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IEO European Institute of Oncology

Scientific Report 2011 Ongoing Research 2012

Scientific Report 2011 Ongoing Research 2012

a Leonardo la Pietra

Chairman’s message

IEO clinical and research activities in 2011 have been carried out in the context of the deep economic crisis which has been affecting the western world for some years now, and the end of which, at this time, is impossible to predict. Obviously, IEO management’s main concern has been to offer to each patient the best service, irrespective of the difficulties of the present situation: in this respect we feel we fully achieved our goal as care and treatment were provided, as usual, with continuity and effectiveness. Unfortunately, we cannot say the same about the financial outcome, which has been the worst since the beginning of IEO activities, because of the restrictive measures issued in July and December 2011 by the Regione Lombardia with resolutions 2057 and 2633 and which led to a significant reduction of income. Nevertheless, IEO has not changed its investment programmes and, at the end of January 2012, inaugurated the ARC - Advanced Center for Radiation Therapy, furnished with state of the art equipment. This center will allow us to substantially improve our radiotherapy activities; in fact, radiotherapy is becoming an increasingly important therapeutic option in the treatment of oncological diseases.

Carlo BUORA Chairman

As regards research, both clinical and basic research activities have been carried out with success in 2011 at IEO, as evidenced by the number of publications in journals with high Impact Factor. We hope that the current critical economic situation will gradually improve. IEO staff, to whom I once again extend warm thanks for what has been achieved, is fully aware of the present difficulties and will certainly support the actions we may take to mitigate the effects of the crisis and relaunch the work of our Institute. We ask our shareholders, to whom I renew my deep thanks for the constant support they give to our activities, to be more than ever near us in these difficult years, helping us to maintain, for our patients, that standard of care and therapeutic activities for which IEO is recognized.

Contents

Colophon Title Publisher Number Month/Year Editor Authors Secretary of editing Graphic Layout Photographer Photographer’s assistants Typographer Paper

IEO Scientific Report 2011. Ongoing Research 2012. IEO European Institute of Oncology©, Milan (Italy) 18 May 2012 Pier Giuseppe Pelicci Elena Belloni Francesco Bertolini Roberto Biffi Lisa Bodei Bernardo Bonanni Fausto Chiesa Nicoletta Colombo Maria Cossu Rocca Giuseppe Curigliano Concetta De Cicco Pier Paolo Di Fiore Oreste Gentilini Barbara Jereczek Fossa Maria Cristina Leonardi Saverio Minucci Franco Nolè Franco Orsi Pier Giuseppe Pelicci Nicole Rotmensz Maria Teresa Sandri Mario Sideri Giuseppe Testa Mario Varasi Giulia Veronesi Roberta Carbone, Mirko Nesurini Juri Weisz Roberto Benzi Sonia Raboini (IEO) Fabio Virdis (Campus IFOM-ieo) Pirovano Srl, Printed in Italy Printed on the uncoated paper On Offset whose elementary chlorine free pulps came from susteinaible forests

Former CEO’s message CEO’s message Scientific Director’s Report

• Ethics Committee • Library • Ecancermedicalscience • The Iberian-Latin American Office The Directive Board General Manager’s Office • Customer Service and Marketing • Finance and Administration • Human Resources • Information Technology • Medical • Operations • Hospital Activities IEO Education Centres of Excellence TTFactor

11 13 15 19 20 22 24 27 29 30 31 32 33 34 35 36 40 42 44

DEPARTMENTS, DIVISIONS AND UNITS Division of Head and Neck Surgery 48 Division of Senology 50 Division of Thoracic Surgery 54 Division of General and Laparoscopic Surgery 56 Division of Abdomino-Pelvic Surgery 58 • Unit of Minimally-Invasive Surgery Division of Gynaecology Division of Urology

Division of Melanoma and Soft Tissue Sarcomas Divison of Plastic Surgery

62 66 68

70 74 Unit of Interventional Radiology 76 Division of Medical Oncology 78 Division of Clinical Pharmacology and New Drugs 80 • Outpatient Clinics 82

• Unit of Research in Medical Senology • Unit for Supportive Care • Psycho-Oncology Unit Division of Cardiology

84 86 88 92 Division of Clinical Haemato-Oncology 104 Division of Radiotherapy 108 Division of Nuclear Medicine 112 Division of Anaesthesiology and Intensive Care 114 Division of Cancer Prevention and Genetics 116 Division of Radiology 120 Breast Imaging Division 124 Unit of Preventive Gynaecology 126 Division of Endoscopy 128 Division of Pathology 130 • Unit of Histopathology and Molecular • Diagnostics 132 • Unit of Diagnostic Cytology 134 • Laboratory Unit of Clinical Haematology-Oncology 136 Division of Laboratory Medicine 138 Medical Physics 140 Division of Epidemiology and Biostatistics 142 Department of Experimental Oncology 144 • Functional Genomics 148 • Oncogenes, Chromatin and Cell Cycle Control 150 • Quantitative Proteomics ato investigate • transcriptional and post-transcriptional • mechanisms that regulate gene expression 152 • Viral Control of Cellular Pathways and Biology • of Tumorigenesis 156 • Bioinformatics and Evolutionary Genomics of Cancer 158 • Molecular Analysis of Kinetochore Composition, Activity • and Regulation during Chromosome Segregation 160 • Molecular Carcinogenesis and Stem • Cell Biology Research 162 • Systems Biomedicine 164 • Cellular and Molecular Pathways Regulating • Melanoma Genesis and Progression 166 • Structural and Functional Studies of the Mitotic

Board of Directors As from May 2012

International Scientific Advisory Board As from May 2012

• Spindle Orientation during Asymmetric Cell Divisions 168 • Chromatin Alterations in Tumorigenesis 170 • Structural and Functional Basis of Mitosis 174 • Transcriptional Control in Inflamation and Cancer 178 • Epigenetic mechanisms in stem cell differentiation • and oncogenesis 182 • Biology and Signal Transduction of Normal and • Cancer Neural Stem Cells 184 • Molecular mechanisms of cancer and ageing 188 • Immunobiology of Dendritic Cells and Immunotherapy 192 • Histone Methylation Dynamics in Stem Cell Renewal • and Lineage Commitment 194 • Drug Discovery Unit 196 • Mechanism Controlling Chromosome Segregation 198

RESEARCH ACTIVITIES Clinical Research

202 Cancer prevention and genetics 203 Anti-VEGF and beyond: Shaping a new generation of anti-angiogenic therapies for cancer 206 Circulating tumour cells 211 Virology 214 Breast Cancer Research Programme 218 Brain Tumours 226 Gynecological Tumours Research Programme 228 Head & Neck Cancer 233 Lung Cancer Reserach Programme 238 (Neuro) Endocrine Tumors 242 Prostate Cancer Research Programme 244 High Intensity Focused Ultrasound (HIFU) 248 Robotic Surgery Research Programme 256 Interventional Radiology 258 Tumour Imaging by Nuclear Medicine 266 Radioimmunotherapy 272 The IEO Tumor Registry 274 Academic Research 278 The Drug Discovery Programme 301 The Molecular Medicine Programme 303

Publications, Clinical TRIALs, Ongoing Grants, Seminars and IEO Foundation Full papers 2011 Clinical Trials IEO foundation Ongoing grants Seminars Acknowledgments

311 342 356 358 364 367

Carlo BUORA Carlo CIANI Piergiorgio Peluso Mauro MELIS

Chairman Deputy Chairman Deputy Chairman MILANO ASSICURAZIONI Chief Executive Officer

Franco Bernabè Alessandro Bertani Rosario Bifulco Ausilia Carena Francesco Chiappetta Maurizio Comoli Davide Croff Paolo Grandi Edoardo Lombardi Piero Melazzini Andrea Novarese Vittorio Ogliengo Antonello Perricone Giampiero Pesenti Umberto Quadrino Giancarlo Scotti Piero Sierra Giuseppe Vita

TELECOM ITALIA MEDIOBANCA SORIN FONDAZIONE CABRINO CARENA IN VIGEVANO PIRELLI & C. BANCO POPOLARE BANCA POPOLARE DI MILANO INTESA SANPAOLO MEDIOLANUM BANCA POPOLARE DI SONDRIO FONDIARIA-SAI UNICREDIT RCS MEDIAGROUP FONDAZIONE ITALCEMENTI ITALCEMENTI EDISON ASSICURAZIONI GENERALI AIRC ALLIANZ

Prof. Mariano BARBACID Prof. Anton BERNS Prof. Peter BOYLE Dr. Julian DOWNWARD Prof. Alexander M.M. EGGERMONT Prof. Carl-Henrik HELDIN Prof. Dr. Heinz HOEFLER Prof. Jan HOEIJMAKERS Prof. Sir David P. LANE Dr. Tomas LINDAHL Prof. D.M. LIVINGSTON Prof. J. Gordon McVIE Prof. Michael NEUBERGER Dr. Liliane OLLIVIER Prof. Pier Giuseppe PELICCI Prof. Ulrik RINGBORG Prof. Walter VAN DEN BOGAERT Prof. Irene J. VIRGOLINI Prof. Karen H. VOUSDEN

Madrid Amsterdam Lyon London Villejuif Uppsala Munich Rotterdam Singapore London Boston Bristol Cambridge Paris Milan Stockholm Leuven Innsbruck Glasgow

Prof. Umberto VERONESI

IEO

Auditors Maurizio Bozzato - Renato Pagliaro - Graziano Visentin Secretary Marco Agnelli Ex officio Umberto Veronesi - Stefano Michelini - Oliviero Rinaldi

IEO — Scientific Report 2011 — Ongoing research 2012

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Former Chief Executive Officer’s Message

During the past 12 months, IEO has obtained exceptional results both in terms of delivering the best quality of patient care and achieving all our scientific and clinical objectives, in spite of the fact that 2011 has been the scenario of unpredictable events that have had a negative impact on the financial performance of all organizations worldwide. As a consequence of the ongoing economic crisis, the NHS (National Healthcare System) has been forced to re-assess the contributions allocated to the institutions providing health care, including IEO. Nevertheless, we maintained the expected high standard of surgical interventions and treatments proper to our Institute. We haven’t modified our investment program in radiation therapy, completing it with an expenditure of approximately 25 million, of which 17 million were allocated for new machinery and 8 for engineering works. Thus, at the end of January 2012, we opened the Advanced Radiation Therapy Centre (ARC), which, implementing machines such as “Trilogy”, “Cyberknife”, “TomoTherapy” and “Vero”, it is going to be one of Europe’s most important centres in the field of radiotherapy.

Carlo CIANI Former Chief Executive Officer

10

IEO — Scientific Report 2011 — Ongoing research 2012

Therefore, regardless of the economic performance, IEO has been equipped with more powerful tools to effectively fight cancer. As for our research activities in 2011, the results have been the best ever achieved. The staff of our Institute has been extremely and constantly hard-working, devoting even more of their time and energy to patient care in order to counteract the impact of the management problems described above as much as possible. We wish to express our sincere gratitude to them all. Finally, we would like to extend a warm greeting and good wishes to all our patients, reassuring them that IEO will continue, with even greater commitment, to be with them on their difficult therapeutic journey, both on their healing and disease stabilisation process.

IEO — Scientific Report 2011 — Ongoing research 2012

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Chief Executive Officer’s Message

As former CEO Carlo Ciani said, IEO economic results in 2011 were not positive, even though we had a good growth in scientific achievements. I’d like to highlight that in 2011 the number of our scientific publications increased significantly. This means that the decision of our shareholders, who contributed to creating the Institute over the years, was an excellent one for all IEO stakeholders and for the scientific community. We strongly believe that our research has to be economically sustainable and progressively applied to the treatment of our patients. For the future we are planning further investments in research and technology aimed at improving even further the effectiveness of our treatments. We are constantly engaged in clinical practice to maintain high quality care for our patients, developing in-house training and external training for many family doctors and specialists who see the Institute as a center of excellence and reference for oncology.

Mauro MELIS Chief Executive Officer

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IEO — Scientific Report 2011 — Ongoing research 2012

Having been the IEO CEO for a few weeks now, I can already say with pride that our staff shows the highest levels of commitment and total dedication to our mission of global excellence in oncology. That’s why my deepest thanks and encouragement for the future go to each and every one of our employees.

IEO — Scientific Report 2011 — Ongoing research 2012

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Umberto VERONESI, MD Scientific Director

Scientific Co-Director: Pier Giuseppe Pelicci, MD, PhD Deputy Scientific Directors: Fausto Chiesa, MD, Aron Goldhirsch, MD, Roberto Orecchia, MD and Giuseppe Viale, MD Research Co-ordinator: Giuseppe Della Porta, MD Strategic Planning: Gordon Mc Vie, MD, DSc Medical Advisors: Stefano Zurrida, MD and Giovanna Gatti, MD Head of the Scientific Secretariat: Lucia Racca Scientific Director’s Secretariat: Francesca Morelli, Eva Bruschini and Anita Larossa Stefano Zurrida’s Secretary: Maria Grazia Villardita Librarian: William Russell-Edu Head of Clinical Trials Office: Atanasio Nonis, MD Clinical Trials Office: Daniela Tamagni Head of Grants Office: Ilaria Foti Grants Office: Lucia Sorrenti, Daniele Calasso and Elena Ottina Press Office and Scientific PR: Donata Francese Ecancermedicalscience Office: Gordon Mc Vie and Linda Cairns

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IEO — Scientific Report 2011 — Ongoing research 2012

The Scientific Directorate is responsible of strategic choices for the future. The foundations of our strategies are: centrality of patient, main importance of prevention, quick transfer of research results from laboratory to clinical research, earlier and earlier diagnoses, more and more conservative therapies with attention to the quality of life. As illustrated in the chart in the following page, the results of the activities in Cancer Research were reported in 337 publications with a full impact factor of 2041,59 and an average impact factor of 6.54. Three hundred twelve of these papers were published in scientific journals with impact factor and twenty-five were published without impact factors. In addition, eighteen were considered as books, chapters and other. The laboratories of the Department of Experimental Oncology in the site adjacent to the IFOM research institute (the FIRC Institute for Molecular Oncology), represent one of the largest European centres for cancer research (the IFOM-IEO Campus), with 24,000 m2 and 450 researchers. The IFOM-IEO Campus was created with the aim of developing and applying genomic research to the field of oncology, providing a substantial contribution to the quick translation of the emerging scientific knowledge into new strategies for the prevention and treatment of tumors. In addition to their intense research activity, in collaboration with the University of Milan and the University of Naples the Campus has established three PhD programs at the European School of Molecular Medicine Foundation (SEMM), these are the Molecular Medicine Programme, the Medical Nanotechnology Programme and the Foundations and Ethics of the Life Sciences Programme. The school enrolls over 150 PhD students from around the world. Our institute devotes much attention to technology updates and to the development of new technologies. Since these activities demand ample investments and critical mass, IEO has decided to join efforts with IFOM and together they created a Consortium dedicated to the development of new technologies in the fields of Structural and Functional Genomics (Cogentech). The main areas of intervention touch every aspect of daily life at the Campus and consist of: DNA Services, Microarrays, Model Organisms, Molecular Pathology Unit, Mass Spectrometry Unit, Protein Chemistry Unit,

Scientific Director’s Office

Scientific Director’s Report Decalogue of patient’s rights I. Right to scientifically proven treatments II. Right to prompt treatments III. Right to a second opinion IV. Right to privacy V. Right to know the truth VI. Right to be informed on treatments VII. Right to refuse a proposed treatments VIII. Right to the living will IX. Right not to suffer X. Right to respect of the personal dignity

European Institute of Oncology Staff MD and PhD post Doctoral Fellows Nurses Technicians Administratives

390 213 470 148 422

Total

1643

Imaging Unit and Crystallization Unit. To accelerate translational research and to create an active interface between the basic research programs of the IFOM-IEO Campus and IEO clinical activities, IEO has launched a Molecular Medicine Program, whose laboratory activities are located within the IEO hospital building with the responsibility of Prof. Pier Paolo Di Fiore. We expect that this new initiative will facilitate the rapid translation of the recent developments of Genomic Sciences into novel approaches to cancer prevention, diagnosis and treatment. Major objectives of the Molecular Medicine Programs are potentiating of the Tumour Bank and Tumour Registry, promotion of early clinical trials for the rapid screening of new drugs or treatment modalities, patient stratification for treatment through the use of genomic screens. This new Program stems from our front line activities in cancer prevention, early diagnosis (for example our programs on cervical, breast and lung cancer)

IEO — Scientific Report 2011 — Ongoing research 2012

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Scientific Director’s Office and novel treatment modalities (targeted peptide- or antibody-guided radionuclide therapy), and will further expand toward molecular imaging. In 2009 we have launched a program of Drug Discovery. Traditionally, basic research on diseases and diseasemechanisms has been conducted in academic institutions, while its application to drug discovery has been the responsibility of the pharmaceutical industry. In recent years, a strong need has emerged for molecularlytargeted drugs that are based on knowledge of diseasemechanisms. This need has exposed the limitations of conducting basic and applied research separately and has created the basis for new interactions between academia and the pharmaceutical industry. To address this problem, IEO launched a new Drug Discovery Program, which is fully intertwined with ongoing basic research at the IFOM-IEO Campus. One important aim of the program is to establish collaborations with private and public institutions whose mission is to cure disease.

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IEO — Scientific Report 2011 — Ongoing research 2012

There seems to be little doubt that the agenda for cancer biology in the next decade will be mainly focused on the understanding of how simple individual molecular functions are integrated into complex pathways and systems, how multiple systems are integrated to govern multifaceted cellular behaviours, and how subversion of these molecular machineries leads to cancer. With this complex picture in mind our scientific efforts are concentrated on three major objectives: 1. Understanding the complex systems controlling cell proliferation. Two major lines of research are being developed aimed at the understanding of exogenouslyoriginated signals (which follow the engagement of surface receptors) and endogenously-originated signals (which follow DNA damage). This is technologically supported by high throughput technologies, including high definition genomic technologies and proteomics, and by a vast array of genetic tools in mammals, fish, nematode and yeast.

2. Understanding complex circuitries in the “actual” picture of naturally occurring cancers. This is being pursued through expression profiling and genetic analysis of cancers with respect to questions of clinical relevance, such as prognostic evaluation, disease classification and patient stratification for therapeutic purposes. These programs are organized within a transversal approach across several ‘task forces’ established around “disease-oriented programs”. Presently, we have programs on breast, lung, ovary, leukemias and melanoma. These programs are centred on first- class clinical resources and supported by high through- put technologies and a vast repertoire of bioinformatics and biostatistics expertise, which provides support to the disease programs in addition to developing its own lines of research. 3. Integrating complex circuitries in higher order cel- lular programs or at the organismal level. Several lines of effort are being pursued here. First and foremost, we are analyzing the impact of stem cells on cancer phenotypes. Efforts are concentrated on leukaemia, breast, melanoma and lung cancer stem cells, with the perspective of integrating these lines of research in the disease-oriented programs. Second, a major effort in system biology has been undertaken to try to develop predictive models, which would unmask non-obvious properties of several

of the molecular machineries studied. Finally, efforts are being directed at the generation of reliable models of mammalian carcino- genesis, by engineering in model systems mutations that mimick those naturally occurring in human cancers (particularly leukaemias, breast cancer, ovary cancer and melanomas). The Basic Research and Molecular Medicine Programs will not detract from our mission of improving prevention, early detection, effective treatments and quality of life of our patients, using at best all the available knowledge. On the contrary, we firmly believe that the best way to cure cancer is by speedy application of the knowledge acquired from research activity to the patient ward. During 2011 the number of patients enrolled in clinical trials was 3034 and we have now 33737 patients in follow-up. 58 new clinical projects were initiated during the year, after approval of our Ethics Committee. The number of visitors and residents from all parts of the world has been very large. Training of young scientists and physicians is a critical component of our mission. Many members of our staff are actively involved in teaching at the University of Milan and at numerous meetings and courses throughout the country. Furthermore, our researchers continued to be a relevant part of the teaching staff of the European School of Molecular Medicine.

Publications 1994 — 2011

1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Full Papers Journals with Impact Factor 36 Total Impact Factor (*) Average Impact Factor

49

103

132

135

153

156

187

167

255

234

2005 2006 2007 2008 2009 2010 2011 245

245

272

234

285

276

312

97,53 180,82 469,13 721,96 717,60 639,73 801,62 782,82 877,81 1’438,12 1’273,42 1’651,96 1’441,55 1874,70 1716,79 1693,28 1849,55 2041,59 2,71 3,69 4,55 5,47 5,32 4,18 5,14 4,19 5,26 5,64 5,44 6,74 5,88 6,89 7,33 5,94 6,70 6,54

Full Papers 6 Journals without Impact Factor

19

25

34

24

18

9

13

Total Full Papers

42

68

128

166

159

171

165

Books, chapters, and others

60

65

79

78

121

80

Total publications

102 133

207

244 280 251

2

17

16

13

26

39

48

49

52

25

200 169

272

250

258

271

311

282

334

328

337

72

43

40

33

38

39

25

38

31

44

18

237

243 203 312

283

296

310

336

320

365

372

355

34

(*) For each year the IF was calculated using the values published in the Journal Citation Reports of the previous year.

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Scientific Director’s Office

Scientific Director’s Office

Ethics Committee

As in previous years, numerous scientific and teaching meetings were directly organized by IEO, both for external and internal attendance. During 2010, IEO continued its scientific collaboration with foreign Countries. Specific efforts have been devoted to establish new relations with Mediterranean Countries, India, Madagascar, Africa, and China. Thanks to a strict cooperation and an intense share of knowledge and expertise, and frequent contacts among IEO and foreign Centers, our successful experience in clinical research, prevention and therapy is becoming a model for the creation of new cancer approaches in many Countries. In particular, we focused on the fight against feminine tumors (breast and cervix) through specific models from training and education till the projects for new comprehensive cancer centers. As in the previous years, we have been very successful in securing funds to support our research activities both at the national and international level. Success in fundraising has been crucial for the steady expansion experienced by IEO during the past several years. Hiring of new research directors has been made possible by competitive start-up packages, including provisions to fund students and postdoctoral fellows and the availability of many state-of-the-art core facilities. Out of a total of 24,870,477 million euro’s, 2,458,467 were from the European Community, 8,487,553 from the Ministry of Health, 253,408 from the Ministry of Research, 1,958,498 from AIRC, 6,051,602 from 5x1000 Fiscal Contribution and 5,660,949 from other sources, including the U.K. Association for International Cancer Research, Human Frontier Science Program, Lega Italiana per la Lotta Contro i Tumori, Umberto Veronesi Foundation, National Institutes of Health, Regione Lombardia and Swiss Bridge. We are very grateful to all the funding agencies, which recognized the validity of our projects. In the middle of 2007 a program of expansion of the clinical activity of the Institute was initiated with the creation of a new building, adjacent to the main Institute. The

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IEO — Scientific Report 2011 — Ongoing research 2012

new building actually host a large new diagnostic clinic, where early detection programs will be implemented together with a program of day surgery and day medicine. In the new premises the Department of Nuclear Medicine is greatly expanded with a new Positron Emission Tomographies and new research laboratories. Finally, I would like to extend our appreciation and gratitude to the President, the CEO, the Board of Directors, all clinical, scientific, technical and administration staff of IEO, for their enthusiasm and their continuous excellent work. I wish to thank Pier Giuseppe Pelicci, Roberta Carbone, Eva Bruschini and the various Writing Committees for their great effort in preparing this annual report. Grants Office The Grants Office acts as a liaison between researchers and sponsoring agencies and is a central source of information on major national and international agencies, foundations, and institutions that support research and scholarship, and assists researchers in identifying appropriate research funding opportunities. It provides assistance to researchers in the preparation of applications, developing proposal narratives and budgets, completing the application forms and interpreting the regulations of the funding agencies, assuring compliance with the sponsor policies and requirements. It negotiates the terms and conditions of awards of successful proposals and provides support for the administration of research grants, including funding allocation, monitoring research expenditures and producing financial statements. It manages research contracts, preparing, whenever necessary, subcontracts or consortium agreements with collaborating institutions, acts as administrative contact point on multicentre research projects, and provides administrative support for IEO research activities. During 2011 through the first months of 2012, 204 grants were funded for a total amount of over 31 million euro.

Clinical research activity in IEO showed a further increase with respect to the previous year. Overall 102 new clinical trial applications were evaluated in 8 plenary sessions of the Ethics Committee (EC). A favourable opinion was expressed in more than 95% of the cases, while the scientific aspects, the expected improvement of the general health and well being and the overall risk/benefit ratio for the participants, were the principal aspects considered by the Committee in the evaluation of these trials. A further significant reduction in the time of evaluation of new trials, as well as of amendments to already approved trials, was achieved. The EC opinion was expressed within 11 days for most of the applications and it was registered in the national clinical trials database in less than 5 working days. The applications for substantial amendments on ongoing trials confirmed the increasing trend of the last few years, and 191 applications were reviewed and approved with a mean evaluation time of 13 days. The therapeutic use of drugs still under investigation in clinical trials, for advanced cancer patients, was approved for 4 out of 5 products and for a total number of 55 patients. The composition of the Committee was partially reviewed and the new composition was accredited at the Ministry of Health, the National Drug Agency as well as the Lombardy Region. The Ethics Committee renewed also the Federal Wide Assurance by the Office for Human Research Protections of the U.S Department of Health and Human Services. With great sorrow we report that Dr Leonardo La Pietra, Medical Officer of IEO and member of the Committee, died on December 2011. SECRETARIAT

Ethics Committee As from May 2012

Luciano MARTINI Chairman Giovanni APOLONE Vice Chairman Atanasio NONIS Secretary Franco BUZZI Natale CASCINELLI Giuseppe GALLUS Stefano GASTALDI Aron GOLDHIRSCH Pasquale MICCINELLI Emanuela OMODEO SALE’ Maurizio PELLEGRINI Enrico RAMBALDI FELDMANN Oliviero RINALDI Nicole ROTMENSZ Umberto VERONESI Daniela TAMAGNI

OBSERVERS Mauro MELIS Stefano MICHELINI

IEO — Scientific Report 2011 — Ongoing research 2012

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Scientific Director’s Office

Scientific Director’s Office

Library

The Library forms part of several extensive networks of medical and scientific libraries, thereby facilitating a rapid exchange of information and knowledge.

enabling literature searches to be taken forward in time, starting from a known key article. Also from here we get the “Impact Factors” whereby a numerical value is assigned to describe the “impact” that a journal has from year to year. These services may be accessed from anywhere in the IEO building, from the Mirror Tower, or from the IFOM-IEO-Campus. Many journals can be accessed from home thanks to Bibliosan. There is also access – again via Bibliosan - to RefWorks, a personal bibliographic management software whereby the user may store and use bibliographic citations. Such citations can be repeatedly used to generate bibliographies in the different formats required by publishers of scientific journals.

Bibliosan – of which the Library is a member – is the health library network of IRCCS (Scientific Institutes for Research, Hospitalisation and Health Care) centres, created within the remit of the Italian Ministry of Labour, Health and Social Policy. It brings together 32 IRCCS Libraries working within the national framework of NILDE, the Network for Inter-Library Document Exchange. The Library is also a member of the Italian National Collective Catalogue of Periodicals (ACNP) linking it to over 110,000 journals and 2300 Libraries throughout Italy. The SBBL consortium (Biomedical Library System of Lombardy), provides comprehensive coverage of literature from resources based in the Lombardy region. In addition, the Library is part of GIDIF-RBM, a consortium of major biomedical and pharmaceutical libraries in Italy, and DOCLINE (via the Italian National Institute of Health and the National Library of Medicine in the USA). Since November 2008 the Library has been located in the Mirror Tower, and offers a quiet space for study and consultation of the literature both online and in print. The Library works closely with SEMM, the European School of Molecular Medicine at the IFOM-IEO-Campus in order to coordinate journal subscriptions and access, thereby avoiding any wasteful duplication. The Library catalogue is therefore shared across both sites. Via the consortium agreements with BiblioSan, SBBL and

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IEO — Scientific Report 2011 — Ongoing research 2012

CILEA, over 7000 journals are accessible 24 hours a day, 7 days a week. BiblioSan also has a “remote access” option enabling Institute staff to access its services to be accessed from outside. So far, 126 users have registered for remote access. Users have access to a user-friendly implementation of the gold-standard medical literature database Medline (via Ovid, to which we subscribe). Users can also access Embase, which is complementary to Medline, focusing more on the pharmaceutical literature and with a more European emphasis. The international nursing literature is covered by CINAHL – the Cumulative Index to Nursing and Allied Health Literature, with the fulltext for over 770 journals and 275 books/monographs in this field. In addition, users may consult sources specialising in Evidence Based Medicine, such as the Cochrane Database of Systematic Reviews and the Database of Abstracts of Reviews of Effects. BiblioSan provides access to the ISI Web of Knowledge and the Journal Citation Reports. These sources yield valuable data on citation patterns from the renowned Science Citation Index, which tells us who has cited whom, thus

Through SBBL over 1000 e-books are available via Springer Publishers. In addition, IEO has online access to 16 books from Lippincott Publishers. These, along with some 6000 journals can all be accessed by the Library “AtoZ catalogue”. In 2011 this Library catalogue was accessed a total of 6081 times. The average session length was 25 minutes and 39,999 pages were viewed in total. An average of 7 pages per session were viewed. Presentations are regularly held, often within Division staff meetings, in which the Library services and BiblioSan are explained and demonstrated in detail. The Librarian offers literature searches and searching assistance in order that IEO personnel may make optimum use of the extensive range of bibliographic and full-text resources available. As the key to a valuable resource, the Library continues to strive towards organising and simplifying the means by which the busy doctor, nurse or research scientist can navigate the ever-increasing ocean of international research literature and bring informed knowledge and state-of-the art discoveries to the service of the patient.

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Scientific Director’s Office

Scientific Director’s Office

ecancermedicalscience

ecancer, the leading oncology channel, is going from strength to strength, and continues to provide a free service to oncologists, including expert videos, news, education and an open access journal . Each month over 40,000 people visit ecancer to read and watch the latest content. The journal of the IEO, ecancermedicalscience, recently became indexed in PubMed and PubMed Central. ecancermedicalscience is already fully indexed in other leading databases, including Embase, Scopus, EBSCO and Google Scholar, so any article published will be highly accessible to other researchers in the field. Over one hundred and twenty papers have now been peer reviewed and published in the journal. ecancer.tv continues to develop its educational platform, reporting and filming from 21 oncology conferences around the globe. There are now more than 800 videos of interviews with eminent oncologists, roundtables and press conferences, which have been watched over 1.5 million times. This is the only site of its kind with such a rich resource for oncology professionals. ecancer also hosted its first CME accredited conference “Blood Cancer in the Elderly: European Expert forum” with over 1100 international attendees. ecancer is actively involved in the management of three European Commission funded FP7 projects in partnership with the IEO. P(ersonalized)Medicine is aimed at developing internet tools to assist clinical research especially clinical trials in the era of genomic led therapies. IEO is responsible for the clinical trials work package while ecancer leads on Patient Empowerment and Education in collaboration with Prof Gabriella Pravettoni (Department of Cognitive Psychology, University of Milan). EurocanPlatform is a new FP7 project linking 23 leading cancer institutes; ecancer is responsible for communication and dissemination of results. EURECA (Enabling information re-Use by linking clinical Research and Care) will look at semantic ontologies of health IT systems.

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IEO — Scientific Report 2011 — Ongoing research 2012

Towards the end of 2011, ecancer.org was redesigned to enable visitors to experience a more user-friendly platform with improved functionality and visibility. As a result the website now has an increased profile on search engines and is compatible for mobile devices. The ecancer iPhone App was also developed to enable content sharing. ecancer continues to place the European Institute of Oncology at the forefront of multi-media communications, looking at innovative solutions to promote equal access to oncology research and learning. The Founding Editors are Professors Veronesi and McVie and Dr. Linda Cairns is the Science Editor. ecancer is supported by the European Institute of Oncology, The Foundation of the IEO, The Umberto Veronesi Foundation, The European CanCer Organisation and Swiss Bridge.

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Scientific Director’s Office

Scientific Director’s Office

The Iberian-Latin American Office Gabriel Farante, MD, PhD Coordinator

Assistant: Nordiana Baruzzi

The Institute of Medicine of the USA defines the quality of healthcare as the “degree to which health services for individuals and populations increase the likelihood of desired health outcomes and are consistent with current professional knowledge”. Following this philosophy, the Iberian Latin American Office (ILAO) organizes educational activities for medical doctors coming to the IEO from Spain and Latin American countries with the aim of achieving this objective. The daily evolution of information applied in their clinical practice obliges doctors to a continuing updating. This applies not only to younger doctors (whose university training is more recent) but also and especially to more experienced doctors. This is the ILAO’s essential purpose and as a result, the volume of educational activities of our office increases year by year considering the growing number of observers, visitors and grant recipients arriving at IEO from the Iberian and Latin American areas. Continuing Medical Education (CME) activities are necessary because oncology changes continuously. Breast cancer today, for example, is not the same as

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IEO — Scientific Report 2011 — Ongoing research 2012

breast cancer in the past; primary breast tumors are at present smaller, involve fewer nodes, and are treated by a multimodal approach. In parallel, results from randomized controlled trials have revolutionized surgical practice, and this has been particularly evident in the treatment of breast cancer. The numerous randomized controlled trials performed by U. Veronesi in Europe and B. Fisher and others in the USA over the last decades have changed breast cancer care, saving countless women from undergoing more radical therapies (both for the breast and the axilla) by adding the revolutionary idea of intraoperative radiotherapy. The following is a summary and update of ILAO activities organized within the Institute and abroad: 1) Educational programme at the IEO: • One-week IEO visit: this particular site-visit has became a standard kind of visit for most medical doctors. In fact, with this educational programme organized by the ILAO, they participate in practical training in the morning and in a theoretical course in the afternoon. 40 Spanish and Latin American medical

doctors coming from the Master in Mastology from the Universidad de Barcelona (50 are already programmed for 2012) and 20 Brasilian medical doctors arrived in two separate groups to visit the IEO in 2011. This training program is always multidisciplinary, as several divisions are involved in each one-week site visit, such as the divisions of surgery, pathology, radiology, radiotherapy, nuclear medicine and the operating theatre, as well as several administrative areas which are involved in the verification of the visitors’ scientific abilities. We can therefore state that for each visiting doctor, over 20 IEO staff are contacted and participate in organising the the site visit, among them the directors or heads of all involved divisions, services and administration. • The agreement with Barcelona University: this agreement dates back 10 years and is aimed at Spanish and Latin American doctors attending the Master in Mastology at Barcelona University. Thanks to this agreement, over 300 Master students from 11 Latin American countries, Spain and Portugal have visited the IEO since 2001 in a specially-organized educational one-week visit. • One-year fellowship: As a sign of the great interest in the educational activities in the IEO, the number is also increasing of doctors from Latin America and Spain who every year apply for a grant from Umberto Veronesi Foundation (FUV). Since 1994, when the grant was created, 64 medical doctors have had the benefit of this important grant that allows Latin American and Spanish physicians to attend the IEO for a year or more, mostly but not exclusively in the Breast Surgery Division. In 2011, the FUV contributed with nine fellowships, selected by the ILAO and elected by the FUV: six in the division of Senology, and one per each Division in Medical Oncology, Plastic Surgery and Head and Neck. • The 10-weeks update course: this course for all internal attendees of the Breast Surgery Division

(grant holders, residents, visitors, etc) met with great success. In weekly 2-hour sessions over a 10-week period, a general overview of innovations in breast cancer management was presented for the visiting doctors. Based on the success of the first course, a second, more complete, 30-hour course has been programmed, where attendees will be awarded 30 CME credits by the Italian Ministry of Health. So, in 2011 almost one hundred medical doctors visited the IEO through the assistance and the organization of the ILAO. This figure is made up of the 40 visitors from the Master in Mastology of the Universidad de Barcelona who come to the IEO for a week, 20 Brazilians doctors who also visited the IEO for a week (10 mastologists in June and 10 in october), 9 grant holders of the FUV who stay at IEO with annual grants and a further 20 students who visit IEO for periods varying from one day to three months. 2) Educational programme abroad: this part of the educational programme was developed in 2011 in the following locations: Buenos Aires, Argentina (April, 21-22), Barcelona, Spain (June, 3-4), Alicante, Spain (September, 23), Goianias, Brazil, (October, 14-15). After having organized hundreds of educational activities in Latin America and having disseminated the scientific programs to thousands of medical doctors firstly through the European School of Oncology (ESO) and then through the ILAO activities, Umberto Veronesi and the IEO can count on a solid scientific network of medical doctors and institutions that follow its scientific and educational philosophy both in Spain and Latin America. New initiatives of the ILAO will soon become a reality as the Newsletter on Breast Cancer and others in collaboration with international Institutions, with the aim of achieving the objective proposed by the Institute of Medicine of the USA, namely that of offering visiting doctors the current professional knowledge to achieve the desired health outcomes.

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The Directive Board

The Directive Board

T. Levitt loved saying that organizations exist and justify their existence insofar as they can create the conditions for “ordinary” people to accomplish, together, extraordinary results (Organizations exist to allow ordinary people to do extraordinary things). At present, more than 1000 people take part into the activities of IEO and many are extraordinary people. The mission of IEO Management is to aim the everybody’s efforts towards the strategic objectives of the Institute and to create the best conditions to allow the operators to put their expertise and knowledge to the patients’ service. There are many ways to play this role in a complex organization. However, we believe that our managers must necessarily own the following principles:

• a continuous thrust towards innovation and an international dimension; ….the best treatments can be found where research is performed …we believe that a research institute can only excel if it is based on mature and advanced management tools;

• a team culture to be searched for and promoted every day in the relationships with all those who operate inside the Institute; • the awareness that, in such no-profit organizations as IEO, the management systems justify their existence insofar as they

strongly contribute to create the conditions to reach and maintain excellence in the fight against cancer.

This is, in synthesis, the “philosophy” that guides the behaviour of the areas that coordinate and support IEO’s clinical and research activities. Our job, our active contribution to the fight against cancer, is to try and put into practice this “philosophy” every day.

Directive Board CEO acting as Chairman General Manager Scientific Director Scientific Co-Director Deputy Scientific Director Deputy Scientific Director Deputy Scientific Director Deputy Scientific Director

26

Mauro Melis Stefano Michelini Umberto Veronesi Pier Giuseppe Pelicci Fausto Chiesa Aaron Goldhirsch Roberto Orecchia Giuseppe Viale

IEO — Scientific Report 2011 — Ongoing research 2012

Chief Customer Service and Marketing Officer Chief Financial Officer Chief HR Officer Chief Information Officer Chief Medical Officer Chief Operating Officer

Flavio Nascé Mario Cesana Daniele Piacentini Claudio Carlo Franzoni Oliviero Rinaldi Nicola Eugenio Spada

This is a tribute to a personal friend and to one of the best friends that IEO has had since its foundation. This is in praise of Prof. Giuseppe “Beppe” Della Porta. Beppe started working at the IEO when this Institution was still a hypothesis, a dream of many of us; its realization is also thanks to him. In December 2011 he decided to retire from everyday work. During his career, Beppe has believed in and encouraged many young people who grew up professionally with him in various sectors. From clinic to research, Beppe has managed to interact with IEO staff at all levels: from primary doctors to administrative workers, from nurses to patients. Beppe has been critical for IEO. It is also thanks to his vision, tenacity and experience, that today IEO is a reference centre for research and treatment in oncology. We have not officially acknowledged Beppe’s retirement from daily activities at IEO or collectively celebrated his achievements because he is still one of us, and we know that he will remain alongside us as he has been for many years, though we cannot enjoy his company every day. Thanks Beppe! Umberto and Pier Giuseppe

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STAFF Head of the General Manager’s Secretariat: Jolanta Orlikowska Project Management Office: Luigi Cassi Occupational Health and Safety Officer: Marco Scuri

Stefano MICHELINI General Manager

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General Manager’s Office

General Manager’s Office

Following the strategic and development objectives established by the Chief Executive Officer, the General Manager heads and coordinates activities of the following Management: • Health • Human Resources • Finance • Operations • Customer Service and Marketing • Information Technology Based on the indications of the Scientific Director visà-vis is areas of responsibility, the General Manager establishes and draws up the operating and economic goals that the heads of the clinical and research departments are expected to achieve, regularly assessing the progress of the individual organizational units in achieving these objectives. The General Manager also attends the meetings of the Board of Directors, reporting on his specific areas of responsibility and implementing the resolutions passed by the Board.

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General Manager’s Office

Customer Service and Marketing

Finance and Administration

Flavio Nascé Chief Customer Service and Marketing Officer

Mario CESANA Chief Financial Officer

CUSTOMER Admittance: Francesco Bernasconi SERVICE STAFF Clinical Secretariat: Fabrizio Di Stefano Clinical Records Administrative Support: Andrea Chiesa Information Management: Adriano Disabella MARKETING STAFF Business Marketing: Marco Vianello Marketing Communication: Emanuela Ottolina New Media: Giovanna Gatti

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IEO — Scientific Report 2011 — Ongoing research 2012

Customer Service involves more than 200 resources. Services are delivered to Patients to support their clinical path and internally to physicians. Marketing: • Business Marketing to develop relations and sales with healthcare insurances • Marketing Communication for Corporate Events and Corporate WEB Site • New Media for social networks communication • Press Offices (Scientific & Corporate) Customer Service: • Booking Call Centre • Clinical Secretariat • Admittance Front Lines • Information Management

STAFF Executive Assistant: Viviana Muggiana Financial and Accounting Office: Angelo Longoni General Accouting: Luigi Romagnoni Legal Affairs: Renato Galasso Purchasing Office: Roberto Benelli

General Manager’s Office

General Manager’s Office

Integration of the clinical, research and administrative areas represents the basic principle underlying the management model adopted by the Finance and Administration Management. The main tasks of the Administrative Directorate are as follows: • Economic - financial management of the Institute • Administrative, legal and tax requirements • Managing the accounting, procurement and stock The Finance and Administration Management is reported to by the following services • Accounting • Legal Affairs • Purchasing Office

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General Manager’s Office

Human Resources

Information Technology

Daniele Piacentini Chief HR Officer

Claudio Carlo FRANZONI Chief Information Officer

STAFF Executive Assistant: Elisabetta Ronchi Performance & Organization: Anna Lauro Recruiting, Education and Training: Elena Mazzoleni HR Research Department: Annalisa Ariesi Payroll Management: Nicoletta Golin

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The central role of the individual and top-quality assistance are the two principles that have always inspired the organizational and management work done by the HRO Office in each of the above areas. These principles have enabled the Institute to achieve its main objective: improving the quality of life of each patient, who must be considered not simply as a person who needs treatment but, above all, as a multifaceted human being. Main tasks of the Human Resources (HR) Department are as follow: • Recruitment and selection • Education and training • Performance evaluations • Salary packages and incentives • Personnel management, planning and costs • Union relations • Work organization The Human Resources (HR) Department reported the following services: • Performance Management & Organization • Pay Roll Office • Recruitment • Education and Training The best example of this approach is the model for managing human resources for professional families “Job Family Model” extended to all roles of the Institute and aims to promote, develop and reward the skills and knowledge of our key employees. The Model Job Family includes all the roles in stimulating professional families, in addition to developing the skills of each professional, interprofessional collaboration and work responsibilities, key elements if we want to ensure clinical excellence and care. This took us years to get major awards, such as the Great Place to Work in 2003-2005-2006-2009, Joint Commission International certification for excellence in 2002 and the Prize Betershamal as one of the 6 best hospitals in the world, demonstration of the quality of work and all our employees. In 2012 We awarded the Top Employers Italy as one of the companies Italian proved excellent in use and management of its human resources.

STAFF Development and Programming: Paolo Zilioli Networks and Systems: Andrea De Grandis IT Research Department: Alessandro Dellavedova

General Manager’s Office

General Manager’s Office

The IT Department handles the whole computing infrastructure: the maintenance and support Office Automation, the management of network infrastructure and telephony, to the design and development of new applications. Main tasks of the Information Systems Management are as follows: • manages, maintains and updates the third party applications, providing user support • designs and develops applications for the management of computerized clinical and administrative processes • deals with the structure, organization and content of data • provides technical support computer software tier. • designs, develops and manages the network infrastructure, servers and telephony • designs, develops and manages the software and the basic application services • deals with the safety and security of data and resources • provides users with computer hardware and software technical support tier • deals with the purchase and installation of information technology The Information Systems Management reported the following services: • Development and Programming • Networks and Systems • Information Systems and Research The Director of Information Services plays the same role for both the IEO for the Monzino Cardiology Centre.

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General Manager’s Office

Medical

Operations

Oliviero Rinaldi, MD Chief Medical Officer

Nicola Eugenio SPADA Chief Operating Officer

STAFF Secretariat: Stefania Piacentini, Evelina Guaragna Nursing Office & Patient Care Services: Giorgio Magon Quality & Accreditation: Pier Luigi Deriu Patient Safety & Risk Management: Massimo Monturano Public Relations: Sergio Carlini Health Information Management & Medical Records: Daniele Dozzo Medical Physics: Guido Pedroli Pharmacy: Emanuela Omodeo Salè Infection Control & Waste Management: Giovanni Grieco

Medical Office oversees all health area, and it not only handles activities related to the Institute’s overall Clinical Governance but also provides its own specific services. Medical serves as a connection and interface for all clinical and health-related organizational and management processes that involve several divisions, operational units and services. The main tasks of the Medical Office are as follows: • Responsibility of all technical, organizational and hygienic aspects in the hospital • Participation in the process of strategic and operational planning of the Institute • Responsibility of Clinical Governance, with the identification and implementation of clinical care pathways • Responsibility of overall quality and technical efficiency - the production of operational performance (“vertical lines”) and distribution

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services through the integration of individual products or services in assistance programs, geared to the individual and the community (“horizontal lines”) • Guarantor of integrated hospital health care, from the organisation and management point of view • Responsibility of the proper organization and execution of welfare programs horizontal, the result of the integration of vertical lines responsible for the production of individual performance • Responasibility of Accreditation (both institutional and excellence) of both facilities and professionals working in the Institute. In particular responsibility of all requirements relating to safety. The Medical Office is also highly oriented towards developing new organizational and management procedures – working alongside Clinical and other IEO Divisions – in order to improve effectiveness, efficiency and appropriateness (i.e. quality) of services. In order to develop plans and policies on Quality and Safety, the IEO Quality Committee (IQC) was created on September 17th 2001. In order to continuously check the surveillance and control of hospital infections and the prevention of pharmacological errors in a perspective of collaborative work, two ad hoc Committees have been created: the Hospital Infections Committee and the Drugs and Medical Devices Committee. The topic of patient safety, part of the Quality Plan of IEO, has been continuously tackled since 2001, with the creation of the Risk Management Office and of the Patient Safety and Risk Management Committee.

STAFF Executive Assistant: Gabriella Peano Operating Planning: Alfonso Lorusso Planning and Budgeting: Nicola Spada (ad interim) Process Engineering: Silvio Pozzi General Service: Bruno Tonon Technical Service and Clinical Engineering: Marco Scuri

General Manager’s Office

General Manager’s Office

The purpose of the Operations is to implement the planning, budgeting and management control process, and assist the General Manager in translating the strategic directions given by the CEO into operational and financial objectives for individual organizational units. Main tasks of the Operations are as follows: • Supervising compliance with the Institute budget objectives; • Hospitalization and outpatient planning; • Acting as a facilitator in the construction process of the Institute industrial plan and annual budget; • Yearly monitoring of cost management trend; • Providing adequate reporting to the company management and organizational units as well as economic parameters on the progress of the Institute; • Acting on the Institute resource planning in order to meet their budget goals (operating sessions, outpatient areas, beds, diaries, etc ...); • Following-up of the authorization process for the investment plan and providing an economic assessment of extra-budgetary activities; • Assisting the General Manager in the analysis of innovative projects cost-effectiveness The Operations Management is reported to by the following services: • Operating Planning • Planning & Budgeting • Process Engineering • General Services • Technical Service and clinical engineering

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Hospital Activities

General Manager’s Office

General Manager’s Office Milan Lombardy except Milan Northern Italy except Lombardy and Milan Central Italy Southern Italy & Islands Outside Italy

Key Case Mix Figures: Impatient Admissions by geographical area At the end of the year the clinical staff of the Institute numbered 390 physicians, 470 nurses, 112 health care assistants, 148 health technicians and 48 medical residents. 126 medical doctors coming from 21 countries spent a significant period (more than 30 days) of clinical training in one or more of our Clinical Divisions. 35225 new patients were enrolled in 2011 adding up to a total patient census (as of 31 December 2011) of 544.478 patients since IEO opened in 1994. Total hospital admissions decreased by 15,54% to 11.925 compared to the previous year, totalling 48.734 hospital

days (47.878 in 2010) with an average length of stay of 4.1 (3.4 in 2010). The ratio between surgical admissions and total admissions (“surgical index”) increased to 63%. Case mix complexity, proxied by the Average Relative Weight, increased to 1.26. Day Surgery cases (surgical treatments which do not require an overnight stay) amounted to 3.336 (1.885 in 2010). Day Hospital admissions, mainly for therapeutic pur poses (chemioterapy or radiation therapy) totalled 8.532 treatments (7.355 in 2010) and involved 2.918 patients (2.035 in 2010)

8

6

4

2

0

1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Inpatients

Medical

2011

Surgical

Key Case Mix Figures: Average Length of Stay (Alos)

2 20.000

1,5 16.000

1 12.000

0,5

8.000

0

4.000 95 96 97

98

99

Key Case Mix Figures: Hospitalisations

00

01

02

03

04

06

07

08

09

Day Hospital

10

100% 90%

80%

80%

71%

40%

36

30%

20%

20%

10%

10% 96

97

98

99

00

01

02

03

04

05

06

07

08 09 10 11

2006

2007

2008

2009

2010

2011

Key Case Mix Figures: DRG Average Relative Weight (ARW)

1000

2000

3000

4000

Gynaecology Urology Thoracic Surgery

50%

95

2005

Other

70%

30%

0%

2004

0

63% 61% 60% 56% 57%55% 56% 56% 58% 59% 55% 54% 54% 54% 52% 52% 53%

40%

Key Case Mix Figures: Surgical Index

2003

11

90%

50%

2002

Inpatient Medical Average Relative Weight Inpatient Average Relative Weight Inpatient Surgical Average Relative Weight

100%

60%

2001

0 05

Inpatients

70%

2000

Medical Surgical

Melanoma Digestive Surgery Head & Neck Surgery Plastic & Reconstructive Surgery Surgical Senology

Key Case Mix Figures: Surgical Interventions

0%

Surgical intervention

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IEO — Scientific Report 2011 — Ongoing research 2012

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General Manager’s Office

150000

30000 112500

22500 Table

75000

15000 37500

7500 0 1995

0

Key Case Mix Figures: Chemotherapy Courses

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

1997

1998

1999

2000

2001

2002

2011

200000

1000

175000

900 800

150000

700

125000

2003

2004

2005

2006

2007

2008

2009

2010

500

75000

400 300

50000

200

25000

100

0

0

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2011

Key Case Mix Figures: Outpatient Visits

Outpatients visit

30000 30000

13000

25000 22500 20000

11000 9000 7000

600

100000

Key Case Mix Figures: Radiation Therapy & IORT

1996

15000 15000

5000

10000 7500 5000

3000 1000

00

1995

1996

1997

1998

1999

2000

2001

2002

2003

Histological

2005

2004

2006

2007

2008

2009

-1000

2010 2011

Key Case Mix Figures: Pathological Examinations

Cytological

1000000

90000

15000

750000

67500 10000

500000

45000 5000

22500

Key Case Mix Figures: Radiodiagnostic & Nuclear Medicine examinations

0

250000 0

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

0 1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

Laboratory Test

2005

2006

2007

2008

2009

2010

2011

Key Case Mix Figures: Laboratory Tests

15000

12000

9000

6000

3000

0 1995

1996

1997

Key Case Mix Figures: Endoscopic Procedures

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1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

Endoscopic procedures

IEO — Scientific Report 2011 — Ongoing research 2012

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IEO Education Pier Giuseppe PELICCI, MD, PhD Scientific Coordinator

STAFF Scientific Secretary: Nicoletta Tradati Exeutive Committee: Annalisa Ariesi, Francesco Bertolini, Roberto Biffi, Bernardo Bonanni, Fausto Chiesa, Marco Colleoni, Giuseppe Curigliano, Pier Paolo Di Fiore, Luisa Lanfrancone, Angelo Maggioni, Giorgio Magon, Oliviero Rinaldi, Giulia Veronesi, Giuseppe Viale Internal Education and Training Activities: Elena Mazzoleni, Ombretta David, Ferdinando Pastrello External Education and Training Activities: Lucia Zigliani, Anna Brandovardi

Daniele Piacentini Management Coordinator

The year 2011 saw the establishment of IEO Education - the IEO School of education and training - set up to coordinate all educational and training activities, integrate them in an innovative manner, and thereby promote, both internally and externally, the Institute’s knowledge. The main developments are a) the introduction of Clinical Science Seminars in Oncology with renowned speakers to visit IEO both for the training of young doctors and for networking; b) the revision of the Grand Round created by Professor Veronesi in order to encourage the participation of all the healthcare staff; c) the design of online surgery courses (e.g. the Esagon Biennial Course), enabling the launch of the WEB Academy in 2012 providing education and training courses online and on demand with a considerable scientific impact; d) the IEO Education website and newsletter designed to circulate and promote the main scientific and training events, e) official recognition of the Institute as Italian CME Provider, playing an active role in the field of continuing medical education, with the aim to create high quality education and training activities, awarding credits to participants in compliance with the criteria laid down by the Ministry of Health. Internal education and training activities The hospital education and training activities in 2011 were: • 38 accredited CME courses. Some courses were repeated, bringing the total number to 60. • 17 non-accredited CME courses. Some courses were repeated, bringing the total number to 21. • 9 behavioural courses linked to needs arising from career development plans, during the annual staff evaluation. Some courses were repeated, bringing the total course number to 12. The courses accredited produced a total of 1147.5 CME credits (690 credits in 2010) The number of

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IEO — Scientific Report 2011 — Ongoing research 2012

hours totalled 1110 for CME-accredited courses, 162 for non-accredited CME courses, and 288 for behavioural courses. The number of participants totalled 1746 for CME-accredited courses, 692 for non-accredited CME courses, and 256 for behavioural courses. Regarding participation in scientific congresses, a total of 1564 participants were registered, of whom 696 attended national events and 868 attended international events. External education and training activities In 2011, 53 educational activities (courses, meetings, and congresses) were organized by the European Institute of Oncology. Some of these were repeated bringing the total number to 63. Of these: • 24 events had both Italian and European CME accreditation (some were repeated and totalled 34 events), • 29 events had no CME accreditation.

• Congress on Non Conventional Imaging in Oncology. • Esagon Biennial Course: a 2-week residential and online course with live surgery and theory sessions, course on the anatomy of the female pelvis. • Multidisciplinary courses in robotic surgery (urology, gynaecology, minimally invasive and abdomino-pelvic surgery, head and neck surgery, and thoracic surgery).

The total number of hours invested in external educational activities was 1517. The total number of attendees (i.e., general practitioners, specialists, and other health care professionals/providers) amounted to 3518 with over 90% coming from national and international institutions. Based on evaluation questionnaires, the satisfaction rate was 90%. Participants in IEO education and training activities gained 5475 Italian CME credits and 622 European CME credits. Following are the main educational activities organized in 2011: • Milan Breast Cancer Conference - New format with 3D projection. • Breast Cancer: Oncologic and Reconstructive Surgery. Interactive course with live surgery - 3D during live surgery as technological innovation.

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Centres of Excellence

At the European Institute of Oncology, there are some important Centres of Excellence, dedicated to the prevention and treatment requirements for special oncological diseases.

of skills, technologies and processes required for prevention, diagnosis and more effective treatment of ovarian cancer.

MCC: Melanoma Cancer Center
 Opened in February 2012 from the development of more then 15 years of dedicated activity, the MCC is a multi-specialty integrated center dedicated to melanoma and other skin cancers. The aim of the center is to offer the patient a specific path for diagnosis and treatment that guarantees access to the most innovative therapies and advanced technologies within the same hospital where a multispecialistic team of dermatologists, surgical oncologists, medical oncologists offers the best integrated approach to all clinical situations. ARC: Advance Radiotherapy Center Opened in January 2012, ARC ranks among the top ten radiotherapy centers in the world thanks to the action of targeted “smart beams”, able to reaching and targeting the tumor, identifying them more precisely. CCC: Cervical Cancer Center Opened in May 2011, the center is a unique facility that combines the latest prevention strategies with the most effective treatments of cervical cancer. Primary and secondary prevention, conservative surgery, imaging, pathology, robotic surgery, tailored chemo-radiation. All the sections of the center are top level and provide patients with the best available solutions from the interdisciplinary team. OCC: Ovarian Cancer Center Opened in September 2008, this center aims to offer patients both medical and surgical support in line with the international standards in order to guarantee access to the most innovative therapies and the full spectrum

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TTFactor srl, the Technology Transfer company Daniela BELLOMO, PhD MBA General Manager

STAFF Chief Financial Officer: Mario Cesana, MSc Business Development and Marketing: Germano Ferrari PhD MBA, Jacopo Franchini MSc Intellectual Property: Marzia Fumagalli PhD Assistant to the General Manager: Aurelia Ramunni BOARD of Claudio Basilico - New York University DIRECTORS Andrea Cuomo - ST Microelectronics Marco Foiani - IFOM Isaac Kohlberg - Harvard University Tomas Lindhal - Cancer Research UK Stefano Michelini - IEO Elisabetta Petrucci - FIRC Domenico Triarico - IEO BUSINESS DEVELOPMENT ADVISORY BOARD

Giulio Draetta - MD Anderson Cancer Centre Isaac Kohlberg - Harvard University Nagesh Mahantappa - Avila Pharmaceuticals Kazumi Shiosaki - MPM - Capital Katherine Turner - Turner Consultant

Piergiuseppe PELICCI, MD Chairman of the Board

In June 2010, TTFactor was formally incorporated as the technology transfer company of the FIRC Institute for Molecular Oncology (IFOM) and the European Institute of Oncology (IEO) for the purposes of advancing scientific results arising from the IFOM and IEO through technology licensing, sponsored research with companies and spin off creation. The Company is composed by a team of professionals with qualified technical/economic background (PhD, MBA) as well as industry experience (pharma & biotech); a Board of Directors composed of top managers of IFOM and IEO, and international representatives as well as a Business Development Advisory Board chaired by the Director of Applied Cancer Science at MD Anderson Cancer Center. This team of experts has been created to serve scientists and ensure both Institutes that their intellectual properties are valued in accordance with fair principles, that means on the basis of their impact on patient’s care and their ability to become commercial products attractive for the industry.

Activities 2011.

In its first months of activity TTFactor has built the basis of a reciprocal trust relation with all of IEO and IFOM’s scientists and clinicians. Through active scouting TTFactor has collected 36 invention disclosures and selected the most promising for patenting and promotion towards the pharmaceutical and biotech industry. TTFactor’s portfolio in oncology now includes: • 10 families of patents created and managed from patent to a potential business opportunity; • A selection of top priority projects with high commercial potential includes: - a tumor vaccine for melanoma, developed by the group of Dr. Rescigno (IEO), nearing proof of concept in humans; - a diagnostic test for early detection of NSCLC cancer, arisen from the research of Prof. Di

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Fiore’s Group stemming from successful results of the IEO sponsored COSMOS clinical study; - a new platform for anticancer biological molecules against the receptor uPAR, developed by Dr. Sidenius lab. (IFOM); - numerous compounds incubated in Drug Discovery Program, IEO. Despite their potential value, these projects will need validation and industrial development and thus TTFactor is looking for industrial partners to “transform” them into a product for patient’s benefit. Since its creation, TTFactor has negotiated and signed approximately 80 agreements with the pharmaceutical & biotech industry, among others with Oncomed and

Genextra, defining the opportunity to receive important future milestones and royalties payments. Furthermore just recently it has entered in negotiation with the food industry world too, opening a new opportunity for corporate collaboration for the two Institutions, IFOM and IEO. TTF has been involved in reviewing the guidelines for sustainable exploitation of IEO and IFOM’s research results: in terms of revenues sharing with the inventors, and for the establishment of the procedure to create spin offs. These IP & Spin Off Policies will shortly be implemented within the set of regulations of each Institute.

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Departments, Divisions and Units

Further details of the programmes of Divisions and Units are available upon request together with copies of published articles. We invite also to visit our website: www.ieo.it

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Fausto CHIESA, MD Director

STAFF IEO Education Director: Nicoletta Tradati, MD Oral Unit Director: Luca Calabrese, MD Thyroid Unit Director: Gioacchino Giugliano, MD Assistants: Roberto Bruschini, MD, Augusto Cattaneo, MD, Luigi De Benedetto, MD, Enrica Grosso, MD, Valeria Navach, MD, Michele Proh, MD, Marta Tagliabue, MD, Stefano Zorzi, MD Consultants: Bianca Gibelli,MD, Endocrinologist, Filippo Cazzulani, DDS, dentist Observers, and Fellows: Koji Atachi, MD (Jap), Zoraida Milena Contreras, MD (Columbia), Samuel Pereira Lima , MD (Br), Thiago Chulam, MD (Br), Kristina Strakova, MD (Cech Republic), Andrei Tibirna, MD (Romania), Debora Ferreira Bibiano Silva, BS (Italy), Giacinto Colicci, nurse ( Italy), Residents: Giulia Danè, MD (Pavia University), Daniele Scelsi, MD (Pavia University), Omar Gatti, MD (Pavia University), Roberto Grigolato (Milan University) Speech Therapist: Valeria Zurlo, BS Data Manager: Maria Angela Massaro, PhD Secretaries: Paola Maggioni, Anna Maria Manti Head Nurses: Silvana Lacapra, Sara Meneghin

Activities 2011.

The clinical research of the Division is focused on early cancer diagnosis, of head and neck cancers, development of new treatment modalities and molecular medicine through a multidisciplinary approach. Main topics are oral and laryngeal precancerous lesions, and cancer of the oral cavity, pharynx, larynx and thyroid. The Division established National and International collaboration with many world-wide Institutions and

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Surgical Area

Division of head and neck surgery Mohssen Ansarin, MD Co-Director

several fellows attend our department for improving their knowledge of Head & Neck Oncology. We are developing organ and function preservation protocols, compartmental surgery of oral cancer; endoscopic robotic- and laserassisted surgery for laryngeal and oropharyngeal malignancies; conservative and video-assisted (MIVAT) thyroid surgery. The clinical activity during 2011 included 1056 patients admitted in the 16 beds of the Division (179 in Day Surgery). Among them 370 underwent mini-invasive oral and laryngeal laser surgery (30 Robot-assisted), 188 thyroidectomies (18 Video-assisted), 93 surgeries for an oral cancer (48 free-flap reconstructions), and 113 for a laryngeal cancer.Patients usually undergo preoperative staging in out clinic regimen and are admitted the day before or the same day of surgery. Mean patients’ stay in Hospital was 6.6 days. About 14500 patients (2300 new patients) were checked in the out-patient head and neck clinic; out of them, 1700 treated for an oral, pharyngeal and laryngeal malignancy and 2100 operated on for a thyroid cancer underwent multidisciplinary checks. Treatment program of 518 patients was discussed and planned in the weekly multidisciplinary meetings (on Wednesday). The division is coordinating 4 prospective trials: The Pioglitazone study S500/409) a multicentric chemoprevention trial on oral precancer lesions involving 10 USA and 2 European Institutes; (the Paccagnella Study (S398), a multicentric European study on cheo-radiotherapy in advanced oropharyngeal cancers; the Multicentric Italian study (IEO S651) on new drugs in metastatic papillary thyroid cancer. Moreover 6 retrospective studies on head and neck cancer are ongoing The Division published 11 papers on peer-reviewed journals, with an overall IF = 20,687, and 3 chapters in books on Head and Neck Oncology and Surgery. Fausto Chiesa was Editor of Acta Oto-rhino-laryngologica Italica, the official Journal of the Italian ENT – Head & Neck

Society (June 2004-May 2011). Acta under his direction became even more and more International. As result of this activty Acta in 2010 achieved the Index Copernicus and the ISI evaluation: ICV=5,6 and Impact Factor=0,427. Fausto Chiesa is President of the Italian Head and Neck Society (IHNS). The Division is involved in the organization of basic and advanced courses on head and neck and thyroid cancer for ENTs, Dentists and GPs in collaboration with Italian ENT Society (SIO) and the IHNS. The Division has an agreement with the ENT post-graduate school of the University of Pavia: each resident attends the Division and participates at the clinical and research activities for 6 months. Physicians of the Division are involved in the didactic activities of the school. A similar agreement is on-going also with the ENT and Head and neck surgery Institute of the University of Praha (Cz), and

the Camargo Oncological Institute of S Paulo (Brasil): a resident spends one year (respectively) in the IEO Head and Neck department and is involved both in clinical and research activities The Division organised: 1) two Resident Courses: in each of them 5 specialists spent a week attending lectures on Head and neck Oncology and observing the clinical activities of the Division: a full immersion experience on head and neck surgical oncology. 2) a Course of head and neck surgical technique on corpses held in Paris in collaboration with the Institute of Anatomy of the University of Paris and the ENT Clinic of Pavia, Ferrara and Brescia (20 participants and a faculty of 10 tutors from the organising Italian Institutes). 3) a Theoretical and Practical Course on Laser surgery in laryngeal cancer for 20 ENTs with 5 tutors

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Surgical Area

Division of Senology Alberto LUINI, MD Director

STAFF Director, Molecular Senology Unit: Viviana Galimberti, MD Director, Integrated Breast Surgery Unit: Paolo Veronesi, MD Director, Diagnosis and Surgical Treatment in Senology Unit: Stefano Zurrida, MD Deputy Directors: Oreste Gentilini, MD, Mattia Intra, MD, Annarita Vento, MD Assistants: Paolo Arnone, MD, Bettina Ballardini, MD, Paola Baratella, MD, Fabio Bassi, MD, Piero Caldarella, MD, Chifu Camelia, MD, Giovanna Gatti, MD, Germana Lissidini, MD, Francesca Magnoni, MD, Simonetta Monti, MD, Paola Naninato, MD, Gianmatteo Pagani, MD, Manuela Sargenti, MD, Antonio Toesca, MD, Silvia Velpidia Ratini, MD Fellows: Bianca Candeloro, MD, Andres Del Castillo, MD, Denise Mattar, MD, Lucilla Moltrasio Salazar, MD, Maybell Nieves Montero, MD, Luannys Rivero, MD, Ivana Romero, MD, Vania Stafyla, MD, Secretaries: Patrizia Bonezzi, Manuela Butti, Elena Cassi, Vittoria Rossi, Anna Maria Corapi, Sonia Raffaele Addamo Data Manager: Claudia Sangalli Fellow Data Manager: Rosaria Gallucci Surgical Area Nursing Coordinator: Luigia Rubio Head Nurse: Denise Santina Bucci

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Activities 2011.

The Division of Senology consists of three operating units which are all concerned with clinical research to develop new surgical techniques to improve the effectiveness of treatments for breast cancer that also preserve the integrity of the woman’s body. The Division of Senology played a major role in the development of sentinel node biopsy for breast cancer, performing pivotal clinical studies demonstrating that this technique is an accurate and minimally invasive method for staging the axilla. Sentinel lymph node biopsy (SLNB) is now a consolidated part of the treatment of breast cancer worldwide, and is indicated for all breast cancer patients, except those in whom axillary metastases are already overt. The fields of application of SLNB have been now enlarged and this procedure can be offered also in clinical scenarios in which it was previously considered contraindicated such as during pregnancy, in multicentric breast cancer, after neoadjuvant chemotherapy or previous breast surgery. Moreover, in a multicentric trial carried out within the International Breast Cancer Study Group (IBCSG) our Division was the leading center with the enrollment of 584 of the 933 randomized patients. The accrual was completed in February 2010. The aim of this prospective randomized trial was to determine whether axillary dissection can be avoided when the sentinel node contains only micrometastasis. Preliminary findings seem to confirm the hypothesis that in presence of a minimal sentinel lymph node involvement axillary clearance can be safely spared. As a further evolution we recently started a new prospective randomized trial (IEOS637/311 Trial SOUND: Sentinel node vs Observation after axillary Ultra-souND) with the aim of improving patients’ quality of life through a further reduction of the extent af axillary surgery. This is a prospective multicentric randomized controlled study leaded by

EIO in which sentinel node biopsy will be compared to observation (no axilary surgery) in patients with small breast cancer (< 2cm) candidate to breast conserving surgery and with negative axillary node at both physical examination and ultra-sound assessment. The estimated accrual is 1560 patients. Treatment of breast cancer needs to be integrated and multisciplinary. The co-operation between surgery, nuclear medicine and radiology led to the development of radio-guided occult lesion localisation (ROLL) and the use of sentinel node biopsy in conjunction with ROLL − a technique called SNOLL.

Sentinel node biopsy employs a radioactive tracer which is injected close to the tumour and moves through the lymph ducts to the sentinel node making it visible on scintigraphy and locatable with a radioactivity-detecting probe during surgery. In ROLL, the radiotracer is not mobile and remains where it is injected. The ROLL technique is therefore used to precisely locate non-palpable breast lesions. The radiotracer is injected directly into the lesion under real time radiographic or ultrasonic control. In the operating room, the surgeon can therefore precisely locate the lesion using a hand-held gamma- detecting probe, and use this probe as an aid to surgical removal. The SNOLL technique combines

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Surgical Area ROLL (injection of immobile tracer to localize the primary lesion) with injection of a mobile tracer to localize the sentinel node. The injections are performed separately but both the non-palpable lesion and the sentinel node are removed in a single surgical session. If the patient has a prior malignant diagnosis (for example by core biopsy) or malignancy is ascertained by intra-operative histological examination of the ROLL specimen, then the entire surgical treatment (lesion removal, sentinel node biopsy and axillary dissection, if necessary) can be completed in a single surgical session. Intraoperative radiotherapy with electrons (ELIOT) was developed by the Division of Senology in conjunction with the Radiotherapy Division. ELIOT delivers a complete radiotherapy course in a single dose during surgery. It can also be used to give a boost dose to shorten the subsequent course of conventional radiotherapy. The breast area from which the cancer has been removed is targeted using special electron beamguiding equipment and chest protection techniques. The first single-centre randomized trial on ELIOT was completed in December 2007. For this trial the Division recruited 1306 patients undergoing conservative surgery for breast cancer, and randomized then either to traditional external radiotherapy or ELIOT. Follow-up is still ongoing. A further example of multidisciplinary treatment is the co-operation carried out during the Nipple-sparing mastectomy which is a surgical technique developed in order to spare the nipple-areola complex in patients requiring mastectomy. During this operation the surgeons of the Senology Division work in close collaboration with the Plastic Surgery Division to achieve the best cosmetic outcome and, thanks to the intervention of the Radiotheraphy and Physics staff, ELIOT is used to irradiate the nipple-areola complex

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to ensure that it can be safely conserved. In patients undergoing conservative surgery in whom a subsequent asymmetry can be estimated, a bilateral reshaping can be performed to ensure the best possible cosmetic outcome. A new technique under development from the beginning of 2008 in conjunction with the Division of Nuclear Medicine is intraoperative avidination for radionuclide therapy (IART). Avidin is injected intraoperatively in and around the tumour bed after cancer removal performed by an usual quadrantectomy. Later, radiolabelled biotin is injected intravenously. The biotin binds to the avidin in the tumour bed, bringing with it a significant quantity of radionuclide whose radioactive decay contributes to killing of residual tumour cells. In preliminary studies, scintigraphic images demonstrated fast and stable uptake of radiolabelled biotin at the site of the operated breast, while the radiation dose released to the breast area was over 5 Gy/GBq, consistent with a boost of 20 Gy. These encouraging results have stimulated a feasibility study for IART. One of the most promising and intriguing new field of clinical research is the use of high intensity focused ultrasound (HIFU) to treat patients with early breast cancer. HIFU is a completely non-invasive method in which ultrasound is used to guide the focusing of high intensity ultrasound energy to a precisely circumscribed area of tissue within the body, resulting in coagulation necrosis within a few seconds. We are now using HIFU on a selected and limited number of patients prior to surgery. Surgical resection of the treated lesion takes place at least a week later, to evaluate residual viable tumour and extent of necrosis. Preliminary and initial findings, which need to be confirmed on a large basis, indicate that HIFU is a feasible method without major side effects.

The Division of Senology collaborates with the Division of Molecular Carcinogenesis and Stem Cell Biology Research in pivotal research on the NUMB protein. NUMB controls the fate of cells during development and differentiation via a mechanism involving inhibition of the activity of plasma membrane receptors of the NOTCH family. Research at the IEO discovered a new function of human NUMB: it is a regulator of p53 cancer protein, and this finding might have potential therapeutic implications for cancer treatment. Our Division strictly cooperates with colleagues of the Medical Oncology department during the multidisciplinary meeting which is weekly held in which every patient operated for either invasive or intraductal neoplasia is discussed to define the postoperative adjuvant treatment along with Radioterapists, Pathologists, and physicians of the Cancer Prevention and Genetics Division. With these latter colleagues it is generally discussed the management of women at “high risk” and the participation of patients to “window of opportunity” trials. We collaborate with other research and clinical Divisions of the IEO on a daily basis. This co-operation has proved very fruitful in research terms, with Senology staff authoring/co-authoring xx medical/ scientific papers in 2010, for a combined impact factor of 172.485. The Division of Senology also remains committed to university and institutional teaching. Division staff teach course for Masters Degrees awarded by the Universities of Milan and Barcelona, and teach various other courses as well. A fruitful collaboration and exchange is ongoing with many countries all over the world and especially with Spain and South America with Dr. Gabriel Farante being the coordinator.

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Lorenzo SPAGGIARI, MD Director

STAFF Director, Research Unit: Giulia Veronesi, MD Deputy Directors: Domenico Galetta, MD, Francesco Petrella, MD Senior Assistants: Alessandro Borri, MD, Roberto Gasparri, MD Assistants: Monica Casiraghi, MD, Stefano Donghi, MD, Adele Tessitore, MD Consultant: Sergio Cavaliere, MD (Pneumologist) Residents: Sava Durkovic, MD, Juliana Guarize, MD Clinical Fellow: Alessandro Pardolesi, MD Data Manager: Daniela Brambilla Secretaries: Enza Adinolfi, Federica Castoldi, Laura Cordini Head Nurse: Ester Spacca

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Surgical Area

Division of Thoracic Surgery Piergiorgio SOLLI, MD Co-Director

Activities 2011.

The clinical activity involved all fields of thoracic oncology including the treatment of early and advanced stage lung cancers, oesophageal tumors, mediastinal and chest wall diseases. The Division has developed innovative protocols of multimodality strategies for lung and pleural malignancies. Surgery of very extended tumors with main vessels involvement and other demanding procedures are fields of interest and of expertise of the Division together with parenchymal sparing procedures and mini invasive approaches for localised early stage lung or oesophageal cancers. During the past years the introduction in the clinical practice of the rigid bronchoscopy with endotracheal stents and laser treatment allowed to obtain good palliation in cases of bronchial obstruction or abextrinseco stenosis due to endobronchial neoplasms or enlarged lymph nodes. All more advanced staging techniques are routinely followed in the preoperative patient evaluation, including CT-PET and video-mediastinoscopy. The development of video-bronchoscopy with trans-tracheal or transbronchial biopsy allowed to obtain diagnosis of neoplastic disease at lymph nodes level in a non invasive manner avoiding often the mediastinoscopy. The use of metal bars has been introduced for the chest wall stabilization as an innovative device for reconstruction after resection of lung cancer or other neoplasms invading the chest wall. The use of laser for lung metastasectomy represents a novel technique to perform nodulectomy and lung resections of multiple lesions with minimal sacrifice of healthy parenchyma with reduced air leak. A multicentric trial with induction chemotherapy, pleuropneumonectomy and radiotherapy for pleural mesothelioma has been concluded in collaboration with other Italian Centres. The preliminary results are under evaluation.

The Division has instituted a program of lung cancer screening with low dose CT scan and biomarkers and developed the minimally invasive approach for the surgery of localised lung cancers and mediastinal tumors including robotic approach and video-thoracoscopic major lung resections. The research activity is involved in different translational research studies such as pharmacogenomic analysis, molecular biology studies of lung carcinogenesis, lung cancer angiogenesis and stem cell experimentation. The Division has developed collaboration with several national and international research Institutions and is coordinator of different multicenter clinical trials. It

is also devoted to educational activity and scientific meeting organisation. In 2011 the Thoracic Surgery Division performed about 1250 surgical procedures. 20 papers have been published on international journals during 2011.

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Surgical Area

Division of General and Laparoscopic Surgery Bruno ANDREONI, MD Director

STAFF Director, HepatoBilioPancreatic Unit: Antonio Chiappa, MD Assistants: Emilio Bertani, MD, Annamaria Minicozzi, MD Resident: Barbara Fiore, MD Medical Fellows: Carlo Corbellini, MD Scientific Secretary: Nordiana Baruzzi Secretary: Paola Italia Data Manager: Darina Tamayo Secretary of the Lu.V.I. Foundation: Rocco Ditaranto Head Nurse: Eleonora Meola

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Activities 2011.

The Division of General Surgery started its activities in 1994 through an agreement with the Milan University School of Medicine (Bruno Andreoni was the Director of the Specialization School in General and Emergency Surgery for 6 years before the reorganization of all Specialization Schools). The medical staff of the General and Laparoscopic Surgery has documented clinical experience in the treatment of upper and lower gastrointestinal tumors (from the esophagus to the anus), including hepatobilio-pancreatic, renal and adrenal cancers, abdominal sarcomas and neuroendocrine digestive tumors. All clinical activities are performed with particular attention to a multimodal, multidisciplinary approach, involving a close cooperation with medical oncologists, endoscopists, interventional radiologists and radiotherapists within the institutional “Digestive Tumors” Task Force. In 2011, 284 major surgical procedures (129 colonic; 81 rectal, 6 ileus,8 esophageal; 49 gastric, 8 pancreatic cancers, 21 liver, 11 retroperitoneal disease), 148 surgical procedures for non oncological diseases (laparocele, explorative surgery, stoma closure) and 71 local anesthesia surgical procedures were performed. The Division has protocols and trials of preoperative treatments for locally advanced gastric and rectal cancers, and for liver metastases (in collaboration with the Divisions of Radiotherapy, Radiology and Medical Oncology). In collaboration with the Divisions of Endoscopy and Pathology, the Division is involved in multicentric trials for the screening and treatment of colorectal cancers. In 2011, the Division of General and Laparoscopic Surgery conducted a number of studies, mostly in collaboration with the Division of Abdomino-Pelvic Surgery, namely: a study to evaluate the appropriateness of surgical procedures; a study to evaluate the “Effect of different mechanical bowel preparations in colorectal-cancer

surgery”; a study on “clinical and biological differences between screen detected versus no screen detected colorectal cancer”, a study to “compare diagnostic and treatment course with administrative source in Lombardia District”, a study on “serum and phosphoproteomic pattern diagnosis for early detection and targeterd patient-tailored treatment of cancer” and a study on the “Detection and evaluation of circulating tumors cells (CTC) in patients undergoing curative surgery (open or minimally-invasive) for advanced rectal cancer detected through routine clinical practice”. Other research programs involve national and international partners in Italy, Europe. USA, Japan,

Australia, Israel. A program of international exchanges for postgraduate doctors and research fellows has been established with a number of National and International Institutions. In 2011, the Division published 8 full papers on peer-reviewed journals, with an overall IF of about 24. The educational activities promoted by the Division include a number of University Courses (for Surgery Specialization Schools, Masters, and Undergraduate Courses in Medicine).

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Surgical Area

Division of Abdominopelvic Surgery Roberto BIFFI, MD Director

STAFF Unit of Minimally Invasive Surgery Director: Paolo Pietro Bianchi, MD (since December 2011) Unit of Integrated Abdominal Surgery Director: Fabrizio Luca, MD (since December 2011) Deputy Director: Simonetta Pozzi, MD Senior Assistant: Sabine Cenciarelli, MD Assistants: Wanda Petz, MD, Manuela Valvo, MD Clinical Researchers: Lorenzo Casali, MD (Fondazione Umberto Veronesi); Massimiliano Zuccaro, MD (Fondazione IEO) Research Fellow: Tiago Leal Ghezzi, MD (Fondazione Umberto Veronesi, until Feb 2011), Matias Parodi (Fondazione Umberto Veronesi) Resident: Daniele Belotti, MD Clinical Fellow: Maria Laura Cossu, MD Data Manager: Simona Musmeci, MSc (until Sept 2011); Davide Pastrello. MSc Secretary: Benedetta Clementelli Cooordinator of Surgical Area and Head Nurse: Liliana Tadini Head Nurse: Marina Mancini (until Sept 2011)

Activities 2011. Established on May 2007, the

Division of Abdomino-Pelvic Surgery included the Unit of Minimally Invasive Surgery since December 2011, becoming Division of Abdomino-Pelvic and Minimally Invasive Surgery; in the same time, a new Unit was established (Integrated Abdominal Surgery). More than 800 major oncology surgical procedures were carried out during 2011, aimed at treatment of the following conditions: oesophageal, gastric, small bowel, colorectal, liver and pancreas carcinomas. In addition, staff Physicians have specific expertise and know-how in integrated surgical treatment of trunk and limb-roots

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sarcomas, gastro-intestinal stromal tumours (GIST), primitive and metastatic tumours located in kidneys and adrenal glands, neuro-endocrine tumours of gastrointestinal tract. A significant portion of routine clinical activity involves close cooperation with other clinical Divisions (Gynaecologic Surgery, Thoracic Surgery, Urologic Surgery, Melanoma) in order to provide comprehensive care for oncologic diseases demanding skills and medical knowledge from different specialties (advanced ovarian carcinomas, oesophageal neoplasms extending into the abdomen, high-grade male pelvis tumours, visceral deposits of melanomas). The activation in 2009 of the Ovarian Cancer Center for Excellence by the Gynaecologic Oncologic Surgery Division offered the opportunity for an even closer cooperation between gynaecologists and abdominal surgeons, as shown by more than one hundred surgical high-complexity procedures per year, carried out with a multidisciplinary approach. Therapeutic choices are routinely made in agreement with other specialists, such as oncologists, radiotherapists, endoscopists and interventional radiologists, by means of team case-discussing within a dedicated task-force. Taking responsabilities for highly complex oncology cases, a strong link with the Anestesiology and Intensive Care Division may be demanded, in order to provide intensive treatments for critically ill surgical patients. Minimally invasive colorectal cancer surgery using a surgical robot started during 2007, and a pilot study on robot-assisted rectal cancer resection using the da Vinci system, a newly developed four-arm robotic device, was concluded. More than 500 patients were so far treated with this technique. A paper, collecting a relevant clinical series of fully robot-assisted rectal and left colon cancer resection, has been published on Annals of Surgical Oncology-2009. On January 2010 a collaborative paper with US and Italian groups was published, again on

Annals of Surgical Oncology, investigating the impact of robotic approach on mesorectal excision for cure of rectal cancer. Extension of this minimally invasive approach to other surgical oncology applications (stomach, adrenal gland, liver, spleen and pancreas) is currently matter of active clinical investigation. Four Editions of a 2-days full immersion Master Course in Robotic Abdomino-Pelvic Surgery were held since October 2010 to February 2011, with participation of attendees of European surgical teams coming from France, Belgium and The Netherlands. Finally, the Division maintained specific expertise on long-term central venous accesses for chemotherapy and total parenteral nutrition administration. A training course for physicians, dealing with diagnosis and treatment of complications associated with central venous access placement and utilization in oncology,

was provided. In addition, a randomized three-arm clinical trial aimed at defining the best insertion site to the central venous system in order to perform long-term chemotherapy was concluded. This study, carried out in collaboration with the Oncology, PsychoOncology and Interventional Radiology Divisions / Units at IEO, received a biennial research grant from AIRC (Associazione Italiana per la Ricerca sul Cancro). Final results have been recently published in a full paper on Annals of Oncology-2009. A second paper, investigating the impact of long-term central venous accesses on quality of life and anxiety-depression was published on Supp Care Cancer in 2011. Dr Biffi was member of the panel of international experts who provided guide-lines for this topic by ESPEN (European Society of Clinical Nutrition and Metabolism, Clin Nutr 2009).

