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Chairman’s message

The improvement in the financial result of the IEO, although still negative, in 2012 – a year of profound crisis for the health/hospital service nationally - is far more than an entry on the balance sheet. It is proof that the clinical and research organisational model of our Institute has been built on solid grounds, able to face even the worst economic situation. This financial improvement was brought about – among other things – jointly with an increase in productivity and in the quality of clinical activity and research. The impact factor, an index of the innovative capacity and worldwide prestige of our research, last year reached its highest level since the Institute opened. Clinical activity - from inpatient and outpatient admissions, right through to radiotherapy and day hospital care – has shown a trend of constant growth, confirming that the quality received by our patients continues to improve. These results are all the more remarkable when seen within the national context of a spending review and an international climate of crisis and of deep uncertainty regarding the availability of resources to overcome it. The main reason for such success resides in the fact the Institute has never ceased to invest in quality: of people – doctors, researchers, managers and personnel as a whole; of technology – diagnosis, treatment and research, of procedures, and of organisational tools.

Carlo BUORA Chairman

Quality and innovation have led to a rationalisation of procedures, with the consequent containment/reduction of costs, from which the IEO model emerges with increased consistency and efficiency. I would particularly like to extend my thanks to our shareholders, who have continued to believe in and support the IEO model, and to all our staff, who have fully appreciated these current difficulties and have also shared in the necessary sacrifices.


Contents

Colophon Title Publisher Number Month/Year Editor

IEO Scientific Report 2012. Ongoing Research 2013. IEO European Institute of Oncology©, Milan (Italy) 19 May 2013 Pier Giuseppe Pelicci

Secretary of editing Graphic Layout Photographer Typographer Paper

Roberta Carbone Andrea Rovatti Roberto Benzi, Marta Rovatti Studihrad Italgrafica Srl, Printed in Italy Printed on the uncoated paper On Offset whose elementary chlorine free pulps came from susteinaible forests

Chief Executive Officer’s Message Scientific Director’s Report • Ethics Committee • Data Management • Unit of International Cooperation in Clinical Trials • ecancermedicalscience • Library The Directive Board CEO’s Office • Communication, Marketing and Customer Service • Finance and Administration • Human Resources • Information & Communication Technology • Medical Office • Hospital Activities • TTFactor • Centers of Excellence

11 13 18 19 20 22 23 25 26 27 28 29 30 32 36 38

Clinical Research

Disease-oriented research Division of Breast Cancer Surgery Division of Plastic and Reconstructive Surgery Medical Division of Breast Tumors Research Activities Division of Thoracic Cancer Surgery Division of Head and Neck Surgery Medical Oncology Division of the Respiratory System Research Activities

42 46 48 50 52 54 56 60

Division of General and Laparoscopic Surgery 66 Division of Abdomino-pelvic Surgery 70

We thank all the authors for their contribution to this report

Division of Endoscopy Medical Division of Gastrointestinal Tumors Research Activities

74 76 78

Division of Urologic Cancer Surgery 82 Medical Division of Urogenital and Head & Neck 84 Research Activities 88 Division of Gynecologic Cancer Surgery Medical Division of Gynecologic Tumors Research Activities

90 92 94

Surgical Division of Melanoma Medical Division of Melanoma Research Activities

100 102 104

Division of Clinical Haemato-Oncology Interdisciplinary research Division of Pathology Division of Laboratory Medicine Division of Laboratory Haematology-Oncology Research Activities

106

Division of Radiotherapy Division of Radiology Division of Breast Imaging Division of Nuclear Medicine Medical Physics Research Activities

120 124 128 132 134 136

Division of Early Drug Development for Innovative Therapies Division of Cancer Prevention and Genetics Applied Reseach Unit for Cognitive and Psychological Science

110 114 116 118

140 146 150


Board of Directors

International Scientific Advisory Board

Carlo BUORA Carlo CIANI Gian Luca SANTI 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 Fabio DAL BONI Paolo Grandi Pietro Scott IOVANE Edoardo LOMBARDI Aldo MAUGERI Piero Melazzini Enrico MIGLIAVACCA Vittorio Ogliengo Giampiero Pesenti Umberto Quadrino Giancarlo Scotti Piero Sierra

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

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

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

Umberto VERONESI

Milan

As from April 2013

Clinical Resources Division of Anaesthesiology and Intensive Care 154 Day Surgery Division 156 Research Activities 158 Division of Cardiology Day Hospital Division

Translational Research

Drug Discovery Program (DDP) The Molecular Medicine Program SmartFood: program in Nutrition Science & Communication Genomic Program Division of Epidemiology and Biostatistics Tumor Registry

Basic Research

160 166 170 174 178 180 182 184

Department of Experimental Oncology 190 Functional Genomics 194 Oncogenes, Chromatin and Cell Cycle Control 196 Quantitative Proteomics to investigate transcriptional and post-transcriptional mechanisms that regulate gene expression 198 Biomedical Humanities 202 Viral Control of Cellular Pathways and Biology of Tumorigenesis 206 Bioinformatics and Evolutionary Genomics of Cancer 208 Studying the regulation of chromosome segregation at centromeres, kinetochores and rDNA 210 Molecular Carcinogenesis and Stem Cell Biology Research 212 Systems Biomedicine 216

Cellular and Molecular Pathways Regulating Melanoma Genesis and Progression 218 Structural and Functional Studies of the Mitotic Spindle Orientation during Asymmetric Cell Divisions 220 Chromatin Alterations in Tumorigenesis 224 Transcriptional Control in Inflammation and Cancer 226 Epigenetic mechanisms in stem cell differentiation and oncogenesis 230 Biology and Signal Transduction of Normal and Cancer Neural Stem Cells 232 Molecular mechanisms of cancer and aging 236 Immunobiology of Dendritic Cells and Immunotherapy 240 Histone Methylation Dynamics in Stem cell Renewal and Lineage Commitment 242 Mechanisms Controlling Chromosome Segregation 246

Educational Programs

SEMM - European School of Molecular Medicine 252 IEO Education 254 Seminars 2012 256

Publications, Clinical Trials, Ongoing Grants, and IEO Foundation Full Papers 2012 Clinical Trials in progress during 2012 Ongoing Grants, Research Agreements and Fellowships - 2012 & 2013 IEO Foundation Acknowledgments

263 292 304 310 313

Auditors Maurizio Bozzato - Renato Pagliaro - Graziano Visentin Secretary Mario CESANA Ex officio Umberto Veronesi - Oliviero Rinaldi


Chief Executive Officer’s Message In 2012 the IEO demonstrated how a period of global crisis may provide the opportunity to improve efficiency and foster innovation capacity. Clear objectives, sound principles and people of merit are called for in order to bring this to fruition. IEO satisfies all three of these characteristics, and it for this very reason it has kept a steady hand on the helm, not without difficulty, navigating through a stormy year in which the financial and economic crisis that has beset the western world has also become a political and institutional crisis in Italy. The consequent health spending review has penalised all hospitals, creating a climate of uncertainty, first and foremost for patients and their families, and additionally for doctors, the health sector and all who work therein. We too have had to reduce costs and make sacrifices. However at the same time we have set ourselves the task of ensuring that no sacrifices whatsoever will be made to our level of excellence, and this has resulted in improvements, both in terms of economic progress and in terms of our clinical and research results. This exceptional feat has been made possible thanks to two factors: ideas and their dissemination. In twelve months we have redefined the clinical facilities, we have reviewed all processes and procedures, striving for optimal efficiency, and we have rationalised the organisation of the hospital.

Mauro MELIS Chief Executive Officer

As a result of this, and going completely against trends, we have implemented all of our strategic development plans. We have expanded our Center for Advanced Radiotherapy with new state-of-the-art technologies that enable more rapid and effective treatments, bringing about an enormous benefit in terms of quality of life for our patients and in terms of healthcare organisation. We have also pursued the process of internationalisation of the Institute, forging agreements with other hospital authorities, including those in the emerging countries, and attesting to our European leadership in cutting-edge research. In the last four years, the IEO has made significant and ongoing investments in genome research and has been amongst the first in Italy and in Europe to invest in the necessary technologies for genome sequencing. Our objective is, within a few years, to sequence the DNA of the tumors of all of our patients – a veritable revolution in the diagnosis and treatment of cancer. The IEO is ready, and possesses all the facilities and expertise to be a driving force and international benchmark center. I would like to thank the personnel of the IEO for all that we achieved in 2012 and for all that we will achieve in the future.

IEO — Scientific Report 2012 — Ongoing Research 2013

11


Scientific Director’s Office

Scientific Director’s Report Umberto VERONESI, MD Scientific Director

Scientific Co-Director: Pier Giuseppe Pelicci, MD, PhD Deputy Scientific Directors: Fausto Chiesa, MD, Aaron Goldhirsch, MD, Roberto Orecchia, MD and Giuseppe Viale, MD Executive Advisor: Stefano Zurrida, MD Medical Advisor for scientific communication: Giovanna Gatti, MD Strategic Planning: J. Gordon McVie, MD, DSc Applied research Unit for Cognitive and Psyconcological Science: Gabriella Pravettoni (Head) Scientific Secretariat: Lucia Racca (Head) Executive Assistants: Eva Bruschini and Anita Larossa Librarian: William Russell-Edu Press Office: Donata Francese Ecancermedicalscience Office: Linda Cairns Grants Office: Ilaria Foti (Head), Lucia Sorrenti, Daniele Calasso and Elena Ottina Clinical Trials Office: Atanasio Nonis (Head), Daniela Tamagni The Iberian-Latin America Office: Gabriel Farante (Coordinator) Data Management Office: Giulia Peruzzotti (Head)

12

IEO — Scientific Report 2012 — Ongoing Research 2013

The Scientific Directorate is responsible for strategic choices for the future. The foundations of our strategies are: the centrality of the patient, the main importance of prevention, quick transfer of research results from laboratory to clinical research, increasingly earlier diagnoses, and increasingly conservative therapies with a focus on the quality of life. We hold to ten principal values for our patients and these are: the right to prompt treatment, the right to a second opinion, the right to privacy, the right to know the truth, the right to be informed about treatment, the right to refuse a proposed treatment, the right to the living will, the right not to suffer, and the right to the respect of personal dignity. From this perspective, during 2013, in agreement with the University of Milan, the Applied research Unit for Cognitive and Psyconcological Science directed by Gabriella Pravettoni was created within the Scientific Directorate. The fundamental concept of this unit consists of multiple research disciplines, including psychology, philosophy, neuroscience, linguistics, and anthropology. It therefore embraces many levels of analysis. This year our scientific activity has yielded many excellent results. We have produced 426 scientific papers with a total impact factor of 2129,47 and an average impact factor of 5,94. Three hundred and fifty-eight of these papers were published in scientific journals with an impact factor, whilst forty-two were published in non-impact factor journals. In addition, twenty-six documents fell into the category of books, chapters, conference papers or other items. 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

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

386 217 451 154 400

Total

1608

daily life at the Campus and consist of: DNA Services, Microarrays, Model Organisms, Molecular Pathology Unit, Mass Spectrometry Unit, Protein Chemistry Unit, 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. We expect this new initiative to facilitate the rapid translation of the recent developments of Genomic Sciences into novel approaches to cancer prevention, diagnosis and treatment. The major objectives of the Molecular Medicine Programs are the potentiating of the Tumor Bank and Tumor Registry, the promotion of early clinical trials for the rapid screening of new drugs or treatment modalities, and patient stratification for treatment through the use of genomic screens. This new Program stems from our front line activities in cancer prevention and early diagnosis (for example our programs on cervical, breast and lung cancer) and from novel treatment modalities (targeted peptide- or antibodyguided radionuclide therapy), and will further expand toward molecular imaging. In addition to its 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

IEO — Scientific Report 2012 — Ongoing Research 2013

13


Scientific Director’s Office 3. Integrating complex circuitries in higher order cellular programs or at the organism level. Several lines of investigation are being pursued here. First and foremost, we are analyzing the impact of stem cells on cancer phenotypes. Efforts are concentrated on leukemia, 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 carcinogenesis, by engineering in model systems mutations that mimic those naturally occurring in human cancers (leukemia, breast cancer, ovary cancer and melanomas).

1. Understanding the complex systems controlling cell proliferation. Two major lines of research are being developed aimed at the understanding of exogenously-originated 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.

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 enrols over 150 PhD students from around the world. The laboratories of the Department of Experimental Oncology in the new site, adjacent to the IFOM research institute (the FIRC Institute for Molecular Oncology), represent one of the largest European centers 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 rapid translation of emerging scientific knowledge into new strategies for the prevention and treatment of tumors. In 2009 we launched a program of Drug Discovery. Traditionally, basic research into diseases and diseasemechanisms has been conducted in academic institutions, while its application to drug discovery has been the responsibility of the pharmaceutical industry.

14

IEO — Scientific Report 2012 — Ongoing Research 2013

In recent years, a strong need has emerged for molecularly-targeted drugs that are based on knowledge of disease mechanisms. 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. 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 behaviors, 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:

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 centered on first-class clinical resources and supported by high throughput 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.

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 the best of 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. From 2012 to the present the number of patients enrolled in clinical trials was 3466, and we have now 37203

Publications 2000 - 2012

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

Full Papers Journals with Impact Factor

156

187

167

255

234

245

245

272

234

285

276

312

358

Total Impact Factor (*) Average Impact Factor

801,62 5,14

782,82 4,19

877,81 5,26

1.438,12 1.273,42 5,64 5,44

1.651,96 6,74

1.441,55 5,88

1.874,70 1.716,79 6,89 7,33

1.693,28 1.849,55 2.040,59 2129,47 5,94 6,70 6,54 5,94

Full Papers 9 Journals without Impact Factor

13

2

17

16

15

26

39

48

49

52

24

42

Total Full Papers

165

200

169

272

250

260

271

311

282

334

328

336

400

Books, chapters, and others

72

43

34

40

33

37

39

25

38

31

44

18

26

Total publications

237

243

203

312

283

297

310

336

320

365

372

354

426

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

IEO — Scientific Report 2012 — Ongoing Research 2013

15


2500 2.042

2000

1.876 1.651 1.438

1500 1000

722

718

640

802

783

1.715 1.696

2.129

2000

1.852

1.450

1500

1.273

1000

878

469

500 98

0

94

500

181

95

96

97

98

99

00

01

02

03

04

05

06

07

08

09

10

11

12

0

Impact Factor 1994-2012 500

500 426

400 311

300

280 244

102

100 0

133

36

49

94

95

103

96

All Papers

patients in follow- up. Many 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 considerable. 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 have continued to make up a relevant part of the teaching staff of the European School of Molecular Medicine. As in previous years, numerous scientific and teaching meetings were directly organized by IEO, both for external and internal attendance. Another source of pride for IEO is the Center for Advanced Radiotherapy (ARC), which is among the top ten centers in the world for treatment, research and technology. It provides radio therapeutic treatment of the latest generation for the treatment of tumors and is able to identify with precision the target tumor, spare the surrounding healthy tissue, preserve the organs and their functions, and significantly reduce the duration of the treatments. The ARC utilizes the most modern equipment in the world. We may mention: 1)

16

237

153

156

207

200

IEO - Research Activity Figures - Publications

251

IEO — Scientific Report 2012 — Ongoing Research 2013

132

135

255

243 187

203

282 236

312 271

245

336

368

373

357

321

328

338

321

272

358

400 300

234

244

200

167

100

97

98

99

00

01

02

03

04

05

06

07

08

09

10

11

12

0

Journals with Impact Factor

the True System, which is a linear accelerator capable of performing so-called “tumor tracking”: the beam of radiation follows the movement of the organs of the patient and synchronizes with extreme accuracy on the latter. Within minutes, the machine can act on more outbreaks. It is used to treat cancers of the prostate, lung and liver 2) the Cyberknife System, which is a sort of virtual scalpel capable of providing, at any part of the body, radiation to ablate the tumor. In the IEO is used for the treatment of brain tumors, primary and secondary, spine, liver, pancreas and lung and for the treatment of relapses 3) the Tomo Therapy System, which is able to adjust the intensity of radiation depending on the organ to be treated. It can intervene in all the anatomical sites and allows treatments to be performed on the whole body. In the IEO it is used for patients operated on for cancer of the breast, even if already treated with intraoperative radiation therapy, to completion of the cure, with mini irradiation global breast 4) Trilogy™ System Integrates, which allows one to irradiate the tumor and visualize the anatomy of the patient immediately before administering the fraction of the dose. It then detects the movement of respiration, allowing the irradiation of the tumor only when it is in

Press Office The Scientific Press Office follows scientific and medical content, falling within the area of the Scientific Directorate, in close cooperation with the Central Directorate of Marketing and Communication. Scientific and medical output provided for the general population or published in scientific newspapers or mutually exchanged with international centers are under the supervision of the Scientific Directorate, through the Scientific Press Office. IEO communication organizes marketing issues and continuous and detailed information for the population and media: the Scientific Press Office works within the global network of institutional communication under the coordinated direction of the Scientific Director, the CEO and the Central Director of Marketing and Communication.

Scientific Director’s Office

2500

the correct position. In the IEO it is used to treat cancers of the head-neck tract where, thanks to its extreme precision, allows the functionality of the parotid gland to be preserved. It is also used in gynecological cancers. IEO continued its scientific collaboration at the international level. Specific efforts have been devoted to establish new relations with Mediterranean countries, and with India, Madagascar, and Africa. In addition to this, the result of the experience of western countries where mortality for breast cancer is now declining with respect to eastern countries, drove us to create an agreement with China. Our goal is to involve other eastern countries to disseminate our surgical techniques and our medical knowledge in the field of oncology. Thanks to a close cooperation and an intensive sharing of knowledge and expertise, and frequent contacts between the IEO and international 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 have focused on the fight against feminine tumors (breast and cervix) through specific models from training and education through to the projects for new comprehensive cancer centers. As in 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. The 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 32.239,00 million Euro, 2.140,00 came from the European Community, 18.000,00 from the ISS (the Italian National Institute of Health), 11.756,00 from the Ministry of Health, 6.280,00 from AIRC, 8.163,00 from 5x1000 Fiscal Contribution and 3.882,00 from other sources, including the Lombardy Regional Authorities, the Umberto Veronesi Foundation (FUV), MIUR (Ministry of Education, Universities and Research), the American Institute for Cancer research, CNR, FIRC, FIEO, Vollaro Foundation, Human Frontier Science Program, and the Lega Italiana per la Lotta Contro i Tumori (LILT). We are very grateful to all the funding agencies, which recognized the validity of our projects. In conclusion, I would like to extend our appreciation and gratitude to the President, the CEO, the Board of Directors, and all clinical, scientific, technical and administrative staff of IEO, for their continuous efforts. I wish to thank Pier Giuseppe Pelicci, Roberta Carbone, Eva Bruschini and the various Writing Committees for their valuable collaboration in 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 and producing financial statements. It manages research contracts, preparing, whenever necessary, subcontracts or consortium agreements with collaborating institutions, acts as administrative contact point on multicenter research projects, and provides administrative support for IEO research activities. Collaboration with the University of Milan Since its launch, IEO has established a very close collaborative relationship with the University of Milan, as regards teaching, research and care activities. In fact, while many university professors lead IEO clinical and research Divisions and Units, IEO hosts PhD students and junior residents specializing in various disciplines, as well as many post-doctoral activities. Significant is the collaboration between IEO and the University of Milan within the SEMM, a school dedicated to the training in Molecular Medicine (see page 255). This partnership is progressively growing, slowing becoming a sharing of strategic objectives between the two institutions. IEO — Scientific Report 2012 — Ongoing Research 2013

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Scientific Director’s Office

Ethics Committee

Data Management

Clinical research activity in IEO decreased during 2012. Overall 76 new clinical trial applications were evaluated in 7 plenary sessions of the Ethics Committee (EC). A favorable 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. The EC opinion was expressed within 15 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 were 154 for this year and were reviewed and approved with a mean evaluation time of 10 days. The therapeutic use of drugs still under investigation in clinical trials, for advanced cancer patients, was approved for 11 products and for a total number of 66 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.

The Data Management (DM) Office is responsible for high quality collection and processing of clinical research data, offers support for their analysis and publishing, and gives an important contribute to the administrative set up, management and reporting of clinical trial results. The main goal is to ensure individualised care to all the patients participating in the trials. The DM Office is composed of 22 data managers, 8 fellowship data managers, and 5 data entry clerks. During 2012 the DM Office has supported the activation of 61 new clinical trials and the conduction of 312 ongoing clinical trials (phase I, II, III, IV, observational and others). 72% of all the clinical trials are sponsored by external agencies, while 28% are financed by IEO; overall, 50% of the studies are international multicenter trials and the remaining ones are either national multicenter trials (26%) or monocenter trials (24%). Specific responsibilities are: screening and overseeing of data accrual, supervision of the scheduling of study interventions and follow-ups, supervision of data collection and processing, compliance with relevant Institutional or governmental regulatory guidelines in the conduct of clinical research, assistance in the interactions with research staff both at the Institute and at other collaborating institutions, coordination of protocol production and submission of regulatory documents to the Ethics Committee and regulatory bodies, monitoring of patients and test result reporting, and assistance in the preparation of research reports and manuscripts for publication and presentations. Several procedures ensure the correct execution of these activities. They include internal Standard Operating Procedures such as the use of Case Report Forms (CRFs) and Database Management, Data Entry process, Patients Registration, Data Manager training. In the last year, we have introduced and developed another important element: the “Collaboration project”; in fact, due to the large amount of clinical trials, the collaboration

Ethics Committee As from May 2013

Luciano MARTINI Chairman Giovanni APOLONE Vice Chairman Atanasio NONIS Secretary Maria Santina BONARDI Natale CASCINELLI Giuseppe GALLUS Stefano GASTALDI Aaron GOLDHIRSCH Pasquale MICCINELLI Francesca MERZAGORA Emanuela OMODEO SALE’ Maurizio PELLEGRINI Enrico RAMBALDI FELDMANN Oliviero RINALDI Nicole ROTMENSZ Umberto VERONESI

SECRETARIAT Daniela TAMAGNI OBSERVER Mauro MELIS

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IEO — Scientific Report 2012 — Ongoing Research 2013

Scientific Director’s Office

Scientific Director’s Office

between data managers, clinical staff and all the people involved in a particular trial has become a key factor to improve the quality of trial management. In addition, a specific tool that allows, directly on the IEO Intranet website, access to the clinical trials and to the relevant documentation is available. By this tool, all the members of a research team can find out which clinical trials are on-going in the different Clinical Units/Divisions and how to access them.

IEO — Scientific Report 2012 — Ongoing Research 2013

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Scientific Director’s Office

Scientific Director’s Office

Unit of International Cooperation in Clinical Trials Aaron GOLDHIRSCH & Marco COLLEONI

Activities 2012. The main international

cooperation of the IEO in a leadership position was held within the International Breast Cancer Study Group. During 2012 the IBCSG was significantly involved in conducting research and in the presentation of results from clinical trials??? in international conferences. Presentations were conducted based on the following study designs: BIG 1-98 (1, 2), HERA (3, 4, 5), IBCSG VIII and IX (6, 7), IBCSG SOFT and TEXT (8). IBCSG Trial 23-01 (examining the need for axillary dissection in the presence of micrometastases in sentinel nodes) closed accrual in February 2010 with 6681 registered and 934 patients randomized. First results were presented at the SABCS in December 2011 and a manuscript has been submitted. IBCSG Trial 36-07 (BIG 2-06: ALTTO testing adjuvant trastuzumab and lapatinib in HER2-positive breast cancer) ended accrual with 8381 patients. Based on results from the parallel neo-adjuvant trial Neo-ALTTO, patients included in this trial who were assigned to lapatinib alone as anti-HER2 therapy were offered crossover to trastuzumab. IBCSG Trial 39-11 (Aphinity) was designed for 3806 patients and compares trastuzumab + pertuzumab to trastuzumab + placebo. Aphinity was the only large trial started within the Group in 2012. IBCSG SOFT and TEXT trials (in premenopausal women with endocrine responsive disease) completed accrual with 3066 (31 January 2011) and 2672 (11 March 2011) patients, respectively. IBCSG Co-SOFT (on cognitive function) accrued 84 and IBCSG SOFT-EST (on estrogen level profiles) 123 patients, respectively. IBCSG Trials 22-00 (CM metronomic therapy as adjuvant) and 35-07 (SOLE: extended letrozole adjuvant therapy after year 5 of adjuvant endocrine therapy for node-positive disease in postmenopausal women) terminated accrual in 2012 with more than the target of 1080 patients and a total of 4884 patients, respectively.

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IEO — Scientific Report 2012 — Ongoing Research 2013

New IBCSG Trials The closure of the SOLE and 22-00 trials leaves the Group with the planning of two small trials, SNAP and TREND. These will accrue patients in 2013. These trials represent successful collaborations with Celgene (for the use of abraxane) and Ferring (for the use of degarelix), respectively. The hope is that these promising agents will become desirable for larger adjuvant trials, and that collaborations will continue. TREND is a neo-adjuvant trial that compares degarelix (a LHRH antagonist) to triptorelin (a LHRH agonist) for premenopausal women with endocrine responsive disease. Another trial, SNAP, evaluates three schedules of nab-paclitaxel for women with advanced breast cancer. Translational Research Gene expression profiling: Collaborative investigations are underway with validations of targeted and exploration of whole-genome gene expression profiles. • RNA extracted from FFPE tumor blocks, using a mutually-agreed upon protocol, was distributed to collaborators, DNA was extracted and distributed, and additional TMAs were prepared from newly-received blocks. • Using IBCSG IX data (Tam vs. CMF+Tam), collaborators thought to determine if a proliferation score could identify a subset of women who benefit differently from the addition of chemotherapy compared to that of Tam. • Genomic Grade (GG): tumor material from BIG 1-98 patients was evaluated for a genomic grade (qRT-PCR GG) to predict distant recurrence. • Our collaborators conducted whole-genome transcript profiling of BIG 1-98 tissue, in order to identify prognostic drivers of ER+ disease, and subsequently used 2 published data sets profiled on a different platform to validate these.

References Metzger O, Giobbie-Hurder A, Mallon E, Viale G, Winer E, Thürlimann B, Gelber RD, Colleoni M, Ejlertsen B, Bonnefoi H, Coates AS, Goldhirsch A, Gusterson B. BIG 1-98 Collaborative Group, International Breast Cancer Study Group. Relative effectiveness of letrozole compared with tamoxifen for patients with lobular carcinoma in the BIG 1-98 Trial. Cancer Res 72 (24 Suppl.): 89s (Abstract S1-1), 2012 Sotiriou C, Ignatiadis M, Desmedt C, Azim Jr. HA, Veys I, Larsimont D, Lyng M, Viale G, Leyland-Jones B, Ditzel H, Giobbie-Hurder A, Regan, M, Piccart M, Michiels S. Independent validation of Genomic Grade in the BIG 1-98 study. Cancer Res 72 (24 Suppl.): 101s (Abstract S4-4), 2012 Goldhirsch A, Piccart M, Procter M, De Azambuja E, Weber H, Untch M, Smith IE, Gianni L, Jackisch C, Cameron D, Bell R, Dowsett M, Gelber RD, LeylandJones B, Baselga J. HERA trial: 2 years versus 1 year of trastuzumab after adjuvant chemotherapy in women with HER2-positive early breast cancer at 8 years of median follow-up. Ann Oncol 23 (Suppl. 9) (Abstract 6-LBA), 2012 http://annonc.oxfordjournals.org/content/23/suppl_9/ixe1. abstract?sid=e548a8f4-adc7-4369-a2ab-6207f504f7c0 Goldhirsch A, Piccart-Gebhart MJ, Procter M, de Azambuja E, Weber HA, Untch M, Smith I, Gianni L, Jackisch C, Cameron D, Bell R, Dowsett M, Gelber RD, Leyland-Jones B, Baselga J, On Behalf of the HERA Study Team. HERA TRIAL: 2 years versus 1 year of trastuzumab after adjuvant chemotherapy in women with HER2positive early breast cancer at 8 years of median follow up. Cancer Res 72 (24 Suppl.): 103s (Abstract S5-2), 2012

trastuzumab for breast cancer: coping with success. Cancer Res 72 (24 Suppl.): 465s (Abstract P5-18-02), 2012 Millar EKA, Coates AS, O’Toole SA, Selinger C, Musgrove EA, Yan M, Viale G, Regan M, Price KN, Castiglione-Gertsch M, Colleoni M, Gelber RD, Goldhirsch A, Sutherland RL. Intrinsic subtype and its clinical significance in early node negative breast cancer: results from two randomized trials of adjuvant chemoendocrine therapy. Abstract #504. Presented at: the 2012 American Society of Clinical Oncology Annual Meeting Sninsky J, Wang A, Gray K, Lagier R, Christopherson C, Rowland C, Chang M, Kammler R, Viale G, Kwok S, Regan M, Leyland-Jones B. Predictive value of a proliferation score (MS) in postmenopausal women with endocrineresponsive breast cancer: results from International Breast Cancer Study Group (IBCSG) Trial IX. Cancer Res 72 (24 Suppl.): 144s (Abstract PD10-03), 2012 Dellapasqua S, Bagnardi V, Regan MM, Rotmensz N, Mastropasqua MG, Viale G, Maiorano E, Price KN, Gelber RD, Castiglione-Gertsch M, Goldhirsch A, Colleoni M. A risk score based on histopathological features predicts higher risk of distant recurrence in premenopausal patients with lymph node-negative endocrine-responsive breast cancer. Breast 21: 621-628, 2012 (IBCSG Trial VIII) (Journal impact factor 2.491).

Regan MM, Dafni U, Karlis D, Goldhirsch A, Untch M, Smith I, Gianni L, Jackisch C, de Azambuja E, Heinzmann D, Cameron D, Bell R, Dowsett M, Baselga J, Leyland-Jones B, Piccart-Gebhart MJ, Gelber RD, on behalf of the HERA study team. Selective crossover in randomized trials of adjuvant

IEO — Scientific Report 2012 — Ongoing Research 2013

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Scientific Director’s Office

ecancermedicalscience

Library

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, is fully indexed in PubMed and other leading databases, including Embase, Scopus, EBSCO and Google Scholar, so any article published will be highly accessible to other researchers in the field. The journal is now receiving a steady flow of submissions with a rejection rate of approximately 60%. ecancer continues to develop its video platform, reporting and filming from all the major oncology conferences around the globe. ecancer.tv now features more than 2200 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 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 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) is looking at semantic ontologies of health IT systems. ecancer’s Education platform continues to grow with new elearning modules being released in the coming months. These include smoking cessation modules (to be launched April 2013); ecancer is working in partnership with the

22

IEO — Scientific Report 2012 — Ongoing Research 2013

BMS Foundation and the International Society of Nurses in Cancer Care to produce 4 educational modules on smoking cessation for nurses. These modules are accredited by the European Oncology Nursing Society (EONS) and are available in English, Czech, Polish, Romanian, Russian and Hungarian. There will also be Palliative Care modules (to be launched November 2013) thanks to a grant from the IAEA. ecancer is working in partnership with Cardiff University to develop 13 elearning modules on palliative care for the African oncology community. ecancer.org was recently redesigned to enable visitors to experience a more professional yet 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. ecancer is also releasing new versions of the ecancer app on iPhone, Android and iPad. ecancerpatient.org was launched in November 2012. It provides information for patients to use while discussing treatment options with their doctor. The videos of cancer specialists talking about the most recent advances in care are designed to empower patients to become more involved in vital treatment decisions. 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.

The Library is based in the Mirror Tower, and offers an area for study and consultation of the literature both online and in print. It is part of Bibliosan – the national network of 59 IRCCS libraries, working within the framework of NILDE, the Network for Inter-Library Document Exchange. BiblioSan also offers remote access, so IEO staff can connect from home. The Italian National Collective Catalogue of Periodicals (ACNP) links it to over 110,000 journals and 2300 Libraries throughout Italy. The Biomedical Library System of Lombardy (SBBL) consortium links it to the regional health libraries.

Scientific Director’s Office

Scientific Director’s Office

Data on “impact factors” (whereby a numerical value represents the yearly “impact” of a journal) and the h-index (a measure of quantifying a person’s scientific research output and impact) is provided by the Journal Citation Reports and the ISI Web of Science respectively. Personal bibliographic management is available whereby the user may store bibliographic citations in a system called RefWorks. This will repeatedly generate bibliographies to suit the different formats required by publishers of scientific journals. Literature searches and assistance are provided to facilitate use of the ever-burgeoning bibliographic and full-text resources available.

The Library is also a member 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). Via these consortium agreements, 8000-plus journals are accessible 24 hours a day, 7 days a week. Through SBBL over 1000 e-books are available via Springer Publishers. All can be accessed via the Library “AtoZ catalogue”. In 2012 this catalogue had a total of 7511 accesses. The average session time was 4 minutes, and a total of 26,647 pages were viewed. The Library offers the key medical literature database Medline, the more pharmaceutically-focused Embase, CINAHL (Cumulative Index to Nursing and Allied Health Literature), along with bibliographic full-text databases specialising in psychology, health administration, science and technology, food science, and systematic reviews (the Cochrane Library).

IEO — Scientific Report 2012 — Ongoing Research 2013

23


The Directive Board

The Directive Board

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 behavior 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 Scientific Director Scientific Co-Director Deputy Scientific Director Deputy Scientific Director Deputy Scientific Director Deputy Scientific Director

24

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

IEO — Scientific Report 2012 — Ongoing Research 2013

Chief Communication, Marketing Barbara Cossetto and Customer Service Officer Chief Financial Officer Mario Cesana Chief HR Officer Daniele Piacentini Chief Information Officer Claudio Carlo Franzoni Chief Medical Officer Oliviero Rinaldi

IEO — Scientific Report 2012 — Ongoing Research 2013

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CEO’s Office

Communication, Marketing and Customer Service

Finance and Administration

Barbara COSSETTO Chief Communication, Marketing and Customer Service Officer

Mario CESANA Chief Financial Officer

Customer Service Staff Admissions Office: Francesco Bernasconi Clinical Secretariat: Fabrizio Di Stefano

Marketing & Communication Staff Marketing: Marco Vianello Communication and PR: Barbara Cossetto (ad interim) Corporate Communication and Website: Emanuela Ottolina New Media: Giovanna Gatti

Head of the Secretariat: Jolanta Orlikowska

Customer Service involves more than 180 resources. Services are delivered to Patients to support their clinical path and internally to physicians. Marketing: • Product Management • Relation and Sales with Healthcare Insurances • International Relationship Customer Service: • Booking Call Center • Clinical Secretariat • Admittance Front Lines Communication and PR: • Brand Image • Website • Corporate Events • Public Relations • New Media

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IEO — Scientific Report 2012 — Ongoing Research 2013

STAFF Executive Assistant: Viviana Muggiana Financial & Accounting Controller: Angelo Longoni General Accouting: Luigi Romagnoni Legal Affairs: Renato Galasso Purchasing Office: Roberto Benelli Budget & Finance Controller: Antonio Di Filippo

CEO’s Office

CEO’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 & Financial • Legal Affairs • Purchasing Office • Budgeting & Financial

IEO — Scientific Report 2012 — Ongoing Research 2013

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CEO’s Office

Human Resources

Information & Communication Technology

Daniele Piacentini Chief HR Officer

Claudio Carlo FRANZONI Chief Information Officer

STAFF Performance Management & Organization: Anna Lauro IEO CCM Recruiting, Training & Development: Elena Mazzoleni HR Research Department: Annalisa Ariesi IEO CCM Payroll & Labor Laws Department: Nicoletta Golin Resources Planning Department: Alfonso Lorusso Organization & Process Engineering Department: Silvio Pozzi Facility Management Department: Gianfranco Piantelli HR Controller: Marco Colombi Executive Assistant: Elisabetta Ronchi

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 HR Office in each of the above areas. In fact, we are convinced that the quality of health services is determined by the quality of those who provide them: this principle has enabled the Institute to achieve its main objective, that is to say the improvement of the quality of life of each patient, who must be considered not simply as a person who needs treatment but, above all, as a human being. Main tasks of the HR Department are as follow: • Recruitment and Selection • Education and Training • Performance Evaluations • Staff Administration and Payroll • Salary Packages and Incentive Policies • Personnel Management, Planning and Costs • Union Relations • Organization

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IEO — Scientific Report 2012 — Ongoing Research 2013

The HR Department reported the following services: • Performance Management & Organization • IEO CCM Recruiting, Training & Development • HR Research Department • IEO CCM Payroll & Labor Laws Department • Resources Planning Department • Organization & Process Engineering Department • Facility Management Department The best example of this approach is the “Job Family Model”, a model for managing human resources for professional families that has been extended to all roles of the Institute and aims to promote, develop and reward the skills and knowledge of our key employees. Through this model IEO wants to realize two fundamental goals for an health organization: 1. To ensure qualified and high skilled employees and constantly develop them 2. To ensure motivated employees who want to apply their knowledge in their everyday job 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 2011-2012-2013 IEO awarded the Top Employers Italy as one of the Italian companies which proved excellent in management of its human resources.

STAFF Head of the Secretariat: Jolanta Orlikowska Clinical ICT Operations: Luigi Grilli Research ICT Operations: Alessandro Dellavedova Applications: Paolo Zilioli Technology & Innovation: Alberto Mancin ICT Governance: Luigi Cassi

CEO’s Office

CEO’s Office

The ICT Department manage the whole computing infrastructure and information services, including the network infrastructure and telephony. Main tasks of the Information & Communication Technology Department are: • support users in the daily information services operations and troubleshooting • 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 Division is also in charge to integrate clinical equipment and medical process with innovativem technology to deploy better quality of care and tomgenerate electronic medical records for e-Heatlth services. The Staff and the Director of Information & Communication Technology Department operate both into IEO and the Cardiologic Center Monzino environments.

IEO — Scientific Report 2012 — Ongoing Research 2013

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CEO’s Office

CEO’s Office

Medical Office Oliviero Rinaldi, MD Chief Medical Officer

STAFF Assistants: Giovanni Grieco, MD, Camilla Ranieri, MD Head of Secretariat: Stefania Piacentini Secretariat: Evelina Guaragna Service for Nursing, Technical and Rehabilitation Staff: Giorgio Magon Quality & Accreditation and Customer Relations Office: Pier Luigi Deriu Patient Safety & Risk Management: Massimo Monturano Health Information Management & Medical Records: Daniele Dozzo, Andrea Chiesa Pharmacy: Emanuela Omodeo Salè

Medical Office oversees all health areas 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 international guidelines and internal clinical care pathways. • Responsibility of overall quality and technical efficiency - the production of operational performance (“vertical lines”) and distribution services through the integration of individual products or services in assistance programs, geared to the individual and the community (“horizontal lines”).

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IEO — Scientific Report 2012 — Ongoing Research 2013

• Guarantor of integrated hospital health care, from the organization 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. • Responsibility of Accreditation (both institutional and excellence) of both facilities and professionals working in the Institute. In particular responsibility of all requirements relating to safety. • Responsibility of the Internal auditing program that aims at verifying the most critical processes of the Institute. 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. Patient safety and quality have been one of the main objectives of Medical Office since the Institute opened. Since 2002 we have achieved Joint Commission International Accreditation; many processes have been Certified ISO 9001. In 2012 we started the Accreditation procedure for OECI (Organization European Cancer Institutes) that will be concluded by the end of 2013.

IEO — Scientific Report 2012 — Ongoing Research 2013

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100% 90% 80% 70% 60% 54% 51% 50% 40%

Hospital Activities

graf 4

52%

57%

55%

54%

56%

54%

54%

03

04

05

06

07

08

56%

63%

68%

30% 20% 10% 00% 01

00

02

Medical

At the end of the year the clinical staff of the Institute numbered 390 physicians, 450 nurses, 106 health care assistants, 154 health technicians and 40 medical residents. 110 medical doctors coming from 18 countries spent a significant period (more than 30 days) of clinical training in one or more of our Clinical Divisions.

52%

CEO’s Office

CEO’s Office

10

11

12

Key Case Mix Figures: Surgical Index

Surgical

Day Surgery cases (surgical treatments which do not require an overnight stay) amounted to 4.550 (3.336 in 2011).

1%

Day Hospital admissions, mainly for therapeutic pur poses (chemotherapy or radiation therapy) totalled 1.647 treatments (8.532 in 2011) and involved 1.169 patients (2.918 in 2010)

35.826 new patients were enrolled in 2012 adding up to a total patient census (as of 31 December 2012) of 579.703 patients since IEO opened in 1994. Total hospital admissions decreased by 5.78% to 11.236 compared to the previous year, totalling 47.950 hospital days (48.734 in 2011) with an average length of stay of 4.27 (4.1 in 2011). The ratio between surgical admissions and total admissions (“surgical index”) increased to 68%. Case mix complexity, proxied by the Average Relative Weight, increased to 1.46.

09

13%

22%

12%

Outside Italy Milan Lombardy except Milan Northen Italy except Lombardia and Milan Central italy Southern Italy & Islands

32% 20%

Key Case Mix Figures: Impatient Admissions by geographical area

6

6

5

5

4

4

3

3

2

2

1

1

0

95

96

97

98

Inpatients

99

00

01

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02

03

04

05

06

07

08

09

10

11

0

12

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

Medical

2,00 1,80 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0

2,00 1,80 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0 2000 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Key Case Mix Figures: Hospitalisations

32

Inpatients

IEO — Scientific Report 2012 — Ongoing Research 2013

Day Hospital

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2012

Inpatient Medical Average Relative Weight Inpatient Average Relative Weight

Inpatient Surgical Average Relative Weight

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

IEO — Scientific Report 2012 — Ongoing Research 2013

33


Key Case Mix Figures: Surgical Interventions

Key Case Mix Figures: Outpatient Admissions for Systemic Therapy

12000 9000 6000 3000 0

0

500

1000

1500

2000

2500

3000

3500

25000

150000

20000

120000

15000

90000

10000

60000

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0

350000

1000 900 800 700 600 500 400 300 200 100 0

300000 250000 200000 150000 100000 50000 0 95

96

97

98

99

Radiation Therapy

00

01

02

03

04

05

06

07

08

09

10

11

95

96

97

98

99

00

01

02

03

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09

10

11

12

95

96

97

98

99

00

01

02

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05

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07

08

09

10

11

12

12

13000

20000

11000 9000

15000

7000 10000

5000 3000

5000 0

1000 95

96

97

98

Histological

15000

Key Case Mix Figures: Outpatient Visits

15000

25000

IORT

120000

Key Case Mix Figures: Endoscopic Procedures

0

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012

Key Case Mix Figures: Radiation Therapy & IORT

CEO’s Office

15000

graf 12

Other Gynaecology Urology Thoracic Surgery Melanoma Digestive Surgery Head & Neck Surgery Plastic & Reconstructive Surgery Surgical Senology

99

00

01

02

03

04

05

06

07

08

09

10

11

12

Cytological

-1000

Key Case Mix Figures: Pathological Examinations

1000000

100000 80000

10000

600000

60000 40000

5000

20000

Key Case Mix Figures: Radiodiagnostic & Nuclear Medicine examinations

34

IEO — Scientific Report 2012 — Ongoing Research 2013

800000

400000 200000

0

0 95

96

97

98

99

00

Radiodiagnostic examinations

01

02

03

04

05

06

07

08

09

Nuclear Medicine examinations

10

11

12

0

95

96

97

98

99

00

01

02

03

04

05

06

07

08

09

10

11

12

Key Case Mix Figures: Laboratory Tests

IEO — Scientific Report 2012 — Ongoing Research 2013

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CEO’s Office

CEO’s Office

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 Leonardo Biondi - IFOM Mario Cesana - IEO Andrea Cuomo - ST Microelectronics Marco Foiani - IFOM Isaac Kohlberg - Harvard University Tomas Lindhal - Cancer Research UK Elisabetta Petrucci - FIRC Domenico Triarico - IEO BUSINESS DEVELOPMENT ADVISORY BOARD

Giulio Draetta - MD Anderson Cancer Center Isaac Kohlberg - Harvard University Nagesh Mahantappa - CEO Scholar Rock LLC Kazumi Shiosaki - MPM Capital, Venture Capitalist Katherine Turner - Turner Consultant, R&D Consultant

Pier Giuseppe PELICCI, MD Chairman of the Board

TTFactor is the technology transfer company of the European Institute of Oncology (IEO) and the FIRC Institute for Molecular Oncology (IFOM). Its mission is in supporting researchers and clinicians in evaluating the commercial potential of their research and promoting relationships with industry to foster further application and development of their research results and inventions. Typical TTF activities include patent filing, licensing, sponsored research and spin off creation. The Company is composed by a team of professionals with qualified technical/economic/legal background (PhD, MBA, LLM) as well as industry experience (pharma & biotech); a Board of Directors composed by the representatives of IFOM and IEO, together with highly reputed international industry and technology transfer professionals, 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 2012. During its third year of

operations, TTFactor has intensified internal service to faculty, increasing invention disclosures, patenting and signing numerous contracts for information and material exchange with top companies worldwide. TTF has achieved a healthy patent portfolio which forms the basis for future license and spin offs transactions, both by internally scouting the best patentable ideas from scientists (17 new invention disclosures received this year) and by in-licensing promising patents from other Universities and Companies. In 2012 TTF filed 3 new patent applications, extended 4 and abandoned 2 old patent applications and now the portfolio includes: 13 patent families. Among the major agreements signed in 2012, an exclusive agreement with Sigma Tau S.p.A around several patent/trademark families related to avidin-biotin

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IEO — Scientific Report 2012 — Ongoing Research 2013

radiopharmaceuticals; the in-licensing from the University of Pisa of cancer metabolism targeting compounds; and sponsored research agreements with Myriad Genetics and Orobics benefiting internal researchers. In addition, TTF entered in negotiation with the food industry signing an important research agreement with Heinz-Plasmon, and an agreement with Agrumigel Italia for the supply of blood orange juice necessary to start an important intervention clinical trial in IEO for the Smartfood program. During 2012 TTF can count also several other agreements with for profit counterparts such as 33 material transfer agreements, 15 non-disclosure agreements, 4 research tools licensing (with opportunity to receive future royalties payments). The company has been awarded an European grant for the performance of specific activities of patenting and tech transfer within the project led by Dr Maria Rescigno. TTFactor, within the limits of its resources, supports the business development of selected priority projects, identified and overseen with the counsel of its international Business Development Advisory Board: - Small molecules incubated in Drug Discovery Program (IEO) targeting cancer epigenetics and enzymatic pathways. The most promising project is focused on a

compound targeting a cancer metabolism enzyme that is highly expressed in poor-prognosis tumors; - A tumor radiotherapy discovered in IEO, for which TTF is seeking investors and capital to continue the clinical development; - A new program for anticancer biological molecules against the interaction of receptor uPAR with the extracellular matrix. Despite their potential value, these projects will need validation and industrial development and thus TTFactor is looking for industrial partners or Venture Capital funds to create value and foster their development. The TTF model of IEO and IFOM has become a unique example in Italy attracting much attention and invitations in several events or seminars at University of Bari, Alma Graduate School in Bologna, University of l’Aquila, Università Bocconi and at University of Milan. TTF has represented the Institute in Assobiotec and in several international biotech meetings including Bio (Boston), Tech Transfer Summit Europe (Paris) and Bio Europe (Hamburg).

