IVD during a series of fractions, for instance at all days in-room imaging is performed.
TREATMENT PLANNING Corine van Vliet-Vroegindeweij, Tomas Janssen, Anne Lisa Wolf, Gijs Franssen, Rob de Graaf, Emmy Lamers, Annemarie Lakeman, Angela Tijhuis, Linda Glaser, Peter de Ruiter, Amber Duijn, Roman Bohoslavsky, Simon van Kranen, Marcel van Herk, Marnix Witte, José Belderbos, Joost Knegjens, Jan-Jakob Sonke, Paula Elkhuizen, Sandra Vreeswijk, Tanja Alderliesten, Sanne Conijn, Peter Remeijer, Eugène Damen
Preoperative accelerated partial breast irradiation: VMAT versus IMRT comparison using Pareto fronts The aim of this study is to compare VMAT with IMRT for partial breast irradiation (PBI). To make an objective comparison we evaluate the techniques based on Pareto fronts. Patients are treated with preoperative accelerated PBI (PAPBItrial), delivering 10x4 Gy to the CTV consisting of the gross tumor volume +2 cm. Both the IMRT and VMAT technique have been used clinically for this treatment. We compare the 2 techniques for 4 patients. The studied VMAT plans use a 6 MV dual arc, with an arc length of up to 232 degrees. In the IMRT plans both 6 and 10 MV beams are used, in up to 9 beams with table rotation for 2 out of
the 4 patients. To make an objective comparison, 1500 plans have automatically been created for each patient and for both IMRT and VMAT using the Pinnacle3 treatment planning system. The planning objectives for these plans are randomly generated from a ‘master set’ of objectives, within a Gaussian distribution with a user-defined list of standard deviations. The set of objectives for the clinical plan are used as the master set for the IMRT plans, while a similar set is used for the VMAT plans. After optimization the plan is prescribed to the correct dose level and all relevant data is saved for evaluation. From the clinically acceptable plans the Pareto front is determined, which is a set containing the points for which a simultaneous improvement in more than one evaluation objective is not possible. The 2 techniques are compared based on the median difference between these Pareto fronts. An example of a 2-dimensional Pareto front for one patient is given in the figure. We found the following mean median difference between the VMAT and IMRT front, where a negative sign indicates a lower value for VMAT compared to IMRT: PTV D1%= -0.9 Gy (s=0.1 Gy, p<0.01), Breast minus PTV Dmean=-0.6 Gy (s=0.2 Gy, p=0.15), Heart D1%=-0.9 Gy (s=0.4, p=0.4) and Lung V5Gy=-4 % (s=4 %, p=0.12). Moreover, for 3 out of 4 patients the VMAT Pareto front was superior to the IMRT Pareto front on all evaluation objectives (figure 1). In the comparison of Pareto fronts for preoperative PBI patients we found a significant improvement on the PTV D1% in favor of VMAT compared with the IMRT technique. The results are an indication that with the VMAT technique an improvement can be obtained in the evaluation criteria Breast minus PTV Dmean and Lung V5Gy, though more patients need to be evaluated for definitive results.
Figure 1: Results for group A (left) and group B (right). Showing Dplan (stars) and DExpected (circles) versus DDelivered. For reference also the unity line is shown.
Seroma volume change as an indicator for the need of replanning during adaptive radiotherapy of breast cancer In the course of radiotherapy (RT) of breast cancer patients with seroma development after breast-conserving surgery (BCS), the seroma volume may shrink considerably. This leads to an excess of unwanted healthy tissue irradiation. Each 100 cc increase in irradiated boost volume is associated with a fourfold increase in risk of fibrosis. Our present adaptive radiotherapy protocol, with replanning during the radiotherapy course, mitigates this problem. However, since seroma reduction differs per patient, replanning is not always necessary. The presently used selection criterion for the decision to enter the adaptive protocol is an initial seroma volume of at least 40 cc. The purpose of this work is to investigate whether the change of seroma volume is a good additional indicator for the need of replanning. 33 patients who developed seroma after BCS were selected for the study, all receiving a simultaneous integrated boost treatment with either 50.7 Gy (elective area) and 64.4 Gy (boost) in 28 fractions or 46.2 Gy (elective area) and 55.9 Gy (boost) in 21 fractions. Initial seroma volume was measured on the planning CT (pCT). Seroma volume changes were evaluated on a repeat CT (rCT) after 13 of 28 or 9 of 21 fractions. A difference in irradiated healthy tissue volume (ΔVhealthy tissue) of 50 cc between pCT and rCT was selected as the clinically relevant threshold for replanning. Three seroma-based anatomical surrogates for ΔVhealthy tissue were investigated: ΔVtumor bed: difference in tumor bed volume;
Figure 2: An example of a Pareto fronts (shown here only in 2 dimensions) for the plans generated using the IMRT – (circles) and VMAT-technique (squares). The fronts are indicated with the dashed and solids lines respectively, showing an improvement in BreastPTV-Dmean and PTC D1% in the VMAT-plan compared with the IMRT-plan.
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ΔVseroma: difference in seroma volume; ΔVseroma, box: difference in volume of the encompassing box around the seroma; These surrogates are consecutively easier to obtain in a clinical setting, but at the same time they are expected to become less accurate surrogates of the irradiated healthy tissue volume. The three surrogates for ΔVhealthy tissue were analyzed in terms of a binary classifier. Receiver operating characteristic (ROC) curves are displayed in figure 2. It can be observed that (for the indicated operating points) ΔVtumor bed and ΔVseroma have false positive rates (FPRs) of, respectively, 0.40 and 0.60 for true positive rates (TPRs) of, respectively, 0.85 and 0.88. The operating points correspond with a threshold value of 20 cc. ΔVseroma, box has even higher FPRs, paired with lower TPRs. ΔVtumor bed and ΔVseroma as surrogates of ΔVhealthy tissue have only a moderate predictive value concerning the effect of replanning on the irradiated healthy tissue volume of breast cancer patients with seroma, with high FPRs for acceptable TPRs. Furthermore, the encompassing box is markedly unreliable as a surrogate. We conclude that there is no clinical incentive to include seroma-based surrogates of ΔVhealthy tissue in the present adaptive protocol. The presently used selection criterion for the decision to replan or not, i.e. an initial seroma volume of at least 40 cc, is adequate (figure 2).
CLINICAL APPLICATION OF IMAGE GUIDED AND ADAPTIVE RADIOTHERAPY Suzanne van Beek, Jose Belderbos, Monique Bloemers, Monica Buijs, Chun Chen, Sanne Conijn, Rianne de Jong1, Birgit Hollmann, Folkert Koetsveld, Simon van Kranen, Corrie Marijnen, Angelo Mencarelli, Jasper Nijkamp, Heike Peulen, Coen Rasch1, Anneke van der Reijden, Peter Remeijer, Simon Rit1, Maddalena Rossi, Peter de Ruiter, Eva Schaake, Marcel van Herk, Baukelien van Triest, Jan-Jakob Sonke
Cervix Opportunities for margin reduction for cervical cancer by application of a population based library of plans
Application of a library of plans can reduce geometrical uncertainties during radiotherapy for cervical cancer. To generate a relevant library of plans, however, sufficient geometrical variation must have been measured during treatment simulation. Moreover, it is unclear how plan selection impacts the required safety margins. The purpose of this study was therefore to develop a population-based library of plans and to quantify impact on safety margins. To that end, 31 patients with cervical cancer were retrospectively selected. Both for pre-treatment imaging and during treatment patients were asked to maintain a ‘comfortably’ full bladder. In the planning CT (pCT) and in weekly cone beam CBCTs, 1) the CTV was delineated, 2) the centerline was automatically extracted and 3) centerline variation was captured with Principal Component Analysis (PCA). Subsequently, new centerlines and corresponding CTVs were created from the planning CT to populate a library. Margins were determined using the conventional margin recipe m=2.5*Σ+0.7*σ, applied to
1 AMC, Department of Radiation Oncology, Amsterdam
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displacements perpendicular to the CTV surface. Without a library of plans, a non-negligible group mean error up to 10 mm in CC direction towards the top of the uterus was found, possibly resulting from decreasing ‘comfortably full’ bladder filling over treatment. Systematic and random errors were up to 12 mm CC and AP. Derived margins typically ranged from 1.6 cm at the base of the cervix to 3.7 cm at the top of the uterus (on average 2.2 cm). The first two PCA components account for 75% of all centerline variation. Generating a library of plans at the optimal loading of PCA components (1.6 SD) as derived by minimizing the PTV volume resulted in nearly uniform margins of 1.6 cm with 3 plans (component 1), 1.5 cm with 5 plans and 1.5 cm with 9 plans (component 1 and 2). In conclusion, considerable margin reduction could be achieved using a population-based library consisting of 2 plans (figure 3).
Head and neck Position variability of the larynx during radiotherapy of stage I/II laryngeal cancer
To determine the position variability of the larynx in relation to rigid bony structures during radiotherapy, 10 stage I/II laryngeal cancer patients were retrospectively selected. To that end, both the larynx and nearby bony anatomy in 85 CBCT scans were registered to the planning CT using local rigid chamfer matching. Residual random and systematic set up errors after corrections based on registration in bony structures instead of registration on the larynx are estimated for LR-direction to be σ=0.5 mm, and ∑=1.3 mm, for CC σ=0.6 mm, and ∑=2.3 mm and for AP σ=0.3 mm and ∑=0.7 mm. Additionally, a multi observer study confirmed a high reproducibility of larynx mobility assessment. In conclusion, measurement and correction of larynx misalignments based on the vertebrae is not recommended for radiotherapy of laryngeal cancer. Cone Beam CT based assessment of the setup reproducibility of two different head supports for the Head and Neck cancer patients
Despite individual five-point thermoplastic masks and Cone Beam CT (CBCT) to the planning CT, residual misalignments are present after the couch correction. Therefore, a constant critical look at the patients positioning and immobilization is necessary. The purpose of this study was to compare the setup reproducibility of a standard head support with an individual H&N support. To that end, 30 head and neck cancer patients were retrospectively selected: 15 patients were positioned using a vacuum cushion (VC) as individual head support and another 15 patients the other with modified standard head support (MSHS). A total of 316 CBCT (11 scan per patient on average) were evaluated using multiple region-of-interest (ROI) registrations. 13 bony structures were identified of which jugular notch (jug) and cervical vertebrae 7 (C7) were the most caudal. No relevant differences in setup reproducibility of the jug and C7 for the groups of patients in the online local setup errors were observed (Table 1). In conclusion, the VC support did not improve the stability of the lower neck and upper thorax compared to our standard H&N support. Automated decision rules for adaptive radiation therapy to reduce differential motion of multiple anatomical regions
Adaptive radiation therapy (ART) has the potential to accurately account for patient’s systematic deformations and differential motion at the cost of re-planning. The purpose of this work was to develop a decision rule (DR) for adaptive interventions
to balance accuracy and workload. To that end, an automated shrinking action level (SAL) decision rule was developed that simultaneously manages the patient's overall setup error, and monitors residual misalignments of multiple relevant anatomical structures relative to the planning anatomy. Fifty head & neck cancer patients were retrospectively selected for which repetitive CBCT scans were available and 7 Regions of Interest (ROI) were placed around the larynx, mandible, four vertebrae and the skull. Subsequently, Monte-Carlo error simulations were made to optimize the parameters of the decision rule. The larynx was the most mobile structure, with systematic misalignment in the CC direction of 4 mm. By allowing an average of one intervention in every three patients, it could be reduced to 2.5 mm using a SAL protocol with a=12 and Nmax=4. In conclusion, automated decision rules to trigger adaptive plan modifications can considerably reduce systematic misalignment of multiple anatomical regions, while the added clinical workload can be kept within acceptable limits.
Lung Factors affecting intrafraction motion and baseline shifts in Stereotactic Ablative Body Radiotherapy for NSCLC
Stereotactic Ablative Body Radiotherapy (SABR) has become the standard treatment for inoperable early stage non-small cell lung cancer (NSCLC) patients. State-of-the-art IGRT systems provide accurate alignment of the target just prior to treatment but do not monitor or correct for intra-fraction motion. The purpose of this study was to find predictive factors for intrafraction motion in NSCLC treated with SABR. To that end, 197 consecutive patients were selected from our SABR database. Most patients received 3x18Gy with an online CBCT guidance protocol. Clinical parameters such as performance status, age, gender, lung function, COPD status, tumor location and weight were analysed. Intra-fraction motion (vector length) was quantified by the difference in target position between post correction CBCT and post treatment scan. Univariate and multivariate analysis were performed to distinguish significant factors, which may predict intra-fraction target motion and baseline shifts. In univariate analysis, the peak-to-peak amplitude derived from the 4DCT (4DCTVECT) (R=0.196, p=0.007), COPD (R=0.148, p=0.043), the average treatment time (R=0.293, p <0.001) and the average baseline shifts observed (R=0.366, p <0.001) were found to be significantly correlated to intra-fraction motion. In multivariate analysis only average treatment time and baseline shifts remained significant (R= 0.243, p <0.001, and R=0.275, p=0.01 respectively). In conclusion, Intra-fraction motion significantly correlated with treatment time, stressing the need to keep this to a minimum. VMAT based delivery techniques facilitate fast delivery times.
Translations (cm) ROI
GM
jug C7 jug
Σ C7 jug s C7
Head support VC
Rotations (°)
LR
CC
LR
LR
CC
AP
0.06
0.0
0.06
0.2
1.2
0.3
MSHS
0.09
-0.01
0.01
0.7
1.5
0.1
VC.
-0.06
-0.02
0.02
0.3
1.0
0.2
MSHS
-0.06
-0.01
-0.01
0.1
1.2
-0.3
VC
0.19
0.27
0.17
2.3
1.3
0.8
MSHS
0.11
0.20
0.17
1.3
0.9
0.8
VC
0.14
0.05
0.12
1.5
0.7
0.7
MSHS
0.09
0.10
0.13
1.8
0.9
0.8
VC
0.13
0.16
0.20
2.2
0.9
1.0
MSHS
0.13
0.17
0.15
1.5
1.0
0.9
VC
0.1
0.08
0.11
1.3
1.0
0.9
MSHS
0.12
0.1
0.10
1.7
1.1
0.8
Table 1 Group mean (GM) systematic error (σ) and random error (Σ) after online overall setup error correction of the jugular notch (jug) and cervical vertebrae 7 (C7) for the vacuum cushion (VC) individual head support and modified standard head support (MSHS).
Figure 3: Roc curves of three proposed surrogates of the volume change of irradiated healthy tissue. Operating points are indicated with dots.
Adaptive radiotherapy for primary lung tumors and mediastinal lymph nodes
Adjustment for geometrical uncertainties during the radical treatment of lung cancer patients is warranted to minimize margins and potentially increase the dose to the target lesions. Differential motion between pathological lymph nodes and primary tumor limits the potential of couch shift based correction strategies to reduce these geometrical uncertainties. Adaptive RadioTherapy (ART), however, has the potential to adequately account for differential motion through one or 167
more re-planning interventions over the course of therapy. The purpose of this study is to geometrically evaluate the potential of ART for locally advanced lung cancer patients. To that end, a small (0.35 x 5mm) gold fiducial markers was inserted in a mediastinal lymph node of NSCLC patients during standard diagnostic transoesophageal endoscopic ultrasound. A respiration correlated 4D planning CT and daily 4D Cone Beam (CB) CT-scans were acquired during radiotherapy (66 Gy/24 fractions) to visualise the primary tumor and mediastinal lymph node marker by the fiducial. Subsequently, patient population statistics of the peakto-peak amplitude and baseline shifts of tumor and lymph node were calculated. An online bone match warrants lymph node margins of 0.98 cm in LR, 1.28 cm in CC and 0.99 cm in AP, and a tumor margin 1.02 cm in LR, 1.75 cm in CC and 1.17 cm in AP. With a single adaptive intervention, the lymph node margins decrease to 0.86 cm in LR (-12%), 0.96 cm in CC (-25%) and 0.87 cm in AP (-12%). For the tumor margins decrease to 0.94 cm in LR (-8%), 1.31cm in CC (-25%) and 1.07cm in AP (-9%). Weekly ART gives the follow margins; 0.71 cm in LR (-27%), 0.76 cm in CC (-40%) and 0.83 cm in AP (-16%), and tumor margins of 0.90 cm in LR (-12%), 1.01cm in CC (-37%) and 0.98cm in AP (-16%). In conclusion, this study demonstrates that substantial margin reduction can be achieved with ART in NSCLC. In clinical practice a single adaptation after week 1 has the optimal margin decrease within the feasibility of daily practise. First clinical results for motion compensated cone-beam CT (CBCT) in Stereotactic Ablative Body Radiotherapy (SABR)
Respiratory motion causes blurring of the anatomy in 3D CBCT. Motion compensated (MC) CBCT using an a priori motion model from the planning CT has shown to improve the image quality while using short acquisition times (figure 4). The purpose of this study was to evaluate the first clinical implementation of MC CBCT in lung cancer patients treated with stereotactic ablative body radiotherapy (SABR). To that end, for six patients and a total of 25 fractions, 4D MC-CBCT scans were reconstructed to assess residual motion of the target. The mean (SD) tumor amplitude derived from the 4D planning CT was 2 (2) mm (LR), 5 (4) mm (CC) and 5 (3) mm (AP). In one patient, the residual tumor amplitude exceeded 8 mm. For the remaining patients, the residual tumor amplitude after motion compensation was 1.4 (0.6) mm (LR), 1.6 (0.7) mm (CC) and 1.7 (0.5) mm (AP), showing the validity of the a priori model. In conclusion, MC CBCT has been successfully introduced in clinical practice while initial results indicate that the a-prior motion model us adequate for about 80% of patients.
acquired for each patient, one for treatment planning (pCT), 5 daily during the first week, followed by 4 weekly scans. CTV shape variation was estimated by calculation of the local surface distances from the pCT CTV to the rCT CTVs. Shape variation was quantified in surface maps representing the group mean error (GM), the systematic error (∑) and the random error (σ). Distances from the average CTV over the first N fractions to the remaining CTVs were calculated to estimate the residual shape variation. Required PTV margins were calculated for adapted and non-adapted strategy. Substantial systematic and random shape variation (figure 5) demanded for a PTV margin up to 2.4 cm at the upper-anterior part of the CTV and a PTV of 993 cc when no ART was used. The average overall treatment PTV volume was minimized for N=4: 870 (p<0.01), with up to 0.7 cm margin reduction. The bowel area volume receiving 15, 45 and 50 Gy was reduced from 372 to 358 cc, 92 to 73 cc and 44 to 27 cc, respectively (p<0.01). In conclusion, adaptive radiotherapy allows considerable reduction of PTV margins bowel exposure.
Positional variation of involved mesorectal lymph nodes during radiotherapy of rectal cancer Pre-operative chemoradiation is generally given for locally advanced rectal cancer. To improve tumor down-staging and increase the chance on a pathological complete response, several boost studies have been initiated recently, in which the tumor and involved lymph nodes are treated to a higher dose. However, knowledge on positional variation of the tumor and involved lymph nodes for margin calculation is lacking. The aim of this study was to quantify the positional variation of pathologically enlarged lymph nodes during chemo-RT of locally advanced rectal cancer using repeat CT scans. To that end, 10 repeat CT (rCT) scans were acquired for each of 6 patients treated with 25x2 Gy, prone on a flat table. Pathologically enlarged lymph nodes (>0.5 cm) were delineated on all planning (p) and rCT scans after bony anatomy registration. The center of mass (COM) was calculated for each delineation, and distances were calculated between the pCT and rCT delineations. Positional deviations increased towards the end of the treatment associated with a caudal group mean error of 0.4 cm. Systematic and random errors were substantial, especially in AP and CC direction (3-4 mm). Required margins for a PTVboost were estimated at 0.7, 1.1 and 1.1 cm in LR, CC and AP direction. In conclusion, substantial positional variation of pathologically enlarged lymph nodes within the mesorectum was observed, requiring generous safety margins in case of a boost.
RECTUM Potential benefit of adaptive radiotherapy during chemo-radiotherapy of rectal cancer
Neo-adjuvant (chemo-) radiotherapy has been established as standard of care for rectal cancer patients. The major geometrical uncertainties during radiotherapy consist of CTV delineation and shape variation. Adaptive radiotherapy (ART) can be used to estimate the average CTV shape during treatment in the first week of treatment, minimizing systematic errors. The aim of this study was to quantify the potential margin reduction and bowel exposure reduction with ART during chemoradiotherapy of locally-advanced rectal cancer. To that end, repeat CT (rCT) scans were acquired for 28 patients treated with 25x2 Gy and Capecitabine. A total of 10 CT scans were 168
QUANTITATIVE FUNCTIONAL IMAGING FOR RADIOTHERAPY DOSE PAINTING Anette Houweling, Anne Lisa Wolf, Wouter Vogel, Olga Hamming-Vrieze, Corine van Vliet-Vroegindeweij, Jeroen van de Kamer, Andrea Holt, Stijn Heijmink, Jelle Teertstra, Saar Muller, Greetje Groenendaal, Birgit Hollmann, Uulke van der Heide
Dose painting uses two properties of radiotherapy that set it apart from other treatment modalities: First, radiotherapy offers a high degree of control over the deposition of radiation
dose by selecting direction and shape of the treatment fields. With in-room imaging devices such as the cone-beam CT, we can ensure that these fields are targeted correctly. Secondly, Intensity modulated radiotherapy (IMRT) and Volumetric Arc Therapy (VMAT) provide a differential capacity, which means that within a single treatment different parts of the target can be irradiated with different dose levels. The implication of these properties is that in radiotherapy the decision about what to treat is not binary. Dose painting is currently tested in several clinical trials. An example is the FLAME trial, where the tumor inside the prostate is boosted based on multi-parametric MRI images. Another example is the Artforce trial for head-neck cancer, where part of the tumor is treated to a higher dose, based on the FDP-PET signal. To guide decisions about what to treat, and to which level, functional imaging techniques can be used to characterize the tumor and its heterogeneity. The research in my group focusses on the development and validation of quantitative imaging methods that allow tumor characterization.
Figure 4: (a) The centerline of the cervix/uterus is automatically determined, rings are placed perpendicular to the centerline at equal distances (b) margin reduction map for a library of 3 plans
Choosing the right imaging modality for dose painting As a variety of functional imaging modalities are available to image and characterize the tumor, the question arises which (combination) of these modalities is optimally suited for dose painting. FDG-PET is currently used in clinical trials of head-neck cancer to identify the part of the tumor that requires a higher radiation dose. Diffusion-weighted MRI (DWI) is a technique that reflects the diffusion of water in tissue. A restricted diffusion is related to an increased cellular density, as found in tumors. While both FDG-PET and DWI could be candidates for dose painting in HN cancer, it is not clear which of these techniques would be most appropriate. Therefore, we investigated in 18 patients the correspondence between the two imaging modalities. Dose painting plans were optimized based on the 50% isocontour of the maximum SUV (SUV50%max). Dose coverage was analyzed in the SUV50%max- and in three ADCtargets using the mean dose and the dose to 2% and 98% of the volume. The average maximum SUV was 13.9 and the mean ADC was 1.17·10−3 mm2/s. The average between SUV and ADC was -0.2 (range: -0.6 to 0.4). The three dose parameters were significantly smaller in the ADC-targets compared to the SUV50%max-target. FDG-PET and DWI contain different information, resulting in different targets. This suggests that it is relevant to consider the dose coverage of these different targets in the evaluation of clinical dose painting trials.
Figure 5: Example of a 3D (top) and 3D motion compensated CBCT scan demonstrating the improved image quality in terms of motion blur.
Reliable quantification of dynamic contrast-enhanced MRI In clinical practice many functional MRI techniques are only used qualitatively. For localizing the tumor, this is usually sufficient. However, for defining the boundaries of a tumor, it is essential to be able to assess if a measured imaging value is high or low. Moreover, conversion of imaging data to dose levels for dose painting requires a consistent quantification. In a pilot study of dynamic contrast-enhanced MRI in prostate cancer, we investigated the influence of an overall increase in injection time on the shape of the arterial input function and the tracer kinetic parameter Ktrans. We increased the injection time from 5s to 30s in 6 steps. Between 5 and 10 seconds, only small changes are observed in the AIF. Indeed, we saw no changes in 169
the tracer kinetics model parameter Ktrans in tumor and normal peripheral zone regions. However, the longer injection times did affect the AIF and lower Ktrans values were found in tumor. On the other hand, no differences were found in normal tissue. A limitation of this pilot was that the different injection times were not measured in the same patient. To exclude that the observed effects can be attributed to patient variation, we have started a study (N12ITC) in which patients receive two exams: first the standard exam with an injection time of 5 seconds, and a week later a study exam with a longer injection time.
Validation of multi-parametric MRI of prostate with histology The interpretation of multi-modality imaging data requires a high level of expertise of radiologists, nuclear medicine physicians and radiation oncologists. To support interpretation and use of the images for dose painting, we are developing automated methods to convert combinations of images into maps that reflect the probability that a voxel contains tumor. Interpretation of images always is subject to uncertainty. We propose to cope with this uncertainty by exploiting the differential capacity of radiotherapy: rather than interpreting the images in binary terms (tumor yes/no), risk levels are derived that can be associated with different dose levels. An example of such a model was developed earlier for prostate cancer. This model is currently validated in a multi-center setting using retrospective data from the KU Leuven and the Netherlands Cancer Institute. Preliminary results show that for 29 patients from Leuven, a voxel-by-voxel area under the curve (AUC) of receiver operating curve analysis was found of 0.78, indicating a high level of accuracy. Further development of a platform to translate multi-parametric MRI data to a dose prescription for radiotherapy dose painting is currently under way in the EU project ‘DrTherapat’ in which our group collaborates with Philips Healthcare, KU Leuven, Aarhus University and others.
BREAST CANCER Femke van der Leij, Marc van de Vijver1, Jelle Wesseling, Kenneth Gilhuijs, Hester Oldenburg, Claudette Loo, Adrian Begg, Wouter Vogel, Sandra Vreeswijk, Corine van Vliet-Vroegindeweij, Harry Bartelink, Paula Elkhuizen
Defining radiotherapy sensitivity (A) Image guided Preoperative Accelerated Partial Breast Irradiation (PAPBI): Radiotherapy is part of breast conserving therapy (BCT) and is known to reduce local recurrence rates in all patients by 60-70%. So far, no patient groups can be defined in whom radiotherapy would not be necessary. It is estimated that in approximately half of the patients whole breast radiotherapy is not necessary, while in others the tumor might be resistant to radiotherapy. If it would be possible to predict tumor response to radiotherapy, a more tailored treatment can be advised to individual patients (higher boost dose or primary mastectomy). To evaluate the in situ breast radiosensitivity, the image guided preoperative accelerated partial breast irradiation (PAPBI) is 1 AMC, Department of Pathology, Amsterdam
170
the best treatment strategy because of (i) low breast α/β ratio suggesting that breast cancer is more sensitive to high dose per fractions; (ii) very precise breast tumor irradiation; (iii) limited volume of irradiated area allowing larger fractions with no extra risk on late toxicities; (iv) short overall treatment time. This trial is directed at implementing pre-operatively given image guided accelerated partial breast irradiation without compromising local control in early breast cancer patients. By assessing tumor response to radiotherapy, the goal of the study is to develop a gene expression profile that predicts the breast cancer radiosensitivity. This gene signature of breast radiosensitivity would further design optimal treatment strategies for individual breast cancer patients treated with BCT. To qualify for the trial, patients must be 60 years or older, and have an unifocal cT1-2 (≤3 cm) pN0 M0 breast cancer; sentinel node procedure before irradiation. Patients will be treated by PAPBI consisting of delivering 10x4 Gy over 12 days. Six weeks after PAPBI, a wide local excision will be performed. As the tumor remains ‘in situ’ during irradiation, accurate tumor delineation and control of accurate radiation dose delivery to the tumor becomes possible by treating these patients with a cone beam CT linear accelerator. To identify a subgroup of breast cancer radiosensitivity, biological studies performed are gene expression profiling from RNA and DNA isolated from biopsies taken of the tumor before radiotherapy and at time of surgery. The mRNA gene expression profiles, the miRNA expression profiles and the DNA copy number changes will be correlated with response to radiotherapy, defined as pathologic response at the time of the lumpectomy (i.e. 6 weeks after the completion of the PAPBI). Response of the tumor will be evaluated by MRI scan and PET (before radiotherapy and before surgery) and classical pathology We evaluated the PET scans of inter alia 31 PAPBI patients before radiotherapy for evaluation of primary tumor visualization and staging in T1 breast cancer patients with FDGPET/CT. In majority of the patients the primary tumor was visible on FDG-PET/CT. The Institut Gustave Roussy (France) and the Karolinska Institute (Sweden) are participating in the trial (Descartes Cancer Consortium) and in January 2012 the UMCU in Utrecht started to participate. Hundred and twenty patients are needed to include in the trial, and 60 patients are included until December 2012. Other important aspects of this study are collections of fresh frozen tumor tissue (i) to assess the radio-induced genetic alterations on the surgical post-radiation specimen compared to the tumor response 6 weeks after the end of radiotherapy; (ii) to study the early changes in gene-profiling and (iii) to evaluate the early functional imaging modifications. Differences in target volume delineation in the PAPBI trial were evaluated by comparing preoperative delineation with postoperative delineation. In a dataset of 24 breast cancer patients we showed less inter-observer variation in the preoperative situation versus the postoperative situation, and comparable target volume sizes.
performed colony assay experiments for 7 breast cancer cell lines (T47D, MCF7, SKBR3, BT 474, Bt 549, SUM 159, BT 20) (figure 6). This will also be implemented in the study described in C. (C) In the completed Young Boost Trial 2500 patients under 50 years have been randomized between normal boost dose vs. a higher boost dose after breast-conserving therapy. From these patients frozen material as well as paraffin embedded material is collected. Follow up is available for the first years. Histological revision of tumor material is performed and tissue micro arrays of these tumors are being made. RNA and DNA isolation is done of paraffin- and frozen material. Gene expression profiling is planned. The profiles found in the studies A-B to be related with radioresponse will be evaluated on this material whether a response profile eventually is associated with local control. In addition, the results from the case-control study will be evaluated on this cohort.