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Surgical Area

Research. Ongoing research activities of 2012 include the following:

Surgical oncology of the gastro-intestinal tract: Staff Physicians actively cooperated in medical oncology research programs investigating the role and effects of adjuvant and neo-adjuvant chemotherapy on colo-rectal primitive tumours and hepatic metastases. Dr Biffi was Principal Investigator in a multi-center international randomized trial aimed at evaluating the impact of pre-operative docetaxel-based chemotherapy on locallyadvanced surgically resectable gastric cancer. This study had the collaboration of the Medical Oncology Division and final data have been published in 2011 on World J Gastroenterol. We concluded that surgery following docetaxel-based chemotherapy was safe and with similar morbidity to immediate surgery in patients with locallyadvanced resectable gastric carcinoma. These data support the rationale for using preoperative chemotherapy in locally advanced, operable gastric cancer, A study involving the detection of HER2 expression in gastric carcinomas is ongoing, aiming at defining a possible therapeutic role of Trastuzumab in the neoadjuvant setting, after publication of a large international trial demonstrating a benefit from this drug for patients suffering from advanced/ metastatic gastric cancer. Full Robot-assisted minimally invasive surgery for colon and rectal cancer: a pilot study was concluded, with the aim to evaluate safety and feasibility of this new system for left colon resection and total mesorectal excision, a recognized standard in rectal resection for cancer. Clinicopathological data of 55 consecutive patients. Who underwent full robotic surgery for the treatment of left colon and mid or low rectal cancer, were prospectively collected in a preliminary study. It showed full safety and effectiveness of this minimally invasive procedure, and was matter of a podium presentation at an International Meeting in

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Toronto and publication of a full paper in Annals of Surgical Oncology-2009. A second collaborative paper with US and Italian groups was published, again on Annals of Surgical Oncology, on January 2010. One hundred forty-three consecutive patients undergoing robotic treatment of rectal cancer in three centers were reviewed. Pathologic data, and postoperative and oncologic outcome measures were prospectively collected and analyzed by an independent researcher. Conversion rate was 4.9%, mean blood loss was 283 ml, and mean operative time was 297 min. The number of harvested nodes (14.1 +/- 6.5) and margin status compared favorably with those of open series (mean distal margin 2.9 +/- 1.8 cm; negative radial margin in 142 cases). The 3-year overall survival rate was 97%, and no isolated local recurrences were found at mean follow-up of 17.4 months. These very promising data were matter of a podium presentation at the Annual Meetings of the most important international robotic surgery societies in Chicago, San Diego, and finally in Durham (Society of Pelvic Surgeons, Sept 14-16 2010). A third paper was published in J Robotic Surg on 2011; full robotic low anterior resection for cure of rectal cancer produced significant less operative blood loss and less drop in postoperative hemoglobin level when compared to open resection in a casematched model, whereas other clinically relevant outcomes were similar or superior to open technique. A phase II trial, investigating the impact of this new approach on genital and urinary functions of patients undergoing this procedure for rectal cancer pretreated with a combination of chemo and radiation therapy is currently run. Finally, an international randomized clinical trial comparing laparoscopic vs robotic mesorectal excision for treatment of rectal cancer (ROLARR trial , RObotic vs LAparoscopic Rectal Resection) is expected to start during 2012. Totally implantable central venous access devices: a randomized three-arm clinical trial aimed at defining the best insertion site to the central venous system in order to perform long-term chemotherapy was concluded and final

results published as a full paper in Annals of Oncology-2009. Four hundred and three patients eligible for receiving i.v. chemotherapy for solid tumors were randomly assigned to implantation of a single type of port through a percutaneous landmark access to the internal jugular, a ultrasound (US)guided access to the subclavian or a surgical cut-down access through the cephalic vein at the deltoid-pectoralis groove. We concluded that central venous insertion modality and sites had no impact on either early or late complication rates, but US-guided subclavian insertion showed the lowest proportion of failures. A paper investigating the impact of long-term central venous accesses on quality of life (QoL) and anxiety-depression was published on Supp Care Cancer in 2010; briefly, we concluded that central venous insertion sites had no impact on patients’ QoL and psychological distress. Patients undergoing palliative therapies showed worse EORTC QLQ-C30 scales. Artificial Nutrition in Oncology: Dr Biffi was IEO-appointed investigator in a multi-center Italian trial aimed at evaluating the effects on surgical morbidity of glutamine parenteral administration in oncology patients undergoing majorsurgical procedures. Trial was closed after reaching the required sample size (428 patients) and an original article was published on Annals of Surgery -2009, indicating that perioperative glutamine does not affect outcome in wellnourished gastrointestinal cancer patients. In addition, Dr Biffi was Coordinator in a multi-center Italian observational study enrolling more than 200 seriously ill oncology patients undergoing oral omega-3 fatty acids supplementation therapy, to evaluate its effects on nutritional status and quality of life. Currently the Division is participating in a multi-center Italian epidemiological observational study on the prevalence of caloric-proteic malnutrition in the oncology outpatient setting (SCRINIO Project). Preliminary data were published on Supp Care Cancer – 2009. Final results were published on Supp Care Cancer .- 2012. Finally, two randomized trial were concluded in col laboration

with Gynecologic Oncologic Surgery Division, and the data were published as full papers in Annals of Surgical Oncology-2009. Early resumption of oral intake was showns feasible and safe in gynecologic oncology patients undergoing intestinal resection as part of a planned surgical procedure. Moreover, significant reduction in length of hospital stay was demonstrated. Venous thromboembolism in surgical oncology: Staff physicians carried out as IEO-appointed investigators a multi-center Italian observational trial, enrolling more then 2000 patients, aimed at defining the prevalence of deep venous thrombosis in surgical oncology patients. They also participated in a multi-center international trial, enrolling more than 25.000 patients, comparing two different strategies for pharmacologic prevention of venous thromboembolism in surgical oncology patients (Fondaparinux vs Dalteparin). Tumor lymphangiogenesis in gastric and colo-rectal cancer: a prospective study in collaboration with the Emato-Oncology Division to investigate whether the extent of tumour lymphangiogenesis (in intratumour and peritumour tissues/ areas) may be related to the risk for lymph node metastasis, distant metastasis and patient survival. Objective of this study is evaluate the clinical and pathological significance of the tumour lymphangio- genesis in intratumour and peritumour area (VEGF-C/ VEGF-D/VEGFR-3 axis) and its correlation with some very important cellular and molecular blood markers (CECs, CEPs, VEGF C-D, VEGF-R, VE-cadherin). All these molecular and cellular biomarkers could play a central role in the development of lymph node metastases. Two studies on surgical site infections prevention in colorectal cancer open surgery and on preoperative bowel preparations are currently run in collaboration with the General and Laparoscopy Surgery Division (see it for details).

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Surgical Area

Division of Abdominopelvic Surgery

Unit of Minimally-Invasive Surgery Paolo Pietro BIANCHI, MD Director

STAFF Assistant: Wanda Petz, MD Research Fellows: Lorenzo Casali, MD (Fondazione Umberto Veronesi), Matias Parodi, MD (Fondazione Umberto Veronesi) Veronica Reusmann (Fondazione Umberto Veronesi) Resident: Daniele Belotti, MD, Fara Uccelli, MD

Activities 2011.

The Minimally-Invasive Surgery unit was created in 2008, to develop advanced Minimally – Invasive techniques in oncology. Minimally-Invasive surgery includes laparoscopic and robotic surgery, these techniques are commonly applied to the treatment of all digestive tumors, especially to colorectal, gastric and liver cancers, adrenal neoplasms and to the staging of advanced malignant diseases. The clinical resarch in 2010 was focused on robotic surgery, on a project of fast-track model apllied to laparoscopic surgery of the colon, on a project of local treatment of rectal cancer and on the minimally-invasive management of focal liver lesions. Robotic surgery represents a field of great interest in minimally-invasive surgery and many national and international cooperations have been established. Paolo Pietro Bianchi, Director of the Unit, is founding member of the Clinical Robotic Surgical Association (CRSA), one of the most important robotic association worldwide. A cooperation with the University of Illinois is active to develop scientific trials on robotic surgery and an international network of robotic schools. The rapid advancement in robotic surgery has highlighted a pressing need for the implementation of highly specialized education, training programs, guidelines and

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protocols so that surgeons can acquire the necessary familiarity and technical know-how to incorporate these advanced surgical therapy in the management of their patients. The Network of robotic schools is formed by highly experienced robotic surgery teaching centre, aiming to create an international didactic network dedicated to organizing theoretical and practical courses to learn basic and advanced robotic techniques. The network intends to define the guidelines and clinical protocols about the application of robotic surgery within different surgical fields and is eager to overcome the boundaries of the various Institutes and widen the range of teaching, enabling international university lecturers to carry out their activity in collaboration with a network of international experts schools-sharing a common purpose-built program. Dr Bianchi, as a member of the Research Committee of the European Association of Endoscopic Surgeons (EAES), is in charge of a European data collection on robotic surgery of the rectum. The Unit is involved in the ROLARR trial (Robotic versus Laparoscopic Resection for Rectal Cancer), this is an international, multicentre, prospective, randomized, controlled, unblinded, parallel-group trial of roboticassisted versus standard laparoscopic surgery for the curative treatment of rectal cancer. The study aims to test the hypothesis that roboticassistance facilitates laparoscopic rectal cancer surgery. On short-term follow-up this should result in a reduction in the conversion rate and no worsening of the circumferential resection margin positivity rate. On longer-term follow-up, the increased accuracy should improve post-operative bladder and sexual function, enhance quality of life, and ensure there is no increase in local diseases recurrence. Thereby the primary endpoint of the study is the rate of conversion to open surgery as an indicator of surgical technical difficulty. Since the robotic surgery is a new technology, it is

essential that a proper evaluation is performed and disseminated prior to its widespread implementation. A timely assessment is imperative and for this reason there is no plan to perform a prior pilot study, which would inevitably delay evaluation by proper scientific methods. The feasibility of robotic-assisted rectal cancer surgery has already been established and preliminary data upon which to base sample size calculations are available. The time is right for a formal randomised controlled trial to provide a definitive answer to the proposed research question. Fast-track laparoscopic colon resection The Unit is part of a multidisciplinary team whose aim is to promote fast track stategies application to surgical patients; in addition to all the surgical procedures normally performed on day-surgery basis, attention is focused on

fast-track laparoscopic colon resection. The application of the principles of fast track strategy to colorectal surgery has demonstrated its efficacity in reducing postoperative morbidity and lenght of hospital stay, permitting a faster functional recovery. Key elements of this strategy are multimodal analgesy without oppioids, limitation of intravenous fluids during surgery, rapid oral feeding after surgery, no routinary use of nasogastric tube, urinary catether, abdominal drainages and avoiding of mechanical bowel preparation. Minimally invasive surgery, decreasing surgical stress, has a predominant role in all the programs of faster patient recovery, but this multimodal/multidisciplinary approach needs the active cooperation between surgeons, anesthesiologists, nurses and physiotherapists. All these professionists interact among them and with the patient to promote and assure a dynamic pain control

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Surgical Area and the early patient mobilisation and oral feeding. A cooperation is going to be est i ablished between our Institution and nearby non-medical structures where, in case of early discharge, patients could be hosted and clinically monitored with the help of a dedicated nurse staff. Local treatment of rectal tumors The Unit is part of a major research project on local treatment of cancer of the rectum. Aim of this study is to evaluate the impact on local control of local excision in patients who had a major clinical response evaluated by magnetic resonance and confirmed by Tumor Regression Grade (TRG) 1-2 scores. The local treatment of tumors is one of the fields where the interdisciplinary nature of the different minimally invasive specialties is moving more intensively. In May 2012 will be held in IEO a workshop on the multidisciplinar innovative treatments in cancer of the rectum. Minimally Invasive management of focal liver lesions The Unit is involved in a clinical project of minimally invasive treatment of focal hepatic lesions, in cooperation with the Interventional Radiology Unit. Laparoscopic radiofrequency ablation of primary or secondary focal hepatic lesions is an original option that is emerging as a valide alternative to surgery. Realizing the radiofrequency thermoablation under laparoscopic control offers the advantage of an accurate staging of the disease and of performing the procedure under direct visualization, thus minimizing the risk of intestinal injury. Current practice of our team is to perform a preoperative superselective transarterial embolization of lesions whose diameter is major than 3 cm; this results in a complete tumour devascularisation, thus reducing the heat-sink effect of radiofrequency. The Resarch activity of the Unit is oriented to study new

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technologies and their clinical application to oncologic diseases. The following trials are ongoing or recentely closed: • sentinel node mapping in colon cancer laparscopically treated. A muticentre international trial on the prognostic value of micrometastases in sentinel node in colon cancer, is ongoing in cooperation with the McLaren Regional Medical Centre at the Michigan State University and the John Wayne Cancer Centre in Santa Monica (US). • laparoscopic radioguided detection of colon cancer with the use of a portable gamma camera. The aim of this trial is to evaluate the utility of radiotracers in detection of small colon cancer lesions during the minimally-invasive surgery and even to map the lymphatic pathway in order to study sentinel lymphnodes. This study is in cooperation with the Divisions of nuclear medicine, endoscopy and pathology • Retrospetcive analyisis of minimally-invasive surgery of the left colonic flexure. In cooperation with Ospedale San Paolo and Istituto Clinico Humanitas. Milano. • Retrospective analysis of clinical and oncological results of minimally-invasive right colectomy performed with intra-corporeal or extra-corporeal anastomosis. This study is realized in cooperation with IRCCS Istituto Clinico Humanitas and Ospedale San Paolo, Milano. • Retrospective comparative study of results of open versus robotic rectal resection for cancer. Aim of this single-center analysis, in coohperation with the Division of General and Laparoscopic Surgery, is to define potential advantages of minimally invasive approach to low rectal cancer in comparison with traditional open surgery. Preliminary results of this study have been presented to the 2012 congress of SAGES (Society of American Gastrointestinal and Endoscopic Surgeons)

The Unit of minimally-invasive surgery is even dedicated to education training inside the IEO Education pro- gramme of laparoscopic and robotic surgery. A training course on advanced digestive robotic surgery was realized in 2011, with national and international experts showing of upper and lower GI live surgical procedures. Training courses of colorectal oncologic laparoscopic and robotic surgery are activated with intraoperative training and direct view of the minimallyinvasive operations. Dr. Bianchi is a teacher of the Special ACOI (Associazione Chirurghi Ospedalieri Italiani) School of Robotic Surgery of Grosseto and Professor of the Postgraduate School of Digestive Surgery, University of Milano. 2011 Peer reviewed pubilshed papers Bonomo G, Della Vigna P, Monfardini L, Orgera G, Chiappa A, Bianchi PP, Zampino MG, Orsi F. Combined Therapies for the Treatment of Technically Unresectable Liver Malignancies: Bland Embolization and Radiofrequency Thermal Ablation within the Same Session. Cardiovasc Intervent Radiol. 2012 Jan 21. . Bianchi P, Petz W, Spinoglio G, Belotti D, Bertani E, Zampino MG, Crosta C, Lazzari R, Andreoni B. [Robotic rectal resection in rectal cancer: short term results in a monocentric prospective study.]. Minerva Chir. 2011 Dec;66(6):527-535. Italian. Spinoglio G, Lenti LM, Maglione V, Lucido FS, Priora F, Bianchi PP, Grosso F, Quarati R. Single-site robotic cholecystectomy (SSRC) versus single-incision laparoscopic cholecystectomy (SILC): comparison of learning curves. First European experience. Surg Endosc. 2011 Dec 17.

Bianchi PP. Reply to: doi: 10.1007/s00464-011-1808-9: Robotic versus laparoscopic total mesorectal excision for rectal cancer: comparative analysis of oncologic safety and short-term outcomes. Surg Endosc. 2011 Jul 30. Bertani E, Chiappa A, Biffi R, Bianchi PP, Radice D, Branchi V, Cenderelli E, Vetrano I, Cenciarelli S, Andreoni B. Assessing appropriateness for elective colorectal cancer surgery: clinical, oncological, and quality-of-life short-term outcomes employing different treatment approaches. Int J Colorectal Dis. 2011 Oct;26(10):1317-27. Epub 2011 Jul 13. Bertani E, Chiappa A, Biffi R, Bianchi PP, Radice D, Branchi V, Spampatti S, Vetrano I, Andreoni B. Comparison of oral polyethylene glycol plus a large volume glycerine enema with a large volume glycerine enema alone in patients undergoing colorectal surgery for malignancy: a randomized clinical trial. Colorectal Dis. 2011 Oct;13(10):e327-34. 2011 Awards: • Best scientific report at the 15th congress of SICE (Società Italiana di Chirurgia Endoscopica) with the oral presentation “Laparoscopic radiofrequency ablation of focal hepatic lesions: technical aspects” • Best video presentation at the 3rd Congress of CRSA (Clinical Robotic Surgery Association) with the video “Full robotic right colectomy with intracorporeal anastomosis” • Best oral presentation at the 3rd Congress of CRSA (Clinical Robotic Surgery Association” with the presentation “Laparoscopic right colectomy: intra or extracorporeal anastomosis”

Bianchi PP, Petz W, Casali L. Laparoscopic lymphatic roadmapping with blue dye and radioisotope in colon cancer. Colorectal Dis. 2011 Nov;13 Suppl 7:67-9

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Angelo MAGGIONI Director

STAFF Senior Deputy Directors: Luca Bocciolone, MD, Fabio Landoni, MD, Gabriella Parma, MD, Vanna Zanagnolo, MD Deputy Directors: Giovanni Aletti, MD Assistants: Annalisa Garbi, MD, Maria Teresa Lapresa, MD, Rosanna Mancari, MD, Research Fellows: Drusilla Rolla, MD, Piergiorgio Rosenberg, MD Data Manager: Maira Biggioggero, ScD Data Manager Coordinator: Sara Boveri, ScD Secretaries: Romina Brugnoli, Romina Manzini, Pina Mariano, Head Nurse: Emanuela D’Anna

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Surgical Area

Division of Gynaecology Nicoletta COLOMBO Co-Director

Activities 2011.

The Division of Gynecology provides all services involving the diagnosis, treatment and follow-up of gynecologic oncology patients. The Division includes the Unit of Gynecologic Oncology, mainly dedicated to chemotherapy treatments, and the Unit of Preventive Gynecology, mainly dedicated to diagnosis and treatment of genital cancer precursors. Each staff member is a fully-trained gynecologic oncologist responsible for various activities within the Division, including surgery (minor, major and minimally invasive: laparoscopic and robotic surgery), chemotherapy treatments, research, clinical trials, and early diagnosis. Clinicians from the Division of Gynecology were principal investigators in several international and national trials, mainly addressing to determine the best treatment for women with ovarian cancer, in co-operation with MaNGO, Mario Negri Gynecologic Oncology group, and Gynecologic Cancer Intergroup, an organization of international cooperative groups for clinical trials in gynecologic cancers. Among the surgical activities particular attention is devoted to fertility preserving surgery in young patients with borderline ovarian tumors and early-stage ovarian, endometrial and cervical cancer. The division has also the facilities and the experience to perform major surgery such as extensive cytoreduction in patients with advanced ovarian cancer and pelvic exenteration with IORT (intra-operative radiotherapy) in patients with recurrent cervical, endometrial and vulvar cancer. Finally the application of minimally invasive robotic surgery in therapeutic approach to the different gynecological malignancies, such as endometrial, and cervical cancer (simple and radical hysterectomy plus lymphadenectomy) is a new field of interest. Recently, we start with a new clinical trial addressing the role of minimally invasive surgery in the treatment of early stage cervical cancer patients versus standard approach (open surgery). In addition, in 2010, we promoted the foundation

of Robotic School in gynecologic oncology for teaching innovative surgical technique. In addition to their clinical and research activities members of the Division also have university and institutional teaching responsibilities that mainly involve training residents and fellows, but they are also involved in Continuing Medical Education (CME) programs. Esagon (European School of Abdomino-pelvic surgery in Gynecologic Oncology), founded in November 2009, arises from the pressing need for advanced post-graduate training, with a particular focus on the recent important progress in the field of oncology surgery. The School’s foremost educational objective is to transmit an approach

to surgery based upon the natural history of the diseases as well as traditional surgical techniques, and what the relevant technology offers. Esagon’s main characteristic is its international nature. The School will involve both institutions and individuals from different countries within the European Union and the rest of the world. The school’s Faculty is composed of renowned experts both in the clinical and the research fields. Finally, in July 2010, the Gynecology Department has been recognized as an accredited European Center in Gynecologic Oncology by ESGO (European Society of Gynecologic Oncology) and EBCOG (European Board and College of Obstetrics and Gynecology).

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Surgical Area

Division of Urology Ottavio DE COBELLI, MD Director

STAFF Senior Deputy Director: Deliu Victor Matei MD Deputy Directors: Danilo Bottero MD, Gennaro Musi MD Assistants: Antonio Brescia MD, Giovanni Cordima MD, Giacomo Galasso MD, Federica Mazzoleni MD Residents: Sara Melegari MD, Marco Rosso MD Secretaries: Elena Collarin, Mariaelisa Ronzino Data Manager: Serena Detti Head Nurse: Eleonora Meola

Activities 2011. The Division of Urology is

concerned with the treatment of all urological tumours, including prostate, bladder, kidney, testis and penis cancer. In 2011, 1145 patients were admitted for surgical treatment. Among these we performed both endoscopic, open and robotic surgery. Among open procedures we performed 15 radical retropubic prostatectomies, 30 radical nephrectomies, 16 nephron sparing procedures and 47 cystectomies. Of these procedures, 15 were with urinary reconstruction. We had a further increase of robotic surgery, with 313 robotic assisted prostatectomies and 47 kidney surgery (22 robotic radical nephrectomies and 25 nephron sparing procedures). Our experience in urologic oncology was extended in all the items such as testis cancer and penis cancer. There were also performed urinary diversions for patients who underwent pelvic exenteration in other divisions (Gynaecology, General Surgery). Many patients underwent endoscopic procedures, like trans-urethral resection of bladder (259 patients) and ureteral stent insertion (80 patients). Accurate follow-up procedures, following international guidelines, are strictly observed. We had a great development of robotic surgery, as it was the more frequent surgical treatment for prostate cancer. The oncological results with a medium follow up of 25 months are similar to the open radical prostatectomy; however, the main advantageof this surgical technique is the shorter time required to reach urinary continence and sexual potency, and the better overall outcome for both functional domains, comparing to the open surgery. Moreover, Robotic Surgery offers better perioperative outcomes: blood loss, catheterization, surgical time. Plans for research projects in 2012/13 include a further increase of robotic surgery in order to validate these kind of treatments in patients with kidney cancer. Moreover, in cooperation with the Department of Medi- cine, trials for Hormone Refractory ProstateCancer are ongoing

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(See specific research chapters on Prostate cancer). The standard treatment for invasive bladder cancer remains radical cystectomy. The indication for orthotopic bladder substitution has greatly increased over the last decade and in suitably selected patients, quality of life is excellent and morbidity is comparable to that with other forms of urinary diversions. The intestinal bladder substitute should be a low-pressure, capacious and highly compliant reservoir, with a state of fullness that can be appreciated by the patient, allowing him to void at a socially appropriate time. For superficial bladder cancer has been carriyng an innovative treatment, “Synergo®”. The Synergo® System is a computer-embedded intravesical irrigation system combined

with an energy-delivering unit. The energy-delivering unit consists of a radio frequency (RF) generator and an antenna. The treatment employs intravesical instillation of a chemotherapeutic agent concomitant with hyperthermia of the bladder wall induced by emission of RF energy, monitored by internal thermocouples. A specially designed combination of hardware and real-time software regulates the operation presenting the processed data on the monitor screen. The object of treatment is to heat the bladder wall while instilling the drug solution, thus providing a synergistic antitumor effect, in patients with intermediate or high risk superficial transitional cell carcinoma of the bladder.

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Surgical Area

Division of Melanoma and Soft Tissue Sarcomas Alessandro TESTORI, MD Director

STAFF Secretaries: Barbara Bottari, Monica Burla Data Managers: Francesco Cataldo, Abbondanza Montinaro SURGERY STAFF Deputy Director: Elisabetta Pennacchioli, MD Assistant: Francesco Verrecchia, MD Fellow: Antonio Intelisano, MD ONCOLOGY STAFF Director Translational Research Unit: Pier Francesco Ferrucci, MD Senior Deputy Director: Emilia Cocorocchio, MD Psyco-oncologist: Beatrice Colombo, Psychologist Fellows: Salvatore Alfieri, MD, Angelo Battaglia, MD Translational Research: Chiara Martinoli, PhD, Biologist (Fondazione Grazia Focacci) DERMATOLOGY Senior Assistants: Federica Baldini, MD, STAFF Massimo Mosconi, MD, Giulio Tosti, MD Assistant: Giuseppe Spadola, MD

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Introduction:

The treatment of melanoma and other skin cancers is tailored on the stage of disease. Procedures include: diagnostic excisional biopsies of the primaries, re-excision plus sentinel node biopsy in stage I-II melanoma patients, complete lymph-node dissection in the case of metastatic spread to the nodes, isolated limb perfusion with TNF and Melphalan or electrochemotherapy with bleomycin in patients with in-transit metastases and systemic treatments within or out of clinical trials for stage IV disease. Interestingly, new technologies have been recently introduced in order to improve the accuracy of the diagnosis of skin tumors. A high resolution digital dermoscopy is available for mapping of nevi and followup of suspicious pigmented lesions. Furthermore, a reflectance confocal laser scanning microscopy (RCSLM) is available for the “in vivo” evaluation of clinically difficult skin lesions. This is is a new diagnostic technique which allows non-invasive imaging of the upper portion of the skin at a resolution that permits visualization of cellular details with near histological resolution in real time. Outlines of cells and their architecture are imaged and may be analyzed both horizontally and vertically to the skin surface. The method has been proved to be highly useful in early melanoma and other skin neoplasms detection. From a surgical point of view, chemosaturation therapy with percutaneous hepatic perfusion is a new technique which represent a unique, repeatable catheter-based regional approach. It is indicated in all those patients with an exclusive hepatic involvement by different cancers but in particular by ocular melanoma, since they do not have any alternative valid therapeutic option. It has many advantages compared to other loco regional treatments since it permits to treat the entire liver (including invisible micro-metastases), it reduces systemic toxicities by drug filtration, thus improving safety, it is repeatable as many as ten times in one single patient and it is minimally

invasive allowing treatment for sicker patients. From a medical oncologist point of view, the availability of 2 drugs, Vemurafenib and Ipilimumab, impacting on survival have revolutionize our therapeutic approach, widening the options and expanding hope for a cure in many patients affected by extended disease. In the future, combination of targeted therapies with chemotherapy and/ or immunotherapies will offer more opportunity to induce durable responses impacting on duration and quality of life of our patients.

Activities 2011. The division is devoted to the diagnosis and treatment of skin cancers and soft

tissue sarcoma. The activity ranges from sophisticated diagnostic procedures, dermatologic and molecular biology researches, to the surgical and medical treatments of melanoma and soft tissue sarcoma patients. Because of the complexity of these items, there is a clear indication for a multidisciplinary approach. That’s the reason why, not only in soft tissue sarcoma but also in melanoma patients, optimal care requires coordination between a variety of specialties, which may include: • Dermatology • Dermatopathology/Surgical Pathology • Surgical Oncology • Medical Oncology

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• Radiology • Radiotherapy • Psycho-oncology • Basic Research and Immunology In our Institution, comprehensive services, available through an out- or in-patient choice, include: • Complete medical, dermatological and surgical evaluation • Diagnosis of suspicious skin lesions with the aid of dermoscopy and digital dermoscopy. • Diagnosis of clinically difficult skin lesions with Reflectance Confocal Microscopy (RCM) • Participation to surgical, chemo- and immunotherapeutic National and International clinical trials. • Pathological review of skin biopsies by dermatologists, surgical pathologists and dermatopathologists. • Surgical treatment of melanomas: excision of the primary, sentinel node biopsy, complete lymphnode dissection, Electrochemotherapy, limb Perfusions and liver Perfusions (1st in Europe). • Genetic counseling and Psychology support. • Multidisciplinary follow-up for patients with a history of melanoma. • Adjuvant immune therapy within clinical trials for high-risk melanoma patients. • Standard and Investigational therapies for the treatment of advanced melanoma, including tumor vaccines. • Molecular biology and immunology research. To coordinate all these activity we created the Melanoma Cancer Center (MCC) within the EIO, with the aim of ameliorating this comprehensive, multidisciplinary approach for the cure of skin cancers and soft tissue sarcoma. The most recent and ongoing Melanoma clinical trials are described in the dedicated chapter of the annual report.

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Concerning soft tissue sarcomas, the mainstay of treatment includes surgical resection and radiation depending on histotype, site, size and location. Also in this case the approach is multidisciplinary. Neoadiuvant chemotherapy options as per histology are under evaluation through participation in a national clinical trial. Brachytherapy as well as intra-operative (IORT) or external beam radiotherapy are available as needed. Loco-regional treatments, as Isolated limb perfusion, are used in locally advanced tumors, by associating TNF to chemotherapeutic agents. During 2011, more than 8000 patients were examined in the outpatient clinics. The surgical procedures conducted on melanoma patients were almost 330. Moreover, approximately 30 sarcoma and 1100 non melanoma skin cancer or various other cutaneous lesions were operated under general or local anesthesia. The Division published 12 articles on peer-reviewed journals with a general impact factor equal to 144,37. Further steps will include the development of new drugs combinations, new surgical procedures and new dermatological diagnostic approaches in order to achieve the best standard of care for patients affected by melanoma and sarcoma. Finally, this vision and the tight work together with devoted basic researchers will allow the growing of the next generation of clinical scientists capable of translating novel concepts from the laboratory to the clinic. Unit of Translational Research on Melanoma In the context of the Multidisciplinary team and the Melanoma Cancer Center, patients with advanced disease are treated mainly through inclusion in chemo-, targetedand immuno-therapeutic clinical trials within the Unit of Translational Research. The group participated to the development of Vemurafenib (an anti BRAF therapy) and Ipilimumab (an anti CTLA4 therapy), the first 2 drugs impacting on survival, by including advanced melanoma patients in the 4 phase II

and III studies that explored their efficacy and toxicity. These drugs revolutionized our therapeutic approach, widening the options and expanding hope for a cure in many patients affected by extended disease. In the future, combination of targeted therapies with chemotherapy and/or immunotherapies will offer even more opportunity to induce durable responses impacting on duration and quality of life of our patients. Recently, the Task Force on Sarcoma started to work involving medical oncologists, surgeons, pathologists, radiotherapists and radiologists in order to offer a comprehensive approach also to patients affected by this disease. Interestingly, our Team include a Psycho-oncologist offering support to all the patients undergoing specific surgical or medical treatments and their family if requested. The most interesting opportunity is for us to bridge basic research to the clinical one. For this reason a molecular biologist is working at IFOM under the Grazia Focacci Foundation support. She reported interesting data on Maspin expression during melanoma progression and metastatization. Moreover, our research program focused on molecular evaluation of the microenvironment and angiogenesis in patients enrolled in a specific protocol and receiving an antiangiogenic drugs combined with chemotherapy. Finally, we are studying the specific effect of various drugs and combinations on the immune system by monitoring the level of expression of target molecules involved in the induction of an immune response.

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Surgical Area

Division of Plastic Surgery Mario RIETJENS, MD Director

STAFF Former Director: Jean-Yves Petit, MD Senior Deputy Director: Cristina Garusi, MD Deputy Director: Francesca De Lorenzi, MD, Stefano Martella, MD Assistants: Benedetta Barbieri MD, Tito Brambullo, MD, Alessandra Gotardi, MD, Giuseppe Lomeo, MD, Andrea Manconi, MD, Francesco Serpi, MD Maria Simona Sommario, MD, Residents: Veronica B. Blanco, MD, Max Cassilha, MD, Vincenzo Giovinazzo, MD, Visnu Lohsiriwat, MD, Specialty: Hubner Arana Gabriel (school of plastic surgery) Consultant doctor: Pierre Rey, MD Secretaries: Sonia Boffini, Manuela Iavarone Data Managers: Ilaria Minotti, Fabio Rossetto Head Nurse: Gina Rubio

Activities 2011. The Division of Plastic Surgery

is dedicated to the improvement of the quality of life of the patients treated for a cancer at the EIO. Most of the reconstructive procedures are well-known and currently applied in our service since many years. However, the recent modification of the cancer protocols such as the irradiation of the thoracic wall in case of extensive involvement of the axilla, led us to question the indications and the techniques of immediate breast reconstruction. We are studying the place of the two stage procedure using the short expansion with an expander introduced at the time of the mastectomy and

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the choice at the second stage between the substitution of the expander by an implant or by an autologous musculocutaneous flap if the histological status of the lymphnodes requires a radiotherapy of the thoracic wall. Also the reshaping of the conservative surgery (so called oncoplastic surgery) with prosthesis implantation can be done more safely now that the protocol of radiotherapy includes frequently, the intra operative irradiation decreasing the risk of post radiation capsular contracture. The Nipple Sparing Mastectomy protocol is going on rapidly. More than 2500 nipple sparing mastectomies have been performed in 10 years. Eleven studies have been published and the more recent ones (2011-2012) are dedicated to the study of local recurrence risk and evaluation of the indication criteriae. Finally two important studies have been published on the psychological improvement for the patientsthanks to the nipple areola preservation. Concerning the technique of reconstruction with latissimus dorsi, a database of 260 flaps has been set up which gave us the opportunity of several national and international presentations and one paper which has been published in 2010 Our database of Tram flaps is made of 712 patients Our study on prosthesis physical and mechanical modifications after explantation is also going on thanks to the good collaboration with the Istituto Politechnico labs. The results will be ready in 2011 Since several years we are developing a new technique of lipofilling to improve the shape of the reconstructed breast without removing the implant. This technique provides many opportunities of clinical research, especially concerning the selection of stem cells. At the end of 2010 a database of around 1000 lipofillings has been set up. More than six recent publications including a multicentric study gave us an opportunity to evaluate the risk of complications and more specially thanks to the first case-control study published todate, to give an answer to the safety of this procedure in breast cancer patients. We are currently working closely with the team of Francesco Bertolini following the principles of the translational research.

Plastic surgery is also required after other cancers such as head and neck, thoracic, gynaecologic and soft tissue tumors where the large defects created by the tumour removal should be reconstructed with a large armamentarium of flaps including microsurgery or endoscopic techniques. Microsurgery at the IEO is developed as a multidisciplinary surgery remaining at the disposal of the different specialties, (head and neck, plastic, thoracic gynaecologic, general surgery and skin and soft tissue surgery. In Plastic surgery Dr Garusi has studied the role of the angio CT scan to improve the localization of the perforator vesssels to improve the quality of the dissection. She studied also the results of specific flaps such as the

gracilis free flap which provides good breast reconstruction with minimal donor site sequelea. Plastic surgery not only improves the quality of life of cancer patients but also provides a better surgical excision of the tumours by the cancer surgeon who knows that the reconstruction is available immediately in the same time of the cancer treatment. Eleven articles have been published in 2011 on peerreviewed journals and one in a book on breast surgery and already 9 articles have been published in 2012.

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Surgical Area

Division of Radiology

Unit of Interventional Radiology Franco ORSI, MD Director

STAFF Deputy Directors: Guido Bonomo, MD, Paolo Della Vigna, MD Senior Assistant: Lorezo Monfardini, MD Technicians: Ubaldo Piccolo Longo, Simone Vito Fasulo Secretary: Mariastella Bottalico

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Activities 2011. Interventional Radiology

(IR) is a modern branch of medicine, with a very high technology content, which deals with the minimallyinvasive management of clinical problems. It represents the union between instrumental diagnostics and clinicalsurgical activity, coming from a super-specialized branch of Diagnostic Radiology. The role of Interventional Radiology in Oncology (Interventional Oncology) has rapidly grown both for invasive diagnostics (e.g. imaging-based percutaneous needle-biopsies) and mainly, for loco-regional therapies as therapeutic alternative to traditional surgical and medical treatments. Diagnostic and therapeutic procedures are performed with percutaneous approach (= through the skin and without surgical cuts), guided by instrumental methodologies as ultrasound, CT, MR and fluoroscopy (X rays). Precision and very low invasiveness represent the main features of this clinical discipline. Interventional Radiologist is, therefore, a Physician specialized in imaging methodologies which are employed for guiding very focused procedures. High Intensity Focused Ultrasound (HIFU) is also a well established non invasive technique for solid tumor treatment atIEO, where it has been added to the IR-armamentarium. Since January 2012, hepatic percutaneous chemoperfusion with extracorporeal circulation is a new intrarterial therapy for advanced metastatic liver involvement. IEO has been the first European Center where this promising technique has been performed within a clinical setting. The Interventional Radiology Unit of the European Institute of Oncology deals with the management of clinical problems, from diagnosis (also by means of biopsy) to the loco-regional treatment of tumors, in multidisciplinary collaboration with surgical and medical depart-ments. Minimally-invasive management

of post-surgical complications, such as the control of hemorrhages and drainage of fluid collections is another field where IR is usually involved. As one of the first examples in Europe, the Interventional Radiology Unit in IEO manages also its own beds, which are reserved to the patients who will need an IR treatment.

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Medical Area

Department of Medicine

Division of Medical Oncology Aron GOLDHIRSCH, MD Director

STAFF Senior Assistant: Marzia Locatelli Assistants: Carmen Criscitiello, Luca Fumagalli, Gino Perri Head Nurse: Enza Dossena Technical Support: Christine Agnu, Sofia Basile, Maria Bivacco, Rosina Lavia, Luciana Mosca Administration and Secretariat: Emanuela Colautti Data Managers: Laura Adamoli, Sabrina Boselli, Raffaella Ghisini, Davide Pastrello Secretary: Maria Cristina Cracas Director of Upper Gastrointestinal and Neuroendocrine Tumors Unit: Nicola Fazio Assistant: Francesca Spada Director Fertility and Procreation in Oncology Unit: Fedro Peccatori Assistant: Maria Anna Sarno Counselor: Chiavari Eleonora

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Giuseppe CURIGLIANO, MD, PhD Co-Director

Activities 2011. The Department of Medicine

incorporates the entire area of care and research for specific domains of cancer medicine: Solid tumors, hematological malignancies and lymphomas, clinical pharmacology and development of new anti-cancer drugs, support and palliative care and psychooncology. The mission of our Department is to provide expert, compassionate care to adults with cancer while advancing the understanding, diagnosis, treatment, cure, and prevention of cancer and related diseases. Our Department also provides training for new generations of physicians, designs programs that promote knowledge particularly among high-risk and underserved populations, and disseminates innovative patient therapies and scientific discoveries to our patients across Italy and throughout Europe. We pursue excellence relentlessly and with integrity in all that we do, adhering always to the highest standards of conduct. We provide compassion and respect for those in our care and for one another. We foster the spirit of inquiry, promoting collaboration and innovation across traditional boundaries while celebrating individual creativity. The Department is organized into divisions and units, which share in-patient wards, and outpatient clinics, including a Day Hospital. Care and research in specific fields characterize the activities of the Division of Clinical Pharmacology and New Drugs Development, the Unit for Supportive and Palliative Care, the Unit of PsychoOncology, the Unit of Upper Gastro-Intestinal and Neuroendocrine Tumors, Unit of Fertility Preservation in Cancer Patients, Unit of Medical Oncology for lung cancer and sarcomas and Unit of Translational research in Melanoma. The Divisions and Units provide care and conduct research in an integrated fashion. Priority of clinical research include tailored treatment for patients with various diseases. Innovative treatments include targeted molecules to specific cancer cellular components, to cancer vessels and experimental use

of anti-cancer vaccines. The cultural principle, which characterizes the attitudinal basis, shared by all the members of the Department, is: “Let innovative research flourish on the ground of the best care available”. During 2011, the Division of Medical Oncology provided 2.831 admissions in its ward (24 beds), and 450 in a singleday hospitalization program. The Division of Clinical Hemato-Oncology provided care for 494 patients in its in-patient ward. The overall outpatient activities of the Department involved at least 38624 consultations at the clinics and 4767 treatments at the Day Hospital Unit. At least 7170 patients were treated within the framework of research trials,

622 newly entered studies during the year 2009. Hemathological and immunological diagnostics and support of treatment, as well as translational research are conducted at the dedicated laboratory of the Division of Hemato-Oncology. Most, if not all, research projects are being curried out in close collaboration with other Divisions and Units both at the clinical and laboratory levels.

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Medical Area

Department of Medicine

Division of Clinical Pharmacology and New Drugs Filippo DE BRAUD, MD (until July 31, 2011) Aron Goldhirsch, MD (since August 1, 2011) Director

STAFF Director, Medical Oncology Unit of Respiratory Tract and Sarcomas: Tommaso De Pas, MD Deputy Director: Cristina Noberasco, MD Senior Assistant: Chiara Catania, MD Assistants: Angelo Delmonte, MD, Monica Giovannini, MD, Gianluca Spitaleri, MD Translational research: Francesca Toffalorio, MD, PhD Research Nurses: Laura Bistocchi, Veronica Brunelli, Livio Libutti Data Managers: Sabrina Boselli, Valentina Sinno, Fabio Vecchio Secretary: Marcella Netti

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Activities 2011. The development of new drugs

in early phases and the promotion of “translational research projects” are the objectives of the Division. Within this remit we propose not only to develop treatments by using new molecular compounds, but also to determine new therapeutic strategies. In both cases the aim is to optimize benefits and minimize toxicity. We are therefore committed to find a clinical way to finalize the attractive hypothesis of being able to personalize treatment on the basis of the biological characteristics of the disease and on pharmacogenomic data of the patient. The number of ongoing studies as well as the implementation of the new Molecular Medicine programs (MolMed) in IEO endorsed the creation of a dedicated Unit for Lung cancer, sarcomas, and rare tumours, within the Division. All “first-in-man studies” as well as early phase II studies and all the clinical studies for lung cancer and sarcomas will be carried out in our Institute, to ensure vigorous quality of monitoring and care during these intensive protocols. In 2011 we designed and/ or conducted dose-finding studies of novel molecules such as antivascular and antiangiogenic agents, cytokine fusion protein, tyrosine kinase inhibitors administered alone or in combination with classic cytotoxic drugs in patients with solid tumour. Detailed information of clinical trials for patients with NSCLC are given in the lung cancer research part. The Medical Oncology Unit of Respiratory tract and sarcomas implemented the research program with the study of the rare tumors of the thorax such as mesotheliomas, thymomas and lung neuroendocrine tumors in order to find new therapeutic approaches for these uncommon diseases (for details, see Lung cancer research). Moreover, a particular attention has been directed towards specific subpopulations such as lung cancers in young patients, patients subjected to a stage shift due to the new TNM edition of lung cancer, and large cell carcinomas.

In collaboration with the Molecular Medicine Program, genetic studies on NSCLC are also currently ongoing. The Division is also carrying out a study on improved health perception due to genetic counselling for women free of disease attending a high risk clinic for breast and/or ovarian cancer and a study about the increasing the awareness of patients with cancer facing with the choice of whether to participate in a phase I clinical trial.

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Medical Area

Department of Medicine

Outpatient Clinics Franco Nolè, MD Director

STAFF Senior Deputy Director: Elisabetta Munzone, MD, Maria Giulia Zampino, MD Deputy Directors: Ida Minchella, MD Senior Assistants: Maria Cossu Rocca, MD, Daniela Cullurà, MD Assistants: Gilda Ascione, MD, Gaetano Aurilio, MD, PhD, Simona Blotta, MD, Angela Esposito, MD, Elena Magni, MD, Angela Sciandivasci MD, Elena Verri, MD Data Managers: Laura Adamoli Secretaries: Daniela Bargiggia, Veronica Bruschi, Monica Croce Nurse Coordinator: Alessio Piredda

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Activities 2011. The Unit is involved in clinical

research on solid tumors. In particular, the Unit is interested in prospective phase II and III trials addressed to investigate the role of novel anti-cancer drugs, innovative strategies or new therapeutic and tailored approaches with particular interest in Advanced Breast Cancer, Head & Neck Cancer, Gastro-intestinal Tumours, Urogenital Cancer. During 2010, the overall outpatient activities involved more than 8500 treatments in patients with solid tumors The scientific activity is also involved in translational research and research of new prognostic and predictive factors to individualize treatment and to guide therapeutic decisions in clinical practice. The collaboration with clinicians and basic researchers in other departments and divisions of the same institution is encouraged and ensured by regular meetings and discussions in the context of Disease Units tumor-specific. Similarly, the national and international cooperation is supported and the unit participates in and promotes a number of prospective multicenter studies. A specific attention is given to quality of life through the investigation of new strategies such as the “metronomic” administration of continuous low dose chemotherapy in collaboration with basic researchers in order to explore possible antiangiogenic mechanisms of this approach. The ongoing research programs include the evaluation of prognostic and predictive value of circulating tumour cells in advanced and operable breast cancer; in castration resistant and in early stage prostate cancer, in gastrointestinal and head & neck cancer, with the aim to better characterize the behaviour of these disease and to individualize the treatment During 2011, the Unit published 11 research papers, with an overall Impact Factor of 68,881.

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Medical Area

Department of Medicine

Unit of Research in Medical Senology Marco COLLEONI, MD Director

STAFF Assistants: Alessandra Balduzzi, MD, Silvia Della Pasqua, MD, Anna Cardillo, MD, Emilia Montagna, MD, Giuseppe Cancello, MD, Monica Iorfida, MD, Manuelita Mazza, MD, Gino Perri, MD. Data Managers: Raffaella Ghisini, Davide Pastrello, Eloise Scarano Secretariat: Simona Puddu, Monica Croce

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Activities 2011. The Unit of Research in Medical Senology provides care and conduct research in an integrated fashion in the field of Medical Senology, mainly in the neoadjuvant and adjuvant setting, The principle shared by members of the Unit is that increased participation in clinical trials would increase learning about the disease and improve patient care. Research projects are being carried out in close collaboration with other Divisions and Units at the EIO and with National and International Cooperative Groups. International trial cooperation, focused on questions relevant for patient care and biological principles represent one of the major commitment for the Unit in particular in the adjuvant setting. A collaborative approach involving the development of new agents and investigation of their optimal integration in therapy programs will best ensure progress for improved patient care. Studies focusing on safety, quality of life, subjective side effects and personal costs are routinely incorporated in the patients care. Assessment of factors, which are associated with response or resistance to therapy and exploration of new therapies accordingly to baseline prognostic features are considered as a priority in the development of the best multi-modal strategy including sequence of local and systemic treatments. Research studies are conducted to define the value of new high-throughput technologies in assessing the level of risk and likelihood of response to specific therapies, in order to improve our knowledge and lead to better tailoring of therapies. Such an approach brings clinical research closer to the individual patient.

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Medical Area

Department of Medicine

Unit for Supportive Care Alberto SBANOTTO, MD Director

STAFF Consultant: (up to 31/03/11) Beatrice Dedor, MD Nurse: (up to 31/12/2011) Angela Cocquio RN Secretary: Cristina Cracas

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Activities 2011.

Supportive and Palliative Care are important in Oncology: prolonged survival of patients, new cancer treatments-related side effects and complications, especially neurological ones, challenge clinicians everyday. The main area of intervention of the Unit are bedside consultations, for all the clinical wards of the hospital, mainly concerning chronic pain problems, symptoms and special syndromes (e.g. dyspnoea, delirium, peritoneal carcinomatosis, inoperable bowel obstruction) and selected postoperative pain items; during 2011 about 750 inpatients visits have been performed. The Outpatients Clinic is mainly devoted to cancer pain syndromes: about 1000 visits have been performed, including a dedicated clinics for radiotherapy patients, where special problems are faced, such as painful radiotherapy-induced stomatitis and bone incidente pain. Continuity of care is so important for cancer patients and their families, most of them living far from the European Institute of Oncology. Telephone calls, emails, faxes are important tools for these purposes. By an agreement with the Fondazione Lucè Onlus, the IEO is implementing a nurse-supervised Call Centre for supporting patients and families faced with clinical problems related to therapies or cancer. During the first 4 years of activity about 5000 telephone calls have been received, concerning about 2000 patients; analysis of customers’ satisfaction questionnaires gave promising results. During year 2011 this kind of intervention has been implemented and more clinical wards involved. Another area of intervention is The Pain Free Hospital Project, where the Unit is closely involved. The main aims of the project are to increase awareness of pain and improve pain and symptoms control at the IEO. The project is involved in teaching, monitoring pain and ensuring better availability of drugs. The first of 4 of a new online Pain Course modules has been implemented and the others are running. In agreement with the Local Health Agency, a complete

Home Care Service Project has been projected and approved. It is devoted to advanced cancer patients living in the south area of Milan and in some of the surrounding small towns. It will be implemented during the year 2012. Scientifical programs Areas of research concern chronic pain syndromes in cancer and supportive models for Continuity of Care in Oncology. Scientifical programs included collaboration in multicentre trials about pain epidemiology, developement in collaboration with the Mario Negri Institute and National Cancer Institute and with the European Palliative Care Society Research Network.

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Department of Medicine

Psycho-Oncology Unit Aron GOLDHIRSCH, MD Director

STAFF Consultants: Andrea Maldifassi-psychologist, Elena Scaffidi- psychologist-psychotherapist Fellows: Paola Arnaboldi- psychologist-psychotherapy student, Beatrice Colombo- psychologist-psychotherapist (Melanoma Division), Stefania Sacchezin-pscyhologistpsychotherapy student, Valeria Vadilonga-psychologistpsychotherapist, Visitors: Beatrice Bianchi (psychology student), Marian de Souza (psychologist), Rossana Mazzeo (psychologist-psychotherapy student), Chiara Mazzola (psychology students), Giada Perinel (psychology student)

Florence DIDIER, psychologist-psychotherapist Co-Director

Activities 2011. Psychological interventions.

Brief crisis counselling, in an individual setting, for patient and family, is typically performed in order to reduce psychological distress and to enhance coping and adaptive skills at every stage of the disease. Cognitivebehavioural and psychodynamic interventions (including relaxation training or relaxation psychotherapy, breathing techniques, guided imagery techniques, hypnosis, E.M.D.R. - Eye Movement Desensitization and Reprocessing) are used by the psychologists of the Unit to help patients cope with the physical, emotional, social and existential traumatic impact of cancer. Difficulties within the couple and psychosexual problems are now included in the routine psychological assessment and discussed with the patient. A specific ‘couple therapy’ or individual sexual therapy are proposed whenever necessary. Psycho-educational groups have been created to help cancer patients face the disease and its treatments. We increased and diversified the type of psycho-educational groups in order to respond to the different needs of patients and their families. For example, women are offered a psycho-educational program, which includes a make-up group session (with the collaboration of the Association La Forza e il Sorriso) and successive supportive group and group relaxation therapy to enhance wellbeing, reduce stress, reinforce self-esteem and improve quality of life. Consultations are performed during the daily medical and surgical routine after referral from physicians, nurses, family members and the palliative care team or as a result of the patient’s self-referral. Psycho-educational groups, expressive-supportive groups and relaxation groups are proposed before or during treatment and at every stage of the disease. Individual psychotherapy or expressive-supportive psychotherapy groups are offered to patients after the end of treatment, to cope with life after cancer and to

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deal with uncertainty, anxiety and fear of the future, body image and relationship and/or sexual/couple difficulties. In 2011 the Psycho-Oncology Unit continued the close collaboration with the Division of Gynaecology and its Ovarian Cancer Center with an ongoing study aimed at .assessing the psychosocial needs and the psychological distress of patients recently diagnosed with an ovarian cancer (34 patients have been interviewed by psychologists of the Unit). Sixty hundred and seven (N= 607) new patients, from all Departments, Divisions and Units, were referred to the Psycho-Oncology Unit. Two thousand nine hundred and twenty (N=2920) consultations were performed during 2011.

After the patient’s discharge, a telephone follow-up is performed by the psychologist of the Unit on a regular basis. In order to improve the psychological support to cancer patients and their families after discharge, we also created a link with the national Foundation Gigi Ghirotti which provides psychological support by phone, performed by external psychologists/ psychotherapists. Educational activities The Psycho-Oncology Unit is involved in several educational activities concerning the recognition, assessment and management of psychological distress of cancer patients. We used different techniques in training in emotional and psycho-physical stress management

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Medical Area dedicated to the oncology staff using; movies-cinema (paper under evaluation. Lupo F. Arnaboldi A et al. Palliative & Supportive Care) and relaxation and guided imagery techniques (accepted as a poster at the National Psycho-Oncology Congress, Brescia, Italy, 2011. Maldifassi A, Didier F et al). Research interests The main research interests of the Psycho-Oncology Unit are the evaluation of the psychological impact of breast cancer and breast surgery on patients and their partners, and patients’ quality of life. In close collaboration with the Divisions of Senology (Dr. A Luini), Reconstructive Plastic Surgery (Prof. JY Petit) and the Department of Medicine (Prof. A Goldhirsch) we study the psychological and body image impact of breast loss, satisfaction with cosmetic results of breast reconstruction, with a specific attention to nipple-sparing mastectomy which is now included in the routine of the Breast Division (JY Petit et al. Nat Rev Clin Oncol, 2011). Following a qualitative methodology, an analysis of the psychological impact of nipple-sparing versus nipple reconstruction is ongoing. We assessed the motivations of patients for accepting a NSM or the nipple reconstruction when NSM was not possible (F. Didier et al, Breast Cancer Res Treat, submitted and under evaluation in 2011) A qualitative analysis of the patient’s and partner’s satisfaction with cosmetic results of breast reconstruction is currently ongoing. The impact of very conservative breast surgery such as BLSN versus ALND on quality of life and psychological distress has been also assessed (Didier F et al, The Breast Journal, 2011). Because of the stressful psychological impact of a breast cancer diagnosis, and the possible role of stress and the role of immunological factors in tumour growth, in 2011 we commenced psychosocial study to determine whether psychosocial stress, in the form of adverse life events and social difficulties, depressive illness, or lack

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of confiding relationships, shortens the postoperative disease-free interval in breast cancer patients. A prospective follow-up was conducted of a cohort of newly diagnosed breast cancer patients for 60 months after primary surgical treatment: 110 patients have been interviewed at baseline, for a total of 238 interviews performed by psychologists. Another study is ongoing aimed at assessing the psychological stressful impact of the breast diagnosis and being on the waiting list, on women who are candidates for mastectomy. 83 patients have been interviewed for a total of 183 interviews performed by psychologists. Bibliography (published 2011) Petit JY, Veronesi U, Lohsiriwat V, Rey P, Curigliano G, Martella S, Garusi C, De Lorenzi F, Manconi A, Botteri E, Didier F, Orecchia R, Rietjens M (2011) Nipple-Sparing Mastectomy –Is it worth the risk? Nat Rev Clin Oncol 8(12):742-7. (Accepted in 2011, under press) F Didier, D Radice, A Maldifassi, G Gatti, A Luini, C Leonardi, N Rotmensz, B Santillo, V Galimberti, A Goldhirsch. Do early breast cancer patients with only sentinel lymph node biopsy experience lower psychological morbidity over time? The Breast Journal, 2011. (Under evaluation in 2011 ) F Didier, P Arnaboldi, S Gandini, A Maldifassi, A Goldhirsch, D. Radice, I Minotti, B Ballardini, A Luini, B Santillo, M Rietjens, JY Petit. Why do women accept to undergo a nipple sparing mastectomy or to reconstruct the nipple areola complex when nipple sparing mastectomy is not possible? Breast Cancer Research Treatment

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Medical Area

Department of Medicine

Division of Cardiology Carlo CIPOLLA, MD Director

STAFF Senior Deputy Directors: Maurizio Civelli, MD, Giuseppina Lamantia, MD Deputy Directors: Nicola Colombo, MD, Carlo Meroni, MD Senior Assistants: Alessandro Colombo, MD, Giulia Bacchiani, MD Consultant: Cesare Fiorentini, MD, PhD, University of Milan Fellows: Marta Beggiato, Student Medical School, Riccardo Ficco, Student Medical School, Damiano Pongan, Student Medical School Secretary: Fabio Farina Chief Nurse: Arnaldo Zanelotti Data Manager: Ines Tedeschi OTA: Maria Iannitelli Consultants Smoke Cessation Center: Elena Calvi, MD, Psychologist and Pneumologist; Mrs Natalia Pozzi (Fondazione Umberto Veronesi). CARDIOLOGY Director: Daniela Cardinale, MD, PhD, FESC UNIT Fellow Research Assistant: Lisa Guzzi, Doctorate Foreign Languages

Activities 2011.

Cardiology Division’s activities relate to pre and postoperative cardiologic assessment, respiratory function evaluations, general internal medicine consultations, antismoking activities, extensive clinical monitoring and therapy for internal wards and treatment of emergencies. The specific cardiological activity is strongly oriented to the diagnosis and therapy of cardiac disorders in order to detect and treat co morbidities (46% of EIO cancer patients present concomitant cardiovascular diseases) as well as potential or evident consequences of oncologic treatments (cardiotoxicity). Cardiological evaluations, either clinical or instrumental, are present in over 140 scientific research protocols of the Institute. In 2011 the Division performed: a) cardiological assessment of 15.413 internal and outpatients; b) complete echocardiographic and Doppler colour evaluations in 3767 patients; c) respiratory physiopathology diagnostic and assistance (2239); d) 343 antismoking activities for patients and employees; e) clinical consultations and/or echocardiographic examinations for over 1700 patients enrolled in different Division’s scientific protocols: f) Overall 54000 written official reports. During 2011 over 1000 patients were treated in urgency/ emergency setting, 79% internal cases, 21% outpatients; the increasing number of treated cases will be one of the elements that will lead the Institute to the opportunity of opening in 2012 a new and original structure: a 24/24 hours ambulatory ward for continued oncologic assistance.

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Cardiology Specific Activities Diagnosis of cardiotoxicity Cardiotoxicity is a common complication of chemotherapy (CT). The clinical manifestation of cardiotoxicity can range, in its more typical form chronic cardiotoxicity - from transient asymptomatic left ventricular dysfunction to cardiac death. This is a growing problem in the setting of clinical oncology due to the tendency in using progressively higher doses of anthracyclines, as well as combined treatments with synergistic cardiac toxic properties. Also newer compounds, as thyroxin kinesis inhibitors, antiangiogenic

drug, and monoclonal antibodies potentially deserve cardiotoxic implications. The clinical implications of cardiotoxicity are particularly relevant in those cancer patients in which onset of cardiac dysfunction, even asymptomatic, seriously limits their therapeutic opportunities and negatively impacts on clinical outcome. At present oncologic guide-lines recommend regular cardiac function assessment (generally by echocardiography or MUGA scan) to detect CT-induced cardiac damage in an early phase. Cardiologic surveillance is required during CT to allow administration of the highest dose without inducing cardiac injury, and after completion of CT to identify

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Medical Area cardiac damage at an early preclinical stage. This is made, in order to limit, by means of pharmacologic intervention, the progression of cardiac dysfunction. The weak point of such an approach is that cardiotoxicity is usually detected when cardiac damage has already occurred. Furthermore, most methods used in clinical practice to detect cardiotoxicity, including echocardiography and radionuclide-angiocardiography, seemed to have shown low sensitivity and poor predictive value. In particular, the evidence of an unaffected heart function does not exclude the possibility of future cardiac deterioration. In our clinical practice we utilize different tools for the early identification of patients at increased risk of cardiotoxicity: biomarkers of myocardial damage, like Troponin I and N-terminal-proB-Type Natriuretic Peptide (Nt-proBNP), and low-dose dobutamine stressechocardiography that allows early detection of left ventricular contractile reserve reduction. For all of them, an accurate predictive value has been demonstrated by our investigations. The possibility of early identifying patients who will develop myocardial dysfunction represents a “golden opportunity” for both oncologists and cardiologists. It allows discriminating, in a very early phase, long before heart function impairment and symptoms manifestation have occurred, patients at high risk of cardiotoxicity from those with a good cardiologic prognosis. As a consequence, most patients can be excluded from a long-term expensive and awkward monitoring program with diagnostic methods like echocardiography and radionuclide angiocardiography. On the other hand, in selected high-risk patients, a strategy based on serial cardiac function evaluations can be planned, and a preventive pharmacological therapy started. Prevention and treatment of cardiotoxicity CT-induced cardiotoxicity can result in a cardiomyopathy generally considered to be irreversible, and leading

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to congestive heart failure and cardiac death. Clinical manifestations of cardiotoxicity may appear months or even years after the end of CT, and are preceded, in most cases, by asymptomatic left ventricular dysfunction. In no monitored patients, symptoms of congestive heart failure usually represent the first manifestation of cardiotoxicity. In our very recently published experience, most patients receiving adequate treatment, including ACE-inhibitors, beta-blockers, diuretics, and anti-aldosterone agents, and closely monitored, experience a relevant improvement in clinical status and cardiac function, in some cases a complete recovery, and a better cardiologic prognosis. For the optimization of cardiologic therapy we usually monitor Nt-proBNP levels, which are related to the clinical and prognostic status of patients with congestive heart failure. Il 2011 the consultancies of the Cardioncology Unit for complex cardiotoxicity problems of external patients were over 150. Diagnosis and management of neoplastic pericardial effusion Malignant pericardial effusion and cardiac tamponade are common complications of several different neoplastic diseases, and have a critical impact on patients’ quality of life and survival. Therefore, the prompt and successful therapy of pericardial disease is crucial in order to increase life expectancy, and improve clinical status. Accordingly, malignant pericardial effusion should not be considered a terminal event, but a treatable condition requiring a true therapeutic intervention. On the basis of our previous studies, we routinely perform pericardiocentesis (PC) as an emergency life-saving procedure. In our experience on almost one hundred patients no clinically relevant untoward side effects were experienced. In this setting however, when PC is utilized only as a drainage technique for symptoms relieve, it is associated with a very high

incidence of early pericardial effusion recurrences. In order to prevent pericardial recurrences, we do not consider PC a mere palliative approach but we associate to the fluid withdrawn also an intrapericardial “oncologic therapy” with both chemotherapeutic and “etiologic” properties. In addition to the reduction of pericardial effusion recurrences, this percutaneous strategy has been proved to be safe and effective in the prevention of neoplastic disseminations that can complicate surgical procedures of pericardial drainage. Furthermore, quality of life and life expectancy of treated patients usually improve. Chemotherapy induced ECG abnormalities and regulatory QT monitoring The evaluation of ECG abnormalities in CT treated patients is routinely performed in our clinical practice. In addition to serial ECG evaluations, we settled an ECG telemetry system to continuously monitor high-risk patients and easily detect arrhythmias and conduction disturbances. Several distinct ECG changes have been described during or soon after the administration of chemotherapeutic drugs: ECG abnormalities may result in ST-segment and T-wave changes, decreased QRS voltage, and prolongation of the QT interval. CT-induced arrhythmias and conduction disturbances include ventricular, supraventricular and junctional tachycardias, and atrioventricular and bundle-branch blocks. In particular, a prolongation in QT interval is associated with onset of severe life-threatening ventricular arrhythmias, named “torsades de points”. In order to more precisely identify a possible proarrhythmic substrate induced by CT drugs, we perform, in selected high-risk patients, also the evaluation of heart-rate variability.

Research Activities 2011 CARDIOTOXICITY: EARLY DIAGNOSIS Markers of myocardial damage: Troponin I. Cardiac toxicity represents one of the most important long-term side effects of high-dose chemotherapy with a strong impact on patient’s quality of life and survival. The clinical course can range from transient asymptomatic left ventricular dysfunction to cardiac death. The possible development of biochemical markers able to identify cardiac damage in an early phase is strategic for both therapeutic and prognostic reasons. In previous studies we demonstrated that troponin I (TNI) is a sensitive and specific marker of myocardial injury after high-dose chemotherapy, and is able to predict, in a very early phase, the development of future cardiac dysfunction, as well as its severity. More recently, we extended this observation to a larger population with a longer follow-up, in which a wide spectrum of cardiologic adverse events was considered. Our study clearly showed that the risk of cardiac events in cancer patients can be predicted by the evaluation of TNI release pattern after chemotherapy. Indeed, most patients with TNI increment soon after HDC developed a significant reduction of left ventricular function in the first year after HDC. A close relationship between the TNI peak value and the degree of cardiac dysfunction during the follow up was found. TNI is also an important predictor of clinical events, as well as anticipating the development of cardiac dysfunction. This finding has important clinical implications and provides an intriguing rationale for the development of pharmacological strategies facing counteracting cardiac dysfunction and cardiac complications occurrence. Hemodynamic markers: Nt-proBNP Natriuretic peptides have recently emerged as biomarkers potentially useful in the diagnosis and

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Medical Area prognostic stratification of patients with heart failure. Particularly, Nt-proBNP is released from the heart in response to a cardiac overload. Preliminary data from our ongoing trial suggest that serial early measurements of plasma Nt-proBNP may represent a useful tool to identify patients at high risk of developing myocardial dysfunction. We planned a further prospective study to clarify either if TNI and Nt-proBNP give the same kind of information, or if their combined evaluation allows a better stratification of the cardiac risk of cancer patients treated with high-dose chemotherapy. Identification of left ventricular contractile reserve impairment: low-dose dobutamine stressechocardiography Dobutamine stress echocardiography (DSE) is widely used in patients with coronary artery disease or dilated cardiomyopathy, and allows the evaluation of left ventricular contractile reserve (LVCR). In cancer patients treated with anthracyclines, we have assessed LVCR by low-dose DSE, in order to detect subclinical left ventricular dysfunction, as a possible early index of cardiotoxicity. In our study, patients with early reduction of LVCR, showed a significant decrease of left ventricular ejection fraction (LVEF) one year after the end of treatment. This information was obtained in a very early phase, when patients were still receiving HDC and when rest LVEF reduction was not detectable yet. Based on these findings, DSE can be considered a safe and useful method for the early identification of patients likely to develop late cardiac dysfunction after HDC. Evaluation of the QT/QTc interval modifications and proarrhythmic potential of chemotherapy in cancer patients An undesirable characteristic of some non antiarrhythmic drugs is their ability to delay cardiac repolarization, an effect that can be measured as prolongation of the QT interval on the surface electrocardiogram. A delay in

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cardiac repolarization creates an electrophysiological condition that can favors the development of cardiac arrhythmias, mostly life threatening as torsade de points and polymorphic ventricular tachiarrhythmias, potentially able to degenerate in ventricular fibrillation and consequently sudden death. In the very early phases of development of new anticancer agents QT/QTc is considered an hallmark of high risk of cardiotoxicity and increased arrhythmogenicity and therefore considered a condition that can stop further interest on these substances, with a great lost of potentially useful compounds. Since the early phases of new drugs development are performed in animals and healthy volunteers, and since we had, over many years of close observation, the strong impression that the effective clinical and arrhythmogenic overall impact of QT/QTc modifications on cancer patients treated with several different chemotherapy agents (alone or in combination) is almost neglectable, we started a study that will consider the impact of electrocardiographic modification induced by chemotherapy in the subset of the “real world” of ten years of consecutive oncologic patients treated with all types of chemotherapeutic schemes. CARDIOTOXICITY PREVENTION: a new prophylactic approach The possibility to identify patients at higher risk of developing late myocardial dysfunction by cardiac biomarkers (TNI, Nt-proBNP) provides a rationale for the development of prophylactic strategies directed against CT-induced cardiotoxicity. Considering the results of our published studies, a possible clinical application of these markers is the evaluation of pharmacological strategies in selected high-risk patients, with the aim to prevent acute cardiac damage, left ventricular dysfunction, and cardiac events. Two different therapeutic strategies could be implemented in order to reduce the clinical

impact of cardiotoxicity: 1) use of specific cardiologic treatments given to cancer patients during CT in the attempt of preventing or blunting the rise of these markers; 2) use of cardiologic treatments given only to those selected cancer patients showing an increase in these markers after CT. This with the aim to interfere with the natural evolution of cardiac toxicity, and prevent the occurrence of left ventricular dysfunction and cardiac adverse events. In particular, the increase of troponin I (TNI) soon after CT is a strong predictor of left ventricular dysfunction and poor cardiologic outcome. We hypothesize that cardioprotective therapies that might limit or prevent the TNI rise after CT, as well as cardiologic treatments that interfere with TNI persistence, could improve cardiac prognosis of these patients. As activation of the renin-angiotensin system has been proved to be involved in the development and progression of cardiac dysfunction in several clinical settings, and has been suggested to have a role in the occurrence of CT-induced cardiotoxicity, we investigated with very positive results the role of treatment with an ACE-inhibitor, enalapril, in the prevention of left ventricular dysfunction in high-risk cancer patients (those with TNI rise after CT). Our data confirm that prophylactic treatment with enalapril effectively prevents the occurrence of asymptomatic left ventricular dysfunction and overt heart failure in these patients. INTRAPERICARDIAL ANTICANCER TREATMENT Pericardial disease and pericardial involvement are increasingly common complications of neoplastic diseases which can be life-threatening, not only in patients with unresponsive or aggressive terminal malignancies, but also in patients with otherwise favourable prognosis. Different methods may be used to treat malignant pericardial effusions, but the gold standard treatment in this subset of patients is yet to be defined. In particular, percutaneous pericardiocentesis

(PC) is associated with a very high incidence of early pericardial effusion recurrences (up to 40%). We performed a prospective, controlled, interventional study in order to investigate short-term safety and effectiveness of PC followed by intrapericardial infusion of an active antiblastic, sclerosing agent, thiotepa, in patients with large malignant pericardial effusion. The results of our study have clearly showed that PC plus thiotepa is a low-cost, low-risk, and safe therapeutic approach, and should seriously be considered as a first choice procedure in approaching neoplastic pericardial effusions. Since 2008 we designed a prospective trial to evaluate clinical and prognostic relevance of intrapericardial TNI determination, and to evaluate the possible role of Nt-proBNP, as a marker of hemodynamic impairment, in defining the clinical indication to PC, in addition to echocardiographic evaluation and in the monitoring of pericardial effusion recurrences. Electronic Cigarette trial for patients affected by acute smoking craving In 2011 we started an original multicenter trial based on the use of a nicotine-free electronic cigarette (TFumo) in different clinical subsets of patients undergoing smoking craving conditions (acute myocardial infarctions, hematoncologic emergencies, urgent cancer treatments) with the participation of three major Institutes of the Milan area (Centro Cardiologico Monzino, Responsible Doctor GianCarlo Marenzi; Ospedale San Raffaele, Responsible Doctor Fabio Ciceri, Istituto Europeo di Oncologia, Responsible and Principle Investigator Doctor Carlo Cipolla, pneumologist Doctor Carlo Meroni, psycocounseling Doctor Elena Calvi, patient’s enrollment and coordination Mrs. Natalia Pozzi). The study protocol will consider over 120 patients and the results will be available at the end of 2012. The activities concerning the nicotine free electronic cigarette TFumo study protocol will integrate the antismoking activities of the IEO Centro

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Medical Area Antifumo (Responsible Doctor Giuseppina Lamantia) concerning either the support to COSMOS 1 population (early preclinical lung cancer diagnostic by Spiral low dose CT scan, Responsible Doctor Giulia Veronesi) and personal direct antismoking external/internal patients therapy. Scientific cooperation for new research avenues Several scientific cooperation have been established in order to study new elements concerning cardiotoxicity not only classically regarding anthracyclines, but also new anticancer drugs (monoclonal antibodies, tyrosine kinesis inhibitors and anti angiogenic agents); some common pathways signalling regarding and their potential translational implications; we deeply thank for great efforts and help: Daniel Lenihan, Cardiovascular Research, Vanderbilt University, Nashville, Tennessee; Fabio Ciceri, Hematology and Bone Marrow Transplantation, Ospedale San Raffaele, Milano; Giancarlo Marenzi, Unità Coronarica, Centro Cardiologico Monzino, Milano; Roberto Latini, Laboratory of Cardiovascular Pharmacology, Istituto Mario Negri, Milano; Marco Giorgio, IFOM-IEO Campus, Milano. With Marco Giorgio we started several research programs and scientific protocols concerning the potential key-role of mitochondrial physiology and dysfunction as a marker of cardiotoxicity induced by anticancer old and new drugs, as monoclonal antibodies, antiangiogenic factors and novel tyrosine kinase inhibitors. A new avenue of research oriented to the study of endothelial modifications possibly related to cardiotoxicity related inflammatory proteins production enhanced by different chemotherapy agents has initiated in collaboration with Istituto Clinico Humanitas – Milan (Cecilia Garlanda, under the Scientific Direction of Prof . Alberto Mantovani).

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Cardiotoxicity Treatment: chemotherapy induced cardiotoxicity can result in a cardiomyopathy to be irreversible and leading to congestive heart failure and death; in our experience most patients receiving adequate treatment (including beta-blockers, ACEinhibitors, diuretics, anti-aldosterone agents) expressed relevant improvement in clinical status and cardiac function; the efficacy of ACE inhibitors and betablockers has been proven in the clinical setting of either high dose chemotherapy and conventional antiblastic treatments; Cooperative protocols were initiated in collaboration with Centro Cardiologico Monzino - Milan (Intensive Coronary Care – Dr. Giancarlo Marenzi) and San Raffaele Hospital – Milan (Hematology and Bone Marrow Transplantation Unit – Dr. Fabio Ciceri). Clinical impact of electrocardiographic QT measurements during chemotherapy: thanks to the interest of Novartis and the interest of the Medical Oncology Division (Dr Tommaso DePas) we started a research study to detect in large series of patients the potential modifications induced by different type of chemotherapy (particularly anthracyclines and alkylating agents) on basal ecg during the treatment; the results of this study will be ready by the end of 2012 and will consider not only QTC changes but also potential arrhythmogenicity of the eventual significant chemotherapy induced QTC changes, if present. In 2011: a) The International Cardioncology Society, that was founded in our Institute in January 2009, was invited to held the Annual International Congress in the headquarters of the FDA and might be useful to report here the press release of that paramount importance event: ICOS and CRSC Explore Cardiac Safety for Cancer Patients

Silver Spring, MD, October 12, 2011—The International Cardioncology Society (ICOS) and Cardiac Safety Research Consortium (CSRC) joined efforts last week at the Food and Drug Administration’s (FDA) White Oak Facilities as part of an annual meeting to explore ways to integrate cardiology and oncology research, focusing on cardiac safety for patients undergoing cancer treatment. “We’re pleased that the CSRC and the FDA cosponsored our annual meeting, because bringing so many world-class experts on cardiology and oncology together at the FDA Headquarters was important. Such collaborations reflect growing awareness of the significant advancements in cardiac safety for cancer patients. Ensuring patient safety is our primary goal, and exploring ongoing opportunities to work with other experts is paramount to achieving that goal,” stated ICOS Chairman (United States and Canada) Daniel L. Lenihan, MD. Dr. Lenihan is Professor of Medicine and Director of Cardiovascular Research at Vanderbilt University, Nashville, TN. When asked about the purpose of the ICOS and last week’s meetings, Dr. Carlo M. Cipolla—ICOS Chairman of Europe/Asia and Director of the Cardiology Division, European Institute of Oncology, Milan, Italy—noted that the organization’s research leads to greater understanding and protocols that minimize the risk of cardiovascular damage while maximizing survival chances for cancer patients. “Before we formed ICOS,” he continued, “cardiologists and oncologists simply did not communicate well. Fifteen years ago, there was no research in this area. So we were proud during this year’s meetings to watch ICOS members, such as my colleagues Drs. Daniela Cardinale and Maria Teresa Sandri, present exciting new scientific findings, which will soon be published in respected peer-reviewed journals.” Attendees showing support for interdisciplinary research included global experts ranging from oncologists,

cardiologists and clinical researchers to government entities such as the National Cancer Institute as well as numerous principals in the medical industry. Representatives from Abbott, Amgen, AstraZeneca, Cordis, Eli Lilly, GE Healthcare, Medtronic, Merck, Sanofi-Aventis, Siemens Healthcare and Theravance were among the many industry leaders at the meeting. Attendees benefited from sessions delivered and moderated by distinguished experts such as Joseph Carver, MD, Chief of Staff, Abramson Cancer Center, University of Pennsylvania, Philadelphia and Michael J. Fisch, MD, MPH, Chair, Department of General Oncology, The University of Texas MD Anderson Cancer Center, Houston. Sessions covered everything from how cardiotoxicity impacts clinicians and patients to the future of Cardioncology research. Jack Lewin, MD, President of the American College of Cardiology (ACC) drew particular interest when discussing his organization’s priorities including International Cardioncology Congress in Bethesda at the Headquarters of the Food and Drugs Administration. b) the Division successfully organized the sixth cycle of the “Incontri di Cardiologia Clinica”, 16 monographic workshops with invited guests discussing specific cardiological or cardio-oncological issues. c) we continue and increased the activity of the first Italian “Unità di Cardioncologia” (Doctor Daniela Cardinale) that treated more than 150 cases of all the comprehensive cardiologic and oncologic co morbidities aspects, statistically increasing in the overall cancer patients population; d) we gave a definite and stable structure and activity to the “ IEO Centro Antifumo” (Doctor Giuseppina Lamantia) as a new antismoking activity strongly integrated to the clinical and research activities of the Cosmos protocol for the early diagnosis of lung cancer in smokers (Doctor Giulia Veronesi).

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Medical Area The clinical and scientific rationale of ICOS is due to the fact that an increasing number of patients is presenting oncologic and cardiologic co morbidities, this because of an increasing number of long-term cancer survivors, the aging of the population, as well as the increased incidence and prevalence of oncologic and cardiologic co morbidities. Accordingly, in order to provide the optimal treatment in every situation, there is a fast growing need for comprehensive and proficient management of patients who host the two co morbidities and for those cancer patients condemned to be located in a scale of higher risks in developing cardiovascular problems because of their clinical history and oncologic treatment. A multidisciplinary approach to avoid the possibility that the development of a second disease leads to a reduction of therapeutic opportunities for cancer patients has become a major clinical goal. Cardioncology, a new medical issue aims at investigating: New strategies for early, pre-clinical diagnosis of chemotherapy related cardiotoxicity Evidence-based indications for cardiovascular follow up of different cancer treatments with “old” and “new” pharmacological drugs Risk stratification and prevention of cardiac effects of cancer multiple, combined and sequential therapeutic strategies Interdisciplinary expertise needed to face combined cardiac and oncologic co morbidities All aspects concerning the concomitant presence of primary or secondary heart (myocardial and pericardial) involvement. Therefore the International Cardioncology Society promotes training and studies in the fields of: Cardiological and oncological co morbidities Cardiological implications of oncological treatments as: Chemotherapy, medical treatments (such as the use of monoclonal antibodies and target therapies) Radiotherapy

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Immunoradiotherapy Locoregional treatments High-dose chemotherapy Multiple, combined and sequential cancer treatments; It promotes basic biology research on anticancer drugs mechanisms of cardiotoxicity focusing on inflammatory and metabolic pathways involved in heart damage Primary heart tumors Metastatic heart tumors Pericardial diseases and intrapericardial chemotherapy for pericardial effusion and more in general all the potential relations between cancer and cardiovascular diseases. The educational purposes of the Association are: To create and promote communication and cooperation between cardiologists and oncologists To create skilled consultants for the evaluation and management of cardiovascular problems in cancer patients To increase awareness of cardiologists and oncologists committees towards the management of cardiotoxicity issue. To promote basic clinical research in the field of cardiac and vascular/endothelial implications caused by oncological treatments. To develop guidelines, which are lacking at the moment, for cardiac monitoring of cancer treatments. There is a need to create and train a new generation of basic scientists and clinicians in the field of Cardioncology, who could build up a “medical synthesis” and a “common sense” and would be able to treat and prevent comorbidities, as well as to remove obstacles from the way to translational medicine.

The 2011 Cardioncology goals How can we further achieve integration between cardiology and oncology with the best practice of medicine?

1) Define cardiovascular risk assessment at the beginning of research protocols: there is burgeoning evidence that cardiovascular risk factors may have a substantial impact on the cardiac toxicity of cancer chemotherapeutic agents and the preventive treatment of CV-related co morbidity conditions can have a significant benefit on all-cause mortality and cardiac-related outcomes; because many easily modifiable CV risk factors are not routinely measured in oncology treatment trials, these efforts must improve and be followed over the course of any carefully done clinical trial. 2) Integrate necessary sophisticated cardiac testing during oncology research into reported findings: improving the standard of cardiology-based data critical to the conduct of a clinical study is essential. 3) Explore optimal screening techniques being mindful of practical issues (such as cost, feasibility, and effect on cancer or cardiacrelated outcomes): screening for undiagnosed CV disease can be an extensive process and has to be viewed in the context of cancer treatment and the underlying cancer diagnosis; screening for asymptomatic cardiac disease in patients with previous cardiotoxic therapy, including mediastinal radiation, requires the cardiologists to have knowledge of previous cancer therapy and its potential impact on the heart; thus, international collaboration to help define these CV risks in cancer patients and report evidence-based screening strategies is needed. 4) Standardize definitions for cardiac-related events during chemotherapy or other cancer therapeutics in a clinically relevant manner: a standardized definition of cardiotoxicity is essential for the conduct of meaningful research; there have been a variety of definitions of “cardiac toxicity” over

the past 3 decades of oncology trials and the majority, if not all of them, are based largely on the measurements of left ventricular ejection fraction. Furthermore, different thresholds for the determination of cardiac toxicity have been used, which makes it difficult to establish the true incidence of “cardiac toxicity” over time; to eliminate such difficulties a common definition needs to be established and one that incorporates clinical findings as well as echocardiographic and laboratory parameters and test into the determination is strongly necessary. 5) Contribute to the creation of an international database with cardiac end points in cancer patients to enhance the potency and relevance of post marketing surveillance. 6) Promoting a stronger prevention of heart failure and other major cardiac events by active cardiology clinical involvement in high risk scenarios: prevention and early detection are principles of cancer therapy that directly apply to the management of cardiac disease encountered in cancer patients: proactive involvement by cardiology in the management of patients with cancer can be critically important; examples of this vital integration includes the management of hypertension during anti-VEGF or antiangiogenic therapy and the detection and treatment of serious arrhythmias in high-risk populations such as those with amyloidosis undergoing stem cell therapy. 7) Active management of preexisting cardiac disease by a cardiologist can promote the most effective cancer therapy: patients who develop major cardiac issues during cancer therapy can be managed and still continue ongoing chemotherapy with appropriate monitoring and treatment. 8) Understanding expected clinical outcomes for each discipline to have shared decision making is crucial: effective communication between the

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Medical Area disciplines of cardiology and oncology in the management of patients can have a profound effect but may not be done consistently; focused education about the overlapping clinical issues - related to patients concomitantly affected by oncologic and cardiac problems and - related to cardiological potential consequences of all cancer therapies are essential steps toward effective communication; in this manner International Cardioncology Society (ICOS) will commit substantial effort to medical school, residency, and fellowship training programs to incorporate these topics in the curriculum. All the element described represent a big challenge and stimulating incentive for both the cardiologist and the oncologist, and the Cardiology Unit of the European Institute of Oncology and the International Cardioncology Society are strongly committed to these crucial clinical and scientific issues. Special acknowledgements to: - Doctor Franco Del Curto, the Lawyer of ICOS, for his friendly and strong support to all legal and ethical aspects of the ICOS activities; - Mrs. Anna Eggertz, ICOS Website Coordinator, for her continuous and professional presence in the development of the website; - Doctor Flavio Pirola, ICOS Treasurer, for his careful activity as a business consultant.