IEO — Scientific Report 2012 — Ongoing Research 2013

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CEO’s Office

CEO’s Office

Centers of Excellence

At the European Institute of Oncology, there are some important Centers of Excellence, dedicated to the prevention and treatment requirements for special oncological diseases. 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 ARC has the latest equipment available for the highprecision radiotherapy like Intensity Modulated Radiotherapy (IMRT, including dynamic arc IMRT using RapidArc technology), Image-Guided Radiotherapy (IGRT), respiratory gating, intra- and extra-cranial 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 Trilogy 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 with electrons, i.e. ELIOT). Molecular Imaging Unit and collaboration with Politecnico of Milan help in the definition of the optimal imaging, clinical and technological aspects of modern radiotherapy. Moreover, Brachytherapy Unit with low-, pulsed- and high dose rate systems allows for the personalized approach in each patient.

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In 2012, 3287 new patients were treated in our Division: 2630, 427 and 230 with external beam radiotherapy, intraoperative irradiation (mainly for breast cancer) and brachytherapy, respectively. The highest proportion of patients has been treated for breast cancer (40%) followed by metastastic disease (30%) and prostate cancer (8%). Special selective RT techniques are employed in the majority of treatments: image guided intensity modulated RT (G-IMRT) was applied in 1046 patients whereas stereotactic radiotherapy – in 327 patients. The patients treated with the last generation linacs include 435 patients treated with Trilogy, 307 – treated with Tomotherapy, 293 – VERO system and 191 – with CyberKnife). The choice of the treatment technique depends on the tumor position and extension as well on the patient characteristics (anatomy, concomitant diseases etc.). Based on the dosimetric and technological features of each linac the institutional rules has been established: VERO is dedicated mainly to prostate tumors and stereotactic body irradiation (thorax and abdomen), CyberKnife – to stereotactic brain and spine radiotherapy, Tomotherapy – to breast tumors and Trilogy to head and neck IMRT and pelvic IMRT (gynecological tumors, gastrointestinal malignancies etc.). 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 chemoradiation. Thanks to clinical studies and experience gained over the years, it has been possible to make a conservative path safe and reliable for young women with cervical cancer. All the sections of the center are top level and provide patients with the best available solutions from the interdisciplinary team.

The Center is involved in experimental clinical studies, national and international to evaluate new treatments for better health and fewer side effects. The center is also actively involved in a prevention program for the developing countries, and in particular for Madagascar. 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 of skills, technologies and processes required for prevention, diagnosis and more effective treatment of ovarian cancer.

The clinical activities of the IEO Cardioncology Center relate to early diagnosis, prevention and treatment of chemotherapy induced cardiotoxicity. In 2012 over 200 outpatients were treated. An official internal clinical IEO protocol for cardiotoxicity early preclinical diagnosis and prevention is currently applied to all the IEO patients undergoing chemotherapy and newer target therapies. Moreover, in the Center are treated pericardial diseases, as neoplastic pericardial effusions by means of intrapericardial chemotherapy (with Thio-tepa) for pericardial effusion and, more in general, are treated all the potential relations between cancer and cardiovascular diseases.

Cardioncology Center The International Cardioncology Society (ICOS, constituted the January 2009 by the Director of the Cardiology Division of the European Institute of Oncology, Dr Carlo Cipolla, Milan-Italy and the actual Director of Cardiovascular Research of the Vanderbilt University, Nashville-Tennessee, Dr Daniel Lenihan) promotes training and studies in the fields of: Cardiological and oncological comorbidities Cardiological implications of oncological treatments as: - Chemotherapy, medical treatments (such as the use of monoclonal antibodies and target therapies) - Radiotherapy - 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.

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Clinical Research

Clinical Research

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Disease-Oriented Research – Breast Tumors DiseaseOriented Research

Division of Breast Cancer Surgery 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: Mattia Intra, MD, Oreste Gentilini, MD, Annarita Vento, MD, Piero Caldarella, MD, Bettina Ballardini, MD Assistants: Paolo Arnone, MD, Fabio Bassi, MD, Paola Naninato, MD, Gianmatteo Pagani, MD, Antonio Toesca, MD, Francesca Magnoni, MD, Germana Lissidini, MD, Paola Baratella, MD, Manuela Sargenti, MD, Silvia Velpidia Ratini, MD, Fellows: Manika Aikaterini, MD, Ivana Lorena Romero, MD, Nieves Montero Maybell Alexandra, MD, Pedro Andres Del Castillo, MD, Marlene Gallardo, MD, Lucila Maria Salazar Moltrasio, MD, Dimitri Aktsalis, Della Caridad Garau Ribeiro Luannys, MD, Pinto Marcio Ivan, MD, Alba Posse Ricardo Sebastian, MD Secretaries: Iliade Lombardi, Manuela Butti Data Manager: Claudia Sangalli Surgical Area Nursing Coordinator: Luigia Rubio Head Nurse: Denise Santina Bucci Auxiliaries: Chiara Agratti, Luana Di Gilio, Patrizia Frigatti, Mercedes Monzo, Alessandra Rubio, Rossana Vangelista

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Activities 2012.

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. In 2012 a total of 3407 operations were performed in the Senology Division with 2545 surgical procedures performed in ordinary admission and 862 procedures performed in Day Surgery, which is one of the new directions in our clinical activity. This management of patients in a Day Surgery setting is currently increasing as 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. 2479 new breast cancer were treated within our Division in 2012. Of these 1485 received a breast conserving surgery procedure. 792 (79,6%) of 994 patients who received a mastectomy underwent an immediate breast reconstruction. 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 tumor 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 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. 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. 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 beam-

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DiseaseOriented Research guiding equipment and chest protection techniques. The first single-center 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 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. 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.

for the International Breast Cancer Study Group Trial 23–01 investigators. Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23-01): a phase 3 randomised controlled trial. Lancet Oncology 2013 Mar 8. pii: S14702045(13)70035 Veronesi U, Stafyla V, Petit JY, Veronesi P. Conservative mastectomy: extending the idea of breast conservation. Lancet Oncology 2012 Jul;13(7): e311-7 Gentilini O, Botteri E, Veronesi P, Sangalli C, Del Castillo A, Ballardini B, Galimberti V, Rietjens M, Colleoni M, Luini A, Veronesi U. Repeating conservative surgery after ipsilateral breast tumor reappearance: criteria for selecting the best candidates. Ann Surg Oncol 2012 Nov;19(12):3771-6 Gentilini O, Veronesi U. Abandoning sentinel lymph node biopsy in early breast cancer? A new trial in progress at the European Institute of Oncology of Milan (SOUND: Sentinel node vs Observation after axillary UltraSouND) The Breast 2012 Oct;21(5):678-81 Toesca A, Spitaleri G, De Pas T, Botteri E, Gentilini O, Bottiglieri L, Rotmentsz N, Sangalli C, Marrazzo E, Cassano E, Veronesi P, Rietjens M, Luini A. Sarcoma of the breast: outcome and reconstructive options. Clin Breast Cancer 2012 Dec;12(6):438-44

Publications Galimberti V, Cole BF, Zurrida S, Viale G, Luini A, Veronesi P, Baratella P, Chifu C, Sargenti M, Intra M, Gentilini O, Mastropasqua MG, Mazzarol G, Massarut S, Garbay JR, Zgajnar J, Galatius H, Recalcati A, Littlejohn D, Bamert M, Colleoni M, Price KN, Regan MM, Goldhirsch A, Coates AS, Gelber RD, Veronesi U.

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Disease-Oriented Research – Breast Tumors DiseaseOriented Research

Division of Plastic and Reconstructive Surgery Mario RIETJENS, MD, PhD Director

STAFF Former Director: Jean-Yves Petit, MD Senior Deputy Director: Cristina Garusi, MD Deputy Directors: Francesca De Lorenzi, MD, PhD, Stefano Martella, MD Assistants: Benedetta Barbieri MD, Alessandra Gottardi, MD, Gabriel Hubner Arana, MD, Marco Iera, MD, Giuseppe Lomeo, MD, Andrea Manconi, MD Consultant doctor: Pierre Rey, MD Secretary: Manuela Iavarone Data Manager: Claudia Sangalli Nurse Case Manager: Katia Venditti

Activities 2012.

The Division of Plastic Surgery is dedicated to the improvement of quality of life in patients treated for cancer at the EIO. We daily collaborate intensively with the breast surgery division, and we also support the divisions of gynecology, thoracic surgery, general surgery and melanoma/ sarcoma unit. Our main contribution to breast surgery includes all techniques of immediate breast reshaping after conservative surgery (so called oncoplastic surgery) and all techniques of total breast reconstructions after mastectomy (both immediate and delayed procedures), including the use of tissue expanders or definitive implants, latissimus dorsii flaps, TRAM flaps and microsurgical flaps. We are also developing new approaches as the use of Acelullar dermal matrix (ADM) in breast reconstruction and the use of fat grafting. Fat grafting is more frequently used as a refinement after reconstruction or, in selected patients, for total breast reconstruction with exclusively adipose tissue.

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Ongoing studies: Several studies are ongoing in our division: - Fat grafting clinical study: In order to verify the incidence of breast recurrence in all patients receiving fat grafting after a breast cancer treatment. Our previous study demonstrated a significant increase of recurrence rate in cases of “in situ” G3 tumors in young patients. - Fat grafting laboratory research: In collaboration with our experimental research lab, we are developing different animal models to investigate the correlation between fat grafting and breast cancer cell lines. - Acelullar Dermal Matrix (ADM): Several different types of ADM are in the market nowadays, deriving from human dermis, bovine pericardium and porcine dermis. This new product can improve the results of immediate implant based breast reconstructions and they probably reduce capsula formation and contracture also in cases of previous radiotherapy. We are currently performing a clinical study to test these different types of ADM and focusing on their post operative complications; in the future our project is to setup a randomized study with different ADM to compare the final results. - Nipple Sparing Mastectomy (NSM): After our last publication with more than 1.000 cases of NSM, we are still following patients within our database, we believe that this new approach will be more powerful in the future with more cases and a longer oncologic follow up. Publications Petit JY, Rietjens M, Botteri E, Rotmensz N, Bertolini F, Curigliano G, Rey P, Garusi C, De Lorenzi F, Martella S, Manconi A, Barbieri B, Veronesi P, Intra M, Brambullo T, Gottardi A, Sommario M, Lomeo G, Iera M, Giovinazzo V, Lohsiriwat V Evaluation of fat grafting safety in patients with intra epithelial neoplasia: a matched-cohort study. Ann Oncol. 2013 Feb 7. [Epub ahead of print] 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. Nipple-sparing mastectomy is it worth the risk? Nat Rev Clin Oncol. 2011 Oct 25;8(12):742-7. doi: 10.1038/nrclinonc.2011.159.

Ahmed Y, Petit JY. Safety of fat grafting in secondary breast reconstruction after cancer. J Plast Reconstr Aesthet Surg. 2011 Apr;64(4):477-83. doi: 10.1016/j. bjps.2010.06.024. Epub 2010 Aug 7.

Petit JY, Botteri E, Lohsiriwat V, Rietjens M, De Lorenzi F, Garusi C, Rossetto F, Martella S, Manconi A, Bertolini F, Curigliano G, Veronesi P, Santillo B, Rotmensz N. Locoregional recurrence risk after lipofilling in breast cancer patients. Ann Oncol. 2012 Mar;23(3):582-8. doi: 10.1093/annonc/mdr158. Epub 2011 May 24.

Petit JY, Veronesi U, Orecchia R, Rey P, Martella S, Didier F, Viale G, Veronesi P, Luini A, Galimberti V, Bedolis R, Rietjens M, Garusi C, De Lorenzi F, Bosco R, Manconi A, Ivaldi GB, Youssef O. Nipple sparing mastectomy with nipple areola intraoperative radiotherapy: one thousand and one cases of a five years experience at the European institute of oncology of Milan (EIO). Breast Cancer Res Treat. 2009 Sep;117(2):333-8. doi: 10.1007/s10549-0080304-y. Epub 2009 Jan 17.

Rietjens M, De Lorenzi F, Rossetto F, Brenelli F, Manconi A, Martella S, Intra M, Venturino M, Lohsiriwat V,

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Disease-Oriented Research – Breast Tumors DiseaseOriented Research

Medical Division of Breast Tumors Marco COLLEONI, MD Director

STAFF Senior Deputy Director: Elisabetta Munzone, MD (since February 2013) Assistants: Alessandra Balduzzi, MD, Silvia Della Pasqua, MD, Anna Cardillo, MD, Emilia Montagna, MD, Giuseppe Cancello, MD, Monica Iorfida, MD, Manuelita Mazza, MD, Angela Simona Sciandivasci, MD (since February 2013), Gino Perri, MD (until December 2011), Gian Luca Cervo, MD (until August 2012), Intrivici Chiara, MD (until September 2012) Fellows: Andrea Francesco Sporchia (since October 2012), Giusy Caponigro (until January 2013) Research Nurse: Claudia Passoni, Carmela Vittoria Arensi Data Managers: Raffaella Ghisini, Volpe Sergio, Sangalli Francesco since November 2012, Davide Pastrello until April 2012, Eloise Scarano until February 2012, Fasano Carlo Giuseppe until January 2013 Secretaries: Simona Puddu, Marcella Netti (since February 2013)

Activities 2012/13. The Division of Medical

Senology, formerly Unit of Research in Medical Senology, provides care and conducts research in an integrated fashion in the field of breast cancer. The Division is aimed to provide comprehensive care of all the types and phases (preoperative, adjuvant, advanced disease) of breast cancer with active clinical and translational research, integrating out-patient and in-patient areas through a common support. The principle of providing the best personalized care for the individual patient with breast cancer, with full respect for quality of life and proper communication, is the best support for competitive and innovative clinical

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research. The principle shared by members of the Division 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 IEO and with National and International Cooperative Groups. International trial cooperation, focused on questions relevant for patient care and biological principles, represents one of the major commitments for the Division. 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 according 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. Publications Ewertz M, Gray KP, Regan MM, Ejlertsen B, Price KN, Thürlimann B, Bonnefoi H, Forbes JF, Paridaens RJ, Rabaglio M, Gelber RD, Colleoni M, Láng I, Smith IE, Coates AS, Goldhirsch A, Mouridsen HT.Obesity and risk of recurrence or death after adjuvant endocrine therapy with letrozole or tamoxifen in the breast international group 1-98 trial. J Clin Oncol. 2012;30(32):3967-75. Iorfida M, Maiorano E, Orvieto E, Maisonneuve P, Bottiglieri L, Rotmensz N, Montagna E, Dellapasqua S,

Veronesi P, Galimberti V, Luini A, Goldhirsch A, Colleoni M, Viale G.Invasive lobular breast cancer: subtypes and outcome. Breast Cancer Res Treat. 2012;133(2):713-23. Coates AS, Colleoni M, Goldhirsch A.Is adjuvant chemotherapy useful for women with luminal a breast cancer?J Clin Oncol. 2012;30(12):1260-3.

Montagna E, Bagnardi V, Rotmensz N, Viale G, Renne G, Cancello G, Balduzzi A, Scarano E, Veronesi P, Luini A, Zurrida S, Monti S, Mastropasqua MG, Bottiglieri L, Goldhirsch A, Colleoni M.Breast cancer subtypes and outcome after local and regional relapse.Ann Oncol. 2012;23(2):324-31.

Colleoni M, Rotmensz N, Maisonneuve P, Mastropasqua MG, Luini A, Veronesi P, Intra M, Montagna E, Cancello G, Cardillo A, Mazza M, Perri G, Iorfida M, Pruneri G, Goldhirsch A, Viale G.Outcome of special types of luminal breast cancer.Ann Oncol. 2012;23(6):1428-36.

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Disease-Oriented Research – Breast Tumors

Research programs are directed to develop personalized and innovative treatment plans for all the phases of the disease, based on individual needs and biological features of the tumor, through an interdisciplinary context. In light of these principles, tailored clinical trials are conducted in the preoperative setting, dedicated to targeted treatment protocols dealing with the different breast cancer subtypes (luminal A, luminal B, triple negative and HER2 positive). In order to minimize the extent of local treatment the Division of Senology started a new prospective randomized trial with the aim of improving patients’ quality of life through the exclusion of axillary surgery. The sample size is 1560 patients with an expected time of 5 years to complete accrual. The trial was designed in collaboration with selected Italian and international centers. Similarly, the value of electron intra-operative therapy (ELIOT) as partial breast irradiation, either as full doses or as boost, is explored. New methodologies in local therapies under investigation include the identification of sentinel node using indocyanin green dye, the use of high intensity focused ultrasound (HIFU) for the treatment of patients with early disease, and breast irradiation through IART (intraoperative avidination for radionuclide therapy). Several trials dedicated to the advancement of Individualized, personalized surgery include testing new types of Acelullar Dermal Matrix (ADM), fat grafting and Nipple Sparing Mastectomy (NSM). ADM deriving from human dermis, bovine pericardium and porcine dermis can improve the results of immediate implant based breast reconstructions and they probably reduce capsule formation and contracture also in cases of previous radiotherapy. The outcome of patients receiving fat grafting and NSM, as well as quality of life related issues, is under evaluation. Translational researches are ongoing in order to investigate the role of stem cells, NumB protein and the impact of fat grafting on breast cancer cell lines. Large cooperative trials focusing on adjuvant therapies are in progress under the umbrella

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DiseaseOriented Research

Research Activities

of the International Breast Cancer Study Group (IBCSG) including endocrine therapy for premenopausal patients, extended endocrine therapies in postmenopausal patients and maintenance therapy in endocrine non responsive breast cancer. In addition to the clinical trials research questions, the IBCSG and IEO are collaborating to carry out extensive research in pathology, database studies, quality of life, and statistical methodology (together with the Harvard Medical School, Boston MA, USA). Research activities are dedicated also to specific aspects of the disease such as special histological types, inflammatory breast cancer, and very young patients. Moreover, in order to develop more effective and less toxic treatment approaches, trials on metronomic chemotherapy are carried out in patients with advanced disease.

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Disease-Oriented Research – Respiratory System Tumors

Lorenzo SPAGGIARI, MD Director

STAFF Research Unit Director: Giulia Veronesi, MD Senior Deputy Director: Domenico Galetta, MD Deputy Directors: Roberto Gasparri, MD, Francesco Petrella, MD Senior Assistants: Alessandro Borri, MD Juliana Guarize, MD (Pulmonologist) Assistants: Monica Casiraghi, MD, Adele Tessitore, MD, Stefano Maria Donghi, MD (Pulmonologist) Fellow: Alessandro Pardolesi, MD Residents: Sava Durkovic, MD, Giorgio Lo Iacono Head Nurse: Ester Spacca Data Managers: Raffaella Bertolotti, Daniela Brambilla

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DiseaseOriented Research

Division of Thoracic Cancer Surgery Piergiorgio SOLLI, MD Co-Director

Activities 2012.

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. Surgery of locally 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 miniinvasive approaches – such as robotic or videoassisted procedures – for localized early stage lung cancers. The rigid therapeutic bronchoscopy under negative pressure ventilation allowed to obtain excellent palliation in cases of tracheo bronchial obstructions and /or compression thanks to laser assisted mechanical resection and stent placement. The development of endobronchial ultrasounds (EBUS) maximized preoperative diagnosis and staging of thoracic neoplasms, often skipping more invasive procedures like mediastinoscopy. The Division has instituted a program of lung cancer screening by low dose CT scan and biomarkers, as well as by the “electronic nose” a new experimental device potentially able to identify distinct characteristics in the exhaled breath of undiagnosed patients with lung cancer. The Division developed the minimally invasive approach for the treatment of early stage lung cancers, including robotic approach and video-thoracoscopic major lung resection. The research activity is involved in different translational research studies such as pharmacogenomic analysis, molecular biology studies of lung carcinogenesis and lung cancer angiogenesis. An experimental study on autologous mesenchimal stem cells transplantation for bronchopleural fistula closure has started in close cooperation with IFOM Campus and the University of Milan School of Veterinary Medicine.

Publications Petrella F, Radice D, Randine MG, Borri A, Galetta D, Gasparri R, Donghi S, Casiraghi M, Tessitore A, Guarize J, Pardolesi A, Solli P, Veronesi G, Spaggiari L. Perioperative blood transfusion practices in oncologic thoracic surgery: when, why, and how. Ann Surg Oncol. 2012 Jan;19(1):82-8.

Multidisciplinary treatment of malignant thymoma. Curr Opin Oncol. 2012 Mar;24(2):117-22.

Galetta D, Solli P, Borri A, Petrella F, Gasparri R, Brambilla D, Spaggiari L. Bilobectomy for lung cancer: analysis of indications, post operative results and longterm outcomes. Ann Thorac Surg 2012 Jan;93(1):215-17 Spaggiari L, Casiraghi M, Guarize J

Veronesi G, Maisonneuve P, Bellomi M, Rampinelli C, Durli I, Bertolotti R, Spaggiari L. Estimating overdiagnosis in low-dose computed tomography screening for lung cancer: a cohort study. Ann Intern Med. 2012 Dec 4;157(11):776-84.

Veronesi G, Agoglia BG, Melfi F, Maisonneuve P, Bertolotti R, Bianchi PP, Rocco B, Borri A, Gasparri R, Spaggiari L. Experience with robotic lobectomy for lung cancer Innovations. (Phila) 2011 Nov 6 (6)355-60

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Disease-Oriented Research – Respiratory System Tumors DiseaseOriented Research

Division of head and neck surgery Mohssen ANSARIN Director

STAFF Oral Unit, Director: Luca Calabrese, MD Thyroid Unit Director: Gioacchino Giugliano, MD Senior Assistant: Roberto Bruschini, MD Assistants: Enrica Grosso, MD, Augusto Cattaneo, MD, Valeria Navach, MD, Stefano Zorzi, MD, Michele Proh, MD, Luigi De Benedetto, MD, Marta Tagliabue, MD Consultants: Bianca Gibelli, MD (Endocrinologist), Deborah Mannavola, MD (Endocrinologist), Filippo Cazzulani, DDS (Dentist) Observers and Fellows: Hamed Hafez, MD, Octavio Iavarone, MD, Bezzera, MD, Hamed Salama, MD, Ignatio Quiroa, MD, Draala Hamza, MD, Kristina Stakova, MD Residents: Niccolò Mevio, MD (Pavia University), Daniele Scelsi, MD (Pavia University) Speech Therapist: Valeria Zurlo, BS Data Manager: Maria Angela Massaro, PhD Secretaries: Paola Maggioni, Anna Maria Manti Head Nurses: Sara Meneghin Auxiliaries: Aghese Codella, Anna Maria Ieronimo, Pietro Grimaldi, Anna Ieronimo

Activities 2012.

The clinical research of the Division is focused on the early diagnosis of head and neck cancers, the development of new treatment modalities and molecular medicine through a multidisciplinary approach. The main topics are oral and laryngeal precancerous lesions, cancer of the oral cavity, pharynx, larynx, salivary and thyroid glands. The Division has established national and international collaboration with many world-wide institutions. Several fellows attend our department in order to improve their

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knowledge of Head & Neck Oncology. We are developing organ and function preservation protocols of the larynx, compartmental surgery of oral cancer; intraoperative ultrasound guide lymphadenectomy of the neck, and endoscopic robotic- and laser-assisted surgery for laryngeal and oropharyngeal malignancies. We are also developing conservative and video-assisted (MIVAT) thyroid surgery. Clinical activity during 2012 included 1029 patients admitted in the 16 beds of the Division (178 in Day Surgery). Among them 400 underwent mini-invasive laryngeal and oral laser surgery (16 Robot-assisted), 175 thyroidectomies (15 Video-assisted), 64 major surgeries for an oral cancer (58 with free-flap reconstructions), and 110 for a laryngeal and hypopharyngeal cancer. Patients usually undergo preoperative staging in outclinic regimen and are then admitted, in most cases the same day of surgery. Mean patients’ stay in Hospital in 2012 was 5.5 days. In 2012 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. Treatment programmes of 551 patients were discussed and planned in the weekly multidisciplinary meetings (held on Wednesdays). The division is coordinating two prospective trials: The first is the Pioglitazone study (S500/409). This is a multicentric chemoprevention trial on oral precancer lesions involving 12 USA institutes and 1 European institute. The second is the Lymphatic Mapping study (S629/411), a monocentric study in which we are studying the lymphatic drainage of the neck with indocyanine dye in advanced oral cavity cancers. Moreover 8 retrospective studies on head and neck cancer are ongoing. The Division published 5 papers on peer-reviewed journals, with an overall IF=12.94, and 3 chapters in books in the head and neck oncology and surgery fields. 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 the 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 in the clinical and research activities for 6 months. Physicians of the Division are involved in the teaching activities of the school. A similar agreement is on-going also with the ENT and Head and neck surgery Institute of the University of Prague (Czech Republic), and the Camargo Oncological Institute of São Paulo (Brazil): a resident spends one year 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 these, 5 specialists spent a week attending lectures on head and neck oncology and observing the clinical activities of the Division: a full immersion experience in head and neck oncology. 2. In collaboration with the Institute of Anatomy of the University of Paris, ENT Clinic of Pavia, Ferrara and Brescia we organize two courses of head and neck surgical techniques using cadavers, which is held in Paris. Publications Navach V, Zurlo V, Calabrese L, Massaro MA, Bruschini R, Giugliano G, Ansarin M, Chiesa F Total glossectomy with preservation of the larynx: oncological and functional results. Br J Oral Maxillofac Surg. 2012 Aug 6. [Epub ahead of print]. Ferrari D, Codecà C, Bertuzzi C, Broggio F, Crepaldi F, Luciani A, Floriani I, Ansarin M, Chiesa F, Alterio D, Foa P

Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a Western population. BMC Cancer. 2012 May 30;12:208. Russi EG, Corvò R, Merlotti A, Alterio D, Franco P, Pergolizzi S, De Sanctis V, Ruo Redda MG, Ricardi U, Paiar F, Bonomo P, Merlano MC, Zurlo V, Chiesa F, Sanguineti G, Bernier J Swallowing dysfunction in head and neck cancer patients treated by radiotherapy: review and recommendations of the supportive task group of the Italian Association of Radiation Oncology. Cancer Treat Rev. 2012 Dec;38(8):1033-49. 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. 2012 Jun 1;130(11):2484-94. 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.

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DiseaseMedical Area Oriented Research

Disease-Oriented Research – Respiratory System Tumors

Medical Oncology Division of the Respiratory System Aaron GOLDHIRSCH, MD Director (ad interim)

STAFF Director Medical Oncology Unit of the Respiratory system: Tommaso De Pas, MD Deputy Directors: Chiara Catania, MD, Cristina Noberasco, MD Consultant: Gianluca Spitaleri, MD, Angelo Delmonte, MD Translational research: Francesca Toffalorio, MD, PhD Clinical Fellow: Mariacarmela Santarpia, MD, PhD Research Nurses: Veronica Brunelli Data Managers: Sabrina Boselli, Sparacio Eleonora, Valentina Liparulo, Letizia Sirica Secretary: Monica Croce

Activities 2012.

The Medical Oncology Division of the Respiratory system was established to guarantee the best of care to patients with thoracic malignancies. This goal is mainly pursued by the creation of a dedicated staff, who accurately takes care of the patient in each stage of disease, and promotes appropriate clinical trials to test new drugs and novel therapies. An intensive collaboration with the Division of Thoracic Surgery make sure the correct follow-up of the patient from the pre- to the post-surgical time. One of the main research fields of the Division is nonsmall cell lung cancer (NSCLC). Several phase I, II and III clinical trials are currently opened to cure this kind

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of disease, both in the metastatic and adjuvant setting. The drugs currently investigated vary among targeted therapy (the drugs target specific proteins of pivotal pathways altered in NSCLC), combined therapy and immunotherapy. In detail, the following clinical trials are currently opened: Target therapies: 1. ALK (Anaplastic Lymphoma Kinase) pathway • Crizotinib (three trials evaluate the efficacy and safety of PF-02341066, Crizotinib, a small-molecule able to inhibit the c-Met/HGFR receptor and the anaplastic lymphoma kinase: a phase II, open-label single arm study to evaluate the efficacy and safety of Crizotinib and two phase III, randomized, openlabel study of Crizotinib versus standard of care chemotherapy, in patients with NSCLC either pretreated and chemonaive, harboring a translocation or inversion involving the ALK gene locus) • LDK378 (phase I, II and III studies with the ALK inhibitor LDK378 in patients with advanced NSCLC progressed to Crizotinib or as first line treatment) • LDK378 plus AUY922 (phase Ib/II heat shock protein 90 inhibitor in patients with advanced NSCLC progressed to ALK-inhibitors) • RO5424802 (phase I/II of RO5424802, a new ALK inhibitor, in patients with ALK mutations who failed crizotinib treatment) 2. EGFR (Epidermal Growth Factor Receptor) pathway • MetMAb (phase III study of Monoclonal antibody directed against the cMet receptor, in combination with erlotinib, in patients with advanced Met positive NSCLC) • INC280 (phase IB/II study of INC280, a Met inhibitor, in association to gefitinib, in patients progressed to EGFR inhibitors carrying cMet amplification) • SAR125844 (phase I study of SAR125844, a Met inhibitor, in patients with solid tumors harboring cMet amplification or in phospho-Met positive tumors)

• Afatinib (phase IIIb study of Afatinib, an irreversible inhibitor of EGFR and HER2, in patients with advanced NSCLC EGFR-TKI inhibitors naive) • AUY922 (phase II study of AUY922 in patients with advanced NSCLC progressed to EGFR-TKI inhibitors) • Dacomitinib (phase III study of dacomitinib versus gefitinib as first line treatment in patients with EGFR mutated tumors) • PROSE study (this phase III study is aimed at prospectively evaluating the value of the proteomic profile as a predictive factor for erlotinib and chemotherapy efficacy in patients with inoperable non-small cell lung cancer)

• TRIGGER (phase II study of erlotinib in patients with locally advanced or metastatic NSCLC who present activating mutation in the tyrosine kinase domain of EGFR) 3. Ras pathway • MEK Inhibitor (phase II study of GSK1120212, a specific MEK1/2 inhibitor, in patients with stage IIIb and IV NSCLC harboring mutations of KRas, NRas, BRaf and MEK1) 4. PI3K/PTEN pathway • BKM120 (tree phase II studies of BKM120, a PI3K inhibitors, in NSCLC advanced patients, mostly of squamous cell type, with activated PI3K, alone or in combination with chemotherapy)

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DiseaseOriented Research • GSK2118436 (phase II study of dabrafenib, a BRaf inhibitor, in patients with advanced NSCLC carrying BRaf mutations) Combined therapy: 1 F16IL2 plus paclitaxel (phase Ib/II, lung cancer expansion, of the tumor-targeting human F16IL2 monoclonal antibody-cytokine fusion protein targeting tenascin C, in combination with paclitaxel in patients with advanced disease) 2. BIBF1120 plus standard chemotherapy (phase I/ II of first-line BIBF1120, a VEGFR, PDGFR and FGFR inhibitor, plus gemcitabine/cisplatin in patients with squamous cell lung carcinoma) 3. Acetyl-L-carnitine plus standard chemotherapy (phase III trial of acetyl-L-carnitine, a neuronal protection, in combination with a cisplatin containing chemotherapy as first line treatment of advanced or metastatic NSCLC) 4. NGR-TNF plus standard chemotherapy (phase II study of NGR-TNF, a targeting vessel peptide, as first line of previously untreated patients with advanced disease) 5 HDAC inhibitor plus radiotherapy (phase I study of S7854, a pan-histone deacetylase inhibitor in association to radiotherapy in patients with advanced solid tumors) Immunotherapy: 1. MAGEA3 (phase III study of recMAGEA3+AS15 antigen-specific cancer immunotherapeutic as adjuvant therapy in patients with resectable MAGEA3 positive NSCLC) 2. PRAME (phase I study of recPRAME + AS15 antigenspecific cancer immunotherapeutic as adjuvant therapy in patients with resectable PRAME positive NSCLC) 3. TG4010 (phase IIb/III of TG4010 immunotherapy, a suspension of recombinant modified vaccinia virus strain Ankara, carrying coding sequences for human

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mucin1 antigen and human IL-2, in association to standard first line chemotherapy in patients with stage IV NSCLC) 4. VX-001 (phase IIb study of optimized criptic human telomerase reverse transcriptase peptide PTERT572Y in patients with local or advanced NSCLC not progressing to chemo- or radio-therapy) The Medical Oncology Division of the Respiratory system implemented the program with several research projects dealing with the study of the rare tumors of the thorax such as lung neuroendocrine tumors (from carcinoids, the most benigne lesions, to the more aggressive lesions as large cell neuroendocrine tumors and small cell lung cancers), mesotheliomas and thymomas, in order to find new therapeutic approaches for these uncommon diseases, in addition to clinical trials specific for these uncommon disease. In detail, the currently open trials are: Thymic neoplasm: Phase II study of everolimus in patients with thymoma and thymic carcinoma previously treated with chemotherapy. Small cell lung cancer: phase II study of TAS-102 as second line treatment in patients with SCLC. Moreover, a particular attention was directed towards specific subpopulations such as lung cancers in young patients, NSCLC patients subjected to a stage shift due to the new TNM edition, and EGFR inhibitors beyond focal progression. In collaboration with the Molecular Medicine Program, genetic studies on NSCLC are also currently ongoing as well as a strong collaboration with the Thoracic Surgery Division has led to the development of a preclinical study on goats with the aim of treating bronchopleural fistula with mesenchymal stem cells (for details, see Lung cancer research). The Division is also carrying out studies about the awareness of patients with cancer facing with the choice of whether to participate in a phase I clinical trial.

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Disease-Oriented Research – Respiratory System Tumors

Thoracic Tumors: Surgery Early detection. Chances of cure of lung cancer 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. 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. 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. 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: 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. MicroRNA (miRNA) are small non-coding RNAs, involved in crucial biological

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DiseaseOriented Research

Research Activities

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. Another approach to fully uncover lung cancer genomes and their genetic defects takes advantage of the recent “next generation sequencing” techniques. 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. 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. Post resectional bronchopleural fistula closure by atologous mesenchymal stem cells endoscopic transplantation. In lung cancer surgery, the incidence of bronchopleural fistula (BPF) ranges from 1 to 4%, but its mortality ranges from 12.5 to 71.2%. It is caused

by incomplete bronchial closure, impediment of wound healing at the stump or destruction of the stump by residual neoplastic tissue; the clinical effects of failure of healing of a bronchial stump after anatomic lung resection can vary from a minor problem to a life - threatening septic and ventilatory catastrophe. In the light of the extremely high mortality rate related to post operative bronchopleural fistula, potentially nullifying the excellent long term result of successful lung cancer surgery, it appears of paramount interst the development of effective bronchopleural fistula treatment procedures. Endoscopic conservative approaches based on fibrin glue or local inflammation development techniques (eg. silver nitrate) have very often failed, in particular in case of larger fistulas where only exteremly invasive surgical salvage procedures may be advocated as rescue treatments. Our project investigates the hypothesis of closing BPF by bronchoscopic injection of autologous mesenchymal stem cells (ASC) into the cavity of the fistula. This project, based on a creation of an animal model, allows to know if and how ASC work when injected into the cavity of the fistula: we may first be able to observe and to predict how ASC interact with the healthy and damaged bronchial tissues, how bronchial secretions and respiratory acts interfere with ASC growing and eventually how ASC can effectively close or at least reduce the entity of the BPF. High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter diseasefree intervals. The increasing relevance of the cancer stem cell (CSC) hypothesis and the impact of CSCassociated markers in the carcinogenesis of solid tumors may provide potential prognostic implications in lung cancer. We propose that a collective genetic analysis of established CSC-related markers will generate data to better define the role of putative CSCs

in lung adenocarcinoma (LAC). Our study reveals that CSC-associated markers: OCT4A, CD133 and ALDH are involved in the initial phase of carcinogenesis of LAC, and can be used as predictors of early stage LAC and poor disease-free intervals. In addition, this work validates the relevance of the CSC hypothesis in LAC. An electronic nose in the discrimination of patients with non-small cell lung cancer. Exhaled breath contains thousands of gaseous volatile organic compounds (VOCs) that may be used as non-invasive markers of lung disease. The electronic nose analyzes VOCs by composite nano-sensor arrays with learning algorithms. It has been shown that an electronic nose can distinguish the VOCs pattern in exhaled breath of lung cancer patients from healthy controls. We hypothesized that an electronic nose can discriminate patients with lung cancer from healthy controls by analyzing the VOC-profile in exhaled breath, thus offering an early diagnosis of lung cancer. Thoracic Tumors: Medical Oncology Pharmacogenetics/pharmacogenomics studies. Predictive biomarkers are the foundation of “personalized medicine” and pharmacogenomics is one of the tools aimed at identify them. Therefore, this approach is a promising one 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 real-time PCR the profile of genes involved in platin 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. Among all of studied genes, 5’-NT was the only gene differently expressed in the responders (complete/ partial response) and no responders (stable/progressive disease) patients (p = 0.016) (unpublished data). In

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DiseaseOriented Research 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. Research about rare diseases of the thorax. • Carcinoids: Lung carcinoids are rare malignant neuroendocrine tumors, accounting for the 1.2% of all lung cancer cases. Histologically, it is possible to distinguish between typical (TC) and atypical carcinoids (AC) according to the number of mitosis and the presence of necrosis. A differential diagnosis between typical and atypical carcinoids is relevant for clinicians in order to correctly discuss prognosis and follow up with patients after surgery, as well as for surgical decision-making in borderline cases and as a main inclusion criteria in experimental studies of systemic treatment. We performed an Affymetrix microarray gene expression profiling to identify a gene signature specific for atypical lung carcinoids. Among the most overexpressed genes in atypical versus typical carcinoids, we selected GC (vitamin D-binding protein or Graves Diseases Susceptibility To-gene) and CEACAM1 (carcinoembryonic antigen family member) that we validated by means of qRTPCR and immunohistochemistry as new potential diagnostic markers. When GC and CEACAM1 are evaluated together, they proved to be a potent tool able to discriminate ACs from TCs, resulting in the definition of an IHC assay possibly suitable for routine diagnostic procedures. • Thymoma: Thymoma is a rare neoplasm of the thymus that originates in the gland’s epithelial tissue with an incidence approximately of 0.15 per 100,000 person/years in the United States of America. We carried out a retrospective analysis to investigate the

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clinical outcome of patients with thymoma treated at our Institute. The study was in cooperation with Tumor Registry and Chest Surgery Division. This retrospective study was planned to investigate the prognosis of thymomas according to the histology and disease extension. In details will try to confirm the activity of ‘standard’ systemic treatment and define which kind of treatment (e.g., chemotherapy, octreotide, biologic treatment, etc.) could be considered better according to tumor histology. The second step of the research will be addressed to a better molecular characterization of tumors with unfavorable features facilitating the identification of prognostic factors and/or predictive factors for more active treatments. • Large cell lung cancer: The low incidence of large cell carcinoma of the lung (LCC) lead to the lack of evidence based data for clinical decision-making. The histological characteristics of 130 patients with an initial diagnosis of LCC at our Institution from November 1995 to December 2009 were retrospectively reviewed by IHC according to 2004 World Health Organization criteria. Considering that survival data of LCNEC in our series are similar to those reported in literature for non small cell lung cancers and that no differences are present with non-LCC when untreated, our results strengthen the indication to peri-operative chemotherapy in stage II and III LCNEC. Research about specific subgroups. • Lung cancers in young patients: We investigated the clinical features, outcome and incidence of drugable targets of lung cancers in patients ≤40 years old. The patients in this retrospective analysis were selected from the Tumor Registry of the European Institute of Oncology. Young patients were compared to 2 different control groups (41-64 and ≥65 years).

Compared with the control groups, the young group contained more women, more never-smokers and more patients presenting with advanced disease. Nineteen of 34 young patients with adenocarcinoma had tumors with specific molecular alterations: nine ALK translocations, five EGFR-, one PI3KCA- and five k-ras-mutations. An EGFR mutation or ALK translocation was found in 7/18 (39%) smokers and in 6/14 non-smokers (43%). Lung cancers in young patients have peculiar features and similar outcome compared to those in older patients. The unexpectedly high rate of driver-gene mutations, which seems unrelated to smoking habit, should be considered in clinical decision-making and suggest an intrinsic susceptibility of young patients with lung cancer to carcinogenesis. • NSCLC patients subjected to a stage shift due to the new TNM edition: Waiting for randomized clinical trials establishing the role of adjuvant therapy in tumors larger than 5 cm without lymph node invasion that shifted from stage IB (6th TNM) to stage II (7th TNM), we derived the rate of shifted patients in our series and analyzed the relationship between specific patient- and tumor- characteristics, and clinical outcome, in order to identify putative prognostic factors. Nearly a quarter of patients shifted from stage I (6thTNM) to stage II (7thTNM), raising a major need for information on the effects of adjuvant chemotherapy in this group of patients. Our findings suggest that randomized clinical trials aimed at addressing this topic should consider only tumor dimensions as principal selection criteria. • EGFR inhibitors beyond focal progression: Recent data show that EGFR pathway and its inhibition maintain their role after progression of disease during EGFR TKI therapy in NSCLCs. We conducted a retrospective study with the aim of evaluating efficacy

and feasibility of prosecution of EGFR TKI therapy beyond focal progression associated to locoregional radiotherapy. The longer disease control observed in our patients suggests that continuation of EGFR TKI beyond focal progression associated to a locoregional treatment is an efficacious therapeutic strategy. Head and Neck Cancer: Surgery Phase IIB Randomized, Placebo Controlled Trial of Pioglitazone for Oral Premalignant Lesions: An InterConsortium Collaborative Study (IEO S500/409). 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 13 participating clinical sites. 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 the mouth) or erythroplakia of any histology. So, the primary objective of this Phase IIB randomized, placebo-controlled trial is to assess the efficacy of pioglitazone 45 mg qd given for 24 weeks in subjects with oral premalignant lesions. Lymphatic mapping in oropharyngeal cancer: integration of dynamic lymphoscintigraphy - lymphoscintigraphy fluorescent indocyanine green (IEOS629/411). Neck treatment in cN0 tongue squamous cells carcinoma is still debated. Sentinel node (SN) technique could help the decision making, but is still unclear which kind of neck dissection must be performed in SN+ cases. In 2006 we published the lymphatic mapping study on 14 N0 patients affected by advanced tongue cancer.

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DiseaseOriented Research Contralateral drainage occurred in 11 patients and in two of them metastatic nodes were found on the contralateral side only. Metastases were found only in radioactive lymph nodes. The limits of this technique were the need of lymphoscintigraphy of specimens as to compare preoperative and postoperative imaging and the impossibility to visualize the lymphatic ways during surgery. Our purpose is to overcome these limits comparing lymphoscintigraphy with Tc-99 and nearinfrared fluorescence imaging using indocyanine green die. Role of human papillomavirus infection and other co-factors in the aetiology of head and neck cancer in Europe and India. (Retrospective multicentric study granted by the CEE VII Framework) (IEO N101/11). 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. We will assemble and analyze a large collection of plasma/sera and HNC tissues from 42 centers in 16 European countries as well as HNC tissues from 7 Indian centers together with epidemiological and clinical data. HPV status in human specimens will be evaluated by different assays in central laboratories. 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. Evaluation of the ecographic score (N90/11). The present study arise from the objective difficulties of the histological examination in some particular and borderline conditions, such as follicular neoplasm, inadequate, false negative or false positive. The main proposal of the present study are: a) to identify the

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echoic features of benign and malignant thyroid nodules, in order to define different group risk and to assign a specific score; b) to correlate echo graphic data with pre-surgical cytological data and with histological data; c) suggest a new score accounting for pre-surgical clinical, cytological and echo graphic evaluations.

main endpoint is the evaluation of the efficacy of the overall treatment strategy. The randomization to one of two chemotherapeutic regimens is aimed at a rough examination of their relative clinical advantages and limits and to the biological evaluation of predictors of response.