Figure 6: Left anterior view of the CTV shape variation when no ART is used (left) and when treatment is adapted based on the first 4 fractions (right). Shape variation is expressed in group mean error (GM, top), systematic SD (S, middle) and random (s, bottom) and expressed in centimeters.
(D) Cohort analysis breast conserving treatment in 10,000 patients treated between 1979 and 2008. All patients were radiated at the Antoni van Leeuwenhoek hospital, surgery was performed at the Antoni van Leeuwenhoek hospital or referring hospitals. Local relapse will be studied in relation to treatment year, patient characteristics and treatment factors. In a subset of 1550 patients, treated between 2002 and 2006, we added pathology information to subdivide patients in different breast cancer subtypes, to study the importance of these subtypes in the prediction of recurrences. (E) A new phase II study is in preparation in the Netherlands comparing the toxicity of 2 different pre-operative Partial Breast Irradiation schedules (10x4 Gy versus 5x6 Gy). (F) This year the long-term results (at least 20 years of followup) were published of the EORTC 10801 breast cancer trial. This trial compared breast-conserving therapy (BCT) with modified radical mastectomy (MRM) in 902 patients with tumors 5 cm or smaller. This multi-centric trial was world wide the first to investigate the efficacy of BCT in early breast cancer up to 5 cm, it was initiated and coordinated by the NKI-AVL. Important was that both in younger and older patients no differences were seen between both treatments. We concluded that the similar survival outcomes found in patients treated with BCT or MRM, confirm that BCT can be safely applied in patients with early breast cancer up to 5 cm (figure 7).
Figure 7: Mean surviving fraction (SF) 6 different cell lines
Figure 8: Overall Survival
The ipsi-lateral breast recurrence rate of the three consecutive breast cancer trials (see above) revealed a dramatic improvement in the most recent young boost trial. The preliminary results of this unblinded trial demonstrated an excellent local tumor control rate even in the high-risk young patients (figure 8).
(B) Radiosensitivity breast cancer cell lines: In addition, with the clinical PAPBI study, 30 human breast cancer cell lines will be investigated for their radiosensitivity profile. Until now we 171
COMBINATION OF RADIOTHERAPY AND CHEMOTHERAPY / BIOLOGICALS Berthe Aleman, Harry Bartelink, José Belderbos, Andre Bergman, Jan Paul de Boer, Henk Boot, Michiel Van den Brekel, Annemieke Cats, Frits Van Coevorden, Ewout Courrech Staal, Rosemarie de Haan, Luc Dewit, Johan Dikken, Ria Dubbelman, Rick Haas, Olga Hamming, Michel Van den Heuvel, Simon Horenblas, Martijn Kerst, Edwin Jansen, Joost Knegjens, Maria Kuiper, Margriet Kwint, Luc Moonen, Arash Navran, Jasper Nijkamp, Heike Peulen, Dick Pluim, Floris Pos, Coen Rasch, Jan Schellens, Jan-Jakob Sonke, Jurriën Stiekema, Anouk Trip, Judi van Diessen, Steven Vanhoutvin, Johanna van Sandick, Baukelien van Triest, Conchita Vens, Vic Verwaal, Thelma Witteveen, Shuraila Zerp, Marcel Verheij
Head and neck In the CONDOR cooperative trial, feasibility of induction chemotherapy with TPF followed by chemoradiation (CRT) was evaluated. Patients below 60 years with stage III/IV head and neck cancer were included. This study closed prematurely after inclusion of 65 of the required 70 patients because 30% of patients could not finish the CRT after induction chemotherapy. The final report concluded that the combination of induction TPF followed by CRT in patients below 60 years with stage III/IV head and neck cancer is not feasible due to toxicity. The final analysis on the 10-year results for patients treated with CRT within the M99RAD trial has started. In this trial patients were randomized between CRT with intra-arterial or intravenous cisplatinum treatment. Preliminary results show no difference in OS, DFS or LR between the intravenous and intraarterial treatment arm. Toxicity data and the occurrence of new primary tumors will be analyzed early 2013. In September 2012, the ARTFORCE trial has started. In this randomized international phase II trial, 268 patients will be randomized between CRT with cisplatin or Cetuximab and between adaptive redistribution of dose or the standard radiotherapy regimen. Furthermore a new type of scan, the 89Zr-labeled Cetuximab PETscan, will be performed for all patients in the study and will be evaluated for its predictive value for treatment outcome. Based on our preclinical research on apoptosis-modulation, a clinical phase I-II trial in locally advanced head and neck cancer combining standard cisplatin-based CRT with dose escalating AT-101 was initiated in 2010. AT-101 is a small molecule inhibitor of anti-apoptotic Bcl-2/Bcl-XL. To date, 13 patients have been included; no DLT has been observed. This study is currently on hold due to unavailability of the AT-101.
Gastroenterology – Esophageal cancer Over recent years a database was set up including data on all patients treated for esophageal cancer in our institute since 1997 (approximately 1500 patients). So far the database was used to evaluate toxicity and efficacy of different chemoradiation schedules and also to study quality of care esophageal cancer patients in a broader perspective. In addition, we performed a review of the literature (20002010) on prognostication and prediction using gene expression analysis in esophageal cancer. We concluded that gene expression profiling has potential clinical applications in esophageal cancer. Especially a signature that is predictive for response to neoadjuvant treatment could be of great clinical value. To date, most published studies suffer from an underpowered training cohort or lack adequate validation. Clinicians should put effort in the collection of high quality tissue samples and should participate in biobank initiatives, considering the increasing availability and possibilities of sequencing technology. Furthermore, we are setting up a single center prospective observational study to evaluate organ motion and early tumor response measurement during neoadjuvant chemoradiotherapy for esophageal cancer. The primary aim of this study is to quantify motion-based variation of the target volume over the course of chemoradiotherapy in esophageal cancer patient, and to use this information to calculate appropriate PTV (planning target volume) margins.
Figure 9: Local recurrence rate in three WBI trials with early breast cancer from 1980 till 2012
Gastroenterology – Gastric cancer In the US Intergroup 0116 study, a significant survival benefit in postoperative CRT was reported in gastric cancer. Furthermore, in the UK MAGIC study, perioperative chemotherapy proved to have a survival benefit in operable gastric cancer. Based on these results we completed three adjuvant chemoradiotherapy (CRT) phase I-II trials in gastric cancer. From these trials we identified a regimen for the experimental arm in the CRITICS study. In this international randomized phase III study patients with operable gastric cancer receive preoperative chemotherapy, surgery and postoperative chemotherapy, or preoperative chemotherapy, surgery and postoperative CRT. At this moment over 520 of the required 788 patients have been randomized, which makes this already one of the largest gastric cancer trials in Western Europe ever. In addition to Sweden that joined the trial in 2008, Denmark participates as well since ultimo 2010. Furthermore, we are running a phase I-II study (NARCIS) together with the AMC, in which neoadjuvant CRT with weekly paclitaxel and carboplatin is applied in patients with inoperable gastric cancer. The results of this trial have been combined with those from a similar study from the University Medical Center Groningen. A microscopically radical resection was achieved in 18 out of 25 patients (72%), 8 of whom had initially irresectable gastric cancer. The pathologic complete response rate was 16% (4/25 patients) and 1 patient died in the postoperative period. These promising results will definitely have an impact on the design of the next phase III (CRITICS-II) study. Currently, we are analyzing the results on the late effects of CRT on kidney, spleen and liver function.
Figure 10: Probability of development of acute esophagus toxicity (AET) grade 2 (grey line) and grade 3 (black line) using the logistic model based on V50. The 95% confidence intervals are plotted in dash dotted lines. The actual incidences, together with its 95% confidence intervals, are plotted in the vertical lines.
Gastroenterology – Rectal cancer Pre-operative capecitabine-based chemoradiotherapy (CRT) has become standard treatment in locally advanced rectal 172
173
cancer with acceptable toxicity and surgical complications. In 2012 results from previous research was implemented in daily practice. The tumor and lymph nodes are more precisely delineated in order to enable a more adaptive margin strategy for patients with locally advanced rectal cancer. MRI-based functional imaging and response monitoring are now the subjects of interest. We will extend these topics to more patients in 2013.
Lung - NSCLC In 2012 we have opened two new clinical trials: one trial involving SBRT to further increase the number of patients who may benefit from this high-dose, high-precision treatment, and one phase I trial with the addition of a new radiosensitizer (the PARP inhibitor Olaparib) to our standard concurrent chemoradiation (CCRT) regime. In 2012, a total of 90 inoperable NSCLC patients treated with CCRT consisting of 66 Gy in 24 fractions and daily Cisplatin 6 mg/m2. From January 2012 we intensified our IGRT protocol from weekly cone beam CT (CBCT) scanning (after scanning the first three fractions) to daily CBCT scanning and online correcting setup errors in all radical lung cancer treatments. We have protocolized a weekly multidisciplinary meeting together with technicians, our chest physicians and dieticians, to discuss toxicity, response/alterations on CBCT and treatment related issues of all combined modality lung cancer patients. The multicenter dose-escalation trial using IMRT by boosting the radiation dose within the primary tumor based upon biological activity on pre-treatment FDG-PET scan - ”PET-Boost trial” (M09PBO) - accrued with steady pace. This PET-Boost study (in collaboration with Prof. De Ruysscher, Leuven) for patients with inoperable stage II or III NSCLC was opened in Denmark in 2012 and is expected to start accruing in Manchester and Leuven in 2013. Patients are randomized to receive the standard 66 Gy in 24 fractions with a dose escalation to the primary tumor as a whole or to the volume with ≥50% SUVmax of the primary tumor. In both treatment arms, the patients are irradiated to the same maximum tolerated dose to the lung and receive concurrent chemotherapy. The primary objective of this study is local progression free survival at 1 year. Especially, recurrence patterns as a function of the local dose and volume will be studied. Until October 2012, 67 patients were registered and 42 randomized. In June 2012 the outcome of our multicenter RADITUX trial (M07CCL) was presented at ASCO. A total of 104 patients were randomized in this multicenter randomized phase II trial patients with inoperable locally advanced NSCLC were randomized to our CCRT regimen with or without the weekly addition of the EGFR monoclonal antibody Cetuximab. The addition of Cetuximab to our CCRT increased the grade 3 toxicities significantly while no improved local progression free survival or overall survival was reached at 29 months follow-up. In the group of NSCLC patients treated with concurrent chemoradiotherapy we continued studying the acute esophagus toxicity using FDG PET-CT SUV information. The V55 was identified as most accurate predictor of AET grade ≥3 (figure 9). The results from this analysis will be tested with clinical data from other centers. We also finished analysis of late esophagus toxicity in this patient group (figure 10). In the N11ORL phase I study that started accrual in 2012 our standard (CC)RT regimen for locally advanced NSCLC is combined with dose escalation of Olaparib, a PARP inhibitor. PARP is involved in the repair of DNA single strand breaks. 174
Preclinical work shows that the inhibition of PARP by Olaparib leads to radiosensitization, including in models for NSCLC. The aim of the current clinical study is to define the recommended dose of Olaparib when combined with (chemo)radiotherapy for locally advanced NSCLC. Since the opening of the trial in May 2012, four patients entered the first dose level, Olaparib 25mg BID. So far, no dose limiting toxicities have been encountered. In November 2012, the phase I “Hybrid study” opened in our institute that investigates the safety of combined stereotactic radiotherapy and conventional fractionation in stage II and III NSCLC with peripheral tumors smaller than 5 cm. The irradiation is combined with daily low dose cisplatin. With SBRT in stage I NSCLC, 3 year local control rates of > 90% with low toxicity are achieved. The aim of this trial is to improve local control in more advanced stages of lung cancer. In the first step we want to assess the Mean Lung Dose (MLD) that can be safely delivered with the combination of SBRT to the peripheral tumor and standard fractionated radiotherapy to the lymph nodes with concurrent chemoradiation. We finalized a quality assurance procedure and expect to include 15 patients in 2 years.
Lung - SCLC Approximately 20% of malignant tumors of the lung are due to small cell carcinoma. In general, these patients carry a worse prognosis compared to NSCLC patients. For limited stage small cell lung cancer (LS-SCLC), the combination of chemotherapy and thoracic radiotherapy is the standard treatment. Two meta-analyses have shown that thoracic radiotherapy given concurrently with chemotherapy improves both local control and survival. Nevertheless, several important questions including the optimal total radiation dose and radiation fractionation are still unanswered. To establish a standard chemo-radiotherapy regimen for LS-SCLC, an EORTC international, multicenter randomized phase III trial started in 2009 comparing twice daily radiotherapy with high dose radiation delivered once daily, both given concurrently with standard cisplatin and etoposide chemotherapy (CONVERT trial). To date, approximately 450 patients were treated within this trial (4 patients from the NKIAVL). A comparable three-arm trial has started accrual in 2008 in the US (CALGB 30610/RTOG 0538). For extensive stage small cell lung cancer (ES-SCLC), chemotherapy is the cornerstone of treatment. However, over 75% of patients have persisting intra-thoracic disease after initial chemotherapy, and about 90% manifest intrathoracic disease progression at 1 year after completing initial chemotherapy. In the absence of promising systemic agents that can improve local response, the role of thoracic irradiation in patients with ES-SCLC is being evaluated in a multicenter phase III randomized trial initiated by the VUMC (CREST trial). The objective of this study is to investigate whether thoracic radiotherapy can improve 1-year survival, following a response to chemotherapy. A total of 496 patients have now been treated (50 patients from the NKI-AVL) and the trial has recently been closed. Provisional results of the trial are expected in 2013. Within the RTOG, a phase II trial to determine the role of consolidation extracranial radiation (thoracic and other extracranial metastatic sites) plus prophylactic cranial irradiation after a response to systemic chemotherapy has been activated in 2010 (RTOG 0937).
Urology – Bladder The standard treatment for muscle invasive bladder cancer is cystectomy. For those not eligible for this type of treatment, or for those wanting to preserve their bladder, the alternative treatment is radiotherapy preferably in combination with cisplatin-based chemotherapy. Although the reported results following cisplatin-based chemoradiotherapy compare reasonably well with those of cystectomy, there is substantial room for improvement. Two thirds of the patients achieve a complete response rate, but the reported 5-year survival rate varies between 30 and 50%. Moreover, the combination of radiotherapy with cisplatin-based chemotherapy appears to be rather toxic. Therefore, we are currently looking for less toxic and more effective radiosensitizers. In recent years, new generations of novel biology-driven drugs have been developed. New molecular targeting agents can interfere with complex cell–cell interactions and microenvironmental factors in the tumor. Some of these drugs can selectively interfere with radiobiological mechanisms of tumor resistance, and thereby potentially increase the efficacy of radiotherapy, for example EGFR-interfering agents. In bladder cancer, EGFR status is associated with poor outcome and seems to be linked to radiation sensitivity. Its inhibition might enhance the radio-responsiveness of bladder tumors and improve the outcome of radiotherapy for bladder cancer. Given the very high affinity for EGFR in combination with the favorable toxicity profile, the combination of radiotherapy with concurrent Panitumumab is very attractive to explore in bladder cancer. In close cooperation between the departments of Urology, Medical Oncology and Radiotherapy, we initiated a phase I trial evaluating the combination of radiotherapy with Panitumumab. A schedule of 66 Gy in 33 daily fractions of 2 Gy is combined with weekly courses of Panitumumab in a dose of 2.5 mg/kg i.v. over 60 m to a total of 7 courses. For this trial a sample size of 31 patients has been chosen. Primary endpoint of the study is the acute toxicity rate during radiotherapy with Panitumumab. Secondary endpoints are the complete response rate at 3 months, local control rate at 6, 12, and 18 months, and at 2 years, bladder preservation rate and any grade 3 or 4 adverse event during and within one month after completion of therapy. Exploratory endpoints are % EGFR expression, RAS mutational status, response rate in correlation with EGFR and RAS status and response rate in relation to treatment path.
Urology – Prostate In a randomized, double-blind multicenter phase 3 trial we are comparing ipilimumab (anti-CTL4 treatment) vs placebo following radiotherapy in patients with Castration Resistant Prostate Cancer (CRPC) who have received prior treatment with docetaxel. The hypothesis tested is that treatment with radiotherapy followed by ipilimumab results in a significant increased survival compared to radiotherapy followed by placebo in patients with CRPC, who have progressed during or following prior docetaxel treatment. The mechanism behind this is that the combination of radiotherapy to symptomatic bone metastasis with an agent able to stimulate T-cell response will amplify the immune response generated by radiotherapy and will result in systemic anti-tumor activity leading ultimately to an improved survival. Subjects receive radiotherapy to metastatic bone lesions at 8 Gy for 1 day with 2 days of the first dose of blinded study drug. The blinded study drug (10 mg/kg ipilimumab
or placebo) will be given every 3 weeks for up to 4 doses. Our site included a significant number of patients, rated number 12 worldwide. The first results are expected in 2013. So far, no severe side effects are reported in this small group of patients.
Soft tissue sarcoma In 2012 we started a multicenter phase I study in sarcomas of the extremities or head and neck, evaluating the safety and feasibility of standard preoperative radiotherapy combined with dose-escalated pazopanib, a small molecule inhibitor of VEGFR. The rationale behind this combined modality treatment is multifactorial: VEGF targeted therapy results in normalization of tumor vasculature which might improve the oxygenation of the tumor and greater efficacy of radiation; both radiation and VEGF-blocking agents target tumor-associated endothelial cells to induce apoptosis; radiation induced upregulation of VEGF is counteracted by VEGF-targeted therapy. So far, 8 patients have been included in the first 2 dose levels. Dose-escalation continues.
TARGETED RADIOSENSITIZATION AND IMPROVED DRUG DELIVERY Wim van Blitterswijk, Gerben Borst, Jannie Borst, Lília Cordeiro Pedrosa1, Saurabh Dayal2, Ben Floot, Rosemarie de Haan, Albert van Hell, Burcu Inanc, Monique de Jong, Jos Jonkers, Gerben Koning1, Siewert Jan Marrink3, Dyane Martins, Rogier Rooswinkel, Jan-Jakob Sonke, Baukelien van Triest, Conchita Vens, Shuraila Zerp, Marcel Verheij
Our translational research program continues to focus on the identification, preclinical testing, clinical evaluation and response prediction of targeted radiosensitization. In a separate project we investigate the patented concept of improved anti-cancer drug delivery by short chain sphingolipid-enriched liposomes (GC-LipoDox®) in vitro and in vivo.
Targeted radiosensitization (1) DNA damage repair inhibitors: In collaboration with C Vens (Division of Biological Stress Response) we explore the interaction between radiation and inhibitors of DNA repair. A first strategy targets NAD+ biosynthesis to enhance radiationinduced cell death. APO866 is a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the regulation of NAD+ biosynthesis. NAD+ is involved in numerous biochemical processes, including regulation of DNA repair, replication, transcription and apoptosis. We investigated the cytotoxic effect of APO866 alone and in combination with radiation on tumor cells in vitro and in vivo. At in nanomolar concentrations, APO866 induces a timeand dose-dependent depletion of cellular NAD+ levels in a panel of human tumor cell lines, including the prostate cancer cell lines PC3 and LNCaP. In vitro, APO866-mediated reduced NAD+ levels resulted in enhanced cytotoxicity and, in combination 1 Erasmus MC, Rotterdam, the Netherlands 2 Beatson Institute, Glascow, UK 3 University of Groningen, the Netherlands
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with ionizing radiation, decreased clonogenic survival. Addition of NAD+ rescued the cells from APO866 mediated cell death, demonstrating the essential role of NAD+ depletion in the induction of tumor cell death. In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD+ levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Collectively, our in vitro and in vivo studies have shown that APO866-induced NAD+ depletion enhances radiation response in prostate cancer. Direct PARP inhibitors are also good candidates for combined use with DNA damaging agents. The main mechanism by which both radiation and cisplatin kill tumor cells is by an accumulation of un- or misrepaired DNA damage. PARP inhibitors increase radiation and chemotherapy (Cisplatin) responses in preclinical studies. We have designed 3 phase I-II studies evaluating the safety and tolerability of Olaparib in combination with (cisplatinbased chemo-) radiotherapy in locally advanced breast cancer, NSCLC and head and neck cancer. The head and neck trial is recruiting and no dose-limiting toxicities (DLT) have been encountered in the first Olaparib dose level (25 mg BID). Novel radio-sensitization strategies can only be successful when radiosensitizing the tumors while sparing normal tissue. Using our recently acquired image-guided mouse irradiator (µIGRT) unit, we recently established a mouse model that assesses radiation damage to the lung. This will be used to validate radiosensitization strategies such as the PARP inhibitor/radiotherapy combination. With these studies we will be able to monitor aggravation or confirm tolerance of the combination treatment and develop schedules to optimize the safe and effective radiosensitizing capacity of novel compounds. (2) Identification of exploitable DNA repair defects: Radiation dose given in clinical radiotherapy schedules is limited by healthy normal tissue toxicity, negatively affecting cancer cure rates. In order to improve radiotherapy, combination strategies should be developed that exploit defects or alterations in the tumors while sparing normal tissue. In a collaborative project with M van den Brekel (Division of Surgical Oncology) and C Vens, we found that DNA repair defects, in particular crosslink repair, are common in head and neck cancers. We analyzed a panel of 30 HNSCC cell lines and determined crosslinker MMC sensitivity as a functional read out of defects in the Fanconi anemia (FA) pathway or in homologous recombination (HR) driven double strand break repair. A considerable proportion of HNSCC cell lines were MMC hypersensitive. MMC induced G2 blocks and FANCD2ubiquitination defects confirmed that FA gene defects might underlie crosslink sensitivity. Sensitivity to the PARP inhibitor Olaparib further revealed DNA repair defects in this panel. Expression analysis and next generation sequencing revealed mutations and alterations in the FA pathway genes that are likely to be the cause of the observed hypersensitivity. HR or FA pathway defects can be exploited by the use of PARP inhibitors that specifically target HR defected cells. Our data reveal such an opportunity for HNSCC in particular when combined with chemoradiation. (3) Exploitation of tumor specific DNA repair defects for tumor-targeted radiosensitization: A proportion of tumors exhibit alterations in the cellular base excision repair/single strand break repair (BER/SSBR) pathway. These are often based on the expression of mutant DNA polymerase beta (polβ) or the overexpression of crucial BER proteins such as 176
AP-endonuclease or polβ. We have previously shown that the expression of truncated polymerase dead polβ inhibits BER/ SSBR upon radiation. We demonstrated that this inhibition caused the formation of replication associated double strand breaks and exposed the involvement of HR in the repair of these secondarily induced damages. As a consequence these polβmutated cells heavily rely on HR for survival upon radiation. This led us to propose a novel tumor-targeted radio-sensitization strategy by targeting the backup HR pathway in these cells. Our recent data confirm the hypothesized BER inhibition by polβ overexpression and further extended this concept to polβ overexpressing tumors. Current efforts focus on the testing and selection of suitable HR inhibitors and an in vivo xenograft validation study (collaboration: J Jonkers, Division of Molecular Pathology). (4) Alkyl-phospholipids (ALPs): We continue to explore underlying mechanisms of cellular uptake and sensitivity of ALP and focus on other promising ALP derivatives with potentially better bioavailability and radiosensitizing properties, like the i.v. compound Erucyl-PC (erucylphosphocholine). In collaboration with W Moolenaar (Division of Cell Biology I) we evaluate the antitumor and radiosensitizing properties of Erucyl-PC, a new and i.v. administrable ALP. We found that expression of the membrane flippase complex CDC50a/ATP8B1 plays an important role in cellular sensitivity towards this and related ALPs. Ongoing studies are aimed at evaluating the performance of this flippase complex as a potential biomarker for ALP efficacy in vivo. (5) Pro-apoptotic death receptor agonists (PARA): In a recently started project with the group of Chalmers at the Beatson Institute, we explore the combination of PARA, notably TRAIL, and radiation in preclinical models of mesothelioma. In addition, in a series of tumor specimens from mesothelioma patients, the expression of TRAIL-R and determinants of synergistic sensitivity to radiation (e.g. c-FLIP, JNK, O-glycosylation) are correlated to response to treatment and prognosis. (6) Inhibitors of Bcl-2 family members: In collaboration with J Borst (Division of Immunology) we study the novel anti-cancer drug ABT-737 that mimics the action of BH3-only proteins, which induce apoptosis by binding to pro-survival Bcl-2 proteins. Our data identified Bcl-B, Bfl-1 and Mcl-1 as mediators of ABT-737 resistance and Noxa-inducing stimuli as optimal agents for combined modality treatment of ABT-737-resistant cancers. We next compared the potency of all pro-survival Bcl-2 proteins to protect against conventional and novel anti-cancer regimens and discovered a striking dichotomy in their anti-apoptotic capacity. Bcl-2, Bcl-xL and Bcl-w consistently provided better protection than Mcl-1, Bfl-1 and Bcl-B. We established a quantitative interaction profile in intact cells of all full-length pro-survival Bcl-2 proteins with all BH3-only proteins, Bax and Bak, which completed and corrected available data. However, binding selectivity did not explain the differential protective ability of pro-survival Bcl-2 proteins. Rather, steady-state or drug-induced degradation of Bcl-2 proteins fully explained the dichotomy in anti-apoptotic potency. We conclude that proteasomal turnover rather than selectivity for BH3-only, Bax, or Bak proteins determines the differential capacity of Bcl-2 proteins to protect tumor cells from apoptosis.
(Division of Biological Stress Response) found that expression of the putative stem cell marker CD44 was the most significant predictor of local control. This was validated in a separate series of larynx tumors in which CD44 staining intensity and frequency on a tissue microarray made from pretreatment biopsies correlated with outcome after radiotherapy. In addition to the mRNA profiling studies, the group of Begg has studied both global mRNA and miR expression in a unique panel of 33 head and neck cancer cell lines with known radiosensitivities. Around 200 mRNAs and 7 miRs were found to correlate with radiosensitivity. The miR data are consistent with other findings in literature, showing that forced expression changes in specific miRs can alter radiosensitivity. Many of the 200 mRNAs and the most strongly associated miRs are known to be involved in epithelial to mesenchymal transition (EMT). Interestingly, CD44 has also been linked to EMT. We plan to carry out miR and mRNA sequencing on formalin fixed paraffin embedded material from patients with T2-3 larynx carcinomas, treated with single modality radiotherapy in the NKI over the last 10 years. In addition, we plan to modify the EMT-inducing miRs in HNSCC cell lines to study the effect on gene expression and radiosensitivity.
Improved drug delivery Design and efficacy of bioactive drugs is greatly restricted by their (in)ability to traverse cellular membranes. Chemotherapy resistance, a major obstacle in cancer treatment, is for example, frequently due to suboptimal intracellular drug accumulation. We previously found that a well-defined class of sphingolipid analogs that effectively overcomes this barrier, catalyzing drug-membrane traversal preferential for tumor cell membranes. A liposomal co-formulation of the short-chain lipid N-octanoyl-glucosylceramide (GC) and doxorubicin was applied in a genetically engineered mouse (GEM) breast tumor model (Wcre; Cdh1F/F; Trp53F/F). These tumors, which are resistant to a variety of conventional and biological antitumor therapies, responded to doxorubicin treatment when combined with the membrane modulation strategy. Co-administration of GC generated a sustained anti-tumor response and significantly improved overall survival. Using a nuclear isolation procedure, we showed that the presence of GC enhanced the intracellular tumor accumulation in vivo by a factor of almost 2. In contrast, the accumulation within normal heart tissue was not elevated. Recently, in collaboration with S Marrink (University of Groningen) we identified the underlying molecular mechanism that promotes this trans-membrane movement of doxorubicin. Our well-defined lipid analogues adapt to the amphiphilic drug doxorubicin, when co-inserted into the cell membrane, and assemble a transient channel that rapidly facilitates the translocation of the drug onto the intracellular membrane leaflet. Molecular dynamic simulations unveiled the structure and dynamics of membrane channel assembly at molecular detail.