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Medical Area

Department of Medicine

Division of Clinical Haemato-Oncology Giovanni MARTINELLI, MD Director

STAFF Director of Clinical Haemato-Oncology Stem Cell Unit: Daniele Laszlo, MD Director of Transplantation Unit: Rocco Pastano, MD Deputy Directors: Alberto Agazzi, MD Senior Assistant: Anna Vanazzi, MD Assistants: Giovanna Andreola, MD, Paola Bertazzoni, MD, Angelo Gardellini, MD, Federica Gigli, MD, Simona Sammassimo, MD Fellows: Laura Cannella MD, Periana Minga MD Data Managers: Liliana Calabrese, Mara Negri Data Manager Fellow: Ambra Malerba Data Entry: Maria Teresa Lionetti Secretaries: Daniela Antoniotti, Tiziana Masala Head Nurse: Laura Orlando Scientific Nurses: Cristina Grossi, Sarah Liptrott

Activities 2011.

Our mainstream is treatment of leukemia, lymphoma, Hodgkin’s disease, multiple myeloma and improvement of the quality of life of patients and their families. In the past, few people affected by haematological diseases survived. Now, thanks to advances in treatments, more than 70 % of patients are successfully cured. Thus, a new challenge arises: the study of the long-term effects of treatments. Our Haematoncology Scientific Committee / Task Force Haemato-Oncology continued to actively meet every month. Multidisciplinary discussion with different specialists including radiologists, pathologists, nuclear medicine doctors, radiation therapists, psychologists, laboratory doctors and nursing staff added new insight to the clinical case management with a dedicated path for patients eligible for clinical trials. Particular attention was given to improve patients quality of life. Within the Division of Clinical Haemato-Oncology, the Allogeneic Transplant Unit performs allogeneic transplants with reduced conditioning or myeloablative regimens, from sibling and unrelated donors, principally for patients with haematologic malignancies. In 2011, 15 allogeneic transplantations and 58 autologous transplants were performed. In order to improve clinical results and reduce acute toxicities, including Graft Vs Host disease, the use of TLI / ATG in malignant haematological diseases was implemented. This regimen, first developed at Stanford University, has the peculiarity to skew graft activity toward host tolerance, maintaining high anti tumour activity and reducing overall transplant related mortality. The same regimen has been proposed for “triple negative” refractory breast cancer, a difficult clinical situation with few treatment alternatives.

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We activated a study to determine if engraftment can be achieved safely in patients with high-risk hematologic malignancies who undergo non-myeloablative BMT from HLA-haploidentical donors. The protocol includes the immunological monitoring of patients with haematological malignancies after allogeneic transplant. The Haemostasis-Clotting Outpatient Unit provides assistance and guidance in solving problems concerning haemostasis and thrombosis in oncology patients at IEO. We are coordinating a research team to study the crossthrombogenicity induced by tumors, chemotherapy used and by hormone therapy, and due to new molecular

medicines (antiangiogenic molecules, monoclonal antibodies), focusing on mechanisms of endothelial damage In collaboration with SAKK we have been investigating the prognostic role of PET in patients affected by Diffuse Large B cell lymphoma. In particular we evaluated if an early positive PET after 2 cycles of dose dense chemoimmunotherapy (R-CHOP 14) can be used to identify a group of patients having a poorer prognosis. The enrolment is completed and we are processing results. Our team is currently working with immunotherapy

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Medical Area protocols in non-Hodgkin lymphomas (NHL). Clinical results showed that the combination of immunotherapy with conventional chemotherapy led to improvements in response rates in NHL. Radio-labelled immunotherapy, which combines the benefits of monoclonal antibody targeting with therapeutic doses of radiation is a promising step forward in the treatment of malignant lymphomas. We concluded the experience of such treatment in marginal extranodal malignant Lymphoma relapsing or resistant to conventional treatment. During 2011 the Hematoncology Division continued its collaboration with different scientific groups. With Southern Europe New Drug Office (SENDO) we took part in the phase I clinical trial, now concluded. The principal objective of this study was to determine the maximum tolerated dose (MTD) and treatment regimen of a new drug (CEP-18770). In this study we enrolled six patients affected by MM. With the International Extra nodal Lymphoma Study Group (IELSG) we have continued the enrolment of patients in already ongoing studies and we have collaborated in elaboration of new projects concerning patients with extra nodal genital lymphoma and extra nodal breast lymphoma.

labelling cellular therapy products. Since 2010 we have been performing extracorporeal photopheresis (ECP) to treat patients with acute/chronic GVHD or cutaneous lymphomas. Early introduced in the treatment of steroid-refractory acute and chronic GvHD, ECP is a well tolerated procedure, with very lowincidence of side-effects that often allows more rapid reduction of concomitant immunosuppressive therapy. In 2011 we have performed 55 ECP procedures in 4 patients affected by acute (1) and chronic GvHD (3). In February 2012 a Gruppo Italiano Infermieri in Mobilizzazione ed Apheresi (GIIMA) group has been formalized with the aim to promote a link between Transplant and Collection Units (www.giima.eu). Communities benefit from a spirit of collaboration and co-operation between local Governments and health institutions. With this in mind, our research group was invited as a member of REL, a network established by the Lombardy Region for optimizing assistance and cure for patients affected by haematological disease. REL also defines criteria for the accreditation of transplant centres operating at regional level.

Productivity Analysis 2009 - 2011 Activity Patients undergoing leukapheresis Total number of procedures performed Donors undergoing leukapheresis Total numbers of procedures performed in donors Total numbers of lymphocytoapheresis procedures performed in donors Total numbers of leukapheresis or lymphocytoapheresis procedures Total number of extracorporeal photopheresis procedures Total number of procedures (LAF + ECP)

2009

2010

2011

40 61* 11 23 2 86 -- 86

52 67 11 18 0 85 50 135

39 51** 11 16 3 70 55 125

* 82,5% of patients collected in only one procedure ** 96% of patients collected in only one procedure

In 2011, our team produced 22 articles published on scientific journals with an overall impact factor of 87 points.

Regarding trials with IEO as sponsor we have continued the enrolment of patients in our first line treatment studies designed for naïve patients with Hodgkin’s Lymphoma or Multiple Myeloma reaching a sample size of 72 and 37 patients respectively. At the Division of Clinical Hematoncology, a JACIE accredited unit Center has been set up for collecting stem cells from peripheral blood. All procedures are managed by physicians and nurses specifically trained in allogeneic and autologous donor evaluation, stem cell and lymphocyte collection by apheresis, coding and

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Medical Area

Division of Radiotherapy Roberto ORECCHIA, MD Director

STAFF Senior Deputy Director: Barbara Alicja Jereczek, MD PhD Deputy Directors: Maria Cristina Leonardi, MD, Andrea Vavassori, MD Senior Assistants: Piero Fossati, Ing MD, Roberta Lazzari, MD, Anna Morra, MD Assistants: Daniela Alterio, MD, Agnese Cecconi MD, PhD, Annamaria Ferrari, MD, Gaia Piperno, MD, Dario Zerini, MD Fellows: Veronica Dell’Acqua, MD, Delia Ciardo Ing. (Bioengineer), Federica Gherardi, MD Residents: Federica Bazzani, MD, Maria Bonora, MD, Isa Bossi Zanetti, MD, Mariangela Caputo, MD, Sara Colangione, MD, Michela Dispinzieri, MD, Silvia Ferrario, MD, Marianna Gerardi, MD, Andrea Maucieri, MD, Roberta Mauro, MD, Alessia Surgo, MD Data Manager: Cristiana Fodor, MSc. MEDICAL Senior Deputy Director: Federica Cattani, MSc PHYSICS Senior Assistants: Raffaella Cambria, MSc, Cristina Garibaldi, MSc, Elena Rondi, MSc, Sabrina Vigorito, MSc Assistants: Stefania Comi, MSc, Rosa Luraschi, MSc Fellow: Floriana Pansini MSc Residents: Alessia Bazani MSc, Edoardo Mastella MSc, Stefania Russo MSc TECHNICIANS (RADIOTHERAPY TECHNOLOGISTS, RTT)

Chief Technician: Massimo Sarra Fiore Technician referent for Brachytherapy Unit: Andrea Guido Technicians: Francesca Baldini, Jennifer Bona Rivas, Enrica Borghetti, Manuela Cannella, Lucia Casanova, Saudia Castagna, Fabiana Castelluccia, Fabio Castellini, Roberto Corea, Guglielmo Gatto, Saverio Greco, Verlie Ann Jones, Olena Kuts, Assuntina Leppa, Francesca Picca, Claudio Pobbiati, Alberto Rampinelli, Daniela Rozza, Elena Strata, Andrea Vaccari, Ilona Vecchio

SECRETARIES Secretary: Mara Crimaldi, Ida Muraca Secretary, Reception: Nadia Zanoni

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Activities 2011. The Division of Radiotherapy

called since the beginning of 2012 Advanced Radiotherapy Center (ARC) is an university department with around 65 employees including a staff of 11 radiation oncologists, 8 physicists and 1 bioengineer committed to the quality care delivery enhanced by research activities and resident and student education. The Division has the convention with the Faculty of Medicine of the University of Milan for postgraduate teaching in radiation oncology. The Division has the latest equipment available for the high-precision radiotherapy like Intensity Modulated Radiotherapy (IMRT, including dynamic arc IMRT using RapidArc technology), Image-Guided Radiotherapy (IGRT), respiratory gating, intraand extracranial stereotactic radiotherapy and 3-D conformal radiotherapy. There are 4 treatment planning systems (with image fusion modality), 2 computer tomography units and 6 linear accelerators for external beam radiotherapy (including RapidARc and 3 accelerators installed at the beginning of 2012: Vero system, Tomotherapy and CyberKnife). Two mobile linear accelerators are installed in the operating theatres for the intraoperative electron beam radiotherapy (IORT). Each external beam linear accelerator is equipped with one in-room Image-Guided Radiotherapy system (based on computer tomography and/or X-ray system combined with a robotic 6-degrees of freedom treatment couch, respectively). The Record & Verify system, connecting all treatment planning systems to the linear accelerators, ensures an automatic, fast and safe treatment delivery. High precision radiotherapy allows for excellent tumor targeting and maximum sparing of normal tissue. In consequence, several clinical protocols with dose escalation and accelerated hypofractionated schedules (higher dose per fraction, leading to the reduction of the overall treatment time) have been activated. In particular, the FAST project (Frazionamenti Accelerati

dello Schema Terapeutico, i.e. Accelerated Fractionation of the Therapeutic Schedule) has been applied to the breast and prostate cancer. Apart from the hypothetical radiobiological advantages, FAST might significantly increase the patient convenience. Molecular Imaging Unit is involved in the research protocols on better definition of biological target volume, allowing for further improvement of radiotherapy precision. Brachytherapy Unit is a full-profile unit equipped with both low-, pulsed- and high dose rate systems. The unit is committed to the integrated approach in the field of radiotherapy. Several clinical protocols are active with brachytherapy as a boost or exclusive treatment.

In particular, prostate cancer patients are treated with exclusive interstitial implant or high dose brachytherapy combined with external beam irradiation, depending on the risk factors. Dedicated planning systems and treatment units allow for image-guided high-precision brachytherapy application. There is an excellent collaboration with other departments within the frame of the disease-specific multidisciplinary teams (see Clinical Research and Management Section). Such multidisciplinary approach allows in many clinical situations for less invasive therapy (organ preservation). Apart from high precision treatment and multidisciplinary

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Medical Area approach, a lot of attention is given to the patient’s quality of life issues. An innovative approach to the advanced disease patients has been created (ARPAR, Ambulatorio di Radioterapia Palliativa Rapida, Out-patient Clinic for Fast Palliative Radiotherapy). The patient is examined by radiation oncologist within 96 hours from the request and, if indicated, the treatment is initiated within 10 days. The Department has access to 10 Day Hospital Beds dedicated to the patients that require intensive supportive care during radiotherapy. The Department collaborates with the National Centre for Oncological Hadrontherapy (CNAO) in Pavia for the definition of the clinical research protocols on the particle therapy in selected cancer patients. There is also an active collaboration with the Department of Experimental Oncology, IEO investigating radiosensitivity of breast cancer stem cells and with Politecnico of Milan working on the bioengineering aspect of high-precision radiotherapy. The Department is involved in several European Projects on improvement in radiotherapy for cancer patients. During 2011 more than 3000 new patients were treated in our Department. The highest proportion of patients has been treated for breast cancer (50%) followed by metastastic disease (16%) and prostate cancer (8%). More than 2100 patients received external beam irradiation, 786 patients - IORT (mainly for breast cancer) and the rest-brachytherapy. Educational activities of the Division include indepartment teaching for pre-and postgraduate medicine, physics and biotechnology students and radiology&radiotherapy technicians (University of Milan and Politecnico of Milan). External educational activities include teaching within the programs of the European School of Oncology (ESO), the European Society for Therapeutic Radiation Oncology (ESTRO), the American Society of Therapeutic Radiation Oncology (ASTRO), the Italian Association of Radiation Oncology (AIRO), and

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the PTCOG (Proton Therapy Co-operative Group) and other national and international societies. Each year the Division hosts numerous visitors from radiotherapy and oncology centers from all over the world (including the fellowship program of the European Agency for Atomic Energy). The Division participates in the establishment of numerous national and international guidelines on cancer treatment. In research activities of the Division the emphasis is placed on breast cancer, prostate tumors and head and neck malignancies. The main aspects include new fractionation protocols, treatment accuracy, side effects and patient’s quality of life, combined modality approach (including organ preservation studies) and new prognostic and predictive factors. Every year the Division publishes about 30 full papers with an overall Impact Factor of about 150.

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Giovanni Paganelli, MD Director

STAFF Radiochemistry Unit Director: Marco Chinol, PhD Radiochemist Assistant: Stefano Papi Fellows: Angela Carollo, Demetrio Familiari, MD, Lucia Garaboldi, Ivan Santi, MD Deputy Directors: Lisa Bodei, MD, PhD, Chiara Maria Grana, MD, Giuseppe Trifirò, MD Senior Assistant: Laura Travaini, MD Assistants: Silvia Melania Baio, MD, Marzia Colandrea, MD, Silvia Lidia Fracassi, MD, Laura Gilardi, MD, Paola Anna Rocca, MD, Stefano Vassallo, MD Data Manager: Giorgia De Luca Chief Technician: Maurizio Fiorenza Technicians: Michele Calabrese, Alfio Severino Cascio, Sebastiano Croce, Riccardo Mei, Domenico Militano, Daniele Paolucci, Gennaro Prisco, Andrea Vertua Scientific Secretary: Deborah Console Secretaries: Valeria Archinti, Laura Brambilla, Karina De Giovanni, Anna Lucia Tusini, Maria Ersilia Viscusi Head Nurse: Gianni Bufi Medical Physicians to Nuclear Medicine: Amalia Di Dia, Francesca Botta, Marta Cremonesi, Mahila Ferrari, Francesco Guerriero

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Medical Area

Division of Nuclear Medicine Concetta De Cicco, MD Co-Director

Activities 2011.

The Division of Nuclear Medicine is devoted to the early localisation and treatment of tumours, by means of functional imaging and targeted radionuclide therapy. The Division offers traditional nuclear medicine techniques as well as new diagnostic exams, such as sentinel node lymphoscintigraphy, radioguided occult lesion localisation (ROLL), peptide-guided whole body scans, and PET scans. 18FDG-PET scans are routinely performed in the diagnosis, staging and follow up of various types of cancer. Since July 2008 PET/TC scans with 68Ga-octreotide are performed. The Division is one of the few in Europe having protocols of radionuclide therapy of solid tumours and lymphomas with new radiolabelled molecules that show high affinity for tumour cells, such as monoclonal antibodies, avidinbiotin and radiolabelled peptides, both as systemic and loco-regional approaches. The Nuclear Medicine Division has a special hospitalisation section with 9 beds in rooms set aside for radionuclide therapy. The Division possesses two PET/CT scanners, one double-head gamma-camera and one single head gamma-camera. Moreover, the Division is fully equipped for the synthesis of 18FDG and 68Ga-peptides and their quality controls and possesses three hot labs for the preparation of radiopharmaceuticals with different types of emission (gamma, beta+ and beta-), both for diagnosis and therapy. In 2011, 11308 diagnostic studies were performed, of which 4368 were PET/CT with 18FDG and 476 with 68 Ga-octreotide, and over 888 outpatients were visited. Radionuclide therapies with Yttrium-90 and Lutetium-177 labelled peptides are carried out every week, both as systemic or loco-regional treatments. During 2011, the Division published 21 articles on peer-reviewed journals, with an overall 98.515 Impact Factor. The Division has an agreement with various Universities in Italy, for the educational activity in the

School of Specialisation in Nuclear Medicine, to spread new therapeutic modalities in the field of Radionuclide Therapy. Nuclear Medicine Division is hosting many young fellows from different countries, for pre-clinical and clinical research activities, to foster future collaborations.

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Marco VENTURINO, MD Director

staff Senior Deputy Directors: Giovanni Francesco Manfredi, MD, Marco Tullii, MD Deputy Directors: Anna Attanasio, MD, Marilia Bedoni, MD, Roberto Capucci, MD, Laura Della Grazia, MD, Francesca De Lucia, MD, Rita Panzeri, MD, Daniele Sances, MD Assistants: Michela Acciaro, MD, Alessandro Acerbi, MD, Pierantonio Beccalli, MD, Ferdinando Bellotti, MD, Marta Maria Bizzarri, MD, Daniele Boninsegna, MD, Gianluca Castellani MD, Raffaella Collini, MD, Lorenzo D’Acquisto, MD, Davide Galli, MD, Antonio Pinna, MD, Tiziana Quattrocchi, MD, Gianluca Spano, MD, Donatella Sparicio, MD, Maria Paola Solinas, MD, Stefano Tredici, MD, Dario Vezzoli, MD Secretary: Maria Rosa Brambilla Operating Theatre Head Nurse: Laura Ogliari Intensive Care Unit Head Nurse: Cristina Simone

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Medical Area

Division of Anaesthesiology and Intensive Care Antonella TOSONI, MD Co-Director

Activities 2011.

In 2011 surgical activity at IEO has performed 12.884 operations. According to different kind of surgery, techniques of either general or locoregional anaesthesia are used. A computerized monitoring system collects data from all devices connected to patients and to records the anaesthetic procedures. An outpatient’s department is activated for the preoperative assessment of patients that need surgical operations: in 2010 more than 70% of the patients submitted to surgical interventions has been checked in this department. In 2010 two operating rooms have been activated for robotic surgery with special integrated equipment: in this year have been performed 716 robotic operations. At the same time three new operating rooms have been organized for day surgery. According to IEO project called pain-free hospital, specific attention is paid to treatment of postoperative pain. The anesthesiologists is also involved in the safety management of operating room and collects data about adverse events, in order to develop always safer protocols. In 2011 has been developed anaesthesiologist activity for HIFU procedures. The activity of the Intensive Care Unit is mainly devoted to post surgical patients. In the 2011 the ICU accepted about 600 patients. The ICU is equipped with eight beds provided with complete invasive and non-invasive monitoring and with ventilators able to support different modalities of invasive and non invasive ventilation. An isolated room is also available for patients affected by immunodeficiency. Specific beds are also available to avoid decubitus problems. In addition to usual invasive and non-invasive hemodynamic monitoring systems, new devices are available for monitoring hepatic and hemodynamic functions like PiCCO system and LiMON. Continuous hemodiafiltration is used as part of treatment of patients with acute renal failure, particularly in patients affected by sepsis. Clinical information about ICU patients is collected by a customized database.

Special attention and studies are payed about new development about the quality of life of ICU patients and their parents. The functional recovery of critical patients is seeked with the dedicated physiokinesitherapists’ collaboration. The anesthesiological staff is trained to perform Transesophageal Echocardiography both in operating theatre and ICU as well and the Division also organizes an annual teaching event about it. The staff is also trained to perform awake intubation with fiberoscopy and to safely manage difficult intubation with many different devices. In this year we have developed a special formation for medical staff about the vessels cannulation by echographic support. Anaesthesiologists also support invasive radiological and endoscopic procedures with Monitored Anaesthesia Care

or sedation techniques as well as regional or general anaesthesia if required. They also perform central venous catheterization for chemotherapy, plasmapheresis and total parenteral nutrition.

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Diagnosis and Prevention Area

Division of Cancer Prevention and Genetics Bernardo BONANNI, MD Director

STAFF Senior Deputy Director: Aliana Guerrieri-Gonzaga, MSc Senior Assistant: Davide Serrano, MD Senior Lab Assistant: Harriet Johansson, MSc Assistants: Monica Barile, MD, Massimiliano Cazzaniga, MD, Matteo Lazzeroni, MD Clinical Monitor: Clara Varricchio, MD Lab Assistants: Valentina Aristarco, MSc, Debora Macis, MSc, Antonella Puccio, MSc Genetic Counselor: Irene Feroce, RN MSc Data Managers: Giorgia Bollani, Chiara Montefrancesco, Serena Mora Research Nurse: Vittoria Arensi RN, Claudia Passoni RN Secretary Coordinator: Alessandra Rossi Secretary: Mariaelisa Ronzino Patient Manager: Angela Maniscalco Scientific Consultant: Andrea DeCensi, MD

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Activities 2011.

The Division of Cancer Prevention and Genetics is essentially dedicated to clinical research on the prevention of solid tumors and clinical management (risk assessment, surveillance and preventive treatment) of subjects at higher-than-average risk for various types of cancer. In order to develop new strategies of cancer prevention the Division’s multidisciplinary staff (including oncologists, geneticist, biologists, research nurses, data managers) is committed to conduct clinical trials with the main aim to validate various drugs, micronutrients, natural compounds as preventive agents. Most of the research efforts are currently focused on chemoprevention trials on breast, ovarian, colorectal, oral and lung cancer. The target population is heterogeneous but includes mainly two groups of at-risk subjects; 1) patients with (previously resected) precancerous conditions (such as breast ductal intraepithelial neoplasia, or colon adenoma); 2) healthy individuals who carry one or more risk factors (such as family history, germline mutations, high levels of androgens or estradiol or IGF-I, use of HRT, metabolic syndrome, insulin resistance, breast lobular intraepithelial neoplasia or athypical hyperplasia, high mammographic density, peripheral lung “ground glass opacities” etc). These at-risk subjects are selected, followed and preferably enrolled in chemoprevention trials. We have an established experience on various types of trials, including: a) phase II studies on surrogate endpoint biomarkers; b) larger phase III, multi-institutional trials on clinical endpoints (cancer incidence); c) pre-surgery, “window-of-opportunity” studies in patients candidate to surgical treatment for primary breast cancer in order to test the efficacy of new and “old” drugs on breast cancer cell proliferation (measured by Ki-67 on baseline biopsy and then on the specimen after 3-4 weeks of drug treatment), and other tissue and circulating biomarkers. Since phase III trials typically last several years before

providing results, we put much effort in the creation and conduction of phase II trials, studying how candidate biomarkers of risk (in different organs and in the blood) are modulated by preventative compounds. We utilize a large spectrum of potentially useful preventive agents, including SERM’s (Selective Estrogens Receptors Modulators), AIs (aromatase inhibitors), retinoids, NSAID’s (Non-steroidal anti-inflammatory drugs), corticosteroids, statins, metformin, Tk-inhibitors, preferably using their minimal active doses. Considerable effort is also being put in pharmacogenomics, studying the Cytochrome P450 enzymes, CYP2-D6 and CYP2-C19 polymorphisms in particular, in order

to stratify patients in different classes of tamoxifen metabolizers, with the ultimate goal of a more effective, less toxic, tailored prevention treatment. Moreover we are studying the polymorphisms of VDR and IGFBP3 (vitamin D receptor and Insulin like Growth Factor Binding Protein 3). Increasing research and clinical assistance have been recently dedicated in our Division to the selection, surveillance, risk-reduction strategies in subjects at very high risk, being carriers of constitutional germline mutations (BRCA1 and 2, MLH1, MLH2, MSH6, APC, MYH, TP53, CDKN2A and CDH1) in strict collaboration with the genetic lab at the IFOM-IEO Campus.

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Diagnosis and Prevention Area We have in fact an established High Risk Clinic (HRC) run by our staff and involving a multidisciplinary group of specialists (radiologists, pathologists, statisticians, endoscopists, surgeons, plastic surgeons and basic researchers). Our High Risk Clinic (HRC) provides to the public the possibility of cancer risk assessment, genetic counseling and testing, tailored surveillance and prevention programs, psychological and counseling support, nutritional and physical activity guidelines, access to chemoprevention trials or off-trial personalized treatment, up to prophylactic surgery in highly selected subjects. During 2011 we performed more than 2,500 visits in our prevention outpatients clinic. In our HRC service we evaluated 465 pedigrees, and we performed 308 first genetic counseling sessions. Among these subjects, 291 underwent a genetic test for BRCA1 and 2 mutations. We found: 50 BRCA1 mutations, 47 BRCA2, 123 wild-type and 36 true negative. One subject was found to be carrier for BRCA1 and 2 mutation. Other 28 subjects were tested for other genes, such as CDKN2A, CDH1 and for Lynch and Cowden syndromes. We coordinate various national research networks collaborating in multicentre phase III studies. We have also a long established research collaboration with international institutions, including: the Division of Cancer Prevention, US National Cancer Institute; the M.D. Anderson’s Cancer Center Consortium for Chemoprevention Trials; Cancer Research UK; the International Breast Cancer Study Group (IBCSG); the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA); the Consorzio per lo studio degli Alleli MMR e dei loro modificatori (CONSAMM); and the Department of Endocrinology, University of Bergen. In 2011, our Division published 26 articles in International peer reviewed journals with a total Impact Factor of over 94. The teaching activities included: Master in Senology, University of Milan; Master in Senology, University of Siena; Postgraduate Course in Pharmaco-oncology, University of Milan; Scuola Italiana Senologia (SIS).

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Diagnosis and Prevention Area

Division of Radiology Massimo BELLOMI, MD Director

STAFF Senior Deputy Directors: Elvio De Fiori, MD, Paolo Della Vigna, MD, Lorenzo Preda, MD, Gaetano Villa, MD Assistants: Giuseppina Calareso, MD, Luigi Funicelli, MD, Lorenzo Monfardini, MD, Giuseppe Petralia, MD, Cristiano Rampinelli, MD, Stefania Rizzo, MD, Fellows: Sarah Alessi, MD Residents: Salvatore Alessio Angileri, Luke Bonello, Sarah Brambilla, Giuseppe Di Pisa, Caterina Giannitto, Maria Carmela Grimaldi, Federica Lanfranchi, Alessandra Ferla Lodigiani, Roberto Lo Gullo, Sara Maccagnoni, Marina Maniglio, Stefano Meroni, Mauro Padrenostro, Francesca Priolo, Laura Rossi, Irina Sosnovskikh, Stefano Viotti, Vecchi Vittoria Research fellows: Lara Durli MD, Fontana Giulia, Paul Eugene Summers Ph.D, Director’s Secretary: Irene Cleopazzo Secretaries: Mariastella Bottalico, Barbara La Mantia, Manuela Lombardo, Paola Lonati, Sabrina Riboni Data Manager: Letizia Sirica Secretary, Clinical Studies: Giovanna Ciambrone Administrative Clerk: Anna Palmeri Chief Technician: Giuseppe Bardo Technicians: Emanuele Addonizio, Dario Ardizzone, Nicola Balestreri, Elena Bollini, Giuseppe Bonfitto, Valerio Cubadda, Roberto Di Filippi, Alessandro D’Incecco, Simone Fasulo, Roberto Labruna, Ferdinando Laserra, Manuela Martino, Valeria Obregon, Gabriella Perrone, Ubaldo Piccolo Longo, Filippo Raccosta, Cinzia Resta, Riccardo Simone, Emanuella Veneziano, Technicians Research Fellow: Gavarini Marco Students, School for Technicians: Alessandro Antinoro, Alessandro Asaro, Giuseppe Buonsanti, Michela Cerutti, Thomas Jamal Daouma, Fabio Di Bella, Salvatore Favara, Ginetta Fazzello, Andrea Malgrati, Luciano Costantino Pertosa, Salvatore Schifano, Liana Tina, Silvia Totaro, Fabio Vaccaro

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Activities 2011.

In 2011 the Department was divided into two and the Diagnostic Senology became an indipendent structure. In the year, 56.700 different diagnostic examinations and interventional procedures were performed, 12% of them in patients enrolled in clinical trials. The number of examination on “heavy” technology, as CT, MRI and Interventional radiology have increased by 9% compared to the previous year, mainly due to the optimization of patients’ workflow and of diagnostic protocols. Cytologic or histologic sampling guided by imaging is greatly increasing, both for diagnosis and for individualized therapy planning and more than 1.400 procedures were performed, mainly under real-time ultrasound control, but 300 of them by CT guidance. The clinical expertise is organ-oriented and different teams are devoted to delve into specific pathologies increasing their knowledge mainly by continuous comparison with the clinicians in multidisciplinary meetings: interventional radiology is committed to the Unit, directed by Dr Franco Orsi, and we have structured teams for head and neck (Dr Preda and Dr De Fiori), Chest (Dr Rampinelli), Liver (Dr Villa and Dr Della Vigna), GI tract (Dr Funicelli), female pelvis (Dr Rizzo and Dr Calareso), while the research team is driven by Dr Petralia. In 2011 we modified the process of reporting and delivering to the patients the results of diagnostic exams by implementing a voice-recognition system allowing the Radiologist to directly dictate the report and introducing a novel office devoted to post-examination patients’ relationship. Prof Bellomi and the senior members of the staff run the Early Diagnosis team (ADO: Anticipazione Diagnostica in Oncologia), who faces and discuss the proposal of institutional guidelines and research projects on the topic. In 2011 we approached a series of topics: the new lung cancer screening programs, that complements

low-dose CT with micro RNA analysis, individual risk evaluation and smoking cessation programs; the proposed implementation of regional colon cancer screening by CT-colonography and of breast cancer screening by Tomosynthesis; the ongoing and planned projects of pharmacoprevention and their possible links with the individual risk analysis and early diagnosis programs. The scientific activity ranges over a wide variety of topics, leading to publish 29 papers, 11 of which derived directly from radiological researches. The Imaging Task Force met regulary to discuss in multidisciplinary setting our research topics, to find common interest

with clinicians in new fields of Imaging and to connect with basic researchers to develop translational research projects. In 2011 we started a cooperation with biophysics, bioengineers and molecular researchers in studying the possible use of gold nanoparticles in MR cancer imaging. We discussed a new face on imaging cellularity by contrast-enhanced Ultrasound Diffusion and the possibility to start an experience on Coherencebased Contrast Ultrasound Diffusion Imaging for Prostate Cancer Detection. One of the resident of the departement started a new co-operation with a multicentric trials studying the new imaging techniques to be used for the screening and monitoring venous multiple sclerosis

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Diagnosis and Prevention Area and our involvement in eventual venous stenting. MRI is rapidly expanding his diagnostic potential due to advancement in technology and improvement by new softwares: his role in planning of intrafascial nerve sparing robotic radical prostatectomy gave unexpected good results in clinical practice. We guested Dr G.Placida, from Universià dell’Aquila, part of the leading team in new MR researches in Italy, who introduced the new studies on the imaging based on ions different from Hydrogen, and Dr L.Grazioli, from Brescia, who reported his wide experience in diagnosing liver adenomas and classifying them on the basis of MR images. Researches are focused on Imaging of neo-angiogenic activity in solid body tumours and the use of different techniques (CT, Ultrasound and MR) in monitoring the response to therapies, imaging of cellularity, based on Diffusion-weighted images by MRI (DW-MR) and early diagnosis of lung cancer in a CT screening setting. Our main contribution to advance in knowledge lied in these fields. We demonstrated a correlation between response of cervical tumours to non-surgical therapy (chemo- and/or radio-therapy) and apparent diffusion coefficient (ADC) values, a quantitative method to measure cellularity by MR, showing a higly significant increase in ADC values following therapy in responder patients, while there was no significant difference in ADC values at the staging MR examination between responders and non-responders, nor between adenocarcinomas and squamous cell carcinomas. MRI can be a valuable addition to the diagnostic work-up of a patient with a breast abnormality or biopsy-proven cancer and it is recommended by the American College of Radiology in selected cases for screening, assessing extent of disease and for additional evaluation of clinical and imaging findings. MRI is therefore increasingly used for breast imaging, and advanced imaging techniques have been proposed and increasingly used in the last few years. The term “advanced imaging” refers to innovative techniques one step beyond morphology,

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able to provide a better insight in tumour biology. These techniques are increasingly investigated in clinical trials, but are not yet used in clinical routine for breast MRI. We contribute to wide the use of these techniques which seem to be closest to clinical application, pubblishing the promising results showing how ADC value is able to predict residual disease after neoadjuvant chemotherapy in patients with locally advanced breast cancer with a high accuracy. From the data of our large series of lung CT examination performed in high risk subjects for early detection of lung cancer, we analysed the asymptomatic extrapulmonary malignancies, that were diagnosed with a frequency of 1 case per 200 individuals screened for lung cancer for five consecutive years and demonstrated that, at the time of diagnosis, most extrapulmonary malignancies detected were at a potentially curable stage and most patients are alive and disease free to date. Lymphomas and renal cancers are the most frequent extrapulmonary cancers detected by low-dose lung CT. Although long-term outcome of extrapulmonary malignancy diagnosed at lung cancer screening with low-dose CT is unknown, early detection of clinically unsuspected extrapulmonary cancers could be considered an additional clinical benefit. The Division is participating to more than 120 clinical trials conducted by clinical departments. The Division of Radiology is member of the European Institute of Biomedical Imaging Research, and strict co-operation, in research and medical education, is maintained with Insespital University of Berne, University of Sussex, the Royal Marsden Hospital in London and Massachusetts General Hospital in Boston. The Division is deeply involved in educational programmes, being part of the teaching activities of the School of Medicine, School of Radiographers and Post-graduate School of Radiology of the University of Milan. In 2010 we organized 7 residential courses, with a total of 108 ECM credits.

Image of breast carcinoma (ROI 1) by Dynamic Contrast Enhanced MRI: perfusioni s evaluated by pixel-by-pixel colour maps with each pixel representing values for microvessels permeability.

The same type of map in a case of fibroadenoma (ROI 2): the colors yellow and green mean a lower permeability, i.e. non malignant lesion.

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Diagnosis and Prevention Area

Breast Imaging Division Enrico CASSANO, MD Director

STAFF Senior Deputy Director: Maria Pizzamiglio, MD Deputy Directors: Francesca Abbate, MD, Anna Bozzini, MD, Brunella Di Nubila, MD, Antuono Latronico, MD, Lorenza Meneghetti, MD Assistants: Chiara Dellabianca, MD, Valeria Dominelli, MD, Serena Ganino, MD, Ourania Papadopoulou, MD, Silvia Penco, MD, Anna Rotili, MD, Chiara Trentin, MD Secretary: Paola Lonati Data Manager: Simona Menna, MSc

Activities 2011. Our primary aim is the early

diagnosis of breast carcinoma. Traditional instruments of detection and diagnosis, as digital mammography, ultrasound examination, stereotactic or ultrasound guided vacuum assisted biopsy and magnetic resonance, are supported and integrated, with new, but wellestablished instrumental technology, in accordance with clinical protocols. A great part of our activity is dedicated to train young or expert doctors, who are improving their knowledge and skills in breast cancer diagnosis by working side by side with our expert radiologists, both on the

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clinical and scientific activities. Breast radiology unit can be proud of many collaborations with clinical and research institutions. Nationally in 2011 we started a work of accreditation and quality control of structures that deal with breast radiology. The division clinical research activity was focused on optimization of traditional breast imaging techniques and setting of innovative procedures. Because of the increasing number of pregnant patients with oncologic disease in IEO, after a careful revision of the literature, we found the need to deepen in diagnostic findings related to pregnancy and breast feeding. Our radiologists staff together with oncologist, senologist and pathologist colleagues constituted a clinic and study group about senology integrated in pregnancy and breast-feeding called SIGA, targeted to analyze clinical and imaging signs and symptoms of breast cancer found during these conditions. The results of our efforts contributed to write the latest edition of FONCaM guidelines. In deepen in of standard procedures study we analysed the data collected about imaging-guided percutaneous biopsy (VABB). VABB allows a high diagnostic accuracy with less invasivity and costs reduction if compared to surgical biopsy, with a slight emotional and aesthetic impact for patients. We published some articles about VABB stereotactic and ultrasound guided procedure highlighting the usefulness of this diagnostic tool. The data about stereotactic VABB indicates also that it may not be considered as a therapeutic procedure, even in the case of complete removal of microcalcifications. However, a complete removal of microcalcifications may result in low rates of underestimation of malignancy and may consequently increase the diagnostic accuracy of the diagnostic procedure. Ultrasound-guided VABB can provide accurate characterization of breast lesions especially classified C1 or C3 on the basis of ultrasound-guided FNAC. The low rate of malignancy observed in our data indicates that

surgical excisional biopsy can probably be avoided in most cases. It is important to recall that VABB findings should be evaluated in light of clinical data and patient history and imaging findings to plan the adequate therapeutic approach. Our unit is collaborating with Politecnico of Milan conducting a clinical trial about Optical Mammography, time-resolved at seven wavelengths (635 to 1060 nm). The aim of this study is to evaluate and define the efficacy of this new device in the non-invasive assessment of breast density as breast cancer risk factor and in the detection and characterization of breast lesion. This trial is actually ongoing and our centre recruited 135 patients.

The breast radiology unit is working with the IEO’s laboratory medicine department in a multicentre international clinical trial which aim is to study the efficacy of a blood test for cancer associated auto antibodies. The aim of this blood test is to validate a new tool available for a better diagnostic management of suspected subjects with breast pathology. 150 patients were enrolled in this trial, which data analysis is ongoing. We are involved in new clinical trials about digital mammography performed after contrast medium injection, we submitted to the attention of Ethical Committee a clinical trial protocol and we are looking for the authorizations to start this study.

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Diagnosis and Prevention Area

Division of Ginaecology

Unit of Preventive Gynaecology Mario SIDERI, MD Director

STAFF Senior Deputy Directors: Dorella Franchi, MD Deputy Directors: Luca Bazzurini, MD Assistants: Eleonora Preti, MD, Michele Peiretti, MD Consultants: Raffaella Di Pace, MD, Simona Moroni, MD, Sabina Oldani, MD, Mariarosa Pittelli, MD, Francesca Sanvito, MD, Laura Spinaci, MD, Noemi Spolti, MD, Aylin Vidal Urbinati, MD, Paola Zamperini, MD Fellows: Sarah Igidbashian, MD, Nadia Caroppo Venturini, MD Data Manager: Sara Boveri, DSc Secretaries: Anna Steinwurzel, Simona Tognetti

Activities 2011. The Unit of Preventive

Gynecology encompasses the fields of prevention, surveillance and diagnosis of gynecologic cancerous and precancerous lesions. The clinical activities involve 16 gynecologists in office and surgical activities exploiting about 4.500 gynecologic consultations, on annual basis. Clinical and research activities are embedded within the frame of the two gynaecologic centers of excellence, CCC and OCC. The Unit has a high experience in laser surgery for cervical, vaginal, and vulvar pre-cancerous and cancerous lesions with approximately 750 laser treatments on lower female genital tract diseases yearly. HPV test

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and HPV genotyping are used for the management, diagnosis and follow-up of cervical pre-cancers and cancers. P16 and other biomarkers are used in screening and triage of borderline lesions; 2.000 colposcopic exams are performed yearly. Primary prevention of HPV related pre-cancerous and cancerous lesions is the goal of our HPV vaccination centre, were both adolescents and women in older ages can receive adequate information on HPV vaccines and all the vaccine doses; during 2011 more than 500. HPV vaccination were performed. A prospective study on HPV vaccination in 18 years old girls is ongoing. The Unit has a central role in the HPV task force, a multidisciplinary group involved in the study of HPV-related genital and non genital cancers. Moreover the Unit is involved in the early detection of ovarian cancer as in general population as in high risk of patients with BRCA1/2 mutation or history of previous breast cancer: during 2011 more than 6.000 transvaginal or transabdominal pelvic US have been performed by highly experienced specialists. Eighty laparoscopies has been performed for prophylactic oophorectomy or suspect ovarian cysts to reduce the risk of ovarian neoplasms. New bio-markers for the early diagnosis of ovarian cancer (HE4 and proteomics) are being evaluated in a prospective ongoing study. Furthermore we are planning a multiinstitutional trial with the aim to evaluate novel tumor markers panel (CA 125; IGF-II; MIF; Leptin; Osteopontin; Prolactin) to diagnose occult ovarian and tubal cancers in BRCA1 and BRCA 2 mutated women undergoing bilateral prophylactic salpingooophorectomy. The Unit is also collaborating with the department of molecular medicine to determine the sensibility and specificity of serum miRNAs as new biomarkers for early diagnosis of ovarian cancer, and in the research of ovarian surface tumor stem cell. The Unit is involved in a multicentric international studies in the ultrasonograpic discrimination of pelvic

masses (IOTA II and III studies) and also to develop new recommendations for standardized measurement technique for both endometrial thickness and uterine cavity lesions (IETA study) Our team is also dedicated to the diagnosis, management, and follow-up of endometrial abnormalities and early endometrial cancer through transvaginal US and hysteroscopy. More than 300 diagnostic and operative hysteroscopies has been performed in 2011 for uterine polyps and fibroids. A particular interest is dedicated to the conservative treatment of early stage endometrial, cervical cancer and ovarian border-line tumors in young patients wishing to

preserve fertility. During 2011, the Unito of Preventive gynecology has submitted several abstracts for internationals meeting and it has published 13 research papers in indexed journals, with an overall Impact Factor of 37,63. The Unit has an active collaboration with the humanitarian project 4AWOMAN in Madagascar, in cervical cancer screening.

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Cristiano CROSTA, MD Director

STAFF Senior Assistant: Davide Ravizza, MD Assistants: Stefania de Lisi MD, Giuseppe de Roberto, MD, Cristina Trovato, MD Fellow Medical Research: Annalisa de Leone, MD Data Manager: Darina Tamayo, PharmD Secretaries: Paola Colli, Elena Degani Head Nurse: Fiorella Zoccatelli

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Diagnosis and Prevention Area

Division of Endoscopy Giancarla FIORI, MD Co-Director

Activities 2011.

The imperative target of the Division is the patient’s satisfaction through the adequate diagnostic-therapeutic pathway/s. The implementation of new diagnostic and therapeutic techniques is an important goal of ordinary clinical and research activity. Advanced techniques are assessed in our department that enable us to offer a minimal invasive treatment for oncologic patients. Over 12.000 endoscopic procedures including mucosectomy and submucosal dissection of early cancer and large adenomas, debulking and stenting of malignancies, treatment of bleeding lesions, endoscopic ultrasonography with fine needle aspiration and management of benign and malignant gastrointestinal diseases were performed during 2011. This Division is a leading center for Colorectal Cancer Screening Programme. Clinical, diagnostic and epidemiological studies are closely integrated with the clinical activity and are developed in collaboration with IEO Divisions and National and International HealthResearch Institutes. A multicentre study, randomised, observer-blind, parallel 2-arm group, comparative was carried out with the aim to assess the efficacy of reduced-volume bowel preparation taken the same day of the procedure in comparison with the standard split dosing. Nowadays, an international multicentre, randomised, double-blind is being developed with the aim to evaluate the sensitivity and specificity in the detection of intraepithelial neoplasia in patients with Ulcerative Colitis. Patient’s monitoring before, during and after endoscopic procedures, together with the reprocessing of endoscopes and endoscopic devices are imperative goals of the Division.

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Giuseppe VIALE, MD Director

STAFF Director, Cytopathology Unit: Chiara Casadio, MD Director, Histopathology and Molecular Diagnostics Unit: Massimo Barberis, MD, MIAC Deputy Directors: Patrizia Dell’Orto, DSc, Giancarlo Pruneri, MD Senior Assistants: Fausto Maffini, MD, Michela Manzotti, DSc, Angelica Sonzogni, MD Assistants: Luca Bottiglieri, MD, Elisa De Camilli, MD, Clementina Di Tonno, MD, Mauro G. Mastropasqua, MD, Eleonora Pisa, MD, Paola Possanzini, Paola Rafaniello Raviele, MD Research Assistants: Elvira Benini, DSc, Olivia Blasi, DSc, Silvestro Carinelli, MD, Caterina Fumagalli, DSc, Daniela Lepanto, MD, Oriana Pala, MD, Leila Russo, MD Data Managers: Stefania Andrighetto, DSc, Francesca Lombardi, DSc Chief Technician: Alessandra Cavallon Technicians: Agnese Baglivo, Enrica Bresaola, Giancarlo Camerino, Carla Camia, Laura Chiapparini, Francesca Ciocca, Gabriele Citelli, Gianluca De Marzo, Silvia Di Vincenzo, Flavia Giarratano, Paola Lento, Luana Lippolis, Mara Lusiardi, Francesca Palumbo, Chiara Scacchi, Konrad Vokrri, Mila Schiavi, Francesco Spinelli, Tania Tamagni, Marilisa Valente, Paolo Lopedote Biotechnologist: Simona Pessina, Viviana Stufano Research Biotechnologists: Chiara Zanetti Scientific Secretary: Luana Balestra Secretaries: Elena Calvi, Giuseppina Figini, Cristina Messina Office Administrators: Laura Arrigoni, Francesca Pellegrino,

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Diagnosis and Prevention Area

Division of Pathology Giuseppe RENNE, MD Co-Director

Activities 2011. The Division of Pathology includes

the Units of Histopathology and Molecular Diagnostics, of Cytopathology, and of Laboratory Haematology-Oncology. This report focuses on the activity of the first two Units, which has included 23,659 histological diagnoses and 15,132 cytologic diagnoses (6,541 fine needle aspiration biopsies or extravaginal exfoliation cytology samples and 8,591 Pap-tests), with 4,399 cases seen in consultation for a second opinion and 4,372 frozen section examinations for intraoperative diagnosis. Among the different tumor types, we have examined 3,627 breast samples, 2,012 biopsies of sentinel lymph nodes, 2,730 surgical specimens of gynaecological pathology, 1,437 specimens of thoracic pathology and 456 malignant melanomas. Besides a diagnostic laboratory supplied with the most updated equipments for histologic and cytologic investigations, the Division includes two functional sections of immunohistochemistry and molecular pathology supplied with automatized instruments that are able to offer extensive immunophenotyping and molecular characterization of normal and tumor tissues by using a large array of monoclonal and polyclonal antibodies, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR) techniques. More than 60,000 immunohistochemical reactions, 1,753 FISH assays, 6,198 PCR analyses and 10,040 direct sequencing have been routinely carried out in 2011 for tumor genophenotyping, including the immuno-histochemical evaluation of estrogen and progesterone receptors, HER-2 and EGFR expression in tumors for tailoring individual therapy; the characterization of malignancies from unknown primary sites; the assessment of gene amplification in carcinomas and gene translocation in malignant non-Hodgkin lymphomas and soft tissue tumors; and the mutational analysis assessment of several genes, including EGFR, K-ras, PDGFRA/B, c-kit, PI3KCA and B-raf, DNA mismatch repair and the methylation status of MGMT. The research activities during 2011 have mainly focused on the modelling of predictive factors for breast cancer,

addressing both the pathological complete remission and the long term survival in patients undergone neo-adjuvant chemotherapy, as well as the long term survival of patients treated in the adjuvant setting. In particular, the value of a model to predict the magnitude of benefit of adjuvant letrozole, as compared to tamoxifen, has been documented in more than 5,000 patients enrolled in the BIG1-98 clinical trial. Furthermore, genetic analyses of the polymorphisms of the CYP2D6 gene have been carried out to assess the response of the patients with endocrine responsive breast cancer treated with tamoxifen. The suitability of needle core biopsy for the diagnosis of malignant lymphomas has been established in a series of more than 400 cases. In primary pulmonary MALT lymphomas we have evaluated prevalence and clinical implications of re-arrangements of the MALT-1 gene.

These activities have required extensive immunophenotyping and molecular characterization of tumor tissues, using automatized immunostainers, PCR-, real time PCR-based and FISH techniques, tissue microarrays, and microdissection for tumor cell enrichment. The research activities of the Division, including the studies performed in collaboration with several Divisions of the European Institute of Oncology, as Experimental Oncology, Senology, Medical Oncology, Head&Neck Surgery, Gynaecology, Thoracic Surgery, and Chemoprevention, have resulted in 63 full articles published during 2011 in peer-reviewed international journals, with an overall IF of 414.198. (mean IF: 6.57). The Division hosts the Postgraduate Medical School in Pathology of the University of Milan.

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Diagnosis and Prevention Area

Division of Pathology

Unit of Histopathology and Molecular Diagnostics Massimo C.P. BARBERIS, MD Director

STAFF It overlaps functionally with that of the Division of Pathology and Laboratory Medicine.

Activities 2011. The 2011 clinical activity of this

Unit has regarded the consultation and revision duties on fellows, residents and staff pathologists working at the Division of Pathology and Laboratory Medicine. Particular attention has been pointed as internal referring pathologist for lung and mediastinal pathology and neuroendocrine tumors of the gastroenteropancreatic tract and lung. A new approach in the role of diagnostic assistance to patients underwenting to trans-bronchial-needle biopsies (TBNA and EBUS) has been defined in cohoperation with the Cytopathology Unit with undoubtful benefit for patients. More than 800 diagnostic procedures have been

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received and successfully treated. The Unit actually works as a full time molecular diagnostic lab for clinical decision making. The activity has been totally optimized with the validation of diagnostic protocols running on the genetic analyzer ABI 3500 Dx and on the 7900 HT fast real time PCR. During this year all the team worked on the same goal: to offer an integrated system of molecular diagnostics based on automatized platforms for immunohistochemistry, in situ hybridization, RT-PCR, qRT-PCR, Sequencing respecting standards, rules, approvals requested for clinical testing. All the reagents, disposables, instruments are validated for IVD according to the 98/78CE directive of the European Council and satisfying the requisites of CE label. Moreover the unit is engaged in developing a robust QC and QA program. In 2011 the number of patients screened for drugable tumors raised of 80%: biopsies or surgical specimens of 2000 patients have been evaluated for gene mutations. Moreover we tried to offer a wide test spectrum for targeted therapies. We report as an example the molecular testing suitable for lung cancer patients: EGFR, k-ras, N-RAS, B-RAF, PI3K mutations, EGFR amplification (FISH), EGFR expression, locus 2p23(ALK) rearrangement (FISH), c-met mutations, c-met amplification (FISH), ERCC1 expression, T.S. expression, PTEN expression, P70S6 kinase, pAKT, pm-TOR expression. Specific panels of molecular tests were prepared for colo-rectal cancer, malignant melanoma, soft tissue sarcomas and malignant lymphomas. During this year, a multi-institutional Italian project has been closed under the guidance of the Unit, the so-called INSPECTOR (Italian Network for the Study of Pulmonary Endocrine Cell TumORs) project that has been funded by an external company. This observational, retrospective, study involving 4 different Italian large hospitals, which raised surgically excised tumors dating back to 1987 and up to the end of December 2007 reached the following goals: full validation of the current WHO diagnostic criteria for neuroendocrine tumors of the lung and robust prognostic

parameters based on multivariate statistical analysis. A validation study on a specific gene signature in patients operated for stage 1 adenocarcinoma of the lung was performed in cohoperation with an American biotechfactory. The results will be presented at the ASCO meeting in Chicago. A large series of pulmonary adenocarcinomas arising in young people has been examined and the genetic markers of potential clinical interest have been reported. The study will be closed in the next weeks. A pilot study on Methylation of O (6)-methylguanine-DNA methyltransferase (MGMT) promoter gene in triplenegative breast cancer patients has been concluded; it

showed that in TNBC with wild-type BRCA1, the direct DNA repair system may be frequently silenced by MGMT methylation. The evaluation of the MGMT status could offer a new adjunct in predicting tumor response to alkylating drugs in these patients. Cohoperative studies on breast cancer and cancer prevention in women living in developing countries with poor income have been continued and the results published. The research activities of the Unit, including studies are performed in collaboration with the Divisions of Pathology and Laboratory Medicine, Thoracic Surgery, Experimental Oncology, Medical Oncology, and Chemoprevention.

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Diagnosis and Prevention Area

Division of Pathology

Unit of Diagnostic Cytology Chiara CASADIO, MD Director

STAFF Senior Cytotechnologist: Laura Chiapparini Cytotechnologists: Enrica Bresaola, Mara Lusiardi, Chiara Scacchi Technologists: Agnese Baglivo, Silvia Di Vincenzo, Giancarlo Scrimieri, Konrad Vokrri

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Activities 2011.

The Unit of Diagnostic Cytology performs cytologic diagnoses for both in-and out-patients. The total number of tests in 2011 was 15,132; 6,541 of them were fine needle aspiration or extravaginal exfoliative cytology samples and 8,591 were Pap tests (mainly liquid based samples). The four technologists are involved in the preparation of the slides and of the cell blocks while all the cytotechnologists perform the screening of the slides. Since March 2011 we started to support thoracic surgeons and endoscopists while performing fine needle aspiration (FNA) samples assessing their adequacy during the endoscopic procedures. Both the technologists and the cytotecnologists are involved in this field together with the pathologist/s intraoperative charged with the diagnosis. 38 adequacy procedures were performed with the digestive endoscopists, mainly on pancreatic lesions, while 264 adequacy procedures were performed with the thoracic surgeons, under fluoroscopy or ultrasound guide. Cytotechnologists are also encouraged to actively participate in updating courses. In the last year all of them prepared a poster which was presented at the European Congress of Cytology and a work which was accepted for oral presentation at the National Congress of Pathology. From October to December 2011, the Unit organized a theorical –practical course on “Mistakes in mammary cytology” which was accredited for continuing medical education. 450 FNA of palpable breast nodules and of superficial lymph nodes were performed by two cytopathologists, while a third cytopathologist performed about 50 ultra sound guided FNA of non palpable breast lesions. 60 FNA of superficial lymph nodes were completed with cell block preparations, stained with immunocyto-chemistry and used for driving therapy in breast cancer follow up. The daily internal quality control system, based on the review of 10% randomly selected cases according to a

computer-mediate selection, guarantees the reliability and accuracy of the test results. A computerized system online connects the department with the wards, so that the diagnoses are immediately available to the physician, just after the validation process. Moreover the Cytology Laboratory has implemented and maintains a quality management system, which fulfills the requirements of JCI. Besides diagnostic cytology, the Unit is involved in different research activities, including the studies performed in collaboration with the Laboratory Medicine Division on circulating tumor cells in breast cancer and with the Division of Chemoprevention on Breast Ductal Lavages (DL).

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Diagnosis and Prevention Area

Division of Pathology

Laboratory Unit of Clinical Haematology-Oncology Francesco BERTOLINI, MD, PhD Director

STAFF Senior Vice-Director: Patrizia Mancuso, Biol Sci D Vice-Directors: Chiara Corsini, Biol Sci D, Cristina Rabascio, Biol Sci D, Senior Assistants: Angelica Calleri, Biol Sci D, Ines Martin-Padura, Biol Sci D, Assistant: Giuliana Gregato, Biol Sci D Fellow: Paola Marighetti, Biotechnol Sci D, Stefania Orecchioni, Biol Sci D Technicians: Pierluigi Antoniotti, Cinzia Massaro, Jessica Quarna PhD Student (SEMM): Chiara Aldeni, Biotechnol Sci D Secretary: Patrizia Passeri

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Activities 2011.

The Laboratory, which is ISO9001, JACIE and JC Lab certified, has two main clinical activities, the diagnosis of haematological malignancies and stem cell processing for transplantation. The lab is now also offering cell sorting and purification for clinicians and scientist interested in translational research. More than 80 stem cell collections were processed in the past year for autologous or allogeneic use and more than 1,100 blood and marrow samples were studied by flow cytometry, PCR, immunohistochemistry, FISH and cytogenetics. The repository of plasma, serum and whole blood samples from leukaemia, lymphoma and myeloma patients includes nearly 7,000 frozen samples from untreated patients at first diagnosis and from patients longitudinally followed after remission or relapse. Trafficking and angiogenic potential of cancer, stem and endothelial cells are the main research interests of the laboratory. We have developed and validated at the preclinical and clinical level a number of surrogate assays of angiogenesis and anti-angiogenic drug activity that are currently used worldwide in many clinical trials where cancer patients are treated with anti-angiogenic therapies. These assays have been found to predict the clinical out-come of breast cancer patients treated with anti-angiogenic therapies (Bertolini et al, BBA Reviews in Cancer 2010) and to be of help to define the most active combination of anti-angiogenic drugs and cytotoxics (Shaked et al, cancer Cell, 2008; Mancuso et al, Clin Cancer Res 2009; Bertolini et al, Drug Discovery Today 2011). We are currently leading an international effort toward the standardization of the measurement of these surrogate markers. In collaboration with IEO Department of Experimental Oncology and IFOM we are investigating novel preclinical models of human haematological malignancies that are used to investigate new drugs and new therapeutic procedures. Along with Pfizer the Laboratory has recently described that targeting ALK1 kinase inhibits

angiogenesis and tumor growth through a mechanism of action complementary to anti-VEGF therapies (Hu-Lowe et al, Cancer Res 2011). Finally, we have described that progenitor cells from the adipose tissue may promote breast cancer growth and dissemination in several preclinical models (Martin-Padura et al, Cancer Res 2012).

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Division of Laboratory Medicine Maria Teresa SANDRI, MD Director

STAFF Deputy Directors: Rita Passerini, DSc, Laura Zorzino, DSc Assistants: Fabio Bottari, DSc, Cristina Cassatella, DSc, Annalisa Cattaneo, DSc, Donatella Gritti, DSc, Paola Lentati, DSc, Giovanna Randine, DSc, Michela Salvatici, DSc Chief Technician: Manuela Sesia Technicians: Giuseppina Facchi, Chiara Gulmini, Lorena Moretti, Adeline Ngounou Ngassa, Nicola Panarese, Marco Picozzi, Teresa Roth, Ermenenziana Soccio, Alessio Tricca, Valentina Urso Fellow: Francesco Cozzolino, DSc, Christian Mauro, DSc Technician Fellow: Matteo Compierchio Secretaries: Elena Campanato, Erika Platano

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Activities 2011.

The Division of Laboratory Medicine encompasses the fields of hematology, biochemistry, coagulation, tumor markers, infectious disease serology, drug monitoring and microbiology. It serves both in- and out-patients, and the total number of tests performed during the 2011 was about 960.000. Moreover, the Division organizes the supply of blood products through a dedicated team. Highly trained technologist perform the tests with automated analyzers, and expert personnel perform manual microscopic and analytical procedures necessary to provide accurate test results. The everyday internal quality controls, and the participation to external quality assessment programs, organized by the Regione Lombardia or by Private Companies, guarantees the reliability of the test results. Moreover the laboratory has implemented and maintains a quality management system, which fulfills the requirements of the ISO 9001:2000 standard, and of the JCI standards specific for laboratories. A very recent computerization system online connects the lab with the wards, so that the results of the tests are very rapidly available for the physician, immediately after the validation process. The lab. is also in charge of the organization of the Transfusional Service, which derives its technical procedure from the Centro Trasfusionale e di Immunologia dei Trapianti di Milano. Besides traditional diagnostic laboratory tests, the Division is involved in different research activities. Studies are ongoing evaluating the prognostic and predictive value of circulating tumor cells in prostate, breast and colon cancer, using innovative and updated equipment that allows not only the separation and enumeration of tumor cells, but also their characterization. Moreover with the Unit of Preventive Gynecology studies are ongoing looking at the role of different high-risk HPV genotypes in the development

of pre-malignant lesions and in the follow-up of patients submitted to conization. We are also involved in the study of the potential use of the newly introduced vaccination against HPV infection for the prevention of cervical cancer. Another field of study is represented by the early detection of chemotherapy-induced cardiotoxicity, with the use of circulating biomarkers for the identification of patients at high-risk of developing myocardial damage. Lastly, studies are ongoing evaluating newly introduced serum tumor markers, both for diagnosis and for triage of patients with suspect lesions.

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Diagnosis and Prevention Area

Medical Physics Guido PEDROLI, Dr. Phys Director

STAFF Senior Deputy Director for Physics in Radiotherapy: Federica Cattani, Dr.Phys Senior Assistants for Physics in Radiotherapy: Raffaella Cambria, Dr.Phys, Cristina Garibaldi, Dr. Phys, Elena Rondi, Dr.Phys, Sabrina Vigorito, Dr. Phys Assistant for Physics in Radiotherapy: Stefania Comi, Dr.Phys, Rosa Luraschi, Dr. Phys Deputy Director for Physics in Nuclear Medicine: Marta Cremonesi, Dr.Phys Senior Assistant for Physics in Nuclear Medicine: Mahila Ferrari, Dr.Phys Assistant for Physics in Nuclear Medicine: Francesca Botta, Dr.Phys Deputy Director for Physics in Diagnostic Radiology and MR Imaging: Daniela Origgi, Dr.Phys Fellows for Physics in Radiotherapy: Alessia Bazani, Dr. Phys, Edoardo Mastella, Dr. Phys, Floriana Pansini, Dr. Phys, Stefania Russo, Dr. Phys Fellows for Physics in Nuclear Medicine: Francesco Guerriero Dr. Phys Fellow for Physics in Diagnostic Radiology and MR Imaging: Marika Guernieri, Dr. Phys, Federica Palorini, Dr. Phys Chief Technician: Annalisa Rossi

Activities 2011.

Many diagnostic, therapeutical and research activities performed in the Institute involve the use of sealed and unsealed radioactive substances and of many types of ionising radiation producing equipments (X-ray tubes for diagnostic radiology, conventional and mobile linear accelerators for external radiotherapy, after- remote-loading equipment for brachitherapy with sealed radioactive sources). The contribution of the Medical Physics team is indispensable in order to guarantee the radiation safety of the exposed personnel and of the patients. Moreover, the team regularly and continuously cooperates with the Divisions of Diagnostic

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Radiology, Nuclear Medicine and Radiotherapy for setting up and optimising protocols for clinical dosimetry, for the development and the introduction into the clinical practice of new and advanced techniques involving the use of ionising and non-ionising radiation sources, for commissioning new equipments and for carrying out a constant quality control on the radiation sources and on the diagnostic imaging systems. In 2011, apart from the radiation protection and quality control procedures, the following routine activities involving the optimisation of diagnostic and therapeutic procedures were performed: • calculation of treatment plans for conventional external radiotherapy and for IMRT, stereotactical radiotherapy, multiple arcs prostate radiotherapy, IORT (Intra Operative Radiation Therapy), conventional brachitherapy, brachitherapy with permanent implant of 125I seeds and TBI (Total Body Irradiation) treatments; • internal dosimetry evaluations for systemic and locoregional radionuclide therapies with agents radiolabelled with ß-emitters, (such as 90Y-avidin-biotin-MoAbs, 90Yavidin-biotin, 90Y and 177Lu-peptides and resin 90Y-microspheres); leakage monitoring in perfusion procedures. The procedures for the commissioning of three new linear accelerators for advanced radiotherapy (CyberKnife, Tomotherapy and VERO) have also been started. Research activities were directed to: • IGRT (Image Guided Radiation-Therapy), RapidArc therapies and extra-cranial stereotactical treatments with “deep inspiration breath hold”; • IART (Intraoperative Avidination Radionuclide Therapy); • radiobiological models in radionuclide therapies; • study of the effect of multi-cycle approach to radionuclide therapy, especially radiopeptide therapy and radioembolization of liver tumours with 90Y-resin microspheres; kidney and red marrow dosimetry in radiopeptide therapy and radioimmunotherapy;

• implementation of a software for the analysis of the activity distribution in scintigraphic images and for the evaluation of dose distribution at the voxel level (voxel dosimetry); • analysis of the dose-volume histograms, Biological Effective Dose histograms and Equivalent Uniform Dose; • comparison of dose conversion factors calculated using different methods (analytical methods and Monte Carlo simulations) for the development of a treatment planning system for nuclear medicine therapies; • implementation of PET/CT imaging for radiotherapy treatment planning; • development of a software for the segmentation of PET volumes and evaluation of standardized uptake value to facilitate patients’ diagnosis and follow-up assessment; • implementation of Monte Carlo methods (PENELOPE and FLUKA codes) to simulate radiation paths and to

calculate absorbed doses in clinical practice (systemic and locoregional therapies); • radiation protection evaluations related to patients undergoing therapies with 90Y- and 177Luradiopharmaceuticals; • development of quality assurance in HIFU (High Intensity Focused Ultrasounds); • optimization of patient dose in screening and follow up procedures with multislice CT; • quality evaluation and dose optimization for the new Contrast Enhanced Spectral Mammography (CESM) system. A new radioluminescent dosimeter, developed by the Department of Materials Science of the Milano-Bicocca University, is being tested with the different radiation sources available in the Institute in order to evaluate its practical use.

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Patrick MAISONNEUVE, Eng Director, Epidemiology Unit

STAFF Senior Epidemiologist Biostatistician: Sara Gandini, PhD Biostatisticians: Edoardo Botteri, MSc, Davide Radice, MSc, Sara Raimondi, MSc, Luigi Santoro, MSc Nutritionist: Patrizia Gnagnarella, MSc Fellow nutritionist: Alessandro Misotti Data Managers: Elena Albertazzi, PhD, Barbara Bazolli, MSc, Barbara Santillo Registrars Tumour Registry: Marina Alfieri, MSc, Laura Manghi, Marco Martinetti, Bruno Montanari, Carolina Quaresmini, BSc Scientific Secretariat: Nadia Bellani Biostatistical consultant: Vincenzo Bagnardi, PhD Visiting Professors: Matthias Löhr, MD (Karolinska Institute), Albert Lowenfels, MD (New York Medical College)

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Research ???Area

Division of Epidemiology and Biostatistics Nicole ROTMENSZ, MSc Director, Data Quality Control Unit

Activities 2011. The division of epidemiology

and biostatistics is conducting epidemiological research activity on a wide range of topics, focusing on patients with cancer or on patients at increased risk of developing the disease. It has throughout its existence, attempted to develop international collaborative research programmes and as a result, the majority of the research activities involve co-operation with scientists from a range of disciplines, both intra-mural and extra-mural. The division is involved in the development and management of the clinical research databases of the hospital and has responsibility upon data quality control. It is running the IEO hospital-based tumour registry. The tumour registry was activated in 2006 and after 6 years of activity, by March 2012, data for 129,592 tumours were retrospectively coded and entered (out of 114,804 individuals presenting for the first time at the IEO over the period 2000-2007). With the publication of its first research projects, the tumour registry has proven to be a valuable source of data for both epidemiological and clinical research. The division also provides consultation in a wide range of areas including the statistical design of experiments and clinical trials, including sample size calculations and randomization schemes (TENALEA web based), protocol development, database management, analysis of data and interpretation of results, preparation of interim reports and manuscripts. In addition, the division has developed a strong expertise in the field of statistical modelling and in the conduct of meta-analyses, providing important information to public-health policy makers and clinicians. In 2011, the division contributed to the statistical analysis of several meta-analyses on the safety of pregnancy following breast cancer diagnosis; on the association between serum 25-hydroxyvitamin D levels and colorectal, breast and prostate cancer and colorectal adenoma; on the association between alcohol drinking and various forms of cancer; on the role of hormonal and reproductive

factors in relation to melanoma in women; and on the effect of various treatment in breast cancer. Based on data from the COSMOS lung cancer screening trial, we developed lung cancer risk prediction models for the selection of high-risk individuals amenable to screening and for assisting clinicians to determine screening intervals. In summary, the statistical support activity of the division led to the publication of 47 peer-review clinical research articles in various fields ranging from cancer screening, chemoprevention, diagnosis, treatment, prognosis and outcome. Most of this activity was related to cancer of the breast, lung, digestive, urogenital organs as well as

hematologic neoplasms. Collaborative activity with other Italian or international institutes led to the publication of 26 additional research papers, with special focus on pancreatic and hepatic diseases, malignant melanoma, urological cancers, vitamin D and alcohol drinking. In total, in 2011 the division contributed to 73 articles published on peer-reviewed journals, with an overall impact factor of 368.

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Research ???Area

Department of Experimental Oncology Pier Giuseppe PELICCI, MD, PhD Chairman

STAFF Operative Director: Domenico Triarico Group Leaders: Myriam Alcalay, MD, PhD, Bruno Amati, PhD, Tiziana Bonaldi, PhD, Susanna Chiocca, PhD, Francesca Ciccarelli, PhD., Peter De Wulf, PhD, Pier Paolo Di Fiore, MD, PhD, Alberto D’Onofrio, PhD, Luisa Lanfrancone, PhD, Marina Mapelli, PhD, Saverio Minucci, MD, Andrea Musacchio, PhD, Gioacchino Natoli, MD, PhD, Diego Pasini, PhD, Giuliana Pelicci, MD, PhD, Pier Giuseppe Pelicci, MD, PhD, Maria Rescigno, PhD, Giuseppe Testa, MD, PhD, MA, Mario Varasi, MChem, Rosella Visintin, PhD. Scientific Writers: Roberta Aina, PhD, Raquel Carvalhosa, PhD, Paola Pierella Dalton, PhD, Rosalind Gunby, PhD Assistant to the Chairman: Roberta Carbone, PhD Chief of Technical Services: Loris Bernard (Genomic Facility), Giuseppina Fiorenza (Kitchen), Giuseppina Giardina (Tissue Culture), Alberto Gobbi (Mouse Facility), Gabriela Grigorean, PhD. (Mass Spectrometry Unit) Technicians: Sara Barozzi, Matteo Dal Molin, Mirko Doni, Manuela Moia, Simona Ronzoni, Mirco Scanarini, Cristina Spinelli, Marika Zanotti. Veterinary Surgeon: Manuela Capillo Laboratory Manager: Samantha Seresini Staff Operative Direction: Liliana Areces, PhD, Paola Durin, Pietro Transidico, Fabio Virdis, Serena Zanfini Management Control: Giovanni D’Angelo, Laura Massardi, Francesca Saba Administrative Services Manager: Annalisa Ariesi Administrative Assistants: Cristina Bonvicini, Alessandra Fossati, Elena Giannotta, Grazia Gioiosa, Alessandra Parolini, Giovanna Rognoni, Daniela Rossi, Monica Salvatore, Cristina Signoroni IT Service Manager: Alessandro Della Vedova IT Service: Alessandro Ogier, Stefano Leva Support Personnel: Giovanna Masarà, Filomena Minafra, Rodolfo Grisorio Maintenance: Andrea Vignati (Responsible), Celso Acacio, Alberto Iannucci

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Activities 2011. The Department of

Experimental Oncology (DEO) is composed of twenty independent research groups, whose scientific endeavours are described under the pages dedicated to the individual Units. The scientific interests of the research groups at DEO include the molecular mechanisms which regulate cell proliferation, differentiation, genomic stability, transcription and DNA repair in normal and cancer cells and stem cells; structural genomics and proteomics; tumour biology with respect to angiogenesis and immunology; the development of bioinformatics tools to characterise the complexity of gene expression unbalance in cancer cells. The European Institute of Oncology actively collaborates with the University of Milan and several scientists of the Department are also University Professors under the terms of a scientific agreement between these two Institutions. The DEO laboratories are located within the IFOMIEO Campus (http://www.ifom-ieo-campus.it), which also hosts the FIRC Institute of Molecular Oncology (IFOM), the Italian Institute of Technology (IIT), the European School of Molecular Medicine (SEMM) and the Consortium for Genomic Technologies (Cogentech). The DEO laboratories moved to their actual location at the beginning of 2007, thus creating together with IFOM one of the largest European research centres for cancer research. Together, the IEO and IFOM research infrastructures occupy about 24,000 square meters and house 40 research groups and 400 researchers. The Campus also hosts the three PhD programmes of SEMM (http://www.semm.it), namely Molecular Medicine, Medical Nanotechnology and Foundations and Ethics of the Life Sciences (FOLSATEC), which enrol over 100 PhD students from around the world.

An open and collaborative research environment. DEO has adopted an open structure model that encourages communication and cooperation between research groups. This open model has indeed created the most favourable conditions for an intensive and productive scientific collaboration among groups, as attested by the establishment of two institutional programmes: the Molecular Medicine Program and the Drug Discovery Program. In this framework, the highly qualified technical support staff that work in the core facilities and provide DEO researchers with an array of centralized high-quality services, deserve a special mention. The Service Units include: the Cell Culture Unit, which manages all departmental cell lines and sets up protocols for the isolation and manipulation of primary cells; the Laboratory Supplies Unit, which provides chemicals, consumables, small equipment items and reagents for the general use of the Department; the Information Technology Unit, which looks after the network and server infrastructure; the Kitchen Unit, which takes care of communal glassware, plasticware, solutions and reagents; the Technical Services Unit, which takes care of the maintenance of departmental instruments; the Chairman’s Office, which includes the scientific secretariat and also deals with general services and administrative procedures. High-profile post-graduate Educational Programs. SEMM is a private foundation established by a joint ministerial decree of the Ministry of Health, the Ministry of Treasure, and the Ministry for Education, University and Research. The SEMM Foundation is an alliance gathering three Italian Universities (Università degli Studi of Milan, Università degli Studi “Federico II” of Naples and VitaSalute San Raffaele University of Milan), two of the largest charities in the country (Telethon Foundation and the Italian Foundation for Cancer Research, both pursuing intra- and extramural research) and three of the most important Italian research institutions (the FIRC Institute of Molecular Oncology in Milan, the European Institute of Oncology in

Milan and the Centre for Genetic Engineering in Naples). The goal of SEMM is to offer advanced training through an array of different tools, including seminar activities, workshops, courses, and above all postgraduate training. SEMM is currently running three PhD programmes: Molecular Medicine (since 2004), Medical Nanotechnology (since 2005) and Foundations of Life Sciences and Their Ethical Consequences (FOLSATEC, since 2006). Characterizing traits of the PhD programmes are strong intedisciplinarity, an international dimension and availability of training platforms based on the development of technical skills through intensive laboratory work and on the acquisition of new theoretical tools through specifically designed courses. Institutional Programs The Molecular Medicine Programme (Director: Pier Paolo Di Fiore). This programme fuses the two great strengths of IEO: excellence in patient care and excellence in basic research. The merger between these two institutional pillars is the ultimate expression of the IEO dictum “Research for care”. The Molecular Medicine Programme at IEO is designed to allow clinical care programmes and basic research programmes to gradually grow into each other, until they become so intimately associated as to be indistinguishable. That is not to say that there will be no boundaries between basic and clinical activities within IEO: clearly, differences between research and clinical activities will always exist. Clinicians need to offer their patients the best possible care today, while researchers wish to develop the best possible treatments for patients tomorrow. Our implicit objective is to provide a medium in which clinicians and researchers are able to recognize each other’s value and respect each other’s priorities, at the same time stimulating them to acknowledge that their combined value is far greater than the sum of their individual strengths. Specifically, work has begun on three scientific programmes: 1) Understanding breast cancer to guide therapeutic choice. Our aim is to identify breast

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Research ???Area

IEO Administration Team at Campus IFOM—IEO with industrial drug discovery experience, and biochemists, cell biologists and in vivo pharmacologists are working together in our state-of-the-art equipped laboratories to identify new drugs against cancer. This biology-inspired, chemistry-driven multidisciplinary effort to provide innovative therapies is sustained by a close collaboration between the Project Originator, the Molecular Medicine Program and the campus technology transfer company (TTFactor S.r.l.). Current projects in the pipeline include Epigenetic targets, inhibitors of the Spindle Assembly Checkpoint (SAC) Kinases and alterations of self-renewal in cancer stem cells (CSCs). State-of-the-art Technological Platforms and Facilities. DEO and IFOM have collaboratively developed a number of technological platforms that have been placed within a Consortium (Cogentech) based at the IFOM-IEO Campus and owned by IFOM and IEO. Cogentech has developed the following technologies: Next-Generation Sequencing (Coordinator: Alcalay M.): The facility has set up protocols to cover most applications that require high-throughput sequencing (including ChIP-seq, RNA-seq, mutational analysis, methylation analysis) using the Illumina platform. stem cell genes that are overexpressed in tumours and that correlate with a negative prognosis, in an attempt to identify markers that will improve our diagnostic/prognostic ability and the clinical management of breast cancer. 2) Development of a blood test for the early diagnosis of lung cancer. Within this program, we are developing a clinical test, based on the detection of circulating serum miRNAs, for the early detection of NSCLC (non-small-cell lung carcinoma). 3) Understanding breast cancer for early diagnosis. This program is focused on the identification of prognostic and diagnostic circulating serum miRNA profiles for breast cancer. The Drug Discovery Programme (Directors: Saverio

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Minucci and Mario Varasi). The primary objective of the Drug Discovery Programme (DDP) is to translate the basic research findings and the technologies developed within the Campus into drug discovery projects. The DDP aims to i) successfully bring these projects up to an Investigational New Drug (IND) application, ii) ensure their development for maximum patient benefit and iii) exploit their potential for the growth of both the DDP and IEO. The DDP is committed to excellence in drug discovery through the creation of a network of fruitful collaborations and the development of educational and training opportunities for talented young people. Medicinal, analytical and computational chemists

Animal Facility: Two facilities currently provide researchers with the possibility to carry out experiments in animal model systems: i) the Mouse Genetics facility (Director: Gobbi A.), which deals with mice housing and caring, colony maintenance and expansion; ii) the Transgenic facility (Director: Allievi E.) which provides support for the generation of transgenic and knock-out mice. Imaging Unit (Facility Director: Parazzoli D.): This facility covers all the main needs of basic and advanced cancer research providing Fluorescence Microscopy, Cell Microinjection, Fluorescence Wide Field, Confocal and Time-Lapse Microscopy, Flow cytometry and Cell

sorting technologies and Digital Image Analysis (including deconvolution and 3D rendering) for all the researchers at the IEO-IFOM Campus. Main responsibilities of the facility are: i) set up, maintenance and development of optical technology for basic and advanced research; ii) competent assistance in sample acquisition and analysis, including training on-site and assistance on advanced imaging applications; iii) independent and collaborative research for the development of advanced imaging-based technology. Crystallization Unit (Coordinator: Mapelli M.): The threedimensional structure of biological macromolecules and their complexes can significantly contribute to the understanding of the biological processes in which they are involved. This Unit has established an automated platform for highthroughput protein crystallization in order to maximize the success rate of initial crystallization trials with minimal amounts of sample. Protein Chemistry Unit (Coordinator: De Marco A.): This facility offers integrated services for the production and characterization of recombinant proteins, maintains a collection of vectors, strains and protocols, and helps users generate monoclonal and polyclonal antibodies. Mass Spectrometry Unit (Coordinator: Grigorean G.): The Protein Analysis Unit aims at providing assistance in the design of experiments and data interpretation as well as scientific and technical knowledge in proteomics by providing tools for protein isolation, identification and characterization using mass spectrometry. DNA Services (Coordinator: Bernard L.): This facility offers DNA sequencing, human cDNA Library Colony Picking and Real Time PCR technologies. Microarray (Coordinator: Minardi S.): This state-of-the-art facility routinely performs complete microarray analysis for internal and external users, using Affymetrix and Nimblegen technology.

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Research ???Area

Department of Experimental Oncology

Functional Genomics Myriam ALCALAY, MD, PhD Director

STAFF Post-doctoral Fellows: Angela De Laurentiis, Alicja Gruszka PhD Students: Elisa Barbieri, Marco Saia

Activities 2011.

High-throughput technologies are the most powerful tools for attempting holistic approaches in the post-genome era, and are rapidly becoming essential for the discovery and analysis of genetic networks underlying cancer. Recent technological advancements, in particular next-generation sequencing, have allowed for a more comprehensive analysis of complex molecular interactions that accompany transformation and tumor progression. The concerted use of these approaches to discover and characterize genetic and epigenetic events that are relevant to oncogenesis is one of the current challenges in the field of oncogenomics. We are using an integrated genomic approach to study the molecular basis of acute leukemias. In particular, we are performing detailed analyses of transcriptional networks underlying normal hematopoietic differentiation and their subversion in the pathogenesis of acute leukemias. Our approach includes the integration of data deriving from gene expression profiling, analysis of transcription factor binding sites through chromatin immunoprecipitation coupled to deep sequencing (ChIP- seq), identification of histone modifications and proteomic analysis of transcriptional complexes containing leukemia-associated oncoproteins. Furthermore, we are specifically analysing the oncogeneinduced activation of stem-cell signalling pathways in acute myeloid leukemia.

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S. Monestiroli, A. Gobbi, M. Alcalay, S. Minucci and P.G. Pelicci: Cell-cycle restriction limits DNA damage and maintains self-renewal of leukaemia stem cells. Nature. 2009 January 1, 457, 51-56.

Publications A. Gardini, M. Cesaroni, L. Luzi, S. P. Minardi, E. Venturini, P. G. Pelicci and M. Alcalay: AML1/ETO oncoprotein is directed to AML1 binding regions and draws E-protein HEB onto its targets. PLoS Genetics, 2008, Nov;4 (11).

S. Licciulli, V. Cambiaghi, G. Scafetta, A. M. Gruszka and M. Alcalay: Pirin downregulation is a feature of AML and leads to impairment of terminal myeloid differentiation. Leukemia, 2010 Feb;24(2):429-37. 71.

A. Viale, F. De Franco, A. Orleth, V. Cambiaghi, V. Giuliani, D. Bossi, C. Ronchini, S. Ronzoni, I. Muradore,

S. Licciulli, C. Luise, G. Scafetta, M. Capra, G. Giardina, P. Nuciforo, S. Bosari, G. Viale, C. Tonelli, L. Lanfrancone and M. Alcalay: Pirin inhibits cellular senescence in

melanocytic cells. American Journal of Pathology, 2011, 178(5):2397-406.

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Research ???Area

Department of Experimental Oncology

Oncogenes, Chromatin and Cell Cycle Control Bruno AMATI, PhD Director

STAFF Scientist: Stefano Campaner Post-doctoral Fellows: Sevgi Bagislar Marcin Gorski Theresia Kress, Alessandra Majorana, Thomas Schleker PhD Students: Heide-Marie Binder, Luana D’Artista, Micol Ravà, Arianna Sabò, Claudia Tonelli Technicians: Mirko Doni, Paola Nicoli, Andrea Piontini, Alessandro Verrecchia Undergraduate Student: Roberta Bonfanti

Activities 2011. Oncogenic signals induce cell cycle progression and malignant transformation, but concomitantly elicit tumor-suppressive mechanisms (including apoptosis, senescence, and/or DNA Damage Responses), which must be bypassed in order to allow tumor progression, and which constitute the main selective pressure for mutation and/or silencing of tumor suppressor genes. Apoptosis and senescence also determine the therapeutic efficacy of genotoxic treatments (whether chemo- or radio-therapy). Hence, the same genetic lesions and/or epigenetic alterations that allow tumor progression also influence therapeutic responses.

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Our group has a long-standing interest in the c-myc oncogene and its product, the Myc protein. In general terms, our research aims at explaining the oncogenic activity of Myc, the tumor suppressor pathways that antagonize it, and their impact on tumorigenesis and therapy. In the last year, we profiled the contribution of Myc to the transcriptional response to serum mitogens in mouse fibroblasts (Perna et al. 2011). This work led to the identification of the core transcriptional program driven by Myc in response to mitogens. We are presently completing a similar study for the identification of genes deregulated by Myc during tumor progression in vivo in a mouse model of Myc-induced lymphoma. In parallel, we pursued our studies on the Myc/ p53-induced DNA Damage Response, a critical event in the modulation of Myc-induced tumorigenesis. In collaboration with the Testa lab, we reported that the histone methyltransferase Setd7 plays no significant role in this process (Campaner et al. 2011). In another project completed last year (Murga et al. 2011), we developed a pre-clinical mouse model to address the therapeutic potential of targeting DDR components in Myc-induced lymphoma. In collaboration with the Fernandez-Capetillo lab (CNIO, Madrid), we showed that the activity of the ATR-Chk1 pathway is essential for tumor cells to bypass Myc-induced replication stress, and hence to survive in the presence of an activated myc oncogene. Most importantly, inhibition of this pathway provided a significant therapeutic window for the treatment of Mycdriven tumors.

Miluzio, A., Beugnet, A., Grosso, S., Brina, D., Mancino, M., Campaner, S., Amati, B., de Marco, A., Biffo, S. (2011). Impairment of cytoplasmic eIF6 activity restricts lymphomagenesis and tumor progression without affecting normal growth. Cancer Cell, 19, 765-775.