Central Neck Dissection in differentiated Thyroid Cancer (N117/12). The aim of this study is to demonstrate that central neck dissection should be limited to the compartment describing a predictable territory of regional recurrences, in order to reduce the associated morbidity.

Surgical organ preservation strategy in advanced III-IVa stage laryngeal cancer (N73/10). In the last 2 decade Chemoradiation in the many countries has been become standard treatment strategy such as nonsurgical organ preservation protocol in respectable stage III and IV cancer of the larynx. The principal aim of this study to evaluate the impact of surgical function-preservation treatment followed by adjuvant radiotherapy or concomitant chemoradiotherapy based on pTNM in advanced stage laryngeal cancer in terms of Overall Survival (OS), Disease Free Survival (DFS) and Organ Preservation.

Predictive models in relationship of postoperative morbidity and length of stay in Hospital in patients with cancer of the oral cavity and oropharynx (N91/11). Squamous cell carcinoma of the head and neck is the sixth most common cancer worldwide, with about 560,000 incident cases and 300,000 cancer-related deaths estimated annually. Surgical removal usually provides the best chance of achieving long-term remission for oral cavity and oropharyngeal cancer. Typically speech, swallowing and respiration are markedly impaired post-operatively. Patients are often elderly, smokers and drinkers (alcohol) and may be undernourished and have comorbidities. These factors seem increase the risk of postoperative complications. Our aim was to obtain a risk assessment nomogram that facilitates better organization of admissions, improves patient care, and gives surgeons pre-warning of high risk cases that may modify therapeutic choices. A phase 2 study of primary chemotherapy followed by laser resection in intermediate stage laryngeal cancer. The clinical study planned is a randomized phase II trial, aiming to evaluate the overall activity and efficacy of the combination of primary chemotherapy followed by laser surgery in patients with selected T2-3 N0 squamous cell carcinoma of the larynx. The

compare the first ten patients (2002-2005) to receive (TLM) for supraglottic cancer with the first ten (20072011) to receive (TORS) for the same disease. Eligibility was cT1-2 (or cT3 with minimal pre-epiglottic space involvement) cN0-cN2c disease in both groups.

Predictive Factors of Postoperative Complications and Long Hospital Stay after Laryngeal Cancer Surgery (N76/10). The treatment of advanced laryngeal cancer has changed significantly in the last two decades, with the less frequent use of total laryngectomy, as this procedure is increasingly employed just as salvage treatment. The frequency of complications is higher in these procedures. The objective of this study was to identify risk related to the incidence of postoperative complications and their association with the length of hospital stay. Transoral robotic surgery versus transoral laser microsurgery for resection of supraglottic cancer. Transoral laser microsurgery (TLM) is well established method for treatment of supraglottic cancer. Transoral robotic surgery (TORS) is an emergent method for the same disease. Present study compares these two methods. The aim of this retrospective study is To

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Disease-Oriented Research – Abdominal Tumors DiseaseOriented Research

Division of General and Laparoscopic Surgery Bruno ANDREONI, MD Director

STAFF Director, HepatoBilioPancreatic Unit: Antonio Chiappa, MD Deputy Director: Maria Laura Cossu, MD Assistant: Emilio Bertani, 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

Activities 2012.

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 hepatobiliopancreatic, 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 2012, 405 major surgical procedures (general anesthesia) were performed (28% more than budgeted for the year) with a total income of 3,015,171 ¤ (27% more than budgeted): very satisfying results, when compared to the limited available resources (number of surgeons, beds and operating sessions assigned to the division). Fifty-three per cent of patients came from outside the region, sure sign of “attractiveness”. In 2012 the personnel’s main clinical aim was to provide appropriate services in accordance with international and local (Oncological Commission Regione Lombardia, COR) guidelines. Hepatobiliopancreatic Unit (Director: Antonio Chiappa) The IEO-HPB Unit proposes novel and multidisciplinary approaches to the treatment of liver, pancreas and biliary tract cancers. The main aim of the dedicated team is an accurate analysis of each and every case in order to offer the best treatment among all possible options. The IEO-HPB works in close collaboration with other departments (medical oncology, diagnostic and interventional radiology, radiotherapy, nuclear medicine,

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endoscopy) to study and treat these particular diseases, offering “tailored” solution that can guarantee long term survival and adequate quality of life. Educational activities Both Directors are University professors, therefore they carry out an intense pre- and post-graduate educational activity as required by the IEO-Milan University agreement. The Division of General and Laparoscopic Surgery of the European Institute is part of the training network of the Specialization School in General Surgery of the Milan University.

Research Activities In his capacity as Coordinator of the COR Group “Appropriateness, quality and costs of surgical procedures for digestive tumors”, the Director of the Division is the Scientific Coordinator of the following observational multicentric studies: • “Clinical pathway (PDTA) in radically-resected rectal tumors, reconstructed through BDA (Regional Patient Database) methodology”. It is a multicenter study by the Regional Oncology Network (ROL), approved by the Ethical Committees of the 49 participating Surgery Units (including IEO). The main aim of the study is to verify if treatments performed in clinical practice

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DiseaseOriented Research for locally advanced rectal cancer comply with ROL guidelines (that were drawn by all local oncologists based on international guidelines). In 2012, 112 patients with locally advanced rectal cancer were enrolled. An interim analysis is planned for September 2013. • “Comparison of clinical results after surgery and biomolecular characteristics of screening detected vs non-screening detected vs interval colo-rectal cancers”. It is a multicenter study approved by the Ethical Committees of the 34 participating Surgery and Pathology Units (including IEO, which is the coordinating Center). The main aim of the study is to verify if colorectal tumors have different clinical and biological characteristics according to the way of diagnosis. In 2012, 98 patients were enrolled. Biomolecular tests (K-RAS and BRAF mutation, microsatellite instability, DNA methylation, Whole Exome Sequencing) are ongoing. • “Comparison of appropriateness, quality and costs of surgical procedures for colo-rectal tumors with open vs laparoscopic vs robotic techniques”. It is a multicenter study by the Regional Oncology Network (ROL) coordinated by IEO aimed at verifying quality and costs of surgical procedures for colorectal cancer. The present protocol is based on a 2009 IEO monocentric study (results published in Int J Colorectal Dis 2011, 26:1317). The IEO Surgery Units have so far enrolled 18 patients. A group of IEO experts (surgeons, anesthesiologists, case managers, ward and operating room nurses, data managers, administrative personnel – Health Services Head Office, Pharmacy, Purchase Department, Business Management, IT Department, etc.) was formed to carry out the present study. An interim analysis is planned for September 2013 to define improvement actions to be implemented during the second part of the study. A final verification of results in terms of procedure appropriateness is planned.

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The Director of the Hepatobiliopancreatic Unit and the other surgeons of the division take part (as principal investigators or co-researchers) in a number of multicentric studies in collaboration with other prestigious Centers for Digestive Surgery, both national and international. The list of these studies can be found in the Clinical Disease-Oriented Research (Abdominal Tumors) section.

Recurrence and prognostic factors in patients with aggressive fibromatosis. The role of radical surgery and its limitations. World J Surg Oncol. 2012 Sep 10;10:184. Corbellini C, Vingiani A, Maffini F, Chiappa A, Bertani E, Andreoni B Retroperitoneal pararenal isolated neuro fibroma: report of a case and review of literature. Ecancermedicalscience. 2012;6:253 Epub 15/5/2012

Publications Biffi R, Botteri E, Bertani E, Zampino MG, Cenciarelli S, Luca F, Pozzi S, Cossu ML, Chiappa A, Rotmensz N, Bazolli B, Magni E, Sonzogni A, Andreoni B. Factors predicting worse prognosis in patients affected by pT3 N0 colon cancer. Long-term results of a monocentric series of 137 radically resected patients in a 5-year period. Int J Colorectal Dis. 2012 Aug 30. [Epub ahead of print] Bonomo G, Della Vigna P, Monfardini L, Orgera G, Chiappa A, Bianchi PP, Zampino MG, Orsi F Combined therapy for the treatment of technically unresectable liver malignancies: bland embolization and radiofrequency thermal ablation within the same session. Cardiovasc Intervent Radiol [epub ahead of print 21/1/2012] Biffi R, Fattori L, Bertani E, Radice D, Rotmensz N, Misitano P, Cenciarelli S, Chiappa A, Tadini L, Mancini M, Pesenti G, Andreoni B, Nespoli A Surgical site infections following colorectal cancer surgery: a randomized prospective trial comparing common and advanced antimicrobial dressing containing ionic silver. World J Surg Oncol. 2012;10(1):94 [epub ahead of print 23/5/2012] Bertani E, Testori A, Chiappa A, Misitano P, Biffi R, Viale G, Mazzarol G, De Pas T, Botteri E, Contino G, Verrecchia F, Bazolli B, Andreoni B

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Disease-Oriented Research – Abdominal Tumors DiseaseOriented Research

Division of Abdomino-pelvic Surgery Roberto BIFFI, MD Director

STAFF Unit of Minimally Invasive Surgery Director: Paolo Pietro Bianchi, MD Unit of Integrated Abdominal Surgery Director: Fabrizio Luca, MD Deputy Director: Simonetta Pozzi, MD Senior Assistant: Sabine Cenciarelli, MD Assistants: Wanda Petz, MD, Manuela Valvo, MD Clinical Researchers: Massimiliano Zuccaro, MD (Fondazione IEO), Igor Monsellato, MD (Fondazione Umberto Veronesi) Research Fellow: Veronica Reusmann, MD (Fondazione Umberto Veronesi) Residents: Fara Uccelli, MD, Marco Marino, MD Clinical Fellow: Maria Laura Cossu, MD (until Oct 2012) Data Manager: Sergio Volpe, MSc Secretary: Benedetta Clementelli Head Nurse: Marina Mancini

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Activities 2012.

Established on May 2007, the Division of Abdomino-Pelvic Surgery included since December 2011 the Unit of Minimally Invasive Surgery (Director: Dr Paolo P. Bianchi), becoming Division of Abdomino-Pelvic and Minimally Invasive Surgery; in the same time, a new Unit was established (Integrated Abdominal Surgery), directed by Dr Fabrizio Luca. One thousand five hundred and sixty oncology surgical procedures were carried out during 2012, aimed at treatment of the following conditions: oesophageal, gastric, small bowel, colorectal, liver and pancreas carcinomas. In addition, staff Physicians maintained specific expertise and know-how in integrated surgical treatment of trunk and limb-roots sarcomas, gastrointestinal stromal tumors (GIST), primitive and metastatic tumors located in kidneys and adrenal glands, neuro-endocrine tumors of the gastrointestinal tract. A significant portion of routine clinical activity usually involves a close cooperation with other IEO clinical Divisions (Gynecologic 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 tumors, visceral deposits of melanomas). Treatment of peritoneal carcinomatosis and pelvic recurrences are regularly part of the surgical activity of the Division; the activation on 2009 of the Ovarian Cancer Center for Excellence by the Gynecologic Oncologic Surgery Division offered the opportunity for an even closer cooperation between gynaecologists and abdominal surgeons, as shown by more than two hundred surgical high-complexity procedures per year, carried out with a multidisciplinary approach. A significant clinical research activity was carried out, and a number of papers were published in close cooperation with Gynecology Oncologists of IEO.

A close collaboration with the Division of Genetics and Oncologic Prevention was established, and clinical and pathology features of the first patient undergoing total prophylactic gastrectomy in Italy for a CDH1 mutation were provided. She was a 41 female patient, operated on for a lobular breast cancer when she was 37, whose pedigree showed cases of gynecological, breast and gastric cancer. She was found positive for E-cadherin (CDH1) mutation. Esophago-gastro-duodenoscopy showed minimal afthoid lesions and a tiny ulcer of the antrum, with negative histology. She was advised to undergo prophylactic total gastrectomy, which was performed on Dec 2, 2012.

The pathology investigation of the surgical specimen showed three localizations of diffuse, signet ring cell gastric carcinoma, with a maximum histological diameter of 0.45 mm.. According to TNM classification, pathology stage was pT1a N0. A significant part of the surgical procedures are carried out with a minimally invasive approach (laparoscopic and robotic), at present time regularly applied to the treatment of tumors arising from colon and rectum, stomach, liver, spleen, pancreas and adrenal glands. Therapeutic choices are routinely made in agreement with other specialists, such as medical oncologists, radiotherapists, endoscopists, nuclear medicine

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DiseaseOriented Research specialists and interventional radiologists, by means of team case-discussing within a dedicated task-force. Taking responsibility for highly complex oncology cases, a strong link with the Anestesiology and Intensive Care Division is demanded, in order to provide intensive treatments for critically ill surgical patients. Minimally invasive colorectal cancer surgery using a surgical robot started during 2007; a study on robotassisted rectal cancer resection using the Da Vinci system, a newly developed four-arm robotic device, was concluded, and a paper, collecting a relevant clinical series of fully robot-assisted rectal and left colon cancer resection, was published in Annals of Surgical Oncology-2009. More than 600 patients were so far treated with this technique. On January 2010 a collaborative paper with US and Italian groups was published, again in 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. More recently, a paper was published in Journal of Robotic Surgery, investigating the pros and cons of robotic approach in treatment of rectal cancer, comparing this innovative technique with open technique. A recent paper was published in Annals of Surgery, aiming at prospectively evaluate the impact of robotic surgery for rectal cancer on sexual and urinary functions in male and female patients. A preservation of urinary and sexual functions was demonstrated, due to the superior movements of the wristed instruments that facilitate fine dissection, coupled with a stable and magnified view that helps in recognizing the inferior hypogastric plexus. Seven editions of a 2-days full immersion Master Course in Robotic Abdomino-Pelvic Surgery were held since October 2010 to December 2012, with participation of attendees of surgical teams coming from Italy, France, Belgium, The Netherlands and

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Pakistan. In September 2012 Dr Luca’s Unit of Integrated Abdominal Surgery has earned the distinction of being named a Colorectal Epicenter, a designation given to hospitals that have excellent surgical outcomes and advanced research and teaching programs. Moreover, Dr Bianchi is Member of the Research Committee of the EAES (European Association for Endoscopic Surgery and other Interventional Techniques). 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 on November 2012. 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) and the International Group who provided clinicians with an evidence-based overview of all topics related to ultrasound vascular access. A multi-center Italian trial aimed at evaluating the effects on surgical morbidity of glutamine parenteral administration in oncology patients undergoing major surgical procedures was carried out. Conclusion was that perioperative glutamine does not affect outcome in wellnourished gastrointestinal cancer patients. A paper was published in Annals of Surgery. In addition, Dr Biffi was Investigator in a multi-center Italian observational study – enrolling more than 1,000 patients – dealing on the prevalence of caloric-proteic malnutrition in the oncology outpatient setting (SCRINIO Project). Preliminary data were published on Supp Care Cancer-2009, whereas final results were published on Supp Care Cancer-2012.

Biffi R, Luca F, Pozzi S, Cenciarelli S, Valvo M, Sonzogni A, Radice D, Ghezzi TL. Operative blood loss and use of blood products after full robotic and conventional low anterior resection with total mesorectal excision for treatment of rectal cancer. J Robot Surg. 2011 Jun; 5(2):101-107. Epub 2010 Dec 16. Biffi R, Fattori L, Bertani E, Radice D, Rotmensz N, Misitano P, Cenciarelli S, Chiappa A, Tadini L, Mancini M, Pesenti G, Andreoni B, Nespoli A Surgical site infections following colorectal cancer surgery: a randomized prospective trial comparing common and advanced antimicrobial dressing containing ionic silver. World J Surg Oncol. 2012 May 23;10:94. doi: 10.1186/1477-7819-10-94. Lamperti M, Bodenham AR, Pittiruti M, Blaivas M, Augoustides JG, Elbarbary M, Pirotte T, Karakitsos D, Ledonne J, Doniger S, Scoppettuolo G, Feller-Kopman D, Schummer W, Biffi R, Desruennes E, Melniker LA, Verghese ST. International evidence-based recommendations on ultrasound-guided vascular access. Intensive Care Med. 2012 Jul;38(7):1105-17. doi: 10.1007/s00134-012-2597-x. Epub 2012 May 22. Luca F, Valvo M, Ghezzi TL, Zuccaro M, Cenciarelli S, Trovato C, Sonzogni A, Biffi R. Impact of robotic surgery on sexual and urinary functions after fully robotic nerve-sparing total mesorectal excision for rectal cancer. Ann Surg. 2013 Apr; 257(4):672-8. doi: 10.1097/SLA.0b013e318269d03b.

Publications Bianchi PP, Petz W, Casali L Laparoscopic lymphatic road mapping with blue dye and radioisotope in colon cancer. Colorectal Dis. 2012 Nov;13 (7): 67-9.

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Disease-Oriented Research – Abdominal Tumors

Cristiano CROSTA, MD Director

STAFF Deputy-Director: Davide Ravizza, MD Senior Assistant: Cristina Trovato, MD Assistants: Giuseppe de Roberto, Stefania de Lisi, MD (until february 2013) Research Fellows: Annalisa de Leone, MD (until October 2012), Ivana Bravi, MD Data Manager: Darina Tamayo, PharmD Secretaries: Paola Colli, Elena Degani Head Nurse: Fiorella Zoccatelli Auxiliares: Ines Chilan, Gabriela Halasag (until October 2012), Ferdinando Ponti (until October 2012)

DiseaseOriented Research

Division of Endoscopy Giancarla FIORI, MD Co-Director

Activities 2012.

The imperative target of the Division is the patient’s satisfaction through the adequate diagnostic-therapeutic pathway/s. Advanced techniques are assessed in our division that enable us to offer a minimal invasive treatment for oncologic patients. During last year a total of 12.497 endoscopic procedures were performed. The main interventional procedures included mucosectomy and submucosal dissection of early cancer and large gastrointestinal lesions debulking and stenting of malignancies, treatment of bleeding lesions, endoscopic ultrasonography with fine needle aspiration. The Division continues as a leading center for Italian Colorectal Cancer Screening Programme. As part of the program of International Medical Education, the division provides the advanced training into the interventional endoscopy and procedures with innovative techniques. Patient’s monitoring before, during and after endoscopic procedures, together with the reprocessing of endoscopes and endoscopic devices are imperative goals of the Division. Publications Trovato C, Sonzogni A, Ravizza D, Fiori G, Tamayo D, De Roberto G, de Leone A, De Lisi S, Crosta C Confocal laser endomicroscopy for in vivo diagnosis of Barrett’s oesophagus and associated neoplasia: A pilot study conducted in a single Italian center. Dig Liver Dis. 2013 Jan 24. [Epub ahead of print] Puntoni M, Branchi D, Argusti A, Zanardi S, Crosta C, Meroni E, Munizzi F, Michetti P, Coccia G, De Roberto G, Bandelloni R, Turbino L, Minetti E, Mori M, Salvi S, Boccardo S, Gatteschi B, Benelli R, Sonzogni A, DeCensi A A randomized, placebo-controlled, preoperative trial of allopurinol in subjects with colorectal adenoma. Cancer Prev Res (Phila). 2013 Feb;6(2):74-81.

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Belsey J, Crosta C, Epstein O, Fischbach W, Layer P, Parente F, Halphen M Meta-analysis: efficacy of small bowel preparation for small bowel video capsule endoscopy. Curr Med Res Opin. 2012 Dec;28(12):1883-90.

De Roberto G, de Leone A, Tamayo D, Fiori G, Ravizza D, Trovato C, De Lisi S, Crosta C. Stent migration after stent-in-stent technique using a biodegradable stent. Endoscopy. 2012;44 Suppl 2 UCTN:E51-2.

Masci E, Mangiavillano B, Crosta C, Fiori G, Trovato C, Viaggi P, Zambelli A, Buffoli F, Staiano T, Manfredi G, Manguso F, Arena M, Santoro T, Viale E, Testoni PA Interobserver agreement among endoscopists on evaluation of polypoid colorectal lesions visualized with the Pentax i-Scan technique. Dig Liver Dis. 2013 Mar;45(3):207-10.

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Medical Division of Gastrointestinal Tumors Aaron GOLDHIRSCH, MD Director (ad interim)

STAFF Medical Unit of Gastrointestinal and Neuroendocrine tumors Director: Nicola Fazio Senior Deputy Director: Maria Giulia Zampino Assistants: Elena Magni, Francesca Spada Data Managers: Sabrina Boselli, Valentina Liparulo, Eleonora Sparacio

Activities 2012.

Our global clinical activity is mainly outpatient- and research-based. More commonly it is included within an integrated multidisciplinary approach, involving other specialities. Intravenous and oral chemotherapy, molecular targeted agents, and biotherapy are usually managed. Regular weekly multidisciplinary meetings have been ongoing for more than ten years, one for GI tumors and one for NET. We have also a regular mono- and multi-disciplinary secondopinion out-patient activity. We consider local and international guidelines and recommendations, and we are included in regional and national clinical networks. Upper GI In locally advanced esophago-gastric and bilio-pancreatic cancers we perform perioperative treatment involving chemotherapy +/- radiotherapy, usually after a baseline multidisciplinary discussion based on biological characteristics of the tumor, clinical characteristics of the patient and goals of treatment. In metastatic stage we usually propose first-line chemotherapy +/- biotherapy possibly within a clinical trials. A close collaboration with the Unit of palliative care is usual. In NET we have a specific team, including the following specialities: medical oncology, surgery, endocrinology, gastroenterology/endoscopy, pathology, nuclear medicine, radiotherapy, clinical pathology. For each specialty there are one or more specific referrals for

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NET. Over the years the number of clinical discussions, new patients, and second opinions have been markedly increasing. For most patients with NET who come to our Institute, spontaneously or, more frequently, referred by other physicians/hospitals, we discuss within the board three main points: diagnosis reliability, staging completeness, and prognosis evaluation. After that we share early and late goals of treatment and a possible global therapeutic strategy. A pathology revision by the referral pathologist is usual. Lower gastrointestinal Systemic treatment of colorectal cancer (CRC) is performed in adjuvant and metastatic setting tailored by biomolecular profile, appropriate instrumental work up and clear and specific objective of cure. Different settings of metastatic CRC are usually differently considered: resectable, potentially resectable or unresectable. In liver-dominant metastatic disease we collaborate with interventional radiologists for liver-directed procedures, including arterial chemoembolisation or chemotherapy. In locally advanced rectal adenocarcinoma we are involved with radiotherapists and surgeons in a preoperative multidisciplinary approach with neoadjuvant intent. In squamous cell anal carcinoma we collaborate with radiotherapists for chemoradiation as first option for cure. We are also particularly involved in treatment of small bowel rare tumors. Publications Everolimus plus octreotide LAR in patients with advanced lung neuroendocrine tumors: analysis of the phase III, randomized, placebo-controlled RADIANT-2 study. Fazio N, Granberg D, Grossman A, Saletan S, Klimovski J, Panneerselvam K, Wolin EM. CHEST Sep 2012; Epub ahead of print Metastatic and locally advanced pancreatic endocrine carcinomas: analysis of factors associated with disease

progression. Francesco Panzuto, Letizia Boninsegna, Nicola Fazio, Davide Campana, Maria Pia Brizzi, Gabriele Capurso, Aldo Scarpa, Filippo G de Braud, Luigi Dogliotti, Paola Tomassetti, Gianfranco Delle Fave, and Massimo Falconi. J Clin Oncol. 2011 Jun 10;29(17):2372-7. Docetaxel, Cisplatin, and Fluorouracil; Docetaxel and Cisplatin; and Epirubicin, Cisplatin, and Fluorouracil As Systemic Treatment for Advanced Gastric Carcinoma: A Randomized Phase II Trial of the Swiss Group for Clinical Cancer Research. Arnaud D. Roth, Nicola Fazio, Roger Stupp, Stephen Falk, Ju¨rg Bernhard, Piercarlo Saletti, Dieter Köberle, Markus M. Borner, Kaspar Rufibach, Rudolf Maibach, Martin Wernli, Martin Leslie, Robert

Glynne-Jones, Lukas Widmer, Matthew Seymour, and Filippo de Braud. J Clin Oncol 2007; 25:3217-3223. Rectal cancer. Zampino MG, Labianca R, Beretta G, Santoro L, Magni E, Gatta G, Leonardi MC, Chiappa A, Biffi R, de Braud F, Wils J. Crit Rev Oncol Hematol 2009; 70(2): 160-82. Concurrent cisplatin, continuous infusion fluorouracil and radiotherapy follone by tailored consolidation treatment in non metastatic anal squamous cell carcinoma. Zampino MG, Magni E, Leonardi C, Santoro L, Petazzi E, Fodor C, Petralia G, Trovato C, Nolè F, Orecchia R. BMC Cancer 2011; 11: 55.

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UPPER GI Confocal Laser Endomicroscopy in early detection of esophagus dysplasia. A trial was activated to investigate the diagnostic potential of Confocal Laser Endomicroscopy in detecting the dysplasia associated to the Barrett’s esophagus. A comparison with the standard Seattle biopsy protocol will be done. Locally advanced resectable gastric cancer. The role of perioperative chemotherapy and post-operative radiation therapy is currently investigated (ITACA-S2 Italian multicenter, 4-arm, randomized trial). Lymphadenectomy in gastric cancer. We analyzed data of 114 patients who underwent gastrectomy and extended lymph node dissection for node-negative adenocarcinoma of the stomach between 2000 and 2005, extracted by our Tumor Registry. As more extended lymph node resection offered survival benefit, lymphadenectomy involving more than 15 lymph nodes should be performed. A paper was published, and a new investigation is foreseen. Metastatic gastric cancer. HER2 positive tumors: we are studying whether pertuzumab can ameliorate the results of trastuzumab (international randomized, JACOB trial). HER2 negative tumors: we are investigating the role of S1 (an oral fluoropyrimidine) in the diffuse histotype (international, multicenter, phase III, randomized trial), and we are comparing two different regimens of threedrug polychemotherapy (GISCAD trial: low TOX vs EOX). Hereditary Diffuse Gastric Carcinoma. In collaboration with the Division of Genetics and Oncology Prevention, a study for detection of CDH-1 germ-line mutation in patients under 45 affected by diffuse gastric carcinoma is currently run.

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DiseaseOriented Research

Research Activities

Pancreatic and biliary tract carcinoma. We participated in the successful phase III study with NAB-paclitaxel combined with Gemcitabine for metastatic patients, and now we are keeping on with the same regimen in the locally advanced study (GISCAD trial). We are participating to a multicenter randomized phase II-III study entitled “Peri- or post-operative chemotherapy in resectable pancreas carcinoma” (PACT-15). In biliary tract carcinoma, we are conducting a retrospective biological study to detect the expression of Hedgehog pathway. Liver metastases. An observational, prospective, multicenter study entitled “Analysis of phosphoproteomics for targeted therapy of colorectal liver metastases (TASK 2)” is now ongoing. LOWER GI Quality assurance in colonoscopy practice. An observational multicenter study is now ongoing, aimed at evaluation of the patients’ compliance for different bowel cleansing preparations for colonoscopy. In collaboration with the Italian Society of Digestive Endoscopy (SIED), a Quality Assurance program was started, with the aim to prospectively assess some quality indicators of good endoscopy practice, like the rate of full colonic examinations (intended as cecum visualization) or detection rate of polyps and adenomas. Moreover, a prospective multicenter study dealing with complications’ rate after endoscopic polypectomy is now ongoing. Chromoendoscopy. A multicenter, prospective, randomized, controlled, single-blind trial is now ongoing with the objective to evaluate whether chromoendoscopy – in conjunction with confocal laser endomicroscopy – is able to identify more accurately neoplasias arising in ulcerative colitis patients as compared to standard video-endoscopy.

Confocal Laser Endomicroscopy in endoscopic surveillance of premalignant colonic lesions. A prospective study on the role of Confocal Laser Endomicroscopy in facilitating the endoscopic surveillance of patients suffering from familial adenomatous polyposis and ulcerative colitis was started. Metagenomics of colorectal cancer. This project is carried out with IFOM-IEO Researchers, and aims to identify new biological markers in the blood of colon cancer patients for diagnosis and prognosis. We postulate a correlation between neutrophils, coagulation defects and HMGB1 in colorectal cancer. HMGB-1 has been recently linked to coagulation. We associated different redox forms of HMGB1 with two exclusive functions of the molecule (chemoattractant or inflammatory mediator), but the identity of the form involved in coagulation or neutrophil polarization is unknown. We want to characterize an unconventional subset of N2 neutrophils found only in the blood of cancer patients and elucidate the role of HMGB-1 in the development of this phenotype. Biomolecular characteristics of colorectal cancers. A prospective study is active, aimed at comparison of clinical results after surgery and biomolecular characteristics of screening detected- vs non-screeningdetected vs interval colorectal cancers. Bowel preparation for elective colorectal surgery. A randomized IEO monocentric study is currently run: Use of polyethilene-glicol-based mechanical intestinal preparation vs enema (glycerin 5%) in patients undergoing colo-rectal resection for malignant neoplasia. 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 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. Robot-assisted minimally invasive surgery for rectal cancer. An international, randomized clinical trial comparing laparoscopic vs robotic rectal resection for treatment of rectal cancer (ROLARR trial, RObotic vs LAparoscopic Rectal Resection) was started on 2012; 15 patients were so far enrolled. Preservation of the genito-urinary function in patients undergoing surgery with robotic technique for rectal cancer. This study is aimed at assessing prospectively the genito urinary function preservation in patients undergoing nerve sparing robotic surgery for the treatment of rectal cancer. Integration of Diffusion-Weighted magnetic resonance imaging in surgical planning for robotic nerve sparing total mesorectal excision. Magnetic Resonance (MRI) is used to stage patients with rectal tumors and new techniques like diffusion-weighted magnetic resonance imaging (DW-MRI) should improve the identification and depiction of the hypogastric plexus. The potential benefits on preservation of genito-urinary function in routine use of this technique integrated with robotic surgery are under investigation in this prospective trial. Low Anterior Rectal Resection-Technique. a randomized trial is now carrying out on reconstruction of bowel continuity after low anterior rectal resection for cancer. It is entitled: Reconstruction with colic J-Pouch versus colo-rectal direct anastomosis after RAR: impact on anastomotic dehiscence, intestinal functions and quality of life.

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DiseaseOriented Research Quality assurance in rectal cancer surgery. Two observational studies are active: a. Clinical pathway (PDTA) in radically-resected rectal tumors, reconstructed through BDA (Regional Patient Database) methodology. b. Comparison of appropriateness, quality and costs of surgical procedures for colo-rectal tumors with open vs laparoscopic vs robotic techniques. Factors affecting prognosis in colon cancer. We retrospectively analyzed the data of consecutive patients, extracted by our institutional Tumor Registry, admitted for adenocarcinoma of the colon (all sites), between 2000 and 2005, and having a final pT3 N0 pathology staging after curative surgery. Data of 137 patients were obtained, with a median follow-up of 77 months (range 6-131). Only histological grade III and mucinous histotype were found to impact on cumulative incidence of colon-related events. The risk of events was found inversely proportional to the number of dissected lymphnodes. These results were published, and an extension of this study is under evaluation. Circulating Tumor Cells (CTC). An observational, prospective, IEO monocentric study is actively run. It is entitled “Identification and possible prognostic significance of Circulanting Tumor Cells (CTC) in peripheral venous blood of patients affected by locally advanced rectal cancer, diagnosed through traditional clinical exams undergoing oncologically radical surgery (open or laparoscopic)”. Medical oncology-adjuvant setting. A prospective study is ongoing, aimed at evaluation of the impact of 3 versus 6 months of FOLFOX/XELOX regimens in patients with colon cancer staged as II-high risk or III (TOSCA trial): we have included 90 patients and we are among the top ten Italian centers for enrollment. Translational research is currently carried out on biological prognostic and

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predictive factors (circulating tumor cells and their characterizations, the role of isolated tumor cells in regional limphnodes in radically resected patients, biomolecular texts as Microsatellite Instability or pharmacogenetic/pharmacogenomic profile).

Squamous cell anal carcinoma. In locally advanced squamous cell anal carcinoma we are activating an international Phase II multistitutional study on new targeted-therapy combined with radiotherapy with a curative intent.

Medical oncology-metastatic setting. The role of immunogenic response after EGFR-inhibitors (in collaboration with IFOM) and the mechanism of primary and acquired resistance to biological agents is investigated. New anticancer drugs and combinations are tested; international multicenter trials: MEHD7945A/FOLFIRI in K-RAS Wild Type, sintuzumab/FOLFIRI in K-RAS mutated cases). Together with IEO interventional radiologists, we are investigating hepatic intra-arterial injection of drug-eluting bead (MIRACLE III trial). Together with IEO radiotherapists we are investigating the administration of selective intra-hepatic radiotherapy treatment (in collaboration with SITILO, trial to be submitted to EC) in pretreated patients. In liver-dominant colorectal cancer patients with unresectable or borderline resectable metastases, we are participating in a multicenter firstline clinical trial (ABOVE study, in collaboration with Niguarda Hospital in Milan) with the aim to evaluate the percentage of conversion to surgery obtained with bevacizumab/FOLFOX regimen.

NEUROENDOCRINE TUMORS (NET) Low-intermediate pancreatic NET. We are studying whether the addition of a somatostatin analog to everolimus gives a benefit; the accrual in the international randomized phase II multicenter trial (COOPERATE-2) is now completed. Moreover we are validating efficacy and tolerability of sunitinib in the phase IV, international, single arm trial (A6181202). We are also evaluating whether inhibition of more targets on the same mTOR pathway may give some benefit; an international, randomized, two-arm, phase II trial comparing everolimus with BEZ235 (CBEZ235Z2401) is ongoing. We are also carrying out a study on the angiogenetic effect of everolimus, focused on three levels: tumor perfusion, circulating factors (e.g. endothelial circulating cells) and tumor tissue. Finally we are studying the role of BEZ235 in patients refractory to everolimus (trial CBEZ235Z2201), whereas in nonfunctioning non-pancreatic NET we are participating in the RADIANT-4 trial, a regulatory, international, randomized, phase III study comparing everolimus with placebo.

Radiation therapy - rectal cancer. We actively participate to the INTERACT trial (Phase III randomized study), regarding patients with limited local disease suitable for neoadjuvant chemo-radiotherapy. A trial evaluating the role of conservative surgery after primary chemoradiotherapy is upcoming, aiming to reduce the morbidity of extended surgery and to contribute to a better patient’s quality of life. Other upcoming trials are investigating the role of adjuvant therapy after radically resected metastases and the role of “induction” chemotherapy in locally advanced rectal cancer with the aim to reduce radiotherapy use.

Low-grade NET Two trials are upcoming: a. NET from different origins exhibiting FDG-PET scan positivity: combination with Lutetium177-Dotatate b. Low-intermediate grade NET from different origins: combination with temozolomide. Both drugs are given metronomically. MGMT, TS, and other biological factors will be studied. Low-grade NET with progression after somatostatin analog: a. a combination with trans-arterial embolization (multicenter Italian trial); b. a higher dose of somatostatin analog (lanreotide 180

mg/4 weeks) in patients progressive at conventional dose of octreotide/lanreotide (multicenter Italian trial). Finally, we are conducting two projects: a. a pharmacogenetic/pharmacokinetic modelling approach to the prediction of everolimus tolerability, in patients with pNET treated with everolimus; b. set-up and molecular analysis of models of tumor xenograft in NET. OTHERS Totally implantable central venous access devices. A cost analysis of a randomized 3-arm trial on best approach to central veins for long-term chemotherapy deliverance is under evaluation. A new revision of the International Guidelines on evidence-based overview of all topics related to ultrasound vascular access is programmed. Home Enteral Nutrition. A randomized prospective trial investigating nutritional and clinical impact of prolonged home enteral nutrition vs dietetic counseling in surgical oncology patients was recently closed. A data analysis is ongoing. Modulation of postoperative insulin resistance. A randomized, prospective multicenter trial is ongoing, aimed at evaluation of the metobolic effects of preoperative oral carbohydrate ingestion on postoperative insulin resistance and infections’ rate in surgical oncology patients. It is entitled PR.O.C.I. (PReoperative Oral Carbohydrate Ingestion).

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Disease- Area Surgical Oriented Research

Disease-Oriented Research – Urogenital Tumors

Division of Urologic Cancer Surgery Ottavio DE COBELLI, MD Director

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

Activities 2012.

The Division of Urology is concerned with the treatment of all urological tumors, including prostate, bladder, kidney, testis and penis cancer. In 2012, 1392 patients were admitted for surgical treatment. Among these we performed both endoscopic, open and robotic surgery. Among open procedures we performed 9 radical retropubic prostatectomies, 21 radical nephrectomies, 5 nephron sparing procedures and 64 cystectomies. Of these procedures, 38 were with urinary reconstruction. We had a further increase of robotic surgery, with 418 robotic assisted prostatectomies and 78 kidney surgery (26 robotic radical nephrectomies and 52 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 transurethral resection of bladder (316 patients) and ureteral stent insertion (100 patients). Accurate followup 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 34 months are similar to

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the open radical prostatectomy; however, the main advantage of 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. The recent development of multiparametric MRI, which combines anatomical T2W images with functional techniques, such as diffusion-weighted MRI and dynamic contrast-enhanced, has significantly improved local staging of prostate cancer, and has shown the potential to influence the decision to preserve neurovascular bundles and the extent of surgical margins in robotic prostatectomy The intraoperative frozen-section procedure, which provides histological assessment of the surgical margin, is attractive as it enables the surgeon to intraoperatively demonstrate the oncologic safety of an nerve sparing radical prostatectomy procedure. In light of the promising results reported for mp-MRI and IFS separately, we hypothesized that their combined use would improve the oncological outcome and functional results. Plans for research projects in 2013/14 include a further increase of MRI and IFS in order to validate these kind of treatments in patients with prostate cancer. Moreover, in cooperation with the Department of Medicine, trials for Hormone Refractory Prostate Cancer are ongoing (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 pr ocessed 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 superfi- cial transitional cell carcinoma of the bladder. Publications De Cobelli O, Brescia A, Mazzoleni F, Musi G, Matei DV. A novel “intuitive” surgical technique for right robotassisted retroperitoneal lymph node dissection for stage I testicular NSGCT. World J Urol. 2012 Dec 16 Brookman-May S, May M, Shariat SF, Xylinas E, Stief C, Zigeuner R, Chromecki T, Burger M, Wieland WF, Cindolo L, Schips L, De Cobelli O, Rocco B, De Nunzio C, Feciche B, Truss M, Gilfrich C, Pahernik S, Hohenfellner M, Zastrow S, Wirth MP, Novara G, Carini M, Minervini A, Simeone C, Antonelli A, Mirone V, Longo N, Simonato A, Carmignani G, Ficarra V Features Associated with Recurrence Beyond 5 Years After Nephrectomy and Nephron-Sparing Surgery

for Renal Cell Carcinoma: Development and Internal Validation of a Risk Model (PRELANE score) to Predict Late Recurrence Based on a Large Multicenter Database (CORONA/SATURN Project). members of the CORONA project and the SATURN project. Eur Urol. 2012 Jun 22. Matei DV, Brescia A, Mazzoleni F, Spinelli M, Musi G, Melegari S, Galasso G, Detti S, de Cobelli O. Robot-assisted simple prostatectomy (RASP): does it make sense? BJU Int. 2012 Dec;110(11 Pt C):E972-9. doi: 10.1111/j.1464-410X.2012.11192.x. Epub 2012 May 18. Simonato A, Varca V, Gacci M, Gontero P, De Cobelli O, Maffezzini M, Salvioni R, Carini M, Decensi A, Mirone V, Carmignani G. Adherence to Guidelines among Italian Urologists on Imaging Preoperative Staging of Low-Risk Prostate Cancer: Results from the MIRROR (Multicenter Italian Report on Radical Prostatectomy Outcomes and Research) Study. Adv Urol. 2012;2012:651061. doi: 10.1155/2012/651061. Epub 2012 May 15. Matei DV, Renne G, Pimentel M, Sandri MT, Zorzino L, Botteri E, De Cicco C, Musi G, Brescia A, Mazzoleni F, Tringali V, Detti S, de Cobelli O. Neuroendocrine differentiation in castration-resistant prostate cancer: a systematic diagnostic attempt. Clin Genitourin Cancer. 2012 Sep;10(3):164-73. doi: 10.1016/j.clgc.2011.12.004. Epub 2012 Mar 7.

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Disease-Oriented Research – Urogenital Tumors

Medical Division of Urogenital and Head & Neck Tumors Aaron GOLDHIRSCH, MD Director (ad interim)

STAFF Medical Oncology Unit of Urogenital Tumors Director: Franco Nolè, MD Senior Assistants: Elena Verri, MD, Maria Cossu Rocca, MD, Daniela Cullurà, MD Assistant: Gaetano Aurilio, MD, PhD Data Managers: Laura Adamoli, Dominique Ronzulli

Urogenital Tumors Medical team of our Division consists of specialists in the area of urogenital tumors. The cooperation among the professionals involved in the urogenital program allow our team to develop a competent and comprehensive treatment program for each patient. Cancers of prostate, bladder, kidney and testis are diseases that are included in the spectrum of clinical and research programs of the Division.

Kidney Cancer Treatment options and recommendations for kidney cancer, depend on several factors, including the type and stage of cancer, possible side effects, the patient’s preferences and overall health. Targeted therapy is a treatment that targets the cancer’s specific genes, proteins, or the tissue environment that contributes to cancer growth and survival. This type of treatment have shown promise in treating metastatic kidney cancer. In 2012, at our Medical Division of Urogenital Tumors we have taken care of 150 patients with advanced kidney cancer treated in clinical research programs with innovative treatments or with standard therapies.

Prostate Cancer Prostate cancer is the most common cancer among males in the Western World and is the second leading cause of male mortality. 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

Bladder Cancer Bladder cancer is the 4th most commonly diagnosed cancer in men and 9th in women. Patients with bladder cancer or with upper urinary tract cancer, have their situation discussed weekly by urologic oncologists, surgeons, radio-oncologists who evaluate x-ray images, pathology reports and patient history to determine the best treatment option for the patient. One of the most

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metastasis) and by response to androgen deprivation. Systemic therapies of prostate cancer, include hormonal treatment, chemotherapy, biologic therapies, targeted approaches, and treatments specifically designed to attack prostate cancers that have spread to the bone. In our Division, patients are considered for treatment after a multidisciplinary discussion and after a clinical evaluation, to determine which treatment or combination of treatments will be most effective, considering the specific features of disease, offering the possibility to participate to national or international clinical studies with innovative treatments or a standard treatment. In 2012, at our Medical Division of Urogenital Tumors we have taken care of 250 patients with prostate cancer treated in clinical research programs or with standard therapies.

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interesting option that we offer to the patient with muscle invasive bladder cancer, who refuse cystectomy or with comorbidities that contraindicate surgery is a bladder sparing program, in collaboration with the Division of Radiotherapy and Urologic Cancer Surgery. In this program, patients with muscle invasive bladder cancer, are treated with IG-IMRT radiotherapy plus chemotherapy with cisplatin. Our preliminary results support this treatment modality in select patients with muscle invasive bladder cancer. In patients with stage IV bladder cancer, platinum-based combination chemotherapy regimens are the standard of care. Together, with the patients with bladder cancer we discuss treatment options, offering

to the patients the best treatment option. In 2012, at our Medical Division of Urogenital Tumors we have taken care of 100 patients with bladder or urinary tract cancer treated in clinical research programs or with standard therapy. Testicular cancer Testicular cancer is the most common solid tumor diagnosed in men between the ages of 15 and 34. In Italy, testicular tumors are the most common malignancy (11%) in males less than 50 years. Thanks to advances in the treatment of testicular cancer, the prognosis is excellent for most men diagnosed with testicular cancer.

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Disease- Area Surgical Oriented Research When found and treated early, more than 95 percent of men are cured. The drugs most commonly used to treat testicular cancer are bleomycin, etoposide and cisplatin. This combination is known as BEP chemotherapy. Other combinations of drugs are also used depending on the stage of the cancer, or if it’s come back after treatment. Chemotherapy for testicular cancer is given: after surgery as adjuvant treatment; to treat testicular cancer with distant metastases; to treat recurrent testicular cancer (salvage chemotherapy). In 2012, at our Medical Division of Urogenital Tumors we have taken care of 130 patients with testicular cancer. Clinical Activity on Head Neck Tumors The Head and Neck Cancer Program of our Division, provides medical treatment for this type of cancer, including tumors affecting tongue, tonsils, mouth, palate, jawbone, sinuses, pharynx, larynx, salivary glands and thyroid. All patient cases are presented at a multidisciplinary tumor board comprised of the patient care team, as well as radiologists and pathologists. We provide state-of-the-art treatment options, including postoperative chemotherapy after surgical treatments or chemo-radiation therapy with curative intents. In addition, programs of chemotherapy are offered to the patients for advanced disease. The Unit participates in innovative multicentric clinical trials investigating new treatments options and new drugs for head & neck cancer. Our programs, are conducted in collaboration with Division of Head & Neck Tumors and with Division of Radiotherapy and are aimed to develop new treatment modalities and to develop molecular medicine. In 2012, treatment programs of 510 patients have been discussed and planned in the weekly multidisciplinary meetings and 160 patients, with different stages of disease have been treated with medical therapy alone or in combination with radiation therapy in programs of “curative” or “post-operative” chemo-radiation therapy.