Response prediction In a recent study of early stage larynx tumors from patients treated with radiotherapy alone, Begg, de Jong and colleagues 177
Division head Otilia Dalesio
Otilia Dalesio MSc Head Harm Van Tinteren PhD Academic staff Andrew Vincent PhD Academic staff Erik Van Werkhoven MSc Academic staff Vincent Van der Noort PhD Academic staff Tinie Benraadt MD Academic staff Jolanda Appelman Technical staff Danny Baars Technical staff Nathalie Barbier Technical staff Frauwkje Bessels Technical staff Judith Boot Technical staff Henk Botma Technical staff Audi Boucher Technical staff Tineke Bruinsma Technical staff Heleen Bussing Technical staff Monique Carreno Technical staff Saskia Cooke Technical staff Jacques Craenmehr Technical staff Gerda De Jong Technical staff Jeltje De Vries Technical staff Marjolijn De Waal MSc Technical staff Ineke De Wit Technical staff Brigitte Dufournij Technical staff Marlou Eilers MSc Technical staff Ernesto Fernandez Salcedo MSc Technical staff Lindsay Grijpink Technical staff Yvonne Groot Technical staff Patricia Hagen Technical staff Christiane Hagenaars Technical staff Song-Hieng Hau MSc Technical staff Annelies Hiemstra Technical staff Paula Hoekstra MSc Technical staff Eduard Ivanov Technical staff Marissa Jansen Technical staff Aarti Jibodh Technical staff Irene Jonkers Technical staff Joop Jonkman MSc Technical staff Josien Kant Technical staff Lies Kolmschate Technical staff Marianne Mahn MSc Technical staff Ingrid Mandjes MSc Technical staff Carla Modder Technical staff Pietje Muller Technical staff Elvira Nuijten Technical staff Caroline Pauwels Technical staff Loes Pronk MSc Technical staff Harriet Rehorst MSc Technical staff Anneke Reinders Technical staff Jolanda Remmelzwaal Technical staff Marielle Roskam Technical staff Helga Schrijver Technical staff Arda Scholtens Technical staff Vincent Scuric Technical staff 178
Lionne Smets Technical staff Dea Storm Technical staff Beata Sznajder PhD Technical staff Ria Tilgenkamp Technical staff Alex Torres Acosta Technical staff Ludy Valkenet MD Technical staff Marjolijn Van den Haak MSc Technical staff Emile Van der Donk Technical staff Marieke Van der Velde Technical staff Tony Van de Velde Technical staff Gabry Van Netten Technical staff Sanne Van Roekel Technical staff Emmie Van Schaffelaar Technical staff Carolien Van Tuijl Technical staff Wil Van Waardenberg Technical staff Steven Vanhoutvin Technical staff Laurien Verkleij Technical staff Marrit Vermeulen Technical staff Sandra Visser Technical staff Nynke Vrolijk Technical staff Anneke Wals Technical staff Lidwina Wever Technical staff Yvonne Wijnands Technical staff Els Willemse Technical staff Jeroen Zandbergen MSc Technical staff
Biometrics Department
Meulenbeld H, van Werkhoven, E, Coenenc J, Creemers G, Loosvelde O, de Jong P, ten Tije A, Fossåh S, Poleei M, Gerritsen W, Dalesio O. & de Wit R. Randomized phase II/III study of Docetaxel with or without Risedronate in patients with metastatic Castration Resistant Prostate Cancer (CRPC), the Netherlands Prostate Study (NePro) European Journal Of Cancer. 2012;48:2993-3000 Koolen B, Peeters MJTFDV, Wesseling J, Lips EH, Vogel WV, Aukema TS, van Werkhoven E, Gilhuijs KGA, Rodenhuis S, Rutgers EJT & Olmos RAV. Association of primary tumour FDG uptake with clinical, histopathological, and molecular characteristics in breast cancer patients scheduled for neoadjuvant chemotherapy European Journal of Nuclear Medicine and Molecular Imaging. 2012;39:1830-1838 Mertens LS, Meijer RP, van Werkhoven E, Bex A, van der Poel HG, van Rhijn BW, Meinhardt W & Horenblas S. Differences in histopathological evaluation of standard lymph node dissections result in differences in nodal count but not in survival World Journal of Urology, 2012
Mertens L, Meijer R, Nunnink C, Bex A, van der Poel H, van Rhijn B, Wassenaar A, van Werkhoven E, Meinhardt W & Horenblas S. Standaard Lymfeklierdissectie Bij Blaascarcinoom: Significante Verschillen In Het Aantal Gerapporteerde Lymfeklieren Tijdschrift voor Urologie, 2012;2:144150
Support for data management and statistical analysis has been obtained from the KWF for 8 studies of the 13 trials that have or are currently being run by the group.
CLINICAL STUDIES AND OTHER COLLABORATIONS We have developed collaborations with other Departments of our Institute, 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 project management, randomization services, development of (electronic) case record forms, data handling, monitoring and quality assurance, and statistical expertise. In addition, the tools that we have developed within our ICT projects attracted new associations
The SIOP-Renal Tumor Study Group Publications
cancer studies. We collaborate in planning, designing, running, monitoring and analyzing their clinical trials in lung cancer and mesothelioma. More than 75 hospitals in the Netherlands participate in these clinical trials.
Since 1993 the Biometrics Department provides data handling and statistical support to the SIOP Renal Tumor Study Group. Almost all children with nephroblastoma or Wilms’ tumor are treated within prospective, randomized multicenter trials conducted either by the International Society of Pediatric Oncology (SIOP-Europe) or by the Children’s Oncology Group (COG-North America). The SIOP Renal Tumour Study Group (RTSG) is an established international collaborative group (21 European countries, the Brazilian national study group and centres in Argentina, Australia, New Zealand, United Arab Emirate, South Africa and Ukraine). Since 1971, prospective randomised clinical trials have been conducted. In 1993, the sixth randomized controlled trial was launched. The SIOP 9301 trial was the first trial to study a reduction of therapy in Wilms’ tumors. The most recent trial, SIOP 2001, continued the SIOP philosophy of applying pre-nephrectomy chemotherapy to allow a response-adapted risk stratification of post-operative treatment intensity. In addition, for the first time the trial collects detailed prospective response data and biological material of all patients. The randomized trial has been closed, the data have been analyzed and a draft manuscript is circulating. The registration of patients continues and currently the database contains over 6500 patients with pediatric kidney tumors. Figure 1 shows the event-free survival of some of the larger groups within the database. Where appropriate, outcomes of histological groups or treatment are discussed and compared at regular meetings with representatives from the Renal Tumors’ Committee of the Children’s Oncology Group (COG), North America. As both study groups are dealing with rare tumors, where feasible the two study groups aim to undertake pooled data analyses to improve the reliability of the conclusions that can be drawn from either trial.
NVALT cancer studies The Department work together with the Dutch Chest Physician Association (NVALT) and functions as Data Centre for their
During 2012, 4 new studies have been designed and are proposed for Medical Ethics Committee approval and for KWF funding or pharmaceutical industry grants. In addition, several ideas are in discussion and would eventually result in new trials The NVALT12, a randomized study of docetaxel, cisplatin, bevacizumb with or without nitroglycerine patches in patients with stage IV non squamous non small cell lung cancer, started in 2011. 17 centers participated and only 1 more patient needs to be randomized to complete the total accrual of 222 patients. With 114 patients entered by 10 participating centers, the NVALT14 could be completed next year. This is a randomized trial comparing longstanding indwelling pleural catheters with pleurodesis as a frontline treatment for malignant pleural effusion. The NVALT 10 is a randomized phase II study comparing erlotinib 150 mg daily (monotherapy) versus erlotinib 150 mg with alternating chemotherapy (docetaxel for squamous (SQ) or pemetrexed for non-squamous (NSQ) pts) in relapsed nonsmall cell lung cancer patients. The planned accrual of two hundred thirty one patients was achieved, the final analysis was performed and results were presented orally at ASCO 2012 and the publication has been accepted by the JCO. The adjusted hazard ratio for progression free survival for all patients was 0.76 (95% CI., 0.58 - 1.02; p=0.06), for overall survival (OS) 0.67 (95% CI., 0.49 - 0.91; p=0.01). This improvement was primarily observed in NSQ subgroup. Common Toxicity Criteria grade 3+ toxicity occurred in 20% versus 56%, rash in 7% versus 15% and febrile neutropenia in 0% versus 6% in mono- and combination- therapy, respectively. In the NVALT11 study the value of prophylactic cranial irradiation (PCI) versus observation is studied in radically treated patients with stage III non-small cell lung cancer. It is done as a cooperation of the NVALT, the Dutch Lung Cancer Research Group (DLCRG) and the National Platform for Radiotherapy in Lung Tumors (LPRL). A total of 155 have been included in this study by 15 centers. Collaboration with the Italian and with the Danish Groups has not succeeded to increase the rate of accrual which is still lower than expected. An Italian cooperative group (GOIRC) was running a study similar to the NVALT 7 comparing Pemetrexed versus Pemetrexed and Carboplatin as Second-Line chemotherapy in advanced non-small-cell lung cancer. The results of Italian study and an analysis of the 2 studies pooled together were published this year in the J Clin Oncol (2012;30:4501-7). This pooled analysis was planned early when studies were starting with the goal of assessing the impact of the addition of carboplatin on the duration of overall survival. In spite of the fact that the 2 studies
Figure 1: Event-free survival of some of the larger groups within the SIOP Renal database.
Lubberink M, Direcks W, Emmering J, van Tinteren H, Hoekstra OS, van der Hoeven JJ, Molthoff CF, Lammertsma AA. Validity of simplified 3’-deoxy-3’-[(18)f]fluorothymidine uptake measures for monitoring response to chemotherapy in locally advanced breast cancer. Mol Imaging Biol. 2012;14:777-782 Graf N, van Tinteren H, Bergeron C, Pein F, van den Heuvel-Eibrink MM, Sandstedt B, Schenk JP, Godzinski J, Oldenburger F, Furtwangler R, de Kraker J. Characteristics and outcome of stage II and III non-anaplastic Wilms’ tumour treated according to the SIOP trial and study 93-01. Eur J Cancer. 2012;48:3240-3248 Verschuur A, van Tinteren H, Graf N, Bergeron C, Sandstedt B, de Kraker J. Treatment of pulmonary metastases in children with stage IV nephroblastoma with risk-based use of pulmonary radiotherapy. J ClinOncol.2012;30:3533-3539 Mertens LS, Meijer RP, Kerst JM, Bergman AM, van Tinteren H, van Rhijn BW, Horenblas S. Carboplatin based induction chemotherapy for nonorgan confined bladder cancer - a reasonable alternative for cisplatin unfit patients? J Urol. 2012;188:1108-1113 Timmers JM, Doorne-Nagtegaal HJ, Zonderland HM, van Tinteren H, Visser O, Verbeek AL, den Heeten GJ, Broeders MJ. The Breast Imaging Reporting and Data System (BI-RADS) in the Dutch breast cancer screening programme: its role as an assessment and stratification tool. EurRadiol. 2012;22:1717-1723 Boogerd W, Groenveld F, Linn S, Baars JW, Brandsma D, van Tinteren H. Chemotherapy as primary treatment for brain metastases from breast cancer: analysis of 115 one-year survivors. J Cancer Res ClinOncol.2012;138:1395-1403 Schaake EE, Kappers I, Codrington HE, Valdes Olmos RA, TeertstraHJ, van Pel R, Burgers JA, van Tinteren H, Klomp HM. Tumor response and toxicity of neoadjuvanterlotinib in patients with early-stage non-small-cell lung cancer. J ClinOncol. 2012;30:2731-2738
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Smets AM, van Tinteren H, Bergeron C, De Camargo B, Graf N, PritchardJones K, de Kraker J. The contribution of chest CT-scan at diagnosis in children with unilateral Wilms’ tumour. Results of the SIOP 2001 study. Eur J Cancer. 2012;48:1060-1065 De Langen AJ, Vincent A, Velasquez LM, van Tinteren H, Boellaard R, Shankar LK, Boers M, Smit EF, Stroobants S, Weber WA, Hoekstra OS. Repeatability of 18F-FDG uptake measurements in tumors: a metaanalysis. J Nucl Med. 2012; 53:701-708 De Weger VA, Turksma AW, Voorham QJ, Euler Z, Bril H, van den Eertwegh AJ, Bloemena E,Pinedo HM, Vermorken JB, van Tinteren H, Meijer GA, Hooijberg E. Clinical effects of adjuvant active specific immunotherapy differ between patients with microsatellitestable and microsatellite-instable colon cancer. Clin Cancer Res. 2012;18:882889 Westermann A, Mella O, Van Der Zee J, Jones EL, Steen-Banasik E, Koper P, Uitterhoeve AL, De Wit R, Van Der Velden J, Burger C, Schem BC, Van Der Wilt C, Dahl O, Prosnitz LR, van Tinteren H. Long-term survival data of triple modality treatment of stage IIB-III-IVA cervical cancer with the combination of radiotherapy, chemotherapy and hyperthermia - an update. Int J Hyperthermia. 2012;28:549-553 De Vries RR, Kauer P, van Tinteren H, van der Poel HG, Bex A, Meinhardt W, van Haarst EP, Horenblas S. Short-term outcome after cystectomy: comparison of two different perioperative protocols. Urol Int. 2012;88:383-389 Wessels R, de Bruin DM, Faber DJ, van Boven HH, Vincent AD, van Leeuwen TG, van Beurden M, Ruersa TJ. Optical coherence tomography in vulvar intraepithelial neoplasia. J Biomed Opt. 2012;17:116022 De Ronde JJ, Lips EH, Mulder L, Vincent AD, Wesseling J, Nieuwland M, Kerkhoven R, Vrancken Peeters MJ, Sonke GS, Rodenhuis S, Wessels LF. SERPINA6, BEX1, AGTR1, SLC26A3, and LAPTM4B are markers of resistance to neoadjuvant chemotherapy in HER2negative breast cancer. Breast Cancer Res Treat. 2013;137:213-23 Oprea-Lager DE, Vincent AD, van Moorselaar RJ, Gerritsen WR, van den Eertwegh AJ, Eriksson J, Boellaard R, Hoekstra OS. Dual-phase PET-CT to differentiate [18F]Fluoromethylcholine uptake in reactive and malignant lymph nodes in patients with prostate cancer. PLoS One. 2012;7:e48430 180
Ardizzoni A, Tiseo M, Boni L, Vincent AD, Passalacqua R, Buti S, Amoroso D, Camerini A, Labianca R, Genestreti G, Boni C, Ciuffreda L, Di Costanzo F, de Marinis F, Crinò L, Santo A, Pazzola A, Barbieri F, Zilembo N, Colantonio I, Tibaldi C, Mattioli R, Cafferata MA, Camisa R, Smit EF. Pemetrexed Versus Pemetrexed and Carboplatin As Second-Line Chemotherapy in Advanced Non-Small-Cell Lung Cancer: Results of the GOIRC 02-2006 Randomized Phase II Study and Pooled Analysis With the NVALT7 Trial. J Clin Oncol. 2012;30:4501-7 Tiseo M, Giovannetti E, Tibaldi C, Camerini A, Di Costanzo F, Barbieri F, Burgers JA, Vincent A, Peters GJ, Smit EF, Ardizzoni A. Pharmacogenetic study of patients with advanced non-small cell lung cancer (NSCLC) treated with second-line pemetrexed or pemetrexed-carboplatin. Lung Cancer. 2012;78:92-9 Wildeman MA, Fles R, Adham M, Mayangsari ID, Luirink I, Sandberg M, Vincent AD, Fardizza F, Musa Z, Armiyanto, Middeldorp JM, Gerritsen G, Suwanto R, Tan IB. Short-term effect of different teaching methods on nasopharyngeal carcinoma for general practitioners in Jakarta, Indonesia. PLoS One. 2012;7:e32756 Spoelstra FO, van der Weide L, van Sörnsen de Koste JR, Vincent A, Slotman BJ, Senan S. Feasibility of using anatomical surrogates for predicting the position of lung tumours. Radiother Oncol. 2012;102:287-9 Joerger M, Burgers SA, Baas P, Smit EF, Haitjema TJ, Bard MP, Doodeman VD, Smits PH, Vincent A, Huitema AD, Beijnen JH, Schellens JH. Germline polymorphisms in patients with advanced nonsmall cell lung cancer receiving first-line platinum-gemcitabine chemotherapy: a prospective clinical study. Cancer. 2012;118:2466-75 Korse CM, Taal BG, Vincent A, van Velthuysen ML, Baas P, Buning-Kager JC, Linders TC, Bonfrer JM. Choice of tumour markers in patients with neuroendocrine tumours is dependent on the histological grade. A marker study of Chromogranin A, Neuron specific enolase, Progastrin-releasing peptide and cytokeratin fragments. Eur J Cancer. 2012;48:662-71 Nijkamp J, Doodeman B, Marijnen C, Vincent A, van Vliet-Vroegindeweij C. Bowel exposure in rectal cancer IMRT using prone, supine, or a belly board. Radiother Oncol. 2012;102:22-9
had a similar design, the results were very different. The NVALT 7 showed a statistically significant improvement in PFS (HR=0.67) in favor of carboplatin plus pemetrexed as compared to permetrexed alone, while the Italian study showed no difference. This could be largely due to differences in the patient populations in particular 75% of patients in the Italian study had adenocarcinoma histology vs only 45% in the NVALT study
Studies with Nuclear Medicine Departments In addition to the studies of the Nuclear Medicine Department at the NKI-AVL, Biometrics Department works together with a similar Department at the VU. NIBIB1 is a collaboration between the NKI Biometrics Department and the VU Nuclear Medicine & PET research departments, the object of which was to assess the association between of FDG-PET metabolic and pathological response in solid extracerebral tumors. A systematic literature review was undertaken resulting in 88 datasets from 69 studies comprising 943 patients, and subsequently an individual level meta-analysis was performed. The meta-analysis consisted of an analysis of heterogeneity, construction of multivariable mixed-effects logistic regression models and summary receiver operating characteristic analyses (figure 2). We found significant heterogeneity in breast cancer studies, possibly due to variability in the definition of response (figure 3). While the association between metabolic and PA response appeared dependant on both therapy type (CT vs CRT) and baseline uptake rates. These results were presented in July 2012 at the Society of Nuclear Medicine’s annual congress, and are currently being prepared for publication. The two departments are following up this research with a subsequent meta-analysis addressing the prognostic value of FDG-PET with respect to survival outcomes.
Other studies We worked together with the Dutch Colorectal Cancer Group (DCCG) on the statistical analysis of several large phase III randomized studies in patients with advanced colorectal cancer. Currently the CAIRO 3 study is being carried out. The study compares maintenance treatment with capecitabine and bevacizumab versus observation after induction treatment with capecitabine, oxaliplatin, and bevacizumab as first-line treatment. Several interim analyses had been prepared and discussed in confidence by the Independent Data Monitoring Committee. The 64 centers participating have registered already 618 patients. We are the data processing and statistical center for the international studies within a project run by a radiotherapy study coordinator supported by the EC (the ART FORCE project). An electronic case record form have been developed based on our ALEA system. An extension has been programmed to be able to visualize the scans (properly anonymized) of every patient entered with the data collected for the patient. We collaborated within the lung CTMM project creating an electronic system to capture data of all participants in the country and we collected the data for the NKI patients. This system will be reviewed and will be maintain for future projects.
with the Radiotherapy Department a large randomized study in young women with early breast cancer (Young Boost trial) carried out by participating centers in the Netherlands, France and Germany. The total accrual of more than 2400 patients has been completed. Translational research and cosmetic results analyses based on serial pictures are currently being prepared. In addition, several phase I/II studies of radiotherapy alone or in combination with chemotherapy in breast, lung, stomach, etc are being coordinated. With the medical oncologists we collaborate in several multicentric studies. In 2012 accrual to the Matador study in breast cancer was completed with a total of 664 patients randomized of which tumor material is available for about 600 patients. Accrual to the neoadjuvant study in breast cancer (TRAIN study) was completed and the final analysis will be possible in 2013. The statisticians have collaborated with other departments of the institute and other, academic and not academic institutes in the region in a variety of studies many of which resulted in coauthorships as can be seen in the publication list.
ICT projects We have developed tools to assist and support us and the participating medical staff in running clinical trials. These developments have often been partly funded within EC projects to which we participated or that we managed during the last 25 years. The most important system we developed is ALEA, which is a comprehensive system to register, randomize and automatically distribute notifications of the randomization; and to allow the development of applications for studies, by which entry of data from the participating centers can be done directly into the Data Center databases (electronic case record forms or eCRF system). The system includes an audit trail in compliance with the requirements of the International Committee for Harmonization and produces standard based export facilities (CDISC, ODM and SDTM) as required by statistical best practice. Last year new features were incorporated into the system like a module to manage the drug supply and more extensive querying and reporting possibilities. In the latest project, (TENALEA Initial Deployment) our system was put into use in several European centers. The most important development of 2012 was the replacement of the form rendering Sharepoint system by a self developed program that has improved the speed by more than 20 times. Following the project, our Institute has given an option to the license of the system to a start up company, FormsVision, under the condition that the Services continued to be available to the Academic groups at a moderate marginal cost. Currently several centers in 12 European countries (Germany, France, UK, The Netherlands and Switzerland) are using the system. In addition, there is increased interest of commercial companies (pharma and CROs) to use the facilities for their own studies making the possibilities of successful continuation of the project more likely.
Figure 2: Multivariable mixed-effects analyses and summary receiver operating curve analyses of the all studies, and those with baseline uptake levels classified as having high repeatability.
Figure 3: Chi-square heterogeneity analyses of studies by treatment and tumor type.
We collaborate with medical departments in our Institute in carrying out, handling data and SAEs and providing statistical support for (single or multi center) clinical trials. We coordinate 181
Research facilities (per ultimo 2012)
Animal facility
Marco Breuer, head
Glassware
Anita de Bois-Bakker
Frits Mulkens
Esther Holman
Roel Sneepers
Francis Makatipu-Kambey
and our animal care-takers
184
Library
Suzanne Bakker
Animal Pathology and Histology Facility
Sumiati Baatje
Irene Benne
Lex de Vrije
Mieke de Mots
Sjoerd Klarenbeek
Meena Kanhai
Ellen Riem
Truud Kroeskamp
Ji-Ying Song
Jelrik van der Meer
Peptide Synthesis Facility
Dris el Atmioui
Martin van der Valk
Henk Hilkmann
Joost van Ooij Protein Expression & Purification Facility
Patrick Celie
Core facility Molecular Pathology & Biobanking
Linde Braaf
John de Widt
Annegien Broeks
Renate de Groot
Radionuclide Center
Theo Lamers
Ingrid Hofland
Desiree Luijten-Verwoerd
Donne Majoor
José Overwater
Robotics & Screening Facility
Roderick Beijersbergen
Denis Peters
Pasi Halonen
Huib Vroege
Cor Lieftink
Marcel Winter
Ben Morris
Cryogenic Storage
Minze Dijkstra
Sequence Facility
Abderrahim Ajouaou
Erwin Kambey
Roelof Pruntel
Digital Microscopy Facility
Lenny Brocks
Technology Transfer Office
Hylke Galama
Lauran Oomen
Rik Grosveld
Bram van den Broek
Frank Hoorn
Marije Marsman
Electron Microscopy Facility
Hans Janssen
Anje Raven
Nico Ong
Koen Verhoef
Flowcytometry Facility
Anita Pfauth
Transgenesis Facility
Rahmen Bin Ali
Frank van Diepen
Tanya Braumuller
Ivo Huijbers
Genomics Core Facility
Shan Baban
Paul Krimpenfort
Erwin Bekema
Colin Pritchard
Wim Brugman
Fina van de Ahé
Ron Kerkhoven
Roel Kluin
Marja Nieuwland
Iris de Rink
Bernd van der Veen
Arno Velds
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Education in oncology
The Netherlands Cancer Institute offers a variety of opportunities for practical and theoretical training to (trainee) technicians, University Master students, PhD students and post-doctoral fellows. Research and clinical staff and their group members are involved in theoretical and practical training. Many staff members have joint appointments as professors at Dutch universities and an even larger number contribute to the regular curriculum at various universities. The research divisions attract students from universities throughout the country. The Netherlands Cancer Institute has a formal affiliation with the Science faculty of the University of Amsterdam (UvA) and is committed to make a contribution to Master student teaching. The institute participates in the Oncology Graduate School Amsterdam, together with the medical faculties of the UvA and the Free University (VU), referred to as Academic Medical Center (AMC) and VU medical center (VUmc), respectively. All educational activities are supervised by the Teaching Committee, which consists of Jannie Borst (chair and dean Master students), Hein te Riele (general affairs), Roderick Beijersbergen (Master course), Fred van Leeuwen (Post-doctoral affairs), John Hilkens (HLO students and publicity), Titia Sixma (dean PhD students) and Fons Balm (clinical teaching). In the course of the year Fred van Leeuwen took over the duty as dean for the post-docs from Peter Peters.
table 1 Course in Experimental Oncology Epidemiology
F van Leeuwen, M Schaapveld
Surgery
E Rutgers
Radiodiagnostics
E Vegt
Pathology
J Wesseling
Molecular diagnostics*
R Kerkhoven, S Linn
Conventional pharmacotherapy
S Rodenhuis
Radiotherapy**
M Verheij
DNA damage response and Apoptosis*
C Vens, J Borst
Telomerase and cancer*
R Beijersbergen
Genomic instability*
H te Riele
Signal transduction*
W Moolenaar
Cell cycle
R Bernards
Oncogene-tumor suppressor gene interactions
D Peeper
Cell division
R Wolthuis
Tumor microenvironment
K de Visser
Mouse models of cancer*
J Jonkers
Immunology and Immunotherapy*
T Schumacher, J Haanen
Analysis of protein structure
T Sixma
Rational drug development
A Huitema
Drug delivery*
A Schinkel
Epigenetics
J Dannenberg
* including tutorial ** including tour
MASTER STUDENTS The program in Experimental Oncology attracts Master students of all national universities (see http://www.nki.nl/Research/Career/Masters+students). Students have backgrounds in (Medical) Biology, Health Sciences, Chemistry, Pharmacology, Medicine, or Psychology. The program offers combined practical and theoretical training in various aspects of experimental oncology. Practical training includes participation in ongoing research projects for a minimum of 4 months. In 2012, 60 Dutch university Master students and 4 students from abroad completed a placement of 5-9 months at the biomedical research divisions. The students came primarily from the Free University Amsterdam (VU) (26) and the University of Amsterdam (UvA) (14), but also from Utrecht University (9), Leiden (6), Wageningen (3), and Twente (2). The institute also provides practical training opportunities to trainee technicians, who stay for similar periods of time as the university students and like these, often make significant contributions to research progress of the PhD students and post-docs who supervise them. There is an increasing demand from Universities for placing Bachelor students for the three month internship that concludes their program and we have accommodated 14 of them this year. The core element of theoretical training is the course in Experimental Oncology, given twice yearly (Table 1). This course is compulsory for Master students who do an internship at the NKI, but in addition attracts many students from master programs throughout the country. We routinely host about 45 students per course. The course comprises a comprehensive overview of the molecular basis of cancer, cancer diagnosis, cancer treatment and the development of novel cancer therapies. It spans the field of cancer from a clinical perspective to translation research to basic research. The subjects are discussed during lectures and tutorials by our highest level clinical and research professionals with expertise in the different subjects of the course. The course is rated very highly in University evaluations and considered by students as a very valuable part in their education in the field of oncology.