Publications Campaner, S., Spreafico, F., Burgold, T., Doni, M., Rosato, U., Amati, B.* and Testa G.* (2011).The methyltransferase Set7/9 (Setd7) is dispensable for the p53-mediated DNA damage response in vivo. Mol Cell 43, 681-688. * BA and GT co-corresponding authors

Murga, M., Campaner, S., Lopez-Contreras, A.J., Toledo, L.I., Soria, R., Montaña, M.F., D’ Artista, L., Schleker, T., Guerra, C., Garcia, E., Barbacid, M., Hidalgo, M., Amati, B. and Fernandez-Capetillo, O. (2011). Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nature Struct & Mol Biol. 18, 1331-1335.

Perna, D., Fagà, G., Verrecchia, A., Gorski, M.M., Barozzi, I., Narang, V., Khng, J., Lim, K.C., Sung, W.-K., Sanges, R, Stupka, E., Oskarsson, T., Trumpp, A., Wei, C.-L., Müller, H. and Amati, B. Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts. Oncogene. 2012 Mar 29;31(13):1695-709. doi: 10.1038/onc.2011.359. Epub 2011 Aug 22.

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Department of Experimental Oncology

Quantitative Proteomics to investigate transcriptional and post-transcriptional mechanisms that regulate gene expression Tiziana BONALDI, PhD Director

STAFF Post-doctoral Fellows: Alessandro Cuomo, Marija Mihajlovich, Mara Colzani, Michael Bremang PhD Students: Monica Soldi, Cecilia Nardini Technician: Elena Vitale Undergraduate Student: Anna Maria Agresta

Activities 2011. The long-standing goal of the

research carried out in the group is to investigate gene expression regulation, at different levels. Historically our research has focused on the epigenetic regulation of gene expression mediated by histone post-translational modifications (hPTMs) and variants. By using massspectrometry (MS), we study: the combinatorial aspects of hPTMs at the level of single histone, mono- or poly-nucleosomes, up to small chromatin regions; the in vivo action of histone-modifying enzymes and the quantitative aspects related to modification dynamics. In addition we are extending the analysis of methylations to non-histonic proteins, to explore the regulatory networks mediated by this modification in the cell. More recently, with the advent of quantitative proteomics that allows accurate and large-scale protein expression profiling, we appreciated the potential of this technology to examine the post-transcriptional events regulating gene expression, with a focus on micro-RNA mediated translational inhibition. Our research elaborates on this observation developing novel strategies to study these processes globally. One quality of the team is the ambition to design and apply innovative and unconventional approaches to investigate various aspects of gene expression, in order to gain original perspectives and contribute new concepts to the field. Two lines of research are developed in the group: Area of Research 1: MS and quantitative Proteomics to explore the plasticity of epigenetic marks and the role of protein methylation Epigenetic information refers to heritable changes of gene expression that do not involve changes in DNA sequence or copy number. Part of epigenetic changes has been ascribed to histone modifications; different

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patterns of PTMs are thought to generate an epigenetic code specifying different patterns of gene expression and silencing. Such information is essential for cells to establish and memorize specific programs of gene expression, which are set during embryonic development and are required for differentiation Aberrant patterns of hPTMs have been observed in several cancer types, often associated with the deregulation of the respective enzymes promoting the reaction. The molecular mechanism behind these events is still poorly defined and consequently the development of drugs targeting such enzymes for epigenetic therapies is still at the concept stage. Thus, it is of general interest to investigate

systematically the “modification-specific proteome of histones”, in relation to differentiation and cancer. In our group we take advantage of high-resolution mass spectrometry (MS) to detect and profile hPTMs and to investigate their functional interplays. In particular, we combine the performance of MS with the quantitation potential offered by SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture), adapted to label histones and to quantitatively compare the abundance of hPTMs. “Heavy methyl SILAC” (hmSILAC) is a variation of SILAC that allows heavy- labeling of methylation on proteins with increased confidence in their identification

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Research ???Area and precise mesurement of their dynamics. Classical and heavy-methyl SILAC, combined with biochemical protocols of purification of histones or chromatin, provide quantitative information about protein and PTMabundance from specific cellular or differentiation states, or in specific genomic loci or domains.

Publications

Area of Research 2: Quantitative Proteomics to study micro-RNA silencing of gene expression According to the central dogma, information contained in DNA is transcribed in mRNA, which is afterwards translated into proteins, the real effectors of cellular functions. Yet, the global methods for gene expression analysis have been focused thus far on measuring changes at the mRNA level, neglecting the relevance of the modulation of the step from transcripts to proteins. However, recent technological achievements in quantitative proteomics make possible a “microarray-like approach at protein level”, with the prospect of profiling changes in protein amounts at the genome-scale within a single experiment. When SILAC is employed, proteomics is competitive with transcriptomics approaches for largescale analysis of gene expression, with the enormous advantage of investigating all those mechanisms that link transcription to translation and include regulation of transcript stability and processing, translation rate, protein half-life, etc. These mechanisms impact directly the final protein production and are now amenable to investigation with integrated approaches intersecting data from transcriptomics and proteomics.

Cuomo A, Bonaldi T. Systems biology “on-the-fly”: SILAC-based quantitative proteomics and RNAi approach in Drosophila melanogaster. Methods Mol Biol. 2010;662:59-78.

Vella P, Barozzi I, Cuomo A, Bonaldi T, Pasini D. Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells. Nucleic Acids Res. 2011 Dec 30.

Cuomo A, Moretti S, Minucci S, Bonaldi T. SILAC-based proteomic analysis to dissect the “histone modification signature” of human breast cancer cells. Amino Acids. 2011 Jul;41(2):387-99. Bonaldi T, Straub T, Cox J, Kumar C, Becker PB, Mann M. Combined use of RNAi and quantitative proteomics to study gene function in Drosophila. Mol Cell. 2008 Sep 5;31(5):762-72.

The group focuses on the gene expression silencing mediated by microRNA (miRNA), small non coding RNAs whose primary regulation of gene expression is believed to occur at the translational level. Great effort has been recently put into miRNA target prediction, by computational studies, and in their identification by transcriptomics; yet Proteomics is now emerging as the elective analytical tool for miR targets screening.

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Department of Experimental Oncology

Viral Control of Cellular Pathways and Biology of Tumorigenesis Susanna CHIOCCA, PhD Director

STAFF Post-doctoral Fellows: Simona Citro, PhD, Domenico Mattoscio, Ph.D, Archana Varadaraj, Ph.D PhD Students: Sara Loponte, Chiara Segré Folsatec Ph.D. student: Paolo Maugeri Technician: Claudia Miccolo Undergraduate Student: Laura Meloni

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Activities 2011.

Viruses reprogram or convert hosts’ metabolic and replication functions in order to obtain a cellular environment more favorable for viral propagation. We have been studying how viral proteins interfere with the regulation of the SUMO (Small Ubiquitin-related Modifier) pathway, a post-translational modification system enzymatically analogous but functionally diverse from the classical ubiquitin system (Ub). As a model system we have been using a peculiar adenoviral protein called Gam1 and demonstrated indeed how a viral protein can degrade the unique SUMO E1 enzyme by hijacking endogenous cellular components of ubiquitin E3 ligases. Protein post-translational modification by ubiquitin and SUMO regulate pathways that contribute to numerous biological processes. An ongoing research theme in our lab is to understand the cross-talk between the Ub and the SUMO pathways. We have also shown that Histone Deacetylase 1 (HDAC1) is post-translationally modified by SUMO. Mammalian histone deacetylases (HDACs) are composed of ubiquitously expressed class I, tissue specific class II, and NAD-dependent class III enzymes. Human HDACs are targets for cancer therapy. In fact, therapeutic efforts with HDAC inhibitors for the treatment of cancer are being pursued and the role of individual HDACs in tumorigenesis is starting to emerge. HDAC1 can also be phosphorylated, ubiquitinated and acetylated. Therefore, this project is based upon our findings that different interdependent modifications might modulate the biological function of HDAC1. Our laboratory is therefore pursuing two major projects: A. The biology of HDAC1 (and HDAC2) and how its post-translational modifications cross-talk and control its activity, also in light of its potential significance as a target for cancer therapy. B. The regulation of the SUMO pathway, its cross-talk to the ubiquitin pathway using the viral protein Gam1 as

a model system. We are also assessing whether other oncogenic viruses exploit the SUMO pathway. Pubblications Regulating the regulators: the post-translational code of class I HDAC1 and HDAC2. Segré CV, Chiocca S. J Biomed Biotechnol. 2011;2011:690848. Epub 2010 Dec 9. Review. PMID: 21197454 [PubMed - indexed for MEDLINE] Free PMC Article A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone

deacetylase inhibitors. Simboeck E, Sawicka A, Zupkovitz G, Senese S, Winter S, Dequiedt F, Ogris E, Di Croce L, Chiocca S, Seiser C. J Biol Chem. 2010 Dec 24;285(52):41062-73. Epub 2010 Oct 14. PMID: 20952396 [PubMed - indexed for MEDLINE] Free PMC Article Listeria monocytogenes: a bacterial pathogen to hit on the SUMO pathway. Citro S, Chiocca S. Cell Res. 2010 Jul;20(7):738-40. Epub 2010 Jun 8. PMID: 20531377 Inhibition of the SUMO pathway by Gam1. Pozzebon M, Segré CV, Chiocca S. Methods Mol Biol. 2009;497:285301. Review.

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Department of Experimental Oncology

Bioinformatics and Evolutionary Genomics of Cancer Francesca CICCARELLI, PhD Director

STAFF Scientist: Anna De Grassi, PhD, Post-doctoral Fellows: Matteo Cereda, PhD, Fabio Iannelli, PhD, Elena Gatti PhD Students: Matteo D’Antonio, Vera Pendino, Shruti Sihna Undergraduate Student: Valentina Melocchi

Activities 2011.

We use next generation sequencing technology (NGS) and systems biology to study the evolution of cancer. In the past years we have applied next-generation sequencing to quantify genomic instability and to detect low levels of constitutional genomic instability in healthy tissues of patients with hereditary non-polyposis colorectal cancer. Our data show that these individuals have hereditary predisposition to acquire the second hit needed to start tumorigenesis (De Grassi, et al PLoS Biology 2010). Using again NGS, we also developed a method to rebuild the proliferation dynamics of single lesions from the mutations that were progressively acquire. The first results on four lesions show that each tumor is formed for the main part of a dominant subclone and that it undergoes latency periods, which could be used for therapeutic interventions. We now intend to enlarge this study to a larger tumor set to infer the relationships between tumor genetics and proliferation dynamics. In the past years we found that cancer genes are fragile points on the protein-protein interaction network, as they engage several connections and avoid gene duplication (Rambaldi et al Trends in Genet 2008). We collected all properties of cancer genes in a public database (http://bio.ifom-ieo-campus.it/ncg/) (D’Antonio et al Nucleic Acid Res 2012). We further found that cancer genes appeared in different times during evolution. Recessive cancer genes are very old and tend to be strictly conserved in several species. Dominant cancer genes are more recent and can undergo gene duplication (D’Antonio & Ciccarelli PLoS Comp Biol 2011). We are now applying these features of cancer genes to search for synthetic lethal partners, i.e. genes whose impairment is detrimental for the cancer cell only in combination with mutations on a particular cancer gene.

Publications De Grassi A et al.: Ultra-deep Sequencing of a Human Ultraconserved Region Reveals Somatic and Constitutional Genomic Instability (2010) PLoS Biology 8(1): e1000275

duplicability during the evolution of protein interaction network (2011) PLoS Comp Biol 7(4):e1002029 Rambaldi D, et al.: Low Duplicability and Network Fragility of Cancer Genes (2008) Trends in Genetics 24:427

D’Antonio M, Pendino V, Sihna S, Ciccarelli FD Network of Cancer Genes (NCG 3.0): integration and analysis of genetic and network properties of cancer genes (2012) Nucleic Acids Res 40:D978-83. D’Antonio M Ciccarelli FD Modification of gene

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Department of Experimental Oncology

Molecular Analysis of Kinetochore Composition, Activity and Regulation during Chromosome Segregation Peter DE WULF, PhD Director

STAFF Post-doctoral Fellows: Cinzia Pagliuca, PhD, Cristina Golfieri, PhD, Maria Iacovella, PhD Undergraduate Students: Enrico Sanna, Lucia Massari

Activities 2011. Our research focuses on

kinetochores; large protein structures that assemble from more than 100 subunits on the centromeric regions of duplicated chromosomes (sister chromatids). Kinetochores regulate sister chromatid segregation during cell division by performing four essential activities: they 1) bind sister chromatids to the microtubules of the mitotic spindle, 2) act as a sensor of microtubule attachment, 3) regulate the onset of sister chromatid segregation via the proofreading spindle assembly checkpoint, and 4) maintain microtubule attachment during cycles of microtubule shrinkage and growth, thereby generating forces required for chromatid movement along the spindle. The presence of abnormal chromosome numbers (an- euploidy) is a hallmark of cancer cells. Precisely how aneuploidy is generated remains unclear. Due to their essential roles in sister chromatid segregation, kinetochore dysfunctions likely contribute to aneuploidy. This hypothesis is further supported by the presence in many tumours of abnormal levels of kinetochore proteins, which may interfere with kinetochore integrity and function. Thus, a better understanding of kinetochore composition, activity, and regulation is bound to reveal new insights into the mechanisms underlying tumorigenesis. We study the kinetochores of budding yeast (S. cere- visiae) and human cells by integrating various methodologies including genetics (mutant suppressor screens, synthetic lethality/viability screens, yeast two-hybrid screens), chemical genetics (identification of compounds that interfere with certain kinetochore or cell cycle activities), biochemistry (chromatography, sedimentation ultracentrifugation, affinity purifications), chromatin immunoprecipitation and hybridization, array-based gene expression analysis, production of recombinant kinetochore proteins/complexes, time-lapse

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videomicroscopy of cells, and single-molecule imaging of proteins. The knowledge obtained from this research will lead to the development of (i) diagnostic tools to screen for cancer susceptibility markers (mutations in or abnormal levels of kinetochore components), and (ii) novel drugs to treat cancer (molecules that interfere with kinetochore assembly, activity or regulation). Publications “Bock L.J., Pagliuca C., Kobayashi N., Grove R.A., Oku Y., Shrestha K., Alfieri C., Golfieri C., Oldani A., Dal Maschio M., Bermejo R., Hazbun T.R., Tanaka T.U., De Wulf P. (2012). Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nature Cell Biology. In press.” Nguyen T.L., Cera M.R., Pinto A., Lo Presti A., Hamel E., Conti P., Gussio R., De Wulf P (2012). Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds. Molecular Cancer Therapeutics. In press. Screpanti E., Santaguida S., Nguyen T.L., Silvestri R., Gussio R., Musacchio A., Hamel E., De Wulf P. (2010). A screen for kinetochore-microtubule interaction inhibitors identifies novel antitubulin compounds. PLoS ONE, 5: e11603. Pagliuca C., Draviam V.M., Marco E., Sorger P.K., De Wulf P. (2009). Roles for the conserved Spc105p/Kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint. PLoS ONE, 4: e7640.

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Department of Experimental Oncology

Molecular Carcinogenesis and Stem Cell Biology Research Pier Paolo Di Fiore, MD, PhD Director

STAFF Staff Scientists: Salvatore Pece, MD, PhD, Daniela Tosoni, Ph.D Scientists: Ivan Colaluca, PhD Post-doctoral Fellows: Macarena Ferrero Gimeno, PhD Postgraduate Students: Letizia Amadori Undergaduate student: Alessio Di Giacomo, Irene Ferrari Technicians: Michele Caccia, Marco Coazzoli, Luca Napolitano Visitors: Blanca Alvarez Moya, Andrea Basile, Angelo Taglialatela

Activities 2011.

Our research focuses on the role of stem cells in cancer. It is generally accepted that cancer stem cells (CSC) are responsible for initiating and maintaining tumour growth. These cells are formed when processes controlling normal stem cell function go awry. Our laboratory is interested in investigating the molecular mechanisms governing the maintenance of the normal stem cell compartment and how these mechanisms are altered in cancer, in particular, in breast and lung. We believe that a high-resolution picture of the stem cell compartment will hold the key to the development of new diagnostic, prognostic and patient stratification tools. Our previous studies have demonstrated that breast cancers can be CSC-rich or CSC-poor, with poor-prognosis (high tumour grade, G3) tumours tending to be enriched in CSC compared to more favorable prognosis (low tumour grade, G1) tumours. An expansion of the stem cell compartment can, therefore, occur in breast tumours and this is associated with a more aggressive cancer phenotype. One well-known regulator of the SC compartment is Numb. This ties in with our recent studies that have established Numb as a tumour suppressor in human breast and lung tumours. Indeed, Numb is degraded in ~50% of breast tumours and ~30% of lung tumours. This has led us to hypothesize the existence of a mechanism, caused by the absence of Numb, which subverts normal stem cell homeostasis thus contributing to tumourigenesis. Three lines of research focusing on Numb are currently being developed in our laboratory: i) We are investigating the exact role of Numb in the regulation of the mammary gland stem cell compartment, and how changes in this protein and in its downstream targets (Notch and p53) can lead to the appearance of CSC; ii) We are performing high-throughput studies to identify molecular players causing Numb degradation in human cancers. These could potentially be novel targets for drug development;

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iii) We are performing a structural and functional characterisation of the Numb-p53-HDM2 tricomplex, which regulates the activity of the tumour suppressor p53, to identify new molecules that can restore p53 function in cancer cells. Publications Tosoni D, Zecchini S, Mazzarol G, Coazzoli M, Galvagno D, Zilian O, Pece S, Di Fiore PP. The Numb/p53 circuitry couples the control of replicative asymmetry and tumour suppression in normal and cancer mammary stem cells, Submitted

S, Di Fiore PP: Alterations of the Notch pathway in lung cancer, Proc Natl Acad Sci U S A 2009, 106:22293-22298 Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S, Bernard L, Viale G, Pelicci PG, Di Fiore PP: Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content, Cell 2010, 140:62-73 Pece S, Confalonieri S, Romano PR, Di Fiore PP: NUMB-ing down cancer by more than just a NOTCH, Biochim Biophys Acta 2011, 1815:26-43 Colaluca IN, Tosoni D, Nuciforo P, Senic-Matuglia F, Galimberti V, Viale G, Pece S, Di Fiore PP: NUMB controls p53 tumour suppressor activity, Nature 2008, 451:76-80

Westhoff B, Colaluca IN, D’Ario G, Donzelli M, Tosoni D, Volorio S, Pelosi G, Spaggiari L, Mazzarol G, Viale G, Pece

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Department of Experimental Oncology

Systems Biomedicine Alberto D’ONOFRIO, PhD Director

STAFF Postdoc fellow: Sebastiano de Franciscis (from Nov 1 2011) Student: Sara Gattoni, MSc (from February 1 2011 to October 15 2011)

Activities 2011.

The research of the lab is focused to the application of a wide spectrum of computational and analytical tools of physics and mathematics in both basic science of cancer and clinical oncology. Indeed, tumors are a family of highly dynamical diseases, so that the nonlinear kinetics of tumor onset and growth shapes both the natural history of neoplasias and their responses to treatments. Moreover, a tumor substantially interacts with its micro-environment, and its nonlinear ‘ecological’ interplay with other systems (e.g. immune system effectors and antibodies, blood vessels etc..) determines its fate. The rationale of the research in this lab follows, essentially, the one of theoretical physics: interpreting and reproducing in silico experiments, connecting through theory apparently unrelated experiments, proposing new hypotheses in order to trigger experimental work that may validate or refuse them. The fundamental step is the critical reading of experimental and clinical papers. When reading of these articles, a theoretical biologist see the described phenomena under the novel light of physics of complex systems. In turn, when applying a physical theory it is obtained a new inference, it is critically read under the light of biology Publications A. d’Onofrio, Spatiotemporal effects of a possible chemorepulsion of tumor cells by immune system effectors, Journal of Theoretical Biology 296 (2012) 41–48 Epub 2011 Nov 19.

A. d’Onofrio (Corr. Auth.) and A. Ciancio, Simple biophysical model of tumor evasion from immune system control Physical Review E 84, 031910 (2011) F. Bertolini, P. Marighetti, I. Martin-Padura, P. Mancuso, D. D. Hu-Lowe, Y. Shaked, and A. d’Onofrio Anti-VEGF and beyond: shaping a new generation of antiangiogenic therapies for cancer Drug Discovery Today (2011), 2011 Dec;16(23-24):1052-1060

G. Caravagna (eq. contr.), R. Barbuti and A. d’Onofrio (eq. contr and corr. auth.) Fine-tuning anti-tumor immunotherapies via stochastic simulations BMC Bioinformatics 2012, 13(Suppl 4):S8

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Department of Experimental Oncology

Cellular and Molecular Pathways Regulating Melanoma Genesis and Progression Luisa LANFRANCONE, PhD Director

STAFF Post-doctoral Fellows: Ewa Aladowicz, Luis Fernandez-Diaz, Leda Ferro, Margherita Yayoi Turco Research technician: Elisabetta Venditti Temporary fellow: Paolo Mariani Undergraduate Students: Andrea Papait

Activities 2011.

Melanoma is an aggressive disease with high metastatic potential and resistance to cytotoxic agents. The molecular mechanisms involved in the progression of the malignancy and the genetic markers associated with metastatic melanoma dissemination and the acquisition of chemoresistance are only beginning to be defined. An understanding of the underlying molecular biology of melanoma provides a necessary basis to enable the generation of more effective therapeutic modalities. Melanoma is amenable to surgical intervention during its early stage and the treatment is for the most part curative, while, at late stage, the metastatic melanoma is refractory to current treatments and still largely incurable. Melanoma thus represents the prototype of a tumor in which the availability of biomarkers predicting metastasis formation could indeed modify the story of the patient. The majority of the melanoma diagnoses are made with great specificity and reproducibility based on established

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histopathologic criteria. However, histopathologic features may not be indicative of tumor aggressiveness and no reliable biomarkers are known to predict which patients will develop metastases after the surgical treatment of a primitive melanoma. The necessity to find appropriate biomarkers that predict metastasis formation and dissemination is mandatory in melanoma, as well as the development of appropriate targeted therapy, to be used alone or in combination, for intervention in melanomas. The main goal of our research is the understanding of the how melanoma cells disseminate to local and distant organs and which are the molecular pathways involved in such dissemination. To do so, we are undertaking various in vitro and in vivo approaches: 1. Development of in vivo authentic models of melanoma to dissect the biology of the tumor and examine the process of metastatization. These models can be used as useful tools for drug discovery and development. Melanoma engraftment in immunocompromised animals is a well established procedure, lacking an in vivo correlate between the hystopathological and clinical features of the human melanoma samples and the corresponding tumor in the animal. A large cohort of patients will be transplanted in the animal and tumor graft analysed for their capacity of phenocopying the original tumor. Genetic lesions will be investigated, to complement the phenotypic analysis. These models will be used to assay for stem cell activity, to predict a patient’s response to a therapy and eventually test and investigate drug resistance. 2. Identification and characterization of novel critical protein kinases involved in the metastatic process to be used as targets for the development of targeted therapy. In this study, we propose to apply an in vivo shRNA-based screening with libraries targeting protein kinases to identify and subsequently validate new potential targets involved in the dissemination of melanoma cells. We will generate metastases from xenografted tumors obtained from highly metastatic melanoma cell cultures and from

primary human melanoma xenografts using a metastatic melanoma cohort of patients. 3. Epigenetic screening of a selected and characterized melanoma cohort of patients to identify and characterize novel determinants of the metastatic process that can be used as prognostic markers and predictive markers of metastasis formation.

to Epiblast Stem Cells is Modulated by the ShcD/RaLP Adaptor Protein. Manuscript under revision, Stem Cells. Bosotti R, Carpinelli P, Healy S, Locatelli G, Cappella P, Lanfrancone L, Calogero R, Moll J, Isacchi A. Transcriptional analysis of the Aurora inhibitor Danusertib leading to biomarker identification in TP53 wild type cells. Gene. 2011 Sep [Epub ahead of print]

Pubblications Turco YM, Furia L, Dietze A, Fernandez Diaz L, Ronzoni S, Sciullo A, Simeone A, Constam D, Faretta M, Lanfrancone L. The Transition of Embryonic Stem Cells

Licciulli S, Luise C, Scafetta G, Capra M, Giardina G, Nuciforo P, Bosari S, Viale G, Mazzarol G, Tonelli C, Lanfrancone L, Alcalay M. Pirin inhibits cellular senescence in melanocytic cells. Am J Pathol. 2011 May;178(5):2397-406.

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Department of Experimental Oncology

Structural and Functional Studies of the Mitotic Spindle Orientation during Asymmetric Cell Divisions Marina MAPELLI, PhD Director

STAFF Post-doctoral Fellows: Andrea Alfieri, Simone Culurgioni, Anna Zoccarato PhD Student: Sara Gallini Research Fellows: Manuel Carminati, Valentina Pendolino

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Activities 2011.

We are interested in the molecular pathways governing asymmetric cell divisions, with emphasis on the role of the mitotic spindle orientation in determining the fate choice of daughter cells. The proper execution of asymmetric divisions is crucial in generating tissue diversity during development, as well as for tissue homeostasis and regeneration in adult organisms. An increasing body of literature supports the notion that certain human cancers arise from abnormalities in adult stem cells asymmetric divisions, able to alter cells’ fate and lead to over-proliferation (the so called cancer stem cell hypothesis). Indeed failures in asymmetric divisions occur when pathways controlling the position of the cytokinesis plane are compromised. To make a cell division asymmetric, the position of the mitotic spindle has to be tightly coordinated to the cortical polarity, so that daughter cells will be properly positioned within the tissue, will inherit unequal sets of fate determinants, and will follow differential fates. This observation sets the stage for our studies, aimed at gaining insight into the structural and functional organization of the molecular machines responsible for spindle coupling to cortical polarity during asymmetric divisions. To address this biological problem, we use a combination of high-resolution X-ray crystallography, biochemical analyses on reconstituted protein complexes, and stem cell biology. Using the detailed molecular information delivered by our structural studies, we formulate molecular models of the mitotic spindle orientation that we challenge in living cells. An emerging concept in the cancer field is that cancer stem cells may be responsible for relapse and resistance to anticancer therapies. In this view, a clear molecular description of processes underlying asymmetric cell divisions will be instrumental in identifying new stem-cell specific drug targets for therapeutical intervention.

Publications

molecular biology 19, 136-144.

Culurgioni, S., Alfieri, A., Pendolino, V., Laddomada, F., and Mapelli, M. (2011). Inscuteable and NuMA proteins bind competitively to Leu-Gly-Asn repeat-enriched protein (LGN) during asymmetric cell divisions. Proc Natl Acad Sci U S A 108, 20998-21003.

Mapelli, M., Massimiliano, L., Santaguida, S., and Musacchio, A. (2007). The Mad2 conformational dimer: structure and implications for the spindle assembly checkpoint. Cell 131, 730-743.

Migliori, V., Muller, J., Phalke, S., Low, D., Bezzi, M., Mok, W.C., Sahu, S.K., Gunaratne, J., Capasso, P., Bassi, C., et al. (2012). Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance. Nature structural &

Mapelli, M., and Musacchio, A. (2007). MAD contortions: conformational dimerization boosts spindle checkpoint signaling. Curr Opin Struct Biol 17, 716-725.

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Department of Experimental Oncology

Chromatin Alterations in Tumorigenesis Saverio MINUCCI, MD Director

STAFF Post-doctoral Fellows: Marco Ballarini (with Pier Giuseppe Pelicci), Marco Cirò PhD (TYM Project), Mara Colzani PhD (with Tiziana Bonaldi), Mohamed Elgendy PhD, (TYM Project) Simona Moretti PhD, Fabio Santoro PhD PhD Students: Giacomo Iros Barozzi, Chiara Biancotto, Gianmaria Frigè, Parinaz Medhipour, Giulia Murari, Pierluigi Rossi Technicians: Walter Gentilini PhD, Isabella Pallavicini, Mauro Romanenghi Undergaduate Students: Luca Morini, Valeria Golli

Activities 2011.

Cancer cells show global changes in chromatin structure (DNA methylation and histone post-translational modifications), that lead to stable alterations in gene expression and potentially other nuclear functions (such as DNA replication and repair). Unlike genetic lesions, those alterations are reversible since the underlying DNA sequence is unchanged: this fundamental difference between genetic and epigenetic alterations makes the epigenome much more amenable to the development of therapeutic strategies. Indeed, small molecules with the capacity to interfere with chromatin modifying enzymes have antitumor activity. The concept of epigenetic therapy has been clinically validated with the approval by regulatory authorities of a small number of drugs for use in selected forms of cancer. In our view, however, drugs interfering with epigenetic enzymes (such as DNA methyl-transferases and histone deacetylases, the most advanced targets in the epigenetic arena) have been used in the vast majority of cases rather aspecifically, without taking into account the context of chromatin alterations occurring in cancer cells. We surmise therefore that one of the major goals of both basic and applied research in this area should be the search of a set of epigenetic alterations in tumor cells, that dictate sensitivity or resistance to epigenetic drugs. We have focused therefore our activities on the study of deregulation of chromatin structure/function in cancer with the goals of: • Identifying sistematically epigenetic alterations in cancer cells; • To exploit this knowledge to optimize epigenetic therapies towards a more targeted approach. To fulfill these goals, we have adopted a combination of experimental strategies: • Mechanistical analysis of chromatin alterations in

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cancer. We have developed new technologies for the study of epigenetic alterations in cancer patients, to reduce the amounts of material required, and to allow access to paraffin-embedded pathology samples: NASeq, and PAT-ChIP (Fanelli et al., Nature Protocols, 6(12), 1905–1919; Fanelli et al., Proceedings of the National Academy of Sciences, 107(50), 21535–21540; Gargiulo et al., Developmental Cell, 16(3), 466–481). Thanks to these new approaches, we are studying acute myeloid leukemias and breast cancer (where mechanistical insights on how epigenetic deregulation takes place are partially available) as a paradigm of the cancer epigenome.

• Functional dissection of the role of chromatin modifiers in leukemogenesis. In parallel, we are undertaking the systematic dissection of the role of individual chromatin modifiers in tumorigesis in murine models of acute myeloid leukemia. By the use of knock-down and conditional knock-out approaches, we are studying the role of histone deacetylases, Polycomb complexes, histone demethylases in both tumor initiation and tumor maintenance. • Epigenetic therapy of cancer. In the same disease model, we are studying the biological and mechanistical effects of epigenetic drugs (histone deacetylase and demethylase inhibitors,

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Research ???Area DNA demethylating agents). In particular, we have developed new assays for the study of the contribution of different subpopulations of tumor cells to cancer growth, focusing on the role of leukemic stem cells (Leiva, M., Moretti, S., et al. Leukemia. doi:10.1038/leu.2012.39). • Optimization of anticancer therapies. The know-how and results gained above are being increasingly useful in other settings, to try to exploit the epigenome and its manipulation for the optimization of anticancer therapies. With this goal, we are: Using yeast as a model system (in collaboration with M. Foiani, Project “TYM”) studying systematically the synthetic lethal interactions of anticancer and epigenetic drugs, and subsequently validating them in mammals; By quantitative chemical proteomics (in collaboration with T. Bonaldi, Project “TIP”), identifying systematically the cell interactors of anticancer and epigenetic drugs; In collaboration with the Novel Therapeutics Program of the IEO, conducting in vivo screenings to identify and validate epigenetic targets in leukemias (in collaboration with PG Pelicci), and analyzing the effect of novel epigenetic drugs being developed against chromatin-associated proteins. Thus, there is an extremely appealing opportunity to perform a mechanistically oriented analysis (“to understand how things happen”) that can immediately be applied to better treat the patients (“to try to change things, when they have gone bad”). The ultimate goal: to go towards a group that considers Man as the primary model system.

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Publications Di Micco, R., Sulli, G., Dobreva, M., Liontos, M., Botrugno, O. A., Gargiulo, G., Dal Zuffo, R., et al. (2011). Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer. Nature Cell Biology, 13(3), 292–302. doi:10.1038/ncb2170 Fanelli, M., Amatori, S., Barozzi, I., & Minucci, S. (2011). Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. Nature Protocols, 6(12), 1905–1919. doi:10.1038/ nprot.2011.406 Occhionorelli, M., Santoro, F., Pallavicini, I., Gruszka, A., Moretti, S., Bossi, D., Viale, A., et al. (2011). The selfassociation coiled-coil domain of PML is sufficient for the oncogenic conversion of the retinoic acid receptor (RAR) alpha. Leukemia, 25(5), 814–820. doi:10.1038/leu.2011.18 Robert, T., Vanoli, F., Chiolo, I., Shubassi, G., Bernstein, K. A., Rothstein, R., Botrugno, O. A., et al. (2011). HDACs link the DNA damage response, processing of doublestrand breaks and autophagy. Nature, 471(7336), 74–79. doi:10.1038/nature09803* *SM co-corresponding author.

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Department of Experimental Oncology

Structural and Functional Basis of Mitosis Andrea MUSACCHIO, PhD Director

STAFF Scientists: Anna De Antoni, PhD Post-doctoral Fellows: Sadasivam Jeganathan, PhD, Jenny Keller, PhD, Stefano Maffini, PhD, Marta Mattiuzzo, PhD, Marion Pesenti, PhD, Arsen Petrovic, PhD, Emanuela Screpanti PhD, PhD Students: Federica Basilico, Veronica Krenn, Stefano Santaguida, Gianluca Varetti Technicians: Tiziana Melis, Silvia Monzani

Activities 2011.

We are interested in the mechanistic aspects of cell division and on perturbations of this process that might cause cell transformation. Mitosis is the process of separation of the genetic material. During mitosis, the replicated chromosomes of the mother cell divide in two equal complements to create two daughter cells with an exact copy of the genetic material. Mitosis is a very complex process. One of its crucial moments is the establishment of strong connections between polymeric structures known as microtubules and kinetochores, specialized structures on chromosomes. It is this interaction that ultimately allows cells to part their chromosomes with the required accuracy. Failures to execute these processes result in aneuploidy, imbalances in chromosome numbers that can be fatal for cell viability, but that can also influence the process of cellular transformation in tumors. After DNA replication, the chromosomes consist of two identical copies of the same chromosome glued together by a macromolecular complex named cohesin. These glued chromosomes are known as sister chromatids. As cells enter mitosis, the kinetochores start interacting with microtubules (prometaphase), and this process continues until all chromosomes have aligned in the middle of the mitotic spindle, the metaphase plate. This marks a phase known as metaphase, which is an obligatory intermediate of mitotic progression. It is only when cells have passed through metaphase, i.e. when all chromosomes are properly aligned on the mitotic spindle, that chromosome segregate to opposite spindle poles in a process known as anaphase. Despite steady progress, how microtubules capturethe sister chromatids and form a stable link with them remains largely unclear. Kinetochores provide the site of attachment for microtubules and host feedback control mechanisms that coordinate this process with

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progres- sion of the cell division cycle. Specifically, mitotic kinetochores host a safety device, the spindle assembly checkpoint (SAC), which is designed to sense the occupancy of microtubules on kinetochores and the forces that result when kinetochores are properly attached (bi-orientation). Tension between sister chromatids can be visualized as an increase in the distance between the sister kinetochores in bi-oriented chromosomes relative to unattached or mono-oriented chromosomes. More recently, it has been shown that kinetochores themselves become stretched when spindle forces are applied. It is believed that tension allows the spindle checkpoint to recognize bipolar orientation from

incorrect configurations, such as syntelic attachment, which fail to generate tension. Before all sister chromatid pairs have achieved biorientation on the mitotic spindle, the spindle assembly checkpoint generates a ‘wait anaphase’ signal that inhibits anaphase, allowing more time for attachment or for the correction of attachment errors. The ‘wait anaphase’ signal originates at kinetochore, consistently with the observation that all the spindle checkpoint proteins are recruited to kinetochores during mitosis (Musacchio and Salmon, 2007). We have been interested in analyzing the interaction

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Research ???Area of the SAC with the kinetochore, and the role of posttranslational modifications, particularly phosphorylation, in this process. Precisely which biochemical steps allow kinetochores to regulate the activation state of the SAC? To answer this question, we have taken a biochemical approach whose main initial goal is the generation of large portions of the kinetochore. For instance, we have recently reported the reconstitution of a 10-subunit assembly known as the KMN network (Petrovic et al., 2010). The KMN is crucial for kinetochore function, as it contains the main site of contact with the mitotic spindle. We discovered that the KMN network interacts with a core kinetochore protein known as CENP-C, and have identified the crucial regions of interaction between subunits in these complexes (Screpanti et al., 2011). Using reconstituted material, we try to identify the requirements for the binding reactions and regulatory steps that normally occur at kinetochores and that are important for the recruitment and regulation of the spindle checkpoint proteins. This strategy of in vitro reconstitution will eventually identify the crucial regulatory elements that allow kinetochores to support microtubule attachment and to regulate the spindle checkpoint. By using these approaches, in 2011 we made important discoveries in the area of checkpoint control (Varetti et al., 2011) and inhibition (Santaguida et al., 2011).

Publications Musacchio A (2011) Travel notes from the equatorial circle. Cell 146:499-501. Santaguida S, Vernieri C, Villa F, Ciliberto A & Musacchio A (2011) Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction. EMBO J 30:1508-19 Musacchio A (2010) Surfing chromosomes (and Survivin). Science 330:183-4 Petrovic A, Pasqualato S, Dube P, Krenn V, Santaguida S, Cittaro D, Monzani S, Massimiliano L, Keller J, Tarricone A, Maiolica A, Stark H & Musacchio A (2010) The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J Cell Biol 190:835-52

We complement this effort of reconstitution with structural analysis with approaches such as X-ray crystallography and electron microscopy, the latter in collaboration with the groups of Prof. Eva Nogales in Berkeley and Prof. Holger Stark in Göttingen. Recently, this effort led to the determination of the crystal structure of the crucial microtubule receptor at the kinetochore, the NDC80 complex. Additional recent work on the MIS12 complex and its complexes is revealing the basis of microtubule attachment at the kinetochores.

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Department of Experimental Oncology

Transcriptional Control in Inflammation and Cancer Gioacchino Natoli, MD, PhD Director

STAFF Post-doctoral Fellows: Liv M. I. Austenaa PhD, Greta Caprara PhD, Francesca De Santa PhD, Serena Maria Luisa Ghisletti PhD, Luca Giorgetti PhD, Flore Mietton PhD PhD Students: Iros Giacomo Barozzi, Xuefen Chen, Agnese Collino, Betsabeh Khoramian Tusi, Samuele Notarbartolo Technician: Elena Prosperini Temporary Fellow: Sara Polletti

Activities 2011.

Chronic inflammation and cancer. Inflammation is a basic response to environmental and endogenous “danger signals” and serves an essential homeostatic and therefore beneficial role. At the same time, as demonstrated by a large body of epidemiological and experimental data, chronic and unresolved inflammation promotes the development of several types of epithelial cancers worldwide, including hepatocarcinomas, colon carcinomas, gastric and prostate cancer. Cancer development in chronically inflamed tissues is often preceded by specific alterations of tissue differentiation, as in the case of intestinal metaplasia of gastric mucosa in patients with chronic gastritis, and Barrett’s esophagus (squamous metaplasia) in patients with gastro-esophageal reflux disease. Such alterations of tissue differentiation are entirely reversible as their mechanistic basis is represented by epigenetic (non DNA sequence-based) alterations of chromatin and a subsequent change in the accessibility and usage of the underlying genome. Experimental data suggest that polyclonal epigenetic changes associated with chronic inflammation may lay the ground for the acquisition of clonal genetic changes leading to tumor development. Defining mechanisms that control inflammatory responses (and particularly sustained inflammation) may lead to the identification of novel cancer chemopreventive treatments, as suggested by the protective role of aspirin towards some tumor types. Research activity in the laboratory is mainly focused on the understanding at the molecular level the mechanisms that control inflammatory gene expression. Transcriptional control of inflammation. Inflammation entails the induction (or repression) of hundreds of genes whose products contribute to different aspects of the response, such as the recruitment of leukocytes, the induction of changes in vascular permeability, the activation of anti-bacterial responses, and in a

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subsequent step the induction of the repair response leading to reconstitution of tissue integrity. The first objective of the lab is the mechanistic understanding of transcriptional regulation of inflammatory genes both in inflammatory cells (like macrophages) and in bystander cells exposed to an inflammatory environment. An indepth understanding of such mechanisms may provide the molecular basis for therapeutic targeting of selected transcriptional events. To achieve these objectives, standard biochemical approaches to transcription are integrated with genomics, computational approaches, physics and in vivo studies.

Mechanisms controlling inflammatory gene expression in macrophages. Most of the research tackled by the laboratory relates to one of the most important cell types involved in inflammation, namely macrophages. Macrophages are highly specialized cells widely distributed in tissues and active both as immune effectors and as housekeeping phagocytes responsible for maintenance of tissue integrity. Macrophages display a striking heterogeneity that reflects a complex interplay between different micro-environmental signals provided by various tissues (as well as by microbial and endogenous stress signals), and a robust differentiation program that determines macrophage identity. The

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Research ???Area main objective of the research activity in this unit is to understand how macrophage identity, functional specialization and plasticity are controlled by their specialized genomic organization, which is encoded in mammalian genomes, controlled by specific transcription factors, and modulated by the microenvironment. Within this area we provided the first genome-wide characterization of the genomic regulatory elements (enhancers) controlling inflammatory gene expression in macrophages. This activity involved the extensive use of chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) and allowed us to determine a general organizational principle of these enhancers, which consists in the combination of binding sites for ubiquitous, stimulus-responsive TFs and binding sites for constitutive cell type-restricted and lineage-determining TFs. Specifically, we have found that in macrophages genomic regulatory elements that control inflammatory gene expression contain two minimal elements, namely a binding site for one or more of the TFs activated in response to stimulation (e.g. NF-kB and AP-1), and a binding site for the major macrophage lineage-determining TF (Pu.1). This combination allows creating a cell type-specific context within which transcription of inflammatory genes is regulated, thus explaining variability among cell types in the inflammatory gene expression program induced by identical stimuli. Interestingly, part of the enhancers controlling inflammatory gene expression were found to undergo transcription, which may be instrumental to the maintenance of an open chromatin configuration and/ or to the production of non-coding RNAs that signal downstream transcriptional events. Ongoing research in the lab is mainly focused on the characterization of the impact of different environmental stimuli on the functional organization of macrophage genome using both in vitro models and ex-vivo analyses on macrophages obtained from tissues and primary tumors.

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As part of this effort we are characterizing the role of a panel of chromatin modifying enzymes, which represent potential drug targets, in the control of inflammatory responses. In this area we have reported a few years ago the first description of a histone demethylase involved in inflammatory gene expression. More recent work allowed us to identify a required role of a histone methyltransferase, MLL4, in the control of macrophage responsiveness to inflammatory stimuli. Publications The histone methyltransferase Wbp7 (MLL4) controls macrophage function through GPI anchor synthesis (L. Austenaa, I. Barozzi, A. Chronowska, A. Termanini, R. Ostuni, E. Prosperini, A. F. Stewart, G Testa, G Natoli) Immunity (36, 572–585, April 20 2012) Transcriptional control of macrophage polarization: enabling diversity with identity (T. Lawrence, G. Natoli) Nature Reviews Immunology 11, 750-761 (2011). The genomic landscapes of inflammation (G. Natoli, S. Ghisletti, I. Barozzi) Genes & Development 25, 101106 (2011). Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages (S. Ghisletti, I. Barozzi, F. Mietton, S. Polletti, F. De Santa, E. Venturini, L. Gregory, L. Lonie, A. Chew, C.L. Wei, J. Ragoussis, G. Natoli) Immunity, 32:31728. Epub 2010 Mar 4 (2010).

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Department of Experimental Oncology

Epigenetic mechanisms in stem cell differentiation and oncogenesis Diego PASINI, PhD Director

STAFF Post-doctoral Fellow: Fulvio Chiacchiera, PhD, Karin Ferrari, PhD PhD Students: Sri Ganesh Jammula, Andrea Piunti, Alessandra Rossi, Andrea Scelfo, Pietro Vella Undergraduate Students: Maria Abbattista, Alessandro Gatti

Activities 2011. Epigenetic mechanisms in

stem cell differentiation and oncogenesis. Organisms’ development and tissues homeostasis is achieved by a precise control of the fate of differentiating cells. Such regulation is influence by several cell autonomous and non-autonomous stimuli that are translated into the establishment of specific transcription programs allowing correct fate determination. Establishment and maintenance of such transcription programs involve several different mechanisms that, by acting at a genetic (i.e., DNA sequence recognition by specific DNA binding transcription factors) and at an epigenetic level (i.e., DNA sequence

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independent mechanisms of transcriptional regulation), resolve the complex three-dimensional structure of chromatin and set up proper transcriptional information. The latter activity involves different enzymes and adaptor proteins that, by remodeling chromatin structure through nucleosome sliding, eviction, histone post translation modifications and DNA methylation, contribute to set up correct transcription. The bypass of cellular identity is a common feature of all human cancers and several mechanisms involved in determining normal cell identity are also essential for tumors development. This also includes the enzymatic activities that are involved in “placing” and “removing” chromatin modifications. The work of our laboratory focuses its attention at different chromatin modifying activities with the aim to understand the molecular basis behind how these influence transcription in normal differentiation and in neoplastic transformation. This includes the characterization of Polycomb Group (PcG) proteins, master regulators of organisms development, whose activities control gene expression by imposing transcriptional repression through the methylation and ubiquitylation of specific lysine residues of Histone proteins. Such activities are frequently deregulated in several human tumors and positively correlate with high grade and poor disease outcome. By combining biochemical, molecular and genetics approaches our laboratory aims to: 1) Characterize the role of different chromatin modifying activities in regulating cell proliferation in normal and cancer cells. 2) Characterize the transcriptional mechanisms by which different chromatin modifying activities contribute to the establishment of cell type specific transcription programs. 3) Characterize the role of DNA binding transcription factor and their oncogenic products in recruiting chromatinmodifying activities to DNA promoting tumors formation.

To achieve this, we implemented cell culture and genetic mouse models to study cellular differentiation and neoplastic transformation using both primary and embryonic stem cell lines. To these models, we apply biochemical approaches to study protein complexes formation and protein-protein functional interactions, complemented by the use of highthroughput technologies such as next-generation sequencing and gene expression profiling.

factors network in embryonic stem cells. Nucleic Acids Res. Stojic, L., Jasencakova, Z., Prezioso, C., Stutzer, A., Bodega, B., Pasini, D., Klingberg, R., Mozzetta, C., Margueron, R., Puri, P.L., Schwarzer, D., Helin, K., Fischle, W. and Orlando, V. (2011) Chromatin regulated interchange between polycomb repressive complex 2 (PRC2)-Ezh2 and PRC2-Ezh1 complexes controls myogenin activation in skeletal muscle cells. Epigenetics Chromatin, 4, 16.

Publications Vella, P., Barozzi, I., Cuomo, A., Bonaldi, T. and Pasini, D. (2012) Yin Yang 1 extends the Myc-related transcription

Piunti, A. and Pasini, D. (2011) Epigenetic factors in cancer development: polycomb group proteins. Future Oncol, 7, 57-75.

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Department of Experimental Oncology

Biology and Signal Transduction of Normal and Cancer Neural Stem Cells Giuliana PELLICCI, MD, PhD Director

STAFF Post-doctoral Fellows: Barbara Ortensi, Cristina Richichi PhD Students: Paola Brescia, Matteo Setti Undergraduate Student: Valeria Alberizzi Technician: Daniela Osti

Activities 2011.

Our lab is performing both basic and translational cancer research in the fields of brain tumors, neural stem cells and cancer stem cells (CSCs). Our final goal is to identify and unravel the molecular mechanisms underlying glioblastoma (GBM) generation and progression. GBM is the most common and lethal type of glioma in adults. There are now emerging evidences in tumor biology validating the hierarchical organization of tumors as abnormal tissues originating from and maintained by a subset of cells with stem cell-like properties able to generate and propagate the tumor and producing differentiated progeny with limited replicative potential. Cancer stem cells have also been isolated in GBMs and although the presence of surface markers selectively expressed on CSCs has been used to isolate these cells, no marker or pattern of markers is sufficiently robust to definitively identify CSCs. In spite of the uncertainty and difficulties related to markers clearly indentifying the CSCs in GBM, it becomes essential to develop multiple strategies for CSCs detection. Consequently the aim of our research is to map cancer stem cells biomarkers from patient derived tumor samples. The end goals of our research are three-fold: 1) to evaluate the efficacy of currently utilized biomarkers (CD133) in the purification of CSCs from multiple patient derived neurosphere cultures; 2) to isolate a pure population of CSCs and identify a universal barcode of cell surface markers 3) to study proteins involved in GBM generation and progression. 1) CD133 function in GBM CSCs. CSCs from human glioblastoma have been isolated by the neurosphere assay and identified by the cell surface protein CD133. Xenograft assays identified as human brain CSCs, the CD133 positive fraction, while CD133 negative cells were not able to produce tumors. However, recently it has been shown that CD133 negative CSCs are capable of forming tumors in nude mice. Thus the notion that CD133

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might be a reliable cancer stem cell marker in brain tumors is currently under revision. Moreover, the functional role of CD133 in cancer stem/progenitor cells is not known. We analyzed the efficiency of CD133 in the identification and isolation of glioblastoma stem cells and we investigated its functional role, studying the biological effects of CD133 down-regulation in GBM-derived neurospheres in vitro and in vivo. Our data demonstrate that CD133 is homogenously expressed in the cytoplasm of the cells composing the neurospheres, while its expression on the cell surface is highly variable. Cloning of single CD133+ and CD133cells proves that CD133 shuttles dynamically between

the plasmamembrane and the cell cytoplasm and no hierarchical relation can be established. However, CD133 is essential to the maintenance and the tumorigenic potential of glioblastoma stem cells, since its silencing reduces both the self-renewal and tumorigenic capacity of glioblastoma CSCs. Furthermore, the block of plasma-membrane CD133 results in growth inhibition of neurosphere cells. Taken together, these data suggest the value of CD133 as a therapeutic target in glioblastomas, and its cell surface expression could be important for diagnosis and staging, for monitoring therapeutic efficacy and influencing therapeutic protocols.

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Research ???Area 2) Marker-independent method to isolate CSCs from GBM patients. We employed a marker-independent method consisting in the cell labeling with PKH-26 fluorescent dye that binds to cell membranes and segregates in daughter cells after each cell division, so that intensity of staining correlates inversely at single cell level, with the number of previous cell divisions. This assay, already validated to identify CSCs in breast tumoral mammospheres, allows to discern progenitors (high dividing cells) from stem cells (slow dividing or quiescent cells). PHK positive and negative cell populations have been isolated from GBM neurospheres by FACS/sorting and characterized in vitro for proliferation capacity and selfrenewal ability, and in vivo for their tumor formation capacity after orthotopic injection in nude mice. Frequency of the number of the putative stem cells by in vivo limiting dilution assays has been evaluated as well as the ability to re-form tumors by in vivo serial xeno-transplantations. Our results show that both PKH positive and PKH negative cells are tumorigenic but the negative fraction does not exhibit stem cell criteria exhausting in culture. The slow-cycling cell pool (PKH+) displays all the hallmarks of a cancer stem cell. 3) Rai/Shc C function in patient-derived GBM CSCs. Rai is an adaptor protein specifically expressed in the adult normal brain in neurons, and absent in glial cells, which becomes ectopically expressed in glioblastomas. We have demonstrated that Rai is expressed in neurogenic areas of the adult brain and its knock-down impairs physiological progenitor migration. Its expression is retained in glioblastoma cancer stem cells where it exerts the same promigratory activity. Rai-silencing in cancer stem cells isolated from different patients causes a significant decrease in cell migration and invasion, in vitro and in vivo, thus increasing survival. Rai depletion is associated with an alteration in multiple signaling pathways, culminating in reduced expression of proinvasive genes (1). The mechanism by which Rai exerts its pro-migratory and pro-invasive effects is still unknown. We are searching for

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the involvement of miRNAs that might mediate Rai function as a modulator of glioblastoma invasiveness. 4) CLIC1 (Chloride intracellular channel 1) function in patient-derived GBM CSCs. Several studies have reported the involvement of chloride channels in cell division, motility and drug resistance, making chloride homeostasis of particular interest in GBM, where proliferation and migration are altered and drug resistance is the main cause of tumor relapse. A marked up-regulation of CLIC1 has been recently reported in human tumors of different origin, including GBMs, where it is enriched in mesenchymal molecular sub-type characterized by patients’ poor prognosis (our data). We are studying the role of CLIC1 in the tumorigenic potential of CSCs isolated from human GBMs. The demonstration of reduced gliomagenesis after CLIC1 interference in tumoral stem/ progenitors cells could identify CLIC1 as a new molecular therapeutic target. Publications Rai is a New Regulator of Neural Progenitor Migration and Glioblastoma Invasion. Ortensi B, Osti D, Pellegatta S, Pisati F, Brescia P, Fornasari L, Levi D, Gaetani P, Colombo P, Ferri A, Nicolis S, Finocchiaro G, Pelicci G. Stem Cells. 2012 Feb 6. The Shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation. Ferro M, Savino MT, Ortensi B, Finetti F, Genovese L, Masi G, Ulivieri C, Benati D, Pelicci G, Baldari CT. PLoS One. 2011;6(12):e29899. Epub 2011 Dec 29. Human glioblastoma tumours and neural cancer stem cells express the chemokine CX3CL1 and its receptor CX3CR1. Erreni M, Solinas G, Brescia P, Osti D, Zunino F, Colombo P, Destro A, Roncalli M, Mantovani A, Draghi R, Levi D, Rodriguez Y Baena R, Gaetani P, Pelicci G, Allavena P. Eur J Cancer. 2010 Dec;46(18):3383-92. Epub 2010 Aug 19.

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Department of Experimental Oncology

Molecular mechanisms of cancer and ageing Pier Giuseppe PELICCI, MD, PhD Director

STAFF Staff Scientists: Mario Faretta, Marco Giorgio, Ph.D., Lucilla Luzi, Ph.D., Enrica Migliaccio, Ph.D., Cristina Moroni Scientists: Marco Ballarini, Emanuela Colombo, PhD, Ivan Gaetano Dellino, PhD, Alessandra Insinga, PhD, Lucilla Titta, Mirella Trinei Post-doctoral Fellows: Alessandra Bigi, PhD, Angela Mariano, PhD, Paul Edward Massa, PhD, Massimiliano Mazza, PhD, Marine Meliksetyan, PhD, Rani Pallavi, PhD, Stefania Rapino, PhD, Linsey Blair Reavie, PhD, Laura Riva, PhD, Chiara Ronchini, PhD, Hanumaiah veena Talagavadi, PhD, Maria Vittoria Verga Falzacappa, PhD PhD Students: Umberto Andrea Cammarata, Petia Doytcheva, Maria Mallardo, Dalia Rosano, Anna Russo, Angela Santoro, Alice Soldà, Thalia Vlachou, Malgorzata Wierzbicka Technicians: Luisa Albano, Alessia Caronno, Errico D’Elia, Giulia De Michele, Laura Furia, Barbara Gallo, Luciano Giacò, Rossana Piccioni, Costanza Savino, Cristina Lynne Sironi, Massimo Stendardo, Mariangela Storto Temporary Fellows: Neethukrishna Kausthubham, Antonella Ruggiero Undergraduate Students: Mario Altieri, Giovanna Pia Castronovo, Silvia Crasto, Giulia De Conti, Matteo Righi, Elisa Romanini, Francesco Santaniello Visitors: Francesca Bernassola, Elisa Memmi

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Activities 2011.

The cancer stem cell (CSC) hypothesis provides an appealing perspective through which we can examine cancerogenesis and tumour progression, and suggests a rationale for therapeutic interventions. A major focus of our group is to generate appropriate mouse model systems of human cancer for the characterization of cancer stem cell properties and their molecular determinants. We are also investigating the effects of metabolism and checkpoint activation in normal and cancer SCs on tissue homeostasis and ageing. Laboratory Research Projects We are currently pursuing several lines of research, focusing on the: 1. Role of quiescent and proliferating normal and cancer stem cells in tumour growth. Our preliminary characterization of the cell-cycle properties of in vitro-growing mammary CSCs, as compared to their normal counterparts, demonstrated that: i) both SCs and CSCs are capable of asymmetric and symmetric divisions: asymmetric divisions generate one quiescent SC and one proliferating progenitor, while symmetric divisions generate two proliferating SCs; ii) mammary SCs divide infrequently, and almost exclusively asymmetrically, while mammary CSCs divide more frequently, alternating asymmetric and symmetric divisions; iii) at the steady-state, mammary SCs are mainly quiescent (>90%), while CSCs exist in two populations: quiescent (~40%) and actively proliferating (~60%); iv) both quiescent mammary SCs and CSCs are able to re-enter the cell cycle and expand clonally to form mammospheres. We are examining whether quiescent CSCs exist in vivo and if they are critical for hematopoietic and mammary tumour growth.

2. Role of the oncogene c-myc in SC divisions and cancer. We are investigating whether c-myc is important for the maintenance of the CSC pool in mammary tumours. Emerging evidence suggests that expression of c-myc in cancer imposes an “embryonic stem cell signature”, which plays a fundamental role in CSCs and which correlates with cancer aggressiveness and poor outcome. We have shown that c-myc is involved in the regulation of self-renewal in mammary SCs, and that its expression alone is sufficient to increase the self-renewing abilities and regenerative potential of mammary SCs, conferring on them an

immortal phenotype. Our recent data suggest that c-myc induces symmetric divisions of mammary SCs and reprogramming of progenitor cells into SCs. However, it appears that the reacquisition of stemness features during reprogramming is associated with the occurrence of genomic rearrangements (Pasi et al., Cell Death and Differentiation, 2011). 3. Role of the NPM mutation in Acute Myeloid Leukemia (AML). AML is the most common form of acute leukemia in adults and it is curable in about 30% of cases. Recently, mutations in one of the two alleles of the NPM gene have been described

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Research ???Area in about 60% of AMLs with normal karyotype (one third of all adult AMLs). We are investigating the molecular mechanisms by which the mutant NPM induces AML and exploring targeting strategies for novel anti-leukemia treatment approaches. 4. Roles of the cell cycle inhibitor p21 and the tumour suppressor p53 in the regulation of DNAdamage response in mammary and hematopoietic stem cells. Our previous published results have indicated that p53 and p21 might exert unique functions in stem cells and that these functions are de-regulated in cancer stem cells as a consequence of primary genetic alterations. We are therefore examining the activation of control systems which are specific to the stem cell compartment after DNA damage caused by X-ray, in order to highlight any distinctive mechanism of regulation of p21 and p53 and investigate the processes that govern the fate of adult stem cells. 5. Identification of PML-RAR cooperating mutations and definition of their biological role in leukemogenesis. We have demonstrated that expression of the oncogenes PML/RAR and AML1-ETO in hematopoietic stem cells (HSCs) induces both DNA damage and up-regulation of p21. This in turn leads to the maintenance of a stem cell pool that harbours a moderate amount of damaged DNA (Viale et al. 2009). Although in these cells p21 up-regulation (like loss of p53 functions in mammary cells) appears critical for the maintenance of the transformed phenotype, it is not sufficient to induce transformation. To assess whether it is the acquisition of secondary mutational events which transforms the preleukemic-HSCs into highly proliferating leukemic stem cells, we are analysing the biological effects of putative cooperatingmutations on the differentiation potential of pretumoural SCs.

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6. Effects of oncogenic fusion proteins, like PML-RAR, on PML localization and chromatin binding, and on DNA replication. DNA replication is a highly orchestrated and regulated process that ensures fidelity of the genome during duplications, as well as its adaptation to variations in cell division, DNA damage and, in multicellular organisms, chromatin changes associated with development and differentiation. DNA replication initiates from multiple chromosomal loci, called replication origins (ORIs). In particular, we are looking at the correlation of replication origins (ORIs) with PMLRAR binding sites through a genome-wide ChIPmapping of human ORIs, and at the alterations in transcription induced by the oncogenic fusion protein. 7. Relationship between stem cell checkpoint competence, aging and cancer development. Ageing is a complex process; in this process, cellular senescence is largely recognized as a tumour suppressor mechanism. However, cancer incidence increases with age, an observation that suggests instead a strong association between cancer and the general degrading of an organism. A common feature of all age-related changes, and a recognized major contribution to cancer, is the increase in oxidative stress induced by excess of free radicals, such as reactive oxygen species (ROS). These are produced in the course of normal cellular metabolism by mitochondria during respiration, but their formation is enhanced by exposure to ionizing radiation, chemical substances and mitochondrial dysfunctions. We are characterizing a novel signalling pathway involving the p53 tumour suppressor, the p66Shc lifespan determinant and reactive oxygen species (ROS). 8. Study of overweight and obesity as main risk factors for breast cancer. Recent findings suggest

that environmental factors and lifestyle are implicated in tumour development and might increase cancer risk by inducing chromatin alterations (epigenomic modifications). In postmenopausal women being overweight or obese strongly correlates with an increase risk of breast cancer, however, no clinical bio-markers are available to measure individual risk in these patients. We are thus attempting to investigate the effects of a high-fat diet on the epigenome of mammary stem cells and on breast cancer development using pre-clinical mouse model systems. Publications Giorgio M, Berry A, Berniakovich I, Poletaeva I, Trinei M, Stendardo M, Hagopian K, Ramsey JJ, Cortopassi G, Migliaccio E, Nötzli S, Amrein I, Lipp HP, Cirulli F, Pelicci PG. The p66Shc knocked out mice are short lived under natural condition. Aging Cell. 2012 Feb;11(1):162-8. doi: 10.1111/j.1474-9726.2011.00770.x. Epub 2011 Dec 28. Pasi CE, Dereli-Öz A, Negrini S, Friedli M, Fragola G, Lombardo A, Van Houwe G, Naldini L, Casola S, Testa G, Trono D, Pelicci PG, Halazonetis TD. Genomic instability in induced stem cells. Cell Death Differ. 2011 May;18(5):745-53. Epub 2011 Feb 11. Bonizzi G, Cicalese A, Insinga A, Pelicci PG. The emerging role of p53 in stem cells.Trends Mol Med. 2012 Jan;18(1):6-12. Epub 2011 Sep 7. Martinelli P, Bonetti P, Sironi C, Pruneri G, Fumagalli C, Raviele PR, Volorio S, Pileri S, Chiarle R, McDuff FK, Tusi BK, Turner SD, Inghirami G, Pelicci PG, Colombo E. The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway. Blood. 2011 Jun 16;117(24):6617-26. Epub 2011 Apr 25.

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Department of Experimental Oncology

Immunobiology of Dendritic Cells and Immunotherapy Maria RESCIGNO, PhD Director

STAFF Staff Scientist: Giuseppe Penna Post-doctoral Fellows: Gerhard Antonie Floris Fransen, PhD, Silvia Guglietta, Paul Edward Massa, PhD, Ana Monegal Granados PhD, Chiara Pozzi, PhD, Fabiana Saccheri, PhD, Tiziana Schioppa, PhD PhD Students: Elisa Mazzini, Ilaria Spadoni, Katerina Tsilingiri, Elena Zagato Technicians: Glenda Bardeli, Erika Mileti

Activities 2011.

Dendritic cells (DC) comprise a family of professional antigen presenting cells unique in their capacity to modulate T cell responses. DC play a primary role in pathogen protection, in central and peripheral tolerance and in anticancer immune responses. Understanding basic mechanisms governing DC function in biology and pathology can be instrumental to unravel how an immune response is initiated and to shape new protocols for immune intervention. In our unit we study the interaction of DC with recombinant bacteria both in vitro and in vivo with the aim of establishing new protocols for cancer immunotherapy. We have proposed bacteria as tumor antigen delivery systems and as infectious agents in order to kill tumor cells both because they are neoplastic and because they are infected. Two lines of research are being developed in this unit. The first one studies a possible use of an attenuated strain of Salmonella to initiate immune responses against tumor cells. We have shown that the intratumoral injection of Salmonella induces the upregulation of a protein called Connexin 43 that generates pores in the tumor cells and allows communication with antigen presenting cells. Through these pores antigenic material can be passed from tumor cells to antigen presenting cells and this results in the activation

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of an immune response to the tumor. This mechanism was exploited to generate efficient vaccination against the tumor and was shown to be more powerful than currently used methods of vaccination. We then evaluated whether also in the human system a similar behaviour is observed in response to Salmonella. We found that indeed Salmonella can induce the upregulation of Connexin 43 in human cell lines and primary cells isolated from human tumors. These studies are fundamental for a translation of the findings to the clinical practice. We are now writing a protocol to treat metastatic melanoma patients. The second line of research studies the interaction of bacteria with dendritic cells in vivo in the gut. The homeostasis of the gut is a crucial step for the well being of an organism. Deregulations of the intestinal flora can lead to severe diseases including, diabetes, inflammatory bowel disease and eventually cancer. Very little is still known on how the immune system distinguishes ‘good’ from ‘bad’ bacteria and how at some point good bacteria are misrecognized as bad bacteria and cause disease. In our laboratory we have identified several factors released by epithelial cells that ‘educate’ dendritic cells and allow the recognition of pathogens versus commensal bacteria. This allows the generation of tolerogenic dendritic cells that contribute to preserving the homeostasis of the gut. We found that under inflammatory conditions this control is missing and this correlates with the development of inflammatory bowel disease. As chronic intestinal inflammation predisposes to colorectal cancer, it will be interesting to address whether intervention at this level can be of benefit to prevent colon cancer development. We have generated a new organ culture system of the intestine to study the interaction of bacteria with healthy and pathological mucosa. This model system will be instrumental to test the effect of different bacterial strains in controlling intestinal homeostasis.

Publications Mittal D, Saccheri F, Vénéreau E, Pusterla T, Bianchi ME, Rescigno M. TLR4-mediated skin carcinogenesis is dependent on immune and radioresistant cells. EMBO J. 2010 Jul 7;29(13):2242-52. Saccheri F, Pozzi C, Avogadri F, Barozzi S, Faretta M, Fusi P, Rescigno M. Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci Transl Med. 2010 Aug 11;2(44):44ra57.

Frittoli E., Matteoli G., Palamidessi A., Mazzini E., Maddaluno L., Disanza A., Yang C., Svitkina T, Scita G., and Rescigno, M. The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration. Immunity 2011, 23;35(3):388-99 Swiatczak B, Rescigno M. How the interplay between antigen presenting cells and microbiota tunes host immune responses in the gut. Semin Immunol. 2011 Nov 30. [Epub ahead of print]

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Department of Experimental Oncology

Histone Methylation Dynamics in Stem cell Renewal and Lineage Commitment Giuseppe TESTA, MD, PhD, MA Director

STAFF Post-doctoral Fellows: Thomas Burgold, Prem Tripathi PhD Students: Sina Atashpaz, Serena Buontempo, Agnieszka Chronowska, Giulia Fragola, Jacopo Sgualdino, Elena Signaroldi,

Activities 2011. Our lab studies the epigenetic

mechanisms that enable lineage commitment and cell fate reassignment and their aberrations in cancer. In his classic representation of the epigenetic landscape, Conrad Waddington depicted development as the progressive channeling of pluripotency (the marble at the top of the hill) down irreversible paths of cell specification (the slopes and canyons available to the marble in its downward rolling). A current version of that same landscape brings to the fore the fate choices of embryonic and tissue-specific stem cells as key transitions for the regulation of self-renewal and

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differentiation that forms and maintains organisms and that goes awry in cancer. In order to understand these transitions we need to uncover how genomic programs are progressively deployed and what are the chromatin regulatory mechanisms that coordinate their deployment. Among these, the methylation of histone H3 on lysine tails 4 and 27, respectively mediated by the Trithorax (Trx) and Polycomb (PcG) protein families, is central to the programming of genomes that underlies the establishment and maintenance of differentiated cell states. Not surprisingly, aberrations in these pathways have also emerged as important determinants or modulators of tumors, hinting at common regulatory circuits that preside over stem cell physiology and that are perturbed or hijacked in oncogenesis. Finally, changes in these posttranslational modifications are also prominent in the epigenetic rewiring that reversed Waddington’s unidirectional slopes, namely the reacquisition of pluripotency from differentiated cells through nuclear transfer or the expression of few pluripotency factors. Consistently, work in our lab pursues the following complementary lines of research: i) the physiology of genome programming during differentiation, using neurogenesis as model system; ii) the aberrant genome programming that accompanies tumorigenesis, focusing on gliomagenesis as the aberrant counterpart of neural fate acquisition; iii) transcription factor-induced genome reprogramming both as a model to understand cell fate reassignment and as a tool to model neurodevelopmental disorders and investigate their epigenetic alterations

G. Testa The time of timing: How Polycomb proteins regulate neurogenesis Bioessays, 2011; 33(7):519-28.

Publications

G. Boniolo and G. Testa The Identity of Living Beings, Epigenetics, and the Modesty of Philosophy. Erkenntnis, 2011; DOI 10.1007/s10670-011-9308-9 Corresponding author

S. Campaner, F. Spreafico, T. Burgold, M. Doni, U. Rosato, B. Amati, and G. Testa The methyltransferase Set7/9(Setd7) is dispensable for the p53-mediated DNA damage response Mol. Cell 43, 2011; 681-688 Corresponding author

C.E. Pasi, A. Dereli-Oz, S. Negrini, M. Friedli, G. Fragola, A. Lombardo, G. Van Houwe, L. Naldini, S. Casola, G. Testa, D. Trono, P.G. Pelicci, and T.D. Halazonetis Genomic instability in induced stem cells Cell Death and Differentiation, 2011; 18(5):745-53

H. Nowotny and G. Testa. Naked genes. Reinventing the human in the molecular age, MIT Press, 2011 (originally appeared as ‘Die gläsernen Gene. Die Erfindung des Individuums im molekularen Zeitalter’, Suhrkamp Verlag, 2009), reviewed in Nature, The Financial Times, Die Zeit, Der Spiegel

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Department of Experimental Oncology

Drug Discovery Unit Mario VARASI Director

STAFF Chemistry Senior Scientist: Luca Sartori Staff Scientists: Raffaella Amici, Daniele Fancelli, Raimondo Fattori, Florian Thaler, Paola Vianello Scientist: Loris Moretti Post-Doctoral Fellows: Maria Carmela Fulco Technicians: Anna Cappa, Giacomo Carenzi Fellows: Giuseppe Meroni, Alessia Romussi, Paolo Trifirò Biology Scientists: Oronza Antonietta Botrugno, Maria Rosaria Cera, Maddalena Donzelli, Fabrizio Villa Post-Doctoral Fellows: Anna De Ponti, Paola Dessanti, Giovani Fagà, Francesca Senic Matullia, Stefania Vultaggio Fellow: Roberto Dal Zuffo, Elisa Zagarri

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Activities 2011.

The Drug Discovery Program (DDP) primary objective is to translate the basic research developed within the Campus into drug discovery projects. The aims of the DDP are to successfully bring these projects up to IND, to ensure their development for maximum patient benefit and to exploit their potential for the growth of the DDP and IEO. Additional goals of the DDP are to create a network of collaborators committed to excellence in drug discovery and to contribute to the education and training of talented young people to develop future leaders in drug discovery and cancer research. Starting from the basic science and the technologies developed at the Campus to understand the molecular basis of cancer, medicinal, analytical and computational chemists with industrial drug discovery experience, and biochemists, cell biologists and in vivo pharmacologists, all operating in state-of-the-art equipped laboratories, work together to identify new drugs and, through innovative research, to create a tradition of excellence in drug discovery. The biology-inspired, chemistry-driven effort to identify innovative therapies against cancer is a team effort developed in strict collaboration in particular with the Project Originators, the Molecular Medicine Program and TTFactor. Current pipeline includes Epigenetic targets, inhibitors of the Spindle Assembly Checkpoint (SAC) Kinases and alterations of self-renewal in cancer stem cells (CSCs).

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Department of Experimental Oncology

Mechanisms Controlling Chromosome Segregation Rosella VISINTIN, PhD Director

STAFF PhD Students: Francesca Montani, Michela Roccuzzo Technician: Clara Visintin Undergraduate students: Maria Nilda Biancardi

Activities 2011. Maintenance of genetic integrity

from one generation to the next requires the accurate replication of chromosomes during S phase and their faithful segregation during mitosis. Accomplishing these tasks requires the coordination of several processes in time and space. Understanding how cells inherit a correct number of chromosomes during cell division is a fundamental goal in biology as all solid tumors contain abnormal numbers of chromosomes. To ensure a correct transmission of chromosomes during cell division, replicated chromosomes (sister chromatids) must be

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separated and then segregated between the daughter cells. Sister chromatid segregation occurs in anaphase and is triggered by a protease known as separase, which severs the linkages that hold the sisters together. Up to metaphase, separase is restrained by its inhibitor securin. At the metaphase-anaphase transition, securin is targeted for degradation by the E3-ubiquitin ligase known as the Anaphase Promoting Complex or Cyclosome (APC/C) in complex with specificity factor Cdc20. The activity of the APC/CCdc20 is controlled by the spindle assembly checkpoint (SAC), a signaling pathway that delays sister chromatid separation (and hence anaphase onset) until all sister chromatids are bipolarly attached to the MTs of the mitotic spindle. Only when all sister kinetochores are correctly bound will the SAC be satisfied and will metaphase proceed into anaphase. Anaphase onset is commonly marked by the dissolution of the linkages holding sister chromatids together, yet it remains a matter of debate whether removal of cohesin is all there is to trigger anaphase. The current model proposes that cleavage of cohesin by separase is sufficient to reduce the forces that oppose the pulling force of the mitotic spindle, thereby promoting sister chroma- tid segregation. However, mounting evidence suggests that cohesinindependent factors may provide additional forces to resist mitotic spindle dynamics. Hence, these activities should also be nullified before sister chromatids can be segregated at anaphase onset. Anaphase segregation of the separated chromatids is mediated by kinetochore motors and spindle dynamics. Our lab studies chromosome segregation to better understand how errors made during this process contribute to the transformation of a healthy cell into a cancer cell. In particular, we investigate how phosphatases balance kinase activities thereby ensuring the correct execution of cell division events. We use the budding yeast Saccharomyces cerevisiae as a model system as the chromosome segregation process, and involved phosphatases and kinases are conserved from yeast to humans.

Publications Manzoni R., Montani F., Visintin C., Caudron F., Ciliberto A. and Visintin R. (2010). Oscillations in Cdc14 release and sequestration reveal a circuit underlying exit from mitosis. JCB, 190: 209-22

Visintin C., Tomson B.N., Rahal R., Paulson J., Cohen M., Taunton J., Amon A. and Visintin R. (2008). Apc/CCdh1- mediated degradation of the Polo kinase Cdc5 promotes the return of Cdc14 into the nucleolus. Genes Dev, 22: 79-90.

De Wulf P., Montani F. and Visintin R. (2009). Protein phopsphatases take the cell cycle stage. Current Opinion in Cell Biology, 21: 806-815. De Wulf P., and Visintin R. (2008). Cdc14B and APC/C tackle DNA damage. Cell, 134: 210-212.

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Clinical Research

Clinical Research

The last year has seen a gradual introduction of a disease-orientated Task Force structure to replace the previous Working Groups at the Institute. These new initiatives have been formed to ensure that Molecular Medicine scientists can integrate seamlessly into the clinical multidisciplinary groups. Also, each Task Force has now got dedicated infrastructural support in the form of statistics and data management. Each Task Force will have a rotating Chairman and vice chairs, plus a professional secretary. The purpose of their meetings is to brainstorm, then formulate new research translational projects, interdigitated with clinical protocols. Funding will be the Task Forces’ own responsibility, together with monitoring and publication of results. The Task Force should also adopt the responsibility of training in research methodology, the clinical and nonclinical research fellows working on the relevant disease site, irrespective of their professional discipline. Inevitably the evolution of the new structure means that the original examplar, the Breast Cancer Task Force, is extremely mature and very productive, whereas several others are still settling down. However, a large number of new projects have been planned and are described in the following pages. Here we are also reporting the summary of some of the results already obtained grouping them either for organ pathologies or for transversal research programmes. One chapter deals with the IEO Tumour Registry. A specific chapter has been added on Biobank which is now operative. Each research chapter had a small Writing Committee composed of scientists often not entirely involved in the described research activities. We are grateful to all of them for their efforts to sintetically describe the many activities developed in IEO with the aim of integrating laboratory and clinical research.

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Our current research lines are focusing mostly on chemoprevention, pharmacogenomics and the effects of lifestyle changes in various study cohorts of subjects at higher risk of developing cancer in order to validate the most effective strategies to reach a tailored prevention. Scientific aims: 1. Chemoprevention in subjects at higher risk for ER negative breast cancer, such as patients with endocrine non-responsive breast cancer and DIN, and BRCA1 mutation carriers. In particular, we are studying two classes of drugs: NSAIDs and Statins; the NSAIDs are tested in pre and post surgical models. 2. The possible role of metformin, raloxifene, very low dose tamoxifen, exemestane and celecoxib as chemopreventive agents in a pre-surgical model (figure1).

Research Activities

Cancer Prevention and Genetics

3. The optimization of the dose of tamoxifen and the selection of the population who can benefit most from tamoxifen as chemopreventive agent, through pharmacogenomics studies on Polyporphisms (SNPs), particularly on CYP2D6 and CYP2C19. 4. Polymorphisms of different genes that may correlate with breast cancer risk: among them we are studing VDR, IGFBP3, MTHFR in a large spectrum of population. 5. The role of aromatase inhibitors in prevention, evaluated in pre- and post-surgery settings. 6. The IGF system and sex-hormones as cancer risk factors and surrogate intermediate biomarkers of response to chemopreventive agents. 7. Ductal lavage as a risk assessment tool and source of new biomarkers studies. 8. Long term effects of radiotherapy in BRCA mutation carriers.

Figure 1: In order to increase the efficiency of drug development in breast cancer treatment and prevention, window of opportunity (WOP), pre-surgical research models are being used to screen the activity of the candidate agents and to characterize their mechanism of action. The different types of surgery offer different portion of tissue: from the pathological one to the adjacent often associated to a intraepithelial neoplasia and the distant one where is not infrequent to find atypical cells. All these variants may give different information to how and when a drug can be active

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Ongoing and starting projects: 1. A randomized phase II prevention trial with anti-COX2 vs. statins in subjects at high risk for hormone non-responsive breast cancer 2. IBIS II - an international multi center study of tamoxifen vs anastrozole in post menopausal women with Ductal Carcinoma in Situ ER+ (DCIS) 3. IBIS II - an international multi center study of anastrozole vs placebo in post menopausal women at increased risk of breast cancer 4. The HOT Study: Hormone Replacement Therapy and low dose Tamoxifen. A phase III trial of breast cancer prevention with low dose tamoxifen in HRT users 5. Pre-surgical study: to investigate the biological activity of metformin in all breast cancer histotypes. The primary endpoint is the KI67 modulation; several other secondary endpoints will be evaluated to study the interaction of the glucose metabolism and breast cancer. 6. The role of radiotherapy in carriers of BRCA mutations after conservative surgery. 7. A phase III study with fenretinide (4-HPR) for primary prevention of breast cancer in subjects at high risk for familial/hereditary breast cancer. 8. A randomized, placebo controlled, phase III clinical trial with low dose tamoxifen (5 mg day) in women with ER positive intraepithelial neoplasia of the breast. 9. DIANA(DIet and ANdrogens)-5: randomized controlled trial to test the efficacy of dietary change and physical activity to prevent or delay the recurrences in breast cancer patients estimated to be at higher risk based on their metabolic milieu. 10. A randomized placebo-controlled phase III clinical trial with vitamin D in colorectal cancer patients after radical treatment. 11. Green tea and silybin for breast cancer: a pilot pre-surgical study 12. Evolution of undetermined ld-CT detected lung nodules among a randomized phase II trial with

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inhaled budesonide 13. Hereditary diffuse gastric cancer study, The aim of this project is to screen the candidate subjects for CDH1 mutation and, if wild type, the allelic expression imbalance of this gene and its possible pathogenetic role will be analyzed. Major achievements and publications: A randomized 3-week presurgical trial of Lapatinib versus placebo in HER-2 positive breast cancer We conducted a placebo-controlled trial of lapatinib, a dual EGFR and HER-2 tyrosinekinase inhibitor, administered for 3-weeks between biopsy and surgery in 60 women with HER-2 positive breast cancer to assess its antiproliferative activity in hyperplastic, dysplastic and malignant tissue. Overall we observed a decrease in Ki67 LI in the neoplastic tissue in the treated group compare with an increased in the placebo arm. The effect of lapatinib was more pronounced in the estrogen receptors negative tumors and in those with an over expression of PTEN in the cytosol (figure2). The effects on Ki67 LI was consistent also looking at the associated intraepithelial neoplasia and the hyperplasic tissue, setting the rational to test lalatinib also in the HER-2 positive intraepithelial neoplasia. Prognostic effect of circulating adiponectin in a randomized 2x2 trial of low-dose tamoxifen and fenretinide in premenopausal women at risk for breast cancer We investigated the associations of plasma leptin and adiponectin with mammographic density and

Figure 2 Response of Ki-67 LI to lapatinib based on Phosphatase and Tensin homolog (PTEN) pattern of expression.

disease status and assessed their prognostic effect on recurrence free survival in premenopausal women at risk for breast cancer. Wihit a chemoprevention traial we studied in 235 premenopausal women with pT1mic/ pT1a breast cancer (n=21) intraepithelial neoplasia (n=160), or 5-year Gail risk of 1.3% or greater (n=54). Median adiponectin levels were lower in affected than in unaffected women (P=.006). After a median of 7.2 years and total of 57 breast neoplastic events, there was a 12% reduction in the risk of breast neoplastic events per unit increase of adiponectin (adjusted hazard ratio, 0.88; 95% CI, 0.81 to 0.96; P=.03). Low adiponectin levels are associated with a history of prior intraepithelial neoplasia or pT1mic/pT1a breast cancer and higher risk of second breast neoplastic events in premenopausal women (figure 3). Our data support the role of adiponectin may be used as biomarkers for chemoprevention trial.

The other two gene CYP2C19 and SULT1A1 with their polymorphisms did not show any correlation with tamoxifen efficacy. Although there are conflicting results in the literature on the role of CYP2D6 polymorphisms, we have further data to support the biological interaction CYP2D6 phenotype and tamoxifen activity

Research Activities

9. Chemoprevention of breast and ovarian cancer with Fenretinide in young healthy BRCA mutation carriers. 10. Diet and physical activity to prevent recurrence after standard treatment in women with invasive breast cancer. 11. Role of an accurate process to select high risk subjects for genetic counseling and their psychological and emotional response.

Efficacy of tamoxifen based on cytochrome P450 CYP2D6, CYP2C19 and SULT1A1 genotype in the Italian Tamoxifen prevention trial Seeking to improve and tailored chemoprevention strategy, we have conduct a comprehensive analysis of allelic variants of CYP2D6, CYP2C19 and SULT1A1 within the Italian Tamoxifen Chemoprevention clinical trial. Tamoxifen is a pro-drug undergoing considerable first-pass oxidative metabolism into more potent active metabolites. The individual genotypes may determine the differential tamoxifen efficacy. The results showed that the subjects with no CYP2D6 enzyme activity, and allocated to the tamoxifen arm, showed a higher risk of developing breast cancer compared to the remaining phenotypes (P=0.035).

Figure 3 Disease-free survival curves according to median adiponectin levels in premoenopausal women.. There was a reduced breast cancer risk with increasing baseline adiponectin level.