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The Unit is also involved in the organization of basic and advanced courses on Head and Neck and thyroid cancer. Publications Curigliano G, Spitaleri G, Magni E, Lorizzo K, De Cobelli O, Locatelli M, Fumagalli L, Adamoli L, Cossu Rocca M, Verri E, De Pas T, Jereczek-Fossa B, Martinelli G, Goldhirsch A and Nolè F. Cisplatin, etoposide and continuous infusion bleomycin in patients with testicular germ cell tumors: efficacy and toxicity data from a retrospective study. J Chemother. 2009 Dec;21(6):687-92. Spitaleri G, Matei DV, Curigliano G, Detti S, Verweij F, Zambito S, Scardino E, Rocco B, Nolè F, Ariu L, De Pas T, de Braud F, De Cobelli O. Phase II trial of estramustine phosphate and oral etoposide in patients with hormone-refractory prostate cancer. Ann Oncol. 2009 Mar;20(3):498-502. Epub 2009 Jan 12 Zampino MG, Verri E, Locatelli M, Curigliano G, Ascione G, Sbanotto A, Rocca A, Verweij F, Matei V, Scardino E, Decobelli O, Goldhirsch A, and Nolè F. Vinorelbine-based chemotherapy in hormone-refractory prostate cancer. Anticancer Res. 2006 May-Jun;26(3B):2375-80. Curigliano G, De Braud F, Teresa Sandri M, Renne G, Zorzino L, Scardino E, Rocco B, Spitaleri G, De Pas T, Noberasco C, Nolè F, Verweij F, Matei V, De Cobelli O. Gefitinib combined with endocrine manipulation in patients with hormone-refractory prostate cancer: quality of life and surrogate markers of activity. Anticancer Drugs. 2007 Sep;18(8):949-54. L. Calabrese, A. Ostuni, M. Ansarin, G. Giugliano, F Maffini, D. Alterio, M. Cossu Rocca, G. Petralia, R. Bruschini, F. Chiesa Future challenges in head and neck cancer: From the bench to the bedside?. on behalf of AROME 1, Crit Rev Oncol Hematol. 2012

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Disease-Oriented Research – Urogenital Tumors

Research Activities

Prostate Cancer Our research activity on prostate cancer addresses the issues commonly encountered by the practicing oncologists. The research themes include: - 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 prostate cancer. - Clinical trials including biological agents targeting different critical points of the signaling cascade or proteins of the mitotic machine. 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®, approved by the FDA for routine clinical use in metastatic breast cancer, colorectal cancer and in castration-resistant prostate cancer (CPRC). We investigated the role of CTCs in: • Patients with CRPC who are starting first or second line systemic treatment for advanced disease. (manuscript in preparation). • Patients with clinically localized prostate cancer eligible for radical prostatectomy (clinical stage T13NxM0, any Gleason score) with the aim to investigate the prognostic value of CTCs before and after curative treatments, the correlation between CTCs and other known prognostic factors and biomarkers. • Patients with PSA failure, following definitive treatment of prostate cancer.

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Ongoing Clinical Trials: • A Phase III study, evaluating Dasatinib, a potent orally active tyrosine kinase inhibitor, or placebo in combination with Docetaxel/prednisone in castrationresistant prostate cancer. This study completed the accrual in 2012. • A phase III double bind, placebo controlled efficacy and safety study evaluating MDV3100 in patients with progressive castration resistant prostate cancer who have previously treated with docetaxel-based chemotherapy. Renal cancer Ongoing Clinical Trials: • Sunitinib either before or after cytoreductive nephrectomy: A phase II trial in patients with metastatic renal cell carcinoma. • Multicenter, phase II evaluation of torisel as II-line treatment for metastatic RCC in patients progressing after cytokine therapy, tyrosine kinase, or angiogenesis inhibitors. • A Study of Pazopanib versus Sunitinib in the Treatment of Subjects with Locally Advanced and/or Metastatic Renal Cell Carcinoma. • “Retry”study - phase II study of Sunitinib as rechallenge third-line therapy in metastatic renal cancer. Bladder Cancer Ongoing Clinical Trials • Randomized phase II study assessing the combination of Vinflunine with Gemcitabine and Vinflunine with Carboplatin in patients ineligible to cisplatin with advanced or metastatic transitional cell carcinoma of the urothelium. • Selective Bladder Preservation Therapy for Patients with Muscle-Invasive Bladder Cancer and who are candidate to Cystectomy.

Head and Neck Cancer • Long term responders to first line chemotherapy plus cetuximab in recurrent/metastatic Head and Neck cancer: a genomic landscape approach to identify predictive biomarkers. • Evaluation of feasibility and efficacy of alternative schedules in patients treated with cetuximab + platinum based therapy for recurrent/metastatic squamous cell carcinoma of Head and Neck. • Collaboration to define national and shared guidelines in the management of main toxicity due to chemoradiation therapies Neoadjuvant docetaxel, cisplatin and 5- fluorouracil (TPF) followed by radiotherapy plus concomitant chemotherapy or cetuximab versus radiotherapy plus concomitant chemotherapy or cetuximab in patients with locally advanced squamous cell carcinoma of the Head & Neck. • A Multicenter, Randomized, Double-Blind, PlaceboControlled, Phase 3 Trial of E7080 in 131I-Refractory Differentiated Thyroid Cancer. • A Randomised, Double-Blind, Placebo-Controlled, Multi-center, Phase III Study of Post-Operative Adjuvant Lapatinib or Placebo and Concurrent Chemoradiotherapy Followed by Maintenance Lapatinib or Placebo Monotherapy in High-Risk Subjects with Resected Squamous cell carcinoma of the Head & Neck. • A randomised, double-blind, placebo-controlled, phase III study to evaluate the efficacy and safety of afatinib (BIBW 2992) as adjuvant therapy after chemo-radiotherapy in primary unresected patients with stage III, IVa, or IVb loco-regionally advanced Head and Neck squamous cell carcinoma.

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Disease-Oriented Research – Gynecological Tumors

Division of Gynecologic Cancer Surgery Angelo MAGGIONI Director

STAFF Senior Deputy Directors: Fabio Landoni, MD, Luca Bocciolone, MD, Vanna Zanagnolo, MD Deputy Directors: Giovanni Aletti, MD Research Fellows: Drusilla Rollo, MD, Tiziana Tomaselli, MD Data Managers: Maira Biggiogero, ScD, Sara Boveri, ScD Secretaries: Gloria Gavioli, Diana Valli Head Nurse: Emanuela D’Anna Unit of Preventive Gynecology Senior Deputy Director: Dorella Franchi, MD Deputy Director: Luca Bazzurini, MD Assistants: Michele Peiretti, MD, Eleonora Preti, MD Consultants: Raffaella Di Pace, MD, Sarah Igidbashian, MD, Simoma Moroni, MD, Sabina Oldani, MD, Mariarosa Pittelli, MD, Francesca Sanvito, MD, Laura Spinaci, MD, Noemi Spolti, MD, Paola Zamperini, MD Resident: Aylin Vidal Urbinati, MD Data Manager: Sara Boveri, ScD Secretaries: Anna Steinwurzel, Simona Tognetti Midwife: Linda Franzini Research Nurse: Eugenia Tomas Roldan Nurse: Patrizia Capodivento

Activities 2012.

The Division of Gynecology provides all services involving the diagnosis, treatment and follow-up of gynecologic oncology patients. The Division includes 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

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Mario SIDERI Co-Director (Unit of Preventive Gynecology)

various activities, including surgery (minor, major and minimally-invasive), research, clinical trials, and early diagnosis. 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 (intraoperative radiotherapy) in patients with recurrent cervical, endometrial and vulvar cancer. Minimally-invasive robotic and laparoscopic surgery is commonly applied to the treatment of different gyneco-logical malignancies. The foundation of Robotic School in Gynecologic Oncology was promoted in 2009 for teaching innovative minimallyinvasive surgical technique. Members of the Division also have institutional teaching responsibilities that mainly involve training residents and fellows, but they are also involved in Continuing Medical Education (CME) programs. The foremost educational objective of European School of Abdomino-pelvic surgery in Gynecologic Oncology (ESAGON), founded in November 2009, 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. The School involves both institutions and individuals from different countries. 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). The Unit of Preventive Gynecology encompasses the fields of prevention, surveillance and diagnosis of gynecologic cancerous and pre-cancerous lesions. The clinical activities involve 16 gynecologists. The results of the research activities are published in peer reviewed journals, IF was 62.7 in 2012. 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 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 center, were both adolescents and women in older ages can receive HPV vaccination. 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 2012 more than 6.000 transvaginal or transabdominal pelvic US have been performed and about one hundred prophylactic salpingo-ophorectomies. 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 350 diagnostic and operative hysteroscopies are performed yearly for uterine polyps and abnormal uterine bleeding. Conservative treatment includes hormonal therapy of endometrial atypical hyperplasia and early endometrial cancer with progestational agents.

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Disease-Oriented Research – Gynecological Tumors

Medical Division of Gynecologic Tumors Nicoletta COLOMBO Director

STAFF Senior Deputy Directors: Gabriella Parma, MD Assistants: Maria Teresa Lapresa, MD, Rosanna Mancari, MD, Annalisa Garbi, MD Research Fellow: Melita Moioli, MD Data Managers: Maira Biggiogero, ScD, Sara Boveri, ScD Secretaries: Gloria Gavioli, Diana Valli Director Fertility and Procreation Unit: Fedro Peccatori, MD, PhD Research Fellow: Maria Anna Sarno Counselor: Leonora Chiavari

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Activities 2012.

The Division of Gynecologic Oncology strictly interacts with the Division of Gynecologic Surgery and Gynecologic Prevention to provide diagnosis, treatment and follow-up of women affected by gynecological malignancies. Each staff member is a fully-trained gynecologic oncologist, responsible for various activities within the Division, including the administration of anti cancer agents and supportive care. All staff members are involved in multidisciplinary patient management, shared decision making within dedicated tumor boards, pathology discussion and radiological review of the most significant cases. Patients and their families access to psychological support and decisional counseling, if needed. A specific focus on translational research and clinical trials characterize the research activities of the Division. Clinicians from the Division of Gynecologic Oncology are principal investigators in several international and national trials, addressing new treatment strategies in ovarian cancer, endometrial cancer and cervical cancer. Most of the clinical trial are run 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. A particular attention is devoted to young patients affected by early stage epithelial ovarian cancer, germ cell tumors, endometrial cancer and cervical cancer. For this specific group of patients a dedicated clinical pathway has been identified, with a special emphasis in preserving their fertility. Recently, the Unit of Fertility and Procreation has joined the Division of Gynecologic Oncology. Young patients with gynecological malignancies, breast cancer, malignant lymphomas and other solid tumor have the opportunity to freeze ovarian cortex or oocytes prior to chemotherapy, surgery or radiation therapy. Gamete storage is performed in cooperation with other Istitutions, thus allowing fertility recovery also after gonadotoxic treatments.

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Research Activities

The treatment of gynecological tumors 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.

and in vivo assays to ascertain whether PKH26-retaining cells obtained from “ovospheres” indeed behave as bona fide CSC. In parallel, we have extended our approach to primary cells derived from normal fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE), namely the two tissues that are thought to give rise to OC. Our efforts are expected to yield an efficient and reliable protocol for the systematic purification of cancer stem cells from fresh surgical OC samples, which will enable us to get a molecular profile of this elusive cell subpopulation.

OVARIAN CANCER Basic research. Many aspects in the clinical evolution of ovarian carcinoma (OC), such as its peritoneal dissemination, relapse and chemoresistance, support the hypothesis that this neoplastic disease is fuelled by a sub-population of tumor-initiating cells or cancer stem cells (OCSC). Thus, the identification and characterization of OCSC would not only advance our knowledge on the biological mechanisms underlying OC development and malignancy, but it would also open new therapeutic options for the eradication of such a devastating disease. So far, the isolation of CSC in OC and other solid tumors has relied on the expression of surface markers. Such an approach, however, has limitations due to the relatively low specificity and heterogeneity of CSC markers. Therefore, we opted for a CSC isolation strategy based on the stemness-related properties of self-renewal and quiescence. In particular, our approach relies on labelling the bulk population of primary tumor-derived cells with the lipophilic fluorescent dye PKH26. The dye is progressively diluted in actively proliferating cells, while it is retained in slowcycling cells (such as stem cells). When combined with the culture of primary tumor cells under non-adherent conditions, this results in the formation of spheroids that contain one or few label-retaining cells, i.e. the putative CSC. We are currently employing a series of in vitro

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 gynecologic 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

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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 long-term survival. We have published our experience (Peiretti et al, Gynecol Oncol, 2012; Zapardiel et al, Int J Gynecol Cancer, 2011; 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 cancer. Patients with advanced ovarian cancer and intra-abdominal complete resection will have clinically/radiologically not detectable lymph node involvement (about 30%). Recently, we participated in a randomized prospective multicenter study (LION), investigating the hypothesis that systematic pelvic and aortic lymph-adenectomy, 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. Since the introduction of platinumbased chemotherapy, further advances in treatment of patients with ovarian cancer have been poor. In this regard, molecular targeted therapeutic agents herald a new era for cancer treatment. In the setting of epithelial ovarian cancer, a growing body of evidence supports the use of anti-angiogenic agents in combination with

standard chemotherapies. In this regard, last years we completed the enrollment in two international studies evaluating the role of BIBF1120 (a potent small molecule triple kinase inhibitor targeting VEGFR 1, VEGFR 3 and FGFR 1) and bevacizumab (ROSiA trial) in combination with carboplatin and paclitaxel in first line therapy. As primary treatment, we have just activated two studies (TRINOVA-3 and MITO-16/MANGO-OV2) to assess respectively the role of AMG-386 (a potent selective inhibitor of angiopoietins) and the clinical and biological prognostic factors of bevacizumab in combination with standard chemotherapy in FIGO stage III-IV ovarian cancers. We recently published the results of a phase III trial testing the efficacy of abagovomab maintenance therapy in patients with ovarian cancer in first clinical remission. The final analysis showed that the administration of abagovomab did not improve neither recurrence–free survival nor overall survival (Sabbatini et al. JCO 2013). We are waiting for the final analysis of a Gynecologic Cancer Intergroup randomized phase III trial regarding the efficacy and the safety of pazopanib, a potent and selective multitargeted receptor tyrosine kinase inhibitor that blocks tumor growth and angiogenesis, as maintenance treatment at the end of primary chemotherapy. In platinum partially sensitive recurring ovarian cancer, we are starting with a multicenter, randomized, non comparative, phase II study on the efficacy and safety of the combination of bevacizumab and trabectedin with or without carboplatin. In addition, should soon restart the enrolment in two randomized Italian studies, temporarily stopped last year for the unavailability of pegylated lyposomal doxorubicin. The MITO-8 (PLD compared to CBDCA-PTX) and INOVATYON (PLD-CBDCA compared to PLD-Trabectedin) trials could make a significant contribution to the long debate whether the extending of the platinum-free interval with a nonplatinum combination prolongs survival in this setting of patients. For resistant/refractory disease, we have

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DiseaseOriented Research recently completed several randomized studies regarding the activity of AMG-386, NGR-hTNF and MM121 (an antiErbB3 human monoclonal antibody) in combination with standard chemotherapy. Still ongoing is a phase II trial evaluating intermittent and continuous administration of OSI-906, an inhibitor of insulin-like growth factor 1 receptor (IGF-1) which plays a significant role in the growth and survival of cancer cells, in combination with weekly paclitaxel. A phase III trial of PLD plus AMG-386 or placebo in patients with relapsing ovarian cancer both platinum resistant and partially sensitive was also recently restarted. Finally, the PENELOPE study, recently approved, will investigate the role of pertuzumab, a monoclonal antibody directed against HER2, in combination with standard chemotherapy, paclitaxel, topotecan or gemcitabine, in recurrent platinum resistant ovarian cancer. 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 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

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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. Over the past three decades, a wealth of scientific evidence has accumulated, showing the importance of angiogenesis in solid tumor growth and metastasis. The addition of Bevacizumab to chemotherapy has been shown to improve PFS and/or OS in several solid tumors. Recently, we started a randomized clinical phase II trial od Carboplatin-Paclitaxel compared to CarboplatinPaclitaxel-Bevacizumab in advanced (Stage III-IV) or recurrent endometrial cancer. In addition, we have just activated a phase II single-arm study to evaluate the efficacy of oral dovinitib, a potent inhibitor of receptor tyrosine kinase with an antiangiogenetic mechanism, as second line therapy in patients with advanced and/ or metastatic endometrial cancer. 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 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. 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. 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 2011). Early cervical cancer: Surgery. Total abdominal radical hysterectomy and pelvic lymph node dissection is the

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DiseaseOriented Research 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. 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 chemoradiation in patients with FIGO stage Ib2-IIA >4 cm, II B cervical cancer. The study is currently ongoing.

It 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 2012 we performed 912 surgical operations with the Da Vinci System (extrafascial hysterectomy: 622; radical hysterectomy: 167; pelvic lymphadenectomy: 39 and, aortic lymphadenectomy: 68).

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. 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.

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Disease- Area Surgical Oriented Research

Disease-Oriented Research – Melanoma

Surgical Division of Melanoma Alessandro TESTORI, MD Director

STAFF Deputy Director: Elisabetta Pennacchioli, MD (Surgical Oncologist) Assistant: Francesco Verrecchia, MD (Surgical Oncologist) Fellow: Antonio Intelisano, MD (Surgical Oncologist) Psyco-oncologist: Beatrice Colombo, (Psychologist) Fellows: Salvatore Alfieri, MD (Medical Oncologist), Angelo Battaglia, MD (School Of Medical Oncology Univ. Catania), Giulio Vitali MD (School Of General Surgery Univ. Genova) Senior Assistants: Federica Baldini, MD (Dermatologist) Massimo Mosconi, MD (Dermatologist), Giulio Tosti, MD (Dermatologist) Assistant: Giuseppe Spadola, MD (Dermatologist) Secretaries: Barbara Bottari, Monica Burla Research nurses: Maria di Leo, Chiara Pari Data Managers: Francesco Cataldo, Concetta Riviello

Activities 2012. The most advanced new

technologies are available in order to improve the accuracy of the diagnosis of skin tumors. A high resolution digital dermoscopy is proposed for mapping of nevi and follow- up of atipical pigmented lesions. Furthermore, a reflectance confocal laser scanning microscopy (RCSLM) is used for the “in vivo” evaluation of clinically difficult skin lesions. This 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

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horizontally and vertically to the skin surface. The method has been proved to be highly useful in early melanoma and other skin neoplasms detection. Surgery is felt as a standard, but daily practice demonstrates that surgical oncology differs center to center, bringing the discussion to the qualitative importance of surgical procedures. These are all represented, safely managed at IEO and 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 or superficial not operable primary and metastatic cancers. First Institute in Europe at the IEO we implemented a new form of treatment of liver cancers, the 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 many times in one single patient and it is minimally invasive allowing treatment for sicker patients. Surgical specimens, with the consensus of patients, may be used both to make tissue available for vaccine studies and to perform molecular analysis to target new therapeutical approaches. 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. Description of clinical practice • During 2012, more than 8000 patients were examined in the outpatients clinics. • The surgical procedures conducted on melanoma patients were almost 400. • 40 sarcoma and 1200 not melanoma skin cancer or various other cutaneous lesions were operated under general or local anesthesia.

Basal Cell Carcinoma Patients affected by basal cell carcinoma usually receive surgery and/or radiotherapy as the only curative approach. When the disease rarely become metastatic or could not be approached by surgery or radiotherapy any more, systemic options were lacking. Recently, a new drug, Vismodegib, is being tested in a clinical trial with interesting results. The study is still ongoing at our Center.

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Disease-Oriented Research – Melanoma

Medical Division of Melanoma Aaron GOLDHIRSCH, MD Director (ad interim)

STAFF Medical Oncology Unit of Melanoma Director: Pier Francesco Ferrucci, MD Senior Vice Director: Emilia Cocorocchio, MD, Biologist: Chiara Martinoli, PhD, (Fondazione Grazia Focacci) Fellows: Salvatore Alfieri, MD Medical Oncologist, Angelo Battaglia, MD (School of Medical Oncology, Univ. Catania)

The Division and the incorporated Unit are devoted to the diagnosis and treatment of skin cancers and soft tissue sarcoma in strict cooperation with the EIO Division of Melanoma and soft tissue Sarcomas. 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, the multidisciplinary approach within the melanoma and sarcoma program allow our Team to develop competent and comprehensive treatment options for each patient which requires coordination between a variety of specialties, including: • Dermatology • Dermatopathology/Surgical Pathology • Surgical Oncology • Medical Oncology • Radiology • Radiotherapy • Psycho-oncology • Basic Research and Immunology

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Activities 2012.

Melanoma. In the context of the Multidisciplinary team and the Melanoma Cancer Center, patients with advanced disease are mainly enrolled in chemo-, targeted and immuno-therapeutic clinical trials and treated within the Medical Oncology Unit of Melanoma. The group participated to the development of the antiBRAF and anti-CTLA4 targeted therapy, the first 2 drugs impacting on survival in advanced melanoma patients. These drugs revolutionized our therapeutic approach, widening the options and expanding hope for a cure in many patients affected by this disease. In the meantime, new clinical trials exploring the combination of targeted therapies with chemotherapy and/or immunotherapies started, offering new opportunity to induce durable responses which could impact on quality of life of our patients. Interestingly, our Team include a molecular biologist working on basic research at IEO-Campus and a Psycho-oncologist offering, if requested, support to all the patients undergoing specific surgical or medical treatments and their family. Finally, adjuvant targeted therapy options as per histology are under evaluation through participation in 2 international clinical trials. Sarcoma The Task Force on Sarcoma started his work at the end of 2011 and involve medical oncologists, surgeons, pathologists, radiotherapists and radiologists in order to offer a comprehensive approach also to patients affected by this disease. Clinical trials and innovative treatments are offered as part of this strict collaboration within different professionals. Publications Ferrucci PF, Tosti G, Pietro A, Passoni C, Pari C, Tedeschi I, Cataldo F, Martinoli C, Testori A. Newly identified tumor antigens as promising cancer vaccine targets for malignant melanoma treatment. Curr Top Med Chem. 2012 Jan 1;12(1):11-31. IF: 4.112

Danielli R, Ridolfi R, Chiarion-Sileni V, Queirolo P, Testori A, Plummer R, Boitano M, Calabrò L, Rossi CD, Giacomo AM, Ferrucci PF, Ridolfi L, Altomonte M, Miracco C, Balestrazzi A, Maio M. Ipilimumab in pretreated patients with metastatic uveal melanoma: safety and clinical efficacy. Cancer Immunol Immunother. 2012 Jan;61(1):41-8. Epub 2011 Aug 11. IF: 4.293

S1470-2045(12)70324-8. Epub 2012 Aug 13. IF: 22.59

Di Giacomo AM, Ascierto PA, Pilla L, Santinami M, Ferrucci PF, Giannarelli D, Marasco A, Rivoltini L, Simeone E, Nicoletti SV, Fonsatti E, Annesi D, Queirolo P, Testori A, Ridolfi R, Parmiani G, Maio M. Ipilimumab and fotemustine in patients with advanced melanoma (NIBIT-M1): an open-label, single-arm phase 2 trial. Lancet Oncol. 2012 Sep;13(9):879-86. doi: 10.1016/

Maio M, Nicolay HJ, Ascierto PA, Belardelli F, Camerini R, Colombo MP, Queirolo P, Ridolfi R, Russo V, Fonsatti E; NIBIT, Parmiani G. Eighth annual meeting of the Italian network for tumor biotherapy (NIBIT), Siena, October 7-9, 2010. Cancer Immunol Immunother. 2011 Jun;60(6):901-7. Epub 2011 Mar 24. IF: 4.293.

Di Pietro A, Tosti G, Ferrucci PF, Testori A. The immunological era in melanoma treatment: new challenges for heat shock protein-based vaccine in the advanced disease. Expert Opin Biol Ther. 2011 Oct;11(10):1395-407. Epub 2011 Jul 30. IF: 3.279.

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Disease-Oriented Research – Melanoma

Research Activities

The research activities on Melanoma are integrated within the Melanoma Cancer Center under the Direction of Dr Testori. Basic Research The most interesting opportunity for us is to bridge basic research to the clinical one. For this reason a molecular biologist is working at IEO-Campus under the Grazia Focacci Foundation support. She reported interesting data on Maspin expression during melanoma progression and metastatization, which could allow to use this protein as a surrogate marker that may have utility in predicting prognosis and monitoring the treatment antitumor effects in melanoma. In this setting, another study is performed in collaboration with the Genova INT on the analysis of CTLA4 polimorphisms which could allow the predict response to Ipilimumab. Moreover, our research program is focused, this time in collaboration with the Milan INT, on molecular evaluation of the microenvironment and angiogenesis in patients enrolled in a specific protocol and receiving an antiangiogenic drugs combined with chemotherapy. In particular, this is a satellite study of a clinical one, exploring the safety and efficacy of the combination of Dacarbazine and Bevacizumab. 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. In the near future we are planning to investigate the role of circulating tumoral cells (CTCs) in melanoma patients receiving different locoregional and systemic treatments. Clinical Trials • A Phase III, randomized, double-blinded study comparing the combination of the BRAF inhibitor, dabrafenib and the MEK inhibitor, trametinib to dabrafenib and placebo as first-line therapy in subjects with unresectable (Stage IIIc) or metastatic (Stage IV)

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BRAF V600E/K mutation-positive cutaneous melanoma. • A Phase III, randomized, open-label study comparing the combination of the BRAF inhibitor, dabrafenib and the MEK inhibitor, trametinib to the BRAF inhibitor vemurafenib in subjects with unresectable (stage IIIc) or metastatic (stage IV) BRAF V600E/K mutation positive cutaneous melanoma. • Phase III, randomized, double blind, placebo-controlled study of vemurafenib (ro5185426) adjuvant therapy in patients with surgically resected, cutaneous braf-mutant melanoma at high risk for recurrence. • A multicenter, open label, randomized Phase II trial of the MEK inhibitor pimasertib (MSC1936369B, formerly known as AS703026) or dacarbazine in previously untreated subjectswith NRAS mutated locally advanced or metastatic malignant melanoma. • Safety and efficacy of intramuscular electrotransfer of plasmid amep in patients suffering from advanced or metastatic melanoma: an open-label phase i/ii clinical trial the aimm study (amep in metastatic melanoma). • A Randomized Double-Blind Phase III Study of Ipilimumab Administered at 3 mg/kg vsat 10 mg/ kg in Subjects with Previously Treated or Untreated Unresectable or MetastaticMelanoma. • Randomized phase III open label study of BMS-936558 versus best investigator choice in patients affected by advanced melanoma patients progressing after antiCTLA4 treatment • A prospective, multicenter, randomized, open-label, active controlled, two-parallel groups, phase 3 study to compare the efficacy and safety of masitinib at 7.5 mg/kg/day to dacarbazine in the treatment of patients with non-resectable or metastaticstage 3 or stage 4 melanoma carrying a mutation in the juxtamembrane domain of c-kit. • A single arm, open label, phase II, multicenter study to assess the safety of vismodegib (GDC-0449) in patients with locally advanced or metastatic basal cell carcinoma.

• Multicentric, open lable of expanded access to assess the safety of RO 5185426 (VEMURAFENIB) in patients affected by metastatic melanoma. • A randomized phase III, double blind study on Vitamine D supplementation for resected stage II melanoma patients. • Adjuvant peginterferon alfa-2b for 2 years vs observation in patients with an ulcerated primary for cutaneous melnamo with T(2-4)bN0M0: a randomized phase III trial of the EORTC Melnaoma Group • BRF115252: dabrafenib (gsk2118436) for compassionate use in braf v600 mutation- positive metastatic melanoma. • Compassionate Supply of Pazopanib for Patients with Advanced Soft Tissue Sarcoma (aSTS) through a Named Patient Program.

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DiseaseMedical Area Oriented Research

Disease-Oriented Research – Leukemia and Lymphoma

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, Niccolò Frungillo, MD Data Managers: Liliana Calabrese, Mara Negri Secretaries: Daniela Antoniotti, Tiziana Masala Head Nurse: Laura Orlando Scientific Nurses: Sarah Liptrott

Activities 2012.

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 2012, 15 allogeneic transplantations and 62 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 tumor activity and reducing overall transplant related mortality. 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.

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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. Our team is currently working with immunotherapy 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. With the International Extra nodal Lymphoma Study Group (IELSG) and SAKK we have continued the enrolment of patients in already ongoing studies. Regarding trials with IEO as sponsor we have continued the enrolment of patients in our first line treatment

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DiseaseMedical Area Oriented Research studies designed for naïve patients with Hodgkin’s Lymphoma or Multiple Myeloma. The study was completed with 38 enrolled patients and results are in press. We are studying the efficacy of prolonged treatment with rituximab and R-2cda in chronic lymphocytic leukemia patients (21 patients enrolled). We also collaborate with GITMO in a multicentric study on donors. 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 aphaeresis, coding and labelling cellular therapy products. Since 2010 we have been performing extracorporeal photopheresis (ECP) to treat patients with acute/chronic GVHD or coetaneous lymphomas. Early introduced in the treatment of steroid-refractory acute and chronic GvHD, ECP is a well tolerated procedure, with very low incidence of side-effects that often allows more rapid reduction of concomitant immunosuppressive therapy. In order to better elucidate the immunomodulating effects of ECP, we are performing in vitro analysis focusing on the apoptosis process and on cytokines expression in patient blood samples before and after the procedures.

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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 centers operating at regional level.

Gallerani E, Zucchetti M, Brunelli D, Marangon E, Noberasco C, Hess D, Delmonte A, Martinelli G, Böhm S, Driessen C, De Braud F, Marsoni S, Cereda R, Sala F, D’Incalci M, Sessa C. A first in human phase I study of the proteasome inhibitor CEP-18770 in patients with advanced solid tumors and multiple myeloma Eur J Cancer. 2013 Jan;49(2):290-6.

Publications Andreola G, Babic A, Rabascio C, Negri M, Martinelli G, Laszlo D. Plerixafor and Filgrastim XM02 (Tevagastrim(®) as a first line peripheral blood stem cell mobilisation strategy in patients with multiple myeloma and lymphoma candidated to autologous bone marrow transplantation. Eur J Haematol. 2012 Feb;88(2):154-8. Renner C, Zinzani P, Gressin R, Klingbiel D, Dietrich PY, Hitz F, Bargetzi M, Mingrone W, Martinelli G, Trojan A, Bouabdallah K, Lohri A, Gyan E, Biaggi C, Cogliatti S, Bertoni F, Ghielmini M, Brauchli P, Ketterer N. A multicenter phase II trial (SAKK 36/06) of singleagent Everolimus(RAD001) in patients with relapsed or refractory mantle cell lymphoma. Haematologica. 2012 Feb 7.

We are also collaborating with the Besta Neurologic Institute of Milan, performing leukapheresis on patients affected by glioblastoma multiforme to be treated with a DC based vaccine immunotherapy. Leukapheresis are necessary to derive autologous DC.

Pastano R, Dell’agn ola C, Bason C, Gigli F, Rabascio C, Puccetti A, Tinazzi E, Cetto G, Peccatori F, Martinelli G, Lunardi C. Antibodies against human cytomegalovirus late protein UL94 in the pathogenesis of scleroderma-like skin lesions in chronic graft-versus-host disease. Int Immunol. 2012 Sep;24(9):583-91.

In February 2012 a Gruppo Italiano Infermieri in Mobilizzazione ed Apheresi (GIIMA) group has been

Scarfò L, Zibellini S, Tedeschi A, Maura F, Neri A, Bertazzoni P, Sarina B, Nalli G, Motta M, Rossini F,

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Cortelezzi A, Montillo M, Orlandi E, Ghia P. Impact of B-cell count and imaging screening in cMBL: any need to revise the current guidelines? Leukemia. 2012 Jul;26(7):1703-7

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Interdisciplinary Research

Interdisciplinary Research – Department of Pathology and Laboratory Medicine

Division of Pathology 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, Benedetta Di Venosa, MD, Maria Cristina Ghioni, MD, Mauro G. Mastropasqua, MD, Valeria Midolo De Luca, MD, Eleonora Pisa, MD, 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, Rocco Olivadese, MD Data Managers: Stefania Andrighetto, DSc, Francesca Lombardi, DSc Chief Technician: Alessandra Cavallon Technicians: Giancarlo Camerino, Carla Camia, Francesca Ciocca, Gabriele Citelli, Manuela D’Autilia, Gianluca De Marzo, Flavia Giarratano, Paola Lento, Luana Lippolis, Paolo Lopedote, Mara Lusiardi, Francesca Palumbo, Mila Schiavi, Francesco Spinelli, Tania Tamagni, Marilisa Valente 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 Cytopathology Unit 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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Giuseppe RENNE, MD Co-Director

Activities 2012.

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 20,795 histological diagnoses and 14,770 cytologic diagnoses (6,372 fine needle aspiration biopsies or extravaginal exfoliation cytology samples and 8,398 Pap-tests), with 3,775 cases seen in consultation for a second opinion and 4,564 frozen section examinations for intraoperative diagnosis. Among the different tumor types, we have examined 3,498 breast samples, 1,966 biopsies of sentinel lymph nodes, 2,744 surgical specimens of Gynecological pathology, 1,406 specimens of thoracic pathology and 408 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, 1,400 FISH assays, 1,427 molecular report with 4,954 PCR analyses and 8,024 direct sequencing have been routinely carried out in 2012 for tumor genophenotyping, including the immunohistochemical 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, methylation status of MGMT, Microsatellite instability, fluoropirimidine and irinotecan response and the N-ras. The research activities during 2012 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 PCRbased and FISH techniques, tissue microarrays, and microdissection for tumor cell enrichment.

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Interdisciplinary Research 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 66 full articles published during 2012 in peer-reviewed international journals, with an overall IF of 377.41. (mean IF: 5.7). The Division hosts the Postgraduate Medical School in Pathology of the University of Milan. Histopathology and Molecular Diagnostics Unit The 2012 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. The Unit play a role as referring center for lung cancer and neuroendocrine tumors. It participates as referral center in the national quality control system for molecular testing. The activity has been totally optimized with the validation of diagnostic protocols running on the genetic analyzer ABI 3500 Dx, on pyrosequencer Qiagen PyroMark and on the 7900 HT fast real time PCR. Recently a MassArray platform has been introduced to support mutational analysis in clinical trials. We can proudly state that our Division proposes to our stakeholders (in and out-patients, oncologists, pharma industries) a fully 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 our ISO-approved Unit is engaged in developing a robust QC and QA program. The main goal for the current year consists in offer a

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wide test spectrum to detect patients with solid tumors candidate to targeted therapies. For the first time guidelines for ALK and EGFR testing produced by Italian referral centers for lung cancer with our Division in leading position, have been produced and accepted for publication on the Journal of Thoracic Oncology. Cytopathology Unit The Unit of Diagnostic Cytology performs cytologic diagnoses for both in- and out- patients. The total number of tests in 2012 was 14,770; 6,372 of them were fine needle aspiration or extravaginal exfoliative cytology samples and 8,398 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 (R.O.S.E.: rapid onsite cytotologic evaluation). Both the technologists and the cytotecnologists are involved in this field together with the pathologist/s intraoperative charged with the diagnosis. 29 adequacy procedures were performed with the digestive endoscopists, mainly on pancreatic lesions, while 602 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 a work was accepted for oral presentation at the European Congress of Cytology in Trieste. 248 FNA of palpable breast nodules and of superficial lymph nodes were performed by two cytopathologists, while a third cytopathologist performed 51 ultra sound guided FNA of non palpable breast lesions. 158 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 and ISO. 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, with the Division of Chemoprevention on Breast Ductal Lavages (DL) and HALO tests and with the Lab of Viral control of cellular pathways and biology of tumorigenesis, trying to understand how oncogenic viruses like HPV exploit the SUMO pathway.

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Interdisciplinary Research

Interdisciplinary Research – Department of Pathology and Laboratory Medicine

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: Christian Mauro, DSc Technician Fellow: Chiara Tigano Secretaries: Elena Campanato, Erika Platano Data Manager: Maria Angela Massaro

Activities 2012. 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 2012 was about 985.000. 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.

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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. 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. To facilitate the management of critically ill patients, a Point of Care Testing (POCT) system has been implemented, with blood gas analyzers and glucometers installed in different guards, controlled and supervised by the lab. The laboratory is 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. The lab. Personnel also provide laboratory support to other clinical divisions for research protocols. Both in terms of aliquoting and storing samples, and in terms of performing esoteric tests, when requested. The laboratory has also organized a Service for external gynecologists and outpatients clinics related to the HPV testing, used for the management and prevention of cervical cancer. Publications Curigliano G, Cardinale D, Suter T, Plataniotis G, de Azambuja E, Sandri MT, Criscitiello C, Goldhirsch A, Cipolla C, Roila F, ESMO Guidelines Working Group. Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy. ESMO Clinical Practice Guidelines. Ann Oncol. 2012 Oct;23 Suppl 7:vii155-66. Cassatella MC, Zorzino L, Sandri MT. Single circulating tumor cell profiling: a new perspective for targeted therapy? Future Oncol. 2012 Oct;8(10):1253-6

Munzone E, Botteri E, Sandri MT, Esposito A, Adamoli L, Zorzino L, Sciandivasci A, Cassatella MC, Rotmensz N, Aurilio G, Curigliano G, Goldhirsch A, Nolè F. Prognostic value of circulating tumor cells according to immunohistochemically defined molecular subtypes in advanced breast cancer. Clin Breast Cancer. 2012 Oct;12(5):340-6 Origoni M, Carminati G, Rolla S, Clementi M, Sideri M, Sandri MT, Candiani M. Human papillomavirus viral load expressed as relative light units (RLU) correlates with the presence and grade of preneoplastic lesions of the uterine cervix in

atypical squamous cells of undetermined significance (ASCUS) cytology. Eur J Clin Microbiol Infect Dis. 2012 Sep;31(9):2401-6 Costa S, Venturoli S, Negri G, Sideri M, Preti M, Pesaresi M, Falasca A, Barbieri D, Zerbini M, Santini D, Sandri MT, Ghiringhello B, Caroppo Venturini N, Syrjänen S, Syrjänen K. Factors predicting the outcome of conservatively treated adenocarcinoma in situ of the uterine cervix: an analysis of 166 cases. Gynecol Oncol. 2012 Mar;124(3):490-5.

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Interdisciplinary Research

Interdisciplinary Research – Department of Pathology and Laboratory Medicine

Division of Laboratory Haematology-Oncology Francesco BERTOLINI, MD, PhD Director

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

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Activities 2012.

The Laboratory has two main clinical activities, both ISO9001, JACIE and JC Lab certified: a) diagnosis of haematological malignancies; and b) stem cell processing for transplantation. Since 2011, the lab is also offering cell sorting and purification for clinicians and scientist interested in translational research. In 2012, the lab performed more than 500 cell sorting procedures, more than 80 stem cell collections were processed for autologous or allogeneic use and more than 1,100 blood and marrow samples were studied by flow cytometry, PCR, immunohistochemistry, FISH, cytogenetics and circulating tumor-specific DNA. The repository of plasma, serum and whole blood samples from leukemia, lymphoma and myeloma patients includes nearly 7,500 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 prognostic markers

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 human adipose tissue may promote breast cancer growth, angiogenesis, migration and metastases in several preclinical models of breast cancer (Martin-Padura et al, Cancer Res 2012).

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Interdisciplinary Research

Interdisciplinary Research – Department of Pathology and Laboratory Medicine

Research Activities

The Department of Pathology involves the Divison o Pathology, Laboratory Medicine and Laboratory of Hematology-Oncology. Divison of Pathology The research activities of the Division 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. Also, 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, has involved collaboration with several Divisions within the European Institute of Oncology, as Experimental Oncology, Senology, Medical Oncology, Head&Neck Surgery, Gynaecology, Thoracic Surgery, and Chemoprevention, and has resulted in 66 full articles published during 2012 in peer-reviewed international journals, with an overall IF of 377.41. (mean IF: 5.7). The Division hosts the Postgraduate Medical School in Pathology of the University of Milan. Division of Laboratory Medicine Circulating tumor cells (CTCs) have been intensely

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investigated as new prognostic and predictive factor in many solid tumors and promising results suggest them as surrogate marker for tumor response and shorter survival, mostly in metastatic breast, prostate and colon cancer patients. Apart from their enumeration, a very challenging filed is represented by their molecular characterization, to gain insight into the metastatic process and to find possible target to candidate patients to personalized therapies. The Division has been involved in two major studies about CTCs: Role of CTCs in (neo)adjuvant setting in patients with triple negative and HER2 positive breast cancer. The aim of this study is to assess the presence of CTCs in non-metastatic breast cancer patients treated with neoadjuvant and/or adjuvant chemotherapy and to evaluate their prognostic and predictive role. The sample size is 112 patients with histologically proven early or locally advanced breast cancer (T1c N1-3; T2-T4 N0-3; M0), ER and PgR <10% and HER2 positive or triple negative. Detection and evaluation of circulating tumor cells (CTCs) in patients undergoing curative surgery (“open” or “mini-invasive”) for advanced rectal cancer detected through routine clinical practice. The aim of this prospective single institution study is to investigate the role of CTCs count in patients with locally advanced RC undergoing neo-adjuvant CT-RT. The sample size of 90 patients was reached on March 2012, and all participants are in follow-in. The human Paillomavirus (HPV) is the causative agent of cervical cancer. Among the more than 100 genotypes isolated, 14 have been recognized as ‘High Risk’, and strictly associated with cancer. Use of new molecular tests in the diagnosis and followup after treatment of precancerous lesions. This study was planned with the aim to find whether a new test based on the detection of the mRNA of the E6 and E7 oncoprotein, which are expressed when the HPV is integrated into the transforming cell, is more specific than HPV DNA-based test during the follow-up of patients treated for a CIN with conservative surgery.

Studies on circulating markers Evaluation of the diagnostic accuracy of serological biomarker (proGRP) in the differential diagnosis and monitoring of small cell lung cancer. The aim of our study is to evaluate the diagnostic utility of plasma levels of ProGRP in patients with different lung malignancies, to help in the distinction between SCLC and NSCLC. Detection of Squamous Cell Carcinoma Antigen with two Systems in the Follow-up of Patients with Cervical Cancer. Approximately 80-85% of cervical cancers are of squamous cell type and the marker of choice for the follow-up of this cancer is the squamous cell carcinoma (SCC) antigen. Recently, an automated assay for SCC determination was released. The objective of this retrospective study was to compare the results generated with the new system with the previous used, in order to evaluate the comparability of SCC measurements obtained with two different analytical systems and to evaluate the concordance of SCC values determined on both systems with clinical state. Division of Laboratory Hematology-Oncology The Division is investigating the role of adipose tissue progenitors in the progression of breast cancer. We recently reported that human white adipose tissue (WAT) progenitors promote breast cancer growth and metastases in preclinical models (Martin-Padura et al, 2012). In 2012 we found that two populations of human WAT progenitors cooperate in breast cancer angiogenesis, growth and metastatic progression. Sorting, electron microscopy, culture and in vivo studies defined human WAT CD45-CD34+CD31+CD13-CCRL2+ endothelial progenitors (EPCs) as small, undifferentiated cells overexpressing endothelial-restricted genes (VECadherin, Claudin 5; Tie-2, ICAM-2, Dll4, etc) and able to generate in vitro and in vivo mature endothelial cells. A second population of purified WAT CD45CD34+CD31-CD13+CD140b+ pericyte progenitors (PPCs) was found to overexpress perivascular genes (Endosialin,

Adam12, PDGF receptors, TGFbeta, CD44, RUNX1, etc). In vivo and in vitro, CD34+ PPCs generated differentiated CD34- pericytes and adipocytes. In co-culture, WAT EPCs and PPCs - together - induced in ductal breast cancer cell lines and primary cells an overexpression of EMT genes (SNAIL2, ZEB1, MAP1b, etc). Similarly, WAT EPCs and PPCs induced - together - an increase in breast cancer cell migration towards chemoattractants. When only EPCs or only PPCs were added to breast cancer cell cultures, their EMT- and migration-induction effects were significantly lower than those observed when both EPCs and PPCs were co-cultured together. In vivo, human WAT EPCs and PPCs increased breast cancer angiogenesis, growth and metastases in several orthotopic models. When only EPCs or only PPCs were injected, their effects on breast cancer growth and metastases were significantly reduced in comparison to the effects observed when EPCs and PPCs were injected together. Z-stack showed that functional cancer blood vessels with a lumen were made of human cells only when EPCs and PPCs were co-injected together. To understand WAT EPCs and PPCs migration potential, we measured WAT EPCs and PPCs in the blood of patients before and after different stem cell mobilization procedures. WAT-EPCs (always <1/mL before mobilization) increased to 2-800/mL after G-CSF administration. The addition of chemotherapy and/or CXCR4 inhibitors did not increase WAT-EPC mobilization. WAT-PPCs were always <1/mL before mobilization. G-CSF administration was associated to a minimal mobilization of these cells (10-70/mL), but only in 3 out of 15 patients. Again, the addition of chemotherapy and/or CXCR4 inhibitors to G-CSF did not increase PPC mobilization. WAT-EPCs, but not WAT-PPCs, were found in large numbers in human lymph nodes (LNs). Our studies indicate that human WAT EPCs and PPCs are able to migrate in LNs and blood and to promote breast cancer angiogenesis, EMT, growth, migration metastases through complementary mechanisms. We are currently investigating several candidate inhibitors of WAT EPCs and PPCs cooperation in vitro and in vivo.