186
PhD STUDENTS The PhD students at the Netherlands Cancer Institute participate in the Oncology graduate school Amsterdam (OOA) together with the oncology departments of the VUmc and the AMC. The number of registered students has been rising rapidly in the past 2 years. In 2012, the institute had ~180 PhD students registered with the OOA. In this year 17 students defended a PhD thesis at a Dutch university. Students participate in research of their group and in interdepartmental work discussions. In addition, the students follow the OOA training program, with courses (Table 2) and an annual retreat. Apart from courses on different topics in cancer research, the OOA offers a course on scientific English. Students with no prior background in cancer research can participate in the Experimental Oncology course. Part of the training of the NKI students are discussions with experts in the field of oncology. The Friday morning seminar speakers take their lunch with a delegation of the students. Twice a year a list of speakers is sent round and each graduate student participates several times per year in these opportunities to exchange views with experts in the field. The PhD student retreat focuses entirely on the research of the graduate students themselves. At this retreat, first year students present their work in the form of a poster and older students give presentations. Moreover students are in charge of chairing sessions and discussions as well as peer review, giving a prize for the best poster and best presentation. In this manner, the retreat provides training in presentation and interaction skills, but it also provides an overview of the research in the OOA at an early stage of the student’s career. This provides a good opportunity for translational interaction and bottom-up research, allowing the graduate students to contribute significantly to interaction between different research groups. Senior graduate students can participate in a joint retreat with other cancer institutes in Europe. In 2012, this event was hosted by at the NKI, organized by a team of NKI 187
graduate students (Michiel Boekhout, Gerjon Ikink, Silvia Ariotti, Wei Chen, Roel Wilting) with participants from among others British CrUK institutes, the Italian IFOM and the German DKFZ contributing to a program of scientific lectures and posters as well as an enthusiastic social session. This retreat gives students the opportunity to compare notes among excellent cancer institutes throughout Europe. The progress of the research is monitored annually by a supervisory committee. Each committee has independent members within and outside the division. The committee discusses progress with the supervisor and student separately and participates in a joint discussion of the research. After two years of PhD research the student, supervisor and committee evaluate the state of the project in a midterm review. At this more elaborate meeting the likelihood of achieving a PhD within a reasonable time frame is discussed. This meeting can be used to redefine goals if necessary. The students are represented in the PhD student-council that meets with the Dean of graduate student affairs on a regular basis, as well as upon special request. They also mediate communication between the graduate students and research manager or board of directors. Table 2 OOA Graduate student courses 2012
March, September and November
English Writing and Presenting in Biomedicine
Total of 36 participants
17-19 October
Annual Graduate Student Retreat, Ermelo
Titia Sixma
126 participants of NKI, VU and AMC
26-30 November
Basic Medical Statistics
Michael Hauptmann, Wilma Heemsbergen,
Marta Lopez Yurda
47 participants
16-23 April
Cancer Genome and Proteome
Daniel Peeper and Roderick Beijersbergen
23 participants
19-28 September
Preclinical assays in cancer therapy
Jos Jonkers and Sven Rottenberg
20 participants
POSTDOCS In 2012 the Netherlands Cancer Institute hosted approximately 140 postdoctoral fellows, almost half of which are from abroad and with equal gender representation. The postdocs at the Netherlands Cancer Institute are represented by the NKI postdoc committee, which is actively involved in improving the Netherlands Cancer Institute as a workplace and in enhancing career development of young scientists at the Netherlands Cancer Institute. To achieve this, the postdoc committee works together with the dean of postdoc affairs, the career coach, and human resources. In 2012, Peter Peters stepped down as dean and Fred van Leeuwen was appointed as the new dean of postdoc affairs. In 2012, the NKI postdoc committee held a survey among their peers and final year PhD students to identify areas in which the institute could further improve to better serve the needs of its young scientists. They obtained constructive feedback from 247 research staff to generate the report and identified several issues that deserve followup by the Netherlands Cancer Institute management, staff, and postdocs themselves. Based on this extensive survey, several changes have already been implemented by the management, such as taking measures to improve formal and informal communication between PIs and postdocs, improving short-stay accommodation for foreign scientists, and updating the intra- and internet pages. Guided by the results of the 2012 survey, the new postdoc committee and dean of postdoc affairs are currently developing career events to be organized at the Netherlands Cancer Institute in the coming years. To further support the personal growth and professional development of the postdocs the NKI embraces the Postdoc Career Development Initiative (PCDI, www.pcdi.nl). The PCDI, which was originally an initiative from the Netherlands Cancer Institute, is now a professional organization that organizes a yearly retreat at Kapellerput in Heeze for postdocs in Health Sciences. There, postdocs can reflect on their current position, explore possibilities for their next career step, discuss developments in the scientific world, broaden their horizons, find out how varied careers in the different areas of the Life Sciences can be and how important it is to think ahead and choose a career path that suits them best. To give postdocs a head start on career development, PCDI offers them three days of keynote lectures by successful (ex)academics on their personal careers, workshops to discover and enhance their talents, a forum discussion and networking sessions with PhDs who pursued different career paths within and outside the Life Sciences. In 2012, the Netherlands Cancer Institute sponsored PCDI participation of 9 postdocs from the Netherlands Cancer Institute.
International PhD Student Cancer Conference 2012, organized for and by PhD students. In 2012 the meeting was organized by PhD students from the Netherlands Cancer Institute.
188
189
Clinical trials
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
All sites
190
M07KUC
A phase I, open-label, study to assess the safety and tolerability of KU-0059436 in combination with Carboplatin, KU-0059436 in combination with Paclitaxel/ Carboplatin doublet and KU-0059436 in combination with Paclitaxel in the treatment of patients with advanced solid tumours
JHM Schellens
I
25-6-2007
M07MKC
A phase I dose escalating study evaluating MK-1775 in both monotherapy and in combination with Gemcitabine, Cisplatin or Carboplatin in adult patients with advanced solid tumors
JHM Schellens
I
21-2-2008
M08HYT
A phase I open-label study of the safety, tolerability and pharmacokinetics of two schedules of oral topotecan in combination with pazopanib in subjects with advanced solid tumors
JHM Schellens
I
15-9-2008 (1-5-2012)
M09BGJ
A phase I open-label, multicenter, dose escalation study of oral BGJ398, a pan FGF-R kinase inhibitor in adult patients with advanced solid malignancies
JHM Schellens
I
10-12-2009
M09DAZ
A phase I open label multicenter study to assess the safety and tolerability, pharmacokinetics, preliminary anti-tumor activity of ascending doses of AZD4547 in patients with advanced solid malignancies
JHM Schellens
I
14-10-2009
M09GDC
A phase Ib open label, dose-escalation study of the safety and pharmacology of GDC-0941 in combination with erlotinib with advanced solid tumours
JHM Schellens
I
12-8-2009 (26-11-2012)
M09NIB
The NIB-Cohort study, therapeutic drug monitoring of tyrosine kinase inhibitors
JHM Schellens
other
9-6-2009
M09RGD
A phase II, open label, non-randomized, multicenter, pilot, efficacy study of [F-18] RGD-K5 Positron Emission Tomography (PET) as a tool to monitor response to an anti-angiogenic drug
TJM Ruers
II
20-5-2010 (26-6-2012)
M10AZD
A phase I open-label, multicenter study to assess the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of ascending doses of AZD5363 under adaptable dosing schedules in patients with advanced solid malignancies
JHM Schellens
I
2-12-2010
M10BBP (CBEZ235A2118)
A phase Ib multi-center, open-label, 4 arm dose-escalation study of oral BEZ235 and BKM120 in combination with weekly paclitaxel in patients with advanced solid tumors and weekly paclitaxel/trastuzumab in patients with HER2+ metastatic breast cancer
N Steeghs
I
14-3-2011
M10BEZ (CBEZ235A2101)
A phase I/Ib, multi-center, open-label study of BEZ235 administered orally on a continuous daily dosing schedule in adult patients with advanced solid malignancies including patients with advanced breast cancer
JHM Schellens
I
17-2-2011 (4-5-2012)
M10FES (LIFE-110)
An open-label, dose escalation, pharmacodynamic, pharmacokinetic and effect of food phase 1 study of E7820 to determine the maximum tolerated dose following twice daily oral administration in subjects with unresectable solid tumours
JHM Schellens
I
5-4-2011
M10IPC (NVALT 14)
A randomized trial comparing longstanding indwelling pleural catheters with pleurodesis as a frontline treatment for malignant pleural effusion
MM van den Heuvel
III
31-1-2011
M10PKS
Use of individual PK-guided sunitinib dosing: A feasibility study in patients with advanced solid tumors
N Steeghs
other
14-3-2011
M11BYL (CBYL719X210)
A phase 1a, multicenter, open label dose-escalation study of oral BYL719, in adult patients with advanced soild malignancies, whose tumors have a PIK3CA gene
JHM Schellens
I
28-7-2011
M11EVE (CPCT-03)
A two parts, biomarker study to identify genetic aberrations predictive for response on Everolimus in solid tumors without regular treatment options
N Steeghs
other
3-10-2012
191
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
N10MTD
Phase I interaction study of docetaxel and tolbutamide with supplementation of Milk Thistle
JHM Schellens
I
22-3-2011
N11ODR
Mass balance of docetaxel after administration of Oral Docetaxel in combination with Ritonavir
JHM Schellens
I
24-1-2012
N12OST (PercuSpect)
Discrimination of benign and malignant human tissue during percutaneous interventions using optical spectroscopy techniques
TJM Ruers
other
13-9-2012
E22051 (SUPREMO)
SUPREMO, an MRC phase III randomised trial to assess the role of adjuvant chest wall irradiation in 'intermediate risk' operable breast cancer following mastectomy
NS Russell
III
27-2-2007
M03RBC (YOUNG BOOST)
Radiation dose intensity study in breast cancer in young women: a randomized phase III trial of additional dose to the tumor bed ("young boost")
GMM Bartelink
III
29-3-2004 (4-1-2012)
M04MAT (MATADOR)
Microarray Analysis in breast cancer to Tailor Adjuvant Drugs Or Regimens
SC Linn
III
26-4-2004 (19-11-2012)
M05BRI (BRIGHT)
Long term risk of breast cancer following treatment of Hodgkin's disease
NS Russell
other
5-1-2006
M05HIR (HIRISE)
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)
M van Beurden
other
31-5-2005 (13-3-2012)
M06TM2 (TEAM II)
A multicentre, prospective phase II trial investigating the efficacy of neoadjuvant hormonal therapy with exemestane for six months B. Randomised, multicentre, prospective, phase III trial investigating the efficacy and safety of the addition of ibandronate to adjuvant hormonal therapy in postmenopausal women with hormone receptor positive early breast cancer
SC Linn
III
26-9-2006
M07CBE
Late effects of chemotherapy on brain functioning in the elderly
SSB Schagen
other
1-7-2008
M08BCP (BOOG 2003-04)
Prospective and Retrospective register study of the German Adjuvant Cancer Study Group (GABG) for diagnosis and treatment of breast cancer in pregnancy
SC Linn
other
27-3-2008
M08HAT
A randomized phase II study of concomitant trastuzumab, bevacizumab with paclitaxel versus trastuzumab and bevacizumab followed by the combination of trastuzumab, bevacizumab and paclitaxel at progression as first-line treatment of patients with metastatic breast cancer with H er2-neu overexpression
SC Linn
II
20-4-2009
M11GLP (GALAPAGOS)
An open-label dose escalating phase 1b study for the assessment of safety, tolerability, pharmacokinetics and pharmacodynamics of multiple intravenous doses of GLPG0187 in subject with solid tumors
JHM Schellens
I
27-9-2011
M11LDK
A phase I, multicenter, open-label dose escalation study of LDK378, administered orally in adult patients with tumors characterized by genetic abnormalities in anaplastic lymphoma kinase (ALK)
N Steeghs
I
5-4-2011
M11MSC (28062)
A multicentre, open label, Phase I trial of the MEK inhibitor MSC1936369B given orally to subjects with solid tumours
JHM Schellens
I
4-7-2011 (1-9-2012)
M11PCT (CPCT-02)
Development of a platform for next-generation DNA sequencing based personalized treatment for cancer patients : protocol to obtain biopsies from patients with metastatic cancer
JHM Schellens
other
24-1-2012
Phase I open label multicenter dose-escalation study to evaluate safety, pharmacokinetics and activity of RO5479599, a glycoengineerd antibody against HER3, administered as IV infusion either alone or in combination with Cetuximab or in combination with Erlotinib in patients with metastatic and/or locally advanced malignant HER3-positive solid tumors of epithelial cell origin
JHM Schellens
An open-label, phase I/IIa dose escalating study of 2B3-101 in patients with solid tumors with brain metastases
D Brandsma
A phase I open label two stage randomized cross over comparative (single dose) pharmacokinetic and safety study of two formulations of CO-1.01 for injections in patients with advanced solid tumors
JHM Schellens
M11TKI
A multicenter open label study to assess pharmacokinetics of TKI258 in adult cancer patients with normal and impaired hepatic function
JHM Schellens
other
24-1-2012
M11TWE (TWEAK)
A phase I Multiple Ascending Dose (MAD) study of RO5458640, a Humanized Monoclonal Antibody against the TNF-like weak inducer of Apoptosis (TWEAK) Ligand, in patients with advanced solid tumors
JHM Schellens
I
5-7-2011
M12DOV
A phase I multi-center open-label drug-drug interaction study to assess the effect of the CYP1A2 inhibitor, fluvoxamine, on dovitinib (TKI258) pharmacokinetics in patients with advanced solid tumors
JHM Schellens
I
21-11-2012
M12SAR (TED12318)
A phase I study to assess the safety, tolerability, pharmacokinetics and biological activity of SAR405838 in patients with advanced cancer
JHM Schellens
I
28-6-2012
M12SEN (senior)
Observational study to evaluate pharmacokinetics and pharmacodynamics of docetaxel,paclitaxel, doxorubicine, gemcitabine, vinorelbine and capecitabine in elderly patients
JHM Schellens
N07DOW
Weekly administration of oral Docetaxel in combinaton with Ritonavir
S Marchetti
I
14-11-2007
N07NEX
Phase I study of gemcitabine and carboplatin plus sorafenib in patients with advanced solid tumors
JHM Schellens
I
29-1-2008
M08MAM (MARI)
An exploratory clinical study for initial validation of a novel small ring device for positron emission mammotomography
RA Valdes Olmos
other
29-4-2009
N08EDO
Phase I interaction study of docetaxel with supplementation of St. John's wort or Echinacea
JHM Schellens
I
3-2-2009 (4-12-2012)
M08MUL (MULTISENT)
Multicenter feasibility study of the sentinel node procedure in patients with multiple breast tumors
HSA Oldenburg
other
16-4-2009
N08NPM
NVALT Palliative care Protocol on malignant pleural effusion
MM van den Heuvel
other
13-3-2008
M08PBI (PAPBI)
Image guided Preoperative Accelerated partial Breast Irradiation (PAPBI): defining radiotherapy sensitivity
PH Elkhuizen
other
1-10-2009
N10BOM
Weekly administration of (bi-) daily Oral Docetaxel in combination with Ritonavir
JHM Schellens
I
17-5-2010 EJTh Rutgers
other
27-10-2009
Proof of principle and pharmacological phase 0 crossover study with controlled release capecitabine
S Marchetti
other
17-11-2011
M09SRB (SNARB)
Sentinal Node and recurrent breastcancer; regional staging and registration
N10CRC (ModraCape001)
7-1-2010
JHM Schellens
I
9-9-2010
Randomized phase II/III study of individualized neo-adjuvant chemotherapy in triple negative breast tumors
II/III
Development and clinical activity of low dose metronomic chemotherapy with oral paclitaxel
M09TNM (Neo-TN)
S Rodenhuis
N10MOP
M11RCE (huMab HER3)
M11TBB
M11TCO
192
Breast I
I/II
I
other
17-11-2011
6-7-2011
4-4-2011
13-9-2012
193
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
A phase Ib/ II, multi-center, open-label study to evaluate the efficacy of AUY922 in combination with Trastuzumab in patients with locally advanced or metastatic HER2-positive breast cancer, that has progressed after or during at least one Trastuzumab-containing regimen
N Steeghs
28-2-2011 (8-3-2012)
N10BMT
Effect of biphosphonates on telangiectasia in irradiated breast cancer patients (biphosphonate modulation of telangiectasia)
NS Russell
other
24-6-2011
N10RDA
Pilot study to determine the utility of a Likert-like scale to assess patients' experience of radiation dermatitis during radiotherapy for breast cancer
NS Russell
pilot
4-8-2011
M10DCI (DCIS)
A randomized phase III study of radiation doses and fractionation Schedules for ductal carcinoma (DCIS) of the Breast
NS Russell
N10RFS (OpSpect)
Observational study into specific in vivo human discrimination between benign and malignant tissue using a combination of diffuse reflectance and fluorescence spectroscopy
TJM Ruers
other
25-10-2010
M10DEN (D-CARE)
A randomized, double blind placebo controlled multi-center phase III study of Denosumab as adjuvant treatment for women with early-stage breast cancer at high risk of recurrence (D-CARE)
SC Linn
N11ISN
Monitoring of the "healthy" immune response in the sentinel lymph node of patients undergoing a prophylactic mastectomy
EJTh Rutgers
other
27-7-2011
M11FAM (FaMRIsc)
Breast density as indicator for the use of mammography or MRI to screen women with familiar risk for breast cancer: a RCT
EJTh Rutgers
N12CLM
Determining the sensitivity and specificity of circulating tumor cells and cytology in cerebrospinal fluid of patients clinically suspected for leptomeningeal metastases
D Brandsma
I
19-6-2012
M12APB (BEECH)
A Phase I/Ib, multicentre, study comprising a safety run-in of AZD5363 when combined with Paclitaxel in patients with advanced or metastatic breast cancer; followed by a randomised expansion of AZD5363 when combined with Paclitaxel vs Paciltaxel plus placebo in patients with ERPpositive advanced or metastatic breast cancer, stratified by PIK3CA mutation status
JHM Schellens
N12DBS
Clinical evaluation of dried blood spots for the determination of tamoxifen and endoxifen levels
SC Linn
other
29-11-2012
M12BRC (BRAVA)
BRCA mutations and ovarian ageing in normo-ovulatery women
LE van der Kolk
other
N12OLG (oligo)
High-dose alkylating chemotherapy in oligo-metastatic breast cancer harboring homologous recombination deficiency
GS Sonke
III
3-7-2012
20-9-2012
M12DEN (DENSE)
Early detection of breast cancer in women with dense breasts
CE Loo
Okselklieroperatie zonder drain postoperatief en met behulp van het LigaSure Precise instrument tijdens de operatie
II
6-6-2012
19-9-2012
N12PRE (PRECISE)
EJTh Rutgers
other
M12LJM (CLJM16X2102)
A multicenter, open-label, dose escalation, Phase I study of LJM716 administered intravenously in combination with trastuzumab in patients with HER2 overexpressing metastatic breast cancer (CLJM16X2102)
N12TCB
pilot
23-11-2012
I
3-10-2012
Transarterial chemoembolization with drug-eluting beads loaded with doxorubicin for the treatment of metastatic breast cancer to the liver: a pilot study
HJ Teertstra
JHM Schellens
M12RAD (BALLET)
An open-label, multicenter, expanded access study for postmenopauzal women with estrogen receptor positive locally advanced or metastatic breast cancer who have progressed following prior endocrine therapy, investigating the treatment of everolimus (RAD001) in combination with exemestane
SC Linn
other
19-9-2012
M12SSU
Detectie van ontstekingsgeassocieerde eiwitprofielen in het serum, speeksel en urine van patiënten met mammatumoren
EJTh Rutgers
other
17-4-2012
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
S Rodenhuis
II
21-3-2005
N06GLB
Phase I study of gemcitabine plus lapatinib (GW572016) in women with advanced breast cancer
JHM Schellens
I
16-5-2007
N07BOS (BOSOM)
Genetic determinants of survival and second breast cancer development in premenopausal breast cancer patients
EJTh Rutgers
other
12-12-2007
N07MAN (Mandjes studie)
Randomized phase II/III study of second-line endrocrine treatment followed by capecitabine versus capecitabine followed by endrocrine treatment in patients with metastatic ER positive breast cancer
S Rodenhuis
II/III
3-4-2008 (11-5-2012)
N08AFT (AFTER)
A randomized prospective trial of 2-6 weeks pre-operative hormonal treatment for hormone receptor positive breast cancer: Anastrozole +/- fulvestrant or tamoxifen exposure - response in molecular profile
SC Linn
II
N08RMB
Tumorresponse monitoring in patients with breast cancer treated with primary systemic therapy: towards predicting response in both the primary tumor and in axillary lymph nodes
MTFD Vrancken Peeters
N09PRF (PRF4PMPS)
Analgesia and nerve function following pulsed radiofrequency for postmastectomy pain
A Lukas
M10ATR (CAUY922A2109)
194
I/II
III
III
other
I
9-6-2010
16-2-2012 (27-7-2012)
30-11-2011
4-10-2012
Gastro Intestinal M06CRI (CRITICS)
A multicenter randomized phase III trial of neo-adjuvant 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)
A Cats
III
11-1-2007
M06SCR (SCRIPT)
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
A Cats
III
9-5-2006
M07CBO (CAIRO3)
Maintenance treatment with capecitabine and bevacuzimab versus observation after induction treatment with capecitabine, oxaliplatin and bevacuzimab as first-line treatment in patients with advanced colorectal carcinoma, a randomised phase III study
A Cats
III
4-9-2007 (1-6-2012)
M07HBT (HerBerT)
Feasibility study of external beam radiation therapy followed by high-dose rate endorectal brachytherapy (HDBRT) in inoperable rectal cancer patients
B van Triest
I/II
25-7-2007
4-8-2008
M07NAR (NARCIS)
Neo-Aduvant Radiotherapy-Chemotherapy In Stomach Cancer. Induction therapy with carboplatin, paclitaxel and radiotherapy in patients with locally advanced gastric cancer.
E Jansen
I/II
28-1-2008
other
23-9-2008
M08ACL (SILENT)
Accelerated growth of synchronous colorectal liver metastases: effects of neo-adjuvant therapy
TJM Ruers
II
21-2-2008
A Cats
other
2-6-2010
M08CEL (CELSIUS)
Capsule endoscopy in Lynch Syndrome for small intestinal tumor screening
other
13-7-2010 (5-11-2012)
195
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
N11BMT
Bevacizumab modulation of telangiectasia in irradiated rectal cancer Patients
NS Russell
other
8-12-2011
N12CLM
Determining the sensitivity and specificity of circulating tumor cells and cytology in cerebrospinal fluid of patients clinically suspected for leptomeningeal metastases
D Brandsma
I
19-6-2012
M08DCS
Tumor destruction and DC activation in situ: towards in vivo loaded DC Vaccines
TJM Ruers
pilot
18-9-2008 (19-12-2012)
M08GPO (PROFOC)
Genetic and protein profiling in patients with oesophageal cancer
JW van Sandick
other
1-9-2008
M08LYN (CHROMOLYNCH)
Chromoendoscopy in Lynch syndrome patiënt
A Cats
other
13-7-2010
N12INT
Pilot study to evaluate the tumor-reactivity of infiltrating T cells in human malignancies
TNM Schumacher
pilot
5-9-2012
M08MEK
Open-label, multicenter, dose-escalation phase I study with extension to evaluate safety, pharmacokinetics and activitiy of RO4987655, a MEK inhibitor, administered orally as monotherapy in patients with advanced tumors
JHM Schellens
I
4-2-2009 (1-12-2012)
N12NSL (NerveSpect)
In vivo identification of peripheral nerve bundles during surgery using optical spectroscopy techniques: a pilot study
TJM Ruers
pilot
19-11-2012
M09OCB
A pilot evaluating response to induction chemotherapy with oxaliplatin, capecitabine and bevacizumab in patients with extensive peritoneal carcinomatosis of colorectar origin
VJ Verwaal
pilot
25-3-2010
M10BDO (B-DOCT)
Phase II study of docetaxel, oxaliplatin, capecitabine with bevacizumab and trastuzumab in case of human epidermal growth factor receptor 2 (HER2)positivity in patients with locally advanced or metastatic gastric cancer or adenocarcinoma of the gastro-oesphageal junction
A Cats
II
8-2-2011
M11BIO (CPCT-01)
Feasibility study of biomarker development for response prediction by large scale DNA mutational analysis of metastatic lesions
N Steeghs
other
M11BLP (SPRINT)
A multicenter randomized open label mechanism of action trial on the biological effects of the therapeutic cancer vaccine Stimuvax (L-BLP25) in rectal cancer subjects undergoing neoadjuvant chemoradiotherapy
TJM Ruers
II
M11CAR (CARTS)
Chemoradiotherapy for rectal cancer in the distal rectum followed by organ – sparing transanal endoscopic microsurgery
A Cats
II
31-1-2012 (24-8-2012)
M11SOM
A multicenter, randomized, blinded efficacy and safety study of pasireotide LAR vs octreotide LAR in patients with metastatic carcinoid tumors whose diseaserelated symptoms are inadequately controlled by somatostatine analogues. CSOM230C2303
M Tesselaar
III
17-11-2011 (1-2-2012)
M12ABL (ABEL)
Tailored patient values elicitation task for rectal cancer treatment decisionmaking
BMP Aleman
other
31-5-2012
M12HAN (HANARO)
A multicenter, prospective feasibility follow-up study to evaluate a fully covered stent with new antimigration properties for the palliation of malignant dysphagia
H Boot
other
4-4-2012 (29-10-2012)
M12HPS (eclipse)
Endoscopic surveillance using narrow band imaging in patients with hyperplastic polyposis syndrome (HPS); a multicenter cohort study
ME van Leerdam
other
24-9-2012
M12LGX
A phase Ib/II multicenter, open label, dose escalation study of LGX818 and cetuximab or LGX818, BYL 719 and cetuximab in patients with BRAF mutant colorectal cancer
JHM Schellens
I/II
15-11-2012
Serum and tissue protein profiling and tumour genetic analysis in patients with potential premalignant conditions or colorectal cancer
A Cats
Pilot study on the use of fluorecence imaging of lymph nodes during colorectal lymphadenectomy using indocyanine green
TJM Ruers
N10ICG
Pilot study on the use of fluorescence imaging of lymph nodes during colorectal lymphadenectomy, using indocyanine green
TJM Ruers
pilot
16-2-2011 (28-11-2012)
N10RFS (OpSpect)
Observational study into specific in vivo human discrimination between benign and malignant tissue using a combination of diffuse reflectance and fluorescence spectroscopy
TJM Ruers
other
25-10-2010
N05STP
N08ICG
196
other
pilot
Gynaecological M05HIR (HIRISE)
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)
M van Beurden
other
31-5-2005 (13-3-2012)
7-12-2011
M05PPO
Proteomic patterns in blood and tissue of ovarian cancer patients
WJ van Driel
other
12-1-2006
M05SNV (GROINSS-V II)
GROningen International study on sentinel nodes in vulvar cancer
WJ van Driel
other
26-3-2007
24-1-2012
M06HRT (NOVARIA)
The effect of hormonal replacement therapy on menopausal complaints related to biochemical changes in surgically and naturally postmenopausal women. A prospective observational comparative study
CM Korse
other
25-9-2006
M06OVH (OVHIPEC)
Phase III randomised clinical trial for stage III ovarian carcinoma randomising between secondary debulking surgery with or without hyperthermic intraperitoneal chemotherapy
WJ van Driel
III
4-1-2006
M06RTE (PORTEC-3)
Randomised phase III trial comparing concurrent chemoradiation and adjuvant chemotherapy with pelvic radiation alone in high risk and advanced stage endometrial carcinoma
B van Triest
III
28-3-2007
M07RCV
Phase II study of definitive radiochemotherapy for locally advanced squamous cell cancer of the vulva: an efficacy study
WJ van Driel
II
26-6-2007
M10MKO
Phase II and pharmacological study with WEE-1 inhibitor MK-1775 combined with carboplatin in patients with p53 mutated epithelial ovarian cancer
JHM Schellens
II
8-7-2010
M11CIM
Immunological aspects of combined chemo-immunotherapy in patients with advanced cervical cancer
GG Kenter
other
15-5-2012
M11CIR (Circle 2)
Charting of immune reactivity against HPV in patients with HPV-induced (pre-) malignant lesions: The Circle 2 study
GG Kenter
other
5-4-2012
M11LOC (LapOvCa)
Laparoscopy to predict the result of primary cytoreductive surgery in advanced ovarian cancer patients
WJ van Driel
other
5-7-2011
M12RTE (PORTEC-4)
Postoperative radiation therapy for endometrial carcinoma. Multicenter randomised phase III trial comparing vaginal brachytherapy (two dose schedules) with observation after surgery in patients with endometrial carcinoma with highintermediate risk features
M Bloemers
III
11-12-2012
M12SOC (SOCcer)
A randomized controlled multicenter phase III cytoreductive surgery followed by chemotherapy versus chemotherapy alone for recurrent platinum-sensitive ovarian cancer
WJ van Driel
III
5-12-2012
19-1-2006
30-12-2008
197
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
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 adv ovarian ca
JHM Schellens
8-9-2005 (28-11-2012)
N07CRH (gossypol)
N10OCT
Optical vulvar biopsy by optical coherence tomography (OCT)
M van Beurden
other
N12INT
Pilot study to evaluate the tumor-reactiviity of infiltrating T cells in human malignancies
TNM Schumacher
pilot
N05CGO
I
Study Phase coordinator
Activated (closed)
Phase I/II study of combined treatment with AT-101, cisplatin and radiotherapy in patients with locally advanced head and neck cancer
M Verheij
I/II
16-2-2010
N07MCP
Microcirculatory changes during photodynamic therapy (PDT) in squamous cell carcinoma of the oral cavity and oropharynx
MP Copper
other
21-5-2007 (14-12-2012)
24-8-2010 (14-12-2012)
N07VIN
Transoral resection of stage I-II carcinomas of the oropharynx by robot-assisted surgery: a feasability study
M van de Brekel
other
12-3-2007 (3-12-2012)
5-9-2012
N08HHF
Validation of hypoxia imaging of cancer in the head and neck area with FAZA-PET
WV Vogel
II
22-10-2008
N09BIO
Prospective study of changes in the oral biofilm and the composition of salivary proteins in patients, being irradiated for a tumour in the head-and-neck area
AJM Balm
other
25-3-2010 (28-11-2012)
N10GVV (EAT)
Phase I-II study of gemcitabine and valproic acid plus valganciclovir in patients with advanced nasopharyngeal carcinoma
JP de Boer
I/II
15-2-2011
N10OPT
Optimising photodynamic therapy for the treatment of head and neck cancer
B Karakullukcu
other
1-11-2010
N10VMO
Evaluation of tumor variability with MRI during radiotherapy treatment in patients with an oropharyngeal or oral cavity carcinoma
O Hamming-Vrieze
other
8-11-2010
N11ADA
Study for optimizing the moment of 'Adaptive Radiotherapy' during curative radiotherapy in Head and Neck cancer
O Hamming-Vrieze
other
18-1-2012
N11HME
Short term effect of heat and moisture exchanger on tracheal mucociliary clearance in laryngectomized individuals
FJM Hilgers
other
11-10-2011
Head and Neck A randomized study of docetaxel/cisplatin/5-fluorouracil (TPF) as neoadjuvant chemotherapy followed by concomitant chemoradiotherapy (CRT) with conventional radiotherapy (RT) versus concomitant CRT with accelerated RT in patients with locally advanced head and neck squamous cell cancer (HNSCC) in good condition
JP de Boer
M08DDL (TYVTAX)
Docetaxel versus Docetaxel and Lapatinib in recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN); an open label multicenter randomized phase II study
JP de Boer
M08EBV
Standardized early detection of primary and recurrent nasopharyngeal carcinome (NPC) using (anti-) EBV based tumor markers in The Netherlands
IB Tan
other
29-4-2009
N12MAC (M&M)
Exploring the contribution of Macrophages in the microenvironment of HPVinduced squamous cell carcinoma of the head and neck
JP de Boer
other
31-8-2012
M08MSH
Measuring shoulder disability after neck dissection; a comparison of 4 self report scales
M Stuiver
other
25-3-2009 (21-11-2012)
N12NSL (NerveSpect)
In vivo identification of peripheral nerve bundles during surgery using optical spectroscopy techniques: a pilot study
TJM Ruers
pilot
19-11-2012
M09HZT (HBOT)
Efficacy of adding hyperbaric oxygen therapy to the treatment of late radiation damage of the lower jaw (osteonecrosis)
LM Smeele
III
23-11-2009
M09PDT