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Anti-VEGF and beyond: Shaping a new generation of anti-angiogenic therapies for cancer

The class of anti-angiogenic drugs has been one of the very few able to gain international approval for clinical use in oncology in the last decade. Most of the biological and clinical activity of the currently available generation of anti-angiogenic drugs targets VEGF and its related pathways. However, the clinical benefit associated with the use of these drugs has been so far limited, and there is an unmet need for biomarkers able to identify patients who are most likely to benefit and to suggest the best schedule and dosage of these drugs. Here we discuss some of the emerging new combination strategies involving the approved anti-angiogenenic drugs, some of the emerging targets associated with neoplastic angiogenesis, and some novel agents used as a paradigm of the next generation of anti-angiogenic drugs. Introduction Anti-angiogenic drugs bevacizumab, sunitinib, sorafenib and pazopanib have been approved in the past 5 years for the therapy of advanced colo-rectal, breast, lung, kidney and central nervous system cancer (Kerbel, 2008). These drugs, alone or in combination with chemotherapy, have shown in randomized clinical trial to be able to improve overall (OS) or progression-free survival (PFS) in cancer patients. However, the clinical benefit associated with these anti-angiogenic drugs has been so far limited to a few months, and the very large majority (if not all) of the treated patients has suffered clinical progression. Two major drawbacks limit the clinical development of this class of drugs. First, it is still unclear whether the limited impact on patients’ OS and/or PFS is due to the presence of a small clinical benefit in every patient or to the presence of a larger benefit in a subpopulation of patients, an effect to be diluted in large clinical trials enrolling unselected patients. The lack of biomarkers able to select the patients who are most likely to benefit from these targeted drugs (as is for instance

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the presence of the BCR-ABL gene rearrangement for leukemia patients to be treated with imatinib, nilotinib or dasatinib), in fact, hampers a rational use of this class of drugs in adequately selected patient populations where a clinical benefit should be relevant. Second, the same lack of validated biomarkers severely limits the ability to determine optimal biologic dose (OBD) and scheduling of these drugs. Most of the biological and clinical activity of the four anti-angiogenic drugs currently approved for cancer therapy is against the vascular endothelial growth factor (VEGF)-related pathways. Here we discuss how to improve the clinical efficacy of the anti-angiogenic therapy of cancer by describing some innovative approaches, some paradigmatic emerging new targets in neoplastic angiogenesis and the related drugs that might be used in the future along, with, or after anti-VEGF drugs. Mechanisms of resistance to anti-VEGF in neoplasia, and the dilemma of anti-VEGF therapy beyond progression Anti- angiogenic drugs are currently thought to act against neoplasia by four major mechanisms: a) pruning of tumor neovessels, b) vascular normalization (which potentially results in improved delivery of chemotherapy drugs to tumor tissue), c) inhibition of the mobilization of pro-angiogenic cells towards the tumor and d) inhibition of autocrine and paracrine growth factor cross talks between tumor, endothelium and stroma cells. The adaptive mechanisms of resistance to the current generation of anti-angiogenic drugs targeting mostly VEGF, as described by Bergers and Hanahan (2008) and summarized in Fig. 1, are likely to be 1) activation and/ or upregulation of alternative pro-angiogenic signalling pathways within the tumor and/or recruitment of proangiogenic cells, both of which can obviate the necessity of VEGF signalling, thereby effecting reinitiation and continuance of tumor angiogenesis; 2) increased

pericyte coverage of the tumor vasculature, in order to support its integrity and attenuate the necessity for VEGF-mediated survival signalling; and 3) activation and enhancement of invasion and metastasis to provide access to normal tissue vasculature without the need for neovascularization. Moreover, there is evidence that certain tumors may have a pre-existing tumor microenvironment supporting intrinsic resistance to antiVEGF therapies. In a recent clinical trial where advanced breast cancer patients were treated with metronomic chemotherapy plus the anti-VEGF antibody bevacizumab (Dellapasqua et al, JCO 2008; Calleri et al, Clin Cancer Res 2009), the risk of relapse was markedly increased in patients who – after two months of therapy – did not achieve a reduction of circulating VEGF levels below a given threshold. This finding – to be confirmed in larger trials - suggests that for the population of patients with high VEGF levels in spite of bevacizumab treatment a possible approach might be to increase to dosage of bevacizumab or to switch towards novel strategies of VEGF inhibition. On the other hand, a large clinical trial has shown that in some cancer patients treated with drug combinations including the anti-VEGF monoclonal antibody bevacizumab, the administration of bevacizumab beyond tumor progression was associated with an improved clinical outcome (Grothey et al, 2008). In the BRiTE study enrolling about 2,000 patients with metastatic colorectal cancer whose first-line therapy consisted of bevacizumab plus chemotherapy, postprogression treatment and survival data for 1,445 patients were obtained, and of those, 642 (44%) continued bevacizumab during second-line treatment. When compared with the 531 patients (37%) who did not receive bevacizumab with their second-line therapy, the median overall survival was 31.8 months versus 19.9 months (hazard ratio 0.48; P <0.001). Even though this OS advantage might well be due to the selection of a subgroup of patients who could tolerate more intense therapy in addition to any therapeutic advantages offered by bevacizumab, these data deserves to be further investigated in a randomized confirmatory trial. Furthermore, adequate preclinical models should be generated and investigated to understand whether a benefit of prolonged anti-VEGF therapy in tumors escaping from therapy does exist and is due to vascular normalization (and better delivery of other antineoplastic drugs) or to an inhibition of cellular and/or molecular pathways other than those directly targeted by the anti-VEGF antibody.

Cell-driven escape from anti-VEGF therapy, and related new therapies in sight. A variety of circulating endothelial and hematopoietic cell populations are known to play a possible role in the generation and maintenance of cancer vessels. Recent evidence also suggests that – at least in mice - a population of myeloid cells may play a crucial role in the escape from anti-VEGF therapies. CD11b+Gr1+ cells are frequently increased in the tumors and in the peripheral blood (PB) of tumor-bearing animals, and have been shown to promote tumor angiogenesis (Shojaei et al, 2009). These cells are also known to suppress immune functions, and have been defined as myeloid-derived suppressor cells (MDSC). In murine models of cancer, MDSCs have a role in mediating refractoriness to anti-VEGF treatment. In fact, MDSCs produce several angiogenic factors including Bv8, a secreted protein previously characterized as an EC mitogen, an hematopoietic growth factor, and as a neuromodulator (Shojaei et al, 2007). Bv8 promotes tumor angiogenesis through increased PB mobilization of myeloid cells such as MDSCs and local stimulation of angiogenesis. Granulocyte colony stimulating factor (G-CSF) was found to be as a strong inducer of Bv8 expression, both in vitro and in vivo. In a recent report, G-CSF and Bv8 were found to have the strongest correlation with refractoriness to anti-VEGF in preclinical tumor models. Treatment with anti-G-CSF antibodies had little or no effect on the growth of tumors sensitive to anti-VEGF therapies, but anti-G-CSF (or anti-Bv8) therapy resulted in reduced tumor angiogenesis and growth, and was additive to anti-VEGF mAb in refractory tumors. Moreover, anti-G-CSF treatment reduced circulating and tumor-associated MDSCs to the levels detected in mice bearing sensitive tumors. Conversely, treatment of mice bearing sensitive tumors with recombinant G-CSF or implantation of G-CSF-transfected cells resulted in increased MDSC numbers and reduced responsiveness to anti-VEGF therapy. These data suggest that the mobilization of proangiogenic cells such as MDSCs involved in escape from anti-VEGF therapy might be targeted with appropriate drugs. This approach needs to be tested in clinical trials, and it remains to be elucidated what is the most appropriate timing (before or after the insurgence of antiVEGF refractoriness?) for such a therapeutic strategy.

Research Activities

Clinical Research

Anti-VEGF and more: the emerging role of combinatory therapies Targeting several interconnected signalling pathways might theoretically result in an increased therapeutic benefit, particularly as the tumor might use alternative

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VEGF VEGFR-TKI. The combination of an anti-VEGF antibody with a VEGFRs-TKI has been suggested to maximize the inhibition of VEGF–VEGFRs signalling by targeting both the ligand and the receptor. Despite increased toxicities and consequently dose reduction, in some phase I and phase II trials in ovarian and kidney cancer the association of bevacizumab plus sorafenib was found to be highly active, suggesting enhanced antitumour activity (Azad et al, 2008). On the other hand, in phase I studies the association of sunitinib, which inhibits a broad range of TKs, plus bevacizumab in patients with kidney cancer was associated to an high incidence of grade 3 to 4 hypertension, thrombocytopaenia and proteinuria, and no substantially different response rate when compared to sunitinib alone. For these reasons, combination trials of the two agents are no longer pursued. This concept however might warrant to be further exploited by sequential administration of bevacizumab with more selective and/or potent VEGFRs TKIs. VEGF and m-TOR. The mammalian target of rapamycin (mTOR) has emerged as a critical effector in cellsignaling pathways commonly deregulated in human cancers. This has led to the prediction that mTOR inhibitors may be useful in oncology, and derivatives of one such molecule, rapamycin (from which mTOR derives its name), are currently in clinical development (Feldman et al, 2009). In preclinical studies, the combination of VEGF and mTOR inhibitors has demonstrated additive or synergistic anti-tumour effects in multiple in vivo models, including hepatocellular carcinoma, kidney, ovarian and pancreatic cancer. VEGF and epithelial growth factor (EGF) receptors. To date, the clinical benefit of dual EGFRs and VEGF inhibition over VEGF inhibition alone has been confirmed only with the combination of bevacizumab and erlotinib. When compared to bevacizumab alone, the combination showed a significant clinical advantage as a maintenance therapy in patients with advanced non-small cell lung carcinoma (Miller et al, 2009). By contrast, this combination did not show a clinical advantage in

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advanced pancreatic, breast cancer and kidney cancer, likely because the molecular synergism occurred only in a small patient population not adequately identified in the latter trials. Although treatment with anti-VEGF and other targeted agents may results in unexpected side effects, there are also some hints of an interesting possible clinical synergy, at least in some types of cancer. Patients should continue to be monitored carefully for evidence of toxicity, particularly because prolonged periods of therapy may be required and side effects may impact treatment compliance. Vascular-restricted antigens as targets for antiangiogenic therapies complementary to anti-VEGF: Activin receptor-like kinase 1 (ALK1) as a paradigm ALK1 is a type I transforming growth factor beta (TGF-beta) receptor showing expression, signalling, and functional properties distinct from other TGFbeta receptors (Massague 2008). ALK1 expression in adults is virtually restricted to proliferating endothelial cells (ECs), but studies in ALK1-/- mice and zebrafish harbouring a loss-of-function mutation demonstrated that ALK1 plays a key role in vessel maturation, in recruitment and differentiation of perivascular cells (PCs), and in the organization and patency of neoangiogenic vessels. Type 2 hereditary hemorrhagic telangiectasia, an autosomal dominant vascular dysplasia syndrome, is linked to LOF mutations in ALK1. ALK1 signaling is context-dependent and can be either pro- or anti-angiogenic. A few different ALK-1 inhibitors have been developed recently, and tested in vivo and in the clinic. Among these, PF3446962, which is a human antibody against ALK1, developed at Pfizer in studies in collaboration with IEO (Hu-Lowe et al, 2011). Mice bearing xenograft tumors, or pancreatic neuroendocrine tumor (the RIP-tag mouse model), treated with anti-ALK1 agents, demonstrated a reduction in tumor burden and increase in survival, through antiangiogenesis. VEGF and FGF stimulate ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and EC tubulogenesis, and a fully human monoclonal antibody specifically targeting ALK1 markedly inhibited these events. Consistently, a murine anti-ALK1 suppressed angiogenesis stimulated by VEGF and FGF in a matrigelbased model in mice, and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, the combination of anti-ALK1 with VEGF RTKI delayed tumor growth and disturbed vascular normalization

associated with VEGF receptor inhibition. Additionally, in a xenograft tumor model grown in a human foreskin engrafted in SCID mice, targeting human ALK1 decreased human vessel density, and improved antitumor efficacy by bevacizumab. Finally, anti-angiogenesis and antitumor efficacy of anti-ALK1 were associated with disrupted colocalization of ECs with desmin+ PCs, and reduction of blood flow primarily in large/mature vessels assessed by contrast-enhanced ultrasonography. These data imply that ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. In the same report, ALK1 was found to be expressed in the blood vessels of many human tumor types and in circulating ECs (CECs) from patients with advanced cancers. Clinical phase I studies of the antiALK1 antibody PF-03446962 and ALK-1-Fc fusion proteins are currently ongoing and showing acceptable toxicity and early sign of clinical activity (Gallo-Stampino C, and Bendell JC, personal communications). Taken together, these findings suggest that ALK1 blockade may represent an effective anti-angiogenesis therapy complementary to the currently available anti-VEGF drugs, and offer a paradigm for the clinical development of novel drugs targeting additional ECrestricted targets in human cancers. Targeting angiogenic growth factors and pathways other than-VEGF: Fibroblast growth factors (FGFs) as a paradigm In addition to VEGF, some other EC-related growth factors are known to play a possible role in cancer vascularization. For the sake of brevity we will not discuss all of them and will use the FGF family as a paradigm. FGFs are involved in many physiological processes, including embryonic development as well as cell proliferation and differentiation, angiogenesis and wound repair in adult organism (Knights et al, 2010). FGFs exert their function through FGF receptors (FGFRs), named FGFR1 to FGFR4, which belong to the family of receptor tyrosine kinases (RTKs). Each receptor comprises a single-pass hydrophobic transmembrane domain, an extracellular N-terminal ligand binding domain, and a C-terminal cytoplasmic region that contains a TK domain which is responsible for the intracellular FGF signal transduction. In addition to the known role of FGFs in tumor vascularization, aberrant FGF signalling have been identified in many types of tumours. FGFR1 amplifications occour mainly in breast and ovarian cancer, while rarely FGFR1 shows missense mutations and chromosomal traslocations. FGFR2 is amplified in 10% of gastric cancer, and in some cases of breast and colorectal

cancer; missense mutations occur in 12% of endometrial cancer and more rarely in other type of cancer. FGFR3 shows missense mutations in 50-60% of bladder cancer. Traslocations of FGFR3 are showed in 15% of myeloma, while FGFR3 amplifications are less common. The last member of the FGFR family is FGFR4 which is mutated in rhabdomyosarcoma and in lung cancer. At the present there are three major approaches to FGF and FGFRs inhibition in human cancer: TKIs, anti-FGFRs antibodies, and FGF ligand traps. All of the TKIs that are in the early phases of clinical trials are multi-tyrosine kinase inhibitors because of the high structural similarity of TKs domain among different RTKs.

Research Activities

signaling pathways as escape mechanisms. In fact, the selective inhibition of multiple receptor and intracellular pathways of tumor cells might overcame drug resistance and improve long term control of malignancies (Kummar et al, 2010). This strategy has led to combine antiVEGF monoclonal antibodies with agents targeting other pathways like VEGFRs-targeting tyrosine kinase inhibitors (TKI), m-Tor- and EGFRs- inhibitors.

Blocking the activity of Angiopoietin1/2 and Tie-2 as an antiangiogenic therapy. In addition to VEGF and its pro-angiogenic effects on endothelial cell proliferation, survival, and tube formation, also the localized expression of angiopoietin 2 (Ang2) on endothelial cells has been shown to play a role in blood vessel remodelling in general, and in tumor vasculature in particular (Dumont et al, 1994). The Ang family has four members (Ang 1-4) and two related receptors (Tie-1-2), whereas only Ang1 and Ang2 are involved in vascular function and integrity. While Ang-1 is important for vascular integrity, Ang2 is found to act as an antagonist and agonist for the Tie-2 receptor, which can mediate signalling of endothelial cell migration, survival, and vessel formation. In clinical specimens, Ang-2 has been shown to act as a predicting prognostic factor to cancer progression and clinical outcome (Huang et al, 2010). Due to the proangiogenic effects of Ang-2 in tumors, drugs which target this ligand or its receptor have been developed. AMG-386 is an antiangiopoietic peptibody comprising a peptide with angiopoetic biding properties that is fused to the Fc region of an antibody and inhibits the interaction between the ligands Anfg-1 and Ang-2 to the receptor Tie-2 (Oliner et al, 2004). In phase I clinical trials, AMG-386 was administered as a single agent or in combination with chemotherapy, and exhibited an antitumor activity with only mild toxicities. Phase II trails are ongoing for the treatment of several malignancies such as breast, ovarian, colorectal, gastric and rencal cell cancers. Another potential drug which interferes with the Ang-Tie signaling is a human monoclonal anti-Ang2 antibody. As opposed to the peptibody, such drug has minimal immunogenic activity, and its half-life is much longer. This antibody blocks Ang-2 activity, and inhibit the signalizing of its Tie-2 receptor. Preclinical studies showed that it can block blood vessel formation in tumor xenografts (Brown et al, 2010), and therefore can serve

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Conclusions VEGF inhibition, alone or (more frequently) in combination with chemotherapy, has showed clinical activity in several types of cancer. When present, though, the clinical benefit was transitory, and tumor growth was eventually observed. To overcome resistance and tumor escape, new strategies are urgently needed. Because of space constraints, we could not discuss all the antigens and pathways currently investigated as possible new targets for the anti-angiogenic therapy of cancer, and used some of these emerging targets as paradigms. One can foresee possible clinical developments in the inhibition of the VEGF pathway beyond clinical progression for patients who had a significant and persistent normalization of the tumor vasculature associated with an improved delivery of chemotherapy. For patients who did not achieve such a vessel normalization by VEGF inhibition, or in tumor types insensitive to chemotherapeutics associated to anti-VEGF, a switch towards drugs targeting different mechanisms of action might be considered, including the inhibition of autonomous tumor cell survival and proliferation

signaling, pro-angiogenic cell enrichment at the site of neoplastic growth, and the inhibition of pro-angiogenic pathways alternative and/or complementary to VEGF such as, e.g., those related to Ang/Tie2, ALK1 or FGFs or – for instance - PHD2 pathways. The clinical development and validation of these new strategies would receive a great and decisive advantage from the availability of biomarkers able to select who, in a given patient population, is most likely to benefit from these new drugs and strategies and what will be the most appropriate therapy after escape. Along a similar line, biomarkers should be of help for the prediction and management of side effects associated with the use of the current and next generation of anti-angiogenic drugs..

Fig.1: Major hypotheses currently used to describe adaptive mechanisms of resistance to the current generation of anti-angiogenic drugs targeting mostly VEGF, as described by Bergers and Hanahan (2008). a) activation and/or upregulation of pro-angiogenic signalling pathways other than-VEGF (eg FGFs) within the tumor; b) recruitment of pro-angiogenic cells. c) increased pericyte coverage of the tumor vasculature to support its integrity in the absence of VEGF-mediated survival signalling; d) activation and enhancement of invasion and metastasis to provide access to normal tissue vasculature without the need for neovascularization.

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Clinical Research

Circulating Tumor Cells

The objective of this group led by Franco Nolè and Maria Teresa Sandri is to understand the role of Circulating Tumor Cells (CTCs) in patients with different malignancies. Cancer dissemination involves CTCs and represents a crucial step for metastasis. CTCs are very rare cells surrounded by billions of hematopoietic cells in the bloodstream. Although the presence of CTC in peripheral blood was first reported in 1869, a lack of sensitive technology precluded the detailed study of these cells until recently. In the last 10 years technological advances have facilitated the identification, enumeration and characterization of CTCs, using different methods such as PCR, flow cytometry, image-based immunologic approach and microchip technology. However, given the rarity of CTCs, the biological and clinical insights into CTCs are strongly dependent on the isolation and detection technique used. Initial studies, using the FDA approved system CellSearch (Veridex LLC, Warren, NJ), have focused on general prognostic implications of CTCs counts above a pre-determined level in patient survival studies (typically 5 CTCs/7.5 ml of blood): patients with metastatic disease who were “CTCs positive” had a worse prognosis than those without detectable CTCs. While these studies have demonstrated the clinical impact of CTC determination, they have not provided information useful for the therapeutic management of the patients. More recently, the focus of the research is toward CTCs characterization in order to reveal new therapeutic options for cancer patients and to provide new insights into the biology of cancer and the process of metastasis. Breast Cancer To date different studies pointed out the prognostic value of CTCs in patients with advanced breast cancer. Using the CellSearch technology, we monitored 80 women with advanced disease starting a new line of treatment and/or treated for the advanced disease with a maximum of two previous lines of therapy.

Research Activities

as a possible new anti-angigoenic therapy candidate. Taken together, growing body of evidence indicates that the blockage of Ang-2 may act as an anti-tumor agents which can inhibit angiogenesis and even invasion and metastases. However, more clinical studies should be performed to test its clinical efficacy, and toxicity.

In this study we observed that CTC were elevated (≥5/7.5 ml blood) in 49 patients at baseline and that the baseline number of CTCs was associated with PFS (hazard ratio [HR] 2.5; 95% confidence interval [CI] 1.2– 5.4). The risk of progression for patients with CTCs ≥ 5 at the last available blood draw was five times the risk of patients with 0–4 CTCs at the same time point (HR 5.3; 95% CI 2.8–10.4). At the last available blood draw, patients with rising or persistent CTCs ≥ 5 demonstrated a statistically significant higher risk of progression with respect to patients with CTCs < 5 at both blood draws (HR 6.4; 95% CI 2.8–14.6). These results confirmed that elevated CTCs levels measured at any time in the clinical course of a patient with metastatic breast cancer predict an imminent progression (Nolè F. et al. Ann Oncol 2008). A subsequent analysis was performed on this population and the results confirmed a clear association between CTCs and OS (P-value <0.01), and, analyzing the CTCs as a continuous variable, an increase in risk with increasing number of CTCs, for both PFS and OS, showing a non-linear increase in risk of both progression and death with increasing number of CTCs with a lessening increase after approximately 5 CTCs (Botteri E. et al Breast Cancer Res and Treat 2010) In addition to enumeration, an exciting area of CTC research involves the phenotyping and expression profiling of CTCs. In this regard, the evaluation of CTCs could be considered as a real-time biopsy allowing, in most patients with metastatic breast cancer, the possibility to evaluate a possible change in tumor genotype and phenotype during the clinical course of the disease. Between June 2007 and October 2008, a total of 76 patients with advanced breast cancer were enrolled in a trial evaluating phenotypic HER2 characterization. Fifty-seven patients had at least 1 CTC in 7.5ml of blood (prevalence 75%). Median CTCs number was 7/7.5 ml (range: 1- 4988). Concordance of HER2 phenotype

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Few reports exist on the role of CTCs in primary breast cancer, and most of them consider patients after surgery and before the start of chemotherapy. We evaluated the role of CTC enumeration in 56 patients with operable breast cancer, before surgery and 5 days after surgery. In case of positivity of one of the perioperative samples, another sample was taken after 30 days. This study provides evidence of the presence of CTCs in approximately 30% of patients with localized breast cancer both before and after surgery, with change from positive to negative and vice versa in 40% of cases. No association with the pathological variables was found, except for vascular invasion and presence of preoperative CTCs. Long-term follow-up will be required to understand the significance of these data. (Sandri MT et al Ann Surg Oncol 2010) Ongoing and starting projects Advanced Breast Cancer Evaluation of acquired overexpression of HER2/neu on CTCs in patients with advanced breast cancer during chemotherapy, and assessment of activity of Trastuzumab-based therapy Several studies demonstrated that the presence of a target antigen can be quantitatively analyzed using CTCs isolated from blood. In particular, there are some evidences of the possibility that HER2 gene amplification can be acquired during progression of cancer, demonstrating a phenotypic conversion from HER2- to HER2+ during the course of the treatment. Based on these data, we are currently running a phase II study with the aim to evaluate the efficacy of Trastuzumab-based therapy in patients with acquired HER2/neu overexpression on CTCs. The study is ongoing and 74 patients have been enrolled. Locally advanced disease Another field of investigation is represented by the possible detection among CTCs of cells bearing stemness characteristics and having acquired the features of mesenchimal cells through the Epithelial Mesenchimal

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Transition (EMT) Process. Some markers are under evaluation to better define this subgroup of cells: ALDK1 (for stemness), Twist, Akt2 and PIk3a (for EMT) are some of the available. We are investigating the detection and the expression of the above mentioned markers on CTCs in a phase II study using epirubicin, cisplatin and infusional fluorouracil (ECF) followed by weekly paclitaxel plus metronomic cyclophosphamide ± Trastuzumab as preoperative treatment in locally advanced ER and PgR negative breast cancer patients. We enrolled 10 patients, who were followed during the therapy, but in none of them any CTC bearing characteristics of EMT or stemness could be identified. Projects in Prostate Cancer Prostate cancer is the second leading cause of cancer death in men in Western countries. Most prostate cancer deaths are caused by haematogenous metastatic spread and subsequent tumour cell growth in distant organs, especially the bones. Currently, treatment of patients with disseminated prostate cancer is based on direct cytotoxicity against tumour cells, via androgen deprivation therapy or docetaxel-based chemotherapy. Prostate cancer does not usually lead to radiologically measurable metastatic disease because the bone is typically the site of metastatic spread. A major problem in the evaluation of the treatment of advanced prostate cancer is the lack of strong surrogate markers for disease outcome and clinical benefit. Prostate-specific antigen (PSA) is therefore currently widely used as a marker of treatment activity although PSA does not always correlate with decreased tumour growth, overall survival (OS) or quality of life. Assessment of CTC using CellSearch has been cleared by the Food and Drug Administration as a prognostic indicator for patients with metastatic prostate cancers. Several groups have shown that CTC are detected at high frequency in castration resistant prostate cancer and are correlated with clinical outcomes. As a result, we conducted a prospective study with the aims to evaluate the predictive role and the prognostic impact on clinical outcome of the baseline level of CTC and the impact of quantitative changes in CTCs during chemotherapy in patients with castration resistant tumor. Fifty-six patients were enrolled in the study. Fortyseven (81%) had detectable CTCs, with a median of 12 cells/7.5mL of blood (range 0-1959). CTC number was significantly correlated with PSA (p=0.05), alkaline phosphatase (p<0.01), bone metastases (p=0.01) and

bone plus visceral metastases (p=0.02). Univariate analysis showed that CTC count was strongly associated with both median PFS (p=0.001,) and median OS (p<0.001, 95%). In this study, we demonstrated the predictive superiority of CTCs over absolute PSA value at baseline and clearly over post-treatment PSA changes from baseline to first evaluation. Patients accrual has been completed in 2010.

Research Activities

between the primary tumor and CTCs in the basal sample was 86% and 82% during treatment. Fourteen percent of the patients with HER2 negative primary tumor acquired HER2 overexpression on CTCs before starting the new chemotherapy regimen. Two patients (13%) with HER2 positive disease in the primary tumor did not show HER2 overexpression on CTCs before atarting the new chemotherapy regimen (Munzone E. et al. Clinical Breast Cancer 2010)

At the end of 2010 a project in patients with clinically localized prostate cancer, eligible for radical prostatectomy was activated. In this population, we will evaluate the prognostic impact of the presence of CTC before any therapy in terms of correlation between presence of CTC, pathological stage, baseline PSA levels, Gleason score and Tumor Volume. In 2011 we enrolled 18 patients, and for some of them we had also the followup samples. The study is ongoing. Projects in colorectal cancer CTC count both at baseline and on therapy in metastatic colorectal cancer patients beginning a new therapy have been shown to be a strong independent predictor of clinical outcome. Patients with favourable CTC at baseline and follow-up have significantly improved outcome compared to patients with unfavourable CTC at baseline and follow-up. Those patients converting from unfavourable to favourable have outcomes that more closely track with patients with favourable counts throughout. In patients with locally advanced rectal cancer few prognostic markers are available to date to better define the impact of chemoradiotherapy. To evaluate if CTC may be a useful marker in this clinical situation in 2008 we started a study aimed at the evaluation of CTCs in patients undergoing curative surgery for advanced rectal cancer. In these patients CTCs are determined before and after chemotherapy and surgery, and the association with Disease Free Survival and Overall Survival will be evaluated. Eighty-seven patients have been enrolled to date: the study recruitment is nearly completed, and final analysis will be performed in 2012.

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Virology

The objective of the group led by Maria Teresa Sandri and Mario Sideri is to understand, cure and prevent the process of virus-induced tumor development, and particularly to study the role of Human Papilloma Virus (HPV) in the development of different tumors like those of the head and neck, anus, vulva, and cervix. Infections with HPV are extremely common, but rarely cause serious primary disease in healthy individuals. Nevertheless, these viruses may persist after primary infection and cause tumor disease later. A cause-effect relationship exist between certain types of HPV, e.i. the oncogenic or “high risk HPV” genotypes, and cervical cancer, and the same types have now been associated with other malignancies, such as head and neck, anus and penis cancer. Nonetheless, a persistent infection is necessary but not sufficient to lead to neoplasia development. Vaccines: a new strategy for prevention and cure? We still lack precise knowledge regarding the pathology of HPV. Why do these viruses sometimes persist? And why does virus persistence lead to tumor development and disease? It is obvious that the immune system plays a crucial role in the interplay between viruses and hosts, since tumor development and disease are more frequently observed in immunosuppressed individuals, like women infected with HIV who are more frequently affected by HPV-related diseases. Many studies have been performed to develop preventive and curative vaccines. In 2007 two preventive vaccines were introduced and are now available: they are both directed at the eradication of infection with HPV 16 and 18, which are known to cause about 70% of cervical cancer. Our group is involved in a study looking at the response to the vaccination of 18 years old adolescents living in the Milano and Monza area who are vaccinated with GARDASIL and followed for a five-year period. The primary endpoint is the incidence of viral infection, looking at the new epidemiology and prevalence of viral

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HPV genotypes in vaccinated girls. The project started in spring 2008 and almost 600 girls have been vaccinated so far. Results at baseline showed HPV positivity in about 10% of sexually active girls. Another interesting question is the value of HPV vaccination in older women; at present the vaccine is licensed for use in women up to 45 years. In particular there is some evidence of vaccine efficacy in women treated for HPV related diseases. A multicentric study has been approved and will start in 2012 directed to the evaluation of the HPV vaccine protection in women submitted to treatment for CIN and condylomata. Cervix cancer screening and HPV The primary research of our group is focused on the role of HPV in cervix cancer. Pap smear screening has revolutionized cervix cancer screening since its introduction into the population. In Italy there is both organized and opportunistic screening: thanks to this approach the prevalence of cervix cancer has dramatically decreased and nowadays about 3500 new cases per year are diagnosed. In recent reports, however, there were observations suggesting an increase in cervix cancer incidence, especially of the adenocarcinoma of the cervix. Apart from those cases observed in women who do not regularly perform a screening test, the Pap test’s insensitivity and lack of specificity have been the main reasons for the remaining cases of cervix cancer despite the introduction of the screening. It is well known that cervical cancer is preceded by a long lag time of 15 to 20 years from early premalignant changes to invasive cancer. The etiologic role of human papillomavirus (HPV) in cervical cancer has been well established by scientific evidence, and more than 99% of these cancers can be attributed to HPV. (zur Hausen, Cancer Res 1989). Although not all women who are infected will develop cervical cancer because other cofactors play a fundamental role, a reasonable approach

is represented by the direct search of the presence of the oncogenic HPV types in cervical samples, both in the screening population and the patients treated for a pre-neoplastic lesion. In particular in this last group of subjects, the persistence of HPV after the treatment during the follow-up would allow the selection of a group of patients bearing a higher chance of relapse and hence needing more intensive controls. On the contrary, the absence of HPV would identify those patients effectively cured by the conservative treatment, in whom intensive follow-up is not needed. The introduction of HPV testing and genotyping in screening requires information on the genotype distribution in positive pap smears and cervical pre-cancerous and cancerous lesions. Our group investigated this aspect in detail and a first paper was published (Sandri et al, J Med Virol. 2009); in a second paper (Sideri et al., Gyenecol Oncol, 2011) a different age distribution was observed between lesion associated with HPV 16-18 and lesions associated with other high risk HPV genotypes; a third paper, the largest Italian series published so far, investigated the distribution of HPV genotypes in invasive cervical cancers in Italy (Sideri et al, Vaccine 2009). New and old diagnostic biomarkers New tests are now being proposed for the detection of the presence of the DNA of HPV in cervical samples. We think that it is very important to evaluate both the analytical and the clinical performance of the newly introduced test, and one of our aim is to present to the scientific community the advantage and disadvantage of the different methods. The results of the comparison of the two methods available have been the topic of a paper recently published (Sandri MT et al, J Clin Microbiol 2006). Apart from the presence of the infection with HPV, we are looking for other diagnostic tool which may help in a better characterization of the lesion, such as evaluation of P16 and chi 67, genotyping, specific mRNA for E6 and E7, viral load. Following the first published studies in which the relevance of bearing HPV 16 or 18 compared to the other oncogenic genotypes leads to a different risk of developing a preneoplastic lesion, we are studying the distribution of the different genotypes in patients with cervical lesions. In fact as it is viral persistence that is considered to be the true pre-cursor of neoplastic progression, DNA genotyping approaches designed to identify type specific persistence, could be more appropriate for identification of those individuals at greater risk of disease progression compared with a presence/absence test. Moreover the prevalence of the different oncogenic

HPV in patients with cervical lesion is still not well known in the Italian population and we are studying the genotype distribution. A key information is related to the performance of the different genotyping systems: we performed a study in collaboration with a group of Marseille showing that two systems commercially available show similar performance (Halfon P, Sandri MT, JMV 2009). A promising marker of progressing lesions is the determination of p16 overexpression. P16 identification by immunostaining can be considered as a marker, not only of HPV infection, but also of activated expression of viral oncogenes and of virusinduced deregulation of the cell cycle. A large multiinstitutional study was concluded in 2009 evaluating double staining on liquid based cytology specimens, in screening and triage of borderline lesions on 27.000 women. The PALMS study was completed in 2010 and included women from screening and borderline cytology triage. The results showed that double stain with the two proteins has a higher specificity than HC2, maintaining the sensitivity of the viral test. A publication of the study results has been submitted. Viral load was investigated in a joint research project with San Raffaele Hospital. More than 700 cases of ASC-US cytology were investigated. The results showed a significant correlation between viral load, expressed as RLU obtained by HC2, and CIN2+ detection (M. Origoni et al., Eur J Clin Microbiol Infect Dis, accepted). The objective of the investigational research is to better characterize women with HPV infection in order to build an algorithm allowing a tailored approach to the different situations. The use of biomarkers gives better positive predictive value as compared to HPV DNA testing alone. With improved diagnostic methods and triage in the detection of precursors and providing efficient treatment, the incidence of cervical cancer can further be reduced from its current incidence of 10/100000 women. Along this line of evidence, a study in collaboration with the Virology Lab at IARC, Lyon (M. Tommasino) is under way investigating the distribution of HPV 16 variants in invasive cervical cancers and cytology negative women in Italy.

Research Activities

Clinical Research

Self collection for HPV testing Although women are encouraged by governments and health authorities to undertake regular Pap smears, the procedure involved in collecting a sample requires the intervention of a medical practitioner. It causes discomfort, is embarrassing, inconvenient, and conflicts with the personal beliefs of some women. Even attending a doctor to discuss sexual matters can be confronting. Non participation in screening

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at IEO showing the accuracy of self sampling in the identification of women withpositive pap smear. As an extension of the concept of self-collection, a protocol has been suggested whereby a woman can purchase a tampon-based kit from a pharmacy or other suitable retail outlet, collect a specimen at her own convenience in private then either mail this to the clinical pathology laboratory or place it into a suitable receptacle, such as a collection box at the pharmacy or supermarket, the samples then being collected by a courier and transported to the laboratory that then does the HPV testing. The result would then be sent to her and/or a doctor she nominates on a form that accompanies the sampling kit. An implementation pilot study is in preparation. HPV investigation at other sites Anal Cancer Anal cancer is also closely linked to HPV infection with studies demonstrating presence of HPV in approximately 90% of squamous cell anal cancers and anal intraepithelial neoplasia (AIN). AIN, especially high-grade, is believed to be the main precursor of anal cancer. The parallels between anal and cervical HPV-associated intraepithelial lesions are many. The HPV types most commonly associated with high-grade anal intraepithelial neoplasia are those most commonly detected in cervical intraepithelial neoplasia. Both the cervix and anus have a transformation zone, and it is at this junction of columnar and squamous epithelium that most intraepithelial neoplasia occurs. Women with a history of high-grade CIN demonstrate nearly a fivefold greater incidence of anal cancer compared to the general population. Even if the data do not support a widespread clinical recommendation that all women with genital intraepithelial neoplasia undergo anal screening, this population deserves clinical and reaserch attention. During 2010 the Virology Group at IEO started a research collaboration with the Division of Endoscopy (dr. Cristina Trovato); an awareness campaign on anal cytologic screening was started, involving patients treated for CIN2+ lesions.

cancers are distinct disease entities are now compelling for a different treatment strategy. Despite the ong evidence linking HPV to a substantial portion of oral cancers and the clinical implication for treatment, much remains unknown about the biology and natural history of HPV infection in the oral cavity. The link between HPV and oropharyngeal cancer has potential implications for prevention. It will be possible to detect precancerous changes in the oropharynx, as in the cervix, with the help of HPV-based screening? Detecting signs of oral HPV infection in oral rinses and brushing is not difficult. However, a barrier to screening is a lack of knowledge on how, or even whether, persistent oral HPV infection progresses to premalignant changes in the oropharynx. Studies are needed to show whether oral HPV infection progresses to dysplasia, as it does in cervical cancer in a prospective, longitudinal study of people with oral HPV infection, followed by visual detection of premalignant changes. A reasearch collaboration with the Head and Neck Division (dr. Luca Calabrese, dr. Valeria Navach) is started to test for the presence of viral DNA in the oral cavity of women positive to cervical HPV.

Research Activities

programmes is consequently higher among certain ethnic and religious subsections of the community, as well as women of poorer socioeconomic status and other women deemed at risk. Apart from this, the Pap smear result can be a false negative if the affected region of the cervix is missed (i.e., inadequate sampling), or the cytological interpretation is incorrect. If a woman is infected with HPV, the viral DNA will be present in cells that are shed from the surface epithelia of the cervix and vagina. Thus testing for HPV in such exfoliated cells is an alternative way of screening, as shown in a number of studies, with good results. In early work collection of exfoliated cells involved a cervico-vaginal lavage. A lavage can be self-administered, but its value is limited due to patient compliance. Subsequent studies found that tampon samples also contained an adequate amount of exfoliated cells for HPV detection. Such sampling can be carried out easily by the woman herself. This can facilitate expanded screening of women and identify infected women without gynecological intervention. It would enable participation by women from geographic localities lacking gynecological services, as well as those who would otherwise be unable or unwilling to attend for a Pap smear. Even women who do attend for their regular Pap smear would likely prefer to take their own specimen, rather than visit their doctor. Selfcollection increases uptake and could make up for loss of diagnostic accuracy, if any. Results from tampons correlate well with results from cervical scrapes. A recent meta-analysis concluded that self-collection may be an appropriate alternative for low resource settings or patients reluctant to undergo pelvic examination. Scrapes and biopsies allow detection of HPV in discrete lesions or sites sampled, so do not yield information representative of the entire cervical epithelium. Scrapes are significantly less sensitive than cervicovaginal lavage specimens. Collecting a scrape requires, moreover, a trained health professional, whereas self-collection does not. Urine self-collected as a dry paper smear for ease of transport, storage and direct HPV PCR testing, gave results that matched those from a cervical scrape, and is an alternate potential sampling approach. A study on women acceptability of the self sampling modality and on a specific self sampling device demonstrated the high impact potential of this new screening modality for developed countries (Igidbashian et al., Journal of Women Health, 2010). A collaborative investigation with the Virology Department of the Statale University of Milano (prof. E. Tanzi) on urine self collected specimens is underway within the frame of the HPV vaccination study. In addition study has been now completed on more than 700 women attending opportunistic screening

Oral Cancer Over the past decade, population studies have established an association between HPV and some tumors in the oral cavity and oropharynx (tonsils, soft palate, posterior pharynx, and base of tongue). In addition, evidence has mounted that the incidence of these cancers is increasing, while the incidence of the remaning tumors of the oral cavity is decreasing. The data indicating that HPVpositive and HPVnegative

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Breast Cancer Research Programme

Future directions for breast cancer research and clinical activity Ideas for clinical research and activities in breast cancer derive mainly from the Task Force for breast cancer, the first multidisciplinary group set up at the IEO. The Main areas of future developments are: 1. The trend of axillary surgery of breast cancer is constantly going towards minimizing lymph node removal. In this view our Division recently started a new prospective randomized trial (IEOS637/311 Trial SOUND: Sentinel node vs Observation after axillary Ultra-souND) with the aim of improving patients’ quality of life through a further reduction of the extent of axillary surgery. 2. A validation trial is ongoing to explore the possibility to identify the sentinel node by a new technique using indocyanin green dye (PDE IEO S562/510). 3. The Division of Senology keeps on collaborating with the Division of Molecular Carcinogenesis and Stem Cell Biology Research in research on the NUMB protein. 4. After a pilot study carried out to improve technical details, the objective is to start the use of high intensity focused ultrasound (HIFU) to treat patients with early breast cancer within a study protocol. 5. One of the new directions in clinical activity is the management of patients in Day Surgery. In fact, many breast operations can be performed either under general anesthesia or under local anesthesia plus MAC (monitored anesthesia care) in total comfort for the patients allowing a discharge on the same day of surgery. This activity will be implemented in the next future. 6. The application of electron intra-operative therapy (ELIOT) as partial breast irradiation, either as full dose or as boost is under strict evaluation.

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After a preliminary analysis of the results from out-trial full dose, according to ASTRO and ESTRO criteria, the single-dose of 21 Gy is bound to be increased to 24 Gy at the 90% isodose. A phase II study will be designed to evaluate side acute and intermediate –term side effects 7. In case of high-risk tumor features on the definitive histologic report, such as extensive vascular invasion, grade 3, negative hormonal receptor status, all patients receive a postoperative whole breast radiotherapy. The scheme consists of 8 fractions of 3.6 Gy delivered to the glandular parenchyma with margin. We are planning to test the feasibility and the safety of this scheme in a pilot trial. 8. The study protocol involving ELIOT boost followed by 13 –fraction whole breast external radiotherapy, specifically addressed to pre-menopausal patients, is continuing. As innovation, we are planning to further shorten the hypofractionated scheme of 13 fractions to 8 fractions: each fraction will be increased from 2.85 Gy to 4 Gy. The eligibility criteria will be the same as the current 13- fraction scheme. A pilot study to investigate the feasibility and the safety of the new protocol is on its way. 9. In patients who develop an in –breast recurrence after being previously treated with ELIOT fulldose for a tumor located in a different breast quadrant, a radiotherapy course to the whole breast except the portion of the breast that has already been treated with ELIOT full dose, will be delivered with arc intensity modulated therapy (Tomotherapy). This new feasibility study called POLO (Partially Omitted ELIOT site) started in February 2012 and consists in delivering 2.25 Gy to the whole breast in 20 fractions, while the ELIOT area is spared as much as possible from having further irradiation, by using strict dose-

constraints. If feasible, an integrated simultaneous boost will be given to the portion of the breast in which tumor reappeared in order to deliver 2.50 Gy in 20 fractions. The primary endpoint is to evaluate the ability to safely deliver irradiation to the breast already treated with a partial treatment. The secondary endpoint is to evaluate in -breast control rate.

10. We are about to carry out a study in noncardiopatic breast cancer patients to estimate dose to the site of a prospective pacemaker during ELIOT to better evaluate the possible use of ELIOT in patients with an actual pacemaker. We are planning to perform in vivo dosimetry with thermoluminiscent radiation detectors (TLD) in noncardiopatic patients receiving ELIOT to the breast after quadrantectomy. The TLDs will be positioned subcutaneously in subclavicular region during quadrantectomy. 11. A new radiotherapy approach will be adopted for elder breast cancer patients as adjuvant treatment. Following the randomised FAST trial preliminary results, 5 once-weekly fractions of 5.7 Gy will be prescribed to the whole breast after radical conservative surgery in over 75 year old patients in order to minimize the drawbacks and the discomfort due to the length of a conventional radiotherapy course. Prone positioning of patients during whole breast irradiation will be considered as a routine set-up and applied when appropriate

Research Activities

Clinical Research

12. A randomized multicenter phase III trial of adjuvant breast radiotherapy (MIRA-SOLE, (MultIcentric RAndomized Study of cOnventionaL and hypofractionatEd RT in adjuvant breast cancer setting) comparing conventional fractionated scheme of 30 fractions with sequential boost to the tumor bed with two hypofractionated schemes, of 20 and 13 fractions each, both with concomitant boost to the tumor bed, started in January 2012. IEO is the principal investigation center and the study is open to Italian radiation therapy divisions. The eligibility criteria include infiltrating tumors staged as pT1-pT3 pN0-pN1a without age limit. The primary endpoint is to assess the incidence of local recurrences, while secondary endpoints include acute and chronic side effects. 13. A phase II trial of partial breast irradiation in patients previously treated with wholebreast radiation started in February 2012. Using arc intensity modulated radiotherapy (Tomotherapy), the target volume corresponding to the recent lumpectomy site is selectively treated, with high dose homogeneity with the PTV and strict dose constraints for critical structures. The scheme is based on hypofractionation and this is a great innovation in the reirradiation field: 2.85 Gy in 13 fractions is delivered over 2.5 weeks. Interstitial brachitherapy consists of 38.5 Gy in 10 fractions over 5 consecutive days is another way of delivering reirradiation to the portion of the breast that developed recurrence.

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Interstitial brachitherapy consists of 38.5 Gy in 10 fractions over 5 consecutive days is another way of delivering reirradiation to the portion of the breast that developed recurrence.

14. After the completion of a feasibility study, the possibility of using IART (intraoperative avidination for radionuclide therapy) within a phase 3 clinical study is under evaluation as a possible method of breast irradiation Medical oncology breast cancer program Medical Oncology research is equally committed to scientific discovery and patient care. Our worldclass clinical investigators work across disciplines, departments, and institutional boundaries to translate research findings into new diagnostics and therapeutics for patients. The cornerstone of translational research of medical oncology staff is collaboration: close interactions among basic scientists, computational biologists,

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The milestones of our clinical research are here summarized: 1. Identification of biological features of disease in early breast cancer predictive of response to a target-oriented approach into the neoadjuvant and adjuvant setting. 2. Identification of biological and genetic features of disease in metastatic breast cancer predictive of response to a target-oriented approach 3. Identification of mechanisms of resistance to trastuzumab in HER2 positive breast cancer disease and development of new strategies to target HER2 positive breast cancer. 4. Molecular screening with next generation sequencing technologies to evaluate potential “molecular drivers” of resistance to standard treatments in patients with luminal B and triple negative breast cancer 5. Role of aromatase inhibitors (AIs) and switching to AIs in the adjuvant setting for the treatment of early breast cancer.

6. Exploring the combination of endocrine therapy with biological agents targeting HER2, src or insulin growth factor receptor (IGFR) 7. Exploring the role of dual targeting (multiple antibodies or antibodies conjugated to chemotherapeutics agents) in patients with HER2 positive breast cancer. 8. Identification of prognostic and predictive molecular markers in metastatic setting of disease (circulating tumor cells) 9. Identification of new strategies (metronomic chemotherapy or combination of metronomics with biological agents) in the treatment of metastatic breast cancer. 10. Identification of surrogate markers predictive of response to antiangiogenic therapies with molecular profiling of patients included in trials with antivascular agents. 11. Identification and development of individualized treatments of specific breast cancer clinical presentations (pregnancy, very young patients, elderly patients). 12. Development of new drugs in the treatment of metastatic breast cancer 13. Selecting cancer vaccine targets for individual cancers. Analyzing the immunogenicity of T-cell and B-cell peptide epitopes and performing cytokine immune assessments to identify epitopes and cytokines that enhance immune responses 14. Exploring the role of antigen specific immunotherapeutics fro patients with triple negative breast cancer with residual disease after a neoadjuvant chemotherapy 15. Identification of mechanisms of resistance to standard therapies by breast cancer stem cells exposure tests. Scientific production of the multidisciplinary team included several high impact peer reviewed manuscripts. Most of them had a major clinical impact, changing practice in the adjuvant treatment of patients with breast cancer; in molecular characterization of subtypes of breast cancer and in the approach of patients in the neoadjuvant setting. Other papers were focused specifically on the impact of oral chemotherapy with a low toxicity profile in the treatment of patients with metastatic breast cancer. Clinical research in the field of breast cancer should be applied for the future to specific subtypes of breast cancer. Trials of the past did not follow such concept of heterogeneity, using extrapolation of data

from retrospective analyses, frequently difficult and imprecise. Future research should achieve the goal to recognizing the diversity of targets in each subtype of breast cancer, taking advantage from molecular characterization tools. New prospective trials will specifically address the questions of targeting multiple pathways in each breast cancer subtype, to maximize response to treatment and minimize the toxicity. Recent large-scale tumor sequencing studies, including wide genome analysis studies, have identified a number of mutations that might be involved in breast cancer tumorigenesis. Analysis of the frequency of specific mutations across different tumors has been able to identify some, but not all of the mutated genes that contribute to tumor initiation and progression. One reason for this is that other functionally important genes are likely to be mutated more rarely and only in specific contexts. Thus, for example, mutation in one member of a collection of functionally related genes may result in the same net effect, and/or mutations in certain genes may be observed less frequently if they play functional roles in later stages of tumor development, such as metastasis. The biggest challenge for the future will be to apply a network reconstruction and coexpression module identification-based approach to identify functionally related gene modules targeted by somatic mutations in cancer. The ultimate goal of this approach is to identify network of pathways and potential crosstalks within pathways. Dual or multiple targeting in order to shutdown “drivers” pathways will be the future of breast cancer treatment within several subtypes. This method was applied to available breast cancer sequence data, and identified several pathways as targets of rare driver mutations in breast. These mutations do not appear to alter genes that play a central role in these pathways, but rather contribute to a more refined shaping or “tuning” of the functioning of these pathways in such a way as to result in the inhibition of their tumor-suppressive signaling arms, and thereby conserve or enhance tumor-promoting processes. We believe a gene network reconstruction, strategy-based approach can successfully identify cancer driver mutations through enrichment of mutations within modules. We should describe highlight a few important caveats in the field. Next generation sequencing technologies used to reconstruct genetic networks can be altered to generate networks of different sizes, or reflecting different coexpression relationships, depending upon the investigators requirements and/ or sample size and likelihood that module enrichment will be observed in different-sized modules. Specifically genome remodelling during cancer progression or

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Research Activities

chemists, clinical investigators, and others. The group also enjoys fruitful partnerships with pharmaceutical and biotechnology companies, which have the complementary resources needed to help transform promising compounds into drugs and biologics. A major departmental research theme is linking knowledge of the genes that cause cancer to the discovery and testing of new therapeutics, involving both small-molecule drugs and immune approaches (vaccines). Other key themes relate to developing personalized medicine strategies by using genetic, epidemiologic, and population-based studies to determine risk and ideal treatment for individual patients. The medical oncology team currently has nearly 60 open adult therapeutic clinical trials. It accrues several hundreds patients to therapeutic and non-therapeutic clinical protocols each year. Disease center members play a major role in the IEO research programs and in international cooperative group trials, such as the International Breast Cancer Study Group (IBCSG) and the Breast International Group (BIG). Department investigators focus on testing new drugs in Phase I and II trials, particularly first-in-human studies that have the potential to move the boundaries of breast oncology care. Technologies being offered include the isolation, enumeration, and genotyping of circulating tumor cells, determination of plasma cytokine levels, and genotypic analysis of plasma-based tumor DNA. All these technologies are applied in clinical trials

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“In the next 20 or 30 years, we will cure or significantly reduce the burden of several subtypes of breast cancer,” Aron Goldhirsch, Breast Cancer Program Director, sums up. “We’ll be able to identify the subtypes and treat them selectively, improving outcomes, and reducing toxic effects. It won’t be easy, but it will be an exciting time in clinical research.” Intraoperative avidination for radionuclide therapy: iart® Intraoperative avidination for radionuclide therapy is a new approach of targeted radionuclide therapy of early breast cancer invented at IEO, which involves two steps: a) inoculation of avidin into residual glandular tissue immediately after primary surgery; b) intravenous injection of 90Y-DOTA-biotin the day after surgery. The 90Y-DOTA-biotin homes in specifically to the avidin

in the breast tissue and provide a therapeutic dose of radiation to the breast. Our studies indicated fast and stable uptake of radiolabelled biotin at the operated site. Data from phase I-II study on 35 patients - who received IART® as a boost to accelerated external beam radiotherapy - revealed that the procedure is safe since no side effects of avidin or radiolabelled biotin occurred. No haematological toxicity was observed. Local toxicity during EBRT was acceptable, with good cosmetic outcomes and quality of life. IART® radiation dose to the heart from the irradiated breast area was negligible, also when left breast was treated, since the mean range of the beta particle of 90Y is 4 mm (Paganelli G. et al. Eur J Nucl Med Mol Imaging 2010;37:203-211). In 2009 in 15 patients who received IART® we compared data derived from standard dosimetry with those obtained using voxel dosimetry. The Biological Effective Dose (BED) and Equivalent Uniform biological effective Dose (EUD) were evaluated in order to assess dose distribution heterogeneity. Absorbed doses from standard versus voxel dosimetry showed a difference of + 12±3%. The mean BED values and related standard deviation resulted to be 21.2±4.3 Gy in standard method versus 24.4±3.2 Gy in voxel dosimetry. The BED-volume histograms showed that 68±25% of voxels received a BED greater than 21 Gy. All voxels received a BED greater than 14 Gy. These results indicated that IART® can deliver into tumor bed an anticipated boost comparable to a EBRT boost. The EUD/BEDvoxel ratio in the target was 0.9. This value, obtained thanks to the crossfire effect of 90Y, indicates a biological efficacy comparable to that of uniform dose distribution, thus the hypothesis of uniform activity distribution is not misleading.

A) Scheme of voxel dose calculation using a dedicated software (Matlab® support, Mathworks). Input data were obtained from SPECT images (activity distribution), biokinetic data (planar images) and S-voxel factors for 90Y and 4.4 mm voxels, specifically calculated for by Monte Carlo simulation (PENELOPE code).

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Research Activities

upon resistance to therapies can up-regulate pathways due to downregulation of current driver pathways. Additionally, this approach probably does not capture all of the secondary driver mutations, which may require either additional complementary systems biology approaches, or larger sample sizes to capture other mutation-enriched coexpression modules. Overall, we believe that this approach shows tremendous promise for the identification of rare tumorigenic driver mutations, which is a crucial task for upcoming large-scale cancer resequencing projects, as it is these more private mutations that may be driving intra-tumor heterogeneity, inter-patient heterogeneity, and ultimately altering response to therapeutic intervention. The future of many investigational therapeutics in breast cancer is therefore linked to our ability to identify the most druggable target in each subtype.

Transaxial slice of the SPECT/CT study and regions of isodose contouring of the breast area

B) Anterior whole body scintigraphic images acquired at 1, 3, 15, and 24 h after intravenous injection of 90Y-biotin.

C) BED-volume histograms showed that 73% of voxels received a BED greater than 21 Gy. All voxels received a BED greater than 14 Gy. BED distribution was evaluated considering radiobiological parameters derived from literature (α/β=10, μrepair=0.5 h-1). Dose-volume and BED-volume histograms were generated, and the corresponding average BED (BEDvoxel) obtained. EUD was estimated considering the surviving fraction of the target (α=0.3 Gy-1). In 2011-2012 a controlled randomized trial comparing IORT vs IART as an anticipated boost in accelerated reduced EBRT has been approved and granted by AIRC.

Radionuclide treatment of very early breast cancer to avoid surgical resection. A phase II study with 90 Y-Labeled Biotin-Avidin. Dosimetric and radiobiologic estimates Non palpable breast lesions (NPBLs) represent approximately 30% of the overall tumoral breast lesions undergoing surgery. At the present time the diagnosis of NPBLs is obtained by mammography and/or ecography and, in case of doubtful malignity, afterwards subjected to VABB (Vacuum-Assisted Breast Biopsy) procedure for diagnostic purpose. VABB is a device for needle breast biopsy highly effective for the diagnosis of NPBLs, representing a valid alternative to the excisional biopsy in case of small, benign lesion. In some cases of malignant tumor the lesion can be totally removed through diagnostic VABB procedure (31.5% according to not published data from our Institute). We designed a study whose rational is the application of a radionuclide treatment following VABB procedure, in order to kill residual tumour cells, so avoiding surgical excision of the tumor. The aim of the therapy in terms of dose prescription is to deliver a 300 Gy dose to the cancer cells, after injecting 1-2 ml of 90Y-avidin-biotin-DOTA. Dosimetric evaluations have been performed in order to determine the 90Y activity needed to fulfil the dose prescription. For comparison, analogue calculation has also been performed considering 177Lu-labelled molecule. Dose calculations have been performed through Monte Carlo simulation, using the PENELOPE (PENetration and Energy LOss of Positrons and Electrons) code simulating the transport of photons and electrons in matter. Hypothesis has been made that the injected

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Y – Dosimetric evaluations The prescription point (either on the sphere edge or 2 mm far from it) would receive a 430 Gy dose instead of 300 Gy. However, the dose would go down to 300 Gy within less than 1 mm. For example, in case of prescription 2 mm far from the sphere egde, the point receiving 300 Gy would be only 0.5 mm shifted (300 Gy at 2.5 mm from the sphere edge, instead of 2 mm).

90

Lu – Dosimetric evaluations In case the 30% wash-out had been foreseen and does not happens, higher doses than the prescribed ones will be delivered. The dosimetric impact of such event has been evaluated.

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The prescription point (either on the sphere edge or 2 mm far from it) would receive a 430 Gy dose instead of 300 Gy. However, the dose would go down to 300 Gy within less than 2 mm. For example, in case of prescription 2 mm far from the sphere egde, the point receiving 300 Gy would be 1.5 mm shifted (300 Gy at 3.5 mm from the sphere edge, instead of 2 mm). Due to the very sharp dose profile of 177Lu with respect to 90Y (177Lu average energy: 130 keV; 90Y average energy: 930 keV), 177Lu turns out to be adequate only for delivering the therapeutic dose of 300 Gy on the sphere edge. Too high 177Lu activities would be required to deliver 300 Gy 2 mm far from the sphere edge. It can thus be concluded that: • 90Y is the adequate isotope when cancer cells are likely to be present in the tissue immediately adjacent the VABB site • both 90Y and 177Lu are adequate if the therapy aim is to irradiate only the edge of the excised volume

the assumptions of Poisson statistic, TCP inside a volume V absorbing an average dose D is defined as

TCP = exp(-TS)

Research Activities

radiopharmaceutical distributes in the tissue assuming a spherical shape after being injected. Thus, 1 ml and 2 ml spheres uniformly filled with 90Y and 177Lu have been simulated. Dose distribution (dose/disintegration, Gy/GBq/s) both inside and outside the spheres has been calculated at voxel level.

(2.2)

where TS (total survivors) is the total number of tumour cells surviving inside V after irradiation. TS is strictly dependent on the initial tumour cell density, ρ, and on the fraction of surviving cells, SF, which is related to D as follows:

{

2 SF (D) = - αD - λβD (λ+μ)

}

(2.3)

α and β being the linear and quadratic radiosensitivity parameters, λ the effective decay constant and μ the time constant for sub-lethal damage repair (assumed to follow monoexponential kinetics). In this study, TCP was calculated as a function of the distance from the sphere centre. Starting from the dose profile, being Di the absorbed dose at progressively increasing distances Ri from the sphere centre (discrete steps), TCP profile was evaluated as TCP(Ri)=exp(TS(R0i)), where TS(R0i) was calculated as:

TS(Ri) = 4πRi2⋅(Ri+1 - Ri)⋅ρ⋅SF(Di)

Conversely, when prescribing 300 Gy 2 mm far from the sphere edge, 177Lu provides in principle a wider controlled area than 90Y thank to the slow trend of the gamma contribution to the dose profile. However, being 177Lu impracticable in this case due to the too high activity needed, the maximum extension of the controlled area (TCP=1), as provided by 90Y, is 4 mm out of the sphere edge.

(2.4)

The α/β ratio for tumours was assumed to be 10 Gy, as frequently reported in literature; μ was chosen to be 0.5 h-1, λ coincided with the physical decay constant, α was set to 0.3 Gy-1 and ρ, assumed to be constant, was set to 109 cells/cm3. Outside the spheres, TCP profiles allow to compare the extension of the controlled area.

These data suggest as 90Y is the more appropriate radionuclide in this protocol. Avidin-biotin-DOTA-90Y will be used to deliver appropriate radiation dose to the tumour site. Avidin, followed by biotin-DOTA-90Y complex will be administrated under ultrasonography (US) guidance few days after VABB at the site of occult carcinoma. Avidin-biotin-DOTA-90Y will be inserted at the site of VABB, destroying any tumor cells possibly present in the tissue.

When prescribing 300 Gy on the sphere edge, 90Y guarantees local control (TCP=1) up to 2 mm out of the sphere edge, to be compared with the 0.5 mm extension of the area out of the sphere controlled with 177Lu. This is due to the higher range of 90Y electrons.

Radiobiological evaluations Tumour Control Probability (TCP) To get a better understanding of the differences among dose profiles including radiobiological interpretation, the parameter TCP (Tumour Control Probability) was calculated. In the framework of linear-quadratic model and under

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Brain Tumours

The prognosis of high-grade glioma is poor and, in most of cases, recurrence invariably occurs within a short period, despite aggressive therapies, such as surgery, radiotherapy and chemotherapy. Antibodies, particularly IgG, have been used frequently in tumour therapy, due to their ability to bind selectively to tumour-associated antigens. The extracellular matrix tenascin molecule represents the most exploited target for radioimmunotherapy in high-grade glioma, both in systemic and loco-regional approach, using a pretargeted method based on the avidin-biotin system [Paganelli G. et al. Neurol Res. 2006;28(5):518-22; Goldenberg D.M. et al. J Clin Oncol. 2006 10;24(5):823-34]. Our group previously assessed the safety profile and therapeutic efficacy of intravenous and loco-regional Pretargeted Antibody-Guided RadioImmunoTherapy (PAGRIT®) in several groups of patients affected by high-grade glioma. In both routes of administration, anti-tenascin monoclonal antibodies and radioactivity had been administered separately, according to the following scheme: on first day biotinylated BC4 MoAbs, on second day avidin, and on third day 90Y-biotin conjugate (intravenously or via a catheter placed into the surgical resection cavity) [Ferrari M. et al. J Nucl Med. 2006;47(1):105-12]. A new GMP-produced anti-tenascin monoclonal antibody ST2146, with the same specificity as old BC4 but lacking the non-functional light chain, was characterised. ST2146 showed a higher affinity and immunoreactivity, and appears to be a good alternative to BC4 for clinical use [Urbano N. et al. Eur J Nucl Med Mol Imaging 2007;34: 68-77]. We are presently working on possible improvements of such protocols, both in chemical synthesis of suitable reagents and in dosimetry planning. A new biotin-DOTA conjugate in which biotin was chemically linked to the DOTA chelator has been investigated by our group. This compound was chosen as the candidate for the development of a therapeutic radiopharmaceutical for

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further clinical investigations. The goal of the study was to optimize the radiometal chelation with 90Y or 177 Lu and to study the stability of the label linked to this new biotin conjugate to support its use in pre-targeting therapy trials. We are studying these possible improvements, as in a retrospective evaluation of our experience in radioimmunotherapy in glioblastoma patients we found interesting results in overall survival. All the patients were affected by progressive glioblastoma and were treated according to our 3-step RIT protocols. We observed 64% progressive disease; 28% a stabilization of disease and in 21 pts (8%) we observed a partial response. The GBM cumulative survival was 98,4% ± 0,9% at 6 months, 79,2% ± 2,1% at 12 months, 51,7% ± 2,6% at 18 months, and 30,7% ± 2,4% at 24 months. The median survival time from diagnosis was 19 months. These results could represent the basis for further prospective trials, which could also exploit new radioisotopes, such as Lu-177 or alpha emitters, and provide a way to assess timing and scheduling of radioimmunotherapy in the therapeutic algorithm of high-grade glioma patients. Moreover, these data suggest that the use of 90Y-biotin guided by anti-tenascin antibodies, interferes with the progression of high grade glioma, prolonging survival in a large series of patients. These results warrant phase III controlled studies although, due to limited budget it is unlikely that such a controlled study will start soon. Anti-cancer therapies with radiolabelled compounds require tumor dosimetry as key information. Standard methods for dosimetry are able to provide average doses in organs and tumours of spherical shape. More sophisticated techniques based on Monte Carlo simulations have been implemented in order to evaluate the dose conversion factors for 90Y, 177Lu, 131I - the most used radionuclides for radionuclide therapy in

locoregional and systemic settings. Moreover, the dose distribution inside spheres and in surrounding volumes was investigated to represent dose in tumours, radiosensitive bordering tissues and/or disease infiltrated tissues. The model studied offers suitable information for dosimetry in several radionuclide therapies. This represents an important tool towards the use of tailored treatments with improved safety and efficacy. The comparison of the results can suggest the choice of a most appropriate radionuclide depending on the clinical situation [Botta F. et al. Eur J Nucl Med Mol Imaging 2008;35(Suppl 2):S201; Strigari L. et al. Eur J Nucl Med Mol Imaging 2008;35(2): P550]. Our group is also active in the treatment of meningiomas. These are generally benign tumours and in most cases surgery is curative. However, high-grade histotypes or partially resected tumours frequently recur and therapeutic options are few. External beam radiation therapy (EBRT) is commonly used, although not always effective. We assessed peptide receptor radionuclide therapy (PRRT) with 90Y-DOTATOC in 29 patients with somatostatin receptor-positive meningiomas recurring after standard treatments. The treatment was well tolerated by all patients. MRI 3 months after treatment completion showed disease stabilization in 19 of 29 patients (66%) and progressive disease in the remaining 10 (34%). Better results, as to morphological response and time to progression, were obtained in patients with grade I meningioma compared to those with grade II–III. We concluded that 90Y-DOTATOC can interfere with the growth of meningiomas and an adjuvant treatment, particularly in atypical and malignant histotypes, deserves further investigation [Bartolomei M. et al. Eur J Nucl Med Mol Imaging 2009; 36:1407-16]. These results, and the absence of side effects, prompt us to propose PRRT after non radical surgery and external radiotherapy, in order to evaluate ORR and PFS in residual atypical and anaplastic/malignant meningiomas. In this setting of patients, PRRT may represent a valuable option and a complementary therapy in the treatment of aggressive meningiomas. On the basis of our previous studies and the existing collaboration between Nuclear Medicine of our Institute, the Neuroncology Division of Istituto Neurologico Besta, Milano in the treatment of brain tumors and the Associazione Italiana Tumori Cerebrali, we are strongly motivated to continue the treatment of these patients, by administering the Peptide Receptor Radionuclide Therapy with 177Lu-DOTATATE, after primary surgery and radiotherapy in residual meningiomas. This protocol has already been submitted for a grant to AIRC.

Research Activities

Clinical Research

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Gynecological Tumours Research Programme

The treatment of gynecological tumours is based on organ of origin and extent of disease. In Italy there are every year 4797 new ovarian cancers, 3418 new cervical cancers and 7759 new endometrial cancers. The main research efforts are against ovarian cancer which represents the number 1 killer among gynecological malignancies. The general aim of our research group is to improve treatment outcome and minimize related morbidities. Great attention is also given to new tools and new biomarkers for the early diagnosis of ovarian cancer. Ovarian cancer Basic research It is now clear that stem cells (SCs) have a critical role not only in the generation of normal tissues, but also in the development of tumors. Recent findings support the concept that cells with the properties of SCs account for malignant properties (e.g., chemoresistance, relapse and metastasis) of several forms of human cancer. A culture system has been recently described that allows for the in vitro enrichment and propagation of SCs (i.e. brain and breast SCs), based on their ability to survive in suspension and produce spherical colonies composed of both stem and progenitor cells. The ability of SCs from different tissues to survive in suspension suggests a common underlying mechanism. Ovarian carcinoma (OC) is the most lethal gynecological malignancy, mostly due to the lack of tools for early diagnosis, the dissemination of cancer cells to peritoneal organs and the relapse which gives rise to chemoresistant secondary tumors. All these features are consistent with the presence of OC-SCs, that would mediate most of the malignant properties of OC. Nevertheless, the existence of OC-SCs has not yet been convincingly demonstrated. In addition, unlike other cancer types, the normal counterpart of OC-SCs, namely ovarian SCs (OSCs) remains to de identified. The

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complexity of the systems is further increased by the fact that the tissue of origin of OC is still controversial, and it appears that both the ovarian surface epithelium and the epithelium of fallopian tubes can exert such a role. We tested the hypothesis that normal and tumor OSCs do exist, developing a suspension culture system to isolate them. After dissociation of human primary OCs, we isolated a population of cells that can be grown in suspension, forming floating spherical colonies, that we named “ovospheres”. We could demonstrate that cancer ovospheres are clonal and grow serially for up to 7 passages. Using the PKH26 staining, we could further isolate cells with stem properties among those constituting the ovospheres. We were also able to obtain ovospheres from normal OSE as well as from normal fallopian tube epithelium, indicating that both tissues contain a SC-like subpopulation of cells. Experiments aimed at verifying the stemness-related features of putative normal and cancer OSCs are ongoing. Our final goal is to define the biological and molecular differences between normal and cancer ovarian SCs, with the aim of identifying more effective cancer diagnostic markers and anti-cancer therapeutic strategies that specifically target the cancer stem cell compartment. Diagnosis and prevention: Transvaginal sonography is still the mainstay in the diagnosis of ovarian cancer. The group is involved in many multicentric international studies in the discrimination of benign and malignant pelvic masses (Histoscanning, IOTAII and III) using 3D sonographic evaluation. The results of the Histoscanning technique have been recently published (Lucidarme O et al, Eur Radiol 2010) and show that the technique is a promising tool to reduce the operator variability in ovarian US diagnosis. IOTA II and IOTA III collaborative studies show that an algorithm based on US characterization

of ovarian masses is helpful to reach the correct diagnosis (Guerriero S et al, Ultras. Ob Gyn, 2011). The most promising approach to ovarian cancer diagnosis is proteomics; new biomarkers for the early diagnosis of ovarian cancer (HE4, Ovalife and proteomics) are being evaluated in prospective ongoing studies. A prospective Italian collaborative study on the accuracy of the R.O.M.A algorithm in the prediction of ovarian malignancy at surgery has been concluded with the accrual of 250 cases. At least 10% of ovarian cancer cases arise in individuals with genetic mutation of BRCA1 and BRCA2. A specific gynaecologic clinic is reserved for the follow up of mutated patients at high risk of ovarian cancer. This population represents an ideal model to study prevention and early diagnosis strategies: a chemoprevention study with HPR has been started recently and a prospective multicentric investigation on Ovalife serum testing in the prediction of occult tubal/ovarian cancer in BRCA1/BRCA2 mutated patients undergoing prophylactic surgery is in progress. Surgery: Surgery represents the cornerstone of treatment for patients with advanced ovarian cancer. Residual tumor is the most important prognostic factor for longterm survival. We have recently published our experience (Peiretti et al, Gynecol Oncol 2010) that seems to confirm that a more extensive surgical approach is associated with prolonged disease-free interval and improved survival in stages IIIC-IV ovarian cencer: the goal of primary surgery should be considered as leaving no macroscopic disease. Patients with advanced ovarian cancer and intra-abdominal complete resection will have clinically/radiologically not detectable lymph node involvement (about 30%). Recently, we are evaluating, in a randomized prospective multicenter study (LION), the hypothesis that systematic pelvic and para-aortic lymphadenctomy, in this setting of patients, has a significant impact on overall survival and progressionfree survival. We have just completed the enrolment and we are waiting for the final analysis. We are also interested in exploring the possible role of surgery at time of relapse prior to second line chemotherapy. A randomized trial will be started to assess whether surgery followed by chemotherapy will improve progression-free survival and overall survival compared to chemotherapy alone in patients with relapsing ovarian cancer. Chemotherapy: Despite aggressive frontline therapy and high initial response rates, more than 50% of women with ovarian

cancer will relapse and develop drug-resistant disease. Addition of a third cytotoxic drug to a platinum-taxane combination did not improve the efficacy but increased the toxicity. Thus, novel therapeutic strategies are warranted and incorporation of targeted agents into standard chemotherapy regimens appears particularly promising. Last year has been activated a multicenter randomized double-blind phase III trial to investigate the efficacy and safety of BIBF 1120 (a potent antiangiogenic agent) in combination with standard chemotherapy and subsequent maintenance therapy in stages IIB-IV ovarian cancer patients. We are also enrolling patients in a global study to assess bevacizumab in clinical practice in combination with carboplatin and paclitaxel in patients with ovarian, fallopian tube or primary peritoneal carcinoma (ROSiA trial). We are waiting for the final analysis of three studies evaluating the efficacy and safety of erlotinib (EORTC trial), sorafenib and abagovomab (MIMOSA trial) as maintenance treatment at the end of primary chemotherapy. In this context, we have just completed the enrollment in a double blind randomized phase III study of Gynecologic Cancer Intergroup to assess the role of pazopanib, a potent PARP-inhibitor. For resistant/refractory disease, were presented the results of a large phase III trial compared patupilone with pegylated liposomal doxorubicin. The primary endpoint was not met: there was no statistically significant difference in overall survival between the two arms (median OS 13.2 and 12.7 months respectively). Median PFS was 3.7 months for both arms. There was a trend toward higher overall response rate in the patupilone arm (15.5% vs 7.9%); however, disease control rates were similar (59.5% vs 56.3%). Is currently analyzing the effectiveness of the combination of pegylated liposomal doxorubicin with velcade (a selective proteasome inhibitor) in platinumsensitive recurrences. The concomitant administration of RAD001 (an mTOR inhibitor) and CaelyxTM showed promising antitumor activity in patients with recurrence of ovarian and endometrial cancer. Data reported 23% of partial response in ovarian cancer patients, also in platinum resistant cases; a stabilization of the disease was observed in about 80% of patients with endometrial cancer, with 40% free of progression at 6 months (Perotti et al., 2011, in press) In platinum-sensitive disease, we have recently closed a phase II trial, but the results of prima analysis of ECHO study did not demonstrated the efficacy of the molecule ZD4054 (an orally active specific endothelin receptor antagonist) which has been used with

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Research Activities

Clinical Research

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Endometrial cancer The endometrial cancers are the most common gynecologic cancers. Endometrial cancer is more frequent in postmenopausal women, however, 25% of cases occur in premenopausal women. In this setting the group is testing the therapeutic effect of

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a levonorgestrel medicated IUD (MIRENA) in patients affected by atypical endometrial hyperplasia or grade 1 endometrioid adenocarcionoma confined to the endometrium in a prospective study; the study has accrued more than 40 patients a first data analysis has been recently published (Minig et al, Ann Oncol, 2010). Most postmenopausal cases are detected early resulting in a good prognosis for cure and survival with a five-year survival of more than 85%. Initial treatment consists of surgery, surgery and radiation, or hormonal therapy. For those women in whom endometrial cancers have progressed despite available treatment options chemotherapy is the only option. Chemotherapy consists of taxanes, anthracyclines, and platinum agents. The response to chemotherapy has ranged from 20 to 35% for single agents and up to 70% for combination regimens with progression free survival (PFS) ranging from 5 to 9 months duration. As a result, further studies are being undertaken to look at multi-drug regimens to determine what combination may improve response rates, increase the length of PFS, and decrease the incidence of side effects (e.g., peripheral neuropathies). Therefore, treatments with advantages over current chemotherapy such as decreased toxicity and improved overall survival are needed. To contribute to this problem, we have recruited some patients in a phase II trial, which recently closed the enrollment, of orally deferolimus, a rapamycin-analog that inhibits mTOR, compared to progestin in patients with recurrent endometrial cancer. Last year was also activated a randomized phase III trial of ixabepilone, a semi synthetic analog of epothilone B, versus paclitaxel or doxorubicin in patients with advanced endometrial cancer previously treated with chemotherapy. The management of patients with uterine leiomyosarcomas poses many difficulties. Patients with metastatic disease at diagnosis or with recurrence after primary treatment have a dismal prognosis and the median survival is less than1 year. Treatment options for recurrent/metastatic disease are limited. A phase II randomized – non comparative study is currently ongoing for patients with metastatic or locally relapsed uterine leiomyosarcoma pretreated with conventional chemotherapy to evaluate the activity of trabectedin, having the standard combination of gemcitabine+docetaxel as a control. Cervical Cancer Prevention The Division is evaluating new modalities of early diagnosis and prevention for cervical cancer.

The introduction of HPV testing and genotyping in screening requires information on the genotype distribution in positive pap smears and cervical precancerous and cancerous lesions. Our group investigated this aspect in detail and a first paper was published (Sandri et al, J Med Virol. 2009); in a second paper (Sideri et al., Gyenecol Oncol. in press) a different age distribution was observed between lesion associated with HPV 16-18 and lesions associated with other high risk HPV genotypes; a third paper, the largest Italian series published so far, investigated the distribution of HPV genotypes in invasive cervical cancers in Italy (Sideri et al, Vaccine 2009). A promising marker of progressing lesions is the determination of the p16 overexpression. A large multi-institutional study (PALMS) was concluded in 2010 evaluating double staining on liquid based cytology specimens, in screening and triage of borderline lesions on 27.000 women. The results showed that double stain with the two proteins has a higher specificity than HC2, maintaining the sensitivity of the viral test (paper submitted). Viral load was investigated in a joint research project on women showing ASC-US cytology.The results showed a significant correlation between viral load, expressed as RLU obtained by HC2, and CIN2+ detection (M. Origoni et al. submitted). The objective of the investigational research is to better characterize women with HPV infection in order to build an algorithm allowing a tailored clinical approach (Castle P, Sideri M et al, 2007). A study in collaboration with the Virology Laboratory at IARC, Lyon (M. Tommasino) is under way comparing the distribution of HPV 16 variants in cytology negative women and in invasive cervical cancer cases. Self-collection of cervico-vaginal samples can facilitate and expand screening as women would likely prefer to take their own specimen, rather than visit their doctor. A study on women acceptability of the self sampling modality from our group demonstrated the high impact potential of HPV self sampling (Igidbashian et al., Journal of Women Health, 2010). In addition a study has been completed on more than 700 women attending opportunistic screening at IEO showing the accuracy of self sampling in the identification of women with positive pap smear. Finally, a collaborative investigation with the Virology Department of the State University of Milano (prof. E. Tanzi) on urine self collected specimen is under way within the frame of the HPV vaccination study in 18 year old girls. Ongoing studies include also a population based project of HPV vaccination with GARDASIL in 18 years old adolescents living in the Milano and Monza area, followed for a five-year period. The primary endpoint is the incidence of viral infection, looking at the new epidemiology and

prevalence of viral HPV genotypes in vaccinated girls. The project started in spring 2008 and almost 700 girls have been vaccinated so far. Results at baseline showed HPV positivity in about 10% of sexually active girls.

Research Activities

standard chemotherapy with carboplatin and paclitaxel. Zibotentan did not improve the progression-free survival compared to standard arm. A study compared the efficacy and tolerability of oral olaparib (a PARP-inhibitor) in combination with paclitaxel and carboplatin versus chemotherapy alone has been activated. The first data review indicated that while maintenance therapy with olaparib significantly extended PFS, this is unlikely to show a benefit in overall survival at the final analysis. As there are no new safety concerns, patients who wish to continue olaparib until progression may do so. For partially platinum-sensitive recurrences, is widely debated whether it is better to re-introduce a platinumbased therapy rather than using a non-platinum treatment. Is still ongoing the MITO-8 study which compares the use of pegylated liposomal doxorubicin versus the combination carboplatin-paclitaxel, precisely in order to clarify this controversy. Also the INOVATYON trial, recently approved, will investigate the role of a non-platinum combination for the treatment of ovarian cancer patients relapsing between six and 12 months after last platinum-based chemotherapy for whom there is need for new treatment options. Specifically, the present trial is aimed at demonstrating that extending the PFI with a non-platinum combination prolongs survival in this setting of patients. We have recently activated a trial for patient with platinumsensitive recurrent ovarian cancer, defined by a relapse more than 6 months after last dose of platinum-based chemotherapy. The aim of this study is to evaluate wether the addition of ombrabulin (AVE8062, antitumor-vasculature agent) to standard chemotherapy of carboplatin and taxol may improve the progression-free survival. It was activated a phase I study of oral administration of S78454 (a pan-HDAC inhibitor) given with a fixed dose infusion of pegylated liposomal doxorubicin in the treatment of primary-resistant and partially platinumsensitive ovarian cancer patients. Finally, a phase II study evaluating intermittent and continuous OSI-906 and weekly paclitaxel in patients with recurrent epithelial ovarian cancer has been activated. OSI-906 is a small molecule potent inhibitor of insulin-like growth factor 1 receptor (IGF-1), which plays a significant role in the growth and survival of ovarian cancer .

Early cervical cancer: Fertility preserving surgery About 40% of patients with stage I cervical cancer will be younger than 40 at time of diagnosis. To preserve the fertility potential in selected and motivated patients, a prospective study has been started in order to evaluate the role of conization and laparoscopic pelvic node dissection in the treatment of young patients with stage IB1 cervical cancer. A subgroup of patients not suitable for this study because of high risk factors will be treated with neo-adjuvant chemotherapy followed by conization and laparoscopic lymphadenectomy. Goals of these two studies will be to provide good oncologic outcome while preserving fertility potential. In the frame of this project an evaluation of diagnostic tools in the preoperative assessment of tumor volume in early cervical cancer cases has been started comparing traditional and contrast enhanced RM, 3D ultrasound, colposcopy. Our preliminary experience was published in 2011 (Maneo et al. Gynecol Oncol). Early cervical cancer: Surgery Total abdominal radical hysterectomy and pelvic lymph node dissection is the current standard treatment for early stage cervical cancer. Laparoscopic techniques have been demonstrated to be feasible and safe in some retrospective and prospective non-controlled series but few data are available on the morbidity and survival. No randomized trials have been completed which directly compare laparoscopic and open radical hysterectomy. To contribute to this issue, we are involving in LACC study, a phase III international clinical trial comparing laparoscopic or robotic radical hysterectomy versus abdominal radical hysterectomy. The trial is an equivalence study with the hypothesis that laparoscopic approach is equivalent to standard treatment in terms of disease-free survival. At the moment, patients enrollment has been suspended in our Institute for the first statistical evaluation. Advanced cervical cancer In collaboration with the EORTC Gynecologic Group we are coordinating a randomized study comparing neo-adjuvant chemotherapy followed by radical surgery against the standard approach consisting of concomitant chemo-radiation in patients with FIGO stage Ib2- IIA > 4 cm, II B cervical cancer. In the study, currently ongoing, we enrolled 29 patients.

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Minimally-Invasive Robotic Surgery - da Vinci System At present we are evaluating the feasibility of conducting routinely gynecologic-oncology surgery using a computer-enhanced robotic surgical system allowing minimally invasive surgery to be more precise and fine-tuned so that more sophisticated procedures can be done endoscopically. The da Vinci Computer-Enhanced Robotic Surgical System is operate by surgeon from a remote workstation in the same room as the patient. Consists of a surgeon’s viewing/control console with an integrated high-performance three-dimensional vision system and a patient sidecart with four robotic arms that respond in real time to the surgeon’s movements. Endo-wrist instruments precisely mimic the surgeon’s hand and wrist movements and allow translation of the movement to operative site. This remarkable technology facilities suturing and dissection and makes skeletonization and ligation of vessels, dissection of pelvic spaces, and lymph node dissection feel more like conventional open surgery than the standard laparoscopic technique. Furthermore the learning curve is shorter than conventional laparoscopy. Finally, the overall increase in the time needed to complete the full procedures is spent in equipment set-up, such as positioning the surgical cart at the bedside, and not on the actual operation. From November 2006 to December 2011 we performed 748 surgical operations with the Da Vinci System. We performed the 50% of extrafascial hysterectomy, the 13% of radical hysterectomy and the 31% of pelvic lymphadenectomy procedures.