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Interdisciplinary Research

Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

Division of Radiotherapy Roberto ORECCHIA, MD Director STAFF Senior Deputy Director: Barbara Alicja Jereczek-Fossa MD, PhD Deputy Directors: Roberta Lazzari, MD, Maria Cristina Leonardi, MD, Andrea Vavassori, MD Senior Assistants: Piero Fossati, MD, MSc Anna Morra, MD Assistants: Daniela Alterio, MD, Agnese Cecconi MD, PhD, Veronica Dell’Acqua, MD, Annamaria Ferrari, MD, Federica Gherardi, MD, Gaia Piperno, MD, Dario Zerini, MD Data Manager: Cristiana Fodor, MSc Fellow: Delia Ciardo (Bioengineer) Residents: Federica Bazzani, MD, Maria Bonora, MD, Isa Bossi Zanetti, MD, Mariangela Caputo, MD, Sara Colangione, MD, Samantha Dicuonzo, MD, Michela Dispinzieri, MD, Silvia Ferrario, MD, Marianna Gerardi, MD, Stefania Gottardo, MD, Andrea Maucieri, MD, Matteo Muto, MD, Roberta Mauro, MD, Sara Ronchi, MD, Ruggero Spoto, MD, Alessia Surgo, MD 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, Floriana Pansini, MSc Residents: Stefania Russo, MSc, Edoardo Mastella, MSc, Alessia Bazani, MSc TECHNICIANS (RADIOTHERAPY TECHNOLOGISTS, RTTs)

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

NURSES E Referent nurse: Claudio Acerbi AUXILIARIES Nurses: Rodolfo Cendamo, Milena Lucic, Loredana Murra, Annamaria Buonfino Auxillaries: Elena Rubio, Cosimo Persichella Secretaries: Ida Muraca, Maria Ersilia Viscusi Secretary, Reception: Nadia Zanoni

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Activities 2012.

The Division of Radiotherapy called since the beginning of 2012 Advanced Radiotherapy Center (ARC) is a university department with about 65 employees including a staff of 14 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, intra- and extra-cranial 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 Trilogy 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 with electrons, i.e. ELIOT). 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 potential radiobiological advantages of high radiotherapy dose per fraction, FAST might significantly increase the patient convenience and compliance. 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 curative brachytherapy application for small tumors (gynecological tumors, selected endocavitary or head and neck cancer etc.) or palliative approach in advanced endocavitary malignancies. There is an excellent collaboration with other departments within the frame of the disease-specific multidisciplinary teams (see Clinical Research and

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Interdisciplinary Research Management Section). Such multidisciplinary approach allows in many clinical situations for less invasive therapy (organ preservation). Apart from high precision treatment and multidisciplinary 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 Center for Oncological Hadrontherapy (CNAO) in Pavia for the definition of the clinical research protocols on the particle therapy in selected cancer patients. Mixed beam protocols (hadrontherapy and photon beam radiotherapy) for selected aggressive tumors have just been undertaken. 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 aspects of high-precision radiotherapy. During 2012, 3287 new patients were treated in our Division: 2630, 427 and 230 with external beam radiotherapy, intraoperative irradiation (mainly for breast cancer) and brachytherapy, respectively. The highest proportion of patients has been treated for breast cancer (40%) followed by metastastic disease (30%) and prostate cancer (8%). Educational Activities Educational activities of the Division include indepartment teaching for pre- and postgraduate

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medicine, physics and biotechnology students and radiology&radiotherapy technicians RTTs (University of Milan and Politecnico of Milan). The Division is the main site of the post-graduate residency program in Radiation Oncology of the University of Milan. The Division is involved in the Teaching Committee of the PhD program of the University of Milan. Internal education events for the staff (continuous medical education) are organized on the regular weekly basis. 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 the PTCOG (Proton Therapy Cooperative Group) and other national and international societies. Radiation oncologists of our Division speak regularly at the numerous national and international educational events. Many of them are members of Scientific Committees, Societies and Editorial Boards including European Society for Therapeutic Radiology and Oncology ESTRO, Italian Society of Radiation Oncology AIRO, Italian Society of Radiobiology AIRB, Italian Society of Urologic Oncology SIURO etc.). They are involved in activities of numerous Research Working Groups of the Italian Society of Radiation Oncology and other scientific societies. Some of the radiation oncologists of our Division serve as teachers and course directors for the ESTRO teaching courses and Masterclass. The Division participates in the establishment of numerous national and international guidelines on cancer treatment. 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).

Publications Riboldi M, Orecchia R, Baroni G. Real-time tumor tracking in particle therapy: technological developments and future perspectives. Lancet Oncol 2012:e383-91. IF-22 Jereczek-Fossa BA, Kaanders JH, Dubray B, Cottrill C, Leer JW. Faculty of Teaching Course on Evidence-Based Radiation Oncology of European Society of Therapeutic Radiology and Oncology. Focal therapy for prostate cancer. Lancet Oncol 2012;13:e280-1. IF-22 Jereczek-Fossa BA, Beltramo G, Fariselli L, Fodor C, Santoro L, Vavassori A, Zerini D, Gherardi F, Ascione C, Bossi Zanetti I, Mauro R, Bregantin A, Bianchi LC, De Cobelli O, Orecchia R. Robotic image-guided stereotactic radiotherapy, for isolated recurrent primary, lymph node or metastastic prostate cancer. Int J Radiat Oncol Biol Phys 2012;82:889-97. IF: 4.1.05 Leonardi MC, Maisonneuve P, Mastropasqua MG, Morra A, Lazzari R, Rotmensz N, Sangalli C, Luini A, Veronesi U, Orecchia R. How do the ASTRO consensus statement guidelines for the application of accelerated partial breast irradiation fit intraoperative radiotherapy? A retrospective analysis of patients treated at the European Institute of Oncology. Int J Radiat Oncol Biol Phys. 2012;83:806-13. IF-4.105 Fossati P, Molinelli S, Matsufuji N, Ciocca M, Mirandola A, Mairani A, Mizoe J, Hasegawa A, Imai R, Kamada T, Orecchia R, Tsujii H. Dose prescription in carbon ion radiotherapy: a planning study to compare NIRS and LEM approaches with a clinically-oriented strategy. Phys Med Biol. 2012;57:7543-54.

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Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

Division of Radiology Massimo BELLOMI, MD Director

STAFF Senior Deputy Directors: Lorenzo Preda, MD, Gaetano Villa, MD, Elvio De Fiori, MD, Paolo Della Vigna, MD Assistants: Lorenzo Monfardini, MD, Giuseppe Petralia, MD, Cristiano Rampinelli, MD, Stefania Rizzo, MD, Luigi Funicelli, MD, Calareso Giuseppina, MD Fellows: Alessi Sarah MD, Ara Alconchel MD Residents: Alessandra Ferla Lodigiani, Roberto Lo Gullo, Caterina Giannitto, Luke Bonello, Irina Sosnovskikh, Sara Maccagnoni, Giuseppe Di Pisa, Maria Carmela Grimaldi, Salvatore Alessio Angileri, Vittoria Vecchi, Giorgio Conte, Francesca Ruju, Riccardo Foà, Emanuela Perucchini, Francesca De Maria, Paola Pricolo Research fellows: Paul Eugene Summers Ph.D, Lara Durli MD, Alessandro Sciarra Director’s Secretary: Irene Cleopazzo Secretaries: Barbara La Mantia, Mariastella Bottalico, Sabrina Riboni Data Manager: Letizia Sirica Secretary, Clinical Studies: Giovanna Ciambrone, Valentina Laudicina Administrative Clerk: Anna Palmeri Chief Technician: Giuseppe Bardo Technicians: Emanuele Addonizio, Dario Ardizzone, Nicola Balestreri, Giuseppe Bonfitto, Valerio Cubadda, Roberto Di Filippi, Alessandro D’Incecco, Simone Fasulo, Roberto Labruna, Ferdinando Laserra, Manuela Martino, Valeria Obregon, Ubaldo Piccolo Longo, Filippo Raccosta, Cinzia Resta, Riccardo Simone, Emanuela Veneziano, Elena Bollini, Marco Gavarini, Gennaro Prisco Technicians Research Fellow: Alberto Carlo Bonissone 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, Davide Isoardi, Lorenzo Pontillo, Federica Galino, Ilaria Milo, Alessandro Asaro, Mattia Benvenga, Andrea Masperi, Gessica Girgenti Vinci, Stefano Mazza, Nelida Omonte, Andrea Malgrati, Federica Susini, Federico Ferrario

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Unit Of Interventional Radiology: Director: Franco Orsi, MD, EBIR Deputy Director: Guido Bonomo, Paolo Della Vigna Senior Assistant: Lorenzo Monfardini Fellow: Nicola Cionfoli Affiliated IR Consultant: Miltiadis E Krokidis MD, PhD, EBIR (Cambridge University Hospitals NHS Foundation Trust) Care Unit Coordinator: Claudio Acerbi Secretary: Bottalico Mariastella Data Manager: Simona Menna

Activities 2012.

Radiology Activities In 2012 the Department of Radiology performed 59.000 differerent diagnostic examinations and interventional procedures, 12% of them in patients enrolled in clinical trials. A new CT, 64 slices, was installed, allowing the 8-slices machine to be devoted to percutaneous biopsy and interventional procedures. 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) and Prostate Imaging (Dr. Petralia). The research team is driven by Dr. Petralia and has a devoted staff with 3 fellows in Radiology, one radiographer, one secretary and one data manager. In 2012 we translated into clinical practice the researches on whole body MR imaging, with diffusion sequenze (WB-DWI), reaching the goal to reduce the scanning time to 30 minutes and demonstrating that the technique can

be used for detecting asymptomatic tumors in healty people, thus increasing the attitude of the Department to be a pioneer and a reference either for early diagnosis of cancer and lung cancer screening, where we started a project in 2000. The scientific activity ranges over a wide variety of topics, leading to publish a number of papers focused on evaluating the response to chemotherapy, the impact of imaging on diagnosing and managing prostate cancer, functional imaging (perfusion and diffusion by CT and MRI) in breast and liver cancer and the diagnose of pulmonary nodules.

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Interdisciplinary Research Interventional Radiology Unit Activities The Interventional Radiology Unit is one of the very few European Units of Interventional Radiology that manages its own beds in a dedicated ward; 225 Ordinary Admissions and 331 Daily Admissions (Day Surgery) were performed in 2012. 1257 main interventions (such as liver, renal and lung thermal ablations; liver embolizations and radioembolizations), 2530 minor procedures (such as image guided biopsies and port placements) were performed in three distinct dedicated interventional rooms. Clinical activity also included around 1000 outpatient consultations and 112 concomitant surgical interventions (both during laparoscopy and open surgery). In January 2012 the Unit performed the first percutaneous hepatic chemoperfusion in extracorporeal circulation in Europe. Innovation, clinical research and development of newer treatment strategy in multimodal management of tumor disease, are the backbone of daily practice of the Unit, which represents today one of the pillars of clinical management at the European Institute of Oncology

graduate School of Radiology of the University of Milan. In 2012 we organized 7 residential courses, with a total of 204 ECM credits.

The Division is participating to more than 120 clinical trials conducted by clinical departments.

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 Dec;35(6):1372-9. doi: 10.1007/s00270-012-0341-0. Epub 2012 Jan 21. PubMed PMID: 22271077

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. Unit of Interventional Radiology is involved into the ESIR (European School of Interventional Radiology) and is an active member of CIRSE (Cardiovascular and Interventional Radiology Society of Europe). The Division is deeply involved in educational programmes, being part of the teaching activities of the School of Medicine, School of Radiographers and Post-

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Publications G. Petralia, P.Summers, S.Viotti, R.Montefrancesco, S.Raimondi, M.Bellomi - Quantification of variability in perfusion CT analysis of HCC: a step towards clinical use - Radiology 2012;265 448-456, doi: 10.1148/ radiol.12111232 (6.069) C.A.Méndez, F.Pizzorni Ferrarese, P.Summers, G.Petralia, G.Menegaz - DCE-MRI and DWI integration for Breast Lesions assessment and heterogeneity quantification - Int J Biomed Imaging 2012; ID 676808, doi 10.1155/2012/676808 G. Veronesi, P. Maisonneuve, M. Bellomi, C. Rampinelli, I.Durli, R. Bertolotti, L.Spaggiari. - Estimating Overdiagnosis In Low-Dose Computed Tomography Screening For Lung Cancer - Ann Int Med 2012;157:776784. (16.733)

Orgera G, Krokidis M, Monfardini L, Arnone P, Bonomo G, Della Vigna P, Curigliano G, Orsi F. Ultrasound-guided high-intensity focused ultrasound (USgHIFU) ablation in pancreatic metastasis from renal cell carcinoma. Cardiovasc Intervent Radiol. 2012 Oct;35(5):1258-61. PubMed PMID: 22011784.

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Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

Division of Breast Imaging Enrico CASSANO, MD Director

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

Activities 2012-13. In the context of

diagnosis and therapy of breast diseases, in recent years it has increasingly affirmed the belief that, are necessary qualified centers which may be a clear reference, in order to guarantee in terms of quality and efficiency an effective service to the female population. In these structures, the patient can be followed from diagnosis, both clinical and instrumental, to define in case of disease, the next therapeutic procedure, whether pharmacological, or surgical, or radiation. Instead, in case of negative diagnosis for breast disease, the patient will be informed relatively to the mode and frequency of the subsequent periodic checkups. It is with this purpose that was born and grew the Breast Imaging Division of European Institute of Oncology (IEO). Here a correct and timely diagnosis is the result of the skills and dedication of the full-time medical and paramedical staff, which makes use of the latest generation instrumentation and always innovative methodologies. 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 (VABB) and magnetic resonance, are supported and integrated, with new, but well-established instrumental technology, in accordance with clinical protocols. In the Division are available to the patient these clinical and instrumental investigations: • Clinical breast examination • Digital mammography • Ultrasound with color Doppler • Magnetic resonance • Fine needle aspiration cytology imaging guided • Micro-histological sampling, carried out with core biopsy or vacuum assisted biopsy • Preoperative localization of non-palpable lesions to be subjected to surgery

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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 clinical and scientific activities. The multidisciplinary approach and the constant exchange of views between the different specialists working within the Institute (radiologists, surgeons, oncologists, radiotherapists), allows to offer a careful and complete service to the patient, from diagnosis to treatment, with all the intermediate stages. The IEO philosophy: “you treat better, where you do research” is reflected in the activity of the Breast

Imaging Division, where constant attention is paid both to the outpatient clinic tasks and to research. In particular, field of research are the new diagnostic technologies, assessed in the light of their clinical impact. In our Division is given the highest attention to the effectiveness and results of studies of diagnostic imaging and all the new possible applications. The research areas of the Division are focused on the study of new methods that allow for a more timely diagnosis in a less invasive mode, with the priority aim of enabling a comprehensive long-term recovery of the patient. Because of the increasing number of pregnant patients with oncologic disease in IEO, after a careful revision of

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Interdisciplinary Research 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 (Senologia Integrata in Gravidanza e Allattamento/Integrated Senology in Pregnancy and Lactation), 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 analyzed 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. In our department VABB procedure is performed under stereotactic, ultrasound and magnetic resonance guidance. We published some articles about VABB stereotactic and ultrasound guided procedure highlighting the usefulness of this diagnostic tool. Ultrasound-guided VABB can provide accurate characterization of breast lesions especially classified C1 or C3 on the basis of ultrasoundguided 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. 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. In women diagnosed preoperatively with ductal

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carcinoma in situ (DCIS) by stereotactic VABB, we investigated the association of clinical and radiological variables associated with invasive component and nodal involvement. We found that patients older age, little lesion dimensions, and no residual image after VABB can predict absence of invasion and no nodal involvement, however it would be imprudent to routinely forego sentinel node biopsy in such patients as non-negligible proportions of them presented invasive disease associated to DCIS. Currently VABB under magnetic resonance guidance is performed only in a few centers in Italy. In our institute we started to perform VABB under magnetic resonance guidance in October 2010, with good results.

Noninvasive assessment of breast cancer risk using time-resolved diffuse optical spectroscopy. J Biomed Opt. 2010 Nov-Dec;15(6):060501 Trentin C, Dominelli V, Maisonneuve P, Menna S, Bazolli B, Luini A, Cassano E. Predictors of invasive breast cancer and lymph node involvement in ductal carcinoma in situ initially diagnosed by vacuum-assisted breast biopsy: Experience of 733 cases. Breast. 2012 Oct;21(5):635-40. Epub 2012 Jul 12.

Publications Di Nubila B, Cassano E, Urban LA, Fedele P, Abbate F, Maisonneuve P, Veronesi P, Renne G, Bellomi M. Radiological features and pathological-biological correlations in 348 women with breast cancer under 35 years old. Breast. 2006 Dec;15(6):744-753. Epub 2006 May 26. Cassano E, Urban LA, Pizzamiglio M, Abbate F, Maisonneuve P, Renne G, Viale G, Bellomi M. Ultrasound-guided vacuum-assisted core breast biopsy: experience with 406 cases. Breast Cancer Res Treat. 2007 Mar;102(1):103-10. Epub 2006 Jul 13. Bozzini A, Renne G, Meneghetti L, Bandi G, Santos G, Vento AR, Menna S, Andrighetto S, Viale G, Cassano E, Bellomi M. Sensitivity of imaging for multifocal-multicentric breast carcinoma. BMC Cancer. 2008 Sep 30;8:275. Taroni P, Pifferi A, Quarto G, Spinelli L, Torricelli A, Abbate F, Villa A, Balestreri N, Menna S, Cassano E, Cubeddu R.

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Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

Division of Nuclear Medicine Giovanni Paganelli, MD Director

STAFF Radiochemistry Unit Director: Marco Chinol, PhD Radiochemist Assistant: Stefano Papi Fellows: Angela Carollo, Fabiola Cocco, MD, Demetrio Familiari, MD, Lucia Garaboldi Deputy Directors: Lisa Bodei, MD, PhD, Chiara Maria Grana, MD Senior Assistants: Silvia Melania Baio, MD, Laura Travaini, MD Assistants: Marzia Colandrea, MD, Silvia Lidia Fracassi, MD, Laura Gilardi, MD, Paola Anna Rocca, MD, Stefano Vassallo, MD Data Managers: Ilaria Minotti, Ines Tedeschi Chief Technician: Maurizio Fiorenza Technicians: Michele Calabrese, Alfio Severino Cascio, Sebastiano Croce, Riccardo Mei, Domenico Militano, Daniele Paolucci, Andrea Vertua Scientific Secretary: Deborah Console Secretaries: Valeria Archinti, Laura Brambilla, Anna Lucia Tusini, Maria Ersilia Viscusi Head Nurse: Gianni Bufi Medical Physicists to Nuclear Medicine: Francesca Botta, Marta Cremonesi, Mahila Ferrari, Francesco Guerriero

Activities 2012.

The Division of Nuclear Medicine is devoted to the early localisation and treatment of tumors, 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

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Concetta De Cicco, MD Co-Director

of the few in Europe having protocols of radionuclide therapy of solid tumors and lymphomas with new radiolabelled molecules that show high affinity for tumor cells, such as monoclonal antibodies, avidin-biotin and radiolabelled peptides, both as systemic and locoregional 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 2012, 10.619 diagnostic studies were performed, of which 4443 were PET/CT with 18FDG and 461 with 68Ga-octreotide, and over 747 outpatients were visited. Radionuclide therapies with Yttrium-90 and Lutetium-177 labelled peptides are carried out, both as systemic or loco-regional treatments. During 2012, the Division published 17 articles on peer- reviewed journals, with an overall 38.838 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 preclinical and clinical research activities, to foster future collaborations. Publications Gilardi L, De Cicco C, Paganelli G. Preoperative FDG PET/CT in breast cancer patients: where are we going? Eur J Nucl Med Mol Imaging. 2012 Nov;39(11):1667-9.

Travaini L, Trifirò G, Vigna P, Veronesi G, De Pas T, Spaggiari L, Paganelli G, Bellomi M. Roles of computed tomography and [(18)F] fluorodeoxyglucose-positron emission tomography/ computed tomography in the characterization of multiple solitary solid lung nodules. Ecancermedicalscience. 2012;6:266. Bodei L, Cremonesi M, Grana CM, Chinol M, Baio SM, Severi S, Paganelli G. Yttrium-labelled peptides for therapy of NET. Eur J Nucl Med Mol Imaging. 2012 Feb;39 Suppl 1:S93-102.

Erba PA, Sollini M, Orciuolo E, Traino C, Petrini M, Paganelli G, Bombardieri E, Grana C, Giovannoni L, Neri D, Menssen HD, Mariani G. Radioimmunotherapy with radretumab in patients with relapsed hematologic malignancies. J Nucl Med. 2012 Jun;53(6):922-7. Montaña RL, González IH, Ramirez AA, Garaboldi L, Chinol M. Yttrium-90 current status, expected availability and applications of a high beta energy emitter. Curr Radiopharm. 2012 Jul;5(3):253-63.

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Interdisciplinary Diagnosis and Research Prevention Area

Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

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, Floriana Pansini, Dr. Phys Senior 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, Anna Cavallo, Dr. Phys, Edoardo Mastella, 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

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Activities 2012.

Many diagnostic, therapeutic and research activities performed in the Institute involve the use of sealed and unsealed radioactive substances and of different types of equipments producing ionizing radiation (X-ray tubes for diagnostic radiology, conventional and mobile linear accelerators for external radiotherapy, after-remote-loading equipment for brachytherapy 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 Radiology, Nuclear Medicine and Radiotherapy for setting up and optimizing protocols for clinical dosimetry, for the development and the introduction into the clinical practice of new and advanced techniques involving the use of ionizing and non-ionizing 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 2012, apart from the radiation protection and quality control procedures, the following routine activities involving the optimization of diagnostic and therapeutic procedures were performed: • set up and commissioning of new high technology accelerators for external beam radiotherapy: Tomotherapy®, for the breast and partial breast cancer treatment; Cyberknife®, for highly conformal treatments of small tumor lesions; Vero®, for stereotactic treatments and tumor tracking; • treatment plan calculation for conventional external beam radiotherapy, Intensity Modulated Radiotherapy and Volumetric Modulated Arc Therapy, stereotactical radiotherapy, multiple arcs prostate radiotherapy, IORT (Intra Operative Radiation Therapy), conventional brachytherapy, brachytherapy with permanent implant of 125I seeds and TBI (Total Body Irradiation) treatments;

• internal dosimetry evaluations for systemic and loco regional radionuclide therapies with agents radiolabelled with b-emitters, (such as 90Y-avidinbiotin-MoAbs, 90Y-avidin-biotin and 90Y and 177Lupeptides, and resin 90Y-microspheres); leakage monitoring in perfusion procedures. Publications Ferrari ME, Cremonesi M, Di Dia A, Botta F, De Cicco C, Sarnelli A, Pedicini P, Calabrese M, Orecchia R, Pedroli G, Paganelli G. 3D dosimetry in patients with early breast cancer undergoing Intraoperative Avidination for Radionuclide Therapy (IART) combined with external beam radiation therapy. Eur J Nucl Med Mol Imaging. 2012 Nov;39(11):1702-11. Lanconelli N, Pacilio M, Lo Meo S, Botta F, Di Dia A, Aroche AT, Pérez MA, Cremonesi M. A free database of

radionuclide voxel S values for the dosimetry of nonuniform activity distributions. Phys Med Biol. 2012 Jan 21;57(2):517-33. Calandrino R, Ardu V, Corletto D, del Vecchio A, Origgi D, Signorotto P, Spinelli A, Tosi G, Bolognesi A, Cariati M, Kluzer A, Muscarella S. Evaluation of second cancer induction risk by CT follow-up in oncological longsurviving patients. Health Physics. 2013 Jan;104(1):1-8. Lonsdale MN, Pedroli G. Technological innovations in nuclear medicine imaging. Q J Nucl Med Mol Imaging. 2012;56:219-20. Ciocca M, Cantone MC, Veronese I, Cattani F, Pedroli G, Molinelli S, Vitolo V, Orecchia R. Application of failure mode and effects analysis to intraoperative radiation therapy using mobile electron linear accelerators. Int J Radiat Oncol Biol Phys. 2012 Feb 1;82(2):305-11.

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Interdisciplinary Research – Department of Medical Imaging and Radiation Sciences

Research Activities

RADIOTHERAPY DIVISION The Division has numerous research activities, including internal studies and institutional multidisciplinary or multicentric studies. The radiation oncologists of our Division are members of the Protocol Review Committee at the European Institute of Oncology, Milan and of the Committee of the Quality of the Clinical Research at the European Institute of Oncology, Milan. The Division is involved in several European Projects on improvement in radiotherapy for cancer patients. The most recent scientific commitments include research projects like ULICE and ALLEGRO project of European Atomic Energy Community’s Seventh Framework Programme [FP7/20072013] and projects of the Italian Ministry of Health, University of Milan, and Italian Association of Cancer Research (AIRC). Active collaboration with Bioengeneers of the Politecnico of Milan and with the National Center for Oncological Hadrontherapy (CNAO) in Pavia result in numerous projects on high precision radiotherapy. In research activities of the Division the emphasis is placed on breast cancer, urological tumors and head and neck and other adult solid tumors. The main accent is focused on the combined modality approach, high precision radiotherapy, hypofractionation and ablative radiotherapy, oligometastatic disease and new prognostic and predictive factors. Last but not least, quality of life and reduction of radiotherapy toxicity is extensively studied. Every year the Division publishes about 30 full papers with an overall Impact Factor of about 150. The most significant research lines include: On-going studies Breast Cancer 1. Randomized phase II clinical study in patients undergoing intraoperative boost the tumor bed with electrons (ELIOT) followed by postoperative accelerated hypofractionated external beam radiotherapy after conservative surgery for early-stage breast cancer. IEO S676/61

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2. Multicenter phase IIb/ III randomized trial in patients with breast cancer, on the postoperative external beam radiotherapy comparing conventional fractionation with 2 accelerated hypofractionation with concomitant boost schemes. MIRA-SOLE trial. IEO S639/311 3. Estimate of the intrauterine dose in non-pregnant patients undergoing intraoperative electron beam radiotherapy (ELIOT) in early-stage breast cancer. IEO S545/210 EU project [FP7/2007-2013] Rectal Cancer 4. Preoperative re-staging for T3 rectal cancer: xeloda + oxaliplatinum + radiotherapy (XELOXRT) vs xeloda + accelerated radiotherapy (XELACRT). Multicentric accelerated study. IEO S417/308 Studies in the phase of activation: Prostate Cancer Short-term high precision radiotherapy for early prostate cancer with concomitant boost on the dominant lesion IEO S768/113. Project AIRC 2012 - IG 13218 Numerous prospective studies are on-going and regard new high precision radiotherapy: IG-IMRT for breast cancer IG-IMRT for gyne malignancies IG-IMRT for prostate cancer VERO image guided radiotherapy for oligometastatic tumors Cyberknife Robotic Image-guided stereotactic radiotherapy - for brain metastases - for spine metastases DIVISION OF BREAST RADIOLOGY The research activity of the Division is focused on optimization of traditional breast imaging techniques and setting of innovative procedures.

The most significant research lines include: On-going studies: 1. New frontiers in breast imaging: contrast enhanced digital mammography. - Diagnostic performance of contrast-enhanced spectral mammography (CESMSenoBright) in pre-surgical evaluation of extent of malignancy in a population of women with breast cancer. At present breast cancer diagnosis is based on three main diagnostic techniques X-ray mammography (MX), ultrasound (US) and magnetic resonance imaging (MRI) the necessity to make multiple tests which results are compared and integrated cause a difficulty to ensure a complete and correct breast cancer diagnosis in a short time. Also interventional procedures can be necessaries to clarify diagnostic doubts. Initial clinical research about CESM showed that it has the potential to improve clinical performance compared to the combination of MX+US. CESM might be an alternative valuable option in routine imaging of dense breasts. Benefits would include shorter examination cycle time and healthcare cost savings. The aim of this study is to evaluate clinical performance of CESM in pre-surgical breast cancer assessment in women with dense breasts, particular attention will be focused on multi-focality cases. 2. New frontiers in breast imaging: optical mammography. The Division 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 detection and characterization of breast lesion and in the non-invasive assessment of breast density, which is a recognized strong and independent risk factor for developing breast cancer. This trial is actually ongoing, but we already published some articles, due to the encouraging preliminary results about breast density assessment, done with a portable clinical instrument for optical mammography.

3. Blood Test for Breast Cancer Associated Auto Antibodies (CAAA). The Division is working with the IEO’s laboratory medicine department in a multicenter 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. DIVISION OF NUCLEAR MEDICINE The most significant research lines include: Diagnostics 1. Sentinel lymph node (SLN) localisation and biopsy represents one of the most important developments in surgery that changed the management of patients affected by early breast cancer and represents today the standard of care for axillary node staging. In the past 15 years we implemented and described the lymphoscintigraphic technique. Currently our studies are devoted to the role of FDG PET in the selection of patients with breast cancer candidate to SLNB after neoadjuvant therapy. Preliminary results on 65 patients demonstrate a relatively high specificity and positive predictive value of FDG-PET for detection of axillary lymph nodes metastases after neoadjuvant therapy (93% and 86%, respectively). These data seem to suggest a role of FDG-PET in selecting patients who, after neoadjuvant therapy, are candidate to axillary lymph nodes dissection, avoiding SNB. 2. Prediction of response to neoadjuvant therapy in patients with locally advanced breast carcinoma with Fluorothymidine (FLT) PET. We investigated the hypothesis that a reduction of 18FLT uptake in the tumor after the first cycle of neoadjuvant chemotherapy may predict the response to the treatment. So far we have enrolled 13 patients. The assessment of FLT biokinetics in breast lesions based on automatic delineation

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Interdisciplinary Research of PET images using RIThM (Recovering Iterative Thresholding Method) has been implemented at our Institution. Therapy 1. Our Institute has been one of the pioneers in the development of peptide receptor radionuclide therapy (PRRT) in neuroendocrine tumors (NETs). Clinical studies have been carried out for over 15 years. First, the studies with 90Y-DOTATOC, with dosimetric and phase I studies, which demonstrated good tolerability and the occurrence of objective response in 26% of patients. Afterwards, studies continued with 177LuDOTATATE, with dosimetric and phase I-II studies, which demonstrated that this radiopeptide is well tolerated from a clinical, hematological and renal point of view and lead to a mean amelioration of clinical status. Objective responses occurred in over 30% of patients with a consistent TTP of 36 months. 2. Phase II studies defining the role of PRRT in NETs, and to evaluating prognostic parameters, such as FDG-PET in NETs. Our study demonstrated that FDG PET is useful for predicting response to PRRT in patients with grade 1-2 advanced NETs and, depending on the positivity or negativity, is able to differentiate aggressive from indolent disease, respectively. 3. The 3-step avidin-biotin pretargeting approach has been applied for almost 20 years in patients with recurrent glioblastoma (GBM), using biotinylated anti-tenascin monoclonal antibody as the first step followed by avidin and 90Ybiotin (PAGRIT®). We recently evaluated the objective response and overall survival rates in 502 GBM patients treated in our Institute from December 1994 to December 2005 at the recurrence after standard treatment. Of the 502 patients, 272 (54%) were evaluable for response and 375 (75%) for overall survival. 174 patients (64%) continued to progress after PAGRIT®, 77 (28%) obtained disease stabilization, and 21 (8%) showed objec-

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tive tumor regression. Survival of the 375 evaluable patients was 98.4% at 6 months, 79.2% at 12 months, 51.7% at 18 months, and 30.7% at 24 months after the first cycle of PAGRIT®. The median survival time from diagnosis was 19 months. These results suggest that PAGRIT® interferes with the progression of glioblastoma, prolonging survival in a larger number of patients. 4. Intraoperative avidination for radionuclide therapy (IART®) is a new approach of targeted radionuclide therapy of early breast cancer invented at IEO, which involves inoculation of avidin into residual glandular tissue immediately after primary surgery and intravenous injection of 90Y-biotin the day after. 90Y-biotin homes in specifically to the avidin in the breast and provides a therapeutic irradiation. Our studies indicated fast and stable uptake of radiolabeled biotin at the operated site. The procedure is safe with no hematological toxicity. Local toxicity during EBRT was acceptable, with good cosmetic results. The BEDvolume 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. MEDICAL PHYSICS The most significant research lines include: 1. IGRT (Image Guided Radiation-Therapy) in order to minimize the treated volumes while increasing the doses to the tumor; 2. VMAT therapies achieved by RapidArc® and Tomotherapy®: rotational radiotherapy allows higher conformality while minimizing the dose to normal tissue; 3. Liver stereotactic treatments with respiratory tracking implementation with Cyberknife®; 4. 4D treatments and tumor tracking treatments implementation on the Vero® system; 5. Multi-modality image fusion with image deformation-

an agreement work in cooperation with the Bioengineering Department of the Politecnico di Milano; 6. IART (Intraoperative Avidination Radionuclide Therapy); study of radiobiological models applied to radionuclide therapies, especially radiopeptide therapy and radioembolization of liver tumors with 90Y-resin microspheres; liver, kidney and red marrow dose-effects in radiopeptide therapy and radioimmunotherapy; 7. Analysis by home-made software (Matlab support) of the activity distribution in scintigraphic images and evaluation of dose distribution at the voxel level (voxel dosimetry); analysis of the dose-volume histograms, Biological Effective Dose histograms and Equivalent Uniform Dose; 8. 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; 9. Implementation of PET/CT imaging for radiotherapy treatment planning; 10. Implementation of a software for the segmentation of PET volumes (RITHM) to use for patients’ diagnosis and follow-up; 11. Assessment of PET parameters (combining standardized uptake value and metabolic volume) to establish the treatment response, e.g. after radioembolization with 90Y microspheres; 12. Implementation of Monte Carlo methods (PENELOPE and FLUKA codes) to model the absorbed doses in loco-regional therapies and to calculate absorbed doses in clinical practice taking into account the specific tissue density; 13. Development of quality assurance in HIFU (High Intensity Focused Ultrasounds); 14. Optimization of patient dose in screening and follow up procedures with multislice CT.

RADIOLOGY DIVISION The research activity is focused on different topics: non conventional imaging, lung cancer, prostate cancer and total-body MRI. Non conventional imaging researches deal with studies on Perfusion (both by CT and MR), Diffusion (DW-MRI) and MR-Spectroscopy, aiming to define standard protocols, validate techniques by assessing their reproducibility, and underline the potential of the integration of different techniques (“multimodality imaging”). Their clinical impact is being confirmed by demonstrating its accuracy in monitoring the changes induced by chemotherapy in patients with different tumors, such as rectal cancer, HCC and SCC of the upper aereodigestive tract. The research on imaging of lung cancer is mainly based on our observational study in a cohort of 6.000 high risk volunteers who are receiving (some from 2000) an yearly low-dose CT for early detection of lung cancer (COSMOS project). We published a number of data demonstrating a relatively high incidence (mean 4.5%), lasting for the 12 years of maximum observation. Five years survival rate of patients with lung cancer detected at screening CT is 63%. The wide field of tumor imaging research is nowdays focused on the role of DW-MRI in diagnosing, localizing and grading the prostate cancer and its impact on patients management. We demonstrated that the use of DWI is able to reduce by 30% the number of positive margins at robotic prostatectomy: the impact on reducing the complications of interventions (surgery and radiotherapy) is under evaluation. After few years of technical researches on Total-body DW-MRI we succeded in reducing the scanning time to 30’: thus the technique has become clinically relevant and its accuracy has been tested as screening test for metastases in high risk young patients (with high grade melanoma). The research is ongoing to test its implementation as screening for cancer in healty people. The Division of Radiology is member of the European Institute of Biomedical Imaging Research.

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Interdisciplinary Research

Interdisciplinary Research

Division of Early Drug Development for Innovative Therapies Giuseppe CURIGLIANO MD, PhD Director

STAFF Deputy Director: Ida Minchella, MD, Marzia Locatelli, MD Assistants: Lucia Gelao, MD, Luca Fumagalli, MD, Angela Esposito, MD, Carmen Criscitiello, MD, PhD Secretariat Coordinator: Emanuela Colautti Chief Research Nurse: Alessandra Milani Research Nurse: Veronica Brunelli Chief Nurse: Enza Dossena Data Managers: Laura Adamoli, PhD, Dominique Ronzulli, PhD

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Activities 2012.

The mission of our Division is to accelerate the development of new anticancer drugs (including biologics, and cellular therapies) that will improve survival and quality of life for patients with cancer. Our drug development program is not only furthering cancer research, but it also offers hope to patients facing the toughest cancer battles. Our personal commitment is devoted to build up an alternative, personalized, disease and pathway oriented model to develop drugs for cancer disease and to ensure equitable access to new and field-relevant health tools. We are committed to develop therapeutics for biomarker-defined subpopulations, to develop new drugs or new less toxic formulations of existing drugs. We are strongly motivated to innovate approach to cancer treatment. Innovation refers to the testing and implementation of novel approaches (clinical trial designs and operations, funding mechanisms, resource utilization, data collection, data analysis, etc) to developing more effective therapeutics more efficiently than existing methods. We need to innovate through interconnectedness. We are involved in a complex international network with cooperative efforts across institutions, industry, and organizations to conduct clinical trials that will have the greatest impact on cancer care more efficiently. The breadth and technical complexity of new technologies that could advance personalized oncology care demand a more interconnected approach to the development of diagnostics and therapeutics. Consortia of institutions that can standardize the acquisition, processing, and shipping of patient specimens may interconnect, with each having a laboratory that specializes in different methods of specimen analysis. Such interconnected facilities may expedite the development of personalized cancer therapeutics more powerfully than single centers. As therapeutics are developed to treat small subsets of individual disease populations, the operations to perform trials in isolation with old methods become inefficient, almost untenable. We cooperate with the internal Drug

Discovery Program to advance unprecedented targets, for orphan indications and high medical needs. We support conduction of pre-clinical and translational trials to enable rational selection of optimal drug candidates for human testing. We are conducting activities in collaboration with basic science labs (involved in drug discovery and target identification, mechanism of action and resistance, and structurefunction analyses). Our Division also provides training for new generations of physicians, designs programs that promote knowledge particularly among highrisk 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 and good clinical practice. 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 Division will be embracing aspects of both academic and industrial research (phase I-II trials): focus on scientific excellence, team working, and hypothesis-driven goaloriented research. It will bring together scientists with complementary expertise and professional backgrounds

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Interdisciplinary Research in the areas of cancer genomics and bioinformatics, cancer biology and genetics, cancer drug discovery and pharmacology as well as clinical trial expertise. Choices of programs will be driven by unequivocal evidence of their role in disease control and treatment in clinically relevant settings. The clinical Division consists of a team of highly trained physicians and staff with extensive experience in internal medicine, cancer treatment, translational medicine, Phase 0-I, early phase II clinical trials and clinical pharmacology research. Our clinical research platform is based on: 1. The speed and efficiency of the design, launch, and conduct of trials; 2. The innovation in science and trial design with strong translational background; 3. Trial prioritization, selection, support, and completion; 4. Dedicated clinical, pathology and laboratory platform integrated with a molecular screening program. Facilities of the Division include Ambulatory Service for patients screening: 3 days per week (24 slots). Outpatients Day Hospital Service for patient treatment: 3 days per week (33 slots). In patients hospitalization: 36 beds for patients on phase I studies and for critical patients with adverse events following experimental treatment. Research Nurses / Study Coordinators The Research Staff consists of Research Nurses and Study Coordinators who are responsible for protocol management and patient care. Our research staff is responsive to both patient and study sponsor needs. Each study is assigned a research team member to ensure continuity of care for study patients as well as the needs of the study sponsor. The research staff is closely involved with patient screening, enrollment, education, and patient follow up. They maintain constant communication with study sponsors, physicians, clinical

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staff, and patients. Annually trained on ICH GCP, Clinical trial procedures, regulatory questions as well as on specific pathologies, our team provides in-depth therapeutic expertise at every level of your study process.

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.

Early Drug Development Research Program Early drug development research program is equally committed to scientific discovery and patient care. Our world-class young 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, 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. 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 early drug development team currently has nearly 40 open adult therapeutic clinical trials. It accrues several 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 firstin-human studies that have the potential to move the boundaries of solid tumors oncology care. Technologies

The milestones of our clinical research are here summarized: 1. Identification of biological features of disease predictive of response to a target-oriented approach within a molecular screening program. 2. Identification of mechanisms of resistance to antiHER2 positive breast cancer disease and development of new strategies to target HER2 positive breast cancer. 3. 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. 4. Exploring the combination of endocrine therapy with biological agents targeting HER2, src or insulin growth factor receptor (IGFR). 5. Exploring the role of dual targeting (multiple antibodies or antibodies conjugated to chemotherapeutics agents) in patients with HER2 positive breast cancer. 6. Generation of human-xenograft models to predict response to targeted agents in patients with metastatic breast cancer. 7. 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. 8. Exploring the role of antigen specific immunotherapeutics for patients with triple negative breast cancer with residual disease after a neoadjuvant chemotherapy.

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.

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Interdisciplinary Research We believe a gene network reconstruction, strategybased 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 upone 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 mutationenriched 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 cancer is therefore linked to our ability to identify the most druggable target in each disease segment.

Molecular pathways: involvement of immune pathways in the therapeutic response and outcome in breast cancer. Clin Cancer Res. 2013 Jan 1;19(1):28-33. Criscitiello C, Curigliano G. HER2 signaling pathway and trastuzumab cardiotoxicity. Future Oncol. 2013 Feb;9(2):179-81. Curigliano G, Cardinale D, Suter T, Plataniotis G, de Azambuja E, Sandri MT, Criscitiello C, Goldhirsch A, Cipolla C, Roila F Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy: ESMO Clinical Practice Guidelines. Ann Oncol. 2012 Oct;23 Suppl 7:vii155-66. Petit JY, Rietjens M, Botteri E, Rotmensz N, Bertolini F, Curigliano G, Rey P, Garusi C, De Lorenzi F, Martella S, Manconi A, Barbieri B, Veronesi P, Intra M, Brambullo T, Gottardi A, Sommario M, Lomeo G, Iera M, Giovinazzo V, Lohsiriwat V. Evaluation of fat grafting safety in patients with intra epithelial neoplasia: a matched-cohort study. Ann Oncol. 2013 Feb 7.

Publications Curigliano G, Locatelli M, Fumagalli L, Brollo J, Munzone E, Nolé F, Criscitiello C, Goldhirsch A. Targeting the subtypes of breast cancer: rethinking investigational drugs. Expert Opin Investig Drugs. 2012 Feb;21(2):191-204. Andre F, Dieci MV, Dubsky P, Sotiriou C, Curigliano G, Denkert C, Loi S.

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Interdisciplinary Diagnosis and Research Prevention Area

Interdisciplinary Research

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 Counserlor: Leonora Chiavari, MSc Data Managers: Giorgia Bollani, Serena Mora Research Nurse: Vittoria Arensi, RN, Claudia Passoni, RN Secretary Coordinator: Alessandra Rossi Secretary: Mariaelisa Ronzino Patient Manager: Angela Maniscalco Scientific Consultant: Andrea De Censi, MD

Activities 2012.