A multi centre cost-effectiveness evaluation of a novel treatment option in the netherlands: photo dynamic therapy with temoporfin for the treatment of advanced incurable head and neck cancers, for whom prior conventional treatments have failed
IB Tan
other
6-8-2009 (14-12-2012)
M11ART (ARTFORCE)
Adaptive and innovative radiation treatment for improving cancer treatment outcome
O Hamming-Vrieze
M11ELN
Diagnostic accuracy of an electronic nose for the detection of early endobronchial squamous cell cancer after prior history of head and neck cancer or lung cancer
AJM Balm
other
29-8-2011
M11FOR (FORECAST)
FORECAST: Functional Outcome of Radiotherapy and Laser in Early Glottic Carcinoma
WMC Klop
III
25-10-2011
M12PCA
An open-label, single arm, Phase II study to evaluate the safety and efficacy of PC-A11 with superficial and interstitial laser light apllication in patients with recurrent head and neck squamous cell carcinoma unsuitable for surgery and radiotherapy and without distant metastases
B Karakullukcu
II
17-7-2012
M12REA (REACTION)
Response evaluation after chemoradiation for advanced oropharyngeal cancer using PET-CT and MRI
M van de Brekel
other
25-7-2012
N05HME
De kortetermijninvloed van een Heat and Moisture Exchanger op de endotracheale temperatuur en luchtvochtigheid bij gelaryngectomeerden
FJM Hilgers
other
1-9-2005
M08CON (CONDOR)
198
II
II
II
2-2-2009 (1-2-2012)
12-2-2009 (25-1-2012)
Leukaemia / MDS M10OMB (HOVON 101)
Ofatumumab maintenance treatment versus no further treatment in relapsed CLL responding to induction therapy
M Kerst
III
10-11-2010
M11APO
Protocol apoptose regulatie van CLL
JW Baars
other
29-9-2011
C11AFA
Named patient use of Afatinib for patients with advanced stage NSCLC: evaluation of response and toxicity profile
J Quispel-Jansen
other
12-5-2011
C12CRI
Compassionate use program Crizotinib
JA Burgers
other
27-3-2012
E08072 (CONVERT)
Concurrent Once-daily Versus twice-daily RadioTherapy: a 2-arm randomised controlled trial of concurrent chemo-radiotherapy comparing twice-daily and once-daily radiotherapy schedules in patients with limited stage small cell lung cancer (SCLC) and good performance status
JL Knegjens
III
11-11-2009
20-12-2011
Lung
199
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
E08092 (MAPPING)
Double blind randomized phase III study of maintenance Pazopanib versus placebo in NSCLC patients non progressive after first line chemotherapy. MAPPING, an EORTC Lung group study
P Baas
III
23-8-2011
N08CPA
A randomized phase I/II study of standard chemotherapy (cisplatin and pemetrexed) with or without Axitinib in patients with malignant mesothelioma: interim biopsy analysis to determine efficacy
P Baas
I/II
22-5-2009 (13-11-2012)
M06NEL (NEL)
Efficacy of neoadjuvant erlotinib in patients with clinical stage I/II non-small cell lung cancer (NSCLC)
HM Klomp
I/II
8-11-2006
N09MLN
Quantification of mediastinal lymph node position variability by implanted fiducial markers using cone beam computer tomogram in non-small cell lung cancer patients treated with radical irradiation
JSA Belderbos
other
8-4-2010
M08MEK
Open-label, multicenter, dose-escalation phase I study with extension to evaluate safety, pharmacokinetics and activitiy of RO4987655, a MEK inhibitor, administered orally as monotherapy in patients with advanced tumors
JHM Schellens
I
4-2-2009 (1-12-2012)
N10ILM
Designing and testing new intervention therapies for lung cancer and Mesothelioma
P Baas
other
30-8-2010
M09CRE (CREST)
Randomized trial on chest irradiation in extensive disease small cell lung cancer
JL Knegjens
III
19-3-2009 (1-12-2012)
N10RFS (OpSpect)
Observational study into specific in vivo human discrimination between benign and malignant tissue using a combination of diffuse reflectance and fluorescence spectroscopy
TJM Ruers
other
25-10-2010
M09LST
Evaluation of molecular sputum test diagnostics for lung cancer
JA Burgers
other
18-5-2009 N11ORL
I
21-2-2012
Dose escalation by boosting radiation dose within the primary tumor on the basis of a pre-treatment FDG-PET-CT scan in stage Ib, II and III NSCLC: a randomised phase II trial
JSA Belderbos
II
26-11-2009
Olaparib dose escalating trial in patients treated with radiotherapy with or without daily dose cisplatin for locally advanced non-small lung cancer
MM van den Heuvel
M09PBO (PET-BOOST)
N12CLM
D Brandsma
I
19-6-2012
M09PCI (NVALT 11)
Prophylactic Cranial Irradiation(PCI) versus observation in radically treated patients with stage III non-small cell lung cancer: a phase III randomized study
JSA Belderbos
III
20-10-2009
Determining the sensitivity and specificity of circulating tumor cells and cytology in cerebrospinal fluid of patients clinically suspected for leptomeningeal metastases
12-7-2012
MM van den Heuvel
III
30-6-2010 (31-5-2012)
Phase I Hybrid study: combined stereotactic radiotherapy and conventional fractionation in stage II and III non small cell lung cancer with peripheral tumors smaller than 5 cm
I
Registration phase III study of LucanixTM( Belagenpumatucel-L) in advanced non-small cell lung cancer: an international multicenter, randomized, doubleblind, placebo-controlled study of LucanixTM maintenance therapy for stages III/ IV NSCLC subjects who have responded to or have stable diseasefollowing one regimen of front-line platinum-based combination chemotherapy
N12HYB (HYBRID)
H Peulen
M10LUC
N12LPR
Early FDG-PET/CT response evaluation of lung cancer during of lung cancer during chemoradiation
WV Vogel
other
10-12-2012
M10N12 (NVALT12)
A randomized phase II study of paclitaxel-carboplatin-bevacizumab with or without nitroglycerin patches in patients with stage IV non-squamous-non-small cell lung cancer
JA Burgers
II
10-3-2011 (1-12-2012)
N12PRO
Pharmacogenomic profiling of short-term cultures of malignant pleural mesothelioma
J Quispel-Jansen
other
21-9-2012
M11ELN
Diagnostic accuracy of an electronic nose for the detection of early endobronchial squamous cell cancer after prior history of head and neck cancer or lung cancer
AJM Balm
other
29-8-2011
M11ETO (lungscape)
A retrospective cohort study of ALK gene arrangement: prevalence and clinical outcomes in patients with non-small cell lung cancer in Europe: ETOP lungspace 001-ALK
P Baas
other
29-12-2011
A phase II Open label, multicenter, randomized study to assess the efficacy and safety of GSK1120212 compared with docetaxel in 2nd line subjects with targeted mutations (KRAS,NRAS,BRAF,MEK1) in locally advanced or metastatic non-small cell lung cancer (NSCLC stage IIIb-IV)
P Baas
M11LUN (lungscape)
Lungscape: a project of European Thoracic Oncology Platform
P Baas
other
M11VOL (VOLUMES)
Treatment of larger tumor volumes or 2 lung metastases simultaneously in lung cancer patients using SBRT in a mean-lung dose escalation study
H Peulen
M12BMS (CA209-017)
An open-label randomized phase III trial of BMS-936558 versus Docetaxel in previously treated advanced or metastatic squamous cell non-small cell lung cancer
P Baas
N04LSN
Feasibility of lymphoscintigraphy and sentinel node biopsy in patients with nonsmall lung cancer
HM Klomp
pilot
N07NRA
A phase I/II study with NAMI-A, a Novel Ruthenium Anticancer Agent, and gemcitabine combination second-line therapy in NSCLC patients
JHM Schellens
I/II
M11GDT
200
Lymphoma - Non-Hodgkin's M07H84 (HOVON-84)
Randomized phase III study on the effect of early intensification of rituximab in combination with 2-weekly CHOP chemotherapy followed by rituximab maintenance in elderly patients with DLBCL
JW Baars
III
22-1-2008 (10-4-2012)
M09ORC (ORCHARRD)
Ofatumumab versus Rituximab salvage chemoimmunotherapy followed by ASCT in relapsed or refractory DLBCL
JW Baars
III
13-7-2010 (21-8-2012)
29-12-2011
M09TAM (TAMIL)
Treatment induced alterations in microenvironment in follicular lymphoma. A multicenter descriptive study
D de Jong
other
27-5-2009
I/II
29-9-2011
M10EXI (Exist)
Exist: Physical exercise to improve fitness and combat fatigue in patients with multiple myeloma and (non-)Hodgkin’s lymphoma treated with high dose chemotherapy and autologous stem cell transplantation
JW Baars
other
3-2-2011
III
9-11-2012 M10H105 (HOVON 105)
Rituximab in primary central nervous system lymphoma. A randomized HOVON/ ALLG intergroup study
JW Baars
III
23-11-2010
8-9-2004
M12AEB (COEB071X210)
An open-label, single arm, Phase I study of AEB071 ( a protein kinase C inhibitor) in patients with CD79-mutant diffuse large B-cell lymphoma
JP de Boer
I
3-5-2012
15-3-2011
M12H110 (ReBeL/HOVON)
ReBel study: a randomized Phase I/II trial of lenalidomide and rituximab with or without bendamustine in patients >= 18 years with a relapsed follicular lymphoma. A HOVON/GLSG study
JW Baars
II
12-6-2012
II
6-12-2011 (2-7-2012)
201
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Melanoma / Skin C12ZEL
Compassionate use Zelboraf (vemurafenib)
JBAG Haanen
other
2-5-2012 (1-7-2012)
M05MSL (MSLT-II)
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
OE Nieweg
III
11-9-2006
Intradermal naked DNA vaccination for mounting tumor-specific immunity in stage IV melanoma patients: a phase I clinical study
JBAG Haanen
M08MEK
Open-label, multicenter, dose-escalation phase I study with extension to evaluate safety, pharmacokinetics and activitiy of RO4987655, a MEK inhibitor, administered orally as monotherapy in patients with advanced tumors
JHM Schellens
I
4-2-2009 (1-12-2012)
M10BRA (BRF113683)
A phase III randomized open label study comparing GSK2118436 to DTIC in previously untreated subjects with BRAF mutation positive advanced (stage III) or metastic (stage IV) melanoma
C Blank
III
31-3-2011 (21-11-2012)
M11BRI (MO25515)
An open-label, multicenter expanded access study of RO5185426 in patients with metastatic melanoma
C Blank
M11MME (CMEK162X2201)
A phase II, open-label study to assess the safety and efficacy of oral MEK162 in adults with locally advanced and unresactable or metastatic malignant cutaneous melanoma, harbouring BRAFV600E or NRAS mutations
C Blank
II
30-3-2011
M11TCR
Feasibility study using T-cel receptor gene therapy in metastatic melanoma
JBAG Haanen
II
17-4-2012
M11VMB (MO25743)
An open label single arm phase II, multicenter study to evaluate the efficacy of vemurafenib in metastatic melanoma patients with brain metastases
C Blank
II
17-2-2012
M12DTD (MEK115306)
A Phase III, randomized, double-blinded study comparing the combination of the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib to dabrafenib and placebo as first-line therapy in subjects with unresectable (Stage IIIc) or metastatic (Stage IV) BRAF V600E/K mutation-positive cutaneous melanoma
JBAG Haanen
M12TFU (MELFO)
Prospective randomized trial for the evaluation of a theoretical follow-up schedule in cutaneous melanoma
M Wouters
other
8-6-2012
N03LAM
Longitudinal analysis of melanoma-specific immunity in stage III and IV melanoma patients
JBAG Haanen
other
22-8-2003
N06TIS
Integrated analyses of melanoma-T cell interactions; relevance for immunotherapy
JBAG Haanen
other
29-8-2006
N10BIO
Novel targets for medication in melanoma patients who are progressive after treatment with BRAF inhibitor RO5185426 or dacarbazine
JBAG Haanen
other
12-7-2010 (14-12-2012)
N10MMI
Local immunotherapy by the synergism of monobenzone and imiquimod cream (MI) for cutaneous metastases in stage III-IV melanoma patients
JPW van der Veen
II
24-1-2011
Pilot study on the use of fluorescence imaging of lymph nodes during melanoma sentinel node procedure, using indocyanine green
M Wouters
Donor leukapheresis for T-cell receptor gene therapy for treatment of metastatic melanoma (skin cancer)
JBAG Haanen
Randomized phase II study using a non-myeloablative lymphocyte depleting regimen of chemotherapy followed by infusion of tumor infiltrating lymphocytes and interleukin-2 in metastatic melanoma
JBAG Haanen
M07DNA
N10MSN
N10TCR
N10TIL (TIL)
202
I
IV
III
pilot
pilot
II
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
N11RFA (SECIRA-UM)
Phase Ib/II study exploring safety and efficacy of the combination of ipilimumab with radiofrequency ablation (RFA) in patients with unresectable uveal melanoma liver metastases
C Blank
I/II
17-4-2012
N12CLM
Determining the sensitivity and specificity of circulating tumor cells and cytology in cerebrospinal fluid of patients clinically suspected for leptomeningeal metastases
D Brandsma
I
19-6-2012
N12NSL (NerveSpect)
In vivo identification of peripheral nerve bundles during surgery using optical spectroscopy techniques: a pilot study
TJM Ruers
pilot
19-11-2012
N12OCT
Optical biopsy by optical coherence tomography (OCT) in melanocytic lesion
TJM Ruers
other
14-3-2012
9-1-2009
Miscellaneous M10RTW (return to work)
To enhance return-to-work in cancer patients - a randomised trial
WJ van Driel
other
14-9-2010 (14-12-2012)
M11COM (COMTT)
Phase III randomized double blind cross-over trial of supersaturated calciumphosphate rinse (Caphosol)versus NACL 0,9% in the relief of oral mucositis in renal cell carcinoma, hepatocellular carcinoma andgastrointestinal stromal tumor patients receiving Targeted Therapy
JBAG Haanen
III
12-12-2011
M12BSC (RADIANT-4)
Randomized, double-blind, multicenter, Phase III study of everolimus (RAD001) plus best supportive care versus placebo plus best supportive care in the treatment of patients with advanced NET of GI or lung origin.
M Tesselaar
III
5-6-2012
N03THY
Therapeutic management of thymoma and thymic carcinoma: - a prospective registration study based on preoperative risk assessment of local failure
LGH Dewit
other
25-11-2003 (28-11-2012)
N09DRF
Intraoperative real time imaging with a dual radioactive/fluorescence modality for sentinel node localization. A feasibility study including reproducibility of multimodality lymphatic mapping with a cocktail tracer containing 99mTc nanocolloid/ICG
RA Valdes Olmos
other
8-6-2009
30-3-2011
19-9-2012
Soft Tissue / Osteosarcoma C12PAZ
Pazopanib early access program
M Kerst
other
30-3-2012
E62091 (TRUSTS)
A phase IIb/III multicenter study comparing the efficacy of Trabectedin administered as a 3-hour or 24-hour infusion to doxorubicin in patients with advanced or metastatic Untreated Soft Tissue Sarcoma
M Kerst
II/III
25-1-2012
E62092 (STRASS)
A phase III randomized study of preoperative radiotherapy plus surgery versus surgery alone for patiens with retroperitoneal sarcoma (RPS)
RLM Haas
III
11-1-2012
M09RTP (PASART-1)
Phase I clinical study of a combined modality treatment of sarcomas of the extremities with radiotherapy (RT) and dose-escalation of Pazopanib
RLM Haas
I
24-6-2010
M10ETD (ET-D-010-10)
An observational, multicenter, open-label study of the management of patients with advanced soft tissue sarcoma after failure of anthracyclines and/or ifosfamide or atients unsuited to receive these drugs
M Kerst
IV
17-2-2011
20-12-2010
5-8-2010 (14-12-2012) 29-3-2011
203
Type of Title cancer study (nick name)
Study Phase coordinator
Activated (closed)
Type of Title cancer study (nick name)
Phase III randomized double blind cross-over trial of supersaturated calciumphosphate rinse (Caphosol)versus NACL 0,9% in the relief of oral mucositis in renal cell carcinoma, hepatocellular carcinoma andgastrointestinal stromal tumor patients receiving Targeted Therapy
JBAG Haanen
12-12-2011
M11PRC (PERCEPTION)
N10DMY (DOREMY)
Dose reduction of preoperative radiotherapy in Myxoid liposarcomas
RLM Haas
II
15-12-2010
N10PPS
Perfusion PET sarcoma; feasability of tumor perfusion quantification with Gallium68-citrate PET/CT in sarcoma and radiotherapy
WV Vogel
other
3-12-2010 (28-11-2012)
N12NSL (NerveSpect)
In vivo identification of peripheral nerve bundles during surgery using optical spectroscopy techniques: a pilot study
TJM Ruers
pilot
19-11-2012
M11COM (COMTT)
III
Uro-Genital
Study Phase coordinator
Activated (closed)
Impact of new approaches to pharmacological management of patients with renal cell carcinoma: a population-based study of process outcomes in The Netherlands
S Horenblas
other
18-8-2011
M11REP (ROPETAR)
A randomized phase II study to explore the efficacy and feasibility of upfront bimonthly rotations between Everolimus and Pazopanib with sequential treatment of first line Pazopanib and second line Everolimus until progression in patients with advanced or metastatic clear cell renal cancer
JBAG Haanen
II
26-9-2012
M11TOO
A European randomised phase 3 study to assess the efficacy and safety of TOOKAD Soluble for low risk localised prostate cancer compared to active surveillance
HG van der Poel
III
4-8-2011
M12CBP (CABARESC)
A phase II study in mCRPC on the pharmacodynamic effects of budesonide on cabazitaxel (Jevtana®): a randomised, open-label multicenter study
A Bergman
II
8-5-2012
M12MAG (MAGNOLIA)
A randomized, double blind, placebo controlled phase II trial to evaluate the safety and efficacy of recMAG-A3 + AS15 ASCI in patients with MAGE-A3 positive muscle invasive bladder cancer after cystectomy
M Kerst
II
2-2-2012
M94SAL
Salvage regimen incorporating repeated ablative chemotherapy with autologous PSCT, a phase II study
S Rodenhuis
II
4-7-1994
C11ABI
Abiraterone compassionate use programma
A Bergman
IV
1-11-2011 (29-3-2012)
14-3-2007
A Bergman
other
13-6-2012
Samarium-153-EDTMP (QUADRIMET) versus docetaxel for multiple painful oseous metastases in prostate cancer
II
Named patient programma met Enzalutamide
N06MPM (QUADRIMET)
HG van der Poel
C12ENZ E22043
Post-operative external radiotherapy combined with concomitant and adjuvant hormonal treatment versus post-operative external radiotherapy alone in pathological stage pT3a-b R0-1 / N0M0 / pT2R1 N0M0, Gleason score 5-10 prostatic carcinoma. A phase III study
F Pos
III
29-12-2010
N08API
Analysis of prostate-specific immunity in stage III and IV prostate cancer Patients
JBAG Haanen
other
22-1-2009 (14-12-2012)
N08SNR
Site and distribution of sentinel lymph nodes in renal cell carcinoma, a phase II study
A Bex
II
19-3-2009
E30073 (SURTIME)
Randomized phase III trial comparing immediate versus deferred Nephrectomy in patients with synchronous metastatic renal cell carcinoma
A Bex
N09IGF
Pilot study on the use fluorescence imaging of lymph nodes during laparos copic pelvic sentinel node dissection for prostate cancer using indocyanine green
HG van der Poel
pilot
14-7-2009
M06LAN (LANCE)
Late Adverse effects in Dutch testicular cancer survivors: a Nationwide casecontrol study on Cardiovascular Events
M Kerst
N10BPA
A Bergman
I
26-1-2011
M06SIL
Phase I dose-escalation trial for the combination of sorafenib with interleukin-2 treatment in patients with clear cell renal carcinoma
JBAG Haanen
I
20-11-2006 (14-12-2012)
Phase I trial evaluating combined image guided radiotherapy with Panitumumab (Vectibix) in patients with muscle invasive transitional cell carcinoma of the bladder
N11DCE
other
11-4-2012
A phase I, open-label, multicenter study to assess the safety, tolerability, pharmacokinetics and preliminary anti-tumour activity of ascending doses of AZD3514 in patients with castration-resistant prostate cancer
JHM Schellens
I
23-8-2010
Determining the reproducibility of quantitative dynamic contrast-enhanced (DCE) MRI is patients with prostate cancer
F Pos
M10AZP (SARAFTip)
N12ITC
Influence of injection time of contrast agent on quantitative Dynamic ContrastEnhanced (DCE) MRI in patients with prostate cancer
F Pos
other
31-8-2012
M10IPI (CA 184-043)
A randomized, double-blind, phase III trial comparing ipilimumab vs placebo following radiotherapy in subjects with castration resistant prostate cancer that have received prior treatment with docetaxel
A Bergman
14-12-2010 (8-2-2012)
N12LAR
Ongitudinal analysis of RCC-specific immunity in renal cell carcinoma patients
C Blank
other
14-12-2012
M10PCM (PCMM)
Prostate cancer molecular medicine
HG van der Poel
other
17-2-2011
N12OPB
Optical penile biopsy by optical coherence tomography (OCT)
TJM Ruers
other
14-3-2012
M11COM (COMTT)
Phase III randomized double blind cross-over trial of supersaturated calciumphosphate rinse (Caphosol)versus NACL 0,9% in the relief of oral mucositis in renal cell carcinoma, hepatocellular carcinoma and gastrointestinal stromal tumor patients receiving Targeted Therapy
JBAG Haanen
III
12-12-2011
M11DPC
A randomized phase 3 study comparing standard fist-line Docetaxel/ Prednisone to Docetaxel/Prednisone in combination with Custirsen (OGX-011) in men with metastatic castrate resitant prostate cancer
A Bergman
III
21-7-2011 (13-11-2012)
M11FLA (FLAME)
Single Blind Randomized Phase III Trial to Investigate the Benefit of a Focal Lesion Ablative Microboost in Prostate Cancer
F Pos
III
5-4-2012
204
III
other
III
17-3-2010
12-2-2007
205
Invited speakers
Paul Nurse, London, UK
Controlling the cell cycle
Eric So, London, UK
Molecular dissection and targeting of epigenetic machinery in acute leukemia
Roel Nusse, Stanford, CA, USA
Wnt signaling and asymmetric stem cell division
Paul Spellman, Portland, OR, USA
Systematically understanding the cancer genome Hiroshi Ohno, Yokohama, Japan
Function and development of specialized epithelial cells, M cells Antoine van Oijen, Groningen, The Netherlands
Ruth Sperling, Jerusalem, Israel
A novel quality control mechanism that regulates alternative 5’ splice site selection
Under the hood: Single-molecule studies of DNA replication Cornelis Albers, Cambridge, United Kingdom
Variant calling by whole-genome assembly with realignment, and exome sequencing of rare inherited platelet disorders
Alfred Goldberg, Boston, MA, USA
New insights into proteasome function: From protein degradation to cancer therapy
Charles Swanton, London, UK Ab Osterhaus, Rotterdam, The Netherlands
Intratumour heterogeneity and personalised cancer medicine
Animal and human viruses: to publish or not to publish Matthijs Verhage, Amsterdam, The Netherlands
Michael Alexis, Athens, Greece
Prognostic and predictive significance of estrogen receptor beta in breast cancer: Towards development of ER subtype specific SERMs
Marcus Groettrup, Constance, Germany
How, where, and why the ubiquitin-like modifier FAT10 targets substrates for degradation by the proteasome
High throughput/content microscopy for systems biology analyses
The protein machines that regulate organelle docking & fusion and cargo delivery at the surface Johannes Walter, Boston, MA, USA
Albert Heck, Utrecht, The Netherlands Francesca Baietti, Leuven, Belgium
Rainer Pepperkok, Heidelberg, Germany
Developing tools for delving into the cancer (phopho)proteome
Stefano Piccolo, Padua, Italy
On the molecular nature of breast cancer stem cells
The mechanism and regulation of DNA replication: from cell-free systems to single molecules
Syndecan-syntenin-ALIX regulates the biogenesis of exosomes Jeroen van Heijst, New York, USA Curtis Barrett, Leiden, The Netherlands
Publish, don’t perish! Essential tips for effective English writing
Probing T cell immunity in clinical settings of immune deficiency and experimental tuberculosis infection
Prem Premsrirut, Cold Spring Harbor, NY, USA
RNAi mouse models for target discovery, validation and toxicology assessment
Lodewyk Wessels, Amsterdam, The Netherlands
Molecular networks in cancer Rob Wolthuis, Amsterdam, The Netherlands
Dirk Busch, Munich, Gemany
Mechanisms of protection towards intracellular pathogens and it's implications for immunotherapy
Mike Hemann, Cambridge, MA, USA
Using mouse models to improve cancer therapy
Exploring the Equation of Cell Division: 1 / 0.5 = 2
Analyzing and targeting networks for combinatorial therapy Anton Wutz, Cambridge, UK
Jonathan Higgins, Boston, MA, USA Edwin Cuppen, Utrecht, The Netherlands
Prahlad Ram, Houston, TX, USA
Regulating mitosis by histone phosphorylation
Sridhar Ramaswamy, Boston, MA, USA
Multi-scale views of cancer heterogeneity
Derivation and characterization of haploid embryonic stem cells from mouse embryos
Detection and decoding of structural variation in genomes Jacqueline Jacobs, Amsterdam, The Netherlands Jan-Hermen Dannenberg, Amsterdam, The Netherlands
Mouse models for HDACs in development and cancer
The mechanisms underlying telomere-dependent control of cancer development and aging
Glen Reid, Sydney, Australia
MicroRNA biomarkers for malignant pleural mesothelioma Carola Ries, Penzberg, Germany
Vishva Dixit, San Francisco, CA, USA
Role of ubiquitin modification in cellular signaling Marileen Dogterom, Amsterdam, The Netherlands
Positioning of microtubule organizing centers by pushing and pulling forces
Olli Kallioniemi, Helsinki, Finland
Individualized systems medicine in cancer: AML and beyond Titia de Lange, New York, NY, USA
How telomeres solve the chromosome end protection problem Peter Lansdorp, Groningen, The Netherlands
Daniel Durocher, Toronto, Canada
Chromatin ubiquitylation and the response to DNA double-strand breaks
Studies of genomic (re-) arrangements in single cells by analysis of DNA template strands
Anselm Enders, Canberra, Australia
Telomerase structure and its regulation at chromosome ends Ultan McDermott, Hinxton, UK
The genomics of drug sensitivity in cancer Shridar Ganesan, New Jersey, NJ, USA
TRIM33 regulates the chromatin association of ALC1 during the PARP-dependent DNA damage response
Hisse Martien van Santen, Madrid, Spain
T cell tuning by oligomeric TCR amplifiers
TLR2 signaling regulates mammary stem cell function and affects tumorigenesis Giorgio Scita, Milan, Italy
Membrane and actin dynamics in the plasticity of tumor cell migration Clemens Schmitt, Berlin, Germany
Senescence and senescence-related vulnerabilities in cancer therapy
Tom Muir, New Jersey, NJ, USA
Chromatin: an expansive canvas for chemical biology
Peter Sicinski, Boston, MA, USA
Cyclins and cancer
Niels Geijsen, Utrecht, The Netherlands
Dazl-in' germ cells and pluripotent stem cells
Johannes Zuber, Vienna, Austria
Finding and probing cancer drug targets using advanced in-vivo RNAi
Ira Mellman, San Francisco, CA, USA
Cancer cell biology: next generation approaches to basic cell biology & drug discovery
Richard Gardner, Seattle, WA, USA
How disorder prevents disaster in nuclear protein homeostasis
Giannino del Sal, Trieste, Italy
Pathways driving aggressiveness in breast cancer
Lars Zender, Braunschweig, Germany
RNAi based functional genomic approaches for accelerated cancer gene discovery in liver cancer
Ferenc Scheeren, Amsterdam, The Netherlands Joachim Lingner, Lausanne, Switzerland
Sppl2a, a novel peptidase involved in processing of MHC invariant chain and B cell maturation
Targeting tumor associated macrophages with a humanized antiCSF1R antibody: A novel approach for anti-tumor therapy
Jonathan Yewdell, Bethesda, MD, USA
How to succeed in science without really trying
Sach Mukherjee, Amsterdam, The Netherlands
Statistical approaches in systems signaling 206
207
Research projects supported by the Dutch Cancer Society
208
Principal investigator
Number Title Started of projects
Ended / Ends
Aaronson, Neil
NKI 2006-3470
Cognitive behavioral therapy (CBT) and physical exercise for climacteric symptoms in breast cancer patients experiencing treatment-induced menopause
5/1/2007
5/1/2012
Aaronson, Neil
ALPE 2009-4299
A-CaRe Project 2: Effectiveness of physical exercise during chemotherapy to improve physical fitness and reduce fatigue: A randomized trial
8/1/2009
8/1/2013
Aaronson, Neil
NKI 2012-5388
A randomized study of an internet-based cognitive behavioral therapy program for sexuality and intimacy problems in women treated for breast cancer
7/1/2012
7/1/2016
Agami, Reuven
NKI 2009-4469
Role of RNA binding proteins in cancer
3/1/2010
3/1/2014
Agami, Reuven
NKI 2009-4498
Bromodomain containing proteins in cancer
9/1/2010
9/1/2014
Bergman, André
NKI 2009-4356
Investigating the Role of Inflammation in Prostate Cancer Development
8/1/2009
8/1/2014
Bernards, René
NKI 2009-4337
Identification of genetic modifiers of sensitivity to mTOR pathway inhibition in breast cancer
8/1/2009
8/1/2013
Bernards, René
NKI 2009-4496
Determinants of the response to retinoic acid in neuroblastoma
1/1/2010
1/1/2014
Bernards, René
BUIT 2009-4429
Synthetisch lethale interacties in kanker
1/1/2013
1/1/2015
Bernards, René
NKI 2012-5401
Finding genetic dependencies in cancer: the missing link in personalized medicine
1/1/2013
1/1/2019
Bernards, René
NKI 2008-4027
Understanding resistance to HER2-targeting therapy in human breast cancer through functional genetics
11/1/2008
11/1/2013
Bernards, René
NKI 2008-4042
Identification of enzymes involved in regulatory ubiquitination in TNF a-stimulated signaling by loss-of-function screens
11/1/2008
11/1/2013
Berns, Ton
NKI 2008-4253
Designing and testing new intervention therapies for lung cancer and mesotheliomas
5/1/2009
5/1/2015
Blank, Christian
NKI 2008-3988
Blockade of PD-1/D-L1 interaction to improve T cell mediated immunotherapy of cancer
1/1/2008
1/1/2012
Bleiker, Eveline
NKI 2008-4016
Identification of psychosocial problems and perceived need for support in cancer genetics
12/1/2008
12/1/2013
Boekhout, Annelies
NKI 2012-5356
Developing (shared-care) breast cancer survivorship programs in the Netherlands. KWF fellowship
9/1/2012
9/1/2016
Borst, Gerben
NKI 2012-5364
A Personalized Approach to Targeted Radiosensitization: Simultaneous Assessment of Tumour Genetics and Microenviromental Heterogeneity
1/1/2013
1/1/2015
Borst, Jannie
NKI 2012-5397
Defining the nature of CD4 T-cell help for the CTL response to optimize immunotherapy of cancer
4/1/2012
4/1/2016
Borst, Jannie
NKI 2008-4110
Impact of TRAIL death receptor trafficking on pro-apoptotic signaling
3/1/2008
3/1/2013
Borst, Jannie
NKI 2008-4028
T cell programming at the dendritic cell interface: impact on anti-tumor immunity
2/1/2008
2/1/2013
Dam, Frits van
KWF Rookpreventie jeugd
Campagnevoering Stichting Rookpreventie Jeugd
8/28/2012
12/31/2013
Dannenberg, Jan-Hermen
NKI 2007-3978
Genetic analysis of class I HDACs in development and treatment of cancer
1/1/2008
1/1/2012
Driel, Willemien van
NKI 2011-5149
Nederlandse Studiegroep Gynaecologische Oncologie. Dutch Gynaecological Oncology Group (DGOG) Startstudie
2/1/2012
2/1/2013
209
Principal investigator
Number Title Started of projects
Ended / Ends
Principal investigator
Number Title Started of projects
Ended / Ends
Driel, Willemien van
CKTO 2006-4176
Phase III randomised clinical trial for stage III ovarian carcinoma randomising between secondary debulking surgery with or without hyperthermic intraperiotoneal chemotherapy (OVHIPEC-1)
3/14/2007
9/14/2012
Lohuizen, Maarten van
NKI 2007-3877
Epigenetic regulation by the deubiquitinating enzyme USP3: impact on genome stability and tumorgenesis
3/1/2007
3/1/2012
Medema, René
NKI 2010-4706
Building Sister Chromatid Cohesion
10/1/2011
10/1/2014
Elkhuizen, Paula
NKI 2009-4389
Preoperative accelerated partial breast irradiation (PAPBI): defining radiotherapy sensitivity
7/1/2010
7/1/2014 Medema, René
UU 2011-5103
Cohesin loading and stripping: two fundamental principles for the maintenance of genomic stability
10/1/2011
10/1/2017
TIL therapy for melanoma: development of a simplified TIL production process using a novel bioreactor
10/1/2011
Medema, René
NKI 2009-4478
Recovery competence after DNA damage
1/1/2012
4/1/2015
Medema, René
NKI 2012-5706
Molecular dissection of ATR activation
7/1/2012
7/1/2014
Medema, René
NKI 2011-5215
Recovery from a G1 arrest
7/1/2012
7/1/2016
Moolenaar, Wouter
NKI 2010-4781
Validation of autotaxin, a metastasis-enhancing exo-phosphodiesterase, as a therapeutic target
8/1/2010
8/1/2014
Nederlof, Petra
NKI 2007-3749
Specific chromosomal imbalance in breast carcinomas as a marker for breast cancer susceptibility
8/15/2007
8/15/2012
Neefjes, Jacques
BUIT 2009-4446
Radiotherapy and immunotherapy in the treatment of cancer
5/1/2012
1/1/2015
Ovaa, Huib
NKI 2010-4781
Validation of autotaxin, a metastasis-enhancing exo-phosphodiesterase, as a therapeutic target
8/1/2010
8/1/2014
Peeper, Daniel
NKI 2007-3957
Towards improved melanoma treatment: gene identification, in vivo modeling and drug target discovery
7/1/2008
7/1/2014
Peeper, Daniel
NKI 2009-4552
Dissecting the essential contribution of Fra-1 to human tumor cell metastasis
1/1/2010
1/1/2014
Peeper, Daniel
NKI 2011-5247
Dissecting the mammalian non-canonical Wnt-pathway
11/1/2011
11/1/2013
Peeper, Daniel
NKI 2012-5732
Functional characterization of TSPYL family genes in oncogenesis
10/1/2012
10/1/2014
Haanen, John
NKI 2011-5162
10/1/2013
Haanen, John
NKI 2007-3943
HPV 16 E6/E7 DNA prime and E6/E7 long peptide boost vaccination for multifocal Vulvar Intraepithelial Neoplasia (VIN)
1/1/2008
1/1/2012
Harten, Wim van
NKI 2010-4854
A-Care 2: ICT supported patient empowerment, return-to-work, telerehabilitation and implementation of Cancer Rehabilitation Programs
11/1/2010
11/1/2015
Herk, Marcel van
NKI 2007-3751
High precision image-guided radiotherapy for bladder cancer
1/1/2008
1/1/2012
Herk, Marcel van
NKI 2007-3895
Probability based treatment planning with biological objectives for high-dose high-precision radiotherapy of prostate cancer
11/1/2007
11/1/2012
Jacobs, Heinz
NKI 2008-4112
A cancer genome atlas of the activation-induced cytidine deaminase: novel insights into the pathogenesis and prognosis of b cell lymphoma.