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Clinical Research

Head & Neck Cancer

Modus Operandi The Head & Neck Multidisciplinary Working Group meets on Wednesday, to discuss each patient referred to IEO with a Head and Neck cancer for plannnig therapy, evaluating treatment outcome, auditing clinical and surgical activities and scheduling follow-up. The last Wednesday of each month the Head & Neck Task Force (which includes Surgeons, Medical Oncologists, Radiotherapists, Radiologists, Pathologists, Nurses, Speech Therapists, Dietologists, and Basic Reseachers) discusses new protocols, reviews on-going trials, diagnostic and therapeutic guidelines. Two/three times a year an invited Visiting Professor attends the department, meets the staff and gives a lecture. Published Clinical researches Gene DOK1 and human cancer The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2’deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human

Research Activities

Recurrent cervical cancer: Surgery We are investigating new techniques for viscera reconstruction after pelvic exenteration: complication rates and quality of life are the major endpoints. We are also evaluating the role of IORT (intra-operative radiotherapy) after exenteration for side pelvic recurrences.

neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt’s lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy. Saulnier A, Vaissière T, Yue J, Siouda M, Malfroy M, Accardi R, Creveaux M, Sebastian S, Shahzad N, Gheit T, Hussain I, Torrente M, Maffini FA, Calabrese L, Chiesa F, Cuenin C, Shukla R, Fathallah I, Matos E, Daudt A, Koifman S, Wünsch-Filho V, Menezes AM, Curado MP, Zaridze D, Boffetta P, Brennan P, Tommasino M, Herceg Z, Sylla BS. Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers. Int J Cancer 2011 Jul 27. doi:10.1002/ijc.26299 [Epub ahead of print] Family history of cancer and the risk of laryngeal cancer Only limited data is available on the relationship between family history of laryngeal and other neoplasms and laryngeal cancer risk. We investigated the issue using data from a multicentre case-control study conducted in Italy and Switzerland between 1992 and 2009 including 852 cases with histologically confirmed laryngeal cancer and 1970 controls admitted to hospital for acute, non neoplastic conditions. Unconditional logistic regression models adjusted for age, sex, study center, education, tobacco smoking, alcohol drinking and number of siblings were used to estimate the odds ratios (ORs) of laryngeal cancer. The multivariate OR

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Garavello W, Turati F, Bosetti C, Talamini R, Levi F, Lucenteforte E, Chiesa F, Franceschi S, La Vecchia C, Negri E. Family history of cancer and the risk of laryngeal cancer: a case-control study from Italy and Switzerland. Int J Cancer. 2012 Feb 1;130(3):665-70. doi: 10.1002/ijc.26055. Epub 2011 Apr 27. Three-dimensional conformal postoperative radiotherapy in patients with parotid tumors Salivary gland malignancies are rare. The aim of our study was to investigate radiotherapy-related toxicity and clinical outcome in patients treated at our division with postoperative radiotherapy (pRT) for parotid tumors. Forty-three consecutive patients (32 with primary parotid tumors, 9 with parotid metastases and 2 with recurrent benign diseases) were retrospectively analyzed. The median follow-up was 28 months. Twenty and 5 patients had a follow-up longer than 2 and 5 years, respectively. Thirty-seven patients were alive and most of them (78%) were free from disease. The local and distant control rates were higher in patients with primary parotid tumors (94% and 87.5%) than in patients with parotid metastases (87.5% and 75%). Grade 3 radiotherapy-related acute toxicity of skin and mucosa was recorded in 20.9% and 28% of patients, respectively. Two patients (4.7%) had grade 4 skin toxicity. Late toxicity data were available for 33 (77%) patients. None of the patients developed severe (grade 3 and 4) late toxicity of soft tissues, skin or temporomandibular joints. Postoperative radiotherapy is a feasible treatment that was found to be effective mainly in patients with primary parotid tumors. Toxicity was acceptable but could probably be further reduced using more advanced radiotherapy techniques. Longer follow-up is required to achieve definitive results.

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Alterio D, Jereczek-Fossa BA, Griseri M, D’Onofrio A, Giugliano G, Fiore MR, Vitolo V, Fossati P, Piperno G, Calabrese LS, Verri E, Chiesa FG, Orecchia R. Threedimensional conformal postoperative radiotherapy in patients with parotid tumors: 10 years’ experience at the European Institute of Oncology. Tumori 2011;97:328-34. Ultrasound dye-assisted surgery (USDAS) The evolution of new techniques for cancer surgery has led to important changes in cancer care in recent years. The endpoint of cancer treatment is now to treat the patient with minimum discomfort while respecting quality of life. New techniques, such as mini-invasive surgery, must respect the correct oncological indications, when technically feasible. The surgery for nodal spread or recurrence of disease, after previous surgery on T or T and N for neck cancer, can represent a diagnostic and therapeutic challenge, especially in the neck, which is characterized by small spaces and noble structures. Often lesions become enveloped in scar tissue and can be difficult to visualize during surgery, representing a genuine problem for the surgeon. Ultrasound dyeassisted surgery is a procedure that combines ultrasound localization of pathological nodes with the use of methylene blue to mark diseased structures to simplify their visualization (and thus removal) in the surgical field. The technique is simple and can be used in surgically and oncologically experienced hands, even in hospitals that do not have sophisticated technology. Giugliano G, DE Fiori E, Proh M, Chulam Celestino T, Grosso E, Cattaneo A, Gibelli B, Massaro M, Ansarin M. Ultrasound dye-assisted surgery (USDAS): a promising diagnostic and therapeutic tool for the treatment of cancer recurrences in the neck. Acta Otorhinolaryngol Ital. 2011;31:222-7. Compartmental tongue surgery Compartmental tongue surgery (CTS) is a surgical technique that removes the compartments (anatomofunctional units) containing the primary tumor, eliminating the disease and potential muscular, vascular, glandular and lymphatic pathways of spread and recurrence. Compartment boundaries are defined as each hemi-tongue bounded by the lingual septum, the stylohyoid ligament and muscle, and the mylohyoid muscle. In this non-randomized retrospective study we evaluated the oncologic efficacy of CTS in patients with squamous cell carcinoma (SCCA) of the tongue treated from 1995 to 2008. We evaluated 193 patients with primary, previously untreated cT2-4a, cN0, cN+, M0 SCCA with no contraindication to anesthesia and able

to give informed consent. Fifty patients treated between October 1995 and July 1999 received standard surgery (resection margin >1cm); 143 patients treated between July 1999 and January 2008 received CTS. Study endpoints were: 5-year local disease-free, locoregional disease-free and overall survival. After 5years, local disease control was achieved in 88.4% of CTS patients (16.8% improvement on standard surgery); locoregional disease control in 83.5% (24.4% improvement) and overall survival was 70.7% (27.3% improvement). The markedly improved outcomes in CTS patients, compared to those treated by standard surgery, suggest CTS as an important new approach in the surgical management of tongue cancer Calabrese L, Bruschini R, Giugliano G, Ostuni A, Maffini F, Massaro MA, Santoro L, Navach V, Preda L, Alterio D, Ansarin M, Chiesa F. Compartmental tongue surgery: Long term oncologic results in the treatment of tongue cancer. Oral Oncol. 2011 Mar;47(3):174-9. Epub 2011 Jan 22. Management and follow-up of thyroid cancer in pregnant women. Thyroid cancer, the most common endocrine malignancy, is often detected in young female patients. Therefore, pregnancy following thyroid cancer is not infrequent, and about 10% of thyroid cancers occurring during the reproductive years are diagnosed during pregnancy or in the early post-partum period. Differentiated thyroid cancer (DTC) in young people generally has an excellent prognosis, and disease-free survival among women with DTC diagnosed during pregnancy may not differ from that in age-matched non-pregnant women with similar disease. However, thyroid cancer detected during pregnancy may cause anxiety about the optimal timing of recommended treatments and about both maternal and neonatal morbidity, as weel as pregnancy following a diagnosis of thyroid cancer obviously needs both maternal and foetal management. The main objectives in clinical monitoring of pregnant thyroid cancer patients are: 1) to reach an adequate balance of maternal calcium and thyroid hormones that is absolutely required by the foetal central nervous system for normal maturation; 2) to maintain optimal levels of maternal thyroxin to avoid possible recurrence or spread of disease; and 3) to perform safe follow-up visits for the mother and to plan further therapy when needed. Data from a review of the literature and the authors’ own experience show that in patients undergoing either suppressive or substitutive thyroxine therapy foetal thyroid growth is normal at ultrasound study, newborn thyroid status is normal, and the incidence of maternal morbidity is not influenced

by the pregnancy. In this review, the authors underline that regular adjustment of levo-thyroxine and calcium therapy is of outmost importance for both maternal and foetal well-being and offer some insight, very interesting from a practical point of view, to provide a clear and simple pathway for the management of pregnancyassociated thyroid cancer

Research Activities

was 2.8 (95% confidence interval [CI], 1.5-5.3) in subjects reporting a first-degree relative with laryngeal cancer, as compared to subjects with no family history. The OR was higher when the relative was diagnosed before 60 years of age (OR = 3.5, 95% CI 1.4-8.8). As compared to subjects without family history, non-smokers, and moderate drinkers, the OR was 37.1 (95% CI 9.9-139.4) for current smokers, heavy drinkers, with family history of laryngeal cancer. Family history of colorectal (OR = 1.5, 95% CI 1.0-2.3) and kidney (OR = 3.8, 95% CI 1.2-12.1) cancer were also associated to an increased risk of laryngeal cancer, while no significant increase in risk was found for family history of cancer at all sites, excluding the larynx (OR = 1.1).

Gibelli B, Zamperini P, Proh M, Giugliano G. Management and follow-up of thyroid cancer in pregnant women. Acta Otorhinolaryngol Ital. 2011;31:358-65. On going clinical researches Locally advanced Head and Neck Squamous Cell Carcinoma: finding new prognostic and predictive factors. Squamous Cell Carcinoma of the Head and Neck (SCCHN) is the sixth most common cancer worldwide, with an estimated annual incidence of approximately 600,000 cases. Patients with locally advanced disease presentation may or may not be amenable to surgery at the time of diagnosis and until now, we have few parameters to design a specific treatment strategy for each patient. What we know is that, compared to radiation, concomitant chemoradiation could offer an 8% improvement of the 5-y Overall Survival (OS), for patients who are candidates for surgical treatment and a 5% improvement for those who are not. Despite great progress in the overall treatment approach and in understanding the mechanisms that lead to head and neck squamous cell carcinoma, many patients (almost 50%) with locally advanced tumor develop a recurrence within three years from treatment, and about 10% of them experience distant metastases. Only 30% of stage III and 15% of stage IV patients are alive and disease-free at 5 years. In order to better select patients who could benefit from non surgical therapies, prognostic and predictive factors for response to chemotherapeutic or biological target drugs and to radiotherapy are therefore required. Actually, only tumor extension (evaluated by TNM parameters) is used in clinical practice to define the therapeutic approach. Recent papers suggest that some defined pathological tumor characteristics, like related-HPV tumor, could be considered as prognostic and predictive factor of response to therapies. In the same way, functional imaging modalities as FDG PET and functional MRI (diffusion weighted MRI -DWI-MRI and dynamic contrast enhanced MRI – DCE-MRI) could improve the correct staging and predict the response

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Phase IIB Randomized, Placebo Controlled Trial of Pioglitazone for Oral Premalignant Lesions: An InterConsortium Collaborative Study. This is an inter-consortium collaboration between two Consortium Lead Organizations (CLO), MD Anderson Cancer Center (MDACC), Houston, TX and University of Wisconsin Paul P. Carbone Comprehensive Cancer Center (UWCCC), Madison, WI with a total of 11 participating clinical sites. (USA cancer Centers (Memorial Sloan-Kettering Cancer Center, New York, NY; University of Minnesota, Minneapolis, MN; University of Wisconsin, Madison, WI; Columbia University Medical Center, New York, NY; Weill Medical College of Cornell University, New York, NY; UT MD Anderson Cancer Center, Houston, TX; University of Iowa, Iowa City, IA; University of Alabama-Birmingham, Birmingham, AL; University of Maryland, Baltimore, MD; Roswell Park Cancer Institute, Buffalo, NY) and in Europe the IEO. The central hypothesis of this protocol is that the PPAR gamma agonist pioglitazone (Actos®) may have activity against tobacco-related intraepithelial neoplasia (IEN) in humans, and this activity may be suggested by clinical or histologic response to pioglitazone treatment of oral premalignant lesions (OPL), namely dysplastic oral leukoplakia, hyperplastic leukoplakia in high risk locations (dorsal, lateral or ventral tongue or floor of

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the mouth) or erythroplakia of any histology. So, the primary objective of this Phase IIB randomized, placebocontrolled trial is to assess the efficacy of pioglitazone 45 mg qd given for 24 weeks in subjects with oral premalignant lesions. Efficacy will be assessed by the proportion of subjects who show complete or partial responses in either clinical or histological outcomes based on the final response evaluation at 24 weeks and the following definitions. If either the clinical or histologic parameters indicate progression, then by default, overall response will be classified as progressive disease. The IEO is supposed to include 9 patients in two years. The study started in March 2011 and as of 31/12/2011, 6 patients underwent randomisation. Radiotherapy vs Transoral Robotic Surgery (TORS) in Stage I-II oropharyngeal cancers: a radomised trial Randomised trial. This ia a multicentric phase III pilot study coordinated by the IEO Head and Neck Division. Criteria of inclusion are patients not previously treated with a cT1 – cT2 cN0 squamous cell carcinoma of tonsil and cT1 cN0 of the base of the tongue and posterior pharyngeal wall. Stage II cancers of the latter sites are not included because there is not yet enough technical and oncological experience in robotic surgery of such a large neoplasias in these regions. Patients randomised in the surgical arm will be closely checked in the 1st year for evaluating possible progression of the disease on the neck. In case of development of nodal metastases they will undergo a neck dissection. The aim of the study is to compare 5-year loco-regional control achieved by transoral robotic surgery vs Radiotherapy alone; second aim is to evaluate how many patients treated by TORS spare neck dissection and post-operative radiotherapy, and third aim is to compare quality of life and function preservation in the 2 arms. The study will randomise 50 patients in each arm in 2 years. It is granted by ROL (Oncological Web of the Regione Lombardia). It started in December 2011 Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in Europe and India. (Retrospective and Prospective multicentric study granted by the CEE VII Framework). Human papillomavirus (HPV) is responsible for approximately 25% of head and neck cancer (HNC) worldwide and appears to be associated with a better response to treatment and improved prognosis. Evidence suggests that HPV-induced HNC has steadily increased in the USA and some European countries in

the last decades. However, whether this is a worldwide phenomenon and specific risk factors are associated with it remains to be proven. In addition, little is known on the natural history and risk factors of oral HPV infection. HPVAHEAD network aims to address these and other unanswered questions on HNC aetiology and epidemiology with a focus on the role of HPV. We will assemble and analyze a large collection of plasma/ sera and HNC tissues from 42 centres in 16 European countries as well as HNC tissues from 7 Indian centres together with epidemiological and clinical data. HPV status in human specimens will be evaluated by different assays in central laboratories. Epidemiological studies will be conducted to establish the overall proportion and type distribution of HPVpositive HNC at different anatomical sites in European and Indian regions as well as the time trend of the proportion of HPV-positive HNC in recent decades. Using the follow-up information on HNC patients, we will further investigate whether HPV positivity confers a better prognosis and survival. We will also conduct a study in HPV-vaccinated and non-vaccinated women in order to determine risk factors and natural history of oral HPV infections. In addition, we will search for new surrogate markers for oral HPV infection to facilitate novel screening strategies. Finally, the HPV-AHEAD consortium aims to transfer technology to Indian centres as well as to develop several strategies for the training of European and Indian researchers in infections and cancers. This study will provide important insights for the screening, diagnosis, treatment and prophylaxis of HPV-associated HNC in Europe, India and elsewhere. This proposal will be focused on the elucidation of the role of HPV types and other environmental risk factors in HNC in Europe and in India. In particular, the proposal will (i) improve our understanding of the role of HNC aetiology in geographical regions with different incidence rates, (ii) lead to the identification of the clinically most useful HPV markers and new biomarkers for different types of HNC, which both can be used for screening and/or therapy strategies, (iii) provide new insights on the natural history of HPV oral infection and its diagnosis, on HNC prognosis and survival by HPV status, and (iv) determine whether prophylactic HPV vaccine may have an impact on oral HPV infections. The study will take 3 years and started in October, 2011. In the first year about 500 slides of patients operated on for an oral cancer at IEO will be evaluated for HPV and DOK1 expression.

Research Activities

to treatments. The aim of our study is to prospectively collect baseline data from both pathological specimens, FDG PET and functional MRI exams in order to correlate these parameters with response to non surgical treatment and clinical outcome (in terms of Overall Survival and Disease Free Survival) in patients with locally advanced head and neck squamous cell carcinoma. This project is organized in tasks: Task 1: The aim of this task is to evaluate, on the initial biopsy specimen, the expression as well as the prognostic and predictive value of some biomarkers potentially involved in treatment failure: ERCC1, CD105, p16INK4,p53 VEGFR, PTEN. All these biomarkers will be evaluated with immuno-histochemical analysis. Task 2-3: The aim of these tasks is to evaluate the predictive and prognostic value of radiological and nuclear medicine parameters. A baseline functional MRI (DCE- and DWI-MRI) and an FDG-PET will be performed before starting the radio-chemotherapy treatment regimens and objective parameters will be collected. Data from baseline pathological specimens, functional MRI and FDG-PET exams will be analyzed and correlated with the response to treatment and patient clinical outcome.

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Lung Cancer Research Programme

Early detection. Lung cancer is a leading cause of cancer death worldwide. The incidence of the disease is increasing among women in resource-rich countries and in both sexes in developing countries. The disease is usually rapidly fatal not only because it is biologically aggressive but also because it is typically diagnosed at an advanced stage. Chances of cure strongly depend on the stage at diagnosis: since smokers show a greatly increased risk of lung cancer, early diagnosis by screening in smokers (high-risk population) is a potentially important way of reducing lung cancer mortality. The introduction of low-dose multidetector computed tomography (LDCT) renewed hope that screening could have a positive impact on survival. Two studies conducted at IEO (a pilot study and the Cosmos study) have permitted the introduction and validation of simple and non-invasive guidelines for the diagnostic work-up of screening-detected lung nodules of unknown nature. These studies showed that the recall rate in the screened population was acceptable (around 7%) and the compliance was high (82% in 5 years). The observed survival rate of lung cancer patients was 63% -72% at 5 years in the pilot and Cosmos study respectively. A potential disadvantage of screening for lung and other tumors is that a fraction of screening-detected cancers is over-diagnosed, i.e. would not have become symptomatic in the patient’s lifetime and would not have caused death. Such cancers are either so slow-growing that the patient dies of other causes before the cancer becomes symptomatic or they never progress, or may even regress. Over-diagnosis can increase screening costs and result in overtreatment, potentially giving rise to unnecessary morbidity; while work-up and treatment unnecessarily stress patients and their families. In a recent study we assessed the growth rate (volume doubling time, VDT) of lung cancers detected by LDCT screening over five consecutive years. We used VDT as a means of identifying slow-growing and indolent

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cancers – which we hypothesized to be in part overdiagnosed – and evaluated clinical outcomes in these cancers compared to faster growing lesions. According to our results slow-growing/indolent cancers constituted about 25% of cases diagnosed after baseline and may have been over-diagnosed. To limit overtreatment of potentially over-diagnosed cases, minimally-invasive limited resection and non-surgical treatments should be investigated. Risk assessment. There is an increasing interest in developing ways of estimating the risk of an individual to develop lung cancer, in order to reduce costs as well as the number of potentially harmful screening procedures and follow-up interventions. While risk modelling has established clinical applications in cardiovascular disease and breast cancer (Gail model), only recently models have been developed to estimate lung cancer risk in high-risk groups and the general population (but not screened populations). Most models rely on epidemiological variables to estimate risk. However Spitz et al. combined epidemiological risk factors with two DNA repair assays, although the improvement in risk assessment obtained by adding the molecular variables was small. We constructed a new lung cancer risk model to quantify individual risk in participants of screening trials, based on the individual risk factors used in the Bach model, plus the results of the baseline CT. The model provided estimates of annual lung cancer detection rate that varied from 0 (first decile of participants) to >3% (top decile) and were compared to the annual lung cancer detection rate derived from subsequent screenings. Although the population consisted of heavy smokers, 40% of participants had a predicted annual risk of lung cancer <0.3% – approximately corresponding to the baseline risk of lung cancer of an unselected population over 50 years. Indeed, only 10% of all lung cancers were diagnosed in this group over the 3 years of follow-up.

By contrast, 90% of all lung cancers diagnosed at annual CT screening were in the group with a predicted annual risk >0.3%; yet individual costs, side effects, and rate of false positives were similar in this group to those in the low risk group. The model therefore appears useful in identifying lower risk persons in whom the time to the next screening CT can be safely increased, reducing unnecessary radiation exposure and lowering the risk of intervention for false positive findings. Limited resections: Lung cancers are detected with increasing frequency as a result of improvements in radiographic imaging technology and the growing use of low-dose Computerized Tomography (LDCT) for screening. The question arises, therefore, as to whether lung lobectomy is always the most appropriate treatment for very small lung cancers. Perhaps a more limited resection, for example segmentectomy, may be appropriate. A number of studies have investigated the role of limited resection in early stage lung cancer (<2 cm). Minimally invasive surgical procedures may also be appropriate in screening-detected lung cancers, since high-resolution multislice computerized thomography detects even smaller nodules that can be localized with great precision. Also metastases to lung from primaries elsewhere can be accurately localized. The advantages of minimally invasive thoracic surgery, compared to open surgery, for both major and sub-lobar pulmonary resection include shorter hospital stay, reduced pain, reduced release of inflammation mediators, better functional results and even improved survival. We offered intentional limited resection with a minimally invasive robotic approach to patients with very initial screening-detected lung cancers as a part of a pilot study [12 patients with a mean age of 68.2 years (range 32 – 82)]. In all cases, robotic anatomical segmentectomy was performed with the Da Vinci Surgical System. Mean operative time was 189 minutes (range 138 minutes to 240 minutes). A preponderance of lower lobe lesions (11 out of 17; 64,7%) was observed. No conversion from robotic to open surgery has been required and no major intraoperative complications occurred. Hospitalization ranged from 2 to 14 days, with a median postoperative length of stay of 5 days. Postoperative mortality was 0%. Postoperative morbidity rate was 17,6%. All complications were minor. The experience of this study showed that robotic surgery for anatomical segmentectomy was feasible, safe and reproducible. The technique appeared well-suited to the delicate dissection required by segmenectomy. Genetic investigations. The COSMOS screening program

has represented an important resource for genetic investigations, through the creation of a serum bank, which is an essential prerequisite for molecular studies. Sera were collected from all the consenting volunteers at the 2nd year of screening as well as from all consenting patients developing lung cancers, before surgery. MicroRNA (miRNA) are small non-coding RNAs, involved in crucial biological processes and contributing to oncogenesis, because acting as either tumor suppressor or oncogenes. The idea that circulating miRNAs with diagnostic potential exist for almost every type of malignant and nonmalignant disease, including lung cancer, has been supported by several evidences. Using sera from the COSMOS population of asymptomatic high-risk individuals, we developed a test, based on the serum detection of 34 microRNAs (miRNAs) that could identify patients with early stage NSCLCs, with 80% accuracy. The signature could assign disease probability accurately either in asymptomatic or symptomatic patients. Moreover, it is able to distinguish between benign and malignant lesions, and to capture the onset of the malignant disease in individual patients over time. This test for NSCLC could be applied in the clinic as a “first line screening test” for high-risk individuals, to identify those who should undergo further testing, including LD-CT. It might prove very useful for highrisk population screening and, due to the simplicity of the procedure, avoid “medicalization” of asymptomatic individuals, encouraging population compliance to largescale screening programs. Another approach to fully uncover lung cancer genomes and their genetic defects takes advantage of the recent “next generation sequencing” techniques. We have started a project regarding the “whole exome sequencing” of NSCLCs in smoker versus non-smoker patients, in order to idenitify i) variants common in the 2 cathegories; ii) smoke-associated variants; iii) variants leading to cancer in individuals not exposed to smoke. The project is presently on-going and data will be analyzed, also taking into account all the available information present at COSMIC, a database collecting all known and/or described somatic mutations in cancers (specifically looking at lung cancers). In parallel, we are continuing our studies on the identification and characterization of lung stem cells, normal and tumoral, taking the murine tissue as a reference for the human counterpart. The “sphereforming assay” has been used in several tissues as an effective way to isolate/enrich for cells with stem properties. We have established that pneumospheres, derived from the normal murine as well as human tissues, can self-renew and, when properly stimulated,

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Clinical Research

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Lung carcinoid tumors. Lung carcinoids are rare malignant neuroendocrine tumors, accounting for the 1.2% of all lung cancer cases. Lung neuroendocrine tumors comprise 4 different tumor types: the low-grade typical carcinoid (TC), the intermediate-grade atypical carcinoid (AC), the high-grade large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung cancer (SCLC). The subclassification of lung carcinoid tumors into typical and atypical is of clinical importance. TC and AC show a different biological behavior, accounting for the different prognosis: the 5-years survival rate is 4069% for ACs, and 87-100% for TCs. As a consequence, distinguishing AC from TC is necessary to correctly discuss patient’s prognosis and follow-up, as well as to select patients suitable for clinical trials to test new drugs in adjuvant settings. Conventional diagnosis is based on histologic parameters (number of mitoses

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Pathology: A validation study on a specific gene signature in patients operated for stage 1 adenocarcinoma of the lung was performed in cooperation with an American biotech-factory. The results will be presented at the ASCO meeting in Chicago. A large series of pulmonary adenocarcinomas arising in young people has been examined and the genetic markers of potential clinical interest have been reported. The study will be closed in the next weeks.

processes show a significant inter-individual variability, leading to remarkable changes in drug efficacy and tolerability. Therefore, this is a promising approach to discover the mechanisms of drug resistance/sensitivity/ tolerability and optimize the clinical outcome of patients treated with cytotoxic agents. In collaboration with researchers of the Institute of Pharmacology of Pisa University, we quantified by realtime PCR the profile of genes involved in platinum and gemcitabine metabolism in patients with non-small cell lung carcinoma to assess the predictive value of this assay in terms of response to drug administration. From 2004 to 2008 a total of 58 patients affected by locally advanced or advanced NSCLC, who have never received any prior treatment for this pathology, were enrolled in the study. Before initiating neo-adjuvant or palliative chemotherapy, patients were subjected to surgical biopsy with collection of fresh tumour specimens. All the patients received a platinum-based treatment in association to gemcitabine and response to chemotherapy was evaluated by mean of CT-scan according to RECIST criteria (Response Evaluation Criteria in Solid Tumours). Resistance/sensitivity to chemotherapy was correlated with the expression levels of genes involved in gemcitabine and cisplatin activity such as excision repair cross-complementing 1 (ERCC1), human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine-kinase (dCK), 5’-nucleotidase (5’-NT), cytidine-deaminase (CDA), and ribonucleotide-reductase subunits (RR1 and RR2). Among them, 5’-NT was the only gene differently expressed in responders (complete/ partial response) versus no responders (stable/ progressive disease) (p = 0.016) (unpublished data). In conclusion, the overexpression of the catabolic enzyme 5’-NT is significantly associated to a poor response to gemcitabine-cisplatin treatment in NSCLC patients and its validation in future prospective trials will offer new tools for treatment optimization of currently available drugs in selected patients.

Pharmacogenetics/pharmacogenomics studies. Predictive biomarkers are the foundation of “personalized medicine” and pharmacogenomics is one of the tools aiming at their identification. Pharmacogenomics examines the genetic bases of variation in drug metabolism, targeting and transport, and provides the molecular bases for rationally choosing most effective drugs. As a matter of fact, most antineoplastic agents undergo enzymatic bio-transformation within tumour cells, becoming either pharmacologically active or inactive. Enzymatic activities involved in these

Clinical trials. Several “target-oriented” and “nontarget-oriented” clinical trials are currently available for patients with advanced disease at the European Institute of Oncology. Among the target-oriented trials: • two trials evaluate the efficacy and safety of PF02341066 (CRIZOTINIB), a small-molecule able to inhibit the c-Met/HGFR receptor and the anaplastic lymphoma kinase (ALK): a phase II, open-label single arm study to evaluate the efficacy and safety of PF-02341066 and a phase III, randomized, open-

and presence of necrosis), which are very difficult to assess, because depending on the size and quality of the specimens. Therefore, discovering a tool that allows a differential diagnosis between TC and AC in preoperative cytological or histological specimens could be extremely useful for an individual clinical decisionmaking, whenever surgery is a high-risk procedure. We have carried out gene expression microarray analyses on a cohort of 6 typical and 7 atypical carcinoids, identifying 273 genes up-regulated in atypical vs typical tumors. We selected two among them, because scoring among the highest fold-changes and arising as interesting candidates for the definition of a new diagnostic strategy. We validated their expression by quantitative PCR (RQ-PCR) on an independent cohort of 12 patients (frozen samples from 6 TCs and 6 ACs), and cross-validated the expression of the two genes on additional 17 tumors (11 TCs and 6 ACs) by immunohistochemistry (IHC). This led to the definition of a new immunohistochemical diagnostic algorithm, based on the expression levels of the two markers, which was able to correctly categorize tumors into the two subtypes, evidencing misdiagnoses given by the conventional procedure. Sensitivity was determined for this algorithm, ranging from ≅ 80% for TC to ≅ 90% for AC, confirming its potential as a new diagnostic tool. Our study will continue with the analysis of the differentially expressed genes, in order to define druggable targets for either existing or new drugs.

label study of PF-02341066 versus standard of care chemotherapy (Pemetrexed or Docetaxel), in patients with NSCLC harboring a translocation or inversion involving the ALK gene locus; • a phase II randomized study to assess the efficacy and safety of GSK1120212, a specific MEK1/2 inhibitor, compared with docetaxel, in second line subjects with mutations of K/N-RAS, B-RAF, MEK1; • an open labelled phase II study of BKM120, a potent and highly specific oral phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with metastatic NSCLC carrying activating mutation of the PI3K pathway. Among the non-target-oriented studies: • a phase III study that considers Erlotinib versus standard chemoteraphy (pemetrexed or docetaxel) as second line treatment. The aim of this study is to prospectively evaluate the value of the proteomic profile as a predictive factor for erlotinib and chemotherapy efficacy; • a randomized phase II trial of NGR-TNF in combination with standard chemotherapy versus standard chemotherapy alone in previously untreated patients with advanced NSCLC (recently opened). In this protocol, the tumour necrosis factor (TNF) is coupled with the vessel-specific NGR peptide in order to increase drug delivery to tumour sites. In addition two studies were carried out in 2011 to evaluate the efficacy of immunotherapy as adjuvant treatment in non-small cell lung carcinoma patients after complete surgical resection: • a double-blind, randomized, placebo-controlled phase III study (MAGRIT) with recombinant MAGE-A3 protein plus an immunological adjuvant system in patients with resected, MAGE-A3-positive non-small cell lung cancer (MAGEA3 is a tumor specific antigen that can be used to stimulate the immune system of the patients against cancer cells expressing this protein); • an open-label, phase I dose escalation study to assess the safety and immunogenicity of the recombinant PRAME antigen plus an immunological adjuvant system (AS15) in tumours positive for the antigen. These two studies are now closed and data under evaluation.

IEO — Scientific Report 2011 — Ongoing research 2012

Research Activities

differentiate into cells of the lung epithelium (Clara cells, for bronchi and bronchioli, and type II pneumocytes, for alveoli). Cells obtained from pneumospheres dissociation can also repair the injured murine lung epithelium (following naphthalene treatment, causing the ablation of Clara cells in bronchi and/or bronchioli), confirming that this assay is able to identify cells with stem properties. Applying this same strategy, we verified that pneumospheres could be obtained also from the human tumoral lung tissue, although with a low frequency. We are presently trying to improve our culturing strategy, in order to increase the assay efficiency. At the same time, we are conducting xenotransplantation experiments (applying serial dilutions) in immunodeficient mice, in order to determine whether cells derived from tumor pneumospheres show an increased tumorigenic potential in comparison with primary tumor cells. Our final goal is to compare normal and tumor human lung cells showing stem properties, in order to identify tumor-stem-cellspecific markers, which could conduct to their specific identification, isolation, and ultimately targeting. Finally, by using mouse models available at the institute, we crossed k-rasV12 with p21 knock-out mice, in order to investigate the role of the cell-cycle inhibitor p21 in the development of k-rasV12 lung adenocarcinomas. As already determined, we know that k-rasV12 lung tumors show a progressively increasing p21 expression, going from benign (hyperplasia and adenoma) to malignant (adenocarcinoma) lesions. We observed that p21 absence results in accelerated tumor onset and progression, leading to more severe phenotypes than those observed in k-rasV12 mice. This model is still under investigation, in order to completely understand the role of p21 in lung cancer initiation and progression.

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(Neuro) Endocrine Tumours

Neuroendocrine tumours (NETs) arise from dispersed neuroendocrine cells, distributed almost ubiquitously in the body. The majority arise from the digestive system and are able to produce a number of specific hormones, such as gastrin, insulin, serotonin, somatostatin, glucagon, pancreatic polypeptide, VIP in gastroenteropancreatic (GEP) tract, catecolamines in adrenal medulla, ACTH, GH, prolactin, FSH, LH or TSH in anterior pituitary, PTH in parathyroid gland tumours. Conversely, the majority of these cells are also able to produce chromogranins and synaptophysin, which are considered aspecific markers. Therefore, from a clinical point of view, NETs can be either functioning or non-functioning. Since 2000, the WHO classification of endocrine tumours has clearly defined the neuroendocrine phenotype. NETs tend to be slow growing (although aggressive forms exist) and are often diagnosed when they have already metastasised, thus when a radical treatment is no longer possible. Treatment of NETs is typically multidisciplinary and should be individualised according to the tumour type, burden, and to symptoms. Therapeutic tools in NETs include surgery, interventional radiology and medical treatments such as somatostatin analogues, interferon, chemotherapy, new targeted drugs and peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues. Surgery is crucial in many phases, from the eradication of the primary to the debulking of metastatic lesions, in view of other therapies. It is also used to control hormonal symptoms. Interventional radiology techniques (TACE, RFA, HIFU, radioembolization) in NETs are important, due to the common spread to the liver with hypervascular metastases. Medical therapy is used for treating symptoms and/ or reducing tumour growth. Traditional chemotherapy is not commonly applied in well-differentiated NETs, since most are slow growing. However, schemes based

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on platinum derivatives and etoposide are considered in poorly differentiated and/or rapidly progressive NETs, but the choice of the regimen is based on the site of the primary and the histopathological differentiation and proliferation index. NETs usually over-express somatostatin receptors on their cell surface, thus enabling the therapeutic use of somatostatin analogues. One of the basic tools for NETs, in fact, is somatostatin analogue biotherapy, which is able to reduce signs and symptoms of hormone hypersecretion, to improve quality of life and to slow tumour growth. Interferons, and particularly α-interferon, have been used in NETs, with similar therapeutic effects. A recent randomized study versus placebo demonstrated a significant impact of octreotide on progression-free survival in patients affected by GEPNETs [Arnold R et al. J Clin Oncol 2009] Nowadays, new molecular drugs, targeting small cellular proteins or messengers involved in proliferation, are being experimented. The most efficient and studied are vascular endothelial growth factor (VEGF), mTOR and tyrosine kinase inhibitors. Recently, the completion of phase III studies with biological targeted agents, such as the tyrosine kinase inhibitor sunitinib, the mTOR inhibitor everolimus, demonstrated an impact of these molecules on survival parameters in patients with pancreatic NETs. Anti-VEGF monoclonal antibody bevacizumab demonstrated an impact on survival parameters in patient with metastatic carcinoids. Radiolabelled somatostatin analogues have been experimented in NETs for more than a decade. Several clinical trials have indicated that PRRT with somatostatin analogues 90Y-DOTATOC and 177Lu-DOTATATE is an efficient tool in the management of NETs. Present knowledge and our experience at the European Institute of Oncology (IEO) indicate that it is possible to deliver high activities, and therefore high absorbed doses, to tumours expressing sst2 receptors, with achievement of partial and complete objective therapeutic responses

in up to 30% of patients. Side effects, involving the kidney and the bone marrow, are mild if adequate renal protection is used [Bodei L. et al. Eur J Nucl Med Molec Imaging 2004]. Nowadays, tumour candidates for PRRT with radiolabelled somatostatin analogues are basically sst2 expressing NETs, mainly of the GEP and bronchial tract, but also pheochromocytomas, paragangliomas, medullary thyroid carcinomas, and, at least theoretically, any other tumour histotype known and documented as over-expressing sst2, as seen at diagnostic OctreoScan scintigraphy [Bodei L et al. J Endocrinol Invest 2009]. Patients are selected on the basis of a somatostatin receptor scintigraphy or, more recently, a receptor PET with 68Ga-octreotide. During 2008, the new automated synthesis module for the radiolabelling of 68 Ga radiopharmaceutical has been in our Institute, along with a GMP 68Ge/68Ga 1.1 GBq generator with high elution yields. Optimization of the synthesis and quality control of 68Ga-DOTATOC has been carried out and new compounds (such as receptor peptides) are under investigation for 68Ga radiolabelling. In order to improve the predictivity of dosimetry as regards renal toxicity, specific studies have been focused on radiobiological models have been applied to PRRT by means of the Biological Effective Dose concept. An important correlation has been found between BED values and renal toxicity, with results overlapping the curves derived for external beam radiation therapy [Wessels BW, et al. J Nucl Med. 2008]. In particular, a threshold BED of 33 Gy for renal damage and a BED50 (associated to 50% probability of manifesting toxicity) of 44 Gy were found. The analysis of clinical results confirmed that multiple cycle approach in PRRT allows to lower the side effects on kidney, as indicated by radiobiology models [Cremonesi M. et al. Q J Nucl Med Mol Imaging. 2010]. Finally, the influence of risk factors (such as hypertension, diabetes, etc.) has been assessed as a lowered tolerability of the renal parenchyma and recovery of renal parameters [Bodei L, et al. Eur J Nucl Med Mol Imaging. 2008]. At IEO Milano, we recently completed the phase I-II study of 177Lu-DOTATATE in 51 patients with sst2 expressing tumours, mainly NETs. Results showed that 177Lu-DOTATATE was well tolerated up to 29 GBq cumulative activity (7.4 GBq/cycle) from a clinical, hematological and renal point of view and lead to a mean amelioration of clinical status and main tumorrelated symptoms. Objective responses occurred in over 30% of patients with a consistent TTP of 36 months [Bodei L. et al. Eur J Nucl Med Mol Imaging. 2011].

In the past few years, some reports indicated the prognostic value of FDG-PET for NETs. We recently completed a retrospective evaluation of 52 patients to assess the role of FDG-PET scan to predict response and progression free survival (PFS) after 177Lu-dotatate receptor radionuclide therapy in patients with well differentiated NETs. Disease control rate (SD+PR+CR) was 100% and 76% with a PFS of 32 and 20 months, in PET- and PET+ patients, respectively. These results suggest that FDG PET evaluation is useful to predict response to PRRT in G1/G2 advanced neuroendocrine tumours. Notably, none of the PET- patients progressed after 177Lu-DOTATATE [Severi S. et al. submitted]. Following an internal guideline, at IEO, patients are discussed weekly by the multidisciplinary team on NETs and, according to radiobiological principles, the indication for PRRT is preferably given in the minimum disease as possible, within a short timing after radiological and/or surgical debulking when feasible [Oyen W.J. et al. Ann Oncol. 2007] Somatostatin analogue biotherapy is used to reduce signs and symptoms of hormone hypersecretion, to improve quality of life and to slow tumour growth. Interferons, and particularly α-interferon, can be used in NETs, with similar therapeutic effects. Presently, the combined use of α-interferon and somatostatin analogues as first-line therapy is not justified by data in literature, while it could be indicated after progression to a single agent [Fazio N et al. Ann Oncol. 2007]. Recently, in order to further increase the objective response to PRRT, particularly in aggressive, metabolically active tumours, a combined treatment of 177 Lu-DOTATATE with the radiosensitizer capecitabine, has been proposed and tested on a small patients’ population with favourable results, in terms of tolerability and efficacy. Based on these preliminary results, a new protocol of combined treatment of 177 Lu-DOTATATE with capecitabine, administered with a metronomic schedule (named LuX), has been recently approved by the Italian regulation authority. As GEP NETs may also express cholecystokinin 2, bombesin, neuropeptide Y, or vasoactive intestinal peptide receptors, even simultaneously, the potential availability and biological stability of radio-analogues of these peptides will improve in the future the multireceptor targeting of the neuroendocrine cell.

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Clinical Research

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Prostate Cancer Research Programme

SURGERY See also the dedicated chapter on robotic surgery RADIOTHERAPY (See also the chapter on the Division of Radiotherapy) Radiotherapy (external beam radiotherapy and brachytherapy), along with radical prostatectomy, active surveillance, and endocrine therapy, constitutes the approved approaches to localized prostate cancer. The conventional external beam radiotherapy schemes include up to 40 fractions given over 8 weeks. The delivery of such a high number of fractions requires increased access to radiotherapy departments leading to important social costs and patient inconvenience. Quicker therapeutic modalities like surgery or brachytherapy are not suitable for a high proportion of the prostate cancer patients and endocrine treatment alone does not represent a curative modality. Therefore, a short-term non-invasive external beam radiotherapy has been attractive but only recently such treatment has become feasible. Modern radiotherapy techniques in use at the Division include high-precision external beam radiotherapy (ERT) in the form of 3D-conformal dynamic arc radiotherapy (3D-CRT), Intensity Modulated Radiotherapy (IMRT), Image-Guided Radiotherapy (IGRT) and brachytherapy with permanent radioactive seed implants. High precision approach is expected to reduce significantly radiotherapy side effects, in particular late proctitis. In prostate cancer, minimizing radiation-induced urinary and rectal complications is of a great value. They represent a dose-limiting factor for dose-escalation and, when present, significantly affect the patient’s quality of life even long time after treatment. Interestingly, recent in vivo and clinical data suggest that prostate cancer may benefit from hypofractionation (higher dose/ fraction, shorter overall treatment time) due to the lower α/β ratio than that of the rectum and other late

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responding tissue (α/β for prostate cancer is 1.5 Gy and for rectum > 4 Gy). The α/β ratio describes the sensibility of the tissue to the fraction size. The low α/β ratio typical to the prostate tumor cells indicates high sensibility to the fraction size. This means that increasing the fraction size over the conventional one (>2 Gy/fraction) should increase the tumoricidal effect of radiotherapy. Such increase in the dose fraction should have minor effect on the normal tissue due to its higher α/β ratio (as mentioned earlier, 4 Gy and 1.5 Gy for rectum and prostate cancer, respectively). Some studies have been launched to investigate the clinical benefit of hypofractionation in the prostate cancer, and majority of them use daily image-guided radiotherapy (IGRT) systems. Apart from hypothetical radiobiological advantages, hypofractionated schedules would significantly increase convenience to patients, patient compliance and quality of life. Moreover, shorter radiotherapy schedules can allow to reduce the waiting lists in busy radiotherapy centers and to increase the number of treated patients. While the role of brachytherapy has been confirmed in the management of patients with favourable prognostic factors, several questions still remain unanswered regarding its efficacy for patients belonging to the intermediate prognostic group. In patients with intermediate or unfavourable prognostic factors, recent findings from dose-escalation studies, based on 3D-CRT, indicate that prostate cancer can be a relatively radioresistant disease. In order to achieve optimal results in terms of local tumour control, therefore, doses above 78 Gy ought to be administered. In that patient population the combination of external beam radiotherapy with High Dose Rate brachytherapy will be tested. In conclusion, the main research activities in prostate cancer include: • Image-guided radiotherapy (IGRT): daily prostate

localization with use of one out of three IGRT systems installed in the Division of Radiotherapy (b-mode acquisition and targeting ultrasound system, X-ray 6D system, cone beam computer tomography system) allowing for the verification of set-up errors and organ motion (fig. 1-3). • Hypofractionated RT with scheme of 2.7 Gy/ day up to 70.2 Gy in 5 weeks (radiobiologically equivalent to the conventional scheme of 84 Gy in 42 fractions over 8 weeks). In all patients IGRT is applied. Various dosimetric and clinical aspects are being investigated including quality of life evaluation. • Intensity Modulated Radiotherapy using dynamic RapidArc modality associated with Image-Guided Radiotherapy (IGRT) verification and hypofraction for post-prostatectomy irradiation. • Institutional dose volume histograms (DVH) constraints has been established based on the follow-up data and review of the literature. Several planning studies on the dose distribution and the optimisation of the treatment plans have been published and the other are ongoing. • Intraoperative radiotherapy IORT (anticipated boost) in intermediate and high risk prostate cancer. • Postoperative radiotherapy vs. salvage radiotherapy: indications, the role of combined RT and endocrine therapy. • Stereotactic radiotherapy for oligometastatic prostate cancer patients. • Artificial neural network use to predict acute rectal toxicity after external beam radiotherapy for prostate cancer (collaboration with and Politecnico of Milan). • Extreme hypofractionation using one of the best available dose delivery systems (two-stage phase II, prospective, single-arm, monocentric clinical trial). Medical Oncology The course of prostate cancer from diagnosis to death is categorized as a series of clinical states defined by extent of disease (localized disease, rising PSA after local therapy, advanced disease with absence or presence of detectable metastasis) and by response to androgen deprivation. Our scientific activity on prostate cancer addresses the issues commonly encountered by practising oncologist. The research themes varied including: • Development of surrogate markers that may have

utility in predicting prognosis and monitoring the antitumor effects of treatment in castration-resistant prostate cancer. • Clinical trials addressing new drugs in patients with castration-resistant prostate cancer • Clinical trials including biological agents targeting different critical points of the signaling cascade or proteins of the mitotic machine.

Research Activities

Clinical Research

Despite its limitations, PSA is the best tumor marker of prostate cancer currently available in clinical practice. We are developing an alternative biomarker strategy, testing the prognostic and predictive value of Circulating Tumor Cells (CTCs) in prostate cancer, using the CellSearch System, an immunomagnetic method for detecting CTCs (see specific chapter) automated, standardized and approved by the Food and Drug Administration for routine clinical use in metastatic breast cancer and recently also in Castration-Resistant Prostate Cancer (CPRC). Two different populations are under investigation in our projects: • Patients with Castration-Resistant Prostate Cancer (CRPC) who are starting first or second line systemic treatment for advanced disease. In this population we are exploring the prevalence of detectable CTCs, the correlation between the presence of CTCs and known independent prognostic factors such as haemoglobin, alkaline phosphatase and PSA values and their predictive and prognostic impact. Beside, a relevant aim of this study is to determine the predictive role of changes in CTCs number during chemotherapy courses as an early surrogate marker of response. Forty-seven (81%) out of 58 pts had evaluable CTCs, with a median of 12 cells/7.5mL of blood (range 0-1959). CTC number was significantly correlated with PSA (p=.05), alkaline phosphatise (p<.01), bone metastases (p=.01) and bone plus visceral metastases (p=.02). At univariate analysis only CTC count was strongly associated with both median PFS (p=.001, 95% CI 5.68.8) and median OS (p<.001, 95% CI 12.4-23.6), alkaline phosphatise was associated with median OS (p=.001), an increase of PSA > 100 ng/mL significantly correlated with shortest median PFS (5.7 months, p=.029) but not with OS (12.4 months) (p=.279). A relevant decrease of median PFS from 0-4 to > 5 as also for median OS starting from >20 cells/7.5mL was reported. Correlation between PFS and OS according to CTC changes compared to baseline was statistically significant at all time points evaluated. These data strengthen the trial

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implicated in oncogenesis as well as the expression and persistence of malignant phenotypes. Besides, complex interactions of the Androgen Receptors with other signalling pathways play a role in hormonal resistence of prostate cancer. Src family kinases, PDGFR-β, c-kit and other receptor tyrosine kinases found to be constitutively activated, overexpressed or activated by androgen receptors in prostate cancer. Primary endpoint of the study is overall survival. The study has been approved by our Ethic Committee and conducted from 2009 to march 2011. Since the enrollment stopped we have continued to follow up patients and data are under evaluation. Due to the recent stop of several research project previously ongoing , we are considering the implementation of further clinical trials with new hormonal and targeted therapy for every stage of prostate cancer disease.

Advanced disease is associated with prostate cancerspecific mortality and morbidity due to disease. First line therapy of metastatic prostate cancer is androgen blockade with bilateral orchiectomy, diethylstilbestrol or luteinizing hormone releasing hormone (LHRH) agonists given with or without an anti-androgen (e.g. flutamide, bicalutamide, or nilutamide) which provides effective palliation, temporary tumor control or regression, and biochemical responses (e.g. decline in serum prostate specific antigen PSA in 80- 85% of patients). Despite the established efficacy of hormonal therapy as first-line treatment of metastatic prostate cancer, virtually all patients will eventually develop disease progression that is resistant to hormonal treatments. Docetaxel and prednisone demonstrated an improvement in survival of approximately 2 months compared to mitoxantrone and prednisone, and provided symptom palliation in less than half of patients in metastatic Castration-Resistant Prostate Cancer. To improve the Docetaxel results, different drugs are under investigation in this setting of patients. • Dasatinib, a potent orally active tyrosine kinase inhibitor, is under investigation in a,randomized, double-blind phase III trial. This trial compares Docetaxel/prednisone and Dasatinib with Docetaxel/prednisone and placebo in castrationresistant prostate cancer. Dasatinib in a potent, orally active tyrosine kinase inhibitor and has been shown to inhibit at least 5 protein kinases families: SRC family kinases, BCR-ABL, KIT, EPHA2 and PDGFβ receptor, all of which have been

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Research Activities

hypothesis about the prognostic and predictive value of CTCs so we closed the enrolment and we are going to complete the paper to submit it for publication. • The second group we are going to study, includes patients with clinically localized prostate cancer eligible for radical prostatectomy or curative radiation therapy. Ninety patients, with biopsy proven prostate cancer, localized disease (clinical stage T1-3NxM0, any Gleason score) suitable for curative local treatment (radical prostatectomy or curative irradiation-external or brachytherapy) will be enrolled In this study. The aims of this project are to investigate the prognostic value of CTCs before and after curative treatments, the correlation between CTCs and other known prognostic factors and biomarkers. We started the enrolment but it’s to early for further evaluations. We will also evaluate the correlation between CTCs levels and acetylation status of primary human prostate tumor cells.

Figure 2. Stereoscopic X-ray imaging

Figure 1

Image guided radiotheraphy systems based on the ultrasound prostate localisation

Figure 3. On-board images

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High Intensity Focused Ultrasound (HIFU)

the advantage of providing temperature data within seconds after HIFU exposure (sonication). However, MRI guidance is expensive, labour-intensive and of lower spatial resolution in some cases, although it is superior to sonography in obese patients. Sonographic guidance provides the benefit of using the same form of energy that is being used for therapy. The significance of this is that the acoustic window can be verified with sonography.

Research Activities

Clinical Research

PET-CT image scan before (a) and after (b) HIFU treatment of bone metastasis from multiple myeloma.

Introduction High intensity focused ultrasound (HIFU) is a highly precise medical procedure using focused ultrasound energy for burning and destroy the tumor tissue at depth within the body, selectively and without harming overlying and adjacent structures within the path of the beam. The possibility that focused ultrasound therapy might be developed as a result of controlling local heating phenomena was introduced by Lynn et al in the 1940s, but the technique was not developed at that time because of inadequate targeting methods. The advent of more sophisticated imaging has led to a resurgence of interest in HIFU. Unlike radiofrequency or cryoablation, which are also used to ablate tumors, HIFU is completely non-invasive and can be used to reach tumoral areas that are deep within the body, if there is an acoustic window for allowing the transmission of ultrasound energy. Preliminary reports underline a reduced toxicity with HIFU ablation compared with other ablation techniques because of the non-invasive nature of the procedure. First devices never used widely in clinical practice were

US-guided HIFU equipment

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Successful treatment of a small liver metastasis from colon cancer close to inferior vena cava. (a). US (ultrasound) image obtained before HIFU treatment (b). US image obtained immediately at the end of HIFU ablation shows hyperechoic region in the treated area.

Focused transducer

trans-rectal probes, which have been used predominantly to treat the prostate cancer. Extracorporeal devices are significantly larger and can be used to treat a variety of problems, most commonly intra-abdominal solid tumors. Background The JC HIFU system (installed since 2007 at IEO), is an extracorporeal device using a 20-cm-diameter plane lead-zirconate-titanate disc transducers with an aluminium alloy lens with focal length of 16 cm, driven at 0.8 MHz. It operates at relatively high intensities up to 20,000 W/ cm2. The imaging probe is situated in the centre of the high-intensity focused ultrasound transducer allowing for real-time US image monitoring during treatment. Another available extracorporeal device uses an MRI-compatible 10-cm-diameter focused transducer with an 8-cm radius of curvature, operating at 1.5 MHz, and only licensed for use with uterine fibroids. Guidance and monitoring of acoustic therapy is most important to ensure that the desired region is treated and to minimize damage to adjacent structures. Real-time imaging, such as ultrasound, ensures that HIFU beam targeting is maintained within the correct area throughout the procedure. Both MRI and Ultrasound methods have their advantages and disadvantages. MRI has

Imaging methods to assess HIFU treatment are similar to those used to assess the response to other methods of ablation such as radiofrequency ablation and include contrast enhanced CT and MRI. In addition, the use of micro-bubbles ultrasound contrast-enhanced is also being examined as a method for evaluating efficacy of HIFU traetment. These methods all examine the change in vascularity of the treated volume. PET scan is another diagnostic tool currently used for oncologic applications; it is used for assessing changes in metabolic activity after HIFU treatment. Diagnostic ultrasound has an excellent safety profile, with no clinically significant deleterious biologic effects having been reported using current diagnostic equipment. However, at high energy, ultrasound can result in tissue heating and necrosis, cell apoptosis and cell lysis. Several mechanisms are involved in the tissue damage induced by HIFU. Localized heat generation due to absorption of the acoustic energy is the main biological effect in tissues where it rapidly raises temperatures from 55°C to 100°C, causing coagulation necrosis within a few seconds. The precise and well delimited US focusing (e.g., 1 mm diameter and 9 mm length), minimizes the potential thermal damage to the tissue located along the acoustic pathway, because the energy is much lower outside the focal region. Mechanical phenomena, in addition to thermal effects, are associated only at high energies.

Cavitation is the most important mechanical phenomenum; it can be defined as the creation or motion of gas bubbles within an acoustic field due to alternating compression and expansion of tissue as ultrasound waves propagate through it. Thermal effects, acoustic cavitation and vessel damage can occur simultaneously within the targeted tissue in HIFU ablation. Therefore, the coagulation necrosis induced by HIFU can be considered as the result of biological effects from a combination of heat, cavitation and vascular destruction on tissue. Combination between imaging and technologies for local therapy has made ablative procedures more reliable and practical, allowing for safe and feasible application of HIFU treatments in clinical practice. US-guided extracorporeal high-intensity focused ultrasound has been recently used to treat patients with various kinds of malignancies, including liver, breast, kidney, bone, and soft tissue both in Asia and Europe. Magnetic Resonance guided Focused Ultrasound is currently used in North America, Europe and Asia for treating uterine fibroids. Investigation and applications of HIFU are growing rapidly worldwide. Although major clinical application for HIFU is in treatment of benign and malignant solid tumors, several other potential therapeutic applications of HIFU are being investigated, including thrombolysis, arterial occlusion for the treatment of tumors and bleeding, haemostasis of bleeding vessels and organs. In China, the first HIFU procedure was performed for male patient with tibia osteosarcoma on December 1997. From December 1997 to December 2007, more than 10,000 patients with primary and metastatic liver cancer, malignant bone tumor, breast cancer, sarcoma, kidney cancer, pancreatic cancer, bone metastatic tumors, uterine fibroids, breast fibroadenoma, and hepatic hemangioma, have been treated with US-guided HIFU ablation in more than 40 HIFU centres. The group from Chongqing University has reported to date the largest series of HIFU clinical applications, in which 1038

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with visceral pain due to pancreatic cancer after treatment with HIFU.

Liver Several studies on HIFU treatment of HCC and secondary liver metastases in human clinical trials have been already published. Wu et al. used an extracorporeal HIFU device to treat 68 patients with liver malignancies showing complete tumor ablation in 30 cases in which surgical excision followed HIFU treatment. HIFU has also been used for palliation in patients with advanced-stage liver cancer by Li CX et al. observing after treatment 87% of symptomatic improvement. In Oxford, UK, in 2005 a total of 22 patients with liver metastases were treated with HIFU revealing a favourable adverse event profile when compared to open or minimally invasive techniques. The only Randomized Clinical Trial is from Wu et al. that randomized patients between transarterial chemoembolization (TACE) alone and HIFU after TACE. The median survival times were 11.3 months in the combined HIFU–TACE group and 4 months in the TACE-only group (p = 0.0042). To date, both animal and human subject investigations show significant promise in the treatment of hepatic malignancies with HIFU. Authors from Chongqing University (China) observed that ablation with HIFU can safely achieve large areas of coagulation necrosis in tumors close to major blood vessels, without damage to vascular integrity. Hence this therapy can be applied to treat many patients who can not undergo conventional treatment techniques due to either absolute and relative contraindication or tumor location.

Bone The HIFU technology has been used also for the treatment of bone and soft tissue malignancies with good functional outcome as a limb-preserving alternative to conventional therapy. In 2004, Wu et al described the use of HIFU system for treating 153 cases of bony malignancy, and 77 cases of soft tissue sarcoma. The interesting aspect observed was the spontaneous regression of lung metastases after HIFU ablation of a primary bone tumor, in the absence of any other systemic therapy. Such findings have not previously been reported and may imply a beneficial immunological process such as specific antitumor immune sensitization. In contrast to other ablative techniques, such as radiofrequency ablation and cryoablation, primary malignancies have been treated more frequently than secondary tumors. In summary, the intention of treatment has been both curative and palliative, depending on clinical circumstances. Hence HIFU has been proposed as a limb-sparing alternative to amputation, and it is implied that it should be used according traditional surgical principles, alongside neoadjuvant and adjuvant chemotherapy where appropriate.

Kidney Treatment of renal tumors has been evaluated in 2006 (Roberts WW) in animal models. HIFU ablation of renal tumors in humans is still within early clinical trials. A clinical feasibility study using HIFU to ablate renal tumors has been performed on eight patients who showed histologic evidence of ablation in the treated areas after excision. Pancreas HIFU for palliative treatment of pancreatic cancer may be useful in patients who develop symptoms that would benefit from local tumor control. Results from an open-label study in China in 251 patients with advanced pancreatic cancer (TNM stages II–IV) suggested that HIFU treatment can reduce the size of pancreatic tumors without causing pancreatitis and thus prolonging survival. All treated patients showed a survival benefit with a symptomatic relief observed in 84% of patients

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Uterine fibroids Clinical trials correlated both treatment volume and symptomatic response to the ablation of uterine fibroids. Stewart et al. reported a series of 109 premenopausal women who underwent MR-guided HIFU ablation of symptomatic uterine leiomyomas. A validated questionnaire, revealed symptom reduction at 6 months in 71%, and 51% at 12 months. The authors concluded that this treatment results in short-term symptom reduction for women with symptomatic uterine leiomyomas with an excellent safety profile. Ieo experience At IEO first study on coagulative lesion formation by high intensity focused ultrasound on ox liver in vitro tests started in 2007; it included a sequence of experiments under different exposure conditions followed by tissue sectioning. We confirmed previously published data about the development of HIFU-induced tissutal lesions, by changing different parameters as acoustic propagation, absorptive heating, power and intensity of sonication in bovine liver as a laboratory model.

cancers and for patients with abdominal tumors close to the bowel. The patients were asked for fasting overnight the day before HIFU treatment. The day of procedure skin surface overlaying the lesion was shaved and purified in every patient, for avoiding the presence of hairs within the acoustic pathway. In order to treat the symptoms and obtain a dimensional reduction we performed HIFU ablation on 16 cases of uterus fibromyoma since march 2009, all in Day Surgery. We obtained symptoms disappearance and uterine myoma ablation in all ladies which were not observed any side effects. Control MRI showed lesion shrinkage in 11 patients after 6 months of follow-up.

At the end of “in vitro Phase” we concluded that HIFU is a feasible and a safe technique to perform a precise tissue ablation at different power and intensity of sonication. As a comprehensive cancer center from November 2007 and December 2009 at EIO, 71 patients (age range 16-77 years) have been treated with ultrasound-guided HIFU, within a feasibility study phase. All patients included in this study were deemed not candidate for surgery, nor suitable for local ablative techniques such as radiofrequency ablation (RFA), transcatheter arterial chemoembolization or embolization (TAE), or were unwilling to have any of those treatments. Among the 71 patients, eight with HCC, 12 with liver metastases from colorectal cancer (CRC), two with chest wall metastasis from CRC, one with liver and lung metastasis from CRC, two with soft tissue metastasis from CRC, one with liver and lung metastases from CRC, two patient with liver metastasis from breast cancer, two patients with 2 liver metastases from neuroendocrine tumor (NET), five patients with bone metastases from breast cancer, one with pubic bone metastasis from bladder cancer, one with osteosarcoma, one patient with leg metastasis from lung cancer, one with iliac metastasis from multiple myeloma, one with abdominal liposarcoma, three patients with pancreatic tail NET. With the aim of palliation we have treated twelve patients with pancreatic cancers, eleven at the advanced stage with no indication for surgical resection; one patient had local relapse after surgery. In these patients we observed disappearance of visceral pain. All of them had chemotherapy and radiotherapy with progressive local disease at the time of HIFU treatment. Patients were evaluated with conventional tests before treatment. MDCT or/and PET-CT or MRI were performed as baseline-imaging. Specific bowel preparations were required for patients with pancreatic

Research Activities

patients have been treated with US guided HIFU in ten centers for the purpose of either cure or palliation.

Pancreatic cancer : PET-CT images. (a). Pre-treatment PET-CT image showing positive of the tumour. (b). One month posttreatment, PET-CT image showing negative of the tumour.

For this study, HIFU therapy has always been performed under general anaesthesia, necessary for preventing the patient from experiencing any pain or discomfort and to ensure immobilization during treatment. As feasibility study IR team considered procedure timing as fundamental. Room time, including preparation time and treatment time, ranged from 1 hours 50 minutes to 7 hours 5 minutes. The sonication time, defined as the exposure time, which was related to the tumor size, tumor site and blood supply, ranged from 1 minute 20 seconds to 172 minutes. Fifty-six patients had one session of treatment, fifteen patients had a second session after a mean period of 6 months from first treatment. PET-CT or/and MDCT showed complete response in about 80 % liver metastases; bone lesions were palliated in symptoms and PET-CT detection; MDCT and PET-CT showed lesions of chest wall, leg muscle, abdominal wall and lung completely ablated; the huge liposarcoma has been almost completely ablated at MRI; pancreatic lesions were palliated in symptoms and at PET-CT or MRI. MRI at 1, 3 and 6 month a complete ablation of all the uterine

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HIFU of Uterine myoma. a) MRI imaging; b) intraoperative UScontrast media showing the vascular feeding; c) US planning before HIFU; b) US-contrast media administered after HIFU procedure showing complete ablation of the myoma.

Early Breast Cancer Over the past decades, there has been a radical change in the surgical treatment for breast cancer. moving towards a more conservative approach, achieving the same clinical efficacy and maintaining the integrity of the woman’s body. The IEO Breast Division is highly involved in clinical research to improve the effectiveness of breast cancer treatments reducing invasiveness and potential side effects of surgical treatments. Many papers has been published on the results on HIFU treatment for breast cancer. Although the results are very encouraging and significant, the small number of cases and the non-homogeneous of enrolling criteria and post histopathological and/or instrumental HIFU evaluation, leave large space for scientific speculation. Along this perspective, from the beginning of 2008, we started a feasibility study on breast cancer treatment by mean HIFU approach, paying particular attention to establish very restrictive criteria for patient recruitment. The study consists on HIFU treatment of selected breast cancer and surgical resection at least one week after treatment, in order to evaluate histological findings such as residual viable tumour, coagulative necrosis extension and so on.

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All patients enrolled for HIFU treatment must meet all of the following inclusion criteria: • Female subject, age > 18 years; • Diagnosis of breast cancer, confirmed by breast corebiopsy; • Single tumour • Clinical stage cT <1,5cm; cN=any; cM=any • Lesion boundaries visualized with US imaging • Lesion circumscribed at least more than 5mm from skin or rib cage • Lesion circumscribed at least more than 20mm from nipple areola complex • passed the HIFU Simulation test • patient able to read, understand and sign the study specific informed consent form; The exclusion criteria were: • Presence of skin ulceration and/or scar • Presence of widespread pathological microcalcifications

as pain, discomfort and involuntary movements. The day of procedure, skin surface overlying the lesion was degassed and degreased with 95° alcohol to remove microbubble of air within the acoustic pathway. The day before surgery, the patient underwent radiological investigations (breast US and digital mammography) for verifying the changes in the tumour area, caused by thermal damage. Surgical specimen was extensively examined histologically in order to verify the thermal damage and confirm the absolute integrity of the surrounding parenchyma. Only 16 out of 58 patients (age range 41-74 years), initially included according to clinical reports at the first medical visit, were selected and then treated by mean of USgHIFU. Tumour lesions ranged from 5 mm to 15 mm. All patients, included in the study, were deemed candidate for breast conservative surgery that was performed after 1-6 weeks from HIFU treatment.

Before HIFU treatment, all patients were subjected to a careful clinical assessment including anamnesis and pre-surgical routine tests. In addition, each patient was re-evaluated by a radiologist with HighFrequency linear array US probe and digital mammography.

Intraoperative image: fascia of muscle pectoralis major has been also involved by HIFU, due to the deep position of treated cancer

Cosmetic result 25 days after HIFU; before surgery

All HIFU treatments were performed in a single session under local anaesthesia plus MAC (Monitored Anaesthesia Care) in order to improve the room time and preserving patient comfort and collaboration, although at the beginning of experience, general anaesthesia was used in order to avoid any possible external effects disturbing the treatment

Great importance was given to the time of the procedure. Room time ranged from 3 hours 55 minutes to 1 hour 20 minutes. The overall treatment time, defined as the time from the beginning to the end of HIFU energy emission, ranged from 1 hours 55 minutes to 8 minutes. Sonication time, defined as the exposure time to HIFU energy, ranged from 15 minute 4 seconds to 38 seconds.

Research Activities

fibromyoma without side effects. Local oedema was observed in 4 patients. No other complications were observed. All the patients returned home 1-3 days after treatment.

No side effects were observed after the HIFU treatment. Only soft local mammary oedema was noted overlying the not allow for dye staining in those cases which had shown receptor expression positivity in the pre HIFU treatment immunohistochemical evaluation. Another method to evaluate the effect of the HIFU was tested: 2,3,5-triphenyltetrazoliumchlorid staining (TTC) was used to estimate the capability of treated cells to absorb a viable dyeing at the gross examination.

From Left to Right (H&E staining): 1-2: coagulation necrosis (x100), ( x20); 3: Thrombosis e microhemorrhages

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Surgical specimen: hemorrhagic ring surrounding the tumour

Haematoxylin & Eosin stain showed that cellular structure looked normal in some cancer cells, without any signs of tumour cell breakdown. The tissues maintained their cytologic staining characteristics giving an appearance similar to viable cells. By using conventional histological techniques, it was difficult to identify whether the treated tumor cells were dead or alive, in this region, under light microscope. Very interesting was the indirect detection of thermal damage in the immuno-histochemical evaluation of oestrogen and progesterone receptors (ER and PR) expression. Protein denaturation indeed, caused by the high temperature, did not allow for dye staining in those cases which had shown receptor expression positivity in the pre HIFU treatment immunohistochemical evaluation. Another method to evaluate the effect of the HIFU was tested: 2,3,5-triphenyltetrazoliumchlorid staining (TTC) was used to estimate the capability of treated cells to absorb a viable dyeing at the gross examination. All lesions evaluated with this kind of method showed to be not stained.

his preliminary research, although limited to a small number of patients, but quantitatively comparable to those presents in literature, showed the USgHIFU treatment appears to be feasible and safe to destroy primary early breast cancer, without side effects. Hence, histopathology evaluation performed on surgical specimen after HIFU treatment, confirmed the correct ultrasound guided targeting and release of thermal energy only within the tumour. Based on these premises, for the next year the Breast Division research activity will be directed towards different targets. Technology upgrade was the other main issue within our research activity: the introduction of a new Real-time Variable-Band Linear Array - 13-4 MHz US probe and a new dedicated HIFU transducer have been developed in order to improve the US guidance/monitoring and reduce treatment time, respectively. Ongoing Research The principal purpose arising from our 2009 experience is addressed to confirm the kind of patients would be the best suitable for this non-invasive treatment. Over this main issue, ongoing research is focused to continue the upgrade of HIFU technology in order to

Regarding the histopathology point of view, specific methods will be improved for assessing cell viability after HIFU through the development of viable colour methods and cell cultures. As a “so called” non-invasive technology, HIFU approach may play a key role in the future management of solid malignancies. The preliminary research activity and application of this new technology in patient care started more than one year ago and was limited to a small number of patients with short-term follow-up, in focusing the feasibility and safety of this new technique. Based on the preliminary results, US imaging-guided focused ultrasound treatment appears to be feasible and safe to ablate primary and metastatic liver tumors, primary and metastatic bone tumors, pancreatic cancers and soft tissue tumours, without significant side effects, and can have a role in “multi-modal” management of patients with tumour disease. The research activity is deeply integrated with the daily clinical work and the principal purpose arising from our studies are addressed to follow up all patients to ascertain which patient group would be the best suited to this non-invasive treatment. Over this main issue, ongoing research is focused also on implementation of HIFU technology in order to optimize the patient’s approach technique.

Research Activities

All lesions evaluated with this kind of method showed to be not stained.

Immuno-histochemical evaluation of oestrogen receptors (ER) expression: Left: before HIFU; right after HIFU

optimizing the technique and reducing the treatment time. From the anaesthesiology point of view, our results indicated that local anaesthesia together with MAC is feasible for HIFU procedure and our encouraging results may change our patient’s management in the future.

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Robotic Surgery Research Programme

Robotic surgery Research Programme Robot-assisted surgery is the latest evolution of minimally invasive surgery; it has been evolving from simple adjustable arms to support cameras in laparoscopic surgery through to the more sophisticated four-armed machines now available. The da Vinci™ operating robot is a telemanipulation system consisting of a surgical arm cart, a master console and a conventional monitor cart. It acts as remote extensions completely governed by the surgeon and thus are best described as master-slave manipulators. At IEO two of da Vinci™ operating robots (Surgical Intuitive, Inc., Mountain View, CA) are active, the first one since October 2006. To maximize utilization and reduce maintenance costs, they are jointly used by the departments of Urology, Gynecology, AbdominalPelvic, Thoracic, General-Laparoscopic and Head and Neck Surgery. The main technological advantages of this system are realistic 3-D imaging, motion-scaling and tremor filtration, facilitating more precise and accurate endoscopic surgery. It makes difficult and previously inaccessible body areas easier for surgeons to access and may lead to decreased morbidity for patients. Various surgical procedures have proved feasible and safe when performed with the da Vinci™ robot. The advantage of the system is best seen in tiny areas difficult of access and when dissecting delicate, vulnerable anatomical structures, like mesorectum, prostate, uterus, pulmonary lobes or larynx. In the light of our present experience, we regard prostatectomy, hysterectomy, pulmonary resection, adrenalectomy, left colectomy, gastrectomy and total mesorectal excision deep in the pelvis as appropriate for a robotic approach, whereas splenectomy, pancreasectomy and liver resection need further evaluation. The steric vision and the intuitive use of the instruments are of great assistance, although the lack of robotic stapler devices and flexible instruments sometimes hamper full robotic performance.

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The robotic approach is significantly more expensive than conventional minimally invasive surgery. This extra cost is due to longer operating times, as well as the high cost of the robot itself and higher costs for the robotic instruments, which are re-usable ten times only. The time delay may be explained by the learning curve. However, with increasing experience on the part of the entire team (surgeons, scrub nurses, and theatre attendants), the setup time has been markedly reduced in our Institution, and no longer involves any time loss. Cost-effectiveness is a major issue; 2 recent studies comparing robotic procedures with conventional operations showed that although the absolute cost for robotic operations was higher, the major part of the increased cost was attributed to the initial cost of purchasing the robot and yearly maintenance. Both factors are expected to decrease as robotic systems gain more widespread acceptance. Decreasing operative time and hospital stay will also contribute to the costeffectiveness of robotic surgery. Other drawbacks to robotic surgery include the bulkiness of the robotic equipment currently in use. Lack of tactile and force feedback to the surgeon is another major problem, for which haptics (ie, systems that recreate the “feel” of tissues through force feedback) offers a promising, although as yet unrealized, solution. The patients’ satisfaction following a robotic approach is high. Ready acceptance of the robotic approach may result from the satisfactory cosmetic and symptomatic results, but also from the patients’ impression that they had taken part in the dawn of a new surgical era. In the single Divisions’ Chapters, a complete list of all ongoing studies is presented. IEO Divisions of Urology and Gynecology are currently runnnig clinical research projects supported by the Italian Ministry of Health, but all surgical Divisions are actively working in research applications of this new surgical tool.

Perspectives Robotic surgery was originally developed to render possible a kind of telesurgery bridging thousands of kilometers or even continents. Although the feasibility of this aspect was proven and gained some media attention, it is not the future of robotic surgery. More probable opportunities for robots are image fusion and surgical training. At this stage the superposition of different radiologic imaging systems permits more precise and detailed surgical planning. The da Vinci™ system will implement this technique in the operating room itself by flashing a patient’s scan images into the virtual three-dimensional view on the console. This will enable the surgeon to more easily detect and identify hidden anatomical structures, and in this way robotic surgery will help to make minimally invasive surgery safer. Another great potential for the da Vinci™ robot probably lies in its impact on surgical training. It will be possible to carry out a particular patient’s complete surgical procedure using his CT scans and robotic virtual-reality training programs. Thus, similar to a pilot on a flight simulator, surgeons in training will perform new operations only after performing them successfully in virtual reality.

reach full potential. The ultimate extrapolation of this progress is the development of intracorporeal robotics, the feasibility of which has been demonstrated. At this moment there are no reports to clearly demonstrate the superiority of robotics over conventional laparoscopic surgery. Further research and more prospective randomized trials are needed to better define the optimal application of this new technology in gastrointestinal oncologic surgery. The challenge for today’s robotic surgeons is to advance the system through clinical research in such a way that it becomes suitable and indispensable for future routine applications.

Research Activities

Clinical Research

Conclusions With the da Vinci surgical robot surgery regains two fundamental tools of surgical procedures: intuitive control over the surgical instruments and steric perception of the operative field. Only several centers are currently using surgical robots and publishing data. In gastrointestinal surgery, robotic surgery is applied to a wide range of procedures, but is still in its infancy. Most studies reported that robotic gastrointestinal surgery is feasible and safe, provides improved dexterity, better visualization, reduced fatigue and high levels of precision when compared to conventional laparoscopic surgery. In a relatively short time, robotic procedures spanning the whole spectrum of surgery have been successfully executed. Initial results show that mortality, morbidity, and hospital stay compare favorably to conventional laparoscopic operations. However, only a limited number of randomized, prospective studies that compare outcomes of robotic techniques with conventional methods exist. While current robotic systems have considerable advantages over conventional laparoscopic techniques, they are not without limitations. Robotics main drawbacks in surgical practice are the absence of force feedback and extremely high costs. Miniaturization of robotic components and systems is feasible and necessary to allow minimally invasive techniques to

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Clinical Research Research Activities

Interventional Radiology A) HCC relapse after left lobectomy B)CT 1 month after TAE C)CT scan 6 months after TAE D) CT scan 15 months after TAE

Clinical needs for local tumor control, by means of minimally invasive approach more than classical invasive, risky and expensive surgical ones, represents the main reason for the very fast developing of new hyper-technological branches in Oncology. Due to the high specialization required for most of the interventional procedures in Oncology, the appellative of “Interventional Oncology” currently defines this field. Image guidance for very precise treatments and low invasiveness, compared with more traditional options, are common elements among all interventional procedures For academic purposes Interventional Radiology may be divided into two different main fields: Vascular treatments and Percutaneous procedures. The use of blood stream for delivering drugs and/or particles directly and selectively to the tumor is the aim of all Vascular treatments in Interventional Oncology and they are mainly oriented for liver tumor control. Percutaneous procedures include all image-guided tumor ablative techniques which require percutaneous insertion of dedicated devices (such as needle for radiofrequency). Moreover, a totally new locoregional therapy for tumor ablation has been very recently added to the IR armamentarium: High Intensity Focused Ultrasound (HIFU-see dedicated chapter). Vascular treatments Introducing the multimodal approach for hepatic lesions Hepatic lesions represent a main challenge in clinical oncology, both for primary and metastatic tumours. It is mainly due to the crucial role this organ represents for the prognosis. In hepatocellular carcinoma, the simultaneous presence of cirrhosis and hepatic tumour masses, in the majority of patients, limits the indication

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for an aggressive local approach, because of the high risk of post-treatment liver failure. Moreover, the high rate in developing new nodules after any local treatment (>80% after 4 years), plays a key role in the decision making on the treatment strategy. Regarding liver metastases, local treatments such as surgery for liver metastases (mainly from colorectal cancer) after systemic chemotherapy seems to induce better results than chemotherapy alone. Minimally invasive local techniques are therefore being continuously developed in order to supplement the surgical resection. Super-selective micro bland arterial embolization with micro-particles. Hepatocellular Carcinoma: Intra-arterial treatments such as transarterial chemoembolization (TACE) and transarterial embolization (TAE), are considered a palliative therapy for multifocal HCC not suitable for surgical resection or percutaneous ablation therapies (mainly indicated for intermediate stage), but the efficacy of intra-arterial treatment in patient with single HCC is not well defined (7,8,9). However, it is still not really clear whether embolization alone gives the same survival advantage (5) nor whether specific patient characteristics affect outcome or any particular technique in performing transarterial therapy is better than any other. A very interesting study on the assessment of feasibility and local response in patients affected by unresectable hepatocellular carcinoma, was carried out in IEO: patients were treated with mbTAE (micro bland TAE) by using very small sized fine-calibrated microparticles (40 and 100 μ). Between October 2007 and september 2009 63 HCC patients underwent 100 TAE, and a total of 87 target lesions were embolized with a super-selective technique. In all cases liver perfusion scintigraphy was performed by injecting Tc-99 labelled albumin macro-aggregates (MAA)

via the main hepatic artery, just before treatment in order to rule out patients with a pulmonary shunt higher than 10%. In four patients a 3-5 % amount of pulmonary shunting was observed and embolization was performed by upsizing microparticles to 100 μ in order to reduce the risk of pulmonary embolism. Twentyfour hours after treatment, upper abdomen MDCT was performed in order to assess the early local result after embolization. Local outcomes on target lesions at 1 year follow-up in these 29 patients are: 6 complete response (CR ), 5 partial response (PR ), 7 stable disease (SD ), and 9 progressive disease (PD ) for new lesions (i.e. new nodules) while 2 PD on treated lesions.

Metastases: The aim of this new field is to assess the feasibility and efficacy in metastatic lesion control with a new kind of coated small particles: 40 and 100 microns EMBOZENE® Between October 2007 and september 2009 42 patients with liver metastatic disease underwent mbTAE: 10 CRC, 17 NET, 5 breast cancer, and 10 from other malignancies. The same technique described for liver HCC for vascular access and microspheres injection was used also for this group of patients. In patients with NET metastases, the

Bland embolization is an effective therapeutic modality in the treatment of unresectable HCC. In our initial experience with Embozene™ 40 and 100 µm we observed a very good handiness in embolization, obtaining good results in lesion and disease control, at a median follow-up of 12 month. Microparticles 40 and 100 µm in diameter are very effective in local response, with good clinical outcomes, compared with other vascular techniques (see Survival Tab), but patients must be very carefully selected in order to reduce major complications. HCC overall survival: TACE vs. DEB-TACE vs mb-TAE

1 year 2 years 3 years

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A) liver NET metastases before treatment B) 3 months after TAE with 40 microns

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Research Activities A) Basal CT-PET performed before SIR-Spheres® therapy in patient with metastases from breast cancer shows some lesions into the hepatic right lobe; B) CT-PET 2 years after treatment confirmed no FDG uptake. CT-PET is completely negative.

Multiple liver metastases from larynx cancer. A) white arrows show one of the huge liver lesions at pre radioembolization CT. B) CT after 11 months show the dramatic shrinkage of the same lesion (yellow arrows)

main purpose for mbTAE was to obtain a “debulking” effect in order to improve the efficacy of the following radionuclide therapy. According to the RECIST criteria, CT scan showed: 12 PD, 15 SD, 8 PR and 3 CR with a mean follow-up of 13 month. 3 pt were lost at follow-up

In most cases, patients well tolerated the procedure and required only brief observation. Side effects were usually limited to nausea, vomiting, abdominal pain and transient elevations in liver enzymes. Concerning the efficacy profile, revaluation CT and PET/CT (6, 12 and 18 weeks after treatment) documented objective response in 83% of patients (fig. 2b), with complete response in 3 of them. At 48 months FU only one patients is still with complete response (see the picture). Relapse of disease was observed in 10 patients, while in 5 patients an extraheaptic progression at disease was registered. In the remaining patients a partial response to treatment or stable disease was evaluated via CT or PET scan. In our experience for whole liver treatment, the optimal dose to the healthy liver is up to 40-50 Gy, but the optimal response to the treatment has been achieved in those lesions with high T/NT ratio at MAA scintigraphy. A more selective administration of microparticles is highly preferred, in order not only to be more safe but mainly for achieving an higher radiation dose to the tumor. In these selective treatments, patients must be carefully selected because only few lesions, located in the same liver area, could be selectively treated. In our experience radioembolization for those patients may have curative purpose thanks to the very high dose delivered. SIRT can be considered as a 2nd or 3rd line therapy for liver secondary lesions, but it must be considered only in a multi-disciplinary and multitreatment behaviour.

Yttrium-90 internal radiation therapy for hepatic malignancy. Surgical resection is the only potentially curative strategy in the treatment of patients with hepatic malignancy. Unfortunately, due to advanced stage, underlying liver disease, or medical co-morbidities, most patients are inoperable at this time of presentation. As a result, various loco-regional therapies have emerged for otherwise unresectable hepatic tumors. Radioembolization (i.e. intra-arterial administration of 90Y microspheres-SIR-Spheres®) is a therapeutic option that allows preferential delivery of radiations into the tumor without significant liver toxicity. SIR-Spheres® (SIRTEX Medical, Australia) is a resinbased microsphere, of 20-40 μm-diameter, and a typical dose of 3-8 milion per injection. 90 Y is a pure β-emitting radioisotope, it has a high average energy (0.936 MeV), limited tissue penetration (mean 2.5 mm; max 11 mm), and short half-life (64 h), making it an ideal transarterial liver-directed agent. 90Y is selectively injected into the whole liver parenchyma, or in one liver lobe, via the main lobar hepatic arterial branches.

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Prior to radioembolization, detailed imaging (CT, PET/CT) and therapy planning must be completed. All patients that are considered for 90Y internal radiation therapy must undergo a preliminary hepatic angiography in order to assess local vascular anatomy. At the end of angiography, simulation of treatment is performed by injecting 99mTc-macroaggregated albumin, in all hepatic arterial branches, and nuclear medicine scan is carried out (MAA scintigraphy) . This work-up aims to determine the presence of hepatopulmonary shunting, and the amount of the dose to administer to the patient. IEO experience. From October 2005 up to now, 60 patients with liver metastases (from colorectal cancer, breast cancer, uterine cancer and other histologies) have been treated with SIR-Spheres® in our Institute. Two patients screened for SIRT, were excluded at preparation time because of mismatch between MAA scintigraphy and CT/PET scan. Once vascular hepatic anatomy was assessed, for each patient a dosimetric evaluation, based on 99mTc-MAA images (WB and SPECT, 30 min p.i.) and CT scans, has been performed in order to estimate the activity to be safely injected. Endovascular embolization of extrahepatic arteries (eg. GDA, right GA, and other extra-hepatic arteries originating from liver arteries) were performed with endovascular metallic coils in order to obtain just one vessel feeding the whole liver parenchyma. Then Sir-Spheres ® were gently administered via the hepatic arteries.