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, counselor, 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

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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, athypical hyperplasia, high mammographic density, peripheral lung “ground glass opacities” etc). These at-risk subjects are screened, followed and possibly 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 usefulpreventive agents, including SERM’s (Selective Estrogens Receptors Modulators), AIs (aromatase inhibitors), retinoids, NSAID’s (Non-steroidal antiinflammatory drugs), corticosteroids, statins, metformin, with particular attention in seeking the minimal active doses. In line with improving subjects characterization we also 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 and less toxic 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, PTEN and CDH1) in strict collaboration with the genetic lab at the IFOM-IEO Campus. 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 the year 2012 we performed 2428 visits in our prevention outpatients clinic. In our HRC service we evaluated 455 pedigrees, and we performed 369 first genetic counseling sessions. Among these subjects, 307 underwent a genetic test for BRCA1 and 2 mutations. We found: 54 BRCA1 mutations, 42 BRCA2, 129 wild-type and 26 true negative. Among the other genes tested we found the following mutations: six CDH1, one APC, four subjects carriers of Lynch syndrome, two with Li-Fraumeni syndrome. We coordinate various national research networks collaborating in multicenter phase

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Interdisciplinary Research 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) and “Gruppo di studio ENIGMA” on Unknown Variant mutations for BRCA1/2 genes; the Consortium for the study of the MMR alleles and their modifier (CONSAMM), and Study group MOMA-CONSAM on Unknown Variant mutations of MMR genes; the Department of Endocrinology, University of Bergen; In-TEF: Italian National Network for “Tumori EredoFamiliari” coordinated by “Istituto Superiore di Sanità”. During 2012, our Division published 28 articles in International peer reviewed journals with a total Impact Factor of over 170. Research activities Our current research lines are focusing mostly on chemoprevention, pharmacogenomics and the effects of lifestyle changes in various cohorts of subjects at higher risk of developing cancer in order to validate the most effective strategies to reach a tailored prevention. Scientific aims: Chemoprevention in subjects at higher risk for ER negative breast cancer, such as patients with endocrine non-responsive 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. The possible role of metformin, raloxifene, very low dose tamoxifen, exemestane and celecoxib as chemopreventive agents in a pre-surgical model. 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. Polymorphisms of different genes that may correlate

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with breast cancer risk: among them we are studing VDR, IGFBP3, MTHFR in a large spectrum of population. The role of aromatase inhibitors in prevention, evaluated in pre- and post-surgery settings. NAF (nipple aspirate fluid) as a risk assessment tool and source of new biomarkers studies. Chemoprevention of breast and ovarian cancer with Fenretinide in young healthy and BC survivor BRCA mutation carriers. Diet and physical activity to prevent recurrence after standard treatment in women with invasive breast cancer. Role of an accurate process to select high risk subjects for genetic counseling and their psychological and emotional response. Ongoing and starting projects IBIS II - an international multi center study of tamoxifen vs anastrozole in post menopausal women with Ductal Carcinoma in Situ ER+ (DCIS) IBIS II - an international multi center study of anastrozole vs placebo in post menopausal women at increased risk of breast cancer. 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 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. A phase III study with fenretinide (4-HPR) for primary prevention of breast cancer in subjects at high risk for familial/ hereditary breast cancer. 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. 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. A randomized placebo-controlled phase III clinical trial with vitamin D in melanoma patients and shortly a phase

II on colorectal cancer patients after radical treatment. Green tea and silybin for breast cancer: a pilot presurgical study Evolution of undetermined ld-CT detected lung nodules among a randomized phase II trial with aspirin and placebo. 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 Our pre-surgical study with metformin analyzed 200 nondiabetic women randomly assigned to receive metformin 850 mg/twice per day or placebo. The primary endpoint was the KI67 modulation. Overall, the metformin effect on Ki-67 change relative to placebo was not statistically significant. However, there was a different drug effect depending on insulin resistance (categorized as homeostasis model assessment [HOMA] index), with a nonsignificant mean proportional decrease in Ki-67 of 10.5% (95% CI, 26.1% to 8.4%) in women with HOMA more than 2.8 and a nonsignificant increase of 11.1% (95% CI, 0.6% to 24.2%) with HOMA less than or equal to 2.8. A similar results were observed according to body mass index. Our collaboration with CIMBA lead to identify three new breast cancer susceptibility loci. Two of them are presumably associated with genes closely related to the mammary gland. The gene PTHLH (12p11) has a crucial role in mammary gland development and it can be correlated also to bone metastasis. It is associated with both ER positive and negative breast cancer. The second one is NRIP1 (21q21), encodes an ER cofactor and has a role in the regulation of breast cancer cell growth. This gene is apparently related only to ER positive breast cancer. To further confirm the effect of low dose tamoxifen we conduct an observational study on women with Er positive intraepithelial neoplasia followed in our Institute. Compared with untreated patients, a significant 30%

reduction in breast cancer risk was observed on low dose tamoxifen with an adjusted hazard ratio (HR)=0.70 (95% confidence interval (CI) 0.51–0.94 see figure 1), with a greater benefit in postmenopausal (HR=0.57; 95% CI 0.34-0.94) than in premenopausal women (HR =0.79; 95% CI 0.54–1.17 see figure 2). Treated patients with ER and progesterone receptor (PgR)>50% DIN had a lower incidence of breast events than untreated ones (HR=0.61; 95% CI 0.40-0.94), whereas no protective effect has been observed in patients with ER or PgR<50% DIN. Drug discontinuation resulted in a doubled risk of recurrence in premenopausal women 25 only (HR=1.95; 95% CI 0.98-3.89 see figure 3). No excess of endometrial cancer occurred. Low dose tamoxifen has to be considered a relatively safe and effective treatment for women with a resected intraepithelial ER/PgR positive neoplasia. Treatment adherence is crucial in premenopausal women. Publications Macis D, et al. Prognostic effect of circulating adiponectin in a randomized 2x2 trial of low-dose tamoxifen and fenretinide in premenopausal women at risk for breast cancer. J Clin Oncol. 2012;30:151-7 Ghoussaini M, et al. Genome-wide association analysis identifies three new breast cancer susceptibility loci. Nat Genet. 2012 Jan 22;44(3):312-8. doi: 10.1038/ng.1049. DeCensi A, et al. Estrogen receptor in breast ductal carcinoma in situ: good cop, bad cop? Clin Oncol. 2012 Apr 20;30(12):1384-6. Bonanni B, et al. Dual effect of metformin on breast cancer proliferation in a randomized pre-surgical trial (Ahead of Print on May 7, 2012 as 10.1200/JCO. J Clin Oncol. 2012 Jul 20;30(21):2593-600 Lazzeroni M, et al. Breast Cancer Research 2012, 14: 214. doi:10.1186/bcr3233

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Interdisciplinary Research

Interdisciplinary Research

Applied Research Unit for Cognitive and Psychological Science Gabriella PRAVETTONI Director

STAFF Researchers: Ilaria Cutica (University Cognitive Psychologist), Alessandra Gorini (University Cognitive Psychologist), Claudio Lucchiari (University Cognitive Psychologist), Ketti Mazzocco (PsychotherapistPsychologist), Marianna Masiero, Ivana Palminteri (Psychologist), Silvia Riva (Psychologist) Fellows: Beatrice Colombo (Psychologist-Psychotherapist -Melanoma Division), Valeria Vadilonga (PsychologistPsychotherapist), Stefania Sacchezin (PscyhologistPsychotherapist) Consultant: Andrea Maldifassi (Psychologist) Visitors: Paola Lumia, Caterina Fanelli (psychologistspsychotherapy students), Giada Perinel, Carolina Platti Scientific Secretary: Maria Grazia Villardita Data Manager: Claudia Sangalli

Activities 2012.

From a psychological perspective, oncology today faces at least three main critical issues. First, the number of available therapies has dramatically increased in recent years, so the choice regarding which treatment is best for a certain patient is more complex than ever before. One of the possible ways to deal with this matter is to adopt a personalized treatment approach based on an analysis and interpretation of the patient’s general values and preferences. From this, the choice of the treatment may be derived that is most consistent with his/her needs. This kind of patient-centered approach promotes a model of shared decision-making and negotiation between doctors and patients. Working on the cognitive processes implicated in decision making, as well as on the communication strategies between the participants involved in the therapeutic journey renders it possible to promote health using non-coercive approaches that influence the patient’s health-related choices, and motivate them to adhere to the treatment. In particular,

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Florance DIDIER Co-Director

effective decision and communication strategies act as the starting point to overcome barriers to understanding, remembering, and implementing effective strategies for reducing exposure to risk factors and making healthenhancing behavior changes. The second important issue is that cancer is becoming a chronic disease. This means that many patients have to cope with their illness for a long time, finding strategies that help them to maintain a satisfactory quality of life. The chronic nature of the illness requires a constant and quantitative evaluation of patients’ behavioral factors (lifestyle, stress, health beliefs) and social conditions (cultural influences, family relationships and social support) in order to understand how psychological, environmental and cultural elements influence their physical and mental health. Psychometric tests and standardized questionnaires can be used to obtain quantitative data that allow a longitudinal evaluation of the patient’s condition, while interventions promoting health through behavioral change combined with the attempt to prevent illness or relapse may also be developed and tested. The third issue regards the patients’ lifestyle. So far, there is considerable evidence suggesting that cancerrelated diseases are related to “unhealthy” choices and behaviors such as smoking, lack of physical activity and poor eating habits. Cognitive psychology analyzes the way in which people make decisions and the reasons for these decisions. It also attempts to find strategies to modify actual behaviors in favor of safer and healthier habits. Cognitive and health psychology can thus be used to empower patients, thereby giving them the possibility to play an active role in the therapeutic process and to choose, in accordance with their therapists, the treatment options that best fit their needs, avoiding biased decisions and maintaining a good quality of life. Research in this field is important not only to improve patient participation, involvement and empowerment but also to promote better work-life balance in

healthcare operators, and therefore takes into account organizational variables such as stress (see DL 81/2008) and well being. Uniting academic with clinical expertise: the Applied Research Unit for Cognitive and Psychological Science The Applied Research Unit for Cognitive and Psychological Science derives from many years of academic experience in which a multidisciplinary team composed of psychologists, physicians, economists, statistics, computer scientists and philosophers have studied the complex topic of human decision making from different, but strictly interconnected perspectives. Thanks to close collaboration with the Faculty of Medicine at the University of Milan, we have developed

specific research and training expertise in the field of medical decision making. In particular, many protocols have been implemented to investigate: - the cognitive and psychological mechanisms necessary to develop patient empowerment - the decision-making processes involving physicians and patients (shared cognition) - the role of heuristics and biases in medical contexts - the measurement of patient needs and quality of life and the promotion of health behaviors - the neurobiological correlates of human decision making - organizational stress - the effect of a disease in the patient’s and family behavior. Such kinds of research-interventions in which scientific elements are woven together from cognitive and

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Interdisciplinary Research health psychology are integrated to promote an active, unbiased and deliberate participation of patients in the therapeutic process representing the core activity of this newly-created Unit. Integrating academic interests with clinical purposes, the present Unit will promote a multidisciplinary approach aimed at developing new training and treatment strategies as well as continually monitoring the cognitive-psycho-social processes which arise from the time of diagnosis communication through to oncology treatment. In particular, the Unit will perform, but will not be limited to, the following activities: - creation of a counseling unit for clinical decisionmaking strategies - administration of periodic and standardized surveys of needs and quality of life of oncology patients - implementation and standardization of advanced instruments for measuring and promoting the patients’ empowerment and health - assessment and evaluation of patients’ cognitive and emotional needs and preferences - implementation and standardization of advanced instruments to develop efficient communication strategies between doctor and patient - implementation of instruments to evaluate and enhance resilience in the patients and their families - creation of a counseling and training unit for patient management. All the activities developed by the Unit will be carried out using a rigorous scientific method applied to the oncologic clinical setting, striving to obtain the best possible integration between academic and clinical expertise. Ongoing researches Within the framework of the FP7 “P-Medicine” project, working together with medical oncologists, Research Unit for Early Diagnosis and Prevention of Lung Cancer, our research group is developing a decision-making tool which offers an active knowledge resource using patients’ personal data and needs to generate case-

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specific advice in support of decision making about individual patients by health professionals, by the patients themselves or by others concerned about them. The tool is designed to aid health professionals and/or oncology patients in the clinical decision making process based on a personalized medicine approach. Within the framework of the “Cosmos” project, we are collaborating with the Research Unit for Early Diagnosis and Prevention of Lung Cancer) to create a cognitive and personality profile of heavy smokers in order to help clinicians understand the possible psychological causes and mental effects of smoking addiction and find the most effective approaches to increase the patients’ compliance to the proposed treatment. The Unit is also very interested in the topic of food choices, considering the considerable bearing it has on cancer-related diseases. Collaborating with oncologists, biologists and nutritionists, we are developing protocols and tools to analyze the way in which healthy people and patients make their choices about food, what factors are involved in such choices and how, if at all possible, to modify “unsafe” habits. In this regard, we have recently been awarded a grant sponsored by Working Capital, to develop a tool to help people to track their eating behaviors and to modify them according to their personal needs and health profile.

Activities 2012.

In 2012 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 (42 patients have been interviewed by psychologists of the Unit). Eight hundred and twelve (N=812) new patients, from all Departments, Divisions and Units, were referred to the Psycho-Oncology Unit. Three thousand three hundred fifty five (N=3355) consultations were performed during 2012.

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. 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, patients’ quality of life (QoL) and sexuality (paper is being draft). A particular attention is paid to the impact of very breast conservative surgery on Quality of life but also to the patient’s satisfaction and Quality of life after breast reconstruction in close collaboration with the EORTC QoL Group. In close collaboration with the Divisions of Senology, Reconstructive Plastic Surgery, and the Department of Medicine we studied 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 and we assessed the motivations of patients for accepting a NSM or the nipple reconstruction when NSM was not possible. Because of the stressful psychological impact of a breast cancer diagnosis, and the possible role of stress and the role of immunological factors in tumor growth, in 2011, thanks to a Grant from the Umberto Veronesi Foundation, we commenced psychosocial study to determine whether psychosocial stress, in the form of adverse life events and social difficulties, depressive illness, or lack 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: 140 patients

have been interviewed at baseline, for a total of 420 interviews performed by psychologists. Another study aimed at assessing the psychological stressful impact of the breast diagnosis and being on the waiting list, on women who are candidates for mastectomy has been terminated. 91 patients have been interviewed for a total of 183 interviews performed by psychologists. Publications Lucchiari, C., and G. Pravettoni. Cognitive Balanced Model: A Conceptual Scheme of Diagnostic Decision Making. J Eval Clin Pract 2012; 18 (1): 82-8. Gorini A, Miglioretti M, Pravettoni G. A new perspective on blame culture: an experimental study. J Eval Clin Pract. 2012, 18(3): 671-675. Cropley M, Michalianou G, Pravettoni G, Millward LJ. The relation of post-work ruminative thinking with eating behaviour. Stress Health. 2012, 28(1):23-30. Gorini A, Pravettoni G. An overview on cognitive aspects implicated in medical decisions. Eur J Intern Med. 2011;22(6):547-53. doi: 10.1016/j. ejim.2011.06.008. Didier F, Arnaboldi P, Gandini S, Maldifassi A, Goldhirsch A, Radice D, Minotti I, Ballardini B, Luini A, Santillo B, Rietjens M, Petit JY. 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. 2012. DOI: 10.1007/s10549012-1983-y

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Clinical Resources – Surgery Resources

Division of Anaesthesiology and Intensive Care Marco VENTURINO, MD Director

STAFF Senior Deputy Directors: Anna Attanasio, MD, Laura Della Grazia, MD, Marco Tulli, MD, Costanza Michela Acciaro, MD Deputy Directors: Costanza Michela Acciaro, MD, Marilia Bedoni, MD, Roberto Capucci, MD, Francesca De Lucia, MD, Davide Galli, MD, Rita Panzeri, MD, Daniele Sances, MD Assistants: 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, Luigi Delunas, MD, Antonio Pinna, MD, Gianluca Spano, MD, Donatella Sparicio, MD, Maria Paola Solinas, MD, Dario Vezzoli, MD Consultants: Giuseppe Susini, MD, Marco Torricella, MD

Activities 2012.

In 2012 surgical activity at IEO has performed 13947 operations of whom 4259 in day surgery regimen. According to different kind of surgery and patients, techniques of either general or locoregional anaesthesia are used, with a large use of Monitored Anesthesia Care (MAC, a form of deep sedation) in day surgery operations. A computerized monitoring system is used in order to collect data from all devices connected to patients and to record the anaesthetic procedures. An outpatient’s department is activated for the preoperative assessment of patients that need surgical operations: in 2012 a large amount of patients submitted to surgical interventions has been checked in this department, with a new modern approach to chemical and instrumental examination requests. In 2012 the robotic surgery has been improved: we have performed 843 interventions with this technique with a very low incidence of complications, developing one of the largest experience in Europe in robotic surgery (more then

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Antonella TOSONI, MD Co-director

3500 from 2006). According to IEO project called pain-free hospital, and to the italian law number 38/2010 introducing a new perspective on pain management, specific attention is paid to treatment of postoperative pain and prevention of its chronicization. The anesthesiologists are also involved in the safety management of operating room. They collect data about adverse events occurred in order to develop even more secure protocols, following the advanced rules of the “Helsinki Declaration on Patient Safety in Anaesthesiology” (2010) sponsored by ESA (European Society of Anesthesiology). Our division is also active member of the ETN (European Trial Network), the research branch of ESA and is involved in the most important large european trials. One of these was published by The Lancet in 20121). Moreover since 2011 our division has started a research collaboration with LABS (Laboratory of Biological Structure Mechanics Department of chemistry, materials and chemical engineering - CMIC - of Politecnico di Milano 2nd, Milano Bicocca University) on development of a novel device for liquid ventilation. The activity of the Intensive Care Unit is mainly devoted to post-surgical patients. In 2012 the ICU accepted 677 patients, 90% postoperative admissions. The mean ICU length of stay was 2 days and the mortality was very low (2.6%). The reported incidence of VAP (Ventilatory Acquired Pneumonia) was 0.2% while the CVC related bacteremia accounted for 0.14%. 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. Our ICU is connected with the major Italian ICUs via the GiViTi network. Special attention and studies are dedicated to new developments about the quality of life of ICU patients and their relatives, so our ICU is a pioneer in applying the “OPEN ICU” theory from many years. Particular care is taken on the functional recovery of critical patients treated by dedicated physiokinesitherapists’. The anesthesiological staff is trained to perform Transesophageal Echocardiography both in operating theatre and ICU as well. The staff is also trained to perform awake intubation with fiberoscopy and to safely manage difficult intubation. In these years we have developed

a special formation for medical staff about the vessels cannulation by echographic support. Anaesthesiologists also support invasive radiological and endoscopic procedures when required. They also perform central venous catheterization for chemotherapy, plasmapheresis and total parenteral nutrition and also support special procedure like HIFU, HILP, HIPEC and Liver Perfusion, developing innovative anesthesiological protocols. Publications Pearse RM, Moreno RP et al. Mortality after surgery in Europe: a 7 day cohort study The Lancet 2012;380;9847; 1059-65

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Clinical Resources – Surgery Resources

Day Surgery Division Giovanni Francesco MANFREDI, MD Director

STAFF Lead Nurse: Liliana Tadini Registered Nurses: Elena Antolini, Marika Comensoli, Diana De Donno, Monica De Piano, Rachida Hazmi, Jelena Pavic, Laura Verdone, Pamela Zirilli, Valentina Zucca Advanced Health Care Assistants: Marisa De Palmas, Antonella Paganoni, Monica Piscopo Ambulatory Surgery Ward Manager: Luca Benatti Registered Nurses: Monia Agostini, Alessandra Marras, Veronica Montagna, Mihaela Paun, Aldina Pavan, Emanuela Pisati, Monica Scotti Health Care Assistants: Lucrezia Piccolomo, Anna Forte

Activities 2012.

Established on May 2010, The Division of Day Surgery deal with all surgical or diagnostic operations/procedures, performed with either local, locoregional, general anesthesia or sedation, where the patient is discharged on the same working day, without a night of hospitalisation. Most people would rather recover from surgery in the comfort of own home, with relatives or friends, than in hospital. Day surgery provides fast come back to family environment, relationships and professional activity. Thanks to the short hospital stay we achieve also clinical advantages do to low rates of complications, such as infections and thromboembolism. Since the opening of the Center, the rate of the activity has steadily grown and during year 2012 over than 4500 patients were admitted for surgical treatments or diagnostic procedures. Patients belong to all of the surgical and diagnostic divisions of Ieo. Moreover 1700 operations were performed in the ambulatory setting. Only 3,5% of the patients require unplanned admission. We pay attention especially to guarantee both patient safety, comfort and satisfaction. Our main goals include the control of postoperative pain as well as quality improvement. Based on the ongoing new government health policy, in the course of year 2013, some procedures will be relocated from Day Surgery to Ambulatory setting, by suitable observational time, without daily hospital stay. Collaborative activity includes the membership in The Day Surgery Study Group of Italian Society of Anesthesia, Analgesia and Intensive Care (SIAARTI).

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Robotic surgery 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, oropharyngeal and supraglottic laryngectomy, adrenalectomy and total mesorectal excision deep in the pelvis as appropriate for a robotic approach, whereas, other operations such as thyroidectomy, pneumonectomy, 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

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sometimes hamper full robotic performance. 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. 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. There is an agreement in the effectiveness of robotic surgery in the treatment of malignant tumors of the pelvis (prostate, uterus, mainly in obese patients, and rectum), and the indication of this procedure together with the laparoscopic surgery is reported in several guidelines (NCNN, and guidelines of the Gynecological and urological societies). In gastrointestinal, head and neck and thoracic oncology, robotic surgery is applied

to a wide range of procedures, but is still in its infancy. Most studies reported that robotic surgery in these fields is feasible and safe, provides improved dexterity, better visualization, reduced fatigue and high levels of precision when compared to conventional laparoscopic, thoracoscopic and mini-invasive oropharyngeal 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. Figure 1 shows the increasing use of robotic –assisted surgery at IEO. 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 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, thoracoscopic and mini-invasive 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.

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Clinical Resources – Medical Resources

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) Secretary: Fabio Farina Chief Nurse: Arnaldo Zanelotti Data Manager: Ines Tedeschi OTA: Maria Iannitelli Consultants Smoke Cessation Center: Elena Calvi, MD (Psychologist and Pneumologist), Natalia Pozzi (Fondazione Umberto Veronesi) Cardioncology Unit Staff Director: Daniela Cardinale, MD, PhD, FESC Senior Assistants: Alessandro Colombo, MD, Giulia Bacchiani, MD Consultant Doctor: Alexandra Gelfi, MD Fellows: Marta Beggiato, Damiano Pongan Data manager: Ines Tedeschi, DSc Secretary Assistant: Alessia Cattaneo Experimental Cardioncology Unit Staff Staff Scientist: Marco Giorgio Scientist: Mirella Trinei Fellow: Elisa Romanini

Activities 2012.

Cardiology Division’s activities relate to pre- and post-operative 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 (51% of IEO cancer patients present concomitant cardiovascular diseases) as well as potential or evident consequences of oncologic treatments - i.e. cardiotoxicity). Cardiological evaluations, either clinical or instrumental, are present in 165 scientific research protocols of the Institute. In 2012 the Division performed: a. cardiologic assessment of 16.665 internal and outpatients; b. complete echocardiographic and Doppler color evaluations in 3954 patients; c. respiratory physiopathology diagnostic and assistance (2343); d. 122 antismoking activities for patients and employees; e. clinical consultations and/or echocardiographic examinations for 18181 patients enrolled in different; f. overall 55639 written official cardiologic, cardioncologic and pneumologic reports. During 2012 1112 patients were treated in urgent emergency setting, 82% internal cases, 18% outpatients. Cardioncology Unit Cardioncology is a novel, interdisciplinary, rapidly evolving area of growing interest, based on a comprehensive approach for the management of cardiovascular problems of cancer patients, pre-existent or induced by anticancer therapy. The Cardioncology Unit of the IEO is the first created in Italy to deal with this need. The main clinical and research areas of the Unit are early diagnosis of cardiotoxicity, cardiac risk

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stratification, prevention, treatment and monitoring of cardiotoxicity during anticancer therapy, including both traditional and new biologic agents. Since the current standard diagnostic methods allow to detect cardiotoxicity only when a functional impairment has already occurred, this precluding any chance of preventing its development, the Cardioncology Unit of the IEO has created specific internal procedures, based on our almost twenty-year-long clinical and research experience. They include the assessment of cardiac biomarkers (Troponin I and NT-proBNP), and an early preventive therapy with ACE-inhibitors, in selected highrisk patients, namely those showing myocardial injury

during the oncologic treatment, revealed by the increase of Troponin I. This approach has proven to be effective in the prevention of the development of cardiotoxicity in more than 1,700 cancer patients, who we have followed-up for more than 7 years. Due to the increasing number of long-term cancer survivors, the aging of the population, as well as the increased incidence and prevalence of oncologic and cardiovascular diseases, the number of patients presenting oncologic and cardiologic co-morbidities are increasing. These patients are often excluded from intensive cardiologic treatment and/or interventions, and are often excluded from a first-line, aggressive - and therefore more effective - therapeutic

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Clinical Diagnosis and Resources Prevention Area oncologic strategy, because considered to be too high a risk for cardiovascular complications. This behavior may have a negative prognostic impact during the course of the two illnesses, whereas an integrated and multidisciplinary approach, involving both the cardiologist and the oncologist, may allow the patient to be effectively and safely treated. To achieve this aim, the IEO Cardioncology Unit has created a specific procedure for these “frail” patients, to allow them to receive an effective oncologic therapy as well. This internal procedure provides a very close cardiac surveillanceincluding the assessment of both cardiac biomarkers, Troponin I and NT-proBNP- and the sharing of all the patient’s information with the oncologist at each step of the way. At present, more than 140 patients, with pre existing cardiac disease, have been treated successfully, without the worsening of the underlying cardiac condition or the occurrence of adverse cardiac events. In 2011 the out-patients clinic of the Unit (Ambulatorio di Cardioncologia – opened in 2009) has performed more than 450 cardioncologic evaluations, both for oncologic patients treated at IEO and at different Italian hospitals, creating active and effective teamwork collaborations with both cardiologist and oncologist colleagues. The Cardioncology Unit is strongly involved in several clinical and translational research projects, in collaboration with the Experimental Cardioncology Unit of our Institute and the Research Center for Vascular Biology and Cardiovascular Regeneration of the Centro Cardiologico Monzino of Milan, mainly focused on the evaluation of new, earlier, biomarkers of cardiotoxicity and the role of angiotensin-converting enzyme inhibitors in cardioprotection against cardiotoxicity, both in animal and human populations. Experimental Cardioncology Unit We study the evolutionary, genetic and molecular links among cellular energetics, fitness and aging. This core of basic science is focusing in particular on the

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mitochondrial-cell crosstalk with the aim of identifying mitochondrial stress markers associated to diseases. Metabolic adaptations to hyperglycemia, dyslipidemia, hypoxia (ischemia/reperfusion) and cardiac toxicity (side effect of antitumor drugs) share common mitochondrial pathways that eventually lead to cardiovascular diseases. Cardiotoxicity limits the efficacy of current chemotherapy. It is induced by different antineoplastic drugs including novel kinase inhibitors and it is characterized by mitochondrial alterations. The mechanisms leading to mitochondrial alterations and whether these are an indirect consequence of cardiomyocyte damage or the cause of cardiac cell injury is unclear. Regarding the kind of stress, due to the abundance of mitochondria in cardiomyocytes and the role of oxidative metabolism on myocardial function, mitochondrial dysfunction is crucial in the heart. On these basis mitochondrial alterations and released components reveal tissue injury. Our mission is to characterize in detail how antineoplastic drugs affect mitochondrial functions including membrane permeability and reactive oxygen species production, to validate the usage of mitochondrial damage and oxidative stress products as markers of myocardial damage and finally to establish the cardioprotective effect of inhibitors of mitochondrial stress. Recently, we have identified circulating cytochrome c as marker of cardiac injury and developed a novel assay to detect cytochrome c for diagnostic purposes. We are now validating novel potential markers of heart injury by studying the transcriptome and the released proteome from cardiomyocyte culture and ex vivo mouse hearts. Then we are investigating the mechanism of cardioprotection of ACE inhibihors and trying to improve other cardioprotective strategy by inhibiting mitochondrial oxidative stress. Cardiology Specific Activities Diagnosis of cardiotoxicity. Cardiotoxicity is a common complication of chemotherapy (CT). The clinical

manifestation of cardiotoxicity can range 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 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 guidelines recommend regular cardiac function assessment (generally by echocardiography or MUGA scan) to detect CT-induced cardiac damage in an early phase. The weak point of such an approach is that these techniques have low sensitivity and poor predictive value. Indeed, cardiotoxicity is usually detected when cardiac damage has already occurred. 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 hemodynamic markers like N-terminal-proB-Type Natriuretic Peptide (Nt-proBNP). For all of them, an accurate predictive value has been demonstrated by our investigations. Cardiotoxicity prevention: a new prophylactic approach. The possibility to identify patients at higher risk of developing late myocardial dysfunction by cardiac biomarkers provides a rationale for the development of prophylactic strategies directed against CT-induced cardiotoxicity. Considering the results of our published studies, a prophylactic treatment with enalapril in patients at high risk to develop cardiotoxicity, namely those showing an increase in troponin during anticancer therapy – including high or standard dose of AC, as well as new biologic targeted drugs – has proven to effectively prevent CD and associated cardiac events.

Cardiotoxicity treatment. CT-induced cardiotoxicity can result in a cardiomyopathy generally considered to be irreversible, and leading 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 recently published experience, most patients receiving early adequate treatment, including ACE-inhibitors, betablockers, diuretics, and anti-aldosterone agents, showed 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. 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. 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 previously investigate short and long-term safety and effectiveness of PC followed by intrapericardial infusion of an active antiblastic, sclerosing agent, thiotepa, in patients with large malignant pericardial effusion.

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Clinical Diagnosis and Resources Prevention Area 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. On the basis of our previous study, we routinely perform pericardiocentesis and we routinely associate an intrapericardial “oncologic therapy” with thiotepa, 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. 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, psycocounselling Doctor Elena Calvi, patient’s enrollment and coordination Mrs. Natalia Pozzi). The study protocol will consider 123 (actual recruitment 62) patients and the results will be available at the end of 2013. The protocol was accepted by Clinicaltrials.gov.The activities concerning the nicotine free electronic cigarette TFumo study protocol will integrate the antismoking activities of the IEO Centro Antifumo 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.

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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. In 2012 an international outstanding Congress on Cardioncology was held in our Institute in Milano (October). The participation was extraordinary, over 150 cardioncologists coming from different 23 foreign Countries. All the cardioncology educational activities are organized by ICOS, the International Cardioncology Society, www.cardioncology. com. The clinical and scientific rationale of ICOS (over 250 members of 45 different countries) 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. We are very grateful to our colleagues Dr Carpi and Prof Di Lisa from Padua University whose invaluable collaboration has greatly benefited Cardioncology translation research. Publications Cardinale D. Cardiac dysfunction after cancer treatment. In: Bonow RO, Mann DL, Zipes DP, Libby P, editors. Braunwald’ Heart Disease E-dition. Chapter 90: Cardiovascular complication of cancer therapeutic agents. Philadelphia: Elservier Inc.,2012:9th edition. Cardinale D, Colombo A, Bacchiani G. Cardioprotection in women with neoplastic disease. G Ital Cardiol 2012;13:461-468. Lenihan D, Cardinale D. Late Cardiac Effects of Cancer Treatment. J Clin Oncol 2012;30:3657-3664. Curigliano G, Cardinale D, Suter T, Plataniotis G, de Azambuja E, Sandri MT, Criscitiello C, Goldhirsch A, Cipolla CM. Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy: ESMO Clinical Practice Guidelines. Annals of Oncology 2012; 23(Supplement 7): vii155–vii166. Cardinale D, Salvatici M, Sandri MT. Role of biomarkers in Cardioncology. Clin Chem Lab Med 2012;49:1937-1948.

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Clinical Diagnosis and Resources Prevention Area

Clinical Resources – Medical Resources

Day Hospital Division Franco NOLÈ, MD Director

STAFF Ward Manager: Silvana Lacapra Registered Nurses: Elena Taraschi, Elena Bocchiola, Barbara Lazzaroni, Laura Poloni, Vanessa Chamizo, Teresa Profeta, Laura Bistocchi, Alice Consoli, Angelica Paoli, Daniela D’Aronzo, Claudia Zencovich, Andrea Tramacere, Enrico Sari, Cristina Berti, Livio Libutti, Federica Macciola, Colette McDonnell Health Care Assistants: Aldo Rossi, Gian Marco Prampolini, Christine Agnu

Activities 2012.

The Outpatient Cancer Care, represent an important part of the activity in oncology in addition to the inpatient approach. Cancer outpatients receive highly complex and technical procedures and treatments, avoiding their in-staying, resulting in an increase of efficiency and in a more satisfying diagnostical-therapeutical process for the patient, helping cancer patients fit their medical care into their lives, instead of fitting their lives into their medical care. The Division of Day Hospital is an essential Department at European Institute of Oncology and provides a range of treatments that can safely be given on a day

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patient basis. The Day Hospital of European Institute of Oncology is open five days a week, from 7:30 AM to 18:00 PM. The Day Hospital has a mixture of beds and chairs for use during treatment in particular: • Sixteen chairs or infusion stations in which patients can receive their therapy in a light, open environment that allows for interaction with others patients. Every care setting includes accommodations for the patient’s family or friends. • Sixteen beds for patients receiving longer treatments or who need more complex support. In this area, chemotherapy patients who would normally need to be hospitalized will be able to receive their medical treatments during the day and then go home in the evening. The Division of Day Hospital has the capability to treat up to 30 patients at a time. All treatment areas are within view of the Nurses’ Station to ensure patients are closely monitored, and each infusion area is connected to a nurse call system for an extra measure of safety. The examination rooms, for the clinical evaluation of the patients before and during the treatment, are located in the treatment area, designed to maximize efficiency. The Division of Day Hospital is composed of a team of a highly skilled oncology nursing. The nursing service is organized according to the model of primary nursing. The primary nurse is responsible for developing the patients’ plan of care, continually assessing progress and outcomes, adjusting accordingly the plan. The primary nurse often provides care to the patient at each visit, and is responsible for directing the care. The Division of Day Hospital will provide medical oncology care including consultation, evaluation and management of patients and the administration of chemotherapy, biotherapy and supportive therapies such as intravenous hydration, electrolyte replacement, blood/ cellular product infusions.

Divisions and Units operating in Day Hospital. Medical Division of Breast Tumors, Division of Clinical HaematoOncology, Division of Early Drug Development for Innovative Therapies, Medical Division of Urogenital and Head & Neck Tumors, Medical Oncology Division of the Respiratory system, Medical Division of Gynecological Tumors, Medical Division of Gastrointestinal Tumors, Medical Division of Melanoma. Adult oncology patients, including hematologic and solid tumor, during all phases of treatment including standard care regimens, cutting edge research and supportive care are treated in Day Hospital. Treatment includes progressive modes of therapy and symptom management. Medical

specialists working in Day Hospital, belong to the individual Divisions and Units of medical oncology. Patients are considered for outpatient treatment after a multidisciplinary discussion and after a clinical evaluation with a medical oncologist specialist, who establishes treatment program, offering to the patients the possibility to participate to national or international clinical studies, which evaluate the advantages that innovative treatments could provide as compared to standard treatments in specific tumor types. During 2012 approximately 17,000 treatments were administered.

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Translational Research

Drug Discovery Program (DDP) Saverio MINUCCI & Mario VARASI Directors

STAFF Target Identification and Validation Unit Unit Director: Luisa Lanfrancone, PhD Post-doctoral Fellows: Daniela Bossi, PhD, Angelo Cicalese, PhD PhD Students: Alessandro Carugo, Carolina D’Alesio Technicians: Elena Cavallaro Drug Discovery Unit 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 Fellows: Giuseppe Meroni, Alessia Romussi, Paolo Trifiro, Maurizio Pasi Biology Scientists: Oronza Antonietta Botrugno, Maria Rosaria Cera, Maddalena Donzelli, Fabrizio Villa Post-doctoral Fellows: Anna De Ponti, Paola Dessanti, Giovani Fagà, Francesca Senic-Matuglia, Stefania Vultaggio Fellows: Roberto Dal Zuffo, Elisa Zagarrì, Silvia Brambillasca

Activities 2012. The primary objective of

the DDP 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 an IND, to ensure their clinical 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. From a starting point embedded in the basic science and technologies developed at the Campus to understand the molecular basis of cancer, the DDP translates basic science into viable therapies for cancer patients. The DDP scientists (medicinal, analytical and computational chemists with industrial drug discovery experience, biochemists, cell biologists and in vivo pharmacologists) work together in state-of-the-art laboratories to identify and develop new drugs through innovative research and excellence in drug discovery. The biology-inspired, chemistry-driven effort to identify innovative therapies against cancer is a team effort developed in strict collaboration with the Group Leaders, the Molecular Medicine Program and the TTFactor. The DDP includes two Units: the Target Identification and Validation Unit (TIV) and the Drug Discovery Unit (DDU). TIV-Target Identification and Validation Unit This part of the Program (started in 2011) is 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 “descriptive” approach

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to the anatomy of tumors, a “functional” strategy has better chances to lead to the identification of immediately validated targets. To this end we have generated an in vivo RNAi screening platform with the aim to screen a large cohort of patients’ samples transplanted in immunocompromised animals (NSG mice) with lentiviral-based shRNA libraries of epigenetic and metabolic targets and kinases. Our interest is mainly focused on melanoma, metastatic breast carcinoma resistant to conventional therapy and adenocarcinoma of the pancreas. Human tumors were transplantated in NSG mice orthotopically, according to well-established techniques. Xenografts in the animal should phenocopy the heterogeneity and the complexity of the tumor of origin. To this end, we have characterized the human tumors immunophenotypically, before injecting it in the mouse, after the first passage in the animal (X1) and after the second and third passages (X2 and X3). A panel of human tumor markers, as well as whole exome sequencing and epigenetic and proteomic profile of the tumors, were used to this purpose. We will then stratify our cohort of patients xeno-transplants according to: i) in vivo growth properties; ii) phenotypic markers; iii) genetic lesions; iv) characterization of intracellular signaling networks and v) epigenetic profile. At the end we will be possibly able to generate a prognostic signature of the patient and the most suitable model for RNAi in vivo screenings. The setting up of the in vivo RNAi screening in our model system requires that we: i) investigate the frequency of tumor initiating cells (TICs) in the tumor samples; ii) choose and test the most appropriate lentiviral-based library; iii) perform a titration and infectivity experiment on our tumor cells; iv) set up the proper amplification and sequencing technique to evaluate hairpins’ representation and v) analyze and validate target genes and pathways. The gold standard technique to evaluate TIC frequency is the limiting dilution transplantation of cancer cells in

the animal. Briefly we have transplanted cancer cells of the three tumor types at scalar doses to determine the minimum number of cells able to generate new tumors. Moreover, seeing that a very high number of cells is injected to simultaneously screen thousands of different hairpins, we set up a “clonal tracking” strategy to evaluate TIC frequency in vivo. Cells from X1 were infected with a lentiviral library, containing a random barcode, at low molteplicity of infection, so that every tumor cell carries a single copy of a randomly integrated, non-targeting, barcoded virus. Transduced cells were orthotopically transplanted in NSG mice at increasing cell doses (millions of cells) and tumor DNA analyzed to track the barcodes along the development of the disease and eventually at each passage of transplantation. The experimental setting will then allow the screening of dedicated shRNA libraries targeting “druggable” genes of our interest. DDU-Drug Discovery Unit Current pipeline includes targets in the area of Epigenetics, Cancer metabolism and Cancer stem cells (CSCs). 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. Chromatin enzyme 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

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Translational Research have been developed, including one to permit high throughput screening. Novel, irreversible, inhibitors of a lysine demethylase have been designed, synthesized and evaluated: the new derivatives, besides having inhibitor potency in the nanomolar range, showed target-related gene modulation in cells. Further, several compounds were profiled for in vitro ADME and in vivo PK characteristics and one molecule was selected for further analysis. This compound is currently undergoing assessment in an in vivo efficacy study. In addition, an alternative strategy to identify reversible inhibitors of the lysine demethylase was established, In collaboration with a Contract Research Organization, a high through put screening campaign against a proprietary library of commercially available compounds was conducted and several interesting hits belonging to different chemical classes were identified (in coll. with S. Minucci, P.G. Pelicci e G. Natoli). Cancer metabolism Altered cancer metabolism is now considered one of the hallmarks of cancer and strategies to target these alternations have become the focus for novel targeted therapies. Many tumors increase glucose uptake and, even in the presence of abundant oxygen, switch from mitochondrial oxidative phosphorylation to perform anaerobic glycolysis, converting pyruvate to lactate (known as the “Warburg effect”). Many of the genes involved in this altered metabolism are regulated by tumor hypoxia and oncogenes, such as Kras and myc. Lactate dehydrogenase is a key enzyme in the glycolytic switch and converts pyruvate to lactate with the subsequent production of 2 molecules of ATP and the regeneration of nicotinamide adenine dinucleotide. LDH-A is up-regulated in many tumor types and its high expression correlates with poor prognosis. Gene ablation studies have shown that the LDH-A gene is required for the proliferation of several cancers in vitro and in vivo. In Collaboration with Prof F. Minutolo (University

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of Pisa) the DDP is developing small molecular weight inhibitors of LDH-A. These inhibitors are competitive with LDH-A substrates in biochemical assays and demonstrate anti-proliferative activity against a panel of tumor cell lines (including, breast, pancreatic and non small cell lung cancer (NSCLC)). In addition, consistent with attenuation of LDH-A activity, these inhibitors are able to reduce the production of lactate in tumor cells. Further, when administered orally once daily to mice, LDH-A inhibitors exhibit good tumor growth inhibitory properties in xenograft models of NSCLC, with no observable toxicity. A potential clinical candidate has been identified and is being advanced as rapidly as possible towards an IND submission and clinical trials. In the meantime, novel chemical classes are being evaluated for LDH-A inhibitory activity in order to develop a second generation of inhibitors. Cancer stem cells: 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 tumor 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 tumors 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 regulators present ideal pharmacological targets for the stabilization of Numb levels and the restoration of its tumor suppressor activity. Two strategies have initially been considered 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 of different gene families have been identified and are currently undergoing validation. In order to facilitate the “reverse strategy” a cellular model, suitable to run a phenotypic High Content Screening campaign, has been established. By evaluating several protocols and reagents to obtain correct image segmentation, optimal experimental conditions in terms of good dynamic range, have been identified. Moreover, the data obtained during uniformity and signal variability assessment were all in the range of acceptance criteria defined by the NIH Chemical Genomic Center guidelines (in coll. with S. Pece and P.P. Di Fiore). Exploratory activities are also developed internally or through external collaboration in order to create a machinery ready to feed the running project pipeline. Exploratory activities include, e.g., approaches to restore functional p53, design of novel scaffolds for innovative chemical libraries and the preparation of kinase-targeted libraries.