3/1/2008
3/1/2012
Jacobs, Jacqueline
NKI 2012-5305
How ubiquitination controls telomere-induced genomic instability
10/1/2012
10/1/2016
Jacobs, Jacqueline
NKI 2007-3907
Dissecting the telomere damage response
8/1/2008
8/1/2012
Jalink, Kees
NKI 2010-4626
Calcium and phosphoinositides in the control of podosome formation and tumor cell invasion
10/1/2010
10/1/2014
Jonkers, Jos
NKI 2011-5232
The effect of the tumor microenvironment on radioresistance of breast cancer
11/1/2011
11/1/2013
Jonkers, Jos
NKI 2011-5197
Analysis of minimal residual disease and therapy resistance in primary tumor xenograft models of triple-negative breast cancer
3/1/2012
3/1/2016
Perrakis, Anastassis
NKI 2010-4781
Validation of autotaxin, a metastasis-enhancing exo-phosphodiesterase, as a therapeutic target
8/1/2010
8/1/2014
Jonkers, Jos
NKI 2007-3772
Preclinical validation of chemical inhibitors of poly (ADP-ribose) polymerase (PARP) in conditional mouse models for BRCA-associated breast cancer.
2/1/2007
2/1/2012
Remeijer, Peter
NKI 2008-4024
Image guided radiotherapy for breast cancer
10/20/2008
10/20/2012
Remeijer, Peter
NKI 2012-5716
3/1/2017
NKI 2008-4116
Impact of BRCA mutations on breast tumorigenesis and treatment response: functional analysis of defined truncation mutants and unclassified variants
1/1/2008
1/1/2012
Optimized targeting for surgery and radiotherapy of breast cancer with a DCIS component
3/1/2013
Jonkers, Jos
Riele, Hein te
NKI 2009-4477
2/1/2014
NKI 2006-3631
Long-term risk of cancer after ovarian stimulation for in vitro fertilization
4/1/2007
4/1/2013
Characterization of unclassified allelic variants of DNA mismatch repair genes to improve genetic counseling
2/1/2010
Leeuwen, Flora van Leeuwen, Flora van
NKI 2008-3994
Cardiovascular morbidity and mortality in breast cancer survivors
10/27/2008
10/27/2013
Riele, Hein te
NKI 2007-3790
Role of the G2 restriction point in tumor development and behavior
4/1/2007
12/1/2012
Leeuwen, Flora van
NKI 2010-4720
Assessment of late effects of treatment for Hodgkin’s lymphoma
1/1/2011
1/1/2016
Rookus, Mattie
NKI 2007-3756
Heterogeneity of risk of breast and ovarian cancer in BRCA1 and BRCA2 mutation carriers
11/15/2007
1/15/2014
Leeuwen, Flora van
NKI 2011-5209
Long-term risk of cardiovascular disease and second malignancies after platinum-based chemotherapy for malignant testicular germ cell tumors
3/1/2012
3/1/2016 Rottenberg, Sven
NKI 2009-4303
Analysis of tumor recurrence in a mouse model for hereditary breast cancer: how do tumor-initiating cells escape eradication by chemotherapy
10/1/2009
10/1/2013
A nationwide survivorship care program for adult (non-)Hodgkin lymphoma survivors
2/1/2012
Rottenberg, Sven
NKI 2011-5220
Resistance to Parp inhibitors:assocciation with DNA damage response alterations
8/1/2012
8/1/2016
Role of the histone methyltransferase Dot1 in gene expression and leukemic transformation
9/1/2010
Schagen, Sanne
NKI 2009-4284
Structural, biochemical and functional indices of chemotherapy-induced cognitive deficits in cancer patients
1/1/2010
1/1/2014
Towards patient-tailored systemic therapy in breast cancer: a combined approached of translational research and mouse model systems
1/1/2007
Schagen, Sanne
BUIT 2010-4894
The application of neuroimaging techniques in studying cognitive impairments and complaints after chemotherapy
1/1/2011
1/1/2013
Deciphering the role of Bmi1 and Ezh2 polycomb group genes in prostate carcinogenesis and metastatis
11/1/2010
Schagen, Sanne
NKI 2010-4829
Molecular and cellular mechanisms of cognitive impairment following chemotherapy for cancer
2/1/2011
2/1/2015
Leeuwen, Flora van
Leeuwen, Fred van
Linn, Sabine
Lohuizen, Maarten van
210
NKI 2011-5270
NKI 2009-4511
NKI 2006-3706
NKI 2010-4757
2/1/2016
9/1/2014
1/1/2013
11/1/2014
211
Principal investigator
Number Title Started of projects
Ended / Ends
Principal investigator
Number Title Started of projects
Ended / Ends
Schagen, Sanne
NKI 2010-4876
An online testing approach to assess cognitive problems associated with cancer and cancer treatment
7/1/2011
7/1/2015
Vens, Conchita
NKI 2010-4877
Tumor-specific radiosensitization by exploiting base excision repair deficiencies
6/1/2011
6/1/2015
Schagen, Sanne
NKI 2012-5495
Prospective predictors of late cognitive decline after anthracycline-based adjuvant chemotherapy for breast cancer: the role of brain white matter
12/1/2012
12/1/2016
Verheij, Marcel
NKI 2011-5155
GC-Lipodox: Liposomal doxorubicin enriched with the sphingolipid analogue N-octanoyl-glucosylceramide(GC)
1/1/2012
1/1/2014
Schinkel, Alfred
NKI 2007-3764
OATP1A/1B (SLCO1A/1B) drug uptake transporters in anticancer drug pharmacokinetics and toxicity risks; possible strategies for therapy optimization
12/1/2007
12/1/2012
Verheij, Marcel
NKI 2008-4113
Short-chain sphingolipids for enhanced cellular uptake of amphiphilic anticancer drugs
11/1/2008
11/1/2013
Verwaal, Vic
KWF
Pixels tegen darmkanker
8/1/2011
2/1/2013
Schmidt, Marjanka
NKI 2009-4363
Breast cancer outcome: genetic destiny
7/1/2009
7/1/2015 Visser, Karin de
NKI 2011-5004
5/1/2015
NKI 2007-3839
Genetics determinants of survival and second breast cancer development in premenopausal breast cancer patients
1/1/2007
1/1/2013
Cancer cell-intrinsic and -extrinsic regulation of breast cancer metastasis formation; implications for adjuvant therapy
5/1/2011
Schmidt, Marjanka
Vogel, Wouter
NKI 2011-5024
10/1/2015
NKI 2009-4535
The Tissue Issue: towards a uniform consent procedure for research with excised (cancer) tissue
8/1/2010
8/1/2014
Recurrent differentiated thyroid cancer: personalized treatment based on evaluation of tumor characteristics with PET (THYROPET). M11TRP
10/1/2011
Schmidt, Marjanka
Wolthuis, Rob
NKI 2008-4135
Divergent Control of Cyclin B1-Cdk1 in Cancer Cells: a Key Role in Therapy
9/1/2008
9/1/2012
Schumacher, Ton
NKI 2009-4282
Preclinical development of TCR gene therapy for prostate carcinoma
12/1/2009
12/1/2013 Zwart, Wilbert
NKI 2009-4435
11/1/2013
NKI 2009-4581
UV-gevoelig MHC peptide uitwisselbare streptameer
7/1/2009
7/1/2015
Estrogen Receptor/chromatin interactions in cell lines and tumor to predict tamoxifen resistance
11/1/2009
Schumacher, Ton Schumacher, Ton
BUIT
Self-renewal in normal and cancer breast stem cells
7/1/2012
7/1/2014
Schumacher, Ton
NKI 2012-5463
Genome-wide analysis of the patient-specific repertoire of mutated antigens in melanoma.
9/1/2012
9/1/2016
Sixma, Titia
NKI 2012-5398
Regulation of the USP7/HAUSP switch. Pj-H1/toekenningsbesluit/5398
7/1/2012
7/1/2016
Sixma, Titia
NKI 2008-4014
Comparative study of Ubiqutin-specific proteases
2/1/2008
2/1/2013
Sonke, Gabe
NKI 2012-5685
High-dose alkylating chemotherapy in oligo-metastatic breast cancer harboring homologous recombination deficiency
7/3/2012
7/3/2016
Sonke, Jan-Jakob
NKI 2009-4568
Optimization of stereotactic image guided radiotherapy for lung cancer through detailed toxicity assessment
1/1/2010
1/1/2014
Sonnenberg, Arnoud
NKI 2009-4485
Dissection of the regulation of hemidesmosome disassembly in tumor cell invasion
9/1/2010
9/1/2014
Sonnenberg, Arnoud
NKI 2008-3996
A mouse stem cell model for skin cancer: Dual role of the integrin a6B4 in tumor initiation and progression
8/1/2008
8/1/2013
Stewart, Fiona
NKI 2008-3993
The role of endoglin in induction and repair of cardiovascular damage after radiation alone or in combination with trastuzumab (Herceptin)
6/16/2008
6/16/2013
Stewart, Fiona
NKI 2009-4480
Modulation of vessel repair to prevent radiation-induced microvascular damage
1/1/2010
1/1/2014
KWF Ontwikkelingssamenwerkingsprogramma
1/1/2008
1/1/2012
Tan, Bin Tan, Bin
NKI 2008-4233
Early detection of primary and recurrent nasopharyngeal carcinoma (NPC) using (anti-)EBV based tumor markers and options for using photodynamic therapy (PDT) in the treatment of local disease
9/1/2008
9/1/2013
Tan, Bin
NKI 2012-5423
Optimizing Nasopharyngeal Carcinoma management through increasing awareness in Yogyakarta
10/1/2012
10/1/2017
Tan, Bin
VU 2012-5554
New therapy for Epstein-Barr virus driven tumours by targeting the virus itself
9/1/2012
9/1/2014
Veer, Laura van ‘t
UU 2010-4893
Towards an optimal set targets for future molecular breast cancer imaging
1/1/2010
3/1/2013
212
213
Research projects supported by other organisations
214
Principal Granting Title Started investigator agency
Ended / Ends
Aaronson, Neil
EORTC
Methods and measures for assessing the HRQL of mid to long term survivors of testicular and prostate cancer
5/1/2009
2/28/2012
Aaronson, Neil
IKA
Onco-Move Paces studie
3/1/2010
1/1/2013
Aaronson, Neil
Pink Ribbon
A prospective, randomized study of the efficacy of an internet-based cognitive behavioral therapy program in alleviating sexuality and intimacy problems in women treated for breast cancer
1/1/2013
1/1/2017
Aaronson, Neil
Pink Ribbon
Cognitieve gedragstherapie voor behandeling geinduceerde menopauzale klachten: ontwikkeling van een internet-based zelfhulpprogramma
3/1/2012
3/1/2015
Aaronson, Neil
St Nuts Ohra
Behavioral and psychosocial effects of rapid genetic counseling and testing in newly diagnosed breast cancer patients: a multicenter study
2/18/2008
11/30/2012
Aaronson, Neil
Stichting Achmea Gezondheidszorg
A-CaRe
1/1/2010
7/31/2013
Agami, Reuven
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Agami, Reuven
EEG-CEC / EU
Indentifying novel regulatory mechanisms of miRNA functions
10/1/2008
10/1/2013
Agami, Reuven
NWO
Combined miRNA profiling and genetic screens to identify and characterize cancer-related miRNAs
10/1/2007
10/1/2012
Agami, Reuven
ZonMw
Genome-wide identification of cancer-induced alterations in protein translation
6/1/2011
6/1/2016
Agami, Reuven
ZonMw
Regulators of alternative polyadenylation
7/1/2012
7/1/2016
Bartelink, Harry
EEG-CEC / EU
Adaptive and innovative Radiation Treatment FOR improving Cancer patients treatment Outcome
4/1/2011
4/1/2016
Beijersbergen, Roderick
Inte RNA Technologies BV
Identification of miRNA’s that control response to Trastuzumab in Breast Cancer.
12/1/2009
1/1/2012
Beijersbergen, Roderick
Merck
Agreement Merck
8/10/2012
8/10/2014
Beijersbergen, Roderick
NWO
Cancer Systems Biology Centre
9/1/2010
9/1/2015
Belderbos, José
EEG-CEC / EU
Adaptive and innovative Radiation Treatment FOR improving Cancer patients treatment Outcome
4/1/2011
4/1/2016
Bernards, René
AstraZeneca
Identification and validation of new targets for colorectal cancer through molecular redefinition of the disease
1/1/2011
1/1/2013
Bernards, René
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Bernards, René
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Bernards, René
CGC
Cancer Genomics Center
8/1/2008
1/1/2014
Bernards, René
EEG-CEC / EU
COLTHERES Modelling and predicting sensitivity to targeted therapies in colorectal cancers
1/1/2011
1/1/2015
Bernards, René
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Bernards, René
EEG-CEC / EU
Functional Genomics Dissecting genetic dependencies in cancer
6/1/2010
6/1/2015
Bernards, René
EEG-CEC / EU
RATHER Rational Therapy for Breast Cancer: Individualized Treatment for Difficult-to-Treat Breast Cancer Subtypes Grant
1/1/2011
1/1/2016
215
Principal Granting Title Started investigator agency
Ended / Ends
Principal Granting Title Started investigator agency
Burgers, Sjaak
CTMM
Personalized chemo-radiation of lung and head and neck cancer. WP5: Imageguided adaptive treatment (evaluation during treatment)
10/1/2008
10/1/2013
Collard, John
EEG-CEC / EU
An integrated concept of tumor metastasis: implications for therapy
4/1/2008
4/1/2012
Dalesio, Otilia
NVALT
Statistical Center NVALT Clinical Trials in oncology
5/1/2004
1/1/2013
Dannenberg, Jan-Hermen
NWO
The role of class I HDACs in normal physiology, tumorigenesis and transcriptional regulation
8/1/2007
8/1/2012
Filippo, Ronald
Koninklijk Nederlands Genootschap voor Fysiotherapie (KNGF)
ontwikkeling KNGF-standaard Beweeginterventie oncologie
10/1/2009
7/1/2012
Gilhuijs, Kenneth
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Haanen, John
AVLKRF (KWF)
TIL project
5/1/2009
5/1/2012
Haanen, John
Bristol-Myers Squibb Company
High-throughput analysis of Ipilimumab-induced Cytotoxic T cell responses: An analysis of blood samples from metastatic melanoma patients treated with ipilimumab
7/1/2011
1/1/2013
Haanen, John
NanoNextNL
Drug Delivery (3-D)
7/18/2011
7/18/2013
Haanen, John
NWO
Treatment of HPV positive penile cancer by induction of HPV E7-specijic T cell immunity through DNA vaccination
9/1/2006
7/1/2012
Harten, Wim van
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Harten, Wim van
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Hauptmann, Michael
AICR
Leukaemia after radiation exposure from pediatric computed tomography
6/1/2012
6/1/2015
Hauptmann, Michael
EEG-CEC / EU
Epidemiological study to quantify risks for paediatric computerized tomography and to optimise doses
2/1/2011
2/1/2016
Herk, Marcel van
CTMM
AIR FORCE
10/1/2008
10/1/2013
Horenblas, Simon
Stichting J.C. van Veen
Bijdrage onderzoek urologie
1/1/2011
1/1/2016
Innocenti, Metello
CGC
Cancer Genomics Center
5/1/2009
1/1/2014
Jacobs, Jacqueline
EEG-CEC / EU
Genome-wide identification of factors controlling the telomere damage response and telomere-driven genomic instability
10/1/2012
10/1/2017
Jacobs, Jacqueline
NWO
A critical role for WHSC1 at telomeres
11/1/2012
11/1/2015
Jacobs, Jacqueline
NWO
Identification of novel mammalian factors involved in telomere-induced chromosome instability and telomere-damage signaling
2/1/2007
2/1/2012
Jalink, Kees
SenterNovem
MEM-FLIM: Modulated Electron-Multiplied all-solid-state camera for Fluorescence Lifetime Imaging Microscopy
3/1/2008
10/31/2013
Jalink, Kees
STW
Labeling strategies for nanoscopy of complex biological specimens
9/1/2011
9/1/2015
Jonkers, Jos
AstraZeneca
Intervention studies with AZD2281, AZD2461 and AZD7762 in mouse mammary tumor model
1/1/2010
1/1/2015
Bernards, René
Merck
Agreement Merck
8/10/2012
8/10/2014
Bernards, René
NWO
Cancer Systems Biology Centre
9/1/2010
9/1/2015
Bernards, René
NWO
Spinozapremie
9/26/2005
12/31/2012
Bernards, René
Overig
Therapie op maat door mutatieanalyse bij kanker
7/1/2011
7/1/2015
Berns, Ton
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
6/1/2012
Berns, Ton
CGC
Cancer Genomics Center
1/1/2008
1/1/2014
Berns, Ton
CTMM
Personalized chemo-radiation of lung and head and neck cancer. WP5: Imageguided adaptive treatment (evaluation during treatment)
4/1/2012
10/1/2013
Berns, Ton
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Berns, Ton
NWO
Mouse Clinic for Cancer and Aging research
10/1/2012
1/1/2018
Blank, Christian
MedImmune Ltd.
Characterization of a new inducible melanoma model. Collaborative Research Agreement
1/1/2011
1/1/2013
Blank, Christian
Melanoma Research Alliance
Development of combined molecular/immunotherapy regimens for human melanoma
5/1/2011
5/1/2014
Borst, Jannie
NWO
A novel type of ubiquitination regulates apoptosis signaling by BH3-only protein Bid.
11/1/2008
11/1/2012
Developing antibodies targeting TNF family members into clinical candidates for treatment of cancer
1/1/2012
Borst, Jannie
TI Pharma
1/1/2014
Borst, Jannie
TI Pharma
TNF ligands in cancer
3/1/2006
7/1/2012
Borst, Piet
EEG-CEC / EU
Molecular mechanisms underlying chemotherapy resistance, therapeutic escape, efficacy and toxicity (CHEMORES)
2/1/2007
8/1/2012
Borst, Piet
ZonMw
Characterization of the physiological roles of Multidrug Resistance Protein (MRP) 1-6 by in vivo screening for their substrates
1/1/2008
1/1/2012
Boven, Hester van
Astra
Neoadjuvante registratiestudie BOOG
1/1/2006
1/1/2014
Brandsma, Dieta
to-BBB technologies BV
An open-label, phase I/IIa dose escalating study of 2B3-101 in patients with solid tumors with brain metastases
8/22/2011
2/22/2014
Brekel, Michiel van den
Verwelius BV
Head and neck cancer research
10/1/2010
10/1/2013
Brekel, Michiel van den
ATOS
Development and evaluation of a third generation ProvoxTM voice prosthesis
6/1/2003
7/1/2015
Broeks, Annegien
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Brummelkamp, Thijn
CGC
Cancer Genomics Center
12/1/2010
1/1/2014
Brummelkamp, Thijn
EEG-CEC / EU
Viral Host Factors, Identification of Host Determinants for Virus Entry using a Haploid Genetic Approach
1/1/2013
1/1/2018
Brummelkamp, Thijn
NIH
The role of the HIppo pathway on mammalian organ size, progenitor cells and cancer
1/1/2009
1/1/2014
Brummelkamp, Thijn
ZonMw
Tissue size control and the regulation of Hippo pathway activity in mammals.
4/1/2011
4/1/2016
216
Ended / Ends
217
Principal Granting Title Started investigator agency
Ended / Ends
Principal Granting Title Started investigator agency
Ended / Ends
Jonkers, Jos
AstraZeneca
Targeting the P13K network in genetically engineered mouse models of invasive lobular breast cancer
11/1/2008
11/1/2012
Leeuwen, Fred van
NWO
Finding readers that can decipher the epigenetic language of the chromatin core
9/1/2011
1/15/2015
Jonkers, Jos
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Linn, Sabine
A Sister’s Hope
Development of a breast cancer BRCA2-like classifier for treatment benefit of DNA-DSB inducing agents
1/1/2011
1/1/2015
Jonkers, Jos
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Linn, Sabine
A Sister’s Hope
Estrogen Receptor interactome from biopsies to guide endocrine treatment
1/1/2012
1/1/2013
Linn, Sabine
A Sister’s Hope
12/1/2013
EEG-CEC / EU
Eurosystem
3/1/2008
9/1/2012
PI3K pathway activation in primary and metastatic estrogen receptor alpha (ERa) positive breast cancer and the association with drug response
12/1/2012
Jonkers, Jos Jonkers, Jos
Merck
Agreement Merck
8/10/2012
8/10/2014
Linn, Sabine
A Sister’s Hope
Toward personalized medicine by using the nationwide population-based breast cancer registry (1989-2009) couple with biobanking: NBCP
6/1/2012
6/1/2014
Jonkers, Jos
NGI
Identification of genes contributing to breast cancer development and therapy resistance in genetically engineered mouse models
10/1/2012
10/1/2016 Linn, Sabine
BBMRI-NL
HEBON - Collection of 1000 BRCA-mutated cancers - DNAs and histopathology
4/1/2012
4/1/2013
Linn, Sabine
EEG-CEC / EU
EUROCANPLATFORM - A European Platform for Translational Cancer Research
1/1/2011
1/1/2016
Linn, Sabine
LSCA Valorisation Fund
Translation of a BAC array-based chip into an oligo array-based with chip to identify hereditary breast cancers with BRCA1 mutations and sporadic breast cancers with a deficient BRCA1 signaling pathway
7/1/2010
3/1/2013
Linn, Sabine
TI Pharma
Applied breast cancer diagnostics
8/15/2011
12/15/2013
Lohuizen, Maarten van
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Lohuizen, Maarten van
CGC
Cancer Genomics Center
10/1/2010
1/1/2014
Lohuizen, Maarten van
EEG-CEC / EU
Eurosystem
3/1/2008
9/1/2012
Lohuizen, Maarten van
EEG-CEC / EU
Functional genetic characterization of a mouse model of Glioma (FGCMOG)
8/1/2010
8/1/2012
Lohuizen, Maarten van
NIRM
NIRM: Epigenetic profiling and lineage tracing of normal and cancer stem cells: Safeguarding normal regenerative stem cells in cancer
1/1/2010
1/1/2016
Lohuizen, Maarten van
NPC
Properties of pluripotency in mouse ESCs and iPS
1/1/2009
1/1/2014
Jonkers, Jos
NWO
Cancer Systems Biology Centre
9/1/2010
9/1/2015
Jonkers, Jos
ZonMw
Ultra-high throughput analysis of insertional mutations to study collaborating cancer genes and genetic networks in mouse tumors
7/1/2010
3/1/2012
Karakullukcu, Baris
PCI Biotech AS
Multi-site study PCIA202/10
6/1/2012
6/1/2013
Kenter, Gemma
EEG-CEC / EU
RAIDs-Rational molecular Assessments and Innovative Drug Selection. FP7Health-2012-Innovation-1
10/1/2012
10/1/2015
Kerkhoven, Ron
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Kind, Jop
NWO
Tracing and mapping of genome-nuclear lamina interactions in single cell
9/1/2011
9/1/2014
Klomp, Houke
Roche
Roche HQ Neo adjuvant Tarceva: Tumor remission rate with neoadjuvant erlotinib in operable patients with clinical stage I/II non-small cell lung cancer (NSCLC) (M06NEL)
7/1/2006
1/1/2013
Linking tumor tissue samples from the OMEGA cohort studies of subfertile women to risk factor data from medical records and questionnaires for advancing molecular and genetic cancer epidemiology
2/1/2012
Leeuwen, Flora van
BBMRI-NL
2/1/2013
Leeuwen, Flora van
CTSU
Heart-breast and heart-Hodgkin case-control studies
11/1/2009
1/1/2013
Lohuizen, Maarten van
NSBC/CGC
Systems Biology of insertional mutagenesis
1/1/2008
1/1/2013
Leeuwen, Flora van
Erasmus Medisch Centrum
Long-term risk of endometrial cancer after ovarian stimulation for in-vitro fertilization. A case-cohort analysis.