Percutaneous Procedures Percutaneous radiofrequency thermal ablation (RFA) Hepatocellular carcinoma and colorectal liver metastases are the two most common malignant liver tumors. While surgical resection remains the gold standard of therapy, only few patients are suitable for curative surgical resection, because of the presence of liver malignancy in unresectable locations, the number and anatomic distribution of tumor lesions, or the presence of extrahepatic disease, poor liver function or medical comorbidities. From experiences reported in literature, reasonable safety of RFA has been established, with mortality and morbidity rates in the largest series of 0.2% and 1.7% respectively. The major complications described are peritoneal bleeding, hepatic abscesses, intestinal perforation, large biloma, acute cholecystitis. Tumor seeding is probably over-emphatisized and can be avoid by a hot withdrawal of the electrode. Candidate patients with HCC and cirrhosis have to be in Child’s class A or B; tumour has to be single or, if multiple, with no more than three nodules, without vein thrombosis or extrahepatic spread. Tumor size should preferably be up to 3.5 cm in order to achieve an 80-90% of “radical” ablation. By using combination therapy (hepatic artery occlusion and RFA; RFA and Pringle manoeuvre during surgery) larger tumours can be treated. RFA is considered a safe bridge to liver transplantation in several recent papers. Pretreatment work-up includes ultrasound (US)

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Research Activities A-B) Cholangiocarcinoma of segment IV at CT and PET-CT examination C) 24 hours after TAE+RFA D) result at CT scan 1 month after E) PET-CT scan 2 month after

Liver metastases from CRC in segment 7 (arrow): A) CT before treatment; B) post-treatment CT shows a low-density area (=necrosis) where thermal ablation has been performed

examination, unenhanced and contrast enhanced CT or magnetic resonance (MR); serum tumor markers, namely alfaphetoprotein (AFP) and carcinoembryonary antigen (CEA) dosage. Recently FDG-PET examination has been introduced in staging liver metastases. Tumors close to the gallbladder or subcapsular adjacent to bowel loops, should be preferably treated by a laparoscopic or open approach; RFA of lesions close to the hilum puts the patient at high risk of biliary injury. Up to now, at IEO, about 400 patients

were treated with percutaneous, laparoscopic and open surgery RFA. Percutaneous RFA is the preferred approach, when technically feasible and safe, and requires general anesthesia and not more the one night of hospital stay. IEO experience in RFA recorded no mortality and complication rate of less than 0.5%. Liver bland embolization associated with radiofrequency thermal ablation Tumor size, site and morphology may affect the local

results of liver radiofrequency ablation (RFA). We evaluated the feasibility, safety and local effectiveness of a single session combined approach for treating difficult liver lesions with bland embolization (TAE) immediately prior to RFA, in order to overcome the common limitations to RFA for achieving a radical local outcome. From May 2006 to January 2011, a total of 30 patients affected by liver metastases with single or multiple unresectable liver-only lesions underwent a combined treatment with TAE followed by RFA in the same session, for a total of 36 treated lesions. Patients were extrapolated from a cohort of patients discussed within the weekly institutional tumor board. TAE was performed by using 100 μm microspheres; RFA was performed immediately after TAE by positioning the electrode needle via ultrasound and/or computed tomographic guidance. Local tumor responses and procedure-related complications were evaluated. Completion of both procedures was obtained in all patients for all 36 lesions. Liver lesions had a maximum axial diameter ranging 16-59 mm. Postintervention unenhanced ablated areas ranged 28-104 mm in maximum axial diameter. Safety margins ranged 1-30.5 mm. Complete response, defined as

complete devascularization at computed tomography, was obtained in all treated lesions for a maximum period of 12 months. Tumor relapse was observed in one patient at 12 months. Sixteen patients developed new liver lesions or progressive systemic disease during follow-up. Nine patients were still diseasefree. Seven patients died as a result of systemic progressive disease. One major treatment-related complication was observed. In our experience, in patients with technically unresectable liver-only malignancies, single-session combined TAE-RFA is an effective and safe treatment. Radiofrequency thermal ablation in RCC With the increasing number of patients with renal cell carcinoma detected at an early stage, nephronsparing approaches are becoming more popular. More recently as an alternative to partial nephrectomy radiofrequency thermal ablation (RFA) has been introduced to treat small renal cell carcinoma, with the rationale of in situ tumor destruction to further reduce morbidity and invasiveness. This technique, destroying tumor tissue by heating, may be an appropriate alternative in patients with RCC detected

a) residual liver metastases after chemotherapy treated with percutaneous RFA b) result after 1 year A-B) CRC met of segment IV at CT examination C) during RFA and after TAE D) 24 hours after TAE+RFA E) result at CT scan 1 month after with lesion shrinkage

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Radiofrequency thermal ablation of lung tumors Percutaneous ablation with use of radiofrequency (RFA) is a minimally invasive technique for local treatment and its use is well established in patients with malignant liver tumors, primary or metastatic, who are not candidates for surgical resection. It induces temperature changes by using high-frequency alternating current applied via electrodes placed within the tissue capable to create large areas of necrosis. Several studies demonstrated that it is an effective and safe technique for treatment tumors with mortality

and morbidity rates in the largest series of 0.2% and 1.7% respectively. Recently RFA has been proposed to treat pulmonary malignancy. In fact this technique can be used in patient that are not suitable for surgical resection for poor pulmonary reserve, comorbid cardiopulmonary disease, number and anatomic distribution of tumor lesions or in patients that refuse to undergo surgery. In patients with metastatic pulmonary disease, especially from colorectal carcinoma, surgical resection can achieve 5-yeatrs survival rates of 2040% but it is indicate in a relatively small percentage of carefully selected patients. If surgery is not indicated these patients would normally receive palliative systemic chemotherapy: RFA can be proposed to obtain a better long-term survival. Also in primary lung tumors RFA can be proposed in patients with unresectable lesions. In fact inpatients with recurrent lung carcinoma or with coexistent chronic obstructive broncho-pneumopathy or other associated disease the only chance is chemotherapy or radiation therapy alone or associated. In these cases percutaneous RFA represents an effective and safe minimally invasive treatment. Are candidate to RFA patients with histological or clinical proof of lung malignancy, maximum diameter of the tumor equal or less than 4 cm. Also for primary tumors haemostasis disorders, location of the lesion adjacent to major pulmonary vessels and to major bronchi are exclusion criteria..

A) Small RCC (arrowhead) very close o the ureter (yellow arrow) at the pre-RFA CT; B) CT at one month after treatment shows complete necrosis (white arrow).

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The procedure is performed under CT guidance, with the patient under general anesthesia and requires almost one night of hospital stay; the RFA needle is positioned in the center of the lesion and than radiofrequency energy applied. The major complications described are pneumothorax, pleural effusion and haematoma of the chest wall.

Research Activities

in a solitary kidney or with marginal renal function in whom surgical resection would likely result in the need of dialysis. RFA should be proposed to patients with a disease limited to the kidney (ie T1N0M0), dimension of the lesion less or equal to 4 cm and with a reasonable life expectancy (more than 12 months), in whom the risk/ benefit ratio is believed to be favorable. Whenever possible biopsy should be performed before ablation, to be sure that the lesion ablated is RCC and not a benign lesion and to determine RCC subtype for systemic chemiotherapy in case of future metastases. Up to now we treated 55 patients with RCC (45 male): tumor size ranged between 11 to 42 mm. We had only 3 minor complications: one subcapsular hematoma spontaneously reabsorbed, one skin-burn at insertional site and a little subcapsular urinoma. We obtained a completely necrosis of the tumor in all cases.

At IEO 34 patients, 30 with colorectal carcinoma metastases (follow up 1 – 48 months, median 23) and 4 with primary lung tumors (follow up 8-31 months, median 20). In our experience we had no complications and no mortality. To assess the results a contrast-enhanced CT and a PET scan is performed after one, three and every six months; the main criteria for complete response are reduction in diameter of the lesion, reduction of density in ct exams and disappearance of 18FDG uptake in PET scan. If a residual non-ablated tumor or tumor re-growth is detected a re-treatment with RFA can be performed.

a) CT shows lung metastasis within the right inferior lobe; b) RFA needle within the nodule, during treatment; c) CT result one year after treatment: the lesion is replaced by scar tissue

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Clinical Research

POSITRON EMISSION TOMOGRAPHY Role of 18F-FDG PET in the selection of patients with breast cancer candidate to Sentinel Node Biopsy after neoadjuvant therapy The main objective of this study is to determine the role of [18F]-2-fluoro-2-deoxy-D-glucose – positron emission tomography (18F-FDG-PET) in the selection of patients with breast cancer candidate to sentinel node biopsy after neoadjuvant therapy. The study foresees the enrolment of patients with primary breast cancer clinically classified as cT2, cT3 or cT4a-c cN0-N2 or cN3 and M0 and with a baseline 18F-FDG-PET scan positive both in the site of primary tumor and axillary lymph nodes. A second PET scan after appropriate primary therapy according to local guidelines and/or protocols is given to the patients. In case of persistent axillary 18 F-FDG-PET uptake, lymph node dissection (ALND) is

performed. Should the second 18F-FDG-PET scan be negative for axilla involvement, sentinel node biopsy (SNB) will be performed in order to evaluate axillary lymph nodes status and eventually spare the axilla. Results on 65 patients demonstrate a relatively high specificity and positive predictive value of 18F-FDG-PET for detection of axillary lymph nodes metastases after neoadjuvant therapy. Figure 1 and 2 show two examples. These data seem to suggest a role of 18F-FDG-PET in selecting patients who, after neoadjuvant therapy, are candidate to ALND, avoiding SNB. However, this issue requires a confirmation on a larger series of patients and the enrolment is still ongoing.

Research Activities

Tumour Imaging by Nuclear Medicine

Figure 2. Coronal CT (A,C) and emission images (B,D) of PET/CT scan before (A and B) and after neoadjuvant treatment (C and D) in a patient with right axillary lymph nodes metastases secondary to ductal breast carcinoma.

18F-Fluorothymidine Positron Emission Tomography as an early predictor of response to neoadjuvant therapy in patients with locally advanced breast carcinoma Neoadjuvant chemotherapy (NAC) is considered aimed to enhance the possibility of breast conserving surgery, avoiding radical mastectomy. Patients achieving a pathological complete remission (pCR) have better outcome, with a better long term survival, than those with incomplete response. However, a pCR is achieved

in a minority of the patients. Assessment of response to treatment by mammography/and or ultrasound requires several cycles of treatment before a change in tumour volume can be detected. Furthermore, changes in size not always identify the responders. Functional imaging techniques could represent a better approach in monitoring tumor response. Although positron emission tomography (PET) with 2-deoxy-2- [18F]fluoro-D-glucose (FDG) FDG plays

Bilateral breast cancer Ki67 dx 12%

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Ki67 sx 70%

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Figure 1. Fused transaxial images of basal 18F-FDG PET/CT scan in a patient with right breast cancer in the lower internal quadrant (white arrow).

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SUV 4,0

FLT-PET

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Figure 3 Comparison between FLT-PET and FDG-PET in a patient with bilateral breast cancer (Ki67 was 12% on the right side and seventy per cent on the left). FDG uptake values do not differ significantly between the two sides, while the left tumor shows an SUV almost five times as high as the right tumor for FLT, that so provides a reflection of the different proliferating rate.

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Movement correction of PET/CT images In thoracic district one of the major limitation regarding PET/CT images and radiotherapy treatment is the movement: one of the possible solution is the acquisition of 4D images through the movement correction. However some artefacts can affect 4D CT images due to breathing irregularities or incorrect breathing phase identification. We conducted a study trying to reduce the artefacts in sorted 4D CT images. The assumption is that the use of multiple respiratory related signals may reduce uncertainties and increase robustness in breathing phase

The calibrated infrared optical localization system used in combination with the RPM system.

identification. Multiple respiratory related signals were provided by infrared 3D localization of a configuration of markers placed on the thoracoabdominal surface. Multidimensional K-means clustering was used for retrospective 4D CT image sorting, which was based on multiple marker variables, in order to identify clusters representing different breathing phases. The proposed technique was tested on computational simulations, phantom experimental acquisitions, and clinical data

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Casiraghi M, Travaini LL, Maisonneuve P, Tessitore A, Brambilla D, Agoglia BG, Guarize J, Spaggiari L. Lymph node involvement in T1 non-small-cell lung cancer: could glucose uptake and maximal diameter be predictive criteria? European Journal of Cardio-thoracic Surgery 39 (2011) e38—e43.

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patients) (p<0.0001). The low probability of lymph node involvement in NSCLC <1 cm or showing glucose uptake <2 suggests lymphadenectomy could be avoided. In conclusion our study confirmed literature reports suggesting that tumor size and SUV could be reliable independent predictors of tumor aggressiveness and lymph node involvement. In the figure we report the 5-year overall survival of patients related to measurement of SUVmax and nodule size. Similar results were reported in Continuous Observation of SMOking Subjects (COSMOS) series where we identified 97 patients with clinical T12N0M0 lung cancer (<3 cm). All patients underwent curative resection with radical mediastinal lymph node dissection: we examined factors associated with hilar extrapulmonary and mediastinal nodal involvement. Nodule size and SUV were evaluated retrospectively in these group of patients and compared to 193 consecutive patients with nonscreening-detected clinical stage I lung cancer. Among COSMOS patients, 91 (94%) were pN0, and six (6.2%) were pN+. All patients with SUV max <2.0 (p = 0.08) or pathological nodule ≤10 mm (p = 0.027) were pN0 (62 cases). Nodal metastases occurred in 6 cases among the 29 (17%) patients with lung nodule >10 mm and SUV max ≥2.0 (p = 0.002 versus the other 62 cases). In the nonscreening series, 42 of 43 cases with negative SUV (<2.0) or nodule ≤10 mm were pN0; 33 of 149 (22%) cases with positive PET-CT (usually SUV max ≥ 2.0) and nodule >10 mm were pN+ (p = 0.001 versus the 43 cases). This limited experience suggests that in early-stage clinically N0 lung cancers with SUV max <2.0 or pathological nodule size ≤10 mm, systematic nodal dissection can be avoided as the risk of nodal involvement is very low. In the figure it is reported the overall survival of COSMOS and nonscreening series.

Research Activities

FDG-PET imaging and lymph node involvement in screening-detected lung cancers The introduction of modern staging systems such as computed tomography (CT) and positron emission tomography/CT (PET/CT) with fluorodeoxyglucose ([(18)F]FDG) has increased the detection of small peripheral lung cancers at an early stage. We analyzed the behaviour of pathological T1 non-small-cell lung cancer (NSCLC) to identify criteria predictive of nodal involvement, and the role of cancer size in lymph node metastases: 219 patients with pathological T1 NSCLC have been analyzed. All patients were staged by high-resolution CT and PET as stage I, and underwent anatomical resection and radical lymphadenectomy. Our data were collected based on pathological nodule size (0-10 mm; 11-20 mm; and 21-30 mm); morphological features of lung nodule and FDG uptake of the tumor measured by maximal standardized uptake value (SUVmax). A total of 190 patients (87%) were pN0, 14 (6%) pN1 and 15 (7%) pN2. No nodal involvement was observed in nodule size less than 10 mm (62 patients), in 17% of nodule size 11-20 mm and in 28% of tumors 21-30 mm in size (p=0.0007). All 55 patients with nodule SUVmax< 2.0 and all 26 non-solid lesions were pN0 (respectively, p=0.0001 and p=0.03). All nodal metastases occurred among the group of 132 patients with size larger than 10 mm and SUV higher than 2.0 with a 22% rate of nodal involvement of (29

an important role in management of patients with cancer, it is not the optimal tracer because changes therapyrelated to inflammation of tumors and surrounding tissue may increase FDG uptake. A proliferation tracer such as 3’-deoxy-3’-[18F]fluorothymidine (FLT) might be more specific for cancer. The FLT is a nucleoside analogue designed as a marker of cell proliferation that can be imaged in vivo by PET. Several clinical studies have obtained promising results for early evaluation of drug efficacy in solid tumors and lymphoma. In this study we are investigating the hypothesis that in breast cancer a reduction of 18F-FLT uptake in the tumor after the first cycle of NAC, compared to the baseline, may predict the response to the treatment. For this purpose we are studying patients with histologically proven primary breast cancer (patients must have had a core biopsy), classified as cT1b,c, T2, T3, or cT4 a-c, cN0-2, N3, M0 after FDG-PET, suitable for preoperative therapy according to EIO guidelines or protocols. Patients undergo FLT-PET scan before (within 7 days) and after the first course of NAC, in order to verify the hypothesis that a reduction of 18F-FLT uptake in the tumor after the first cycle of NAC, compared to the baseline, may early predict the response to the treatment. Secondary endpoints of the study are the comparison of the proliferation index (Ki 67) with the uptake value of 18F-FLT and the evaluation of the differences between 18F-FLT uptake and 18-FDG uptake at the baseline.

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Veronesi G, Maisonneuve P, Pelosi G, Casiraghi M, Agoglia BG, Borri A, Travaini LL, Bellomi M, Rampinelli C, Brambilla D, Bertolotti R, Spaggiari L. Screening-detected lung cancers: is systematic nodal dissection always essential? J Thorac Oncol. 2011;6: 525–530.

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RPM phase sorting

by the described clustering technique. Artifact reduction was clearly observable on both data set especially in the lower lung region. The implemented multiple point method demonstrated the ability to reduce artifacts in 4D CT imaging. In the figure we report qualitative comparison between the RPM phase sorting (upper panel), RPM amplitude sorting (central panel), and clustering technique (lower panel) for a patient case (patient 2). Clustering technique was performed with uniform in time initialization and set to set reassignment. The reported images are relative to the breathing phase identified with symbol º (expiration after inspiration peak). Sentinel node imaging in multicentric breast cancer The study was designed to present the 5-year results of patients with multicentric breast cancer who underwent sentinel lymph node biopsy (SLNB) in a single institution. Between June 1999 and December 2007, 337 patients with multicentric breast cancer and a clinically negative axilla underwent lymphatic mapping by a single periareolar/peritumoral (n = 306) or a double peritumoral or subdermal injection (n = 31) of 99mTc-HSA nanocolloids. The sentinel lymph node (SLN) was evaluated by intraoperative frozen section and axillary dissection was performed only in cases of positive SLNB. The median age of the patients was 48 (range, 22-81) years. The mean number of hot spots

identified was 1.4 in the whole series, 1.3 in patients who received a single injection, and 1.7 in those who received a double injection (P < 0.001). The mean number of removed SLNs was 1.7 (median, 1; range, 1-7) with an identification rate of 100%. A total of 138 patients with negative SLNB (n = 134) or isolated tumor cells in the SLN (n = 4) did not receive completion axillary lymph node dissection (CALND). In these latter patients, a total of 27 events (19.5%) occurred with 3 patients (2.2%) developing axillary recurrences after a median followup of 5 years (range, 17-134 months). Axillary lymph node reappearance was infrequent among patients with multicentric breast cancer, having negative SLNB and no CALND. We recommend SLNB as the standard procedure for nodal staging in patients with multicentric breast cancer and a clinically negative axilla.

Survival probability

Research Activities

coming from two patients. Computational simulations provided a controlled and noise-free condition for testing the clustering technique on regular and irregular breathing signals, including baseline drift, time variant amplitude, time variant frequency, and end-expiration plateau. Specific attention was given to cluster initialization. Phantom experiments involved two moving phantoms fitted with multiple markers. Phantoms underwent 4D CT acquisition while performing controlled rigid motion patterns and featuring end-expiration plateau. Breathing cycle period and plateau duration were controlled by means of weights leaned upon the phantom during repeated 4D CT scans. The implemented sorting technique was applied to clinical 4D CT scans acquired on two patients and results were compared to conventional sorting methods. For computational simulations and phantom studies, the performance of the multidimensional clustering technique was evaluated by measuring the repeatability in identifying the breathing phase among adjacent couch positions and the uniformity in sampling the breathing cycle. When breathing irregularities were present, the clustering technique consistently improved breathing phase identification with respect to conventional sorting methods based on monodimensional signals. In patient studies, a qualitative comparison was performed between corresponding breathing phases of 4D CT images obtained by conventional sorting methods and

Overall survival

1,0 5-years OS: 96,9% 0,8

0,6

RPM amplitude sorting

0,4

0,2

0

1

2

3

4

5

6

Years

clustering technique

Gianoli C, Riboldi M, Spadea MF, Travaini LL, Ferrari M, Mei R, Orecchia R, Baroni G. A multiple points method for 4D CT image sorting. Med Phys. 2011 Feb;38(2):656-67.

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Overall survival. Gentilini O, Veronesi P, Botteri E, Soggiu F, Trifirò G, Lissidini G, Galimberti V, Musmeci S, Raviele PR, Toesca A, Ratini S, Del Castillo, A, Colleoni M, Talakhadze N, Rotmensz N, Viale G, Veronesi U, Luini A. Sentinel lymph node biopsy in multicentric breast cancer: five-year results in a large series from a single institution. Ann Surg Oncol (2011) 18:2879–288.

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271

Radioimmunotherapy

The concept of targeting radionuclides to tumours using radiolabeled monoclonal antibodies (MoAbs) against tumour associated antigens (Radioimmunotherapy – RIT) was proposed more than a century ago. With the development of the hybridoma technology and the availability of MoAbs against tumour-associated antigens, this concept was investigated in animal models and in cancer patients. RIT combines the specificity of a monoclonal antibody in targeting the cancer antigen with the activity of a radioactive molecule. Despite more than 30 years since the introduction of radioimmunotherapy, this technology has attracted interest of oncologists only in the last few years. This interest can be attributed to the availability of two clinically approved RIT drugs and their remarkable therapeutic efficacy in the management of non-Hodgkin’s lymphoma (NHL). The success of RIT in B-cell NHL therapy is, at least in part, related to the characteristics of the disease, such as the radiosensitivity and the abundance of the target antigens on its surface, and the deliver of a cytotoxic radiation to the tumour by the presence of a suitable therapeutic radionuclide. So, the conjugation of a monoclonal antibody with a radioisotope represents a valid option to strengthen the efficacy of MoAbs, supplying doses of radioactivity to specific targets. Yttrium-90 (90Y) ibritumomab-tiuxetan (Zevalin®) is a beta-emitter, radio-conjugated, anti-CD20 antibody which includes ibritumomab, a murine parent of the humanized anti-CD20 MoAb Rituximab, conjugated by tiuxetan to 90 Y. Zevalin® has been already demonstrated to be safe and effective, as a complementary approach, in the treatment of follicular and diffuse large B-cell NHLs, as well as in cases resistant or refractory to Rituximab. We evaluated the efficacy and safety of a single dose of Zevalin® in elderly patients in first relapsed or primary refractory diffuse large B cell Non-Hodgkin Lymphoma,

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ineligible for stem-cell transplantation, and demonstrated high response rates to Zevalin® in this setting of patients, in a prospective multicenter non randomized phase II trial. The overall response rate (ORR) was more than 50% with complete response rates of more than 25%. When administered at the conventional prescribed activity of 14.8 MBq/kg (0.4 mCi/kg), the red marrow is the critical organ and toxicity is primarily haematological but reversible. The activity of 1.18 GBq (32 mCi) has been identified as the maximum tolerable, in spite of the single patient absorbed dose to the red marrow. In order to increase its efficacy, Zevalin® has been investigated at myeloablative activities: a high dose (HD) trial based on 90Y-ibritumomab-tiuxetan activities up to 55.5 MBq/kg, followed by autologous stem cell transplantation (ASCT) is ongoing in our Institution for the treatment of resistant/refractory follicular B-cell non Hodgkin’s Lymphoma. To date, 39 patients affected by resistant/refractory NHL have been enrolled in the trial. In this trial, the patients’ recruitment is based on patient specific dosimetry, in order to evaluate safety and toxicity and confirm the justification of such an aggressive therapy. Before RIT all patients underwent dosimetry, and therapy was administered only if a 20-Gy limit dose to normal organs (except red marrow) was guaranteed. The dosimetric analysis highlighted the need of deepening the red marrow absorbed dose, as its evaluation by the standard method applied - based on the blood curve – might underestimate the dose in these patients- showing spine marrow uptake. Alternative methods based on imaging and bone aspirates have indicated higher values with a factor of 2–2.5 and have been applied to assess also safe time for transplantation. Studies are ongoing on this matter. Two of the patients monitored for this therapy showed atypical pharmacokinetics and biodistribution, with prevalent high liver uptake. In both patients, liver absorbed doses higher than 50 Gy would have resulted

with administration of the prescribed activity; following these dosimetric results, both patients did not receive the planned therapy because the treatments would have been unjustified and with possible serious adverse effects. The importance of highlighting these clinical cases enables to enrich an evidence-based discussion about drug use and development in clinical practice, especially for protocols based on the administration of high activities of radiopharmaceuticals. Another field of investigation has been the optimization of the radiolabelling and quality control of Zevalin® at high activities, to simplify the procedure and to decrease the finger radiation exposure of the operator. Studying the parameters of the reactions, it has been possible to reduce the steps of preparation and to improve the handling of high activity samples, maintaining the quality and the safety of the radiopharmaceutical. In another study, we considered marginal-zone lymphomas (MZL), a subgroup of disease in which this treatment could offer an alternative option when they relapse or do not respond to conventional treatment. Seventeen patients were enrolled in a prospective, singlearm, open-label, pilot phase II trial of RIT with Zevalin®. Seven out of 17 patients had Helicobacter Pylori-negative gastric MALT (mucosa-associated lymphoid tissue) NHL. The remaining 10 patients had non gastric extranodal MZL. At time of treatment 10 out of 17 patients had disseminated disease (stage III/IV); bone marrow biopsy showed disease localization in 4 out of 17 patients. Median number of previous therapies was 2, including prior chemotherapy, Rituximab, and radiotherapy. Patients received standard activities of 0.4 mCi/Kg, after preloading with Rituximab. The therapy was well tolerated without acute and subacute side effects. G3 and G4 neutropenia and thrombocytopenia in 10 out 17 patients, with transfusional support in 2 patients and infections in 3 patients were observed. Twelve out 17 patients obtained a complete response (71%); partial response was observed in 2 out 17 patients (12%). Three patients (18%) obtained a stabilization of disease; one of them progressed after 4 months from RIT. Complete responses are still ongoing (35 months). The high observed overall response and complete response rates indicate that Zevalin® in MZL is efficient. If these preliminary results will be confirmed in larger number of patients, one single administration of 90 Y-Ibritumomab Tiuxetan delivered at conventional activity (0,4 mCi/kg) could be considered a possible alternative option in the treatment of such an indolent disease. During 2008, we participated in a phase I trial using an iodine-131 labelled antibody fragment raised against the EDB fragment of fibronectin in patients with advanced solid and haematological malignancies. Biodistribution and dosimetric

studies showed a rapid pharmacokinetics, especially as compared with other radiolabelled MoABs, indicating low risk of haematological toxicity For therapeutic activity of 3.7 GBq, median absorbed doses (Gy) were: 3.70 (kidneys), 3.0 (liver), 2.0 (lungs), 4.20 (testes), 0.85 (red marrow), 12.2 Gy (tumor lesions). The favourable dosimetry results indicated the feasibility of therapeutic protocols with further increased activities (> 3.7 GBq).

Research Activities

Clinical Research

Despite the use of aggressive combination chemotherapy regimens, 30% to 40% of pts with aggressive NHL do not respond or relapse after first line therapy. High dose chemotherapy is a widely accepted procedure in this setting of pts. However, this option is precluded to elderly because of morbidity and mortality. Moreover, high dose Zevalin has the main limit in ASC mobilization: elderly and pre-treated pts failing mobilization need alternative options. So, new therapeutic approaches are certainly needed. Besides neuroendocrine tumours that over-express somatostatin receptors on their cell membrane (SSR), somatostatin receptors were identified also in lymphatic tumours, that owing to their radiosensitiveness, might be suitable targets for radionuclide labelled somatostatin analogues for diagnostic and therapeutic purposes. On this basis, we have submitted and already approved by our EC, a phase II study to evaluate the efficacy of Peptide Receptor Radionuclide Therapy (PRRT) with 90 Y-DOTATOC in aggressive NHL patients. This protocol is supported by a grant from Fondazione Umberto Veronesi.

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Clinical Research

1. Description of the registry The Tumour Registry (TR) was activated in March 2006 with the aim to collect data on all those consulting at the European Institute of Oncology (IEO), at risk of developing or already presenting with a tumour. It has actually become a supporting tool for the current practice as well as for epidemiological/basic research, guaranteeing a quick analysis of the IEO clinical activity and playing a key role in the production of scientific publications. Eligible to enter the Registry are all those coming to the IEO for consultation since its opening, with unique identification number (patients’ record) and at least one episode accessible from Institute’s intranet. A minimum data set of variables was defined and data entry was divided in 4 forms (see Variables collected section for details). Briefly, on the first form personal data (i.e., sex, address, date of birth) and information on follow-up (i.e., date of last contact, date of last visit, vital status, cause of death) are recorded. The following types of record are assigned: 1. Visit: a healthy individual comes to IEO for either visit or genetic counselling 2. Anamnesis: the patient, at the moment free of disease, reports on a tumour diagnosed in the past and already treated and cured. 3. Diagnosis: diagnosis of tumour is made at IEO. The patient decides to be treated elsewhere. 4. Second Opinion: the patient or the patient’s parents come to IEO and ask for a second opinion on a diagnosis and/or a treatment proposed elsewhere. 5. Long: the patient receives at least one treatment at IEO. Detailed information on patient’s tumour(s) (i.e., date of diagnosis, morphology, topography, TNM staging) is recorded on the second form, together with some

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Research Activities

The IEO Tumour Registry

epidemiological information (i.e., familiarity, height and weight at diagnosis and smoking habits). The third form is dedicated to the treatment strategy, where every therapy is classified as administered or proposed. The fourth is dedicated to the chronology of events, in order to better describe the history of the disease and to take note of the tissues preserved in the Biobank of IEO. Sources of information for data collection are: 1. database of patients’ administrative data (personal information is automatically downloaded); 2. files accessible on intranet; 3. online databases (surgery, laboratory medicine); 4. patients’ clinical dossier digitalized and accessible on e-Paper. The Registry was implemented using the interface software Argos™, based on Oracle™ database system. The implementation, completed in February 2006, was managed in the division of Epidemiology and Biostatistics, with the help of the IT division. Although it was decided that data from the TR must not be directly accessible to researchers or clinicians, we have established a system to collect and answer requests. Data extrapolation and analysis are managed in the division of Epidemiology and Biostatistics on the advice of the Scientific Direction of IEO. For detailed information on the TR structure, materials and methods please refer to a paper published in the IEO Journal ecancermedicalscience [1]. 2. Six years of activity After a 6 months pilot period, from March to August 2006, which involved the training of the operators, ad hoc improvements to the structure of the registry, data quality control and editing of the user guide, from September 2006 the data entering has been running at

top speed. We started entering individuals who came for the first time to IEO in the year 2000 (dossier number CC00) in a sequential fashion. By March 2012, 203,479 individuals who visited IEO for the first time in the years 2000-2007 were entered in the Tumour Registry. These were not necessarily presenting with a tumour. Individuals’ characteristics are reported in Table 1 of the Data section. By March 2012, 129,592 tumours, out of 114,804 individuals presenting with 1 or more tumours, were entered (see Data section, Tables 2 and 3).

Table 1. Characteristics of individuals  

Gender

Age

Place of residence

Type of record Long: the patient receives at least one treatment at IEO. Second Opinion: the patient or the patient’s parents come to IEO and ask for a second opinion on a diagnosis and/or a treatment proposed elsewhere. Anamnesis: the patient, at the moment free of disease, reports on a tumour diagnosed in the past and already treated and cured. Diagnosis: diagnosis of tumour is made at IEO; the patient decides to be treated elsewhere. Visit: a healthy individual comes to IEO for either visit or genetic counselling.

Total

Classification

No. Patients (%)

Male

66,221 (32.5)

Female

137,258 (67.5)

< 20 years

2,988 (1.5)

20-34 years

21,543 (10.6)

35-49 years

60,252 (29.6)

50-64 years

71,251 (35)

65-79 years

43,240 (21.3)

> 80 years

4,205 (2.1)

Northern Italy

131,929 (64.8)

- Lombardy

107,543 (52.9)

- Milan

38,772 (19.1)

Central Italy

38,377 (18.9)

Southern Italy

31,487 (15.5)

Foreign countries

1,686 (0.8)

Long

47,547 (23.4)

Second Opinion

64,380 (31.6)

Anamnesis

2,877 (1.4)

Diagnosis

2,176 (1.1)

Visit

86,499 (42.5)

 

203,479 (100,0)

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Table 2. Tumors by sitea Collecteda Overall

Total

Invasive tumors IEO diagnosis

IEO Surgery

Head and Neck

3,780

3,334

970

690

Lip

121

110

32

28

Tongue

680

652

290

261

Major salivary glands

671

338

113

75

Gum

48

45

24

22

Floor of mouth

113

109

25

24

Other and unspecified parts of mouth

581

544

161

125 86

Oropharynx

552

541

127

Nasopharynx

425

422

83

9

Hypopharynx

280

279

50

32

Other and other ill defined sites Digestive organs and peritoneum b Oesophagus Stomach Small intestine, including duodenum

294

65

28

19,534

3189

1903

561

547

116

62

3,439

3,412

636

473

373

363

90

24

Colon

7,080

6,904

1,092

758

Rectum, rectosigmoid junction and anus

3,064

3,013

598

456

Liver and intrahepatic bile ducts

1,469

1,447

192

44

Gallbladder and extrahepatic bile ducts

1,120

1,090

81

18

Pancreas

2,433

2,415

292

54 11

Retroperitoneum and peritoneum

166

155

48

Other and ill-defined sites

204

188

44

3

16,505

15,836

3,806

2,464

Respiratory and intrathoracic organs Nasal cavities, middle ear and accessory sinuses

95

93

9

3

Larynx

1,702

1,517

478

438

Lung

13,686

13,280

3,063

1,907

Pleura

663

625

132

38

Thymus, heart and mediastinum

353

315

124

78

6

6

0

0

52,967

44,064

22,788

15,324

Other and ill-defined sites Bone, connective tissue, skin and breast Bone and articular cartilage Connective and other soft tissue

19

13

3

1

2,772

2,641

933

397

Skin Melanoma

3,543

3,343

1,365

453

Skin Non Melanomac

3,250

3,080

1,404

1,231

Breast

43,383

34,987

19,083

13,242

Genitourinary organs

25,445

20,931

5,420

3,748

Cervix uteri

4,378

1,694

627

443

Uterine corpus

1,951

1,752

557

399

Ovary and other uterine adnexa

3,795

3,461

1,145

663

838

564

202

130

7,594

7,453

1,613

1,133 158

Other and unspecified female genital organs Prostate Testis

861

813

243

Penis and other male genital organs

124

105

45

37

2,913

2,265

428

331

Bladder Kidney and other and unspecified urinary organs

2,991

2,824

560

454

Other and unspecified sites

4,337

3,456

618

493

Eye

19

15

2

1

Brain

1,633

1,608

45

0

Other and unspecified parts of nervous system Thyroid gland Other endocrine glands and related structure Lymphatic and haematopoietic tissue Hodgkin lymphoma Non-hodgkin lymphoma

50

46

2

0

2,454

1,658

543

480

181

129

26

12

4,392

4,361

1,361

80

807

807

241

11

2,333

2,333

911

68

Multiple myeloma

433

433

83

1

Leukaemia

663

663

95

0

Other and unspecified

156

125

31

0

Non-skin melanoma

165

165

27

21

2,092

2,019

508

66

129,592

113,700

38,687

24,789

Malignant neoplasm without specification of site Total

276

309 19,909

IEO — Scientific Report 2011 — Ongoing research 2012

3. Developments Four recent papers represent the first published studies conducted on the data of the TR. The first one [2] consisted of a multi stage analysis of local, regional and distant recurrences, performed on data of 2,784 women treated for early breast cancer by quadrantectomy and whole breast irradiation at IEO. The second paper [3] described the survival patterns of 114 patients affected by pN0 stomach cancer. The third one [4] consisted of an investigation on the relationship between ABO blood group and the risk of cancer based on data from 15,359 IEO patients. The last one [5] aimed at studying the impact on survival of the biopsy of liver metastasis in women with breast cancer (see Publication section for details). Many other studies and projects based on the TR data are now arising from the collaboration of our and other divisions of the Institute (see Some ongoing projects section for details). Also, through a link with the IEO bio bank, new molecular features will be available soon for better clinical investigations. The IEO TR has been proven functional and reliable in monitoring the activity of the Hospital, allowing extraction of data from any subpopulation with characteristics of interest. This structured and centralized Registry represents an important tool for our research-oriented Institution.

4. Iodice S, Maisonneuve P, Botteri E, Sandri MT, Lowenfels AB. ABO blood group and cancer. Eur J Cancer. 2010 Dec;46(18):3345-50. 5. Botteri E, Disalvatore D, Curigliano G, Brollo J, Bagnardi V, Viale G, Orsi F, Goldhirsch A, Rotmensz N. Biopsy of liver metastasis for women with breast cancer: Impact on survival. Breast. 2011 Dec 31. [Epub ahead of print]

Research Activities

Tumour site

4. Cited papers 1. Botteri E, Iodice S, Maisonneuve P, Alfieri M, Burzoni N, Manghi L, Martinetti M, Montanari B, Albertazzi E, Bazolli B and Rotmensz N. “Case mix at the European Institute of Oncology: first report of the Tumour Registry, years 2000-2002” doi: 10.3332/ecancer.2009.149 2. Botteri E, Bagnardi V, Rotmensz N, Gentilini O, Disalvatore D, Bazolli B, Luini A, Veronesi U. “Analysis of local and regional recurrences in breast cancer after conservative surgery.” Ann Oncol. 2010 Apr;21(4):723-8 3. Biffi R, Botteri E, Cenciarelli S, Luca F, Pozzi S, Valvo M, Sonzogni A, Chiappa A, Leal Ghezzi T, Rotmensz N, Bagnardi V, Andreoni B. “Impact on survival of the number of lymph nodes removed in patients with node-negative gastric cancer submitted to extended lymph node dissection.” Eur J Surg Oncol. 2011 Apr;37(4):305-11. a

Including all invasive and in situ tumours. Benign neoplasia/negative histology after radical surgery in IEO are also collected. Polyps are not collected; cBenign nevi are not collected.

b

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277

Academic Research

STAFF The personnel involved in the Academic Research of the Department of Experimental Oncology has been already described in the section dedicated to individual groups.

Identification and validation of new molecular targets for drug development Successful cancer treatment requires an understanding of the cascade of molecular lesions that underlie the acquisition of the tumor phenotype and that can therefore constitute meaningful therapeutic targets or diagnostic and prognostic indicators. The search for these molecular lesions has received great impetus over the last decades, and we now have an ever expanding catalog of genetic and epigenetic alterations that are being associated to various types of cancer and to various stages of its development. In parallel there has been a conceptual reframing of cancer as a developmental problem, which is enabling to position the list of molecular lesions within a pathological history of the disease and a hierarchy of cell lineages. This conceptual reframing of cancer allows then to test experimentally, thanks to new cancer models and to novel high-throughput technologies, how the various cell compartments, the genetic and epigenetic lesions that affect them, and the range of cancer cell-host interactions ultimately shape the tumor phenotype. Consistent with this conceptual and experimental paradigm, and in line with the objective to investigate cancer as a complex disorder of development gone awry, we have focused on: i) the alterations in genetic and epigenetic regulatory networks; ii) the actual aberrations in cell division; iii) the emerging links between cancerous growth and the molecular environment of the host; and iv) the predictive relevance of molecular markers for clinical prognosis. This structure is meant to follow our refined understanding of the disease, in which the derangement of growth and differentiation programs (through genetic and

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epigenetic mechanisms) leads to abnormal cell division in the context of the host tissue and its microenvironment, enabling a molecular dissection that is a prerequisite for the experimental validation of therapeutic interventions.

1. Probing the cancer networks with genome wide approaches (Myriam Alcalay) The main focus of the lab is to understand the molecular basis of acute leukemias by exploiting the integrated use of high-throughput technologies, which represent powerful tools for the discovery and analysis of genetic networks underlying cancer. We are focusing on two main lines of research: a. Transcription factor networks in Acute Myeloid Leukemia Acute myeloid leukemias (AML) are characterized by the clonal expansion of myeloid precursors that present an increased proliferative potential and the incapacity to differentiate into mature white blood cells. One of the most frequent translocations in AML is t(8;21), which involves the genes coding for AML1 and ETO, and gives rise to the AML1/ETO fusion protein. AML1 is involved in the maintenance of the hematopoietic stem cell (HSC) pool whereas ETO is a transcriptional co-repressor required to silence the expression of a variety of genes by recruiting N-Cor/Sin3/HDAC complexes to gene regulatory sequences. AML1/ETO is able to bind AML1 responsive genes as it retains the AML1 DNA-binding domain and to repress them through the ETO moiety. Moreover, AML1/ETO interacts with other transcription factors, such as PU.1, RUNX1 and C/EBPα, known key regulators of haematopoiesis. The major effect of AML1/ ETO is to block terminal differentiation, buts it is not sufficient for full leukemic transformation. Previously, our group found that the fusion protein preferentially binds to genomic regions containing a specific sequence signature that includes consensus binding sites for AML1, the E-protein HEB, AP.1 and Ets1.

We showed that the fusion protein heavily interferes with the genomic binding pattern of AML1 and HEB, but the relevance of the Ets and AP.1 motifs for AML1/ETO function remained unexplored. We, therefore, decided to analyze the interplay among transcription factors during myeloid differentiation and its subversion during AML1/ETO-induced leukemogenesis. The EML C1 cell line chosen for this purpose, as it is able to differentiate into all haematopoietic lineages (myeloid, lymphoid and eryhroid-megakaryocytic). We found that AML1/ ETO efficiently blocks myeloid differentiation of EML C1 cells. Chromatin immunoprecipitation (ChIP) experiments coupled to Next Generation Sequencing (ChIP-Seq) for PU.1, RUNX1 and C/EBPα in EML C1 cells was performed to map their genomic distribution during myeloid differentiation, and in EML C1 cells expressing AML1/ETO to assess how the fusion protein disrupts their binding pattern. Furthermore, mRNA and miRNA expression profiling experiments were performed to detect changes in the transcriptome during differentiation and in the presence of AML1/ETO. Data analysis is ongoing. b. Comparative analysis of genomic and epigenomic alterations induced by leukemogenic AML1/RUNX1 fusion proteins Structural abnormalities of the AML1 gene, including chromosomal translocations and point mutations, are frequently found in acute leukemias and myelodysplasias. More than 50 different translocations involving the AML1 gene have been described, but only few are recurrent events, the most frequent being t(12;21) in pediatric B-cell acute lymphoblastic leukemia (B-ALL) and the above mentioned t(8;21) in AML, which encode for the TEL/AML1 and AML1/ETO fusion proteins, respectively. Other recurrent albeit rare rearrangements result in expression of the AML1/ MECOM and AML1/PRDM16 fusion proteins, respectively, and are associated with both de novo and therapyrelated myeloid neoplasms. AML1 fusion proteins share important features, including the capacity to bind specific consensus sequences through the AML1 DNA-binding domain, to maintain protein-protein interactions through the AML1 moiety, and to function as oncogenic transcription factors by recruiting corepressor complexes to their target sequences on DNA. There are, however, relevant differences, including the prevalent occurrence in different types of hematopoietic malignancies, association with diverse protein partners, structural differences and specific functions contributed to each fusion protein by domains deriving from the AML1 partner protein. Although acute leukemias caused by TEL/AML1 and

AML1/ETO have a favorable prognosis with current therapeutic regimens, there are two important problems that remain unresolved: i) the toxicity of the drugs that are employed is relevant, and ii) the risk of disease relapse. Both these issue are particularly critical in the case of pediatric malignancies. The prognosis of therapy-related malignancies is instead poor due to the scarce therapeutic efficacy in this setting. These problems could be addressed by the use of drugs that directly target AML1 fusion protein functions. Many studies have analyzed the mechanism of action of different AML1 fusion proteins, but a parallel and comparative analysis of their functions in a controlled model system is lacking. We are currently performing a comparative study of four recurrent AML1 fusion proteins (TEL/AML1, AML1/ETO, AML1/MECOM and AML1/PRDM16), with the aim of identifying specific and common functions. Using cell lines expressing these AML-fusion proteins, we will study their DNA binding pattern and associated chromatin status, their capacity to interfere with the binding profiles of other regulators of hematopoiesis such as CEBPα and PU.1, as well as their effects on nuclear architecture and regulation of mRNA and miRNA expression. We expect that our findings may be extended to other acute leukemias bearing more rare AML1 abnormalities.

Research Activities

Department of Experimental Oncology

c. Activation of Wnt signalling in AML with mutant nucleophosmin In previous work, we showed that the tumor suppressor Prdm5 negatively modulates both canonical Wnt/βcatenin and planar cell polarity (PCP) Wnt pathways during zebrafish development2. We found that PRDM5 physically interacts with nucleophosmin (NPM), a nucleolar protein that shuttles between the nucleus and the cytoplasm and that acts both as proto-oncogene and as tumor suppressor. NPM is the most frequently mutated gene in AML3. In zebrafish, npm1 strongly expressed during embryogenesis. The effects of npm1 targeted knock-down resembles an impairment of morphogenetic movements during gastrulation due to a block of PCP Wnt pathway, accompanied by a reduction of the cephalic region. In agreement, overexpression npm1 causes a “dorsalized” phenotype, resembling that obtained by overexpression of Dkk1, a key negative regulator of Wnt signaling. Expression of the NPM mutant isolated from human AML in zebrafish embryos results in a phenotype similar to that obtained through knock-down experiments, suggesting the mutant protein acts as a dominant negative and may exert its oncogenic activity at least partly through activation of Wnt signaling.

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279

2. The role of the Chk1 kinase in Myc-induced tumorigenesis (Bruno Amati) Activation of the c-myc proto-oncogene is a primary driving event in some tumors, most notably via translocation in Burkitt’s B-cell lymphomas, as well as gene amplification in various carcinomas. In other cancers, c-myc is not structurally altered, although it is frequently over-expressed due to oncogenic activation of upstream signaling pathways (e.g. Ras, Wnt, Notch), and contributes to their growth- and tumor-promoting potential. The common denominator of these genetic alterations is aberrant expression of the c-myc protein product, Myc. Myc, is a transcription factor of the “basic helix-loophelix leucine-zipper (bHLH-LZ) family, and like all bHLH-LZ proteins must dimerize in order to bind DNA. Myc has only one known bHLH-LZ partner, Max. Myc/ Max dimers bind to the DNA sequence CACGTG (E-box) and can either activate or repress gene expression. The transcriptional programs driven by Myc are highly complex: Myc associates with thousands of loci in cellular genomes and regulates subsets of those genes, largely in a cell- and context- dependent fashion. Identifying the transcriptional programs driven by Myc will be essential to explain its activities in growth control and oncogenesis. In work published last year (Perna et al. 2011), we profiled the contribution of Myc to the transcriptional response to serum mitogens in mouse fibroblasts. To this aim, we used an immortalized cell line homozygous for a conditional knockout allele (c-mycflox/flox) expressing a conditional CreER recombinase. Deletion was induced in quiescent cells, followed by serum stimulation and mRNA profiling. In parallel, we used Chromatin Immunoprecipitation coupled with next-generation DNA sequencing (ChIP-seq) to generate the genome-

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wide Myc-binding map in serum-stimulated cells. We thus identified a group of 282 Myc-dependent Serum Response (MDSR) genes, most of which (224, or 79%) were directly targeted by Myc in their promoter region. However, MDSR genes also constituted a surprisingly small fraction (ca 5,7%) of all Myc-bound loci in those cells (Perna et al. 2011). This work thus led to the identification of the core transcriptional program driven by Myc in response to mitogens. We are presently completing a similar study for the identification of genes deregulated by Myc during tumor progression in vivo in a mouse model of Myc-induced lymphoma. In parallel, we pursued our studies on the Myc-induced DNA Damage Response, a critical event in the modulation of Myc-induced tumorigenesis. In collaboration with the Testa lab in our department, we reported that the histone methyltransferase Setd7 plays no significant role in the Myc-induced DDR and is dispensable for the activity of p53 (Campaner et al. 2011), in contrast to what reported in a previous study. In another project completed last year (Murga et al. 2011), we developed a pre-clinical mouse model to address the therapeutic potential of targeting DDR components in Myc-induced lymphoma. In collaboration with the Fernandez-Capetillo lab (CNIO, Madrid), we showed that the activity of the ATR-Chk1 pathway is essential for tumor cells to bypass Myc-induced replication stress, and hence to survive in the presence of an activated myc oncogene. Most importantly, inhibition of this pathway provided a significant therapeutic window for the treatment of Mycdriven tumors.

3. Quantitative Proteomics to investigate transcriptional and post-transcriptional mechanisms that regulate gene expression: projects developed in 2011 (Bonaldi) a. Chromatomics: proteomics of chromatin domains by a combined ChIP~ MS approach Chromatin is a highly dynamic, well organized, nucleoprotein complex of DNA and proteins that controls various DNA-dependent processes. Chromatin plasticity is regulated by different associated proteins, posttranslational modifications on histones (hPTMs) and DNA methylation, which all act in a concerted manner to create a specific “chromatin landscape”, with regulatory effect on gene expression. Until now the characterization of the proteomic component of chromatin at specific regions has been held back by the difficulty in enriching such domains at purity and amount sufficient for the in depth analysis of proteins and hPTMs associated. We have optimized a method for a preparative-ChIP to

isolate specific functional chromatin regions and the subsequent mass spectrometric analysis to identify their interactome and hPTMs patterns. Our approach led to the characterization of transcriptionally silent, proximal (promoter) and distant (enhancers) gene regulatory regions, marked by H3K9me3, H3K4me3 and H3K4me1, respectively. Interestingly, we found that H2A.X and the WICH complex are enriched in the heterochromatic territory. We hypothesized that the recruitment of WICH, with consequent H2A.X-Tyr142 phosphorylation, represents an additional level of modulation of the DNA damage response (DDR) in this chromatin compartment; we are currently working to validate this hypothesis. By combining state-of-the-art biochemical and analytical approaches, we gained a detailed description of the features (proteins and PTMs) of certain chromatin functional sub-domains and expanded the arsenal of analytical strategies to understand how hPTMs combinations and their specific interactors mediate the locus-specific structure-mediated function of chromatin. b. Deciphering the code of protein methylation by qProteomics: the cellular methylome. Protein methylation at Arginines and Lysines (R and K) is emerging as an important regulatory element in several cellular processes, such as signal transduction, protein translocation, processing of mRNA and transcriptional control. So far this modification has been extensively studied on histones, but the global cellular “methylome”, including methylation of non-histonic proteins, remains poorly explored. In 2011 this project focused on the compilation of a comprehensive and unbiased list of novel methylated non-histonic proteins through a combination of biochemical approaches for the enrichment of modified proteins and MS-based proteomics to unambiguously detect modification sites. We successfully established a protocol for immunopurification of intact methylated proteins (Protein-IP) by screening a panel of antibodies antimethyl- Lysine and anti-methyl-Arginine from various companies, as well as from collaborators at IMCB, Singapore. We took advantage of the heavy methyl SILAC strategy to increase the confidence in PTM identification, specifically discriminating between in vivo methylated sites from either chemical artefacts or amino acids substitutions with the same deltamass. Applying this set of biochemical and analytical methods, we have so far determined in HeLa cells a list of 200 methylated R-sites and 92 methylated K-sites on 90 unique proteins, many of which are novel. This significantly expands the current notion of the mammalian “methylome”.

c. Identification of miR17~92 targets in myc- driven B cell lymphoma by qProteomics The microRNA cluster miR-17-92 is transcriptionally activated by the proto-oncogene c-MYC and is one of the best-known oncogenic miRNAs. The aberrant expression of the cluster is observed in various human malignancies and this cluster synergizes with c-MYC in the acceleration of disease onset in a mouse model of Burkitt’s lymphoma. Interestingly, recent literature supports the model of an unexpected structured network of functional interactions between c-MYC and the cluster, in which the same molecules are transcriptionally induced by c-MYC, and translationally repressed by members of the cluster (feed-forward loops). Hence, we were interested in the role of miR1792 in lymphoma maintenance, in which the expression level of a large panel of mRNAs – that represent a milieu of possible miRNA targets- is strongly stimulated by c-myc.

Research Activities

We next analyzed the effect of the NPM mutant on the hematopoietic compartment in developing zebrafish through in situ hybridization experiments of using early hematopoietic markers (tal1, runx1, lmo2, pu.1, and gata1). Expression of the NPM mutant in one-cell stage embryos leads to the increased expression of hematopoietic markers (runx1, lmo2 and tal1) in the intermediate cell mass (ICM), which is the region of primitive hematopoiesis in teleost embryos. This effect is rescued by the co-injection of NPM mutant and Dkk1, with embryos showing a wild type phenotype. Interestingly, knockdown of Npm1 resulted in increased expression of runx1, lmo2 and tal1, suggesting that NPM mutant exerts a dominant negative effect upon the wild-type form of Npm1.

We examined the effect of the cluster over-expression in primary tumour cells from the Burkitt’s lymphoma mouse model: we observed an increased rate of apoptosis and the block in G1/S transition which – together- cause a reduced proliferation of miR- overexpressing cells as compared to controls, both in vivo and in vitro. To understand at the molecular level this unexpected phenotype, we carried out and extensive SILAC-based qProteomics analysis to profile global changes in protein levels upon mir17-92 induction. Intersection of significantly down-regulated proteins established by proteomics and in silico predicted targets generated a list of experimentally identified 288 miR-targets. Their functional analysis revealed that miR-17-92 affects various pathways relevant for the cellular homeostasis, including cell cycle and apoptosis. Interestingly, we observed that 40% of identified miR-17-92 targets meets the criteria of feed-forwardloops with c-MYC, and moreover, the level of MYC itself was decreased upon cluster over-expression (though indirectly – in fact there are no miR-17-92 binding sites in c-myc mRNA), unravelling a novel and unpredicted level of control of MYC expression and new downstream effects if its activity. d. Chemical proteomics to identify targets of anticancer drugs: the E-3810 case A key step during drug discovery and development is the identification of drug’s targets, crucial to define drug selectivity and for the early detection of possible side effects. Target identification is conventionally achieved by screening in vitro a number of selected

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4. Biology of Histone deacetylase 1, HDAC1 (and Hdac2) (Chiocca) HDAC1 and HDAC2 are deregulated in many cancers and are emerging as the main deacetylases involved in neoplastic transformation. In fact chemical inhibitors of HDACs are a relatively new class of drugs with anticancer potential. HDACS are not only protein-modifiers, but are in turn regulated by post-translational modifications (PTMs): phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation and carbonylation. Some of these PTMs crosstalk specifically on HDAC1 or HDAC2, creating a rational “code” for a differential, contextrelated regulation. We have been attempting to decipher a part of the PTM code of HDAC1 and HDAC2: we now know that HDAC1 and HDAC2 are hyperphosphorylated specifically in mitosis in a variety of different cell types. This study has unveiled the kinases responsible as well as the target sites and is currently rounding up its biological significance. Concurrently, we have recently observed an HDAC1 serum and growth factor dependent phosphorylation. Furthermore, we have uncovered that conjugation to the two main SUMO paralogues (SUMO1 and SUMO2) has a different outcome on HDAC1 stability and protein turnover. The SUMO (Small Ubiquitin-related Modifier) pathway is

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implicated in diverse critical cellular processes. Finally, in a collaborative project (with Saverio Minucci and the Pathology Division at IEO), we have seen that primary human tumors respond differently to HDAC inhibitors depending on their grade of acetylation.

Viral interplay with key cellular proteins involved in carcinogenesis (Chiocca) The Gam1 adenoviral protein has been an exceptional tool to uncover novel and peculiar strategies viruses adopt to bypass host cellular defenses. Recently, our studies have highlighted Gam1 as a model for viral BCbox domain containing proteins, revealing a new viral mechanism to degrade certain host tumor suppressors. The function of the BC-box domain was first described for SOCS proteins, a family of downstream effectors of cytokine signaling cascade. Previously, our group had demonstrated that the viral protein Gam1 is a BC-box containing protein able to reconstitute active Ubiquitin E3 ligase complexes and target the SUMO E1 enzyme for degradation. Furthermore, our interest on viral exploitation of the SUMO system continues: we have been focusing on other oncogenic viral proteins interaction with SUMO enzymes. The SUMO (Small Ubiquitin-related Modifier) pathway is a post-translational modification system enzymatically analogous but functionally diverse from the classical ubiquitin system (Ub). Much scientific evidence is indeed suggesting that pathogen modulation of the host SUMO pathway is quite a common mechanism.

5. Evolutionary Genomics of cancer (Ciccarelli) The research focus of our group is to study how the tumors evolve and to describe the properties of cancer genes that drive this evolution. To this aim, we pursue two main lines of investigation: a. Rebuilding tumor evolution using genomics instability as a marker: in these years we gained a deep expertise in the analysis of rare (subclonal) mutations to be used for measuring genomic instability (De Grassi PLoS Biology, 2010) and for rebuilding the proliferation dynamics of individual tumors (De Grassi, under review). Since there is no gold standard in the analytical procedures for calling rare variants, we developed our own methodology, which is one of the few pipelines available so far for the detection of subclonal variants. We could infer that each lesion tends to develop following its own dynamics, although there are recurrent properties shared among tumors. One of those common properties is the presence of a dominant subclone that contributes for most of the cells of the tumor population. Mode and tempo for the

establishment of the dominant subclone are however cancer specific. We are now verifying whether and to which extent this specificity depends on the genetics and/ or epigenetics landscape of the tumor. 2. Evolutionary and Network Analysis of Cancer Genes: we developed and maintain the Network of Cancer Genes (http://bio.ifom-ieo-campus.it/ncg), a web resource that collects the properties of ~1500 genes mutated in cancer (D’Antonio et al Nucleic Acid Res 2012, Syed et al Nucleic Acid Res 2010). We discovered that these genes are mainly single copy-genes and engage several physical connections with other human proteins. We have interpreted these peculiar properties as a sign of fragility of these genes towards systems perturbations (Rambaldi Trends in Genetics 2008). Starting from these observations, we rebuilt the evolution of protein interaction networks taking into account gene duplicability and evolutionary origin (D’Antonio PLoS Comp Biol 2011). We now plan to use all the data that we accumulated on the properties of cancer genes to interpret the results of large-scale screenings of cancer genomes to distinguish driver from passenger mutations. In addition to the main research lines of my lab, we have established several fruitful collaborations with internal and external laboratories. Our role in these collaborations concerns data analysis and evolutionary studies. For example, we are involved in the analysis of mutations and structural rearrangements both in mouse models of liver cancer and in human carriers of hereditary breast cancer. The aim of the first project, which is part of a big European network led by the IEO, is to provide a systematic overview of the genetics and epigenetic modifications that are progressively acquired during liver cancer progression. In the second project, we aim at identifying the germline predisposing mutations in a cohort of fifty Italian women that are affected by hereditary breast cancer, but that are BRCA1 and 2 negative.

understanding of kinetochore composition, activity, and regulation is bound to reveal new insights into the mechanisms that underlie tumorigenesis. Also, advancing kinetochore biology will allow kinetochore factors to be converted into cancer susceptibility markers as abnormal levels of kinetochore components are regularly identified in myriad tumors. Furthermore, an improved knowledge of kinetochores will allow us to develop new anticancer therapies, for example by using molecules that interfere with kinetochore assembly, activity or regulation. To study kinetochores, we use the budding yeast Saccharomyces cerevisiae as a model species. As kinetochore composition, function and control are highly conserved we use this species as its genome and cell cycle can easily be manipulated in the lab. Two research projects are the current focus of our lab.

6. Molecular analysis of kinetochore composition, activity and regulation during chromosome segregation (De Wulf)

a. Role of kinetochore protein Cnn1CENP-T in chromosome segregation During the purification of kinetochores from yeast, we recently identified Cnn1 as a novel and evolutionary conserved kinetochore protein (CENP-T in humans). Cnn1 regulates the physical interaction between the complexes that form the framework of each kinetochore thereby modulating the activity in time and space of kinetochores themselves. Cnn1 activity is controlled by a number of cell cycle kinases. A lack of kinase activity acting on Cnn1 negatively affects chromosome segregation fidelity. Also, a lack or elevated levels of Cnn1 interfere with faithful kinetochore activity, resulting in chromosome missegregation and aneuploidy. This is an important finding as in many tumors, CENP-T is pathologically expressed. During the last years we have studied at molecular level the role of Cnn1 at kinetochores. More recently, we have focused on identifying the regulatory mechanisms that ensure correct Cnn1 concentrations and activity at kinetochores in dividing cells. These efforts have led to the identification of a new and conserved E3 ubiquitin ligase that monitors at kinetochores, in trans with the mitotic spindle checkpoint, the correct concentrations of Cnn1 and of other kinetochore proteins at centromeres.

Our lab studies kinetochores, large protein structures that assemble from >100 proteins on the centromeric region of replicated chromosomes. During cell division, kinetochores orchestrate the segregation of chromosomes from the mother to the daughter cells. Errors made during this progress result in offspring with abnormal chromosome numbers (aneuploidy). As all solid tumors are aneuploid, it has been suggested that chromosome missegregation initiates and/or drives the cancer transformation process. Thus, a better

b. Role of novel kinetochore kinase Rio1 in chromosome segregation While purifying kinetochores from S. cerevisiae, we identified a conserved and hitherto poorly studied cell cycle kinase named Rio1, which is overexpressed in myriad cancers. Till now, Rio1 had only been known to be involved in rRNA maturation. We mapped Rio1 to kinetochores and to a specific complex within the kinetochore structure. It dependence on a specific complex

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Research Activities

substrates. Chemical proteomics is an alternative strategy, where whole proteomes are screened by drug affinity chromatography to detect interactors, possibly in physiological-like conditions mimicking the cellular environment. In the last decade, quantitative proteomic strategies based on stable isotope labelling allowed the development of a more advanced version of chemical proteomics. In this project, quantitative proteomics has been combined to chemical proteomics to identify proteins interacting with the new multi-kinase inhibitor E-3810. Different SILAC-based experimental setup has been applied to obtain quality filters necessary to confidently identify and characterized proteins interacting with the inhibitor in lysates obtained from cancer cells. Our strategies led to the identification of different E-3810 interactors, to estimate their dissociation constant (Kd) and to draw IC50-like curves. While in vitro screening initially performed on E-3810 identified VEGRs and FGFRs as primary targets, our chemical proteomic analyses identified a broader number of interactors, providing information potentially useful to define the mechanism of action of the inhibitor and to prevent possible sideeffects.

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Figure 1: A) Loss-of-Numb expression in Numb-deficient breast tumour cells is due to enhanced ubiquitinmediated proteasomal degradation. Numb protein was immunoprecipitated (IP) from Numb-proficient and Numbdeficient primary breast tumour cells treated with the proteasome inhibitor MG132, or left untreated. IP proteins were then analysed by immunoblotting (IB) with anti-Numb and anti-ubiquitin antibodies

for localization to kinetochores suggests that this complex is a main substrate of Rio1. As the role of this conserved kinetochore complex is well known, it suggests a specific contribution of Rio1 to kinetochore function. We have also shown that Rio1 localizes to another chromosome region -known as the rDNA array- that is very important for the timely segregation of chromosomes. Interfering with rDNA maintenance or activity during chromosome segregation leads to genetic instability and aneuploidy. We are currently studying at molecular level how Rio1 regulates kinetochore and rDNA activity in time and space. Cells suffering from reduced or elevated levels of Rio1 die due to aneuploidy. Hence, our study will show how this cell cycle kinase helps to avoid that a healthy cell turns into a cancer cell. Secondly, information about its roles will also allow Rio1 to be converted into a novel anticancer drug target, thereby extending the oncologist’s toolbox to treat cancer in the future.

7. Molecular carcinogenesis and stem cell biology research (Pier Paolo di Fiore, M.D., Ph.D.) Numb is a multifunctional protein that is involved in diverse cellular processes, such as cell fate developmental

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• Figure 1: B) High-throughput siRNA screening to identify E3-ligases that might be responsible for loss-of-Numb in Numb-deficient tumours. We established the Numb-deficient breast cancer cell line MDA-MB361 as a suitable model system to perform the siRNA screening. Restoration of Numb was achieved in these cells by MG132 treatment, while normal breast epithelial cells, MCF10A, were not affected. Alterations in Numb protein levels following transfection with siRNA were detected using a capture ELISA assay. Preliminary results of the screening identified 14 negative regulators and 80 positive regulators of Numb, which are now being confirmed.

• addition, we also identified ~80 E3-ligases, the silencing of which results in loss of Numb protein. These hits are candidates for ligases that act as indirect positive regulators of Numb that might be aberrantly repressed in Numb-deficient tumours. We are now in the process of confirming hits identified in the high-throughput screening. Confirmed candidates will then be validated through high-resolution studies in Numb-deficient tumour cells. These studies will shed light on the molecular events responsible for Numb degradation in human tumours, and possibly lead to the identification of novel molecular targets for the development of new strategies to treat Numb-deficient cancers.

8. Systems Biomedicine (D’Onofrio) Our main research topics are the following: • Spatial and temporal patterns in cells and cell

• •

aggregates. The breaking of symmetry in biochemical networks of reactions is at the base of human life. Cancer deranges those phenomena. For example, cellular differentiation can be read as a loss of spatial symmetry, which in cancer, instead, is partially preserved through, for example, the increase of symmetric division of stem cells. Cell division cycle is an example of breaking of temporal symmetry, leading to periodic phenomenon, whose control in tumors is deranged. Intracellular effects of antitumor drugs Effects of antitumour drugs on a tumour mirror the effects that the employed drugs have on a single cancer cell. We study with mathematical models how drug molecules disrupt pathways essential for the cancer onset and growth. Interplay between stem and progenitor cells in early tumor growth, and its influence on antitumor therapies. The macroscopic growth of a tumor is ruled by changes of the proliferation rate of both stem and progenitor cells. These cells nonlinearly interact, which modulates the expansion of the neoplasm. Tumor angiogenesis and anti-angiogenic therapies. A landmark of tumor growth is its ability to produce chemicals that attracts blood vessels, which are ‘embedded’ in order to bring nutrients, i.e. energy. Biophysical modeling of those phenomena may be of help in improving various antitumor therapies. Tumor-Immune System (IS) interplay. Immune surveillance and tumor evasion from it, tumor-IS equilibrium, immuno-therapies: those phenomena are all complex, nonlinear and to some extent adaptive. However, we showed how their main features can be captured by some simple mathematical models. Role of intrinsic and extrinsic biological ‘noise’ in shaping intra- and inter-cellular phenomena, including chemoresistance. A biological system is affected by large internal statistical fluctuations, and by external fluctuations that are ‘transmitted to it by the other systems with which it interacts’. Our models suggests that those fluctuation can sometime be ‘positive’ for the tumor (e.g. fluctuations of adaptive immunity could help tumor immunoevasion; fluctuations in drug clearance and pharmacodynamics might induce nongenetic patterns of resistance to chemotherapies), and in some case ‘negative’ (e.g. internal fluctuations of the number of tumor cells could significantly help the tumor suppression by the immune system). In-population spreading of tumor-related infectious diseases (ID). Philosophy and Sociology of Science. The activity of biomedical research must not be uniquely confined

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Research Activities

decisions, maintenance of stem cell compartments, regulation of cell polarity, and cell adhesion and migration. Recent studies in our laboratory have also established Numb as a tumour suppressor in human cancers. We observed that loss of Numb expression occurs frequently in breast, lung and salivary gland cancer, and that it is associated with clinico-pathological parameters of aggressive disease and poor prognosis. Importantly, Numb-deficient tumour are dependent on loss-of-Numb, as re-expression of Numb in Numbdeficient, but not Numb-proficient, primary tumour cells inhibits their growth. Thus, we hypothesized that restoration of Numb expression in Numb-deficient cancers might represent a successful therapeutic strategy. We have, therefore, investigated the mechanisms responsible for loss-of-Numb in breast and lung cancer. We determined that loss-of-Numb occurs post-transcriptionally, as a consequence of enhanced ubiquitination of the Numb protein, which targets it for degradation by the proteasome. Indeed, treatment of Numb-deficient primary tumour cells with proteasome inhibitors, such as MG132, restores Numb expression to normal physiological levels (Fig. 1A). Loss-of-Numb in tumours is, thus, likely due to the functional alteration of components of the ubiquitination machinery, where the obvious candidates are E3 ubiquitin ligases: enzymes that catalyse the final step in the transfer of ubiquitin to the protein substrate. To investigate whether deregulation of a Numb-specific E3-ubiquitin ligase is responsible for aberrant Numb degradation in Numb-defective tumours, we have employed a high-throughput RNA interference-based screening approach to identify candidate ligases, the silencing of which is able to restore Numb levels in Numb-negative tumour cells (Fig. 1B). For the screening, we used the breast cancer cell line MDA-MB361, which we have shown to be a suitable cell model system for Numb-deficient breast tumours: these cells display low basal levels of endogenous Numb, however, upon treatment with the proteasome inhibitor MG132, Numb protein is restored. We silenced E3-ligase expression in these cells using a commercially available small interfering (si)RNA library targeting ~500 E3-ligase genes. The screening was performed in 384-well plates and restoration of endogenous Numb protein was measured using a capture ELISA assay, set-up and optimized in our lab. From this screening, we identified 14 E3-ligases, the silencing of which results in restoration of Numb expression. These E3-ligases are putative components of the machinery responsible for Numb ubiquitination that might be deregulated in Numb-deficient tumours. In

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9. Cellular and Molecukar pathways regulating Melanoma genesis and progression (Lanfrancone) The goal of our research is the understanding of the cellular and molecular pathways involved in melanoma dissemination. We have identified and structurally characterized a new member of the Shc family of adaptor proteins (RaLP), whose expression is confined to the cells of melanocytic origin. RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies. The biological function of RaLP in cells of melanocytic origin and its role in tumor progression is being investigated in a large cohort of primary melanomas. We have verified that the expression of RaLP significantly correlates with the most important prognostic markers of melanoma (Breslow thickness, Clark’s level of invasion, ulceration, mitotic index and presence of metastasis in lymph nodes) and that patients with RaLP expressing tumours had reduced disease-free survival and overall survival, suggesting that RaLP can be identified as a novel prognostic molecular marker and an independent prediction factor of melanoma progression. We have shown that persistent RaLP silencing does not affect proliferation of melanoma metastatic cells in vitro, but reduces their migratory and invasive potential and their extracellular matrices-adhesive capabilities. Conversely, RaLP overexpression in RGP and VGP melanoma cell lines increases their migration and invasion while reducing spreading and cell to cell contacts, suggesting a role of RaLP in helping cells to leave the tumoral mass and disseminate. To investigate the molecular mechanisms of this phenomenon we have recreated melanoma development in mature melanocytes. We have genetically engineered normal human melanocytes and subsequently in vitro and in vivo tested their migratory and metastatic potential. Immortalized melanocytes were generated by hTERT expression in normal human adult melanocytes derived from skin biopsies. Following hTERT expression, Rb gene was silenced, leading to long-term proliferating melanocytes that still retain normal features of growth factor requirement and apoptosis sensitivity. Indefinte life span was tested over a period of six months. Karyotypic analysis showed a normal karyotype in the majority of

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the melanocyte lines that were generated. Mutated BRAF and NRas oncogenes were transduced in immortalized melanocytes to transform them. While BRAF-expressing melanocytes show accelerated proliferation rate and are capable of growing in soft agar, NRAS-expressing ones are not, suggesting a stronger role of BRAF in transforming immortalized melanocytes in vitro. Both cell cultures have been injected in immunocompromised animal to evaluate their tumorigenic and metastatic potential. RaLP function in sustaining migration in vitro and metastasis in vivo will be tested, in order to define a cellular model in which novel therapeutic agents could be appropriately tested according to melanoma progression. The physiological role of RaLP is still largely uncharacterised. Adaptor proteins play an important role in the regulation of specific signaling pathways by linking activated cell-surface receptors to their downstream intracellular targets and mediating crosstalk mechanisms between pathways. We have previously shown that RaLP has a unique expression pattern, being expressed early during embryogenesis in the pluripotent epiblast and being re-expressed in the developing central and peripheral nervous system. Its expression is then confined to the brain in the adult. RaLP is implicated in the differentiation of neural stem cells to neuronal fate. We found that the neural stem cells derived from RaLP KO animals showed severe defects in neuronal formation and we are currently analysing our RaLP KO mouse model to obtain a further understanding of the physiological role of RaLP. We have also shown that RaLP expression was recapitulated in vitro as in vivo as it was tightly regulated during the formation of the epiblast in several differentiation programs and then re-expressed when neuronal commitment intiated. Using the recent protocol that allows the transition of pluripotent ESCs to another pluripotent stem cell type, epiblast stem cells (EpiSCs), we have found that the genetic deletion of RaLP resulted in an impairment of the establishment and maintenance of EpiSCs due to defective proliferation capacity upon differentiation. Furthermore, we have also described for the first time, the emergence of Cdx2 expressing cells during ESC commitment to EpiSC fate and we observed that the absence of RaLP results in an enrichment of this population due to enhanced levels of MAPK/ Erk activation. Our data suggests that RaLP plays an important role in the switch of key pathway/s involved in determining EpiSC identity and that the absence of RaLP perturbs the commitment process.

10. Structural and functional basis of asymmetric cell divisions (Mapelli) We are interested in the molecular pathways governing asymmetric cell divisions, with emphasis on the role of the mitotic spindle orientation in determining the fate choice of daughter cells and their proliferative potential. Our activity is organized in three main research lines:

of the intervening components. An emerging concept in the stem cell field is that differential cues instruct the position of the cytokinesis plane. For this reason, to unveil the molecular network coupling force generators to cellular polarity in different environments, we are also pursuing the identification of new tissue specific interactors of NuMA and LGN.

a. Structural and functional characterization of cortical force generators. Cortical force generators are molecular motors orchestrating the correct placement of the mitotic spindle within the cell. To achieve this result, they perform different tasks: 1) they localize at well defined spatially-restricted cortical sites in conjunction with polarity cues; 2) they capture astral microtubules emanating form the spindle poles; 3) they generate microtubule pulling forces whose resultant is responsible for the final position of the mitotic spindle. The core components of force generators are evolutionary conserved from nematode to mammals. Their central module consists of heterotrimeric NuMA/ LGN/Gαi complexes, assembled on GDP-loaded Gαi species. From a topological point of view, LGN acts as the molecular link between Gαi subunits anchored at the plasma membrane via a myristoyl group and the microtubule associated protein NuMA. Intriguingly, in the apo form LGN behaves as a molecular switch, held together by head-to-tail interactions. We are interested in understanding the molecular events triggering the LGN conformational transition required to assemble the NuMA/LGN/Gαi complexes, including the Gαi GTP-cycle controlled by the GEF RIC-8A.

c. Implications of the mitotic spindle orientation pathway in mammary stem cell asymmetric divisions. In multicellular organisms, stem cells and progenitors balance asymmetric versus symmetric cell divisions to generate tissue diversity and regulate homeostasis. The genetic pathways affecting the interplay between spindle position and asymmetric divisions have been first discovered in Drosophila neuroblasts, and only a few studies documented the implications of similar pathways in skin and neural progenitors. Recent reports highlighted the involvement of oriented divisions in progenitor differentiation during mammary gland morphogenesis. However, very little is known to about the molecular mechanisms sustaining asymmetric divisions in this system, and how they are deregulated in cancers. On these premises, we started investigating how cortical polarity and spindle alignment pathways affect the asymmetric outcome of mammary stem cell divisions in mice. We also study the relevance of these pathways on the stem cells regenerative potential and proliferation, which we believe will ultimately pertain to breast cancer progression.

b. Molecular characterization of the interplay between polarity and cell division plane. Our second research line deals with the issue of how force generators are specifically recruited at sites of polarization. In several model systems, cortical polarization is established by the asymmetrical distribution of the evolutionary conserved Par3/Par6/aPKC complex, which in turn defines the asymmetrical localization of fate determinants. During asymmetric divisions of Drosophila melanogaster neuroblasts and vertebrate skin progenitors, Par3/ Par6/aPKC localize at the apical site, and recruit force generators via an adaptor named Inscuteable (Insc). We have recently solved the crystallographic structure of the LGN/Insc complex, and discovered that Insc and NuMA are mutually exclusive partners of LGN [1]. This unexpected finding challenges the established model of force generators assembly, which we revised on the basis of the newly discovered biochemical properties

We summarize here the activities that have reached advanced results during 2011.

Research Activities

in our labs. We have to reason on the ethical and philosophical aspect and implications of our research. • …who knows?
 Theoretical research is strongly driven by curiosity… ;-D

11. Role of chromatin alterations in tumor initiation and maintenance (Minucci)

Mechanistical studies Mechanism of action of HDACs/HDAC inhibitors. We found a novel link between HDACs, the DNA damage response and autophagy. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA–RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. In our studies (in collaboration with M. Foiani, IFOM), we have showed that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strandDNA–RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles

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Biological studies Effect of Valproic acid on leukemia initiating cells (LICs). Aberrant histone acetylation was physiopathologically associated with the development of acute myeloid leukemias (AMLs). Reversal of histone deacetylation by histone deacetylase inhibitor (HDACis) activates a cell death program that allows tumor regression in mouse models of AMLs. We have used (in collaboration with H. de The, Paris) several models of PML-RARA-driven acute promyelocytic leukemias (APLs) to analyze the in vivo effects of valproic acid, a well-characterized HDACis. Valproic acid (VPA)-induced rapid tumor

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regression and sharply prolonged survival. However, discontinuation of treatment was associated to an immediate relapse. In vivo, as well as ex vivo, VPAinduced terminal granulocytic differentiation. Yet, despite full differentiation, leukemia-initiating cell (LIC) activity was actually enhanced by VPA treatment. In contrast to all-trans retinoic acid (ATRA) or arsenic, VPA did not degrade PMLRARA. However, in combination with ATRA, VPA synergized for PML-RARA degradation and LIC eradication in vivo. Our studies indicate that VPA triggers differentiation, but spares LIC activity, further uncouple differentiation from APL clearance and stress the importance of PML-RARA degradation in APL cure. Novel Technologies PAT-ChIP. Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. We have set-up pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPEsamples (in collaboration with M. Fanelli and PG Pelicci). Technically, the main challenge of PATChIPis the preparation of good-quality chromatin from FFPEsamples. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIPseq. PAT-ChIPprovides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPEsamples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.

12. Molecular mechanisms of cell division (Andrea Musacchio) Our studies address the molecular mechanisms of cell division, the process whereby a single mother cell creates two identical daughter cells. Our approach is rooted in biochemical reconstitution, structural cell biology, and chemical biology. We study the reactions that ensure correct chromosome segregation, and the perturbations that prevent the correct execution of the cell division process. In the long run, these studies will shed light on the molecular basis of carcinogenesis. During cell division, the name mitosis denotes the process of physical separation of the genetic material. Cells enter mitosis with replicated copies of their chromosomes, known as sister chromatids. In mitosis, the sister chromatids form stable connections with molecular “cables” named microtubules. Microtubules

and microtubule motors are proteins that harness chemical energy to perform physical work. They are crucial components of the so-called mitotic spindle, a self-organizing structure that subtends to the entire process of cell division. The process of attachment of sister chromatids to the spindle is error-prone. Errors need to be identified and corrected if unbalances in the division of chromosomes are to be avoided. Two feedback control mechanisms, known as the spindle assembly checkpoint (SAC) and error correction (EC) ensure that each and every cell is given enough time to divide its chromosomes in two equal masses, preventing premature chromosome segregation. Furthermore, the EC mechanism sorts good attachments from bad ones, correcting the latter and selectively stabilizing the former. Together, EC and the SAC prevent improper cell division, protecting our cells from aneuploidy, unbalances in chromosome numbers that are all too frequently found in tumours. A fundamental question in the field of mitosis regards the relationship between feedback mechanisms and their regulation at kinetochores. Kinetochores are large protein scaffolds (>100 different proteins, each in multiple copies) that mediate the interaction of the sister chromatids with the mitotic spindle. Precisely how kinetochores regulate the SAC and EC is unclear, but the creation of intrakinetochore stretch, a structural deformation of the internal structure of the kinetochore when microtubules capture kinetochores, seems to be important. A 10-subunit protein assembly at the kinetochore, the KMN network, is crucially implicated in the control of the SAC and EC responses. The KMN network contains the main microtubule binding activity of the kinetochore, and it is therefore the logical site of control of the EC and the SAC. We have recently reconstituted most of the KMN network complex using recombinant proteins, and are now in the process of testing models for the interplay of these components on kinetochores under different conditions that simulate in vitro the attachment, or lack thereof, of microtubules. Small molecules are crucial tool for the characterization of the feedback mechanisms acting in mitosis. They allow acute inhibition of their targets, which in turn allows the observation of the effects of relevant perturbations in real time. In recent years, we characterized a small-molecule named reversine as a potent inhibitor of Mps1, a prominent SAC kinase. Cells treated with reversine exit mitosis prematurely, as they fail to maintain the SAC. Interestingly, we have recently shown that the inhibition of Aurora B and of Mps1 leads to a strongly synergistic effect on checkpoint inhibition that allows lowering the doses of inhibitor

used by a factor of 20 or more. The network properties underlying this behaviour are currently unclear and their identification represents a priority of our studies. Our long-term vision is that drug combinations will be exploited as a means to target cancer cells selectively and with low toxicity. To exploit the full potential of this approach, however, the molecular mechanism inhibited by different small molecules will need to be known in much greater detail.

Research Activities

in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. These findings led to a model where Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy, and point to new, ongoing studies in mammalian model systems. An additional, novel link has been established with the process of cell senescence. Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescenceassociated heterochromatic foci (SAHF). In our studies (in collaboration with F. D’Adda di Fagagna, IFOM), we have showed that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogeneinduced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogeneexpressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.

13. Chronic inflammation and cancer: Transcriptional mechanisms as potential therapeutic targets (Gioacchino Natoli) Epidemiological and experimental data demonstrated a direct link between chronic inflammation and the development of several types of cancers. Moreover, cancers often contain an inflammatory infiltrate that is hijacked by tumor cells to promote angiogenesis, tissue invasion and cell proliferation. In vivo experiments in mouse models have demonstrated that modulation of tumor properties by inflammatory cells requires the transcription factors of the NF-kB family, which are essential to mount the transcriptional program underlying the inflammatory response. In turn, this has led to the suggestion that anti-NF-kB drugs may be used in tumor therapy. Drugs disabling the whole NF-kB signaling pathway are already being tested in clinical trials, but several concerns about their safety have been raised because of the many physiological responses in which NF-kB is required, most notably the defense against microbial infections. Conversely, therapies blocking the induction of subsets of inflammatory genes relevant to disease (and specifically cancer) pathogenesis regulated genes should provide more restricted and predictable effects, but the molecular bases for their design are not available. Our unit is interested in understanding the mutual relationships between the transcriptional response underlying the inflammatory response and the epigenome, namely the collection of chromatin modifications that maintain and propagate across mitosis the program of gene expression characteristic of a given cell type. On the one hand chromatin controls recruitment of transcription factors to inflammatory genes and generates tissue-specific and temporal profiles of inflammatory gene expression. On the other, inflammation affects epigenetic control in bystander tissue cells, thus leading to alteration of tissue differentiation in chronically inflamed tissues (e.g. intestinal metaplasia in chronic gastritis and Barrett’s esophagus in reflux esophagitis). Such alterations often precede and are associated with the

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14. Epigenetic mechanisms in stem cell differentiation and oncogenesis (Pasini) An adult human body contains over 200 different cell types that despite bearing and identical genetic information exert totally different functions. In general, different cells activate specific transcription programs in order to acquire and perform specific functions.