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The Molecular Medicine Program Pier Paolo DI FIORE, MD, PhD Director

STAFF Staff Scientists: Elena Carla Belloni, Giovanni Bertalot, MD, Ugo Cavallaro, PhD, Salvatore Pece, MD, PhD, Daniela Tosoni, PhD Scientists: Ivan Nicola Colaluca, PhD Scientific Responsible: Fabrizio Bianchi, PhD, Giuseppina Bonizzi, PhD, Stefano Confalonieri, PhD, Manuela Vecchi Post-doctoral Fellows: Francesca Biagioni, PhD, Giovanni Corso, MD, PhD, Macarena Ferrero Gimeno, PhD, Michela Lupia, PhD, Stefano Marchesi, PhD, Alessandra Micaela Villa, PhD, Elena Miranda, PhD, Francesca Montani, PhD, Davide Rambaldi, PhD, Angelo Taglialatela, PhD PhD Students: Letizia Amadori, Francesca Angiolini, Claudia Baccichet, Maria Elena Bicchieri, Fabio Dezi, Pietro Lo Riso, Eleonora Lusito, Valentina Melocchi, Simona Monterisi, Silvia Restelli, Chiara Tordonato Data Entry: Maria Capra Technicians/Technologists: Stefania Amodio, Rossana Bettolini, Michele Caccia, Rose Mary Carletti, Marco Coazzoli, Ivan Cuccovillo, Elena D’Acunzo, Alessia Delli Carli, PhD, Francesca De Santis, Stefano Freddi, PhD, Tiziana Fumagalli, Fulvia Fusar Imperatore, Donatella Genovese, Elvira Gerbino, Barbara Giulini, Giovanna Jodice, Chiara Luise, Elena Marino, Valentina Mattei, Luca Napolitano, Stefania Pirroni, Flavia Troglio, PhD, Andrea Ugetti, Emanuele Villa Carlo, Silvia Zecchini, PhD Undergraduate Students: Susanna Diamanti Visitors: Blanca Alvarez Moya, PhD, Andrea Basile, MD, Elisa Dama, Dyvia Purushothaman, PhD, Alicia Rubio Garrido, PhD Molecular Medicine-Program Manager: Bronislava Matoskova, MD Laboratory Manager: Natalia Meani, PhD Administrative Assistant: Semhare Berhane Support Personnel: Domenico Gualandi

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Activities 2012. The “Molecular Medicine for

Care” (MMC) program fuses the two strengths of IEO: excellence in research and excellence in patient care. The MMC program provides a work environment that nurtures the direct collaboration between clinicians and researchers. It acts as an interface that allows clinical care and basic research programs to gradually grow into each other in a way that will boost the successful and efficient transfer of scientific results from the laboratory to the clinical setting. MMC Scientific Programs Our scientific programs focus on breast and lung cancer, the two most commonly diagnosed cancers worldwide. In spite of their high incidence, our biological knowledge about these diseases has had little impact on the way these diseases are managed in the clinic. Our research programs are designed to address this problem. 1. Understanding breast cancer to guide therapeutic choice Our first scientific program is based on our discovery that ‘breast stem cell’ genes can stratify breast tumors according to their pathological, molecular and clinical characteristics. We have previously identified a gene profile of normal mammary stem cells with more than 1,000 genes. 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. This study will allow us to assess the ability of these genes to stratify patients according to tumor aggressiveness, metastatic risk and therapeutic response to hormonal and/or chemo-/ radio-therapy. We are currently developing this set of genes as a clinical grade parameter to refine our current diagnostic/prognostic ability and improve the

clinical management of breast cancer. The success of this program 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 Tumor Registry. 2. Development of a blood test for the early diagnosis of lung cancer The absence of national screening programs for lung cancer, and its lack of symptoms means that early detection of this disease is a rare and sporadic event. Therefore, the development of clinical tools for lung cancer early diagnosis is a pressing clinical necessity, particularly for at-risk subjects (smokers or exsmokers). We have previously 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. We now aim to prove the clinical applicability of this test through its largescale validation in a prospective trial that will recruit some 20,000 patients. This is an essential study, which will permit the transfer of our results to the clinic, should our initial results be confirmed. To date, 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. The large-scale prospective screening study is currently underway. This program is possible thanks to the close relationship that has developed, through the MMC program, between our researchers and lung clinicians at IEO. 3. Understanding breast cancer for early diagnosis Our third scientific program is designed to investigate whether prognostic/diagnostic miRNA profiles exist for breast cancer. We have set up a trial to analyze the miRNA profiles of breast cancer patients, with the

aim of identifying a circulating breast cancer-specific miRNA profile that is diagnostic for breast cancer. Such a profile would be specifically present within the sera of breast cancer patients, but would be absent in healthy subjects. If successful, this can be used as a comparatively inexpensive first level ‘diagnostic’ test for breast cancer that can easily be implemented within national breast screening programs. We have previously shown that more aggressive breast cancers tend to have a larger number of stem cells than less aggressive tumors. Within the context of this program, we are therefore analyzing the miRNA profile of breast stem cells. We will determine if such a miRNA profile exists within the sera of breast cancer patients, and whether it can be used to improve patient prognosis. MMC Laboratory Infrastructures The following facilities provide enabling conditions for the MMC scientific programs: • Tissue Bank and xenotransplantation Unit • Molecular Pathology Laboratory • Primary Epithelial and Stem Cell Culture Facility • Clinical Biomarkers Laboratory The IEO Tissue Bank (Biobank and Biomolecular Resource Infrastructure - IBBRI) collects, catalogues and stores surgically excised tissue samples that are nonessential for diagnosis as well as plasma/serum, total blood, DNA and RNA, according to specific protocols and standard operating procedures (SOPs). This ensures optimized and standardized treatment of all samples for research purposes. In collaboration with the Cell Culture Facility, the IBBRI also ensures the storage of purified normal and cancer stem cells and primary cell cultures. The IBBRI provides high quality biospecimens required for biomarker discovery and xenotransplantation experiments, the latter being essential for testing

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Translational Research new combination therapies or new drugs that will be developed in the future through MMC. 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 has been developed specifically for this purpose, thanks to the collaboration of clinicians, researchers, and bioethicists, and has been approved by the Ethics Committee of IEO. The IBBRI is tightly connected with the IEO Tumor Registry, which 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 IBBRI. Furthermore, the IBBRI is deeply integrated with the daily workings of IEO clinical and surgical wards for the delivery of informed consent forms to patients and for sample collection. Importantly, collaboration between the IBBRI and the Department of Pathology and Laboratory Medicine guarantees that patient diagnosis remains the primary objective, and provides pathologist expertise for sample classification and dissection. The IEO Molecular Pathology Laboratory relies on highthroughput screening technology (Tissue Microarrays, TMA) in combination with in situ detection methods (ISH and IHC) to identify novel putative cancer targets for drug discovery, diagnostic and/or prognostic applications. TMA technology allows performance of 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 have a pipeline between the Pathology Department at IEO and our service, which is essential for the replenishment of our TMAs. Through the Tissue Bank collaboration with the Tumor 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

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outcome, a necessary step for the validation of new diagnostic and prognostic markers.

embedded tissue specimens and selects appropriate tissue samples for prospective and retrospective studies.

The Primary Epithelial and Stem Cell Culture Facility has the specific task of deriving bulk primary epithelial cells and stem cells, whenever possible from human biopsy specimens of matched normal and tumor 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. We have developed protocols and SOPs 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 Tumor Registry for the collection of clinico-pathological information linked to human samples.

Publications Tosoni D, Di Fiore PP, Pece S. Functional purification of human and mouse mammary stem cells. Methods Mol Biol 2012; 916:59-79.

The Clinical Biomarkers Laboratory acts transversally with clinical and research activities and with the different enabling platforms of the program to optimize cancer biomarker candidates and facilitate the clinical development of research results. 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. In collaboration with the IBBRI (Tissue Bank), the Clinical Biomarkers Laboratory optimizes 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-

Boniolo G, Di Fiore PP, Pece S. Trusted consent and research biobanks: towards a “new alliance” between researchers and donors. Bioethics 2012; 26:93-100. Bianchi F, Nicassio F, Marzi M, Belloni E, Dall’Olio V, Bernard L, Pelosi G, Maisonneuve P, Veronesi G, Di Fiore PP. A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO Mol Med 2011; 3:495-503. 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. Boniolo G, Di Fiore PP. Deliberative ethics in a biomedical institution: an example of integration between science and ethics. J Med Ethics 2010; 36:409-14.

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Translational Research

SmartFood: program in Nutrition Science & Communication Pier Giuseppe PELICCI Director

STAFF Laboratory activities: Marco Giorgio, PhD Dietary intervention trials: Lucilla Titta, PhD Communication: Marco Bianchi

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Activities 2012. Cancer is a disease of genes,

which are vulnerable to harmful mutations, especially over the long human lifespan. Some substances present in foods play a role in determining the likelihood of mutations, as well as in changing gene function even without mutation. Epidemiological and experimental evidence demonstrate that only a small proportion of cancers are inherited; environmental factors are the most important cause of genetic modification. These factors include food poisons such as mycotoxins or alcohol as well as smoking, infectious agents, radiation, drugs, industrial chemicals and pollutants. Thus, habits (including healthy diet) aimed to minimize the exposure to these carcinogens are known to protect from tumorigenic mutations. More recently some nutrients and physical activity have been demonstrated to prevent cancer and other aging associated disease by altering the functions of specific genes (belonging to longevity genetic pathways) and inhibiting fat accumulation that increases the cancer risk. Essentially, these are good news. It means that a healthy environment can stop cancer before it starts. In this context nutrition represents an extraordinary tool to prevent cancer by reducing the intake of food dirties and increasing the consumption of beneficial “smart” foods. Validating healthy effects of food components and selecting foods enriched for such components represent the new target of nutrition research together with understanding the molecular mechanisms how diets work. However healthy nutritional guidelines are partially known and poorly practiced, others (smart diet) could be established and implemented.

The aim of our program “SmartFood” is devoted to developing nutritional improvement at different levels taking advantage of our already existing network. The potential goals of the project include: • Identify compounds in foods that interact with longevity genetic pathways (smart food compounds) • Select those foods enriched for such compounds (smart foods) • Validate the effects of “smart food compounds” and “smart foods” in disease prevention and cure, in model systems and humans (smart trial 1) • Develop “smart food compounds” into dietary supplements or pharmaceutical drugs • Promote good health through nutrition and the primary prevention of nutrition related illness in the population (science communication, societal interventions).

Titta L, Trinei M, Stendardo M, Berniakovich I, Petroni K, Tonelli C, Riso P, Porrini M, Minucci S, Pelicci PG, Rapisarda P, Recupero GR, Giorgio M. Blood orange juice inhibits fat accumulation in mice. International Journal of Obesity (London), 2009 Dec 22 Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nature Biotechnology, 2008 Nov 26

The potential benefits of the program include: increased focus on diet and life span, motivate positive behavior change, increased awareness of risk of certain conditions, improve health and healthy aging, focus on prevention, reduce health care costs, better understanding of the mechanism involved in disease susceptibility. Pubblications Salamone F, Li Volti G, Titta L, Puzzo L, Barbagallo I, La Delia F, Zelber-Sagi S, Malaguarnera M, Pelicci PG, Giorgio M, Galvano F. Moro orange juice prevents fatty liver in mice. World Journal of Gastroenterology. 2012 Aug 7

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Genomic Program

Activities 2012. The new Genomic Research at

IEO: bridging the gap from research to patients. The availability since 2007 of innovative technological platforms for genome sequencing (collectively indicated as Next-generation sequencing) has enabled studies investigating genome structure and regulation at an unprecedented speed and coverage. These technologies are evolving in an exponential manner in terms of ability of individual instruments to rapidly process samples, thus generating huge amounts of data at a speed that could not even be imagined even a few years ago. At the same time, although the equipments that are required for these approaches require significant investments as well as specialized technical personnel and experimental and computational infrastructures, the cost required for sequencing individual biological samples is rapidly dropping. Estimates from the National Human Genome Research Institute (NHGRI, USA) indicate that the initial human genome cost over 10 million dollars; in 2001, complete sequencing of a genome cost about 1.3 million dollars, while the current cost is about 6 thousand dollars and is expected to drop to less than one thousand dollars in less than three years. Although these technologies are currently used mainly for research purposes or for very selected diagnostic approaches, they will become part of the routine diagnostic assessment of tumors in just a few years from now. These genomic analyses will allow determining at very high resolution the structure of aberrant human genomes in cancers, thus allowing clinicians to classify apparently similar tumors (based on standard histopathological classifications) in homogeneous subgroups characterized by the same ensemble of genomic changes. Since the properties of each tumor, in terms of growth, invasiveness, ability to metastasize and response to therapy, are encoded by its abnormal genome, this will lead to the definition of homogeneous groups that will enable tailored therapeutic approaches. In fact an in depth knowledge of tumor genomes is a crucial pre-requisite for a truly

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personalized medical treatment. In this scenario, IEO considers research in genomics a strategic priority for the next decade. Since 2009 IEO has made significant investments in Genomics in collaboration with the Center for Genomic Sciences of the Italian Institute of Technology (IIT) hosted at our Campus. Currently, about ten research groups of the DEO-IEO and additional groups at the IIT are actively using Next Generation Sequencing technologies to investigate genome regulation and structure in the context of different aspects of tumor biology. This has led to the development of a highly integrated genomic research community including more than one hundred wet biologists, about 30 computational scientists, and a Technological Unit dedicated to genomic platforms and run in partnership by IEO and IIT. During these years, genomic research carried out at IEO has already led to a number of high-impact scientific publications and patents. Moreover, IEO is founding member of the two most important international scientific consortia dedicated to the use of Next Generation Sequencing technologies to investigate modifications of DNA and its associated proteins in normal and pathological tissues, Blueprint (www.blueprint-epigenome.eu/) and IHEC (International Human Epigenome Consortium, http://ihec-epigenomes.org/) References Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells (Vella P, Scelfo A, Jammula S, Chiacchiera F, Williams K, Cuomo A, Roberto A, Christensen J, Bonaldi T, Helin K, Pasini D.) Mol Cell. Feb 21;49(4):645-56 (2013). Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma (Shukla R, Upton K, Muñoz-Lopez M, Gerhardt D, Fisher M, Nguyen T, Brennan T, Baillie T, Collino A, Ghisletti S, Sinha S, Iannelli F, Radaelli F, Dos Santos A, Rapoud D, Guettier C, Samuel D, Natoli G, Carninci P, Ciccarelli F, Garcia-Perez JC, Faivre J, Faulkner G.) Cell in press (2013)

Latent enhancers activated by stimulation in differentiated cells (Ostuni R, Piccolo V, Barozzi I, Polletti S, Termanini A, Bonifacio S, Curina A, Prosperini E, Ghisletti S, Natoli G.) Cell. 152: 157-71 (2013).

reveals somatic and constitutional genomic instability (De Grassi A, Segala C, Iannelli F, Volorio S, Bertario L, Radice P, Bernard L, Ciccarelli FD) PLoS Biol. Jan;8(1):e1000275 (2010).

Hdac3 requirement for the inflammatory gene expression program (X. Chen, I. Barozzi, A. Termanini, E. Prosperini, A. Recchiuti, J. Dalli, F. Mietton, G. Matteoli, S. Hiebert, G. Natoli) Proc Natl Acad Sci USA 109:E2865-74 (2012).

Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing. (Dellino GI, Cittaro D, Piccioni R, Luzi L, Banfi S, Segalla S, Cesaroni M, Mendoza-Maldonado R, Giacca M, Pelicci PG) Genome Res. Jan;23(1):1-11 (2013) Genomics of acute myeloid leukemia: the next generation (Riva L, Luzi L, Pelicci PG.) Front Oncol. 2012;2:40 (2012)

Transcript dynamics of pro-inflammatory genes uncovered by RNA-Seq analysis of subcellular RNA fractions. (D.M. Bhatt, A. Pandya-Jones, A.J. Tong, I. Barozzi, M. Lissner, G. Natoli, Black D.L., Smale S.T.) Cell 150, 279-290 (2012). The histone methyltransferase Mll4 controls macrophage function through glycosylphosphatidylinositol 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 (2012). The genomic landscapes of inflammation (G. Natoli, S. Ghisletti, I. Barozzi) Genes & Development 25, 101-106 (2011). A large fraction of extragenic RNA Pol II transcription sites overlap enhancers (F. De Santa, I. Barozzi, F. Mietton, S. Ghisletti, S. Polletti, BK Tusi, H. Muller, J. Ragoussis, CL Wei, G. Natoli) PLoS Biology 8(5): e1000384. doi:10.1371/journal.pbio.1000384 (2010). 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, 32:317-28. Epub 2010 Mar 4 (2010). Modification of gene duplicability during the evolution of protein interaction network (D’Antonio M, Ciccarelli FD) PLoS Comput Biol. Apr;7(4):e1002029 (2011) The (r)evolution of cancer genetics (Ciccarelli FD) BMC Biol. Jun 11;8:74 (2010) Ultradeep sequencing of a human ultraconserved region

Fanelli M, Amatori S, Barozzi I, Soncini M, Dal Zuffo R, Bucci G, Capra M, Quarto M, Dellino GI, Mercurio C, Alcalay M, Viale G, Pelicci PG, Minucci S. Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples. Proc Natl Acad Sci U S A. Dec 14;107(50):21535-40 (2010) Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue (Fanelli M, Amatori S, Barozzi I, Minucci S.) Nat Protoc. Nov 10;6(12):1905-19 (2011). Fish the ChIPs: a pipeline for automated genomic annotation of ChIP-Seq data (Barozzi I, Termanini A, Minucci S, Natoli G.) Biol Direct. Oct 6;6:51 (2011). Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts (Perna D, Fag G, Verrecchia A, Gorski MM, Barozzi I, Narang V, Khng J, Lim KC, Sung WK, Sanges R, Stupka E, Oskarsson T, Trumpp A, Wei CL, Muller H, Amati B) Oncogene Mar 29;31(13):1695-709 (2012) Cell reprogramming requires silencing of a core subset of polycomb targets (Fragola G, Germain PL, Laise P, Cuomo A, Blasimme A, Gross F, Signaroldi E, Bucci G, Sommer C, Pruneri G, Mazzarol G, Bonaldi T, Mostoslavsky G, Casola S, Testa G.) PLoS Genet. Feb;9(2):e1003292 (2013)

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Translational ??? Research

Translational Research

Division of Epidemiology and Biostatistics Patrick MAISONNEUVE, Eng Director, Epidemiology Unit

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

Activities 2012.

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 programs 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 tumor registry. The tumor registry was activated in 2006 and after 7 years of activity, by March 2013, data for 154,099 tumors were retrospectively coded and entered (out of 240,440 individuals presenting for the first time at the IEO over the period 2000-2008). With the publication of its first research projects, the tumor registry has proven to be

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Nicole ROTMENSZ, MSc Director, Data Quality Control Unit

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 randomisation schemes, 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. Achievements 2012 The Division is maintaining large institutional clinical databases, which have been already in the past, the basis for numerous studies, particularly on breast cancer. During 2012 a major research focus was made on the outcome and clinical aspects of breast cancer subtypes defined either by their molecular of morphological characteristics. A series of reports were published on the outcome of special types of luminal breast cancer, on triple-negative breast, on primary and secondary angiosarcomas of the breast, on invasive lobular breast cancer subtypes and on sarcoma of the breast. Other research interests during 2012 included the prognosis of breast cancer patients who underwent plastic or reconstructive surgery, such as nipple sparing mastectomy or lipofilling, and of patients with breast cancer diagnosed during pregnancy. Finally the division contributed to an important study on the prognostic effect of circulating adiponectin in a randomised 2 x 2 trial of low-dose tamoxifen and fenretinide in premenopausal women at risk for breast cancer. In summary, the statistical support activity of the division led to the publication of 45 peer-review clinical research articles in various fields ranging from cancer screening, chemoprevention, treatment, prognosis and outcome. Most of this activity was related to cancer of

the breast, lung, digestive tract, urogenital organs as well as haematological neoplasms. The division is also largely involved in collaborative research with other Italian or international institutions: In 2012, results from nine meta-analyses and reviews on the association between alcohol drinking and cancers of the stomach, oesophagus, bladder, kidney, prostate, ovary, breast, Hodgkin disease and non-Hodgkin lymphomas were published, conducted, among others, in collaboration with the University of Milan-Bicocca, the Mario Negri Institute for Pharmacological Research, Milan; the International Agency for Research on Cancer, Lyon, France; The International Prevention Research Institute, Lyon, France; the Karolinska Institutet, Stockholm, Sweden; the Tehran University of Medical Sciences, Tehran, Iran; and The Tisch Cancer Institute, Mount Sinai

School of Medicine, New York, USA. The Division is also part of the PANC4 consortium, which last year published results from large multicenter pooled analyses on the association between alcohol drinking, tobacco smoking and pancreatitis and pancreatic cancer risk. Other research areas in which the division had a long-standing interest and reputation include the role of vitamin D, including vitamin D supplementation, on cancer and mortality risk and the epidemiology of malignant melanoma. In 2012, special research focus was made on the role of sun-bed use and melanoma risk. In total, in 2012 the division contributed to 83 articles published on peer-reviewed journals, with an overall impact factor of 350.

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Translational ??? Research

Translational Research

Tumor Registry

The project The Tumor 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 tumor. 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. The purpose is also to provide global information on the activity of the hospital, to document the cancer burden borne by the hospital for specific periods of time, to provide background information useful for the design of clinical studies, and to encourage clinicians and researchers to enquire about data and run new projects on the population of our patients 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. 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. Data Collected 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 tumor diagnosed in the past and already treated and cured. 3. Diagnosis: diagnosis of tumor is made at IEO. The patient decides to be treated elsewhere.

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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 tumor(s) (i.e., date of diagnosis, morphology, topography, TNM staging) is recorded on the second form, together with some 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 ArgosTM, based on Oracle TM database system. The implementation, completed in February 3 2006, was managed in the division of Epidemiology and Biostatistics, with the help of the ICT IEO/CCM 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.

Confidentiality All cancer case information included in the Tumor Registry is considered confidential. Data that identify patient-specific information are not included in the database. Use of the data has been authorised by the Institutional Ethics Committee and renewed on March 7th 2013. First 7 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 2013, 240,440 individuals who visited IEO for the first time in the years 2000-2008 were entered in the Tumor Registry. Individuals’ characteristics are reported in Table 1.

Table 1. Characteristics of individuals  

Gender

Age

Place of residence

Type of record

Total

Classification

No. Patients (%)

Male

78,169 (32.5)

Female

162,271 (67.5)

< 20 years

3,566 (1.5)

20-34 years

25,642 (10.7)

35-49 years

71,645 (29.8)

50-64 years

83,047 (34.5)

65-79 years

51,426 (21.4)

> 80 years

5,114 (2.1)

Northern Italy

155,099 (64.5)

- Lombardy

126,322 (52.5)

- Milan

45,204 (18.8)

Central Italy

45,668 (19)

Southern Italy

37,636 (15.7)

Foreign countries

2,037 (0.8)

Long

55,993 (23.3)

Second Opinion

77,108 (32.1)

Anamnesis

3,252 (1.4)

Diagnosis

2,561 (1.1)

Visit

101,526 (42.2)

 

240,440 (100,0)

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Collecteda Overall

Total

Invasive tumors IEO diagnosis

IEO Surgery

4,484

3,976

1,162

823

Lip

137

125

36

31

Tongue

802

772

339

306

Major salivary glands

795

416

146

100

Gum

56

53

26

25

Floor of mouth

133

128

32

30

Other and unspecified parts of mouth

704

662

195

149

Oropharynx

666

652

155

99

Nasopharynx

490

485

96

10

Hypopharynx

330

329

62

40

Tumor site Head and Neck

Other and other ill defined sites Digestive organs and peritoneumb Oesophagus Stomach

354

75

33

2,3388

3,782

2,276

672

657

135

72

4,084

4,051

746

551

Small intestine, including duodenum

444

433

101

29

Colon

8,501

8,282

1,294

909

Rectum, rectosigmoid junction and anus

3,636

3,572

740

560

Liver and intrahepatic bile ducts

1,744

1,720

219

54

Gallbladder and extrahepatic bile ducts

1,368

1,331

97

21

Pancreas

2,932

2,906

330

61

194

182

58

16

Retroperitoneum and peritoneum Other and ill-defined sites Respiratory and intrathoracic organs Nasal cavities, middle ear and accessory sinuses

271

254

62

3

19,680

18,902

4,594

2,936

109

106

11

3

Larynx

2,027

1,811

587

533

Lung

16,338

15,862

3,698

2,275

Pleura

791

750

160

47

Thymus, heart and mediastinum

409

367

138

78

6

6

0

0

62,817

52,606

26,901

18,147

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

20

14

3

1

3,344

3,207

1,097

466

Skin Melanoma

4,391

4,140

1,681

546

Skin Non Melanoma c

4,003

3,787

1,690

1,472

Breast

51,059

41,458

22430

15,662

Genitourinary organs

30,566

25,095

6,547

4,559

Cervix uteri

5,360

2,036

747

533

Uterine corpus

2,357

2,142

693

507 818

Ovary and other uterine adnexa

4,639

4,261

1,393

Other and unspecified female genital organs

1,007

674

238

161

Prostate

9,012

8,846

1,955

1,391

Testis

1,011

959

284

172

Penis and other male genital organs

152

128

53

43

Bladder

3,479

2,696

505

386

Kidney and other and unspecified urinary organs

3,549

3,353

679

548

Other and unspecified sites

567

4,980

3,945

712

Eye

23

19

3

1

Brain

1,792

1,767

48

0

Other and unspecified parts of nervous system

54

50

2

0

2,901

1,960

632

554

Other endocrine glands and related structure

210

149

27

12

Lymphatic and haematopoietic tissue

5,175

5,144

1,499

93

Hodgkin lymphoma

951

951

261

11

2,716

2,716

1,007

78 2

Thyroid gland

Non-Hodgkin lymphoma Multiple myeloma

537

537

94

Leukaemia

796

796

104

2

Other and unspecified

175

144

33

0 25

Non-skin melanoma Malignant neoplasm without specification of site Total

186

371 23,846

IEO — Scientific Report 2012 — Ongoing Research 2013

Translational ??? Research

Table 2. Tumors by sitea

At that time, 154,099 tumors, out of 138,679 individuals presenting with 1 or more tumors, were entered (Table 2) Developments Nine studies based on the data from the TR have been published so far. Many other studies in collaboration between our division and other divisions of the IEO are ongoing. The TR will soon be linked with the Biobank, allowing new molecular features to be available. The IEO TR has proven to be functional and reliable in monitoring the activity of the Hospital, allowing extraction of data from any subset of patients with characteristics of interest. This structured and centralized Registry represents an important tool for our research-oriented Institution. References Botteri E, Iodice S, Maisonneuve P, Alfieri M, Burzoni N, Manghi L, Martinetti M, Montanari B, Albertazzi E, Bazolli B, Rotmensz N. Case mix at the European Institute of Oncology: first report of the Tumor Registry, years 20002002. Ecancermedicalscience 2009;3:149.

Iodice S, Maisonneuve P, Botteri E, Sandri MT, Lowenfels AB. ABO blood group and cancer. Eur J Cancer 2010;46(18):3345-50. 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 2012;21(3):284-8. Bertani E, Testori A, Chiappa A, Misitano P, Biffi R, Viale G, Mazzarol G, De Pas T, Botteri E, Contino G, Verrecchia F, Bazolli B, Andreoni B. Recurrence and prognostic factors in patients with aggressive fibromatosis. The role of radical surgery and its limitations. World J Surg Oncol 2012;10:184. Toesca A, Spitaleri G, De Pas T, Botteri E, Gentilini O, Bottiglieri L, Rotmentsz N, Sangalli C, Marrazzo E, Cassano E, Veronesi P, Rietjens M, Luini A. Sarcoma of the breast: outcome and reconstructive options. Clin Breast Cancer 2012;12(6):438-44.

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;21(4):723-8.

Biffi R, Botteri E, Bertani E, Zampino MG, Cenciarelli S, Luca F, Pozzi S, Cossu ML, Chiappa A, Rotmensz N, Bazolli B, Magni E, Sonzogni A,Andreoni B. Factors predicting worse prognosis in patients affected by pT3 N0 colon cancer. Long-term results of a monocentric series of 137 radically resected patients in a 5-year period. Int J Colorectal Dis 2013;28(2):207-15.

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;37(4):305-11.

Botteri E, Munzone E, Intra M, Bagnardi V, Rotmensz N, Bazolli B, Montanari B, Aurilio G, Sciandivasci A, Esposito A, Pagani G, Adamoli L, Viale G, Nolè F, Goldhirsch A. Role of breast surgery in T1-3 breast cancer patients with synchronous bone metastases. Breast Cancer Res Treat 2013;138(1):303-10.

193

193

32

2,358

2,297

584

73

a

154,099

135,546

45,813

29,499

b

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

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Basic Research

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Basic Research ???

Basic Research

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, Giovanni Boniolo, 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, PhD, 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), Paolo Soffientini, (Mass Spectrometry Unit), Alberto Gobbi (Mouse Facility), Sebastiano Pasqualato (Cristallography Unit) Technicians: Sara Barozzi, Federica Baldi, Matteo Dal Molin, Mirko Doni, Manuela Moia, Simona Ronzoni, Mirco Scanarini, Cristina Spinelli, Marika Zanotti Veterinary Surgeon: Manuela Capillo Laboratory Manager: Samantha Seresini Staff Managing Direction: Paola Durin, Pietro Transidico, Liliana Areces, PhD, 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, Maria Grazia Gioiosa, Alessandra Parolini, Giovanna Rognoni, Daniela Rossi, Monica Salvatore, Cristina Signoroni IT Service Manager: Alessandro Dellavedova IT Service: Alessandro Ogier, Stefano Leva, Carlo Ferretti Support Personnel: Oscar Ignacio Bautista, Filomena Minafra, Rodolfo Grisorio, Tamara Brunetti

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Activities 2012. The Department of

Experimental Oncology (DEO) is composed of about 200 scientists and 19 independent research groups, whose scientific activities are described under the pages dedicated to the individual Units. Scientific interests of our research -groups comprise several aspects of tumor biology and tumor-host interactions, mainly focused on molecular mechanisms of cell proliferation, differentiation, genomic stability, immune evasion, gene transcription and DNA repair in normal and cancer cells or stem cells. Particular emphasis is given to applications of highthroughput screening-technologies (proteomics, genomics and epigenomics) and development of dedicated computational tools and approaches. The European Institute of Oncology actively collaborates with the University of Milan and several scientists of the DEO are also University Professors under the terms of a scientific agreement between these two Institutions.

scientific interests are mixed. As part of our training programs, and to maximize efficiency in lab activities, we encourage all researchers at every stage of their career to actively participate in the decision-making process and lab management. This open model has indeed created the most favorable grounds for the establishment of two institutional research-programs, both focused on translational and clinical objectives and rooted in the strong basic science environment of the DEO: the Molecular Medicine and the Drug Discovery Programs.

Since 2007, the DEO laboratories are located within the IFOM-IEO Campus (http://www.ifom-ieo-campus. it), which also hosts two other research institutions, the FIRC Institute of Molecular Oncology (IFOM) and the Center of Genomic Sciences of the Italian Institute of Technology (IIT), and the Milan headquarter of the European School of Molecular Medicine (SEMM), a foundation for higher education that directs our international postdoctoral program and four PhD programs (http://www.semm.it). Currently, over 100 PhD students from all over the world are enrolled in these programs.

An essential component of the DEO philosophy is to maximize the ability of scientists to carry out their research activity. In this light, over the years we selected a highly qualified technical staff that works in core support units and provides DEO researchers with an array of centralized high-quality services, including 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 and Kitchen Units, which provide chemicals, consumables, small equipment items and reagents for the general use of the Department; the Technical Services Unit, which takes care of the maintenance and repair of departmental instruments. The computational needs of the DEO are managed by the Information Technology Unit, which takes care of our IT network and server infrastructures and interfaces with DEO and IIT computational biologists for their software needs and handling of the massive amount of data generated by our Next Generation Sequencing platforms.

An open and collaborative research environment. The DEO has adopted an open structure model that fosters communication and cooperation between researchers. This model is based on open-space labs in which researchers from different groups but similar

High-profile post-graduate Educational Programs. SEMM is a private foundation for higher education established by a joint decree of the Ministry of Health, the Ministry of Treasure, and the Ministry of Education, Scientific and Technological Research.

The SEMM Foundation is an alliance gathering two Italian Universities (Università degli Studi of Milan and Università degli Studi “Federico II” of Naples), 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 (IEO, IFOM and the Center for Genetic Engineering in Naples). The goal of SEMM is to offer advanced training through an array of different tools, including toplevel international seminars, workshops, courses, and, above all, postgraduate training. SEMM is currently running four PhD programs: Molecular Medicine (since 2004), Medical Nanotechnology (since 2005), Foundations of Life Sciences and Their Ethical Consequences (FOLSATEC, since 2006) and bioinformatics Characterizing traits of the PhD programs are their strong interdisciplinarity, 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 competences through specifically designed courses. State of the art technological platforms and facilities. Advanced technical services are provided either by the Technological Units of the DEO or by the facilities run by Cogentech (Consortium for Genomic Technologies, owned by IEO and IFOM). The technological Units of the DEO include the Genomic Unit, the Crystallization Unit and the Mass Spectrometry Unit. Genomic Unit: This unit is jointly run by IEO and the Center for Genomic Sciences of IIT and has set up protocols to cover most applications that require highthroughput sequencing (including ChIP-seq, RNA-seq, mutational analysis, methylation analysis).

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IEO Administration Team at the IFOM—IEO Campus Protein Chemistry Facility: 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. DNA Services: This facility offers DNA sequencing, human cDNA Library Colony Picking and Real Time PCR technologies. Microarray Facility: This facility routinely performs complete microarray analysis for internal and external customers, using both Affymetrix and Nimblegen technology.

Crystallization Unit: The three - dimensional 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. Mass Spectrometry Unit: 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,

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identification and characterization using mass spectrometry. The Cogentech facilities include the Mouse Facility, the Protein Chemistry facility, the DNA Sequencing and quantitative-PCR facility and the microarray facility. Mouse Facility: Two units 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.

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Basic Research

Functional Genomics Myriam ALCALAY, MD, PhD Director

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

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Activities 2012.

High-throughput technologies have become essential for the discovery and analysis of genetic networks underlying cancer. Recent technological advancements, in particular next-generation sequencing, allow 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 caused by chromosomal translocations involving the AML1 gene (AML1/ETO, TEL/AML1, AML1/MDS1, AML1/PRDM16). Our approach includes the generation of cell lines expressing AML1-fusion proteins, analysis of their DNA binding patterns and associated chromatin status, the study of their capacity to interfere with the binding profiles of other regulators of hematopoiesis such as CEBPα and PU.1, and of their effects on nuclear architecture and regulation of mRNA and miRNA expression. - we are studying the activation of stem-cell signalling pathways in acute myeloid leukemia (AML) bearing mutations of the nucleophosmin (NPM1) through the analysis of the hematopoietic compartment in developing zebrafish expressing mutant NPM1. - we are investigating the molecular basis of chemoresistance in AML through mutational analysis of matched tumor samples from patients at diagnosis and relapse. Our results will be integrated in a database of genomic data from AML that is being compiled in our laboratory.

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). N. Meani and M. Alcalay: Role of nucleophosmin in acute myeloid leukemia. Expert Reviews in anticancer Therapy, 2009, Sep;9(9):1283-94 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. A. M. Gruszka and M. Alcalay (2012). Clinical and Biological Relevance of Gene Expression Profiling in Acute Myeloid Leukemia, Myeloid Leukemia - Clinical Diagnosis and Treatment, Steffen Koschmieder and Utz Krug (Ed.), ISBN: 978-953-307-886-1, InTech. A. M. Gruszka and M. Alcalay (2013). PML/RARα Fusion Gene and Response to Retinoic Acid and Arsenic Trioxide Treatment. Handbook of Therapeutic Biomarkers in Cancer. Edited by Sherry X. Yang and Janet Dancey. Pan Stanford Publishing, ISBN: 9789814364652.

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Basic Research

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

STAFF Post-doctoral Fellows: Sevgi Bagislar, PhD, Aleco D’ Andrea, PhD, Marcin Gorski, PhD, Theresia Kress, PhD, Alessandra Majorana, PhD PhD Students: Heidi Binder, Luana D’Artista, Micol Ravà, Claudia Tonelli Technicians: Mirko Doni, Paola Nicoli, Andrea Piontini, Alessandro Verrecchia Undergraduate Student: Roberta Bonfanti

Activities 2012.

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 Perna et al. (2012), we profiled the contribution of Myc to the transcriptional response to serum

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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 (ChIPseq) to generate the genome-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. 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.

In parallel work, we reported that Myc is a substrate of the tyrosine kinase c-Abl (or Abl) (Sanchez-Arévalo Lobo et al. 2013). Our work stemmed from the observations reported in the literature that Abl and the prolylisomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the p300 acetyl-transferase. As Myc was known to be regulated by Pin1 and p300, we hypothesized that it might also be targeted by Abl. The action of Abl, however, was more complex than predicted based on p73, and was twofold. First, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were indirect, however, occurring via phosphorylation of substrate(s) different than Myc, and yet to be identified. Second, Abl interacted with the C-terminus of Myc and phosphorylated its N-terminus on up to five tyrosines, the principal of which was Y74. Immunofluorescent and immunohistochemical staining indicated that the Y74-phosphorylated form of Myc (Myc-pY74) was cytoplasmic. Myc-pY74 coexisted with active Abl in a minority of mammary carcinomas and, most importantly, with the Bcr-Abl oncoprotein in chronic myeloid leukemia (CML). Thus, Abl has two potential effects on Myc: first, Abl indirectly augments acetylation of Myc by p300, most likely enhancing its transcriptional activity in the nucleus; second, Abl directly phosphorylates Myc: the resulting form of Myc occurs in the cytoplasm, and correlates with Abl activation in cancer.

Publications Migliori, V., Muller, J., Phalke, S., Low, D., Bezzi, M., Mok, W. C., Sahu, S. K., Gunaratne, J., Capasso, P., Bassi, C., Cecatiello, V., De Marco, A., Blackstock, W., Kuznetsov, V., Amati, B., Mapelli, M. and Guccione, E. Symmetric dimethylation of H3R2 is a newly identified histone mark that supports euchromatin maintenance. Nature Struct & Mol Biol. 19, 136-44 (2012). 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 31, 1695-1709 (2012). Adv. Online Publ.: 2011 Aug 22. doi: 10.1038/onc.2011.359. Campaner, S. and Amati B. Two sides of the Myc-induced DNA damage response: from tumor suppression to tumor maintenance. Cell Div. 7, 6 (2012) [review]. Sanchez-Arévalo Lobo, V.J., Doni, M., Verrecchia, A., Sanulli, S., Piontini, A., Bianchi, M., Conacci-Sorrell, M., Mazzarol, M., Peg, V., Hernandez Losa, J., Ronchi, P., Ponzoni, M., Eisenman, R.N., Doglioni, C. and Amati, B.* Dual regulation of Myc by Abl. Oncogene (2013) Jan 14. doi: 10.1038/onc.2012.621. [Epub ahead of print]

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Basic Research

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

STAFF Post-doctoral Fellows: Alessandro Cuomo, Michael Bremang, Marija Mihailovic, Roberta Noberini (@IIT), Monica Soldi (PhD defense in March 2013), Mara Colzani (until March 2012) PhD Students: Gianluca Sigismondo, Valeria Spadotto Technician: Alessio Silvola

Activities 2012.

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 mass-spectrometry (MS) technology, 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 histonemodifying enzymes and the quantitative aspects related to modification dynamics. In addition, we are extending the analysis of methylations of 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. Chromatomics: proteomics of chromatin domains by ChroP approach Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational

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modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. We have optimized and published a modified protocol for chromatin immunoprecipitation, combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture, to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics (ChroP) strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as

a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH (Soldi and Bonaldi, 2013). On-going work: we are developing the ChroP 2.0 approach, namely using ChroP to characterize enhancers and TSSs of inflammatory genes, at basal level and upon LPS stimulus, using H3K4me3 to enrich TSSs and H3K4m1 and PU.1 for enhancers. The comparison of distinct inflammatory stimuli will allow identifying stimulus- specific determinants. Deciphering the code of protein methylation by MSProteomics Protein methylation is a post-translational modification

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Basic Research ??? by which a variable number of methyl groups is transferred to Lysine and Arginine residues within proteins. Despite increased interest in this modification due to its reversible nature and its emerging role in a diverse set of biological pathways beyond chromatin, global identification of protein methylation has remained an unachieved goal. To characterise sites of Lysine and Arginine methylation globally, we employed an approach that combines heavy methyl stable isotope labelling by amino acids in cell culture (hmSILAC) with high-resolution mass spectrometry-based proteomics. Through a broad evaluation of immuno-affinity enrichment and the application of two classical protein separation techniques prior to mass spectrometry, to nuclear and cytosolic fractions separately, we identified a total of 501 different methylation types, on 370 distinct Lysine and Arginine sites, present on 127 unique proteins. Our results considerably extend the number of known in vivo methylation sites and indicate their significant presence on several protein complexes involved at all stages of gene expression (Bremang et al. in press in Mol. BioSystems). On-going work: while optimizing the analytical methods to extend the annotation of the methylome, we are investigating: the role of methylation in regulating the Microprocessor complex and the modulation of the methylome upon PRTM5 and PRMT7knock-out, in collaboration with E. Guccione at IMCB. Identification of miR17-92 targets in myc- B cell lymphoma by qProteomics The functional effect of microRNA activity depends on its molecular environment: the same miRNA may play different roles at different stages of any particular biological process, including tumorigenesis, on the basis of the changing gene expression landscape. An oncogenic role of miR-17-19b, a truncated version of the miR-17-92 cluster, has been previously associated to B-cell lymphoma. In this project, we aim at elucidating

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the contribution of the cluster to the maintenance of the malignant phenotype, using an established MYCdependent B-cell lymphoma as a model. We found that enforced expression of the cluster impaired lymphoma growth, both in vitro and in vivo, as a consequence of decreased tumor proliferation and increased apoptosis, indicating that in MYC-dependent cancer cells, miR-17-19b represses MYC function. Thus, we used a systems biology approach to dissect the gene expression regulatory network mediated by miR-17-19b: SILAC-proteomics led to the identification of over 200 novel targets of miR-17-19b. Analysis of these targets revealed that 40% are under the transcriptional control of the MYC oncogene, uncovering a widespread silencing effect operated by these miRNAs on the MYC-centered regulatory network (Mihailovic et al., in preparation). On-going work: among the identified miR targets we found E2F1 and Chek-2, transcriptional and translational regulator of MYC, respectively. We are assessing these two aspects of MYC regulation. In particular, MYC translational repression via suppression of Chek-2 represents a completely original mode of miR17-92 action. Chemical proteomics to identify targets of anticancer drugs 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 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.

- Target deconvolution for the multi-kinase inhibitor E-3810: different SILAC experiments have been designed to confidently identify putative targets of E-3810 in cancer cells lysates. We have identified a set of E-3810 interactors and estimated their Kd and IC50. While in vitro screening initially performed on E-3810 identified VEGRs and FGFRs as primary targets, our analysis identifies a broader set of interactors, with novel information to define the mechanism of action of the compount and to understand possible sideeffects (Colzani et al., in preparation) - On-going: the mPTP dissected by chemical proteomics: mitochondrial permeability transition is a process characterized by a sudden increase in inner mitochondrial membrane permeability, which represents a crucial step in cell death and is involved in various pathological processes, such as ischemia/ riperfusion injuries and neurodegenerative diseases. Mitochondrial permeability transition is mediated by the opening of a pore located in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP), whose structural nature remains controversial. Inhibitors targeting this pore may have important therapeutic applications. At the DDU a set of acrylamido derivatives is available, which inhibit the mitochondrial permeability transition, in vitro and in vivo. We are using chemical proteomics to analyze the proteins isolated from mitochondrial extracts in pull-down experiments using acrylamido derivative, coupled to beads. The IC50 and KD values calculated will identify the specific interactors of the compounds, thus leading to the biochemical characterization of the mPTP.

Vella P, Scelfo A, Jammula S, Chiacchiera F, Williams K, Cuomo A, Roberto A, Christensen J, Bonaldi T, Helin K, Pasini D. Tet Proteins Connect the O-Linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells. Mol Cell. 2013 Feb 21;49(4):64556. doi: 10.1016/j.molcel.2012.12.019. Epub 2013 Jan 24. Soldi M, Bonaldi T. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components. Mol Cell Proteomics. 2013 Mar;12(3):764-80. doi: 10.1074/mcp. M112.024307. Epub 2013 Jan 14. 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. doi: 10.1007/s00726-010-0668-2. Epub 2010 Jul 9. 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. doi: 10.1016/j.molcel.2008.07.018.

Publications Soldi M, Cuomo A, Bremang M, Bonaldi T. Mass spectrometry-based proteomics for the analysis of chromatin structure and dynamics. Int J Mol Sci. 2013 Mar 6;14(3):5402-31. doi: 10.3390/ijms14035402.

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Basic Research

Biomedical Humanities Giovanni BONIOLO Director

STAFF Deputy Director: Giuseppe Testa PhD Students: Marco Annoni, Federico Boem, Tommaso Bruni, Luca Chiapperino, Maria Damjanovicova, Lorenzo Del Savio, Pierre-Luc Germain, Fridolin Gross, Alma Linkeviciute, Luca Marelli, Cecilia Nardini, Zsuzsa Pavelka, Emanuele Ratti, Virginia Sanchini, Giuseppe Schiavone, Bettina Schmietow, Christopher Wareham Visiting professor: Mark Bedau (Reed College, Portland, USA), Matteo Mameli (King’s College, UK) Visitors: Marta Bertolaso (Campus Biomedico, Roma), Sara Casati (Università Bicocca) Marco Nathan (University of Denver, USA) Research fellow: Paolo Maugeri

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Activities 2012.

Recent advances in molecular biology are drastically changing our perceptions of oncological disease, diagnosis, and therapy. This is possible thanks to progress in understanding the molecular basis of cancer, at the level of genetic predisposition as well as of its interaction with individual lifestyles and environments. Such understanding is in turn enabling the development of new diagnostic and therapeutic approaches. Given this new scientific scenario, innovative foundational, ethical, sociological and political analyses are needed. It is time for Biomedical Humanities. This approach consists in addressing within a humanistic framework that iceberg whose tip is the care of individual oncological patients in clinical practice and whose basis is formed by the huge numbers of scientists working on the molecular basis of cancer, on how to detect it before it becomes lethal, and on how to cure it by taking into account individual patients’ genetic makeups, lifestyles and aspirations. Biomedical Humanities is a multi-disciplinary approach that puts together biomedical research, clinical practice, and humanistic disciplines. It does so, both at the level of training and at level of research and practice, by producing a fruitful and useful humanistic analysis of the many steps and facets that underpin the transition from the scientist’s lab bench to the oncological patient’s bedside (via translational research and considering the use of the latest biotechnologies). In the era of molecular medicine, Biomedical Humanities is a way to reflect on biomedical research and on clinical practice to improve their humanistic awareness, in order to draw their implications, and in order to ameliorate the quality of life of actual and potential patients. One important aspect of this is the cooperation required to establish a democratic knowledge-based society, where citizens have the right tools to participate in the making of biomedical policies and in choosing what is best for them.