1/1/2011
1/1/2013
Lohuizen, Maarten van
NWO
Maintenance of genome integrity by histone (de)ubiquitinating enzymes
11/1/2008
11/1/2013
Medema, René
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Leeuwen, Flora van
NIH
Prediction Model: Breast cancer in women irradiated for a pediatric malignancy
4/1/2010
1/31/2013 Medema, René
CGC
Cancer Genomics Center
1/1/2012
1/1/2014
Medema, René
TI Pharma
Kinases in Cancer 2
6/6/2012
12/31/2013
Medema, René
UMC Utrecht
CGDB Graduate Rotation Programme Lenno Krenning
1/1/2012
10/1/2013
Medema, René
ZonMw
A phosphoproteomic approach to uncover regulators of checkpoint recovery
1/1/2012
1/1/2014
Medema, René
ZonMw
Motor proteins in spindle assembly: targets for cancer therapy?
1/1/2012
1/1/2015
Michalides, Rob
TI Pharma
Nuclear receptors in targeted cancer therapy: improved methods for candidate selection
11/1/2006
1/1/2012
Moolenaar, Wouter
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Moolenaar, Wouter
NWO
Autotaxin, a secreted phosphodiesterase with diverse roles in disease: structural and functional studies
2/1/2011
2/1/2017
Leeuwen, Flora van
Pink Ribbon
Cardiovascular morbidity and mortality in breast cancer survivors: identifying high risk subgroups
1/1/2013
1/1/2017
Leeuwen, Flora van
Pink Ribbon
Pink Ribbon Voorlichting over en sreening op mammacarcinoom bij jonge vrouwen die met radiotherapie zijn behandeld voor Hodgkin lymfoom
2/1/2011
2/1/2013
Leeuwen, Flora van
Pink Ribbon
Prevention of job loss and unemployment of breast cancer survivors. A population- and hospital-based studie in the Netherlands
4/1/2012
4/1/2014
Leeuwen, Flora van
Pink Ribbon
The role of physical activity in the primary prevention of premenopausal breast cancer
4/1/2013
4/1/2014
Leeuwen, Flora van
Vlissingen Lymfoomfonds
Polikliniek overlevers Hodgkin
8/1/2009
8/1/2012
NPC
Chromatin dynamics and epigenetic changes
Leeuwen, Fred van
218
1/1/2009
1/1/2014
219
Principal Granting Title Started investigator agency
Ended / Ends
Principal Granting Title Started investigator agency
Ended / Ends
Ovaa, Huib
NWO
How proteoglycan receptors affect antigen cross-presentation and immunity
10/1/2010
9/1/2012
Ovaa, Huib
NWO
Large Scale Peptide Synthesis and Purification
5/7/2012
9/7/2012
Ovaa, Huib
NWO
Pan- Dub Inhibitors to delineate DUB dependent biology and tools to study the ubiqultinated proteome
8/1/2009
8/1/2013
Ovaa, Huib
NWO
Post-translational transpeptidation and immunity
10/1/2009
10/31/2012
Ovaa, Huib
STW
Development and Commercialization of High Throughput Assays for Drug Discovery in the Ubiquitin Field
7/1/2011
1/1/2014
Ovaa, Huib
STW
Targeting Hypoxia-Inducible Factor-1alpha for Degradation to Inhibit Tumor Cell Proliferation
8/1/2012
8/1/2016
Peeper, Daniel
A Sister’s Hope
Screening and in vivo validation of pharmalogical inhibitors of human breast cancer metastasis
4/1/2011
4/1/2013
Mukherjee, Sach
NIH
The Integrative Cancer Biology Program (ICBP): Centers for Cancer Systems Biology
3/1/2012
2/28/2015
Mukherjee, Sach
NWO
Cancer Systems Biology Centre
9/1/2010
9/1/2015
Neefjes, Jacques
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Neefjes, Jacques
CGC
Methotrexate variants for more selective anti-immune effects in reuma and psoriasis
7/1/2012
12/31/2013
Neefjes, Jacques
EEG-CEC / EU
MHC Class II-omics Towards understanding and manipulation of MHC class II antigen presentation
9/1/2010
9/1/2015
Neefjes, Jacques
EEG-CEC / EU
Systems biology of phagosome formation and maturation - modulation by intracellular pathogens
11/1/2008
5/1/2012
Neefjes, Jacques
EMBO
EMBO Long-Term Fellowship Ilana Berlin
1/1/2012
1/1/2013
Neefjes, Jacques
NPC
Kinome and phosphatasome knock-down libraries
1/1/2009
1/1/2014
Peeper, Daniel
Merck
Agreement Merck
8/10/2012
8/10/2014
Neefjes, Jacques
NPC
Valorisation Voucher. Identification of the methotrexome for drug development
9/1/2012
12/31/2013
Peeper, Daniel
NWO
Synthetic lethality: a novel tactic for selective anticancer drug target discovery
6/1/2007
6/1/2012
Neefjes, Jacques
NWO
Manipulating the MHC class II system to control immune responses in autoimmunity
10/1/2007
10/1/2012
Peeper, Daniel
NWO
The role of nuclear lamina in oncogene-induced senescence and cancer progresssion
6/1/2012
6/1/2016
Neefjes, Jacques
NWO
Systemic approach to define the MHC class I and II modifying secretome
11/1/2012
11/1/2015
Peeper, Daniel
Overig
The role of oncogene-induced senescene in long-latency leukemia
6/30/2010
6/30/2014
Neefjes, Jacques
NWO
The molecular mechanism of the degradation of large molecular complexes
1/1/2010
5/1/2013
Perrakis, Anastassis
NWO
Autotaxin, a secreted phosphodiesterase with diverse roles in disease: structural and functional studies
2/1/2011
2/1/2017
Oldenburg, Hester
Pink Ribbon
Werkhervatting en seksualiteit na borstkanker
9/1/2008
3/1/2013 Perrakis, Anastassis
NWO
11/30/2014
CGC
Methotrexate variants for more selective anti-immune effects in reuma and psoriasis
7/1/2012
12/31/2013
PDB_REDO: procedures for rebuilding and refinement of macromolecular models in X ray crystallography
12/1/2011
Ovaa, Huib
Peters, Peter
1/1/2011
12/31/2014
CTMM
Design and use of enhanced T cell antigens and T cell detection technology for human disease.
10/1/2010
10/1/2014
Aeras Global TB Vaccine Foundation
Research to improve recombinant BCG
Ovaa, Huib
Ovaa, Huib
EEG-CEC / EU
Decoding the ubiquitin code
11/1/2011
11/1/2016
Peters, Peter
EEG-CEC / EU
NOTOX Predicting long-term toxic effects using computer models based on systems characterization of organotypic cultures
1/1/2011
1/1/2016
Ovaa, Huib
EEG-CEC / EU
EU-OPENSCREEN European Infrastructure of Open Screening Platforms for Chemical Biology
11/1/2010
11/1/2013 Peters, Peter
EEG-CEC / EU
Protecting the food chain form prions: shaping European priorities through basic and applied research
10/1/2009
10/1/2014
Peters, Peter
Leprastichting
How mycobacteria lyse the phagosomal membrane
1/1/2011
1/1/2015
Peters, Peter
MIN OCW
E-cadhering and cadhering-11 and their role in cancer migration” binnen het SmartMix programma NIMIC
1/1/2009
7/1/2013
Peters, Peter
NanoNextNL
FES HTSM Towards a Sustainable and Open Innovation Ecosystem
1/1/2010
12/31/2016
Peters, Peter
NIH
Pathways of Antigen Presentation by CD1
7/1/2010
6/30/2015
Ovaa, Huib
EEG-CEC / EU
Protein Stabilization by chemistry: total synthesis of an MHC class I
1/1/2011
4/21/2013
Ovaa, Huib
EEG-CEC / EU
UPStream-European Research Training in the Ubiquitin Proteasome System
11/1/2011
11/1/2014
Ovaa, Huib
NGI
An Integrated approach to immunomodulatory agents and T-cell tumor vaccines
10/1/2011
10/1/2015
Ovaa, Huib
NGI
Commercial development of ubiquitinated peptides and screening assays produced by high throughput robotic peptide synthesis
1/1/2010
1/1/2012
Ovaa, Huib
NPC
Commercial development of synthetic Nedd8 and SUMO conjugates
1/1/2009
1/1/2014
Poel, Henk van der
CTMM
Prostate Cancer Molecular Medicine (PCMM)
12/1/2009
12/1/2014
Ovaa, Huib
NPC
Tyrosine phosphatase inhibitors, baits and ABPs
1/1/2009
1/1/2014
Riele, Hein te
MAAG LEVER DARM
Modifiers of tumor development in Lynch syndrome
11/1/2010
11/1/2012
Ovaa, Huib
NPC
Valorisation Voucher. Identification of the methotrexome for drug development
9/1/2012
12/31/2013 Riele, Hein te
NWO
A novel procedure for enzymatic production of low-complexity RNAI Libraries to identify coding- and non-coding tumor suppressor genes
8/1/2009
8/1/2012
Development of conditional protein-ligand exchange applied to immune technology; Onderzoeksprogramma: Integration of Biosynthesis & Organic Synthesis (IBOS)
9/1/2008
Riele, Hein te
NWO
Oligonucleotidedirected gene targeting: evading mismatch repair
1/1/2013
1/1/2017
Ovaa, Huib
220
NWO
9/1/2012
221
Principal Granting Title Started investigator agency
Ended / Ends
Principal Granting Title Started investigator agency
Ended / Ends
Riele, Hein te
NWO
Sister chromatid cohesion: lessons from yeast, mice and men
12/1/2012
12/1/2015
Schmidt, Marjanka
BBMRI-NL
HEBON – ExomeChip for genotyping of non-BRCA1/2 carriers
1/1/2012
1/1/2014
Riele, Hein te
Universiteitsklinikum Dusseldorf
Generation of FAAP24 knockout and T212M mutant mice
5/1/2012
8/1/2012
Schumacher, Ton
ACTS (NWO)
Development of conditional protein-ligand exchange applied to immune technology
9/1/2008
9/1/2012
Schumacher, Ton Oligonucleotide-directed gene modification in human induces pluripotent stem cells
6/1/2011
6/1/2016
Development and clinical evaluation of new strategies for adoptive cell transfer (ACT) in the treatment of cancer
1/1/2014
ZonMw
Centre for Cancer Immune Therapy (CCIT)
1/1/2009
Riele, Hein te
Rodenhuis, Sjoerd
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Schumacher, Ton
CTMM
Design and use of enhanced T cell antigens and T cell detection technology for human disease.
10/1/2010
10/1/2014
Rookus, Matti
BBMRI-NL
HEBON - Plating of 10.000 DNA samples of women tested for BRCA 1/2 mutations
5/1/2010
5/1/2012
Schumacher, Ton
EEG-CEC / EU
ATTRACT - Advanced Teaching and TRaining for Adoptive Cell Therapy
10/1/2009
10/1/2013
Schumacher, Ton
EEG-CEC / EU
Barcoding approach for dendritic cells differentiation (BARDIF)
4/1/2011
4/1/2013
Rookus, Matti
Pink Ribbon
Website voor families met een hoog risico op borst- en/of ovariumkanker
9/1/2010
9/1/2012 Schumacher, Ton
EEG-CEC / EU
Mapping the life histories of T cells
5/1/2011
5/1/2016
Rookus, Matti
ZonMw
HEBON research facility for individuals tested for BRCA 1/2 mutations
11/1/2009
11/1/2013 Schumacher, Ton
EEG-CEC / EU
11/1/2013
EEG-CEC / EU
Collaborative Oncological Gene-environment Study
5/1/2009
5/1/2013
SPHINX - Spontaneous clearance in Patients acutely infected with HCV Immune profiling, Novel biomarkers and X-omics approaches
11/1/2010
Rookus, Matti Rookus, Matti
Universiteit Utrecht
Shift work and risk of breast cancer, a prospective cohort study among Dutch nurses
9/1/2008
9/1/2016
Schumacher, Ton
H.F.S.P.O.
Single cell lineage tracing to understand hematopoietic development and differentiation. HFSP grant
8/1/2012
8/1/2015
Rottenberg, Sven
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Schumacher, Ton
Landsteiner Stichting
Graft versus Host disease by TCR-engineered T cells: mechanisms and means for prevention
1/1/2009
1/1/2012
Rottenberg, Sven
ZonMw
Predicting and targeting primary chemotherapy resistance in conditional mouse models of breast cancer
1/1/2011
12/31/2015
Schumacher, Ton
LLS
Career Development Program
7/1/2010
1/1/2013
Schumacher, Ton
ZonMw
Intravital imaging and computational modeling of skin immunity
3/1/2011
3/1/2015
Ruers, Theo
CTMM
Non-invasive treatment of cancer by MRI-guided high-intensity focused ultrasound ablation
12/1/2009
12/1/2014 Schumacher, Ton
ZonMw
T cell receptor gene therapy of metastatic melanoma: a phase I clinical trial
4/1/2005
2/1/2013
Sinaasappel, Michiel
LSCA Valorisation Fund
Clinical validation of a Phase-sensitive Gamma Detector
7/1/2010
3/1/2013
Sixma, Titia
CBG
Identification of cancer-relevant genes using a functional genetic approach
1/1/2009
1/1/2014
Sixma, Titia
EEG-CEC / EU
Mismatch2model-Characterization and quantitative modeling of DNA mismatch repair and its role in the maintenance of genomic stability and cancer avoidance
11/1/2008
11/1/2012
Sixma, Titia
EEG-CEC / EU
Neurotransmitter Cys-loop receptors: structure, function and disease
2/1/2008
8/1/2012
Sixma, Titia
EEG-CEC / EU
The balance of ubiquitin conjugation and deconjugation
2/1/2010
2/1/2015
Sixma, Titia
NWO
Initiation of DNA mismatch repair
8/1/2011
8/1/2016
Sixma, Titia
NWO
Regulation of H2A ubiquitination
6/1/2012
6/1/2018
Sixma, Titia
NWO
The regulatory role of Ubl domains in ubiquitin specific protease function
10/1/2010
10/1/2015
Sonke, Jan-Jakob
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Sonnenberg, Arnoud
DEBRA
Studying Kindler Syndrome in an in vivo model system
10/1/2011
10/1/2013
Sonnenberg, Arnoud
Nierstichting
Function of tetraspanin-integrin complex Cd151- a3B1 in development and maintenance of the glomerular filtration barrier
10/1/2008
5/1/2013
Sonnenberg, Arnoud
NWO
Mechanical control of chromatin organization and gene expression
9/1/2011
9/1/2014
Steensel, Bas van
EEG-CEC / EU
Principles of Chromatin Organization
3/1/2012
3/1/2017
Ruers, Theo
Philips
Research collaboration Philips
4/1/2010
7/1/2013
Sandick, Johanna van
Vrolijk
Slokdarmkankeronderzoek
1/1/2008
5/1/2013
Schagen, Sanne
Fonds NutsOhra
Analyse en reductie van neurocognitieve afwijkingen bij hersenbestraling bij kankerpatienten
10/1/2011
10/1/2014
Schellens, Jan
Merck
Merck OncoNet Member Agreement
12/1/2009
1/1/2012
Schellens, Jan
Roche
Support Roche clinical research associate Candersartan study M06HER
2/1/2009
8/1/2014
Schinkel, Alfred
Malaysian Government
Fellowship SC Tang: Impact of apical ABC transporters and Cytochrome P4503A on novel tyrosine kinase inhibitor anticancer drugs
Schmidt, Marjanka
EEG-CEC / EU
CAncer Risk and INsulin analoGues
11/1/2011
11/1/2015
Schmidt, Marjanka
EEG-CEC / EU
Collaborative Oncological Gene-environment Study
5/1/2009
5/1/2013
Schmidt, Marjanka
BBMRI-NL
Regenboogproject 6 Towards a joint strategy for the return of results and optimal communication with biobank donors
1/1/2012
1/1/2014
Finding the balance between overdiagnosis and undertreatment: risk of ipsilateral and contralateral invasive breast cancer and breast cancer death after primary ductal carcinoma in situ
3/1/2012
Assessment of genetic, lifestyle and environment information for geneenvironment-tumor subtype interaction studies within the randomized breast cancer trial ‘MINDACT’ (EORTC 10041/BIG 3-04)
1/1/2012
BOSOM – ExomeChip for genotyping of young breast cancer patients
1/1/2012
Schmidt, Marjanka
Schmidt, Marjanka
Schmidt, Marjanka
222
Pink Ribbon
EORTC 10041/BIG 3-04
BBMRI-NL
3/1/2014
1/1/2014
1/1/2014
223
Principal Granting Title Started investigator agency
Ended / Ends
Principal Granting Title Started investigator agency
Visser, Karin de
ZonMw
The inflammatory tumor microenvironment and its impact on breast cancer development and therapy
2/1/2009
6/1/2014
Wesseling, Jelle
Pink Ribbon
Finding the balance between overdiagnosis and undertreatment: risk of ipsilateral and contralateral invasive breast cancer and breast cancer death after primary ductal carcinoma in situ
3/1/2012
3/1/2014
Wesseling, Jelle
TI Pharma
Applied breast cancer diagnostics
8/15/2011
12/15/2013
Wesseling, Jelle
ZonMw
Automated histology slide imaging and biomarker and biomarker recognition for standardized, quantitative and high-throughput tissue evaluation
9/1/2011
9/1/2015
Wessels, Lodewyk
AstraZeneca
Identification and validation of new targets for colorectal cancer through molecular redefinition of the disease
1/1/2011
12/1/2013
Steensel, Bas van
NGI
Chromatin Protein Discovery Project
2/1/2008
2/1/2012
Steensel, Bas van
NSBC/CGC
Systems biology of insertional mutagenesis
1/1/2008
1/1/2013
Steensel, Bas van
NWO
Nuclear lamina genome interactions in mammalian cells
7/1/2009
7/1/2014
Steensel, Bas van
ZonMw
Control of chromosome architecture by nuclear lamins: role in laminopathies.
9/1/2012
9/1/2017
Tan, Bin
Biolitec
The cost-effectiveness study
1/1/2009
7/1/2012
Tan, Bin
ZonMw
A multi-centre cost-effectiveness evaluation of a novel treatment option in the Netherlands: Photo Dynamic Therapy with temoporfine for the treatment of advanced incurable head and neck cancers, for whom prior conventional treatments have failed.
1/1/2009
7/1/2012
Ended / Ends
Tan, Bin
ZonMw
New therapy for Epstein-Barr virus driven tumours by targeting the virus itself N10GVV
1/1/2011
1/1/2013
Wessels, Lodewyk
AstraZeneca
Pathway-based tumour characterization & alignment of cell panels against tumour samples
2/1/2010
11/1/2012
Tellingen, Olaf van
Stop Hersentumoren
Promising drug combinations in the treatment of high-grade glioma
12/1/2011
12/1/2013
Wessels, Lodewyk
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014
Valdes Olmos, Renato
Ministerie van Economische Zaken
Real-time fhSpect
7/1/2012
7/1/2014
Wessels, Lodewyk
EEG-CEC / EU
Collaborative Oncological Gene-environment Study
5/1/2009
5/1/2013
Wessels, Lodewyk
EEG-CEC / EU
EuroTARGET
3/1/2011
3/1/2016
Veer, Laura van ‘t
CGC
Cancer Genomics Center
8/1/2008
1/1/2014
Wessels, Lodewyk
NBIC
NBIC-II BioRange BR4.9
10/1/2009
12/31/2013
Veer, Laura van ‘t
EEG-CEC / EU
Breast Cancer Somatic Genetics Study Grant
7/1/2010
7/1/2014
Wessels, Lodewyk
NGI
NCSB AddOn projecten CGC en CMSB
11/1/2010
1/1/2014
Verheij, Marcel
AstraZeneca
Olaparib-Radiotherapy combination trials: optimization, normal tissue toxicity evaluation and biomarkers for toxicity and tumour response
1/1/2012
1/1/2014
Wessels, Lodewyk
NSBC/CGC
Systems biology of insertional mutagenesis
9/1/2009
1/1/2014
Wessels, Lodewyk
ZonMw
5/1/2013
EEG-CEC / EU
Adaptive and innovative Radiation Treatment FOR improving Cancer patients treatment Outcome
4/1/2011
4/1/2016
Signaling pathways and gene regulatory networks responsible for th17 cell differentiation
5/1/2010
Verheij, Marcel
Wolthuis, Rob
H.F.S.P.O.