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Intra and extra-cellular signaling tightly regulates the activity of cell-type specific transcription factors (TFs) that, by binding precise elements of the DNA, set the gene expression profiles of cells. However, TFs occupy only few potential binding sites in the genome highlighting an higher level of complexity in binding site selection and suggesting the existence of different levels of regulation above the DNA sequence that falls into the concept of epigenetic. In general, epigenetic mechanisms involve potentially heritable modifications of chromatin that do not imply changes in the underling DNA sequence. This mainly involves covalent modifications of both DNA and histone proteins, which together assemble into a nucleosome forming the basic unit of the chromatin. Such modifications are controlled by specific enzymatic activities that result essential for several differentiation and developmental processes and are frequently deregulated and directly implicated in cancer development. Indeed, in the recent years, a lot of attention has been put in the inhibition of chromatin-modifying enzymes for cancer treatment and several compounds are nowadays in advanced phases of clinical trials or already approved for treatment. The main focus of our laboratory is to characterize transcriptional mechanisms that control cell fate during differentiation as well as the pathological implications that these mechanisms have in tumor formation. In this, we focus on chromatin modifying activities and their interactions with signaling, DNA binding transcription factors and transcriptional machinery. We combine biochemical, genetic and highthroughput next-generation sequencing (NGS) approaches in the following main lines of research: Polycomb Group proteins in proliferation and cancer development Polycomb group (PcG) proteins are master regulators of development that function as transcriptional repressors. PcGs exist in two different polycomb repressive complexes, PRC1 and PRC2 that respectively ubiquitylates (ubq) Histone H2A on K119 and tri-methylates (me3) Histone H3 Lysine (K) 27. The activity of PcG proteins is required for cell proliferation and is found frequently deregulated (hyper-activated) in many different tumors. In general, the current models of PcG oncogenic activity suggest that increased PcG activity represses the expression of genes with tumor suppressive function. Indeed, one of the most characterized PcG target is the INK4A-ARF locus that produce the tumor suppressive proteins p16 and ARF, upstream regulators of pRB and p53 activities. Despite this, some evidences have shown that PcG oncogenic and proliferative control is

likely more complex than simple INK4A-ARF regulation. Using genetic tools and in vivo or tissue culture models of cellular transformation in combination with NGS approaches, we aim to dissect the normal mechanisms of proliferation control mediated by PcG proteins and the relevance that these mechanisms and pathways have in cancer cell proliferation ad survival. Chromatin modifiers in stem cells and cancer Using biochemical approaches applied to both pluripotent and adult stem cells, we aim to characterize the structure of multiprotein complexes that play essential developmental and pathological roles in human tumors. Often, proteins that exert essential function, due to their structural proprieties are difficult or impossible to drug target. Understanding how their regulatory subunits modulates their activity might be extremely important not only to comprehend important regulatory mechanism but also to identify critical novel factors that can be more easily targeted for therapeutic purposes. In general, this line of research uses the biochemical characterization of novel chromatin modifying complexes as a screening platform to further characterize the role of novel interactions in regulating stem cells functions and cancer formation using both genetic and NGS approaches. Role of Histone and DNA Modifications in stem cells and cancer: Starting from NGS approaches, the lab is interested at characterizing the role that different histone and DNA modification have in regulating transcription as well as the regulatory relationships that exist between these different modifications and how they interact with non-histone proteins. For this, we are using models of pluripotent stem cells and in vivo adult stem cell as well as models of tissue culture cellular transformation or in vivo cancer development.

15. Biology and signal transduction of normal and cancer neural stem cells (Giuliana Pelicci) Our lab is performing both basic and translational cancer research in the fields of brain tumors, neural stem cells and cancer stem cells (CSCs). Our final goal is to identify and unravel the molecular mechanisms underlying glioblastoma (GBM) generation and progression. GBM is the most common and lethal type of glioma in adults. There are now emerging evidences in tumor biology validating the hierarchical organization of tumors as abnormal tissues originating from and maintained by a subset of cells with stem cell-like properties able to generate and propagate

the tumor and producing differentiated progeny with limited replicative potential. Cancer stem cells have also been isolated in GBMs and although the presence of surface markers selectively expressed on CSCs has been used to isolate these cells, no marker or pattern of markers is sufficiently robust to definitively identify CSCs. In spite of the uncertainty and difficulties related to markers clearly indentifying the CSCs in GBM, it becomes essential to develop multiple strategies for CSCs detection. Consequently the aim of our research is to map cancer stem cells biomarkers from patient derived tumor samples. The end goals of our research are threefold: 1) to evaluate the efficacy of currently utilized biomarkers (CD133) in the purification of CSCs from multiple patient derived neurosphere cultures; 2) to isolate a pure population of CSCs and identify a universal barcode of cell surface markers 3) to study proteins involved in GBM generation and progression.

Research Activities

development of malignant tumors. We have identified a number of enzymes acting on chromatin and regulated by inflammatory stimuli that may provide both a mechanistic link between inflammation and altered differentiation, as well as potential targets for pharmacological intervention. One such gene is Jmjd3, which is inducible by inflammatory stimuli and erases a histone covalent modification that controls cellular differentiation by keeping repressed the genes that specify alternative cell fates (trimethylation of histone H3 at lysine 27). Data obtained in our lab demonstrated a role of Jmjd3 in the induction of acute inflammatory responses, which suggests the possibility that it may represent a relevant target of anti-inflammatory drugs. Another enzyme we recently found to be involved in inflammatory gene expression in macrophages is the histone methyltransferase MLL4. In this case the mechanism of action is indirect and relates to the MLL4 requirement for the activation of a gene, Pigp, whose product participates in the first steps of the enzymatic synthesis of the GPI anchor. In MLL4-deficient cells, GPI anchor synthesis is defective. As a consequence, macrophages cannot load on the membrane CD14, which acts as coreceptor for macrophage activators of microbial origin, such as lipopolysaccharide (Austenaa et al, in press). An additional area of research tackled by the laboratory related to the identification of genomic regulatory elements (enhancers) controlling inflammatory gene expression. This activity involved the extensive use of chromatin immunoprecipitation coupled with highthroughput sequencing and allowed us to determine a general organizational principle of these enhancers, which consists in the combination of binding sites for ubiquitous, stimulus-responsive TFs and binding sites for costitutive cell type-restricted and lineage-determining TFs. This combination allows creating a cell type-specific context within which transcription of inflammatory genes is regulated, thus explaining variability among cell types in the inflammatory gene expression program induced by identical stimuli. The impact of different environmental and stimuli on the macrophage specific enhancer repertoire is currently a most active research area in the lab.

a. CD133 function in GBM CSCs CSCs from human glioblastoma have been isolated by the neurosphere assay and identified by the cell surface protein CD133. Xenograft assays identi fied as human brain CSCs, the CD133 positive fraction, while CD133 negative cells were not able to produce tumors. However, recently it has been shown that CD133 negative CSCs are capable of forming tumors in nude mice. Thus the notion that CD133 might be a reliable cancer stem cell marker in brain tumors is currently under revision. Moreover, the functional role of CD133 in cancer stem/ progenitor cells is not known. We will analyze the efficiency of CD133 in the identification and isolation of glioblastoma stem cells and we will investigate its functional role, studying the biological effects of CD133 down-regulation in GBMderived neurospheres in vitro and in vivo. b. Marker-independent method to isolate CSCs from GBM patients We will employ a marker-independent method consisting in the cell labeling with PKH-26 fluorescent dye that binds to cell membranes and segregates in daughter cells after each cell division, so that intensity of staining correlates inversely at single cell level, with the number of previous cell divisions. This assay, already validated to identify CSCs in breast tumoral mammospheres, allows to discern progenitors (high dividing cells) from stem cells (slow dividing or quiescent cells). PHK positive and negative cell populations will be isolated from GBM neurospheres by FACS/sorting and characterized in vitro for proliferation capacity

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c. Rai/Shc C function in patient-derived GBM CSCs Rai is an adaptor protein specifically expressed in the adult normal brain in neurons, and absent in glial cells, which becomes ectopically expressed in glioblastomas. We have demonstrated that Rai is expressed in nerogenic areas of the adult brain and its knockdown impairs physiological progenitor migration. Its expression is retained in glioblastoma cancer stem cells where it exerts the same promigratory activity. Raisilencing in cancer stem cells isolated from different patients causes a significant decrease in cell migration and invasion, in vitro and in vivo, thus increasing survival. Rai depletion is associated with an alteration in multiple signaling pathways, culminating in reduced expression of proinvasive genes (1). The mechanism by which Rai exerts its pro-migratory and pro-invasive effects is still unknown. We will specifically search for the involvement of miRNAs that might mediate Rai function as a modulator of glioblastoma invasiveness. d. CLIC1 (Chloride intracellular channel 1) function in patient-derived GBM CSCs, Several studies have reported the involvement of chloride channels in cell division, motility and drug resistance, making chloride homeostasis of particular interest in GBM, where proliferation and migration are altered and drug resistance is the main cause of tumor relapse. A marked up-regulation of CLIC1 has been recently reported in human tumors of different origin, including GBMs, where it is enriched in mesenchymal molecular sub-type characterized by patients’ poor prognosis. We will study the role of CLIC1 in the tumorigenic potential of CSCs isolated from human GBMs. The demonstration of reduced gliomagenesis after CLIC1 interference in tumoral stem/progenitors cells could identify CLIC1 as a new molecular therapeutic target. We will demonstrate the relevance of these proteins (Rai, CD133, CLIC1) in gliomagenesis by silencing them directly in tumors established in immunocompromised mice.

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16. Understanding the molecular changes that lead to the development of cancer (Pier Giuseppe Pelicci). Within the field of cancer biology, one of the challenges for the next decade is to understand how distinct, simple molecular functions may be part of complex pathways and systems, how multiple systems may come together to control complicated cellular behaviours and how alteration of this composite molecular machinery may ultimately lead to cancer. Our group is trying to investigate these molecular mechanism/interactions with research that spans from regulation of cellular division and proliferation to control of DNA transcription and replication, to the role of tumour-associated oncogenes and suppressors in tumour development and progression, and to the links between cancer and aging. It is now generally thought that tumour cells may be different with regard to tumour-initiating ability and only a few of them have the capacity to unlimitedly divide and form new tumours. These rare tumour-initiating cells have characteristics similar to those of stem cells (SCs) and are thus called cancer stem cells (CSCs). An accepted distinctive property of normal SCs is that they are mostly “quiescent” or “infrequently dividing”. If the rare CSCs are also dormant this could help explain the lack of long-term efficacy of many current anticancer drugs, which are mostly anti-proliferative drugs; consequently, these chemo-resistant CSCs would be capable of bringing back the tumour. Understanding the basic mechanisms of SC behaviour has therefore an important effect on therapeutic approaches and tumour relapse prognosis. Accordingly, a considerable part of our research effort is devoted to the characterization of normal SCs, to the analysis of the similarity and difference between normal SCs and the rare CSCs, and to study whether common mechanisms are controlling the growth and maintenance of both these types of cells across different normal and cancer tissues. To this end we generate accurate models of carcinogenesis in mammals, creating, in these model systems, mutations that mimic those that occur spontaneously in human cancers (especially leukaemia and breast). Stem cell quiescence: development of advanced protocols. To understand the role of quiescence in the maintenance of the self-renewal ability of normal and cancer SCs we require ad-hoc tools to target these small subpopulations and to provide a quantitative description of their phenotypes in relation to proliferative history and cell cycle progression. Thus, alongside our studies on animal models, we have developed a new automated

microscopy platform which allows the reconstruction of the proliferative history of targeted cell populations and their progression throughout the cell cycle. Specifically, we have produced a series of protocols for targeting cell subpopulations based on automated widefield and confocal microscopes to work both on cell cultured mono-layers and on more complex biological culture models, such as, for example, mammospheres; we have also developed a high-content approach based on the simultaneous observation of 7 fluorescence parameters that allow the measurement of i) DNA content, ii) DNA replication through Ethinyl-deoxy-Uridine (EdU), iii) phosphorylated histone H2AX, a marker of DNA damage iv) 53BP1, a DNA damage response (DDR) mediator v) KI67, a proliferation marker, vi) p53 and vii) p21. Finally, we have developed a multi acquisition approach based on computer controlled analysis-driven data collection, which allows the identification of the cells of interest (Faretta et al., submitted). DNA damage repair and therapeutic strategies. Recently, we have been trying to understand the complex systems that control cell proliferation, studying how signals of endogenous origin (as a result of damage to DNA) affect cell growth and renewal. Adult proliferating cells deal with excessive accumulation of genomic damage with the elimination of the damaged cell through apoptosis (programmed cell death) or senescence (irreversible cell growth arrest), processes mediated by the tumour suppressor p53 and its transcriptional target p21. Our past research, described in previous editions of this report, highlighted the importance that these molecules also have for the modality of division of normal and cancer SCs (Cicalese et al., Cell 2009) and for the maintenance of the leukemic clone (Viale et al., Nature 2009), respectively. In addition, by analysing the behaviour of both normal hematopoietic and mammary stem cells exposed to different doses of radiation (compared to more mature cells, such as progenitor and common myeloid precursor cells) we found that SCs respond to DNA damage by activating a unique checkpoint system that is p21dependent and leads to inhibition of apoptosis and induction of symmetric division and DNA damage repair. It appears that up-regulation of p21 causes inhibition of the activation of p53 (an early event in a cell tumour suppression mechanism). The described results have implication for cancer therapy. As increasing evidence suggests that CSCs are largely chemo- and radioresistant, thus surviving in vivo treatments and initiating tumour regrowth, is essential to know whether p21 is also implicated in CSC resistance to therapy and

relapse. One potential therapeutic approach could be to render a dormant CSC sensitive to chemotherapy by the use, in combination, of anti-proliferative drugs and agents that boost proliferation of dormant tumour cells. Alternatively, since we found that in pre-leukemic SCs the abrogation of DNA damage repair mechanisms might be synthetic lethal with oncogene expression (Viale et al., Nature 2009), inhibition of different DNA repair pathways could lead CSC accumulation of genomic damage to such an extent that it would result in the loss of self-renewal and consequent tumour exhaustion. We described our efforts in this direction in the 2010 report (Falzacappa et al., in preparation). Accumulation of DNA damage can be also achieved by the enforced expression of activated oncogenes in primary cells in vitro. This induces damage and activates the ARF/p53 tumour-suppressive pathway, leading to senescence. Accumulating evidence indicates that the ARF/p53 axis also functions as a barrier to tumour development in vivo, inducing senescence of pre-malignant cells carrying oncogene mutations. On the other hand, several leukaemia/lymphoma-initiating oncogenes lead to functional attenuation of p53 or ARF: for instance, PML-RAR in Acute Promyelocytic Leukaemia (APL) and cytoplasmic NPM in Acute Myeloid Leukaemia (AML) or NPM-ALK in Anaplastic Large Cell Lymphomas (ALCL). These oncogenes are thought to represent the initiating event of tumourigenesis because of their ability to inactivate the ARF/p53 tumour-suppressive pathway. However, these tumours have a low frequency in the human population, suggesting that they activate other, p53/ARF-independent, protective pathways to hamper the development of a tumour. We have studied the effects of the expression of the fusion protein NPM-ALK in primary cells. NPM-ALK is composed of the N-terminal portion of Nucleophosmin (NPM) and the intracellular domain of Anaplastic Lymphoma Kinase (ALK), and is found in ~50% of ALCLs. We found that NPM-ALK induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumour-suppressive pathway (Martinelli et al., Blood 2011). Our results are important for the treatment of ALCLs and other hematopoietic tumours where the ARF/p53 pathway is not altered but functionally suppressed by the expression of oncogenes, since, in these tumours, restoration of oncogene-induced senescence might be instead achieved by re-activation of the p16INK4a/pRb pathway.

Research Activities

and self-renewal ability, and in vivo for their tumor formation capacity after orthotopic injection in nude mice. Frequency of the number of the putative stem cells by in vivo limiting dilution assays will be evaluated as well as the ability to re-form tumors by in vivo serial xeno-transplantations.

Cooperative events that lead to the development of leukaemia. Cancer development and progression

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We have been investigating what these cooperating mutations and their biological functions might be, primarily in the presence of PML/RAR. Genetic screening for mutations which contribute to tumour formation is a very efficient tool for the identification of genes involved in tumorigenesis. Slow transforming retroviruses, such as Moloney Murine Leukemia Virus (MLV), can mutate genes in a cell by the integration of the provirus into the host genome. Through proviral mutation of cellular genes, transformed cells can acquire a proliferative

advantage and eventually give rise to tumours. We have used insertional mutagenesis screening and identified 264 putative cooperators of PML/RAR in leukemogenesis by high-throughput sequencing of viral integration sites from 48 PML/RAR-dependent MLV infected leukaemias. We have found that infection with MLV cooperates with PML-RAR during promyelocytic leukemogenesis, by both increasing the penetrance and reducing the latency of leukaemia in mice that overexpress PML/ RAR (PML/RAR knockin, KI, mice) with respect to wild type mice infected with the same virus. Interestingly, MLV-infected animals develop multiple and independent leukemic clones, each harbouring few and unique retroviral integrations. Around 67% of the CIS (common insertion site)-associated genes appear transcriptionally activated by the proviral integrations (promoter insertion and enhancement), while the remaining 35% are transcriptionally downregulated or express abnormal transcripts. Well-known oncogenes, such as Myb, Myc, Evi1, Gfi1, Sox4, and Raf1, are among the putative targets but the majority of the genes we found are novel cancerrelated candidate genes. We then identified tumourspecific mutations by comparing MLV infected PML/RAR KI and wild type mice after whole-exome sequencing. Importantly, we have found that expression of the CIS-associated genes is consistently deregulated during murine promyelocytic leukemogenesis, irrespective of the initiating genetic alterations (PML/RAR or AML1/ETO) and follows similar patterns of de-regulation regardless of whether the PML-RAR cooperating mutations

Figure 1. Relative distribution of Retroviral Integration Sites (RIS) and Common Integration Sites (CIS) in the mouse genome. The mouse genome is divided per chromosome (vertical gray-dashed lines). The black upward lines indicate how many reads have been sequenced for each RIS. The putative target genes and the leukaemia in which that particular RIS is the prominent clone (pr) are indicated on the top of the major peaks. The light blue downward lines represent CIS; the length of each line indicates how many leukaemias bear that CIS.

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occurred spontaneously or were induced by retroviral integrations. In addition, deregulation of CIS-associated genes appears predictive of the human disease as well. The observation of a generalized deregulation of a high number of genes in every tumour, suggests that the targets of the genetic lesions are part of an interactive network. Notably, we have found that most of the identified CIS-target genes are involved in cellproliferation control (70%) and in canonical pathways such as p53 signalling (Figure 1). DNA replication origins. The process of DNA replication, when a cell copies its DNA in preparation for cell division, initiates from multiple chromosomal loci called replication origins (ORIs), which are selected in the G1 phase of the cell cycle by the sequential recruitment of the origin recognition complex (ORC), the proteins CDC6 and CDT1 and the MCM complex (together constituting the pre-replicative complex; pre-RC). However, little is currently known about the mechanisms regulating the initiation of replication on DNA strands, unlike the highly conserved basic copying mechanism which leads to their correct duplication. We have thus set-up an experimental procedure called ChIP-seq (ChIP: chromatinimmunoprecipitation; Seq: sequencing) that has allowed, for the first time in mammalian cells, the identification of replication origins in their natural chromosome context. We performed ChIP on ORI-enriched chromatin fractions, followed by parallel sequencing of the precipitated DNA. This new method has made possible the genome-wide analysis of Orc1, a component of the pre-RC, which, among all pre-RC components, is the most involved in the selection of active ORIs. Genomic mapping of the Orc1sites and analyses of their association with transcription has shown that nearly all of them are localized to active

transcription start sites (TSSs) of coding or non-coding RNAs. We have found a tight correlation between amplitude, expression, timing and genomic position of the Orc1 sites: the highest and most expressed (efficient ORIs) are located at the Transcription Start Sites (TSSs) of known genes and replicate early in the S-phase; the lowest and less expressed (sporadic ORIs) are instead located at TSSs of non-coding RNAs and replicate throughout the entire S-phase. These data suggest that transcription initiation is critical for the recruitment of the pre-replication complex and determines the probability of origin firing during the S phase (Dellino et al., submitted to Nature Genetics).

Research Activities

requires the accumulation inside a cell of genetic and epigenetic changes, each conferring a selective advantage to the cell at a specific stage of tumorigenesis. In animal models, expression of the oncogenic fusion proteins PML/RAR and AML1/ETO in hematopoietic stem/progenitor cells results in the development of myeloproliferative disorders and in the induction of a pre-leukemic state; the latter, in the case of PML/RAR, progresses to AML with incomplete penetrance, long latency and after acquisition of other karyotypic abnormalities, or, in the case of AML1/ ETO, results in AML development only after chemical mutagen challenge. Thus, although expression of PML/ RAR or AML1/ETO makes the hematopoietic SCs (HSCs) prone to the accumulation of secondary mutations, it is not sufficient per se to induce full transformation. Acquisition of secondary mutational events (cooperating mutations) are, hence, a requisite for leukaemia development.

Aging. Genomic instability is considered a hallmark of most cancers; however, genomic instability also appears to be a hallmark of aging. It is not surprising then that germline mutations in the genes for proteins that have been linked to DNA damage control (such as p53, ATM and BRCA1) have also been linked to cellular or organismal aging. Indeed, many scientists think that cancer and aging are two different sides of the DNA damage response. While cancer can affect people of all ages, the risk of developing cancer generally increases with age; however, it is still unclear, mechanistically, how and where these two outcomes, cancer and aging, coalesce and/or diverge. Aging is in fact associated with a number of events at the molecular, cellular and physiological levels that might influence carcinogenesis, thus understanding these events will help cancer prediction and treatment. Cellular senescence, for example, is considered as a process that limits the proliferative potential of damaged cells; indeed, recent data have shown the presence of many senescent cells within a tumour.

Figure 2. Activation of distinct p53 pathways in the progression of aging and cancer development. Oxidative stress activates a specific p53 transcriptional response mediated by the p44/p53 isoform.

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17. Dendritic cell biology in health and disease. (Maria Rescigno) Dendritic cells (DC) comprise a family of professional antigen presenting cells unique in their capacity to modulate T cell responses. DC play a primary role in pathogen protection, in central and peripheral tolerance and in anticancer immune responses. Understanding basic mechanisms governing DC function in biology and pathology can be instrumental to unravel how an immune response is initiated and to shape new protocols for immune intervention. In our unit we study the interaction of DC with recombinant bacteria both in vitro and in vivo with the aim of establishing new protocols for cancer immunotherapy. We have proposed bacteria as tumor antigen delivery systems and as infectious

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agents in order to kill tumor cells both because they are neoplastic and because they are infected. In the laboratory we study the interaction of dendritic cells and bacteria in the context of two pathologies: cancer and inflammatory bowel disease. Within cancer, we try to understand whether bacteria can be used to successfully exploit antigen presentation and to develop new ways to target tumor antigens into dendritic cells. For this we have shown that bacteria can induce the upregulation of gap junctions in tumor cells. These gap junctions pair with those of dendritic cells and allow the transfer of antigenic material from tumor cells to dendritic cells. This mechanism of antigen loading is very efficient and allows for the generation of a very strong immune response. The same data have been confirmed in the human system, indicating that we can transfer this technology to the clinical practice for the generation of a potent vaccine against melanoma. We are also using bacteria to target tumor cells via the generation of intelligent missiles that will recognize only tumor cells. Finally, we are trying to understand the mechanism of resistance to tumor targeted therapy that involves the activation of the immune system. In particular we are evaluating the role of immune cells in antibody-dependent tumor targeting. In the second line of research, as a dysregulation in bacterial handling has been associated to the development of inflammatory bowel disease, we want to understand the basis of bacterial handling in the gut. We found that even though dendritic cells encounter bacteria in the gut they are inhibited in their inflammatory potential. This characteristic is conferred by the local microenvironment and in particular by epithelial cells. We also studied the molecular factors involved and identified TGF-b, retinoic acid and TSLP as important mediators. Interestingly all of these factors are highly reduced in epithelial cells isolated from inflammatory bowel disease patients. We are also trying to understand whether beneficial microbes like probiotics can re-establish the generation of a homeostatic environment via the upregulation of these factors or via a direct anti-inflammatory activity on DCs. We found that some Lactobacilli have indeed an anti-inflammatory activity. We are now studying the molecular bases of this activity.

18. The epigenetics of genome programming and reprogramming: Research Activities (Testa) Activities 2011. The focus of the lab is on the epigenetic mechanisms that enable cell fate transitions and on their aberrations in cancer. The goal is to uncover how genomic programs are deployed during physiologic and pathologic development and what are the

chromatin regulatory mechanisms that coordinate their deployment. Specifically, our work is articulated in three complementary lines of research that investigate: i) the physiology of genome programming during differentiation, focusing on neurogenesis as model system; ii) the aberrant genome programming that accompanies gliomagenesis, the pathological counterpart of normal neural and glial differentiation; and iii) the controlled genome reprogramming that enables the reacqusition of pluripotency or the direct cell fate reassignment of somatic cells. a. The physiology of genome programming: exploring the role of silencing chromatin in neural fate acquisition The methylations of histone H3 on lysine tails 4 and 27, respectively mediated by the Trithorax (Trx) and Polycomb (PcG) protein families, have emerged as central regulators of the establishment and maintenance of differentiated cell states. The connection between histone lysine methylation and developmental fate became apparent with the realization that Ezh2, a member of the Polycomb group (PcG) of proteins first discovered in the fly as stable repressors Hox genes, catalyzes the trimethylation of histone H3 on lysine 27 (H3K27me3) while Trx (and its mammalian homologs of the Mll family), identified in the fly as astable activator of Hox genes, catalyzes the trimethylation of histone H3 on lysine 4 (H3K4me3). In ES cells and some adult stem cells most PcG target genes are kept in a repressed state but poised for activation by a bivalent chromatin signature that features both H3K4me3 and H3K27me3. Upon differentiation many of these bivalent domains are resolved and their genes become either completely active (marked solely by H3K4me3) or definitely repressed (marked solely by H3K27me3) in a lineage specific fashion. Contrary to the long held assumption that histone lysine methylation (HLM) was irreversible and that this irreversibility underlined lineage stability, research over the last years revealed the existence of histone lysine demethylases (HDMs) as key effectors of the dynamic regulation of HLM, suggesting that the establishment and maintenance of cell lineages involves a regulated process of addition and removal of methyl marks (Natoli, Testa and De Santa, Curr Opin Drug Discov Devel 2009). In particular, we showed that Jmjd3, a histone H3 lysine 27 demethylase, is required for the neural commitment of ES cells by regulating the expression of key drivers and markers of neurogenesis (Burgold et al. PLoS One 2008). In the past year, our efforts focused on how the epigenetic axis centered on H3K27me3 and its

downstream silencing steps regulate distinct phases of neurogenesis as well as the maintenance of the differentiated state in neural and glial cells. Specifically, we carried out a critical systematization of existing data that ushered in the development of a model of Polycomb-mediated neurodevelopmental timing that reflects both the epigenetic transmission of the mark and its dynamic relocation to new targets during corticogenesis, and that frames our current research questions (Testa, Bioessays, 2011). We then harnessed the power of conditional mutagenesis to establish conditional murine strains and embryonic stem cell (ESC) systems in which to modulate the activity of key effectors of the epigenetic axis centered on H3K27me3, which we are currently characterizing to dissect how the dynamics of this histone mark regulates neurodevelopmental transitions.

Research Activities

Oxidative stress is a potent inducer of the tumoursuppressor p53 discussed above, which mediates all the anti-proliferative cellular responses to oxidative signals. In mammals, increased resistance to oxidative stress is consistently associated with delayed aging, resistance to aging-related diseases and enhanced longevity. Interestingly, in vitro, primary cells from longlife mouse models and p53 null transgenic mice seem to have similar resistance to oxidative stress (such as H2O2 exposure), suggesting the inactivation of selected p53-downstream pathways. Notably, attenuation of p53 function is invariably associated with increased tumour formation in mammals; however, tumorigenesis is absent or reduced in long-lived mice. We have investigated p53 regulation in mice which lack the gene for the protein p66, a condition which retards aging in these animals and confers cellular- and systemic- resistance to oxidative stress. We have found that p53 transcriptionalresponse to oxidative-stress involves down-regulation of a set of more than 200 genes that encode for determinants of progression through the cell cycle or suppression of senescence. These genes are selectively down-regulated in cultured fibroblasts after oxidative stress, in proliferating tissues, and during physiological aging. Our results indicate that expression of p66 leads to activation of an isoform of p53, p44/p53, which in turn accelerates aging and prevents mitosis after protein damage; on the contrary, p66 deletion retards aging and increases longevity of p44/p53 transgenic mice. Thus, activation of p66 in proliferating cells, following oxidative-stress, might favour up-regulation of selected p53-downstream pathways, through p44/p53, leading to transient cell-cycle arrest and repair of damaged proteins, or, if protein damage is severe or prolonged, cellular senescence or apoptosis (Gambino et al., submitted) (Figure 2).

b. Aberrant genome programming in tumorigenesis: Polycomb in gliomagenesis Consistent with the role of PcG in lineage choices, alterations in H3K27me3 are likely to be early events in the cascade of epigenetic aberrations of cancer, particularly the hypermethylation of CpG promoters that is an important mechanism of tumor suppressor inactivation. Several strands of evidence indicate that CpG hyper-methylation in cancer cells is the result of an instructive process through which altered developmental programs determine aberrant hypermethylation at multiple loci. The majority of genes that are hypermethylated in cancers are premarked by H3K27me3 in ES cells and the recent observation that most epithelial cancers share a core transcriptional signature with ES cells corroborates this model and suggests that the PcG-dependent gene expression program that orchestrates development in normal cells is hijacked in cancer cells as the main template for cancer DNA methylation. Finally, the oncogenic role of H3K27me3 imbalances is underscored by the direct involvement of various PcG members in human and experimental tumors, of which the two best characterized examples are Ezh2 and Bmi1. Hence, this line of research in the lab investigates the pathologic counterpart of the neurogenic and gliogenic program, namely the development of malignant gliomas, with the aim of elucidating the epigenetic basis of the lineage aberrations that characterize this disease. To this end, we test the proposition that loss of the physiologic regulation centered around H3K27me3 is important for the initiation and/or maintenance of gliomas, combining the conditional modulation of this epigenetic axis in advanced murine models of glioblastoma with its

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Set7/9 in p53-mediated DNA damage response This line of research was originally triggered by the observation that the enzyme Set7/9, first identified as a H3K4 methyltransferase involved in lineage specific gene activation, was found to methylate also lineage specific transcriptional regulators as well as p53. In particular, methylation by Set7/9 had been proposed as a critical layer of post-translational p53 regulation, contributing to its stabilization and activation. Together, these observations provided a potentially unifying mechanism for the coordination of p53 responses with transcriptional programs involved in lineage determination, including those entailing gene-specific H3K4 methylation. This prompted us then to investigate whether Set7/9 could physiologically link aberrant genome programming to cell cycle control during tumorigenesis (Campaner et al. Molecular Cell 2011). Contrary to what was previously claimed however, we found that deletion of Set7/9 had no effect on p53dependent cell cycle arrest or apoptosis following sublethal or lethal DNA damage, induced either by radiation or genotoxic agents. Consistently, Set7/9 was also dispensable for p53 acetylation following irradiation and for the execution of the p53-dependent gene expression program, indicating that Set7/9 is surprisingly dispensable for p53 function in the mouse. Finally, a thorough biochemical analysis also established that the antibody raised against methylated K372 in human p53, the critical tool used to define the in vivo relevance of p53 methylation, is not suitable to discriminate the methylation state of K369 in murine p53, leading to a thorough revaluation of the biology of this enzyme in the mouse.

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relative contribution to cell fate reassignment, defining what are the epigenetic windows of sensitivity during reprogramming. To this send we use both experimental paradigms of induced pluripotent stem cells (iPSC) and induced neuronal cells (iNC). In this context, in collaboration with the Pelicci group (Pasi et al., Cell Death and Differentiation, 2011), we also contributed to the definition of oncogene-induced replicative stress during transcription factor-induced reprogramming, an observation that led, along with several other parallel studies, to a major reappraisal of genomic integrity during cell fate reassignment.. Modeling neurodevelopmental disorders through somatic cell fate reprogramming One of the most tangible outputs of somatic cell reprogramming has been a paradigm shift in our ability to model human diseases. The fundamental limitation in the study of human diseases has been so far the scarce availability of primary diseased tissues, which is particularly salient for disorders of the nervous system. The consequence has been the use of transformed cell lines that often have no physiologically meaningful relationship to the cell types targeted in the diseases. Transcription factor (TF)-induced cell reprogramming is overcoming this bottleneck at an unprecedented pace through the possibility of obtaining from any patient a virtually endless supply of disease-relevant cell-types. Progress has been particularly promising for several neural diseases, for which patient-specific iPSC are proving to be meaningful models to elucidate disease pathogenesis and test new therapies. On the basis of the above considerations, we are using somatic cell reprogramming to develop physiopathologically meaningful in vitro models of neurodevelopmental disorders characterized by epigenetic disfunction, in which to investigate the molecular basis of mental retardation and cognitivebehavioral impairment.

c. Somatic cell fate reprogramming and induced pluripotency Building on the recent derivation of pluripotent stem cells (iPSC) from adult somatic cells, we pursue the following two lines of research.

19. Mechanisms controlling chromosome segregation (Visintin)

Epigenetic mechanisms of cell fate reassignment Here we take transcription factor-induced cell-fate reassignment as an experimental system that makes the epigenetic rewiring of differentiation states amenable to genetic and biochemical studies and that complements the approaches described above. Specifically, widespread changes in H3K4 and H3K27 methylation have been shown to accompany the reprogramming process, and our objective is to dissect functionally their

Our laboratory is interested in understanding the molecular mechanisms that control cell division, the process by which a cell generates two genetically identical daughter cells. For this to occur, cells need to replicate their chromosomes and faithfully distribute each copy into the daughter cells. To ensure that each cell receives only one copy of each chromosome, cell cycle events need to be coordinated in time and space. If these mechanisms fail then genomic integrity is lost, which can lead to cell death or the acquisition

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Figure 1: The metaphase to anaphase transition is triggered the cleavage of cohesin complexes. Anaphase onset is conventionally marked by the dissolution of the cohesin linkages that hold the sister chromatids together and oppose the pulling forces of the spindle microtubules. Cleavage of cohesin by separase (Esp1 in budding yeast) gives way to spindle forces, resulting in sister chromatid segregation. Separase activity is restrained by securin (Pds1 in budding yeast), which is held in check by the anaphase promoting complex or Cyclosome. Courtesy of Rosella Visintin

of proliferation abnormalities. In particular, we focus on mitosis, the phase of the cell cycle during which replicated genomes are separated and packaged into daughter nuclei. We study chromosome segregation to better understand how errors made during this process contribute to the transformation of a healthy cell into a cancerous one. Mitosis Mitosis is comprised of a highly choreographed sequence of events that lead to dramatic cellular reorganization. Although it is a continuous process, cytological changes allow it to be arbitrarily divided into sub-phases including prophase, prometaphase, metaphase, anaphase and telophase. Three major transitions take place during mitosis: 1) the G2/M

Research Activities

functional dissection in primary cells isolated from human tumors.

transition, where entry into mitosis is controlled; 2) the metaphase-anaphase transition, at which sister chromatid separation is triggered; and 3) the M/G1 transition, at which cells reverse the processes that led to mitotic entry and reset the conditions for a new round of cell division. Specific projects: Metaphase-anaphase transition: Chromosome segregation To ensure the correct transmission of chromosomes during cell division, replicated chromosomes (sister chromatids) must first be separated and then segregated between the daughter cells. Sister chromatid segregation occurs in anaphase and is triggered by the dissolution of the cohesin complexes that hold the sister chromatids together.

Figure 2: The phosphatase Cdc14 is regulated by changes in its subcellular localization. In budding yeast, the phosphatase Cdc14 promotes exit from mitosis by triggering the inactivation of CDK complexes and by reversing the phosphorylation events mediated by these kinases. Cdc14 activity is restrained by Cfi1 that sequesters and inhibits Cdc14 in the nucleolus from G1 until metaphase. At anaphase two regulatory networks, FEAR and MEN, bring about the sequential release of the phosphatase thereby guaranteeing the correct execution of the anaphase program. Courtesy of Rosella Visintin

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M-G1 transition: Mitotic exit Mitotic exit initiates with the down-regulation of cyclindependent kinase (CDK) activity, a family of kinases whose activity controls cell cycle progression. Next, the phosphate groups that CDKs added to their targets to allow cells to enter mitosis must be removed so that the cells can exit mitosis. In budding yeast the Cdc14 phosphatase is important for both CDK down-regulation and the reversal of mitosis-promoting phosphorylation events. Cdc14 activity is controlled by changes in its subcellular localization. The phosphatase is sequestered in the nucleolus by its inhibitor Cfi1/Net1 for much of the cell cycle. At anaphase, two regulatory networks; the Cdc Fourteen Early Anaphase Release network (FEAR) and the Mitotic Exit Network (MEN) sequentially release Cdc14 from Cfi1. This sequential activation of Cdc14 triggers, in a wave-like manner, the dephosphorylation of distinct populations of CDK substrates and thus mitotic events at different stages of anaphase. Indeed the FEAR and MEN networks coordinate mitotic exit with different cell cycle events (Figure 2). Although we have established a framework of how mitotic exit is controlled in budding yeast, important questions remain to be addressed. For instance, what is the molecular mechanism that regulates the Cdc14Cfi1 interaction is still unclear? What is the significance of the double kinases requirement underlying Cdc14 release? How is Cdc14 activation orchestrated and coordinated with different cell cycle events? Other questions regarding the pathways regulating Cdc14 activity are also unresolved: Why does the FEAR network consist of at least two branches? Which signals are sensed by the FEAR network? How does the FEAR network promote Cdc14 activation? We wish to provide answers to these questions.

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Department of Experimental Oncology

The Drug Discovery Programme

DDU STAFF The personnel involved in the Drug Discovery Unit has been already described in the dedicated section. TIV STAFF Post-doctoral Fellows: Daniela Bossi, PhD, Angelo Cicalese, PhD PhD Students: Alessandro Carugo, Carolina D’Alesio Technician: Elena Cavallaro

An attractive alternative to low productivity in drug development (especially in terms of truly innovative drugs) and the almost exclusive role played by industry is to enhance the drug R&D efforts in “academic” biomedical research institutions. These institutions are able to attract the sharpest minds and bright young people and create an appropriate climate where cutting-edge, creative and innovative science is performed that is not dictated by marketing or commercial objectives. In June 2009, the European Institute of Oncology launched a Drug Discovery Programme (DDP) located within the IFOM-IEO Campus, to translate the basic research developed within the Campus into drug discovery projects. The aim of the DDP is to successfully bring these research projects up to the filing of an Investigational New Drug application, to ensure their development for maximum patient benefit and to commercially exploit these discoveries for the growth of the DDP, the IFOM-IEO Campus and IEO. Additional objectives of the DDP are to create a network of collaborators aiming at excellence in drug discovery, to contribute to the education and training of talented young people and to create leaders in drug discovery and cancer research. Starting from the basic science and the technologies developed at the Campus to understand the molecular basis of cancer, medicinal, analytical and computational chemists with industrial drug discovery experience work together with dedicated biochemists, cell biologists and

Research Activities

Cohesin is cleaved by separase whose activity is restrained by securin (Figure 1). Securin, in turn, is controlled by a surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC is a signaling pathway that delays sister chromatid separation until all sister chromatids have correctly attached to the microtubules of the mitotic spindle. When the SAC is satisfied cells can proceed into anaphase. Progression through anaphase is mediated by mitotic spindle activities. A focus of the lab is to obtain a molecular understanding of the regulatory networks that control sister chromatid separation and spindle dynamics. We recently found a budding yeast mutant that cannot proceed through anaphase regardless of having degraded securin and cleaved cohesin. Elucidating the molecular defects characterizing our double mutant will allow us to define a novel pathway that is essential for sister chromatid segregation.

in vivo pharmacologists to identify new drugs. Through multidisciplinary innovative research the DDP intends to build a tradition of excellence in drug discovery at IFOMIEO Campus. During 2011, the Department focused on the following scientific projects: • Epigenetic targets • SAC kinases • NUMB modulation • Exploratory activities and Target identification and validation activities Epigenetic targets Emerging evidence suggests that epigenomic changes are causally linked to oncogenesis and tumor progression. Histones are a key component of the machinery regulating the dynamics of eukaryotic genomes. The elaborate set of post-translational modifications found in their protruding “tails” has been proposed to form a “histone code”, that dictates the accessibility of other factors to chromatin, imposing alterations in chromatin structure, and subsequent changes in nuclear functions. These modifications are carried out by macromolecular complexes containing components endowed with enzymatic activities. Prioritarized chromatinenzyme targets belonging to the Lysine demethylase (KDM) family have been selected for the epigenetic platform in the drug discovery pipeline. The Screening funnel has been set up and related assays have been developed, including one to permit high throughput screening. Chemical hits are being sought using an integrated approach including traditional medicinal chemistry tools and computer-assisted drug design: small molecule-hits have been identified and are the object of active exploitation. Furthermore, commercially available chemical libraries have been evaluated and high quality compounds have been selected in order to build up a chemical collection ready to be screened in a high throughput fashion against targets of interest. (in coll. with S. Minucci, P.G. Pelicci e G. Natoli)

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Numb modulation Loss of Numb expression concomitantly results in two major effects: deregulation of a potent oncogene (the Notch receptor) and loss of function of a tumour suppressor (the p53 protein). The combined dysfunction of the Numb/Notch and Numb/p53 axes most likely accounts for the particularly aggressive phenotypes displayed by Numb-defective tumors (e.g breast and lung cancers). Numb expression in tumours is most likely determined at the post-transcriptional level where posttranslational modifications (ubiquitination, phosphorylation) target the protein for degradation. Hence there is an urgent need to identify these upstream mechanisms as their inhibition could restore Numb levels and counter the imbalance in both the Numb/ Notch and Numb/p53 axes. These potential Numb

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regulators present ideal pharmacological targets for the stabilization of Numb levels and the restoration of its tumor suppressor activity. Two strategies will initially be employed to identify the “upstream regulators” of Numb: “target identification” by siRNA screening (forward strategy-ongoing); “phenotypic screening” against a small molecule library (reverse strategy).The ”forward” strategy has been implemented and candidates have been identified and are currently undergoing validation. (in coll. con S. Pece and P.P. Di Fiore) Exploratory activities Exploratory activities are developed internally or through external collaboration in order to create a machinery ready to feed the running project pipeline. Target identification and validation activities This part of the Program (started in 2011) ia based on the assumption that there is a fundamental biological heterogeneity among tumors, and these differences must be understood and exploited to identify cellular pathways governing the biology of the tumor within a specific patient, that can be targeted pharmacologically. In our view, rather than taking a uniquely “descriptive” approach to the anatomy of tumors, a “functional” strategy has better chances to lead to the identification of immediately validated targets. We therefore have planned to perform a discovery study based on functional in vivo genetic screenings to knock-down a large subset of genes (5001,000) that can be targeted pharmacologically in primary tumor samples, and analyze their growth in orthotopic xenotransplantation models. At the same time, those samples (and their primary counterpart, stored in our pathology archives) will undergo an extensive characterization through high-throughput approaches (genomic sequencing, transcriptome, epigenome) of the global tumor anatomy. The screening will therefore lead to two outcomes: 1) identification of targets that have been validated in the most advanced preclinical setting; 2) identification of the molecular context (gene, chromatin, transcript) in which the target is validated. As a secondary outcome, this will lead to the identification of candidate biomarkers for patient treatment. We will focus –among drugable enzymes- on kinases and epigenetic enzymes. Our choice is based on the fact that there are clinically available drugs for several members of these enzyme classes, and at the same time there are several other members that have not been validated as cancer targets, and for which drugs have not been developed. For these latter targets, we plan to start (within the Drug Discovery Program) drug discovery projects to identify antagonizing drugs.

Department of Experimental Oncology

The Molecular Medicine Programme

MOLMED Staff Scientists: Elena Carla Belloni, Ugo Cavallaro, STAFF Giovanni Mazzarol, Salvatore Pece, Daniela Tosoni Scientists: Ivan Nicola Colaluca, Francesco Nicassio Scientific Responsibles: Fabrizio Bianchi, Giuseppina Bonizzi, Stefano Confalonieri, Daniele Galvagno, Manuela Vecchi Post-doctoral Fellows: Macarena Ferrero Gimeno, PhD, Stefano Marchesi, PhD, Matteo Jacopo Luca Nicolò Marzi, PhD, Davide Rambaldi, PhD, Alessandra Micaela Villa, PhD PhD Students: Letizia Amadori, Maria Elena Bicchieri, Pietro Lo Riso, Eleonora Lusito, Simona Monterisi, Chiara Tordonato Data Entry: Fabio Dezi Technicians/Technologists: Antonio Bai, Rossana Bettolini, Michele Caccia, Maria Capra, Rose Mary Carletti, Marco Coazzoli, Elena D’Acunzo, Francesca De Santis, Tiziana Fumagalli, Fulvia Fusar Imperatore, Donatella Genovese, Elvira Gerbino, Barbara Giulini, Giovanna Jodice, Chiara Luise, Elena Marino, Valentina Mattei, Luca Napolitano, Micaela Quarto, Flavia Troglio, Andrea Ugetti, Silvia Zecchini Undergraduate Students: Alessio Di Giacomo, Irene Ferrari, Chiara Giacomelli, Salvatore Lorenzo Renne Visitors: Blanca Alvarez Moya, PhD, Andrea Basile, MD, Alicia Rubio Garrido, PhD, Maria Antonietta Sabatino, PhD, Silvia Sterpone, PhD, Angelo Taglialatela Molecular Medicine-Programme Manager: Bronislava Matoskova Laboratory Manager: Natalia Meani Administrative Assistant: Semhare Berhane Support Personnel: Domenico Gualandi

Research Activities

SAC Kinases One of the “hallmarks” of cancer is the occurance of aneuploidy, a state whereby cancer cells contain extra chromosomes. At mitosis, normal cells have two centrosomes which migrate to opposite poles of the cell and generate a bi-polar mitotic spindle which functions to segregate the newly replicated chromosomes into two daughter cells. Aneuploidy is often accompanied by the presence of extra centrosomes and this can lead to the formation of a multi-polar spindle. Mitosis attempted with a multi-polar spindle would lead to miss-segregation of the chromosomes and would be catastrophic for the cancer cell. To avoid this cancer cells must prolong mitosis long enough to rearrange the supernumery centrosomes into a pseudo bi-polar spindle that will permit proper segregation of the chromosomes. This is achieved by maintaining the activity of the spindle assembly checkpoint (SAC), a mechanism which prevents mitotic exit until bipolar chromosome attachment has been achieved. Several kinases are involved in SAC function and inhibition of these kinases is expected to override the SAC and afford premature mitotic exit. Thus SAC kinase inhibitors are attractive targets as novel cancer therapeutics, especially in cancer cells characterized by aneuploidy and supernumery centrosomes where a prolonged SAC activity is essential for viability. Two SAC kinases have been prioritized within the drug discovery program, biochemical and cellular assays have been developed to set up the screening funnelincluding one for high throughput screening. Several approaches are being taken to identify chemical hits. A high throughput screening campaign against one of the Sac kinases, utilizing the DDP kinase-targeted chemical collection, together with a classical medicinal chemistry approach and structure-based drug design (SBDD) has led to the identification of starting small molecule hits. (in coll. with A. Musacchio)

We have named the Molecular Medicine Programme “Molecular Medicine for Care” (MMC). The three words that make up MMC bear equal weight. “Molecular” derives from the technological advances of the last decade that finally permit researchers to understand cancer at the deepest molecular level. “Medicine” is of course the science and art of healing, the daily task in which our clinicians at IEO are unrivalled. Better “Care” is what both clinicians and researchers taking part in MMC aspire to provide to all patients. We need the whole-hearted participation of both clinicians and researchers for the boundaries between the first two objectives to become so blurred as to become irrelevant. Without this, MMC would simply remain a (high quality, no doubt) translational research department. We have much higher expectations of ourselves. We have finally begun to travel the path that will allow us to reach our goal.

Development of MMC Laboratory Infrastructures A number of infrastructures provide the enabling conditions for MMC. Many of these were resources that were already serving our basic research programmes before the inauguration of MMC (such as the bioinformatics unit and the technological platforms). However, the roles of some services are gradually being adapted so that they can also cater to the specific needs of MMC. We focus here on the most significant developments for our MMC programme since its inauguration. Tissue Bank and Xenotransplantation Unit The IEO Biobank and Biomolecular Resource Infrastructure (IBBRI) is a prestigious element of our Institution and a necessary step for the realization of the MMC program. It was established to collect, catalogue and store surgically excised tissue samples (non neoplastic and neoplastic) that are non-essential for diagnosis (biological material in excess of that needed

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The IBBRI is tightly connected with the IEO Tumour Registry that collects and compiles all IEO clinical data (see below), with IEO clinical and surgical wards, upon which it relies for sample collection, and with the Department of Pathology and Laboratory Medicine, which provides pathologist expertise for sample classification and dissection. The IBBRI has developed the necessary infrastructure to provide high quality biospecimens required for biomarker discovery and xenotransplantation experiments. This latter technology, which involves the transplantation of human tumours into immunodeficient mice, is vitally important as it constitutes an ex-vivo model system that retains the morphology and biological features of the tumour of origin. Thus, it is essential for testing new combination therapies or new drugs that will be developed in the future through MMC. Tumour Registry The IEO Tumour Registry is an independent data collection unit for IEO, which will play a critical role in the success of the MMC programme. Upon informed consent, the Registry collects all available personal data, clinical data relating to diagnosis and treatment, epidemiological data, and patient clinical course, which it links to the identity of the samples preserved in the tissue bank. Because of the sensitive nature of this information, we have developed safeguards for patient privacy, in close collaboration with the Tumour Registry. Researchers, therefore, do not have direct access to Registry data, which must necessarily be retrieved through Tumour Registry researchers, who filter out any patient identifying information. The expertise and resources of the Tumour Registry, act as guarantee

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that researchers can link clear clinical information to the samples present in the Tissue Bank. This resource is thus a vital part of our MMC programme. As well as contributing to the success of MMC, the Tumour Registry plays an important role in monitoring clinical activity and analysing IEO clinical data. This function, which is currently independent of its supporting role in MMC, is described separately below. Molecular Pathology Laboratory The goal of the Molecular Pathology Laboratory is to identify novel putative cancer targets for drug discovery, diagnostic and/or prognostic applications. For this purpose, we use a high-throughput screening technology (Tissue Microarrays, TMA) in combination with in situ detection methods (ISH and IHC) for the rapid translation of early discoveries into clinical applications. TMA technology allows us to perform gene expression analysis on thousands of patient tissues simultaneously, and is thus a powerful tool for the validation of basic research findings, and to establish their relevance to cancer. We routinely assess the deregulation of target genes, based on the comparative expression analysis between the tumour tissues and their normal counterparts. We have a pipeline between the Pathology Department at IEO and our service, which has become much more regular since the establishment of the IEO Tissue Bank. This pipeline is essential for the replenishment of our TMAs, which are rapidly exhausted through large-scale screening studies. Through the Tissue Bank collaboration with the Tumour Registry, all arrayed tissues are linked to clinico-pathological and follow-up data. We can therefore link the expression patterns of genes of interest to clinical evaluation and outcome, a necessary step for the validation of new diagnostic and prognostic markers. The Molecular Pathology Laboratory has a long history in TMA screening, which guarantees excellence of service. Our service offers screening studies on multi-tumour microarrays, consisting of tissue cores for each of a wide selection of tumours. These arrays contain a series of matched (where possible) and unmatched normal tissue counterparts (eg. [1]). We also perform screening studies on single tumour microarrays, which we have available for i) breast (~1300 samples are currently being arrayed); ii) lung (~800 tumour samples, arrayed in two specific TMAs); iii) colon (a “progression TMA” consisting of ~250 samples of normal tissues, hyperplastic tissues, adenomas and carcinomas); iv) melanomas (a series of three independent TMAs of 150-250 samples, containing benign and dysplastic nevi, primary tumours and metastatic melanomas); v) ovary

(~250 tumour samples of which ~200 matched with a metastatic sample from the same patient, arrayed in two different TMAs). Primary Epithelial and Stem Cell Culture Facility The Primary Epithelial and Stem Cell Culture Facility is one of the enabling infrastructures of the Molecular Medicine for Care Programme at IEO. The Facility has the specific task of deriving bulk primary epithelial cells and stem cells, whenever possible from human biopsy specimens of matched normal and tumour pairs from the same patient. These model-systems recapitulate ex-vivo the physiopathology of naturally occurring human cancers, and they can therefore be used to derive information that can be directly extrapolated to the clinic. Standardized operative procedures (SOP) are currently in place for processing of tissue biopsy specimens, and for the efficient preparation of bulk primary epithelial cell and stem cell cultures from breast, lung, ovary and prostate tissues. We have established a continuous pipeline of samples from the operating theatres to the laboratory through coordination with the IBBRI (Tissue Bank). We have also established a close connection with the IEO Tumour Registry for the collection of clinical-pathological information linked to human samples. During the last year, our Facility has provided material for a number of basic studies aimed at elucidating molecular mechanisms of breast cancer and mammary stem cell biology. In addition, in particular for breast and lung cancer, a number of different studies are being conducted at IEO, based on the use of primary epithelial cell cultures as a ‘pre-clinical’ model to assess the in vitro efficacy of different types of drug treatment. Clinical Biomarkers Laboratory The Clinical Biomarkers Laboratory acts transversally with clinical and research activities and with the different enabling platforms of the program with the aim of optimizing cancer biomarker candidates and facilitating clinical development. The laboratory performs screening, pre-clinical validation, and optimization of biomarker candidates, as well as the design and execution and technological development of cancer diagnostic assays to aid their translation into the clinical setting. In this role, we also support collaborative study programs between biotech companies, researchers and clinicians for the co-development of biomarkers and novel therapies. We have collaborated with the IBBRI (Tissue Bank), to optimize protocols for the extraction of nucleic acids (including total RNA, DNA and microRNAs) from human

tissues, such as blood, plasma, fresh bioptic tissues and paraffin-embedded tissue specimens. We collaborate with the Tissue Bank, the Tumour Registry and the Pathology Department at IEO to select tissue samples for prospective and retrospective studies.

Research Activities

for diagnosis, that would otherwise be discarded) as well as plasma/serum, total blood, DNA and RNA. Within the framework of MMC, we have set up specific protocols and standard operating procedures (SOPs) to ensure optimized, standardized treatment of all samples for research purposes. In collaboration with the Cell Biology Unit (see below), the IBBRI also ensures the storage of purified normal and cancer stem cells and primary cell cultures. Biological samples are collected only if donors have provided informed consent for sample use for research purposes. A new informed consent form for research purposes (including biobanking of tissues and biological material) has been developed specifically for this purpose, thanks to the collaboration of clinicians, researchers, and bioethicists. This form has been approved by the Ethics Committee of IEO.

Our current projects include: 1) the prognostic evaluation of a breast stem cell signature identified in the lab through an extensive meta-analysis of the expression profile of normal stem cells and their progenitors recently generated in house [2] and of publicly available breast cancer data sets; 2) the technological validation, in paraffin-embedded tissue samples, of a 10-gene predictive model of overall survival in early stage NSCLC that was recently identified in the lab [3]; 3) the identification of novel candidate prognostic microRNAs in G2 breast tumours; 4) microRNA profiling in a retrospective cohort of breast cancer tumours with complete follow up to identify novel biomarkers and novel therapeutic targets; 5) the analysis of prognostic microRNAs from plasma of both in NSCLC and breast cancer; 6) the design of a PCR-based assay for a rapid and cost-effective detection method of the ALK translocation in non-small cell lung cancer patients. Several high-throughput technology platforms have been established in the Clinical Biomarkers Laboratory, including the recently released ViiATM7 real-Time PCR system machine with the Micro Fluidic Card Upgrade (Applied Biosystems) and the OpenArray Real-Time PCR System for gene expression, genotyping and digital PCR applications in a mid-density format, which will help streamline genomic screening and validation, absolute quantitation, and biomarker identification and screening at a scale previously unattainable. We have also purchased the EnVision Multilabel Reader (Perkin-Elmer) for cell-based drug discovery and enzyme assays. Recently, we have also established an automated robotic workstation for medium-high-throughput automation of sample preparation procedures, methods and assays. The workstation displays a modular and flexible system configuration, composed of two independent platforms: one dedicated to the set up of the different molecular assays and one to the purification of nucleic acids. Each workstation is configured with multiple pipetting and labware gripping devices. Pipetting channels and labware grippers move independently of each other, supporting the use of a wide range of labware. The autoload option provides barcode tracking of samples, labware, racks and carriers and data can be tracked and processed within the application as well as interfaces to internal and external databases. Thanks to the scalability

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MMC Scientific Programmes Our scientific programmes are currently focused on the two most commonly diagnosed cancers across the globe: breast cancer and lung cancer (specifically, non-small cell lung carcinoma, NSCLC, which makes up some 80% of all lung cancers) [4]. In spite of their high incidence, we know relatively little about the biology of these diseases. However, technological advances of the past decade, and molecular expertise in both breast cancer and NSCLC that our scientists have gained in recent years have allowed us to design research programmes that will, hopefully, dramatically change the way these diseases are managed in the clinic. PROGRAMME 1. Understanding breast cancer to guide therapeutic choice Since the introduction of nationwide screening programmes for early breast cancer by mammography, we have undisputedly witnessed a reduction in the mortality associated with this disease. Furthermore, diagnostic assessment based on pathological parameters (tumour size/grade, lymph node status, sentinel lymph node involvement) has now been improved by the use of biomarkers (e.g., hormone receptor or ERBB2 status) for patient stratification. Notwithstanding these advances - and due to the molecular and clinical heterogeneity of breast cancer - currently available diagnostic and prognostic tools do not have sufficient power to permit fully tailored disease management in individual patients. We have reached a plateau in our ability to cure, which now seems to depend more on how early the disease is detected than on a real understanding of its biology. To make radical improvements in the cure rate, we need to devise new strategies that complement our ability of early detection and improve the range of treatment strategies. We will achieve this with a deeper understanding of breast cancer biology. In recent years we have witnessed the emergence of a new concept in cancer biology, namely that cancer stem cells (CSCs) sit at the heart of the tumorigenic process for many tumours, including breast cancer. CSCs are a small subpopulation of cells within the tumour that possess stem cell-like properties including self-renewal, quiescence, unlimited replication potential and the ability to produce differentiated progeny [5,6]. These cells appear to be the only ones capable of sustaining tumour

306

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growth within the bulk non-tumorigenic population. It is our belief, therefore, that a high-resolution picture of the stem cell compartment will hold the key to the development of new diagnostic, prognostic and patient stratification tools. Our first scientific programme builds on our discovery that ‘breast stem cell’ genes can stratify breast tumours according to their pathological, molecular and clinical characteristics [2]. We have previously identified a broad molecular profile of normal mammary stem cells (SCs) [2]. The large number of genes (more than 1000) means, however, that this stem cell signature has extremely limited clinical applicability. Our aim is to reduce this number to 10-20 candidate genes that retain the predictive features of the stem cell profile, and that can therefore be handled easily in a clinical setting as a diagnostic kit by immunohistochemistry (IHC) or quantitative PCR analysis. We have compared our SC profile to a number of publicly available datasets and have identified 20 candidate genes, the clinical relevance of which is currently under validation by high-throughput tissue microarray analysis of a large number of breast cancer samples (complete with clinical history). This study will allow us to assess the ability of these genes to stratify patients according to tumour aggressiveness, metastatic risk and therapeutic response to hormone and/or chemo/radio-therapy. Our aim is thus to develop this set of genes as a clinical grade parameter that will refine our current diagnostic/ prognostic ability and improve the clinical management of breast cancer. The success of this programme rests, in no small part, on the expertise of the MMC Clinical Biomarkers and Molecular Pathology Laboratories and will rely on resources collected by the Tissue Bank and Tumour Registry. Needless to say, this work would not be possible without a deep level of integration between basic researchers, breast clinicians and pathologists. PROGRAMME 2. Development of a blood test for the early diagnosis of lung cancer Unlike breast cancer, no national screening programme exists for the early diagnosis of NSCLC. Therefore, the early detection of lung cancer is a sporadic (and, for patients, highly fortunate) event. When it occurs, the 5-year survival rate leaps from 15% (for all NSCLCs diagnosed, most of which at a late stage) to 60-80% (for stage I NSCLCs) [7]. With only 20-25% of all NSCLCs being diagnosed early today [8,9], the development of clinical tools that permit the early diagnosis of this disease is clearly a key clinical necessity, particularly for at-risk subjects (smokers or ex-smokers).

Recent studies have demonstrated that low-dose spiral computerized tomography (LD-CT) can efficiently identify small (less than 5 mm diameter) nodules and asymptomatic lung cancers, most of which at stage I [10-13]. However, this technique suffers from a number of disadvantages: firstly, it cannot distinguish between benign nodules and frank tumours; secondly, it requires sophisticated and costly instrumentation that is not necessarily available in all clinical structures, and, finally, it requires the presence of specialized personnel trained in the execution of the clinical exam and in the subsequent evaluation of test results.

with the large-scale prospective screening study we have designed. This programme is possible thanks to the close ties that have been constructed between researchers and lung clinicians, as a result of the MMC programme.

It is therefore apparent, that there is a need to identify new strategies for the early diagnosis of lung cancer that can more easily be applied on a nationwide scale, but that have the same diagnostic capability as LD-CT. As part of our basic research programme, we identified a circulating miRNA signature (based on 34 miRNA species) that can be used in a blood test to detect early stage lung cancer in asymptomatic patients [14]. The stability of miRNAs in biological fluids, including human serum, makes them ideal targets for detection in a diagnostic blood test [15-17]. This test can detect early lung cancer with the same frequency as LD-CT, and presents an additional number of advantages: in particular, blood sample collection is inexpensive and requires no specialized instruments or personnel, and the actual blood test can easily be performed in any pathology laboratory. Furthermore, unlike LD-CT, our blood test is capable of distinguishing benign nodules from tumours. We have developed our test using a small number (< 100) of patient samples. To prove the clinical applicability of our test, we now need to validate it in a large-scale prospective trial (which will involve the recruitment of some 20,000 patients). This is an essential step necessary for the transfer of our results to the clinic: our goal is to integrate molecular and radiological methods in a sequential protocol to be used in lung cancer early detection programmes.

PROGRAMME 3. Understanding breast cancer for early diagnosis On the strength of our discovery of a diagnostic blood miRNA profile for lung cancer, we investigated whether an equivalent miRNA profile might not exist for breast cancer. Our preliminary results suggest that this is the case, but we have not yet identified a precise profile. Our third scientific programme is designed to achieve this aim. We will analyse circulating breast cancer-specific miRNA profiles that are diagnostic for breast cancer. In addition to this, we will also analyse the miRNA profile of breast stem cells. The rationale for doing this lies in the fact that our work has demonstrated that clinically more aggressive breast cancers tend to have a larger number of stem cells than less aggressive tumours [2]. We will investigate, therefore, whether it is possible to identify a ‘prognostic’ circulating miRNA profile (based on the specific detection of a stem cell miRNA signature in the sera of patients with poor prognosis tumours, but not in the sera of those with better prognosis tumours), as well as a ‘diagnostic’ circulating miRNA profile (based on the specific detection of a breast cancer miRNA signature in the sera of breast cancer patients, but not in healthy subjects). We have set up the necessary collaborations with the breast surgery, and breast screening departments at IEO, and have obtained the necessary ethical clearance from the Institutional Review Board. We are also working closely with our colleagues from the Tissue Bank who are involved in explaining the informed consent details to patients who require further clarifications, before accepting to participate (or deciding not to participate) in this study. We are therefore ready to initiate this new research programme in 2012.

For our large/scale trial, we have set up a semiautomatized platform for the large-scale analysis of miRNA, which is able to process up to 12 samples per day, and we will soon install a completely automatized platform, capable of running 48 samples per day. More than 200 serum samples have already been screened to set the test parameters, and define the accuracy, sensitivity (proportion of correct positive diagnoses), and specificity (proportion of correct negative diagnoses) of the test in a clinical setting. Once the baseline parameters are established, we will proceed

Cited References 1. Luise C, Capra M, Donzelli M, et al. An atlas of altered expression of deubiquitinating enzymes in human cancer. PLoS One 2011; 6: e15891. 2. Pece S, Tosoni D, Confalonieri S, et al. Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 2010; 140: 62-73. 3. Bianchi F, Nuciforo P, Vecchi M, et al. Survival prediction of stage I lung adenocarcinomas by expression of 10 genes. J Clin Invest 2007; 117: 3436-3444.

IEO — Scientific Report 2011 — Ongoing research 2012

Research Activities

of these instruments, the widest possible range of throughputs can be accommodated: additional pipetting channels, a 96 or 384-probe head or an integrated robotic arm can be fitted to existing configurations.

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4. Pass HI, Mitchell JB, Johnson DH, et al. Lung cancer, principles and practice. (3rd edition ed). Lippincott Williams and Wilkins, 2005. 5. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008; 8: 755-768. 6. Zhou J, Zhang Y. Cancer stem cells: Models, mechanisms and implications for improved treatment. Cell Cycle 2008; 7: 1360-1370. 7. Jemal A, Thun MJ, Ries LA, et al. Annual report to the nation on the status of cancer, 1975-2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 2008; 100: 1672-1694. 8. Greenlee RT, Murray T, Bolden S, et al. Cancer statistics, 2000. CA Cancer J Clin 2000; 50: 7-33. 9. Porter JC, Spiro SG. Detection of early lung cancer. Thorax 2000; 55 Suppl 1: S56-62. 10. Henschke CI, Yankelevitz DF, Libby DM, et al. Survival of patients with stage I lung cancer detected on CT screening. N Engl J Med 2006; 355: 1763-1771. 11. Pastorino U, Bellomi M, Landoni C, et al. Early lungcancer detection with spiral CT and positron emission tomography in heavy smokers: 2-year results. Lancet 2003; 362: 593-597. 12. Swensen SJ, Jett JR, Hartman TE, et al. Lung cancer screening with CT: Mayo Clinic experience. Radiology 2003; 226: 756-761.

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Publications, Clinical Trials, Ongoing Grants, Seminars and IEO Foundation

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Publications

Full Papers 2011

AUTHORS

TITLE

JOURNAL

VOLUME

PAGE

I.F.

ABRAHAMS J. P., APWEILER R., BALLING R., BERTERO M. G., BUJNICKI J. M., CHAYEN N. E., CHENE P., CORTHALS G. L., DYLAG T., FORSTER F., HECK A. J. R., HENDERSON P. J. F., HERWIG R., JEHENSON P., KOKALJ S. K., LAUE E., LEGRAIN P., MARTENS L., MIGLIORINI C., MUSACCHIO A., PODOBNIK M., SCHERTLER G. F. X., SCHREIBER G., SIXMA T. K., SMIT A. B., STUART D., SVERGUN D., TAUSSIG M.

4D Biology for health and disease workshop report.

NEW BIOTECHNOL

28

291 - 293

1,843

AEBI S., SUN Z., BRAUN D., PRICE K. N., CASTIGLIONE-GERTSCH M., RABAGLIO M., GELBER R. D., CRIVELLARI D., LINDTNER J., SNYDER R., KARLSSON P., SIMONCINI E., GUSTERSON B. A., VIALE G., REGAN M. M., COATES A. S., GOLDHIRSCH A.

Differential efficacy of three cycles of CMF followed by tamoxifen in patients with ER-positive and ER-negative tumors: Long-term follow up on IBCSG Trial IX.

ANN ONCOL

22

1981 1987

6,452

AGLIANO A., MARTIN PADURA I., MARIGHETTI P., GREGATO G., CALLERI A., PRIOR C., REDRADO M., CALVO A., BERTOLINI F.

Therapeutic effect of Lenalidomide in a novel xenograft mouse model of human Blastic NK cell lymphoma/blastic plasmacytoid dendric cell neoplasm.

CLIN CANCER RES

17

6163 6173

7,338

ALEXANDER J., LIM D., JOUGHIN B. A., HEGEMANN B., HUTCHINS J. R., EHRENBERGER T., IVINS F., SESSA F., HUDECZ O., NIGG E. A., FRY A. M., MUSACCHIO A., STUKENBERG P. T., MECHTLER K., PETERS J. M., SMERDON S. J., YAFFE M. B.

Spatial Exclusivity Combined with Positive and Negative Selection of Phosphorylation Motifs Is the Basis for Context-Dependent Mitotic Signaling

SCI SIGNAL

4

RA42

6,12

ALTERIO D., JERECZEK B. A., GRISERI M., D'ONOFRIO A., GIUGLIANO G., FIORE M. R., VITOLO V., FOSSATI P., PIPERNO G., CALABRESE L., VERRI E., CHIESA F. G., ORECCHIA R.

Three-dimensional conformal postoperative radiotherapy in patients with parotid tumors: 10 years' experience at the European Institute of Oncology

TUMORI

97

328 - 334

1,014

AMSON R., PECE S., LESPAGNOL A., VYAS R., MAZZAROL G., TOSONI D., COLALUCA I. N., VIALE G., RODRIGUES-FERREIRA S., WYNENDAELE J., CHALOIN O., HOEBEKE J., MARINE J. C., DI FIORE P. P., TELERMAN A.

Reciprocal repression between P53 and TCTP

NAT MED

18

91 - 99

25,43

ANDREONI B., CAMELLINI L., SONZOGNI A. M., CROSTA C., PIROLA M. E, CORBELLINI C.

Multicentric GISCoR Study "Intensive clinical follow-up versus surgical radicalization after complete endoscopic polypectomy of a malignant adenoma" (SEC-GISCoR)

UPDATES SURG

63

171 - 177

0

IEO — Scientific Report 2011 — Ongoing research 2012

311

312

Common alleles at 6q25.1 and 1p11.2 are associated with breast cancer risk for BRCA1 and BRCA2 mutation carriers.

ARCAINI L., LASZLO' D., RIZZI S., BALZAROTTI M., ANTONIAZZI F., ZILIOLI V.R., GUGGIARI E., FARINA L., TODISCO E., BONFICHI M., MIQUELEIZ ALAMOS S., ROSSI G., MARTINELLI G., MORRA E.

Plerixafor and G-CSF for PBSC mobilization in patients with lymphoma who failed previous attempts with G-CSF and chemotherapy: A REL (Rete Ematologica Lombarda) experience.

LEUKEMIA RES

35

712 - 714

2,555

ARGENZIO E., BANGE T., OLDRINI B., BIANCHI F., PEESARI R., MARI S., DI FIORE P. P., MANN M., POLO S.

Proteomic snapshot of the EGF-induced ubiquitin network.

MOL SYST BIOL

7

462

9,667

ARISTEI C., LEONARDI M. C., STRACCI F., PALUMBO I., LUINI A., VIALE G., CRISTALLINI E. G., CAVALIERE A., ORECCHIA R.

Risk factors for relapse after conservative treatment in T1-T2 breast cancer with one to three positive axillary nodes: results of an observational study

ANN ONCOL

22

842 - 847

6,452

ARUMUGAM M., RAES J., PELLETIER E., LE PASLIER D., YAMADA T., MENDE D. R., FERNANDES G. R., TAP J., BRULS T., BATTO J. M., BERTALAN M., BORRUEL N., CASELLAS F., FERNANDEZ L., GAUTIER L., HANSEN T., HATTORI M., HAYASHI T., KLEEREBEZEM M., KUROKAWA K., LECLERC M., LEVENEZ F., MANICHANH C., NIELSEN H. B., NIELSEN T., PONS N., POULAIN J., QIN J., SICHERITZ-PONTEN T., TIMS S., TORRENTS D., UGARTE E., ZOETENDAL E. G., WANG J., GUARNER F., PEDERSEN O., DE VOS W. M., BRUNAK S., DORE' J., METAHIT CONSORTIUM, RESCIGNO M., WEISSENBACH J., EHRLICH D., BORK P.

Enterotypes of the human gut microbiome

NATURE

473

174 - 180

36,101

IEO — Scientific Report 2011 — Ongoing research 2012

HUM MOL GENET

20

3304 3321

8,058

AURILIO G., MACARULLA T., RAMOS J. F, FAZIO N., NOLE' F., IGLESIAS C.

Successful treatment with GEMOX in patient with metastatic pancreatic adenosquamous carcinoma.

TUMORI

97

239 - 242

1,014

AZIM H. A. JR, PECCATORI F. A.

Managing cancer during pregnancy: what evidence do we have?

POLSKIE ARCHIWUM MEDYCYNY WEWNETRZNEJ

121

29 - 33

0

AZIM H. A. JR, SANTORO L., PAVLIDIS N., GELBER S., KROMAN N., AZIM H., PECCATORI F. A.

Safety of pregnancy following breast cancer diagnosis: A meta-analysis of 14 studies

EUR J CANCER

47

74 - 83

4,944

AZIM H. A. JR., PECCATORI F. A.

Leczenie nowotworow zlosliwych u kobiet w ciazy: na jakich danych naukowych mozna sie oprzec?

OKIEM EKSPERTA

2

49 - 55

0

AZIM H. A. JR., PECCATORI F. A., DE AZAMBUJA E., PICCART M. J.

Motherhood after breast cancer: searching for la dolce vita

EXPERT REV ANTICANC

11

287 - 298

2,976

AZIM H.A. JR., DEL MASTRO L., SCARFONE G., PECCATORI F. A.

Treatment of breast cancer during pregnancy: Regimen selection, pregnancy monitoring and more ...

BREAST

20

01-06'

2,089

AZIM JR H.A., GENTILINI O. D., LOCATELLI M. A., CIRIELLO E., PECCATORI F. A.

Managing pregnant women with cancer: personal considerations and a review of the literature

ECANCERMEDICALSCIENCE

5

204

0

BAGNARDI V., ROTA M., BOTTERI E., SCOTTI L., JENAB M., BELLOCCO R., TRAMACERE I., PELUCCHI C., NEGRI E., LA VECCHIA C., CORRAO G., BOFFETTA P.

Alcohol consumption and lung cancer risk in never smokers: a meta-analysis.

ANN ONCOL

22

2631 2639

6,452

BAGNARDI V., SORINI E., DISALVATORE D., ASSI V., CORRAO G., DE STEFANI R., COLLABORATIVE 'ALCOHOL, LESS IS BETTER' GROUP

'Alcohol, less is better' project: outcomes of an Italian community-based prevention programme on reducing per-capita alcohol consumption.

ADDICTION

106

102 - 110

4,145

BALZANO D., SANTAGUIDA S., MUSACCHIO A., VILLA F.

A general framework for inhibitor resistance in protein kinases.

CHEM BIOL

18

966 - 975

5,838

BAROZZI I. G., TERMANINI A., MINUCCI S., NATOLI G.

Fish the ChIPs: a pipeline for automated genomic annotation of ChIP-Seq data.

BIOL DIRECT

6

51

3,737

BECKFORD VERA D. R., EIGNER S., BERAN M., HENKE K. E, LAZNICKOVA A., LAZNICEK M., MELICHAR F., CHINOL M.

Preclinical Evaluation of 177Lu-Nimotuzumab: A Potential Tool for Radioimmunotherapy of Epidermal Growth Factor Receptor-Overexpressing Tumors

CANCER BIOTHER RADIO

26

287 - 297

1,873

BEESLEY J., JOHNATTY S. E., CHEN X., SPURDLE A. B., PETERLONGO P., BARILE M., PENSOTTI V., MANOUKIAN S., RADICE P., AUSTRALIAN OVARIAN CANCER STUDY GROUP, KATHLEEN CUNINGHAM CONSORTIUM FOR RESEARCH IN FAMILIAL BREAST CANCER, CHENEVIX-TRENCH G.

No evidence for an association between the earwax-associated polymorphism in ABCC11 and breast cancer risk in Caucasian women.

BREAST CANCER RES TR

126

235 - 239

4,859

BELLONI E. C., SHING D. C., TAPINASSI C., VIALE A., MANCUSO P., MALAZZI O., GERBINO E., DALL'OLIO V., EGURBIDE I., ODERO M. D., BERTOLINI F., PELICCI P. G.

In vivo expression of an aberrant MYBGATA1 fusion induces leukemia in the precence of GATA1 reduced levels.

LEUKEMIA

25

733 - 736

8,966

BELLONI E. C., VERONESI G., MICUCCI C., JAVAN S., MINARDI S. P., VENTURINI S., MAISONNEUVE P., VOLORIO S., RIBONI M., BELLOMI M., SCANAGATTA P., TALIENTO G., PELOSI G., PECE S., SPAGGIARI L., PELICCI P. G.

Genomic characterization of asymptomatic CT-detected lung cancers

ONCOGENE

30

1117 - 1126

7,414

IEO — Scientific Report 2011 — Ongoing research 2012

Publications

ANTONIOU A. C., KARTSONAKI C., SINILNILOVA O. M., SOUCY P., MCGUFFOG L., HEALEY S., LEE A., PETERLONGO P., MANOUKIAN S., PEISSEL B., ZAFFARONI D., CATTANEO E., BARILE M., PENSOTTI V., PASINI B., DOLCETTI R., GIANNINI G., PUTIGNANO A. L., VARESCO L., RADICE P., MAI P. L, GREENE M. H., ANDRULIS I. L., GLENDON G., OZCELIK H., THOMASSEN M., GERDES A. M, KRUSE T. A., JENSEN U. B., CRUGER D. G., CALIGO M. A., LAITMAN Y., MILGROM R., KAUFMAN B., PALUCH-SHIMON S., FRIEDMAN E., LOMAN N., HARBST K., LINDBLOM A., ARVER B., EHRENCRONA H., MELIN B., SWE-BRCA., NATHANSON K. L., DOMCHEK S. M., REBBECK T., JAKUBOWSKA A., LUBINSKI J., GRONWALD J., HUZARSKI T., BYRSKI T., CYBULSKI C., GORSKI B., OSORIO A., RAMON Y CAJAL T., FOSTIRA F., ANDRES R., BENITEZ J., HAMANN U., HOGERVORST F. B., ROOKUS M. A., HOONING M. J., NELEN M. R., VAN DER LUIJT R. B., VAN OS T. A. M., VAN ASPEREN C. J., DEVILEE P., MEIJERS-HEIJBOER E. J., GOMEZ GARCIA E. B., HEBON, PEOCK S., COOK M., FROST D., PLATTE R., LEYLAND J., EVANS D. G., LALLOO F., EELES R., IZATT L., ADLARD J., DAVIDSON R., ECCLES D., ONG K. R., COOK J., DOUGLAS F., PATERSON J., KENNEDY M. J, MIEDZYBRODZKA Z., EMBRACE, GODWIN A., STOPPA LYONNET D., BUECHER B., BELOTTI M., TIRAPO C., MAZOYER S., BARJHOUX L., LASSET C., LEROUX D., FAIVRE L., BRONNER M., PRIEUR F., NOGUES C., ROULEAU E., PUJOL P., COUPIER I., FRENAY M., CEMO STUDY COLLABORATORS, HOPPER J. L., DALY M. B., TERRY M. B., JOHN E. M., BUYS S. S., YASSIN Y., MIRON A., GOLDGAR D., BREAST CANCER FAMILY REGISTRY, SINGER C. F., TEA M. K, PFEILER G., DRESSLER A. C., HANSEN T. V. O., JONSON L., EJLERTSEN B., BARKARDOTTIR R. B., KIRCHHOFF T., OFFIT K., PIEDMONTE M., RODRIGUEZ G., SMALL L., BOGGESS J., BLANK S., BASIL J., AZODI M., EWART TOLAND A., MONTAGNA M., TOGNAZZO S.

313

International expert consensus on primary systemic therapy in the management of early breast cancer: highlights of the Fourth Symposium on Primary Systemic Therapy in the Management of Operable Breast Cancer, Cremona, Italy (2010).

J NATL CANCER INST MONOGR

BERTANI E., CHIAPPA A., BIFFI R., BIANCHI P. P., RADICE D., BRANCHI V., CENDERELLI E., VETRANO I., CENCIARELLI S., ANDREONI B.

Assessing appropriateness for elective colorectal cancer surgery: clinical, oncological, and quality-of-life short-term outcomes employing different treatment approaches.

INT J COLORECTAL DIS

Comparison of oral polyethylene glycol plus a large volume glycerine enema with a large volume glycerine enema alone in patients undergoing colorectal surgery for malignancy: a randomised clinical trial

COLORECTAL DIS

Anti-angiogenesis in cancer, met and unmet goals. An interview with Robert Kerbel.

INT J DEV BIOL

BERTOLINI F.

Response to anti-angiogenesis: An ever changing feature.

BREAST

20 S3

s61 - s62

2,089

BERTOLINI F., MANCUSO P., SHAKED Y.

Circulating endothelial cells as biomarkers for patients receiving bevacizumab

LANCET ONCOL

12

217 - 218

17,764

BERTOLINI F., MARIGHETTI P., MARTIN PADURA I., MANCUSO P., HU-LOWE D. D., SHAKED Y., D'ONOFRIO A.

Anti-VEGF and beyond: shaping a new generation of anti-angiogenic therapies for cancer

DRUG DISCOV TODAY

16

1052 1060

6,422

BERTUCCIO P., LA VECCHIA C, SILVERMAN D. T., PETERSEN G. M., BRACCI P. M., NEGRI E, LI D., RISCH H. A., OLSON S. H., GALLINGER S., MILLER A. B., BUENO-DE-MESQUITA H. B., TALAMINI R., POLESEL J., GHADIRIAN P., BAGHURST P. A., ZATONSKI W., FONTHAM E. T., BAMLET W. R., HOLLY E. A., LUCENTEFORTE E., HASSAN M., YU H., KURTZ R. C., COTTERCHIO M., SU J., MAISONNEUVE P., DUELL E. J., BOSETTI C., BOFFETTA P.

Cigar and pipe smoking, smokeless tobacco use and pancreatic cancer: an analysis from the International Pancreatic Cancer Case-Control Consortium (PanC4).

ANN ONCOL

1420 1426

6,452

BERTUCCIO P., LA VECCHIA C., SILVERMAN D. T., PETERSEN G. M., BRACCI P. M., NEGRI E., LI D., RISCH H. A., OLSON S. H., GALLINGER S., MILLER A. B., BUENO DE MESQUITA H. B., TALAMINI R., POLESEL J., GHADIRIAN P., BAGHURST P. A., ZATONSKI W., FONTHAM E. T., BAMLET W. R., HOLLY E. A., LUCENTEFORTE E., HASSAN M., YU H., KURTZ R. C., COTTERCHIO M., SU J., MAISONNEUVE P., DUELL E. J., BOSETTI C., BOFFETTA P.

Reply to Are cohort data on smokeless tobacco use and pancreatic cancer confounded by alcohol use?

BIANCHI F., NICASSIO F., MARZI M. J., BELLONI E. C., DALL'OLIO V., BERNARD L., PELOSI G., MAISONNEUVE P., VERONESI G., DI FIORE P. P.

A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer

EMBO MOL MED

3

495 - 503

8,833

BIANCHI P. P.

Reply to: doi: 10.1007/s00464-011-18089: Robotic versus laparoscopic total mesorectal excision for rectal cancer: comparative analysis of oncologic safety and short-term outcomes.

SURG ENDOSC

25

3957 3958

3,436

BIANCHI P. P., PETZ W. L., CASALI L.

Laparoscopic lymphatic roadmapping with blue dye and radioisotope in colon cancer.

COLORECTAL DIS

13 s7

67 - 69

2,728

BERTANI E., CHIAPPA A., BIFFI R., BIANCHI P. P., RADICE D., BRANCHI V., SPAMPATTI S. G., VETRANO I., ANDREONI B.

BERTOLINI F.

314

IEO — Scientific Report 2011 — Ongoing research 2012

ANN ONCOL

43

26

13

55