Research Unit 1: Philosophical questions in biomedicine and oncological practice There are at least two ways of addressing biomedicine and clinical practice from a philosophical perspective: one is more attentive to the philosophical side and one more attentive to the scientific side. Concerning the former, we are interested in the conceptual analysis both of terms belonging to biomedical research or clinical practice (e.g., gene, epigenome, susceptibility, health, disease, therapy, cancer, stem cell, model organism, model disease, etc.) and of terms belonging to philosophy but that may be explicated by means of biomedical knowledge and clinical competence

(e.g., life, death, individuality, organism, action, etc.). Regarding the latter, we propose an investigation that has a real impact on research and clinical practice. This means pursuing a philosophical enquiry that could find an application in the scientists’ and clinicians’ activity. For example, i) we are developing a formal language that should permit to write intra- and infra-cellular processes as computable theorems; ii) we are approaching the issue of the bio-ontologies which, in these years, have a great relevance relatively to the elaboration, storage and retrieval of the enormous bulk of data coming from the lab and from the clinical research. All of our investigations are focused on, or have as source of

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Basic Research ??? inspiration in, the researches on the molecular basis of cancer and the ways of diagnosing and curing it. Research Unit 2: Individual and public ethical implications of biomedicine and oncological practice Each step of the chain from the scientist’s lab bench to the oncological patient’s bedside raises a host of ethical issues, both at the individual level and at the collective level. Our research focuses on some of these (e.g., the ethical problems raised by biobanks, consent, clinical trials, human embryonic stem cells, vaccination, etc.). Our aim is to improve the quality of the public discussion on these important issues as well as to promote responsible individual choices and effective public policies (e.g., we touch issues such as democratic legitimation of public policy concerning health matter, responsible and active citizenship in the health domain, freedom of choice and expression in relation to research and treatment, how to integrate biomedical and ethical expertise with wide democratic participation, etc.). We support a thoroughly democratic, anti-dogmatic and deliberative approach and we believe only by focusing on the ethical issues in an intelligent and humane way it will be possible to improve the quality of life of actual and potential oncological patients. Research Unit 3: Science and Technology Studies and biomedicine and oncological practice Here we focus on the mutual shaping of epistemic and normative orders that arise at the interface of biomedicine and society, with a particular focus on cancer research and care. The momentous developments of molecular biomedicine are unfolding in a space of experimentation that is not only technical and epistemic but also, and importantly, social, ethical and legal. In its steep acceleration the production of biomedical knowledge is also being redistributed to a host of new sites that extend well beyond academia, and all the while the public space is itself changing rapidly, evolving new

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institutions and accommodating new relationships among citizens along with new distributions of power. Weaving together epistemological, historical and sociological insights, the field of Science and Technology Studies (STS) has developed a rich array of analytical tools to investigate the settlements that enable biomedical novelties to circulate through society and to dissect the mutual constitution of their epistemic and social viabilities. From cells to sequences, from regulatory agencies to patent offices, we focus on the objects and sites of contemporary biopolitics, and harness STS to trace their implications for health care policy. Pubblications G. Boniolo, T. Vaccari, Alarming shift away from sharing results, Nature, 488 (2012): 157. G. Boniolo, G. Testa, The Identity of Living Beings, Epigenetics, and The Modesty of Philosophy, Erkenntnis, 76(2012): 279-298. G. Boniolo, P.P. Di Fiore, M. D’Agostino, Zsyntax: a formal language for molecular biology with projected applications in text mining and biological prediction, PLOsONE, 5(3)(2010): e9511. G. Boniolo, L. Chiapperino, Rethinking Medical Humanities, Journal of Medical Humanities, 2013, forthcoming. G. Boniolo, P.P. Di Fiore, S. Pece, Trusted Consent and Research Biobanks. Towards a “new alliance” between researchers and donors, Bioethics, 26(2012): 93-100. G. Boniolo, The Art of Deliberating. Democracy, Deliberation and the Life Sciences Between History and Theory, Springer, Heidelberg 2012.

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Basic Research

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

STAFF Post-doctoral Fellows: Simona Citro, PhD, Domenico Mattoscio, PhD, Chiara Segrè, PhD, Archana Varadaraj, PhD PhD Student: Sara Loponte Technician: Claudia Miccolo

Activities 2012.

Viruses are known hijackers of the host’s cellular machinery: several viral proteins are known to utilize, interfere, and/or augment cellular proteins and signaling pathways, in order to replicate and/or enter latency in cells. One of the most famous examples of this interaction consists of the adenoviral E1A interaction with cellular pRB that pushes quiescent cells into the cell cycle so that the infecting adenovirus can utilize the cell cycle machinery for its own replication. 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 how viral proteins impact SUMOylation of cellular proteins. We have also shown that Histone Deacetylase 1 (HDAC1) is post-translationally modified by SUMO. Mammalian histone deacetylases (HDACs) are composed of

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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 posttranslational 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, using the viral protein Gam1 as a model system. We are also assessing how other oncogenic viruses exploit the SUMO pathway. Biology of Histone Deacetylase 1, HDAC1 (and HDAC2): 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, context-related 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 and are 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. As mentioned above, SUMO plays diverse roles in many important cellular processes. Finally, in a collaborative project (with Saverio Minucci and the Pathology Division at IEO hospital), 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: 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 BC-box 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, in collaboration with Dr. Chiara Casadio and Dr. Mario Sideri, in one study and Dr. Fausto Chiesa’s division, and Dr. Fausto Maffini in another study. Much scientific evidence is indeed suggesting that pathogen modulation of the host SUMO pathway is quite a common mechanism.

Pubblications Viral manipulation of cellular protein conjugation pathways: the SUMO lesson Mattoscio D, Segrè CV, Chiocca S. World Journal of Virology. 2013 May 12. A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance. Santoro F, Botrugno OA, Dal Zuffo R, Pallavicini I, Matthews GM, Cluse L, Barozzi I, Senese S, Fornasari L, Moretti S, Altucci L, Pelicci PG, Chiocca S, Johnstone RW, Minucci S. Blood. 2013 Feb 25. [Epub ahead of print] Sumo paralogs: redundancy and divergencies. Citro S, Chiocca S. Front Biosci (Schol Ed). 2013 Jan 1;5:544-53. The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNAdependent manner. Sinigalia E, Alvisi G, Segré CV, Mercorelli B, Muratore G, Winkler M, Hsiao HH, Urlaub H, Ripalti A, Chiocca S, Palù G, Loregian A. PLoS One. 2012;7(11):e49630. doi: 10.1371/journal. pone.0049630. Epub 2012 Nov 15. Lifting the threshold between life and death: SUMO and HDAC fine-tune HIPK2 to sense redox status. Chiocca S, Seiser C. Mol Cell. 2012 May 25;46(4):382-3. doi: 10.1016/j. molcel.2012.05.010.

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Bioinformatics and Evolutionary Genomics of Cancer Francesca CICCARELLI, PhD Director

STAFF Post-doctoral Fellows: Matteo Cereda, PhD, Matteo D’Antonio, PhD, Fabio Iannelli, PhD, Elena Gatti, PhD, Fiorella Guerra, PhD PhD Students: Omer An, Vera Pendino, Shruti Sihna VisitorS: Marco Gentilini, Emanuele Ratti

Activities 2012.

Our group studies the effects of genomic instability in the development of human cancer. We tackle this issue using a combination of experimental and computational methods, with the aim of: 1. tracing the progressive acquisition of mutations during cancer development; 2. identifying systems-level properties of cancer genes; 1. Measure of somatic and constitutional genomic instability In addition to searching for cancer-specific mutations, we exploit deep next generation sequencing (NGS) to re-sequence several thousands single DNA filaments in parallel and unravel different aspects of cancer progression. For example, we developed a procedure for the quantification of somatic and constitutional genomic instability that is based on the detection of random mutations. We performed an ultradeep screening to identify random modifications that occur in a tiny fraction of cells, even prior to the establishment of the tumoral clone. To account for the occurrence of sequencing errors, we developed a statistical framework that relies on the ultraconserved elements of the human genome as error normalization. Using this method we were able to measure the constitutional genomic instability in individuals with heterozygous mutations in MMR genes, thus suggesting a predisposition of these individuals to acquire the second hit needed for tumor initiation. Our study constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability. We further used this feature of NGS to rebuild the

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proliferative tree of cancer clonal expansion. Mutation frequency indeed reflects the proportion of cells that bear each individual mutation while the number of somatic mutations is informative of the relative occurrence of cell death, cell proliferation, and cell quiescence during the clone formation. Following this idea, we reconstructed the proliferation trees of four colorectal tumors using their mutation profiles. We showed that the majority of the tumor mass in all four tumors is formed of a dominant subclone that started to prevail very early, although its establishment varied over time between and within tumors and seemed to be correlated with tumor genetics and clinical aggressiveness. 2. Systems biology of cancer genes We undertook a systematic study of the properties of cancer genes in the attempt of rationalizing the genetic heterogeneity of human cancer. We set out to analyze the relationship between the propensity of cancer genes to duplicate (i.e. gene duplicability) and the network properties of the encoded proteins, because connectivity and duplicability are usually indicative of gene fragility towards perturbations. We showed that cancer genes are mostly singletons and tend to encode central hubs at the crossroads of multiple biological processes. Although these properties are rare within the human gene repertoire, they are recurrent within known cancer genes, thus confirming the existence of systems-level properties – not detectable from the individual gene function – that explain the role of these genes in tumor development in terms of systems perturbation. We also discovered that most cancer genes appeared at two time points in evolution: caretakers and tumor suppressors are ancient genes that have orthologs also in prokaryotes, while gatekeepers and oncogenes were acquired with metazoans. These two time points correspond to two main transitions in evolution that led to an increase in complexity of the whole protein interaction network.

Web Servers and Public Databases a) Network of Cancer Genes: a web resource to analyze duplicability, orthology and network properties of cancer genes (http://bio.ieo.eu/ncg/) This public resource collects and integrates data on systems-level properties of cancer genes. It provides information on duplicability, orthology, evolutionary appearance and topological properties of the encoded protein in a comprehensive version of the human proteinprotein interaction network. NCG also stores information on all primary interactors of cancer proteins, thus providing a complete overview of 5357 proteins that constitute direct and indirect determinants of human cancer. b) FancyGene: dynamic visualization of gene structures and protein domain architectures on genomic loci http:// bio.ieo.eu/fancygene/) FancyGene is a web-based interactive tool for producing representations of one or more genes directly on the corresponding genomic loci. It is extremely flexible and allows the user to change the resulting image dynamically, to modify colors and shapes and to add and/or to remove objects. FancyGene is a useful tool to draw scientific pictures for scientific publications and presentations.

Publications De Grassi A, Segala C, Iannelli F, Volorio S, Bertario L, Radice P, Bernard L, Ciccarelli FD: Ultradeep sequencing of a human ultraconserved region reveals somatic and constitutional genomic instability. PloS Biol 2010, 8:e1000275. Rambaldi D, Giorgi FM, Capuani F, Ciliberto A, Ciccarelli FD: Low duplicability and network fragility of cancer genes. Trends in Genetics 2008, 24:427-430. Ciccarelli F: The (r)evolution of cancer genetics. BMC Biology 2010, 8:74. D’Antonio M, Ciccarelli F: Modification of gene duplicability during the evolution of protein interaction network. PLoS Comput Biol 2011, 7:e1002029. D’Antonio M, Pendino V, Sihna S, FD C: Network of Cancer Genes (NCG 3.0): integration and analysis of genetic and network properties of cancer genes. Nucl Acids Res 2012.

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Basic Research

Studying the regulation of chromosome segregation at centromeres, kinetochores and rDNA Peter DE WULF, PhD Director

STAFF Guest Professor: Tony Hazbun, PhD (Purdue University, USA) Post-doctoral Fellows: Maria G. Iacovella, PhD, Cinzia Pagliuca, PhD Undergraduate Students: Lucia F. Massari, M.Sc.

Activities 2012. Our lab studies how

replicated chromosomes segregate from the dividing mother cell into its two daughters. As most tumor cells contain abnormal chromosome numbers it has long been hypothesized that chromosome missegregation contributes to cancer transformation. By identifying, studying and understanding at the molecular level the proteins involved in chromosome segregation we will be able to convert them into novel cancer biomarkers and new anticancer drug targets. As the chromosome segregation process is highly conserved from yeast to humans we study this process in the budding yeast Saccharomyces cerevisiae, a model organism that is highly amenable to imaging, genetic, biochemical and cell division (cell cycle) research. Segregation of the replicated chromosomes during mitosis depends on the timely activity at two genomic regions, the centromeres and rDNA. Kinetochores, large protein complexes containing >100 subunits, assemble on the centromeres of each replicated chromosome pair (sister chromatids) to bind the sister chromatids to the microtubules of the mitotic spindle. After the cohesion rings that hold the sister chromatids together are resolved, the kinetochores move the chromatids along the spindle into the daughter cells. Importantly, the last region of the genome to segregate is the rDNA array, which must be actively transcribed to generate ribosomes and ensure cell viability. Only at the end of mitosis does rDNA transcription become repressed, allowing for the segregation of this region and completion of the chromosome transmission process. The focus of our lab lies on kinetochore protein Cnn1 and on the kinetochore kinase Rio1, which also regulates the transcriptional repression of the rDNA region. Cnn1 (CENP-T in humans) is a centromere-binding protein whose N-terminal tail inhibits in a cell cycledependent manner the interaction between the

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kinetochore Ndc80 and Mtw1 complexes, which establish the contact between kinetochores and the spindle microtubules. We have shown that Cnn1 concentrations at centromeres change through the cell cycle, as directed by phosphorylation via a set of conserved kinases. Cnn1 becomes highly enriched at centromeres during anaphase (mitotic stage during which chromosomes segregate) resulting in a less compact kinetochore, which allows for an efficient transduction of forces required to move the separated chromosomes. In myriad tumors, CENP-T is expressed excessively. Our work with yeast indicates that this pathology disturbs kinetochore structure and function resulting in chromosome missegregation and, consequently, support of the cancer transformation process. By combining yeast genetic screens, biochemistry and high-resolution imaging we recently identified a novel ubiquitin-mediated response pathway that evolved to neutralize abnormally high levels of Cnn1/CENP-T thereby preventing cells from producing daughters with abnormal chromosome numbers. The hitherto poorly characterized Rio1 kinase (RioK13 in humans) was identified in our lab as a novel kinetochore kinase that also regulates rDNA transcription in anaphase. These findings indicated a primary role for Rio1 in chromosome segregation. Rio1 activity represses RNA polymerase I thereby halting rDNA transcription and permitting condensin to be recruited to the region. This promotes the condensation and segregation of the rDNA array into the daughter cells. We also found that Rio1 targets the condensin complex itself to support its localization to and activity at rDNA. We believe that Rio1 functions in similar fashion at centromeres; a timely inhibition of centromere transcription would allow the well-timed assembly of kinetochores and attachment of sister chromatids to the mitotic spindle. As we found that Rio1 also targets proteins within the kinetochore, its role in chromosome segregation likely is more

complex than anticipated. By studying how Rio1 directs chromosome segregation we will obtain new insight into how this essential kinase underlies chromosome transmission, both in healthy and cancer cells, in which Rio1 is often found misexpressed or mutated. Publications Bock L.J., Pagliuca C., Kobayashi N., Grove R.A., Oku Y., 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, 14:614-624. Nguyen T.L., Cera M.T., Pinto A., Lo Presti L., 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, 11:1103-1111. 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. De Wulf P., Ernshaw W.C. (2009). “The Kinetochore: from Molecular Discoveries to Cancer Therapy”. Springer Publ., New York City, pp. 509.

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

STAFF Staff Scientists: Salvatore Pece, MD, PhD, Daniela Tosoni, PhD Scientist: Ivan Colaluca, PhD Post-doctoral Fellows: Macarena Ferrero Gimeno, PhD, Francesca Biagioni, PhD, Angelo Taglialatela, PhD PhD Students: Letizia Amadori, Claudia Baccichet, Silvia Restelli Undergaduate student: Susanna Diamanti Technicians: Michele Caccia, Marco Coazzoli, Luca Napolitano Visitors: Blanca Alvarez Moya, PhD, Andrea Basile, MD, Divya Purushothaman, PhD

Activities 2012. Understanding the

molecular mechanisms that govern tumor initiation and progression is crucial to improve current cancer treatments. In recent years, increasing evidence supporting the involvement of stem cells in the tumorigenic process has emerged. According to the cancer stem cell (CSC) model, tumor growth is fueled by a small subpopulation of cells with stem cell-like properties, including self-renewal and the ability to produce differentiated progeny. However, these CSCs lack control mechanisms of normal stem cell function and, thus, show unpredictable behavior and uncontrolled growth. Our research is focused on investigating the molecular mechanisms involved in 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 highresolution picture of the normal stem cell compartment will allow us to develop new diagnostic, prognostic and patient stratification tools. We have previously identified a normal breast stem cell molecular signature that is predictive of the biological, molecular and pathological features of human breast cancers. Indeed, we showed that breast cancers can be CSC-rich or CSC-poor, with poor-prognosis (high tumor grade, G3) tumors tending to be enriched in CSC compared to more favorable prognosis (low tumor grade, G1) tumors. By doing so, we have demonstrated that an expansion of the stem cell compartment occurs in breast tumors. Currently, we are following two separate lines of research, designed to gain a deeper understanding of the molecular mechanisms involved in the maintenance of the stem cell compartment and in tumor progression.

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Role of Numb in tumorigenesis Numb is a well-known regulator of the stem cell compartment. Recently, we have also established that Numb acts as a tumor suppressor in human breast and lung tumors. Our results show that Numb is degraded in ~50% of breast tumors and ~30% of lung tumors. In addition, Numb-deficiency is associated with clinico-pathological parameters of biological aggressiveness and, at least in breast cancer, with poor prognosis. 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 tumorigenesis. We aim to unravel the role of this protein in tumorigenesis by:

i) Analyzing the involvement of Numb in the regulation of the mammary gland stem cell compartment. To this aim, we have developed an in vitro model based on the ablation of Numb expression in mouse mammary stem cells using a lentiviral vector expressing short-hairpin RNAs and an in vivo mouse model (K5-Cre/Numbflox/flox), in which Numb expression has been ablated specifically within the basal/myoepithelial layer of the breast parenchyma, where breast stem cells reside. We are interested in determining if changes in Numb expression and in its downstream targets (Notch and p53) can lead to the development of CSC.

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Basic Research ??? ii) Identifying molecular players causing Numb degradation in human cancers. Aberrant Numb degradation occurs as a consequence of enhanced ubiquitination of the Numb protein. Thus, we have investigated whether deregulation of specific components of the ubiquitination machinery, with special focus on E3-ubiquitin ligases that catalyze the transfer of ubiquitin to the protein substrates, is responsible for loss of Numb expression in Numbdeficient tumors. Through a high-throughput RNA interference-based screening approach, we have identified several candidate ligases, the silencing of which affects Numb expression. These negative and positive candidate regulators of Numb expression are currently being validated through high-resolution studies in Numb-defective tumor cells. We aim to identify novel molecular targets for the development of new strategies to treat Numb-deficient cancers. iii) Characterizing the Numb-p53-HDM2 tricomplex. We have shown that Numb can regulate the activity of the tumor suppressor p53. Numb forms a complex with p53 and the E3-ubiquitin ligase HDM2 (also known as MDM2). In the context of the NumbHDM2-p53 tricomplex, Numb prevents HDM2mediated ubiquitination and degradation of p53, which translates into increased p53 protein levels and activity. Understanding the interaction between Numb-p53-HDM2 will allow us to design new molecules that can restore p53 function in cancer cells through inhibition of HDM2 activity. Mechanisms driving tumor progression and metastasis formation A necessary step to reduce cancer mortality is to understand the natural evolution of tumors that leads to metastasis formation. This is particularly true in the case of breast cancer, where our ability to completely cure the disease depends mainly on how early diagnosis is performed and on the

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absence of secondary tumors. While our findings, which demonstrate that more aggressive tumors are characterized by an expansion of the CSC compartment, argue for a supportive role of CSCs in tumor progression, the origin and identity of the molecular/genetic alterations driving this process are still unknown. Thus, we are currently focused on tracing the origin of the genetic lesions responsible for tumor expansion and metastasis formation by i) identifying cancer driver mutations and ii) verifying if these genetic lesions are harbored in CSCs. To this aim, we have developed an experimental approach that combines high-throughput genomics with a technological platform to study CSC biology. Our goal is to determine whether cancer driver mutations present in breast tumors and in their matched metastases are also found within CSCs. Through this approach, we expect to determine whether the metastatic potential of tumors stochastically arises from cell clones within the bulk tumor population or whether it resides in the stem cell compartment. In addition, we envision that some of the identified cancer driver mutations will function as clinically useful prognostic markers.

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. Colaluca IN, Tosoni D, Nuciforo P, Senic-Matuglia F, Galimberti V, Viale G, Pece S, Di Fiore PP: NUMB controls p53 tumor suppressor activity. Nature 2008; 451:76-80.

Publications Amson R, Pece S, Marine JC, Di Fiore PP, Telerman A. TPT1/TCTP-regulated pathways in phenotypic reprogramming. Trends Cell Biol 2013; 23:37-46. Pece S, Confalonieri S, Romano PR, Di Fiore PP. NUMBing down cancer by more than just a NOTCH. Biochim Biophys Acta 2011; 1815:26-43. Amson R, Pece S, Lespagnol A, Vyas R, Mazzarol G, Tosoni D, Colaluca I, Viale G, Rodrigues-Ferreira S, Wynendaele J, Chaloin O, Hoebeke J, Marine JC, Di Fiore PP, Telerman A. Reciprocal repression between P53 and TCTP. Nat Med 2011; 18:91-9.

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Systems Biomedicine Alberto D’ONOFRIO, PhD Director

STAFF Postdoc fellow: Sebastiano de Franciscis (from Nov 1 2011)

Activities 2012.

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 G. Caravagna (Eq. Contr.), G. Mauri, A. d’Onofrio (Eq. Contr. and Corr. Auth.). The interplay between intrinsic and extrinsic noise in biomolecular networks PLoS ONE 8(2): e51174 (2013). doi:10.1371/journal.pone.0051174 G. Caravagna, R. Barbuti and A. d’Onofrio (Corr. Auth.) Fine-tuning anti-tumor immunotherapies via stochastic simulations, BMC Bioinformatics 12 (Supp 4), S8 [18 + 2 pages] (2012)

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M. Al-Taamemi, Mark Chaplain (co-corr Auth) and Alberto d’Onofrio (co-corr auth) Evasion of tumors from the control of the immune system: consequences of brief encounters. Biology Direct 7, art.n. 31 (2012) S. de Franciscis and A. d’Onofrio (Corr. Author). Spatiotemporal Bounded Noises, and transitions induced by them in Ginzburg-Landau model. Physical Review 86, 021118 (2012)

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Basic Research

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

STAFF Post-doctoral Fellows: Ewa Aladowicz, Leda Ferro, Margherita Yayoi Turco Research technician: Elisabetta Venditti Undergraduate Students: Andrea Papait

Activities 2012.

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 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

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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 group is the understanding of 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 metastatic 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 metastases from melanoma patients was transplanted in NSG mice with a very high take rate. Tumor grafts phenocopy the original human tumors and their mutational and epigenetic profile will be further investigated to complement the phenotypic analysis. Metastases formation is followed in the animal. 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. Pubblications Aladowicz E, Ferro L, Vitali GC, Venditti E, Fornasari L, Lanfrancone L. Molecular networks in melanoma invasion and metastasis. Future Oncol. 2013 May;9(5):713-26.

L. Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells is Revealed by the ShcD/RaLP Adaptor Protein. Stem Cells. 2012 Nov;30(11):2423-36. 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. 2012 Feb 25;494(2):202-8.

Turco MY, Furia L, Dietze A, Fernandez Diaz L, Ronzoni S, Sciullo A, Simeone A, Constam D, Faretta M, Lanfrancone

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

STAFF Post-doctoral Fellows: Greta Bonetto, Anna Zoccarato PhD Students: Manuel Carminati, Sara Gallini

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Activities 2012.

In multicellular organisms, asymmetric cell divisions regulate the position and the fate choice of daughter cells, with impact on developmental programs and tissue homeostasis. We are interested in the molecular pathways that govern asymmetric cell divisions, with emphasis on the role of the mitotic spindle orientation. 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 leading 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 [1]. 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.

Our activity is organized in three main research lines: 1. 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: a) they organize contacts with specialized cortical domains; b) they coordinate in space and time pulling forces acting on astral microtubules; c) they transduce cytosolic and extracellular stimuli instructing the spindle orientation. 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. Recently, LGN has also been shown to associate with the actin-binding protein Afadin, hinting at an active role of the acto-myosin cytoskeleton in stabilizing spindle placement in mitosis. 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 NuMA/ LGN/Gαi complexes, and required to maintain them at

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Basic Research ??? the cortex, including the Gαi GTP-cycle controlled by the GEF RIC-8A. 2. 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) [2]. We have recently solved the crystallographic structure of the LGN/Insc complex, and discovered that Insc and NuMA are mutually exclusive partners of LGN [3]. This unexpected finding challenges the established model of force generators assembly, which we revised on the basis of the newly discovered biochemical properties 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. 3. 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

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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 Publications Mapelli M., Gonzalez C. (2012). On the inscrutable role of Inscuteable: structural bases for the competitive binding of NuMA and Inscuteable to LGN. Open Biology 2, 120102. Culurgioni S., Mapelli M. (2013). Going vertical: functional role and working principles of the protein Inscuteable in asymmetric cell divisions. Cellular and Molecular Life Sciences (in press). 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. USA 108, 20998-21003. 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 & molecular biology 19, 136-144. 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.

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Basic Research

Chromatin Alterations in Tumorigenesis Saverio MINUCCI, MD Director

STAFF Post-doctoral Fellows: Marco Ballarini (with Pier Giuseppe Pelicci), Chiara Biancotto PhD, Marco Cirò PhD (TYM Project), Mohamed Elgendy PhD (TYM Project), Lorenzo Fornasari, Gianmaria Frigè PhD, Simona Moretti PhD, Fabio Santoro PhD Temporary Fellows: Amir Hosseini PhD Students: Giacomo Iros Barozzi (with Gioacchino Natoli), Parinaz Medhipour, Pierluigi Rossi, Amal Saadeldin Technicians: Marco Gentilini PhD, Isabella Pallavicini, Mauro Romanenghi Undergraduate Student: Elena Ceccacci

Activities 2012.

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 methyltransferases 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

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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 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. 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, DNA demethylating agents). In particular, we have developed new assays for the study

of th contribution of different subpopulations of tumor cells to cancer growth, focusing on the role of leukemic stem cells. • 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), identifying systematically the cell interactors of anticancer and epigenetic drugs; In collaboration with the Drug Discovery Program of the IEO (TIV), 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.

Publications F Santoro, O. A. Botrugno, R Dal Zuffo, I Pallavicini, G. M. Matthews, L Cluse, I Barozzi, S Senese, L Fornasari, Simona Moretti, Lucia Altucci, PG Pelicci, S Chiocca, R. W. Johnstone and S Minucci. Blood. 2013 Feb 25. “A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance”. M Soncini, F Santoro, A Gutierrez, G Frigè, M Romanenghi, O. A. Botrugno, I Pallavicini, PG Pelicci, L Di Croce, S Minucci. BBA, Volume 1832, Issue 1, January 2013, Pages 114–120. “The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia”. S Saeed, C Logie, KJ Francoijs, G Frigè, M Romanenghi, FG Nielsen, L Raats, M Shahhoseini, M Huynen, L Altucci, S Minucci, JH Martens, HG Stunnenberg. Blood. 2012 Oct 11;120(15):3058-68. “Chromatin accessibility, p300, and histone acetylation define PML-RARα and AML1-ETO binding sites in acute myeloid leukemia”. M Leiva, S Moretti, H Soilihi,I Pallavicini, L Peres, C Mercurio, R Dal Zuffo, S Minucci*, H de Thé. Leukemia. 2012 Jul;26(7):1630-7. “Valproic acid induces differentiation and transient tumor regression, but spares leukemia-initiating activity in mouse models of APL”. OA Botrugno, T Robert, F Vanoli, M Foiani, S Minucci. Clin Cancer Res. 2012 May 1;18(9):2436-42. “Molecular pathways: old drugs define new pathways: non-histone acetylation at the crossroads of the DNA damage response and autophagy”. *SM co-corresponding author.

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Basic Research

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

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Activities 2012.

Chronic inflammation and cancer. Inflammation is triggered by both external (such as microbes) and endogenous (necrotic cells) danger signals and serves an essential homeostatic and therefore beneficial role. However, 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. Analysis of the links between chronic inflammation and hepatocellular carcinomas in mouse models and human patients led us to identify in collaboration with F. Ciccarelli’s group a distinct profile of genomic changes specifically triggered by chronic inflammation and leading to amplification and increased expression of components of signaling pathways involved in tumor progression and amenable to pharmacological inhibition (Iannelli et al., ms. submitted). This effort is part of the activity of a large research consortium funded by the European Community and entirely dedicated to the

understanding of molecular mechanisms controlling liver cancer development (MODHEP, Modeling Hepatocarcinoma). 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

subsequent step the induction of the repair response leading to reconstitution of tissue integrity. The main 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.

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Basic Research ??? 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 innate immunity and 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 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 and work in the last years has led to additional major findings in this field. This activity involved the extensive use of chromatin immunoprecipitation coupled with highthroughput 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

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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. Subsequent work on the characterization of the impact of different environmental stimuli on the functional organization of macrophage genome allowed us to identify a specific subset of genomic regulatory element that remain in an inactive state and are undetectable before stimulation using all currently available tools. These latent enhancers are activated in a stimulus- and cell type-specific manner and many of them remain active or poised for activity for a few days after removal of the primary stimulus that caused their emergence, thus representing a sort of short term memory of the stimuli a cell has been exposed to. 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, and a histone deacetylase (HDAC3) in the control of macrophage responsiveness to inflammatory stimuli.

Hdac3 requirement for the inflammatory gene expression program (X. Chen, I. Barozzi, A. Termanini, E. Prosperini, A. Recchiuti, J. Dalli, F. Mietton, G. Matteoli, S. Hiebert, G. Natoli) Proc Natl Acad Sci USA 109:E2865-74 (2012). Transcript dynamics of pro-inflammatory genes uncovered by RNA-Seq analysis of subcellular RNA fractions. (D.M. Bhatt, A. Pandya-Jones, A.J. Tong, I. Barozzi, M. Lissner, G. Natoli, Black D.L., Smale S.T.) Cell 150, 279-290 (2012). 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 (2012). Transcriptional control of macrophage polarization: enabling diversity with identity (T. Lawrence, G. Natoli) Nature Reviews Immunology 11, 750-761 (2011).

Publications Latent enhancers activated by stimulation in differentiated cells (Ostuni R, Piccolo V, Barozzi I, Polletti S, Termanini A, Bonifacio S, Curina A, Prosperini E, Ghisletti S, Natoli G.) Cell. 152: 157-71 (2013).

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Basic Research

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

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

Activities 2012. Organisms’ development

and tissues homeostasis is achieved by a precise control of the fate of differentiating cells. Such regulation is influenced 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 independent mechanisms of transcriptional regulation), resolve the complex three-dimensional structure of chromatin to 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 establish correct transcription.

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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 which are frequent targets of genomic alterations in a large variety of human tumors (i.e. mutations, deletions, translocations and amplifications) that strongly suggests a selective pressure, during cancer development, for altering the proper epigenetic control of normal cells. Thus, the characterization of the molecular mechanisms underling these activities in normal and pathological contexts as well as studying their role in different neoplastic environments, will help not only to better comprehend the molecular basis of cancer development, maintenance and evolution, but also to highlight novel potential pathways that can be pursued for therapy and prognosis. Ongoing research activities The work of our laboratory focuses its attention on different chromatin modifying activities that are potentially implicated in tumor development. To achieve this, we take advantage of biochemical, cell culture and mouse genetic approaches in order to characterize the role and the molecular mechanisms behind different chromatin modifying activities in normal cellular differentiation and in different contexts of neoplastic transformation. This work can be summarized in the following lines of research. Regulation of cell proliferation by chromatin modifiers. The mechanisms by which different chromatin modifiers can control cellular proliferation are diverse. Among these, the connection between chromatin modifications and cell proliferation remains in large part poorly understood. This line of research focuses its attention on the activity of Polycomb group proteins (PcG), frequent targets of deregulation in different tumors. Our goal

is to study the role of PcGs in regulating normal and cancer cells proliferation as well as to characterize the mechanisms controlled by these activities in proliferating cells. We combine cell culture and in vivo studies taking advantage of mouse genetics in order to elucidate the relevance of PcG inhibition for cancer treatment and to characterize the mechanisms triggered by the loss of PcG activity in normal and cancer cells. Mechanisms of epigenetic control in multi-protein complexes Most, if not all, chromatin modifying activities are found associated in large multiprotein complexes. This line of research uses biochemical approaches combined to analyses with mass spectrometry to characterize the diverse composition of protein complexes associated to chromatin in different cellular contexts. For example, this approach allows to study the composition of different chromatin remodeling complexes during normal cell differentiation or to characterize how oncogenic stimuli can alter the structure and activity of specific chromatin modifiers. All together, this approach allows to discover novel functional epigenetic interactions and to characterize how the activity of specific chromatin modifying complexes can be modulated to regulate cell fate in normal and neoplastic conditions. Role of Histone and DNA Modifications in transcriptional control and genomic integrity. Although a large number of different histone and DNA modifications have been identified in the last two decades,

they role in controlling transcriptional processes and genomic integrity still requires a lot of effort for a full comprehension. This part of the work of my laboratory combines next-generation sequencing (NGS), mouse genetic and mass spectrometry approaches to characterize the role of different histone and DNA modifications in regulating chromatin dynamics and gene transcription. Publications Chiacchiera, F., Piunti, A. and Pasini, D. (2013) Epigenetic Methylations and their Connections with Metabolism. Cell Mol Life Sci. [Epub ahead of print] Vella, P., Scelfo, A., Jammula, S., Chiacchiera, F., Williams, K., Cuomo, A., Roberto,, A., Christensen, J., Bonaldi, T., Helin, K. and Pasini, D. (2013) Tet Proteins Connect the O-linked N-acetylglucosamine Transferase Ogt to Chromatin in Embryonic Stem Cells. Mol Cell. 49, 645-656 Ferrari, J.K. and Pasini, D. (2012) Regulation and Function of DNA and Histone Methylations. Curr Pharm Des. 19, 719-733. Vella, P., Barozzi, I., Cuomo, A., Bonaldi, T. and Pasini, D. (2012) Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells. Nucleic Acids Res. 40, 3403-3418 Piunti, A. and Pasini, D. (2011) Epigenetic factors in cancer development: polycomb group proteins. Future Oncol, 7, 57-75.

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Basic Research

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

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

Activities 2012.

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. In human GBM CSCs have been isolated mostly on the basis of the expression of the cell surface protein CD133. We have recently described that CD133 is essential to the maintenance and the tumorigenic potential of human glioblastoma stem cells (Brescia et al 2013). Moreover, we have shown the property of CD133 to re-cycle dynamically between the plasma-

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membrane and the cytoplasm of GBM-derived neurosphere cells. The clonogenic potential of cells not expressing CD133 on the plasmamembrane is reduced, even if CD133 is re-localized from the cytoplasm to the cell surface. As a consequence, we hypothesize that the localization of CD133 on the plasmamembrane is functionally important for the stem properties of the cell. We investigated whether the block of cell surface CD133 with anti-CD133 monoclonal antibodies induces an inhibition in cell growth and in the self-renewal of GBM neurosphere cells, mimicking the effects of the gene silencing. Our results suggest that the functional role of CD133 in the stem cell compart-

ment is strictly related to its cell surface localization, giving a new insight in the biological function of this protein in glioblastoma stem cells. 2. Marker-independent method to isolate CSCs from GBM patients. We choose to apply a marker-independent method consisting in the cell labeling with PKH-26 fluorescent dye of GBM neurospheres, since they represent a good surrogate for in vitro study of glioma stem and progenitor cells. The intensity of PKH-26 staining correlates inversely with the number of previous cell divisions discriminating then highly cycling cells from

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Basic Research ??? slow-dividing (putative stem cells) responsible for tumor growth and progression. PHK-26 positive and negative cell populations are isolated from GBM neurospheres by FACS/sorting and characterized in vitro for proliferation capacity and self-renewal ability, and in vivo for their tumor formation capacity after orthotopic injection in nude mice. Putative stem cells frequency is evaluated by in vivo limiting dilution assays. Our results show that both PKH-26 positive and negative cells are tumorigenic but the negative fraction does not exhibit stem cell criteria exhausting in culture. The slow-cycling cells (PKH positive) display all the hallmarks of a cancer stem cell. 3. RAI and microRNA We recently demonstrated that RAI is a novel regulator of glioblastoma invasion. RAI expression in cancer stem cells obtained from human glioblastoma tumors allows their infiltration throughout brain parenchyma, preventing complete surgical resection and allowing relapse (Ortensi et al., 2012). Uncovering the molecular mechanisms regulating cancer infiltration would permit to set up new therapeutic strategies to block glioblastoma invasion. We are now performing microRNA arrays to identify potential microRNAs involved in RAI regulation in cancer stem cells, aiming at the identification of clinically relevant targets. 4. CLIC1 (Chloride intracellular channel 1) function in patient-derived GBM CSCs. Chloride intracellular channel-1 (CLIC1) belongs to a class of chloride channels that exists as both soluble globular protein and integral membrane protein with ion channel function. CLIC1 protein is overexpressed in several human solid tumors including gliomas: we have demonstrated that CLIC1 is variably expressed in patient-derived GBM neurospheres and is enriched in the stem/progenitor compartment of the neurosphere. Moreover, its silencing in GBM stem/progenitor cells

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negatively influences both proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. In addition to its role inside the tissue, CLIC1 has been identified as a secreted protein and detected in exosomes released from different cell types. We aim to elucidate the biological and clinical impact of CLIC1 secreted protein. Publications Ortensi B, Setti M, Osti D, Pelicci G. Cancer stem cell contribution to glioblastoma invasiveness. Stem Cell Research & Therapy 2013, 4:18 Savino MT Ulivieri C, Emmi G, Prisco D, De Falco G, Ortensi B, Beccastrini E, Emmi L, Pelicci G, Milco D’Elios M, Baldari CT. The Rai adaptor inhibits Th17 cell development in lupus autoimmunity. Journal of Leukocyte Biology 2013 Jan 23 Brescia P, Ortensi B, Fornasari L, Levi D, Broggi G, Pelicci G. CD133 is essential for glioblastoma stem cell maintenance. Stem Cells 2013 Jan 10 Brescia P*, Richichi C*, Pelicci G. Current strategies for identification of glioma stem cells: adequate or unsatisfactory? J Oncol. 2012;2012:376894 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. Rai is a new regulator of neural progenitor migration and glioblastoma invasion. Stem Cells. 2012 May;30(5):817-32

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Basic Research

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

STAFF Staff Scientists: Mario Faretta, Marco Giorgio, PhD, Lucilla Luzi, PhD, Enrica Migliaccio, PhD, Cristina Moroni Scientists: Marco Ballarini, Emanuela Colombo, PhD, Ivan Gaetano Dellino, PhD, Alessandra Insinga, PhD Post-doctoral Fellows: Alessandra Bigi, PhD, Angela Mariano, PhD, Paul Edward Massa, PhD, Massimiliano Mazza, PhD, Marine Meliksetyan, PhD, Rani Pallavi, PhD, Cristina Pasi, PhD, Stefania Rapino, PhD, Linsey Blair Reavie, PhD, Hanumaiah Veena Talagavadi, PhD, Maria Vittoria Verga Falzacappa, PhD, Malgorzata Wierzbicka, PhD PhD Students: Umberto Andrea Cammarata, Giulia De Conti, Nikolay Dobrev, Maria Mallardo, Dalia Rosano, Anna Russo, Francesco Santaniello, Angela Santoro, Thaleia Vlachou 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 Undergraduate Student: Silvia Crasto Visitors: Francesca Bernassola, Elisa Memmi, Maria Giulia Sanarico, Alice Soldà

Activities 2012.

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 behaviors and how alteration of this composite molecular machinery may ultimately lead to cancer. Our group is trying to investigate these molecular mechanisms/interactions with research that extends from the regulation of cell division and proliferation to the control of DNA transcription and replication, to the role of tumor-associated oncogenes and suppressors in tumor development and progression, and to the links between cancer and metabolism and cancer and aging. Accumulating evidence suggests that only rare cancer cells [which possess characteristics associated with normal stem cells (SCs) and are thus called cancer stem cells (CSCs)] have the capacity to maintain tumor growth, so a considerable part of our research efforts is specifically devoted to the characterization of normal and cancer SCs, 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 leukemia and breast). We use these model systems in combination with primary patient derived samples to identify biological markers of disease and to develop innovative strategies to target CSCs in a clinical setting. The activities of the past year can be grouped into 4 main lines of research: 1. Molecular and biological mechanisms underlying the effects of p53-loss and p21-activation on normal and cancer SCs, and the role of quiescence, DNA damage repair, and reprogramming in tumor progression and relapse.

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Adult proliferating cells deal with excessive accumulation of genomic damage by the elimination of the damaged cell through apoptosis (programmed cell death) or senescence (irreversible cell growth arrest), which are processes mediated by the tumor suppressor p53 and its transcriptional target p21. We have discovered that SCs, however, respond to DNA damage by activating a unique checkpoint system that is p21-dependent and leads to inhibition of apoptosis by preventing activation of p53 (an early event in a cell tumor suppression mechanism), and induction of symmetric division and DNA damage repair. The outcome is the expansion of a pool of

functional SCs, which can support regeneration of injured tissues. However, repair of DNA damage in hematopoietic and mammary SCs is not faultless, since SCs with low levels of damage can be found after irradiation and physiological aging, and, crucially, these SCs show diminished self-renewal potential. We think that this imperfect repair might function physiologically as a mechanism of tumor suppression in these cells, as the boundary between an immortal stem cell and a cancer stem cell might be very thin. These results have implication for cancer therapy as increasing evidence suggests that CSCs are largely chemo- and radio- resistant

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Basic Research ??? (likely because there are dormant), survive in vivo treatments and initiate tumor regrowth: p21 and p21dependent mechanisms of DNA damage repair are being investigated as potential therapeutic targets. Also of importance for cancer therapy is the possibility that progenitor cells, early descendants of SCs that can differentiate to form one or more kinds of cells but cannot divide and reproduce indefinitely, may be reprogrammed into SCs. We found that transduction of the oncogene Myc in normal mammary-progenitors generates cells with mammary SC characteristics. We are investigating whether loss of p53 leads to Myc activation in mouse models of mammary tumors, and whether this event is responsible for the expansion in the number of mammary CSCs via increased symmetric division and for progenitor-reprogramming. 2. State of the art technology to study the molecular basis of chemoresistance and replication stress. We have also begun testing the hypothesis that relapse acquired chemoresistance in acute myeloid leukemia (AML) is the consequence of the selection of rare tumor cell populations (which we assume are CSCs) harboring specific gene-mutations or abnormalities in the epigenetic regulation of chromatin structure and function (epimutations). We have established next generation sequencing (NGS) and RNA interference (RNAi) protocols and libraries to identify genome wide, relapse-specific DNA mutations and epimutations in AMLs. In vivo genetic screens using RNAi libraries are performed in growing tumors after xenotransplantation. We have also developed, in house, a robust pipeline for the analysis of the data generated from the different protocols. The process of DNA replication, when a cell copies its DNA in preparation for cell division, initiates from multiple chromosomal loci called replication origins

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(ORIs), which are selected in the G1 phase of the cell cycle by the sequential recruitment of the proteins constituting the pre-replicative complex. However, little is known about the mechanisms regulating the initiation of replication on DNA strands, and, up to now, the identification of ORIs in metazoans has been hampered by the lack of stringent and sensitive genome-wide techniques for identifying these sites. We thus set-up an experimental procedure, based on the anti-Orc1 ChIP-seq (ChIP: chromatinimmunoprecipitation; Seq: sequencing) that has allowed, for the first time in mammalian cells, the identification of ORIs in their natural chromosome context. These results are important as they will allow assessment of the extent of DNA-replication stress at genomic level in oncogene-expressing stem cells. 3. Cancer and Aging. Genomic instability, a hallmark of most cancers, 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, as mentioned above, cancer and aging may be two different aspects of the DNA damage response. While cancer can affect people of all ages, the risk of developing cancer generally increases with age. 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. Oxidative stress is a potent inducer of the tumorsuppressor p53, which mediates all the antiproliferative 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. We

have found that the p53 transcriptional-response 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 downregulated in cultured fibroblasts after oxidative stress, and, in vivo, in proliferating tissues and during physiological aging. Our results indicate that activation of the redox protein p66 in proliferating cells, following oxidative stress, might favor upregulation of selected p53 downstream pathways through activation of p44/p53, an isoform of p53, leading to transient cell cycle arrest and repair of damaged proteins or, if protein damage is severe or prolonged, to cellular senescence or apoptosis. 4. Cancer and metabolism. This line of research is closely connected with the previous one as it is now well established in animal models that reducing calorie intake can increase life span and lower the risk of various age related diseases, including cancer. Growing awareness that diet and environmental factors have a profound effect in the initiation, promotion, and progression of cancer implies that cancer is a “preventable” disease. We are investigating, in animal models, the role of p53 or p53-independent mechanisms in mediating the tumor suppressor activity of caloric restriction by testing the hypothesis that SCs are critical targets of calorie reduction (CR) and analyzing the functional effects of CR on SCs (self-renewal and differentiation); also, at the same time, we are investigating, in human AML patients (and mouse models), the hypothesis that obesity generates