Cycle-Quant: Defining Cell Cycle Progression and Responses to Perturbations
10/1/2010
10/1/2013
Verheij, Marcel
EOS / voorheen Philips
Framework Research Agreement between Elekta and NKI/AvL
8/10/2010
8/10/2020 Zwart, Wilbert
A Sister’s Hope
A genomics-based tool to predict tamoxifen success
1/1/2012
1/1/2014
Verheij, Marcel
LSCA Valorisation Fund
Dose-finding study for a novel liposomal doxorubicon formulation
7/1/2010
3/1/2013
Zwart, Wilbert
A Sister’s Hope
Estrogen Receptor interactome from biopsies to guide endocrine treatment
1/1/2012
1/1/2013
Zwart, Wilbert
Pink Ribbon
3/1/2014
NGI
Ceronco: Enhancing drug membrane translocation in cancer treatment
8/29/2012
8/29/2014
An individualized prediction for the risk of tamoxifen-induced endometrial cancer
3/1/2012
Verheij, Marcel Verheij, Marcel
STW
Catalyzing drug membrane translocation
5/1/2012
5/1/2013
Zwart, Wilbert
ZonMw
Spatial/temporal regulation of the Estrogen Receptor transcription complex and its role in breast cancer
5/1/2012
5/1/2016
Vijver, Marc van de
CTMM
Neoadjuvant drug treatment for breast cancer - response-prediction and response monitoring (BREAST CARE)
2/1/2009
2/1/2014 Zwart, Wilbert
TI Pharma
Estrogen Receptor deubiquitination by otubains as a novel drug target
12/1/2012
6/1/2014
Visser, Karin de
AICR
Tumor-associated macrophages as therapeutic targets for metastatic breast cancer
6/1/2011
6/1/2014
Visser, Karin de
AstraZeneca
Functional assessment of CSF-1 receptor signaling in de novo breast cancer development and metastatis information
11/1/2009
12/31/2012
Visser, Karin de
EEG-CEC / EU
BMDCMET - Innate and adaptive immune cell contribution to the premetastatic niche
6/1/2011
6/1/2013
Visser, Karin de
EEG-CEC / EU
Tumour Infiltrating Myeloid Cell Compartment. Marie Curie
11/1/2012
11/1/2016
Visser, Karin de
Roche
Research Agreement Roche / NKI Amsterdam Combichemo
5/1/2011
5/1/2013
Visser, Karin de
Roche
Research Agreement Roche / NKI Amsterdam Metastasis
5/1/2011
5/1/2013
224
225
Personnel index
A Aalbers, Arend 144 Aalbersberg, Else 118 Aaronson, Neil 12 Abdullah, Kahairi 144 Abrao Possik, Patricia 66 Achachah, Mohamed 119 Adriaansz, Sandra 130 Afanasyev, Pavel 70 Agami, Reuven 14 Ahrends, Tomasz 28 Ait Moha, Daoud 119 Ajouaou, Abderrahim 119, 185 Akthar, Waseem 102 Albers, Harald 64 Alderlieste, Yasser 130 Alderliesten, Tanja 158 Aleman, Berthe 158 Alendar, Andrej 22 Alexi, Xanthippi 114 Alpay, Erdenay 158 Amendola, Mario 104 Amore, Alessia 64 Angelopoulos, Nicos 110 Appelman, Jolanda 178 Aprelia, Melinda 56 Argenzio, Elisabetta 58 Ariese-Beldman, Karin 145 Ariotti, Silvia 84
B Baank, Saskia 118 Baars, Danny 178 Baars, Jessica 26 Baars, Joke 130 Baas-Vrancken Peeters, Marie-Jeanne 144 Baas, Paul 130 Baatje, Sumiati 184 Babala, Nikolina 28 Baban, Shan 184 Badhai, Jitendra 22 Bakker, Jeroen 62 Bakker, Martine 118 Bakker, Suzanne 185 Balague Ponz, Olga 118 Balm, Alfons 144 Banishki, Nikola 30, 72 Barbier, Nathalie 178 Bartelink, Harry 158 Batteau, Lukas 118 Beekman, Chantal 118 Beelen, Karin 54, 130 Beijersbergen, Roderick 16, 185 Beijnen, Jos 76, 130 Bekema, Erwin 184 Belderbos, José 74, 158 Beltman, Joost 84 Bendle, Gavin 84 Benne, Irene 185 Benraadt, Tinie 178 Bentin Toaldo, Cristiane 118 Bergman, André 18, 130 Berkhof, Marianne 12 Berlin, Ilana 62 Bernards, René 20 Berns, Anton 22 Berns, Katrien 20 Bes-Gennissen, Annemiek 20 Besnard, Peter 118 226
Bessels, Frauwkje 178 Betgen, Anja 158 Bex, Axel 144 Bies, Laura 84 Biewenga, Petra 144 Bin Ali, Rahmen 22, 185 Bins, Adriaan 130 Blank, Christian 24, 130 Bleiker, Eveline 26, 119 Bloemers, Monique 158 Blom, Marie-José 96 Blom, Marleen 102 Blomen, Vincent 32 Bochove, Glenda 158 Boekel, Naomi 96 Boekhout, Annelies 130 Boekhout, Michiel 112 Boer, Mandy 42 Boerrigter-Barendsen, Lucie 118 Boersma, Vera 48 Bohoslavsky, Roman 158 Bol, Mijke 118 Bombardelli, Lorenzo 22 Bonzanni, Nicola 110 Boogerd, Willem 74, 130 Boon, Ute 52 Boot, Henk 130 Boot, Judith 178 Borst, Gerben 158 Borst, Jannie 28 Borst, Piet 30, 72 Bos, Arnold 14 Boshuizen, Rogier 130 Bosma, Astrid 20 Bosma, Libertje 119 Botma, Henk 178 Boucher, Audi 178 Boutmy-de Lange, Majella 119 Bouwman, Peter 52 Braaf, Linde 118, 184 Brandenburg, Alexander 82, 96 Brandsma, Dieta 130 Braumuller, Tanya 52, 185 Breuer, Marco 184 Brinkman, Eva 104 Brocks, Lenny 184 Broeks, Annegien 82, 118, 184 Brohet, Richard 96 Bronsveld, Heleen 82 Brood, Monique 144 Brouwer, Oscar 118, 144 Brugman, Wim 184 Bruin, Natascha 118, 158 Bruining, Annemarie 118 Bruinsma, Tineke 178 Bruinsma, Wytse 56 Brummelkamp, Thijn 32 Brunen, Diede 20 Bucholtz, Karina 130 Buckle, Tessa 118 Buikhuisen, Wieneke 130 Buil, Levi 118 Buitelaar, Dick 145 Buma, Sannine 145 Buning-Kager, Marian 118 Bunschoten, Anton 118 Burgers, Sjaak 130 Burylo, Artur 76 Bussing, Heleen 178
C Caesar, Lisa 144 Caillat, Christophe 68 Campbell, Andrew 44 Canisius, Sander 110 Cantelli, Erika 92 Carreno, Monique 178 Cats, Annemieke 26, 130 Celie, Patrick 185 Chen, Chun 158 Chen, Tao 104 Chen, Wei 158 Chin, Patrick 118 Chuan Tang, Seng 80 Ciampricotti, Metamia 36 Citterio, Elisabetta 102 Clapham, Renee 144 Clerici, Marcello 86 Clijsters, Linda 112 Coffelt, Seth 36 Cooke, Saskia 178 Cordeiro Pedrosa, Lília 106 Cornelissen, Lisette 52 Cornelissen, Paulien 102 Cornelissen, Sten 82 Coumou, Annette 130 Cozijnsen, Miranda 22 Craenmehr, Jacques 178 Crijns, Marianne 26, 145
D Dackus, Gwen 54 Dalesio, Otilia 178 Damen, Eugene 158 Dannenberg, Jan-Hermen 34 Dayal, Saurabh 106 De Boer, Jan Paul 130 De Boer, Johan 158 De Boer, Maurits 26 De Boer, Roel 158 De Bois-Bakker, Anita 185 De Bois, Josien 158 De Cortie, Karin 90 De Graaf, Carolyn 104 De Groot, Renate 118, 184 De Haan, Rosemarie 106, 158 De Haas, Marcel 104 De Jong, Annemieke 64 De Jong, Gerda 178 De Jong, Jeroen 118 De Jong, Johann 110 De Jong, Monique 106, 158 De Jong, Rianne 158 De Jong, Trees 38 De Krijger, Inge 48 De Lange, Judith 96 De Lint, Klaas 16 De Mots, Mieke 185 De Punder, Karin 70 De Ridder, Mischa 144 De Rink, Iris 184 De Ronde, Jorma 108, 110 De Ruiter, Julian 52, 110 De Ruiter, Michiel 74 De Visser, Karin 36 De Vries, Evert 28 De Vries, Hilda 22 De Vries, Jeltje 178 De Vries, Kim 158 De Vries, Nienke 102
De Vries, Sandra 104 De Vrije, Lex 184 De Waal, Marjolijn 178 De Widt, John 185 De Wit-van der Veen, Linda 118 De Wit, Ineke 178 Decker, Nicky 96 Dees-Ribbers, Hermine 158 Deken, Marcel 24 Dekker, Marleen 92 Delzenne-Goette, Elly 92 Derissen, Ellen 130 Derks, Elin 144 Dewit, Luc 158 Dihalu, Dosti 158 Dijkgraaf, Feline 84 Dijkstra, Minze 184 Djajdiningrat, Rosa 144 Dohmen, Amy 62, 144 Dondelinger, Frank 60 Donker, Mila 144 Doodeman, Barry 158 Doornebal, Chris 36 Dorlo, Thomas 130 Douma, Sirith 66 Droog, Marjolein 114 Drost, Brigitte 26, 145 Drost, Jarno 14 Drost, Rinske 52 Drukker, Caroline 82 Dubbelman, Anne Charlotte 130 Dubbelman, Ria 130 Dufournij, Brigitte 178 Duppen, Joop 158 Durmus, Selvi 80 Duursma, Anja 56
E Efthymiou, Katina 145 Eijkenboom, Dion 144 Eijzenga, Willem 12, 26 Eilers, Marlou 178 Ekkebus, Reggy 64 El Atmioui, Dris 64, 185 El Oualid, Farid 64 Elbatsh, Ahmed 56 Elbers, Jos 158 Elkhuizen, Paula 158 Elkon, Rani 14 Elouarrat, Dalila 58 Elshof, Lotte 108 Epping, Mirjam 66
F Fan, Lin 118 Fanchi, Lorenzo 84 Fase, Sandra 96 Feenstra, Christel 118 Feenstra, Heleen 74 Feringa, Femke 56 Fernandez Salcedo, Ernesto 178 Fish, Alex 86 Flach, Koen 114 Fles, Renske 144 Floot, Ben 90 Foekema, Joke 130 Frantzen, Marloes 158 Frederikx, Geert 76, 130 Fruijtier, Agnetha 74
227
G Gadiot, Jules 28 Galama, Hylke 185 Gallenne, Tristan 66 Gambino, Valentina 20 Gardeniers, Danielle 118 Gargiulo, Gaetano 102 Garska, Gosia 62 Gasparini, Alessia 158 Gerritsma, Miranda 12, 96 Geumann, Ulf 28 Geurink, Paul 64 Gilhuijs, Kenneth 118 Gisler, Santiago 102 Godsave, Sue 70 Goey, Andrew 130 Gogola, Ewa 30, 72 Gomez, Raquel 84 Greig, Kylie 66 Grernrum, Wipawadee 20 Grijpink, Lindsay 178 Groen, Wim 94 Groenendaal, Greetje 158 Groenendijk, Floris 20 Groenewegen, Jan 158 Groot, Harmke 96 Groot, Yvonne 178 Groothuizen, Flora 86 Grootscholten, Cecile 130 Grosveld, Rik 185 Guislain, Aurelie 24 Guyader, Charlotte 30, 72
H Haanen, John 38, 130 Haarhuis, Judith 56 Haas, Rick 158 Hage, J Joris 145 Hagen, Patricia 178 Hagenaars, Christiane 178 Hagendoorn, Jeroen 144 Hahn, Cristoph 145 Hahn, Daniela 26, 119 Halim, Vincentius 56 Halonen, Pasi 16, 185 Hameed, Dharjath 64 Hamming-Vrieze Olga 158 Hanegraaf, Maaike 22 Harinck, Femme 26 Haringhuizen, Annebeth 130 Harms, Emmy 130 Harmsen, Tim 92 Hau, Song-Hieng 178 Hau, Tisee 36 Hauptmann, Michael 40 Haussman, Jens 68 Heemsbergen, Wilma 40, 158 Heemskerk, Bianca 38 Heidebrecht, Tatjana 68 Heideman, Richard 34 Heijmink, Stijn 118 Helgason, Helgi 130 Hendrikx, Jeroen 76, 80, 130 Henneman, Linda 52 Henneman, Roel 144 Heukelom, Jolien 158 Heynen, Guus 20 Hibbert, Rick 86 Hiemstra, Annelies 178 Hijmans, Marielle 20 228
Hilgers, Frans 144 Hilkens, John 42 Hilkmann, Henk 64, 185 Hill, Steven 60 Hillebrand, Michel 130 Hinnen, Karel 158 Hoefnagel, Cornelis 118 Hoekstra, Paula 178 Hofland, Ingrid 118, 184 Hogenbirk, Marc 46 Hogervorst, Frans 118, 119 Hollenstein, Andreas 84 Hollmann, Birgit 158 Holman, Esther 185 Holt, Andrea 158 Holtkamp, Marjo 130 Honnef, Joeri 158 Hooijkaas, Anna 24 Hoorn, Frank 185 Hoornweg, Marije 145 Hoppes, Rieuwert 64 Horenblas, Simon 144 Horlings, Hugo 20 Houben, Anna 58 Houlleberghs, Hellen 92 Houweling, Anette 158 Hoving, Saske 90, 118 Huang, Sidong 20 Huijbers, Ivo 22, 185 Huissoon, Sandra 145 Huitema, Alwin 130 Hulshoff, Lenie 145 Hulsman, Danielle 102 Hummel, Lisanne 12 Hunneman, Daoin 118
I Ibrahim, Tihana 144 IJpelaar, Annemarie 158 Ikink, Gerjon 42 Inanc, Burcu 106 Innocenti, Metello 44 Iskit, Sedef 66 Isogai, Tadamoto 44 Iusuf, Dilek 80 Ivanov, Eduard 178
J Jacobi, Irene 144 Jacobs, Bart 76, 130 Jacobs, Heinz 46 Jacobs, Jacqueline 48 Jae, Lucas 32 Jager, Nynke 130 Jakobs, Wendy 74 Jalink, Kees 50 Jansen, Edwin 158 Jansen, Marissa 178 Jansen, Robert 30 Janssen, Aniek 56 Janssen, Esther 96 Janssen, Hans 70, 184 Janssen, Lennert 62 Janssen, Tomas 158 Janssens, Soe 145 Janssens, Tine 130 Jaspers, Janneke 52, 72 Jastrzebski, Kathy 16 Jeanson, Kiki 119 Jenal, Mathias 14
Jessurun, Nico 158 Jibodh, Aarti 130, 178 Johnson, Jackie 20 Jolink, Casper 144 Jongsma, Maikel 58 Jongsma, Marlieke 62 Jonker, Marcel 158 Jonkers, Irene 178 Jonkers, Jos 52 Jonkman, Joop 178 Joosten, Krista 68 Joosten, Robbie 68 Juan Xu, Yan 56
K Kaasenbrood, Tim 158 Kaiser, Andrew 24 Kalisvaart, Robin 158 Kambey, Erwin 184 Kanhai, Meena 185 Kant, Josien 178 Kaplon, Joanna 66 Karakullukcu, Baris 144 Kas, Sjors 52 Kasiem, Mobien 119 Kedziora, Kasia 50 Keeman, Renske 82 Keessen, Marianne 130 Kelderman, Sander 84 Kemper, Kristel 66 Kemperman, Myrle 74 Kenter, Gemma 144 Kerkhoven, Ron 184 Kersbergen, Ariena 30, 72 Kerst, Martijn 130 Kersten, Kelly 36 Kersten, Kim 74, 96 Ketema, Mirjam 88 Kieffer, Jacobien 12, 26, 74 Kieft, Mariette 40 Kim, Robbert 86 Kind, Jop 104 Kist, Jacob 118 Klarenbeek, Jeffrey 50 Klarenbeek, Sjoerd 52, 184 Klomp, Houke 144 Klompmaker, Rob 56 Klop, Martin 144 Kluijt, Irma 119 Kluin, Roel 184 Klümpen, Heinz Josef 130 Knegjens, Joost 158 Knegt, Cuna 144 Knijnenburg, Theo 110 Knockaert, Bart 144 Knol, Cora 96 Kobus Monica 74 Koch, André 56 Koenen, Annemieke 130 Koetsveld, Folkert 158 Kolhate, Lies 178 Kooijman, Karen 96 Koolen, Bas 118 Koops, Wim 118 Koornstra, Rutger 54 Kooy, Raymond 64 Koppelmans, Vincent 74 Koppens, Martijn 102 Korse, Tiny 118 Kort, Anita 130 Krap, Menno 144 Kreeft, Anne Marijn 144
Kreft, Maaike 88 Kregel, Eva 52 Krenning, Lenno 56 Krimpenfort, Paul 22, 185 Kristel, Petra 108 Kroeskamp, Truud 185 Kröger, Robert 118 Kromdijk, Wiete 130 Kruis, Matthijs 158 Krul, Inge 96 Küçükosmano lu, Asli 30, 72 Kuenen, Marianne 12, 26, 74 Kuhlmann, Koert 144 Kuijpers, Wilma 94 Kuiken, Johan 16 Kuil, Joeri 118 Kuiper, Maria 130 Kujala, Pekka 70 Kumar, Prasanth 20 Kvistborg, Pia 38 Kwint, Margriet 158 Kwon, Min-chul 22
L Lambooij, Jan Paul 22 Lamers, Theo 185 Lammens, Chantal 12 Lammerts van Buren, Alicia 70 Lancini, Cesare 34, 102 Lange, Charlotte 118 Lange, Jan 144 Langhout, Niels 144 Lankheet, Nienke 130 Lansaat, Liset 144 Lebesque, Joos 158 Leijen, Suzanne 76, 130 Lenain, Christelle 66 Léveillé, Nicolas 14 Leyton Puig, Daniela 50 Lieftink, Cor 16, 185 Lieshout, Michiel 144 Linders, Dorothé 118 Linn, Sabine 54, 130 Linnemann, Carsten 84 Lips, Esther 108 Loayza Puch, Fabricio 14 Lodder, Mels 84 Lodder, Wouter 144 Lohuis, Peter 144 Lok, Christianne 144 Loo, Claudette 118 Lopes, Rui 14 Lopez, Marta 40 Luijten-Verwoerd, Desiree 185 Luimstra, Jolien 64 Lukas, Anne 130, 145
M Mahn, Marianne 178 Maia, Rita 56 Maiburg, Merel 119 Majewski, Ian 20 Majoor, Donné 118, 184 Makatipu-Kambey, Francis 185 Maletta, Massimiliano 70 Mallo, Henk 130 Mandjes, Ingrid 178 Mans, Anton 158 Marchetti, Serena 76, 130 Margadant, Coert 88
Marsman, Marije 185 Matas-Rico, Elisa 58 Mattiroli, Francesca 86 Medema, René 56 Mehta, Akash 144 Meijer, Richard 144 Meijnen, Philip 158 Meinhardt, Wim 144 Meissl, Katrin 66 Melis, Jacoline 12 Melis, Monique 18 Melo, Carlos 14 Mencarelli, Angelo 158 Menning, Sanne 74 Merkx, Remco 64 Merqui-Roelvink, Marja 130 Mertens, Laura 144 Meuleman, Wouter 104 Meulenaar, Jelte 130 Meulendijks, Didier 76, 130 Meulepas, José 40 Mewes, Janne 94 Mezzadra, Riccardo 84 Michalak, Ewa 52 Michaut, Magali 110 Michels, Samira 38 Mijnheer, Ben 158 Milojkovic, Bojana 76, 130 Miquel Cases, Anna 94 Mittempergher, Lorenza 20 Modder, Carla 178 Moes, Johannes 130 Mooij, Thea 96 Mook, Stella 158 Moolenaar, Wouter 58 Moonen, Luc 158 Morphey, Mary 70 Morris, Ben 16, 185 Mueller, Judith 66 Mukherjee, Sach 60 Mulder, Lennart 108 Mulder, Monique 64 Mulkens, Frits 184 Muller, Pietje 178 Muller, Saar 118 Muris, Jettie 118 Mylvaganan, Chelvi 118
N Nacerdine, Karim 102 Nahidi, Leila 50 Naik, Shalin 84 Navran, Arash 158 Nederlof, Petra 108, 118, 119 Neefjes, Jacques 62 Neuzillet, Yann 66 Nichol, Kiri 158 Nieuweboer-Krobotova, Inka 145 Nieuwenhuis, Joppe 32 Nieuwland, Marja 184 Nieweg, Omgo 144 Nijkamp, Jasper 158 Nijkamp, Wouter 16 Nixon, Iain 144 Nol, Annemarie 130 Nooijen, Willem 118 Nowee, Marlies 158 Nuijen, Bastiaan 130 Nuijten, Elvira 178 Numan, Rachel 144 Nyst, Heike 144
O Oates, Chris 60 Ogink, Janneke 112 Oldenburg, Hester 74, 144 Olszewska, Agnieszka 158 Onderwater, Suzanne 130 Ong, Nico 70, 184 Oomen, Lauran 184 Oosterhuis, Koen 38 Opdam, Frans 130 Opdam, Mark 54 Opstal-van Winden, Annemieke 96 Oude Vrielink, Joachim 14 Ouwens, Gabey 96 Ovaa, Huib 64 Overwater, José 184
P Paalman, Carmen 96 Paape, Anita 118 Pagie, Ludo 104 Pang, Baoxu 62 Panneman, Carmen 158 Pasca, Edoardo 118, 158 Paul, Petra 62 Pauwels, Caroline 178 Pawlitzky, Inka 102 Peen, Kirsten 118 Peeper, Daniel 66 Pelders, Saskia 96 Pengel, Kenneth 158 Perez, Arturo 94 Perie, Leila 84 Perrakis, Anastassis 68 Peters, Carolien 158 Peters, Dennis 118, 184 Peters, Peter 70 Peulen, Heike 158 Peuscher, Marieke 48 Pevenage, Philip 118 Pfauth, Anita 184 Philips, Daisy 84 Piek-den Hartog, Marianne 144 Pigot, Garry 144 Pijpe, Anouk 26, 96 Pindyurin, Alexey 104 Ploeger, Lennert 158 Pluim, Dick 76 Pluister, Yvonne 118 Poell, Jos 16 Polders, Daniel 158 Pool, Bert 118 Pos, Floris 158 Prahallad, Anirudh 20 Pramana, Jimmy 144 Prevoo, Warner 118 Pritchard, Colin 22, 185 Pronk, Loes 178 Proost, Natalie 22 Pruntel, Roelof 119, 185
Q Qi, Wen 118 Qiao, Xiaohang 62 Quispel-Janssen, Josine 62, 130
r Raaijmakers, Jonne 56 Radhakishun, Nalini 130 Rajan, Abinaya 94 Rana, Anas 60 Raspe, Marcel 50 Raven, Anje 185 Ravesloot, Madeline 144 Rebers, Susanne 82, 96 Rehorst, Harriet 178 Reinders, Anneke 178 Relyveld, Germaine 145 Remeijer, Peter 158 Remmelts, Andrea 144 Remmelzwaal, Jolanda 178 Retèl, Valesca 94 Ridderbos, Jan-Nico 118 Riem, Ellen 184 Rijpkema, Tjitte 118 Rodenhuis, Sjoerd 108, 130 Rodenko, Boris 64 Rohr, Jan 84 Rooijers, Koos 14 Rookus, Matti 96 Rooswinkel, Rogier 28 Rooze, Lyandra 118 Rosado, Arantxa 104 Rosenberg, Efraim 118, 119 Roskam, Marielle 178 Rossi, Maddalena 118, 158 Rottenberg, Sven 30, 72 Rowland, Benjamin 56 Rozendaal, Roel 158 Rucktooa, Prakash 86 Ruers, Theo 144 Ruijs, Marielle 119 Ruijter-Schippers, Henrique 118 Rumpf, Cornelia 112 Russell, Nicola 90, 158 Rutgers, Emiel 144
S Sahtoe, Danny 86 Salguero, Javier 158 Salinas, Pilar 118 Salomon, Izhar 54 Salverda, Govert 158 Sanders, Joyce 118 Sani, Musa 70 Sapmaz, Aysegul 64 Sapthu, Sunny 30 Saveur, Lisette 130 Sawicki, Emilia 130 Scanu, Tiziana 62 Schaake, Eva 144, 158 Schaapveld, Michael 96 Schagen, Sanne 74 Scharpfenecker, Marion 90 Scheenstra, Alize 158 Scheeren, Ferenc 84 Scheerman, Esther 118, 119 Schellens, Jan 76, 130 Schinkel, Alfred 80 Schlicker, Andreas 110 Schmidt, Marjanka 82 Schneider, Christoph 158 Schoenmakers, Noortje 130 Schol, Joke 130 Scholtens, Arda 178 Schot, Margaret 130
Schouten, Philip 54, 130 Schreuder, Pim 144 Schrier, Mariëtte 14 Schrijver, Helga 178 Schrijver, Lieske 96 Schumacher, Ton 84 Schunselaar, Laurel 62 Schutte, Peter 145 Scuric, Vincent 178 Secades, Pablo 88 Seemann, Ingar 90, 118 Seigers, Riejanne 74 Serresi, Michela 102 Severson, Tesa 54 Shaltiel, Indra 56 Sidharta, Grace 12, 26 Siedschlag, Christian 118 Sinaasappel, Michiel 118 Sivro-Prndelj, Ferida 118 Sixma, Titia 86 Slobodin, Boris 14 Smeele, Ludi 144 Smets, Lionne 178 Smit, Judith 86 Smit, Marjon 66 Smits, Fokko 144 Sneepers, Roel 184 Snijder, Dominic 130 Snoek, Margriet 22 Sol, Wendy 30, 72 Sombroek, Cherita 96, 158 Song, Ji-Ying 184 Sonke, Gabe 74, 130 Sonke, Jan-Jakob 158 Sonneborn, Mariska 118 Sonnenberg, Arnoud 88 Soueidan, Hayssam 110 Spaan, Mandy 96 Spaapen, Robbert 62 Sparmann, Anke 102 Spliethoff, Jarich 144 Spreeuw, Hanno 158 Srámek, Michael 145 Städler, Nicolas 60 Staiger, Christine 110 Stam, Barbara 158 Stankovic, Uros 158 Staring, Jacqueline 32 Steeghs, Neeltje 76, 130 Stelloo, Suzan 114 Steuten, Lotte 94 Stewart, Fiona 90 Stoker, Sharon 144 Stokkel, Marcel 118 Storm, Dea 178 Stornaiuolo, Mariano 86 Stouthard, Jacqueline 130 Straetmans, Jos 144 Stuiver, Martijn 144 Stulemeijer, Iris 100 Stuurman, Rik 76, 130 Sun, Chong 20 Sutherland, Kate 22 Sznajder, Beata 178
T Taal, Babs 130 Tame, Mihoko 56 Tan, Bing 144 Tanger, Ellen 102 Te Poele, Johannes 90 Te Riele, Hein 92 229
Teertstra, Jelle 118 Tefsen, Hannah 144 Ten Cate, Julia 145 Ten Hoeve, Jelle 110 Ten Oever, Diederik 130 Ter Brugge, Petra 52, 108 Terweij, Marit 100 Tesselaar, Margot 130 Teunissen, Bas 130 Theunissen, Noortje 144 Tibben, Matthijs 130 Tielen, Ivon 118 Tielenburg, René 158 Tilgenkamp, Ria 178 Timmermans, Jacqueline 144 Toebes, Mireille 84 Tolhuis, Bas 102 Tomasoa, Brenda 158 Topolnjak, Rajko 158 Torres Acosta, Alex 178 Trip, Anouk 158 Tulner, Linda 130
U Uckelman, Michael 86 Urbanus, Jos 84 Uyterlinde, Wilma 130
V Valdés Olmos, Renato 118 Valkenet, Ludy 178 Valstar, Matthijs 144 Van ‘t Veer, Mars 96 Van ’t Veer, Laura 119 Van Alphen, Maarten 144 Van Amerongen, Renée 66 Van Arensbergen, Joris 104 Van As-Brooks, Corina 144 Van Bemmel, Joke 104 Van Berkel, Peter 94 Van Berlo, Damien 106 Van Beurden, Marc 144 Van Boven, Hester 118 Van Breugel, Pieter 14 Van Burgsteden, Johan 18 Van Buuren, Laurens 158 Van Buuren, Marit 84 Van Coevorden, Frits 144 Van de Ahé, Fina 22, 185 Van de Kamer, Jeroen 158 Van de Kasteele, Willeke 38 Van de Kooij, Bert 28 Van de Poel, Henk 144 Van de Velde, Tony 178 Van de Ven, Marieke 52 Van de Vrugt, Henri 92 Van de Wetering, Koen 30 Van de Wiel, Bart 118 Van Deemter, Liesbeth 30 Van Delft, Foke 130 Van den Belt-Dusebout, Sandra 96 Van den Berg, Joost 38 Van den Berg, Nynke 144 Van den Berk, Paul 46 Van den Boer, Cindy 144 Van den Brekel, Michiel 106, 144 Van den Broek, Bram 50, 184 Van den Broek, Daan 118 Van den Broek, Sandra 82, 96 Van den Haak, Marjolijn 178 230
Van den Hengel, Lisa 32 Van den Heuvel, Michel 106, 130 Van den Hoven, Jolanda 130 Van der Baan, Frederieke 96 Van der Berg, Marieke 145 Van der Burg, Eline 52 Van der Donk, Emile 178 Van der Eerden, Paul 144 Van der Geld, Ylanga 158 Van der Gulden, Hanneke 52 Van der Hage, Jos 144 Van der Heide, Uulke 158 Van der Heijden, Ingrid 52 Van der Heijden, Iris 130 Van der Heijden, Michiel 130 Van der Hiel, Bernies 118 Van der Kammen, Rob 44 Van der Kant, Rik 62 Van der Kolk, Lizet 26, 119 Van der Laan, Elsbeth 130 Van der Leij, Femke 158 Van der Maas, Martin 38 Van der Meer, Jelrik 184 Van der Molen, Lisette 144 Van der Noll, Ruud 76, 130 Van der Noort, Vincent 178 Van der Reijden, Anneke 158 Van der Riet, Emma 119 Van der Sar, Jana 130 Van der Steeg, Wim 130 Van der Torre, Jaco 48 Van der Valk, Martin 184 Van der Veen, Bernd 184 Van der Veen, Fientje 145 Van der Veen, Wietze 145 Van der Velde, Marieke 178 Van der Velden, Sophie 119 Van der Wal, Anja 92 Van der Weide, Marchien 130, 145 Van der Wel, Nicole 70 Van Deventer, Sjoerd 62 Van Diemen, Ferdy 32 Van Diepen, Frank 184 Van Diessen, Judi 158 Van Dijk, Laura 84 Van Dijk, Pim 86 Van Dongen, Miranda 118 Van Driel, Willemien 144 Van Dyk, Ewald 110 Van Eggermond, Anja 96 Van Esch, Anita 80 Van Geel, Robin 76, 130 Van Geer, Michael 130 Van Gijn, Roel 130 Van Harn, Tanja 92 Van Harskamp, Marieke 130 Van Harten, Wim 94 Van Hasselt, Coen 130 Van Heesbeen, Roy 56 Van Hell, Albert 106 Van Herk, Marcel 158 Van Huizum, Martine 145 Van Kasteren, Sander 64 Van Kouwenhove, Marieke 14 Van Kranen, Simon 158 Van Leerdam, Monique 130 Van Leeuwen, Fijs 118 Van Leeuwen, Flora 96 Van Leeuwen, Fred 100 Van Leeuwen, Marieke 12 Van Lent, Wineke 94 Van Lohuizen, Maarten 102 Van Luenen, Henri 30 Van Meerbeeck, Jan 130
Van Miltenburg, Martine 52 Van Monsjou, Hester 144 Van Mourik, Anke 158 Van Netten, Gabry 178 Van Nieuwenhuizen, David 130 Van Nimwegen, Rianne 96 Van Ooij, Joost 184 Van Pel, Renée 118 Van Rens, Anja 26, 119 Van Rhijn, Bas 144 Van Riel, Candy 118 Van Roekel, Sanne 178 Van Rooij, Nienke 84 Van Sandick, Johanna 144 Van Schaffelaar, Emmie 178 Van Son, Rob 144 Van Steensel, Bas 104 Van Tellingen, Olaf 74, 118 Van Thienen, Hans 130 Van Tilburg, Gabrielle 64 Van Tinteren, Harm 178 Van Triest, Baukelien 106, 158 Van Tuijl, Carolien 178 Van Veen, Michiel 58 Van Velthuysen, Loes 118 Van Vliet-Vroegindeweij, Corine 158 Van Vliet, Mariska 130 Van Vugt, Huub 102 Van Waardenberg, Wil 178 Van Waart, Hanna 12 Van Welsem, Tibor 100 Van Werkhoven, Erik 178 Van Winden, Annemieke 130 Van Wouwe, Merian 96 Van Zeijl, Leonie 68 Van Zon, Maaike 70 Vanhoutvin, Steven 178 Vanneste, Ben 158 Veenstra, Corine 158 Veenstra, Hidde 144 Vegt, Erik 118 Velds, Arno 184 Vens, Conchita 106 Veraar, Elise 28 Verbrugge, Inge 62 Vergouwe, Ingeborg 145 Verhagen, Caroline 106, 144 Verheij, Marcel 106, 158 Verhoef, Koen 185 Verhoef, Senno 119 Verhoeven, Els 118 Verkleij, Laurien 178 Verloop, Janneke 96 Vermeulen, Eric 82 Vermeulen, Marrit 178 Verwaal, Vic 144 Vidal-Rodriguez, Jordi 16 Vincent, Andrew 178 Visser, Daan 50 Visser, Dick 130 Visser, Nils 66 Visser, Sandra 178 Vissers, Joep 102 Vlaming, Hanneke 100 Voets, Erik 112 Vogel, Celia 66 Vogel, Wouter 118, 158 Von Castelmur, Eleonora 68 Voncken, Francine 158 Vos, Matthijn 70 Vossen, David 106, 110, 144 Vreeswijk, Sandra 158 Vrijaldenhoven, Suzan 130 Vroege, Huib 118, 184
Vrolijk, Nynke 178 Vroonland, Colinda 118
W Wagemakers, Sander 130 Wagenaar, Els 80 Wals, Anneke 178 Wals, Kim 64 Wang, Liqin 20 Warmerdam, Daniel 56 Wesseling, Jelle 108, 118 Wessels, Lodewyk 110 Wessels, Ronnie 144 Westerveld-de Zwart, Ingrid 118 Wever, Lidwina 178 Wevers, Marijke 12, 26 Wielders, Camiel 92 Wientjens, Ellen 52 Wieringa-Ariaens, Aafke 118 Wijdeven, Ruud 62 Wijnands, Rosemarie 96 Wijnands, Yvonne 178 Wildeman, Maarten 144 Willemse, Els 178 Wilting, Roel 34 Winter-Warnars, Gonneke 118 Winter, Marcel 118, 184 Winterwerp, Herrie 86 Wit, Niek 46 Witte, Marnix 158 Witteveen, Thelma 158 Witting, Katharina 64 Wittkämper, Frits 158 Woerdeman, Leonie 26, 145 Wojciechowicz-Grzadka, Kamila 92 Wolf, Anne Lisa 158 Wolthuis, Rob 112 Wolzak, Hilde 130 Wouters, Michel 144 Wu, Li-en 62
X Xiao, Yanling 28 Xu, Guotai 30, 72 Xu, Ning 80 Xue, Zheng 20
Y Yalçin, Zeliha 48 Yazdi, Amir 118
Z Zandbergen, Jeroen 178 Zander, Serge 30, 72 Zerp, Shuraila 106 Zevenhoven, John 22 Zhang, Hua 158 Zijlmans, Henry 144 Zijp, Lambert 158 Zimmerman, Marion 130 Zupan-Kajcovski, Biljana 145 Zuur, Charlotte 144 Zwart, Wilbert 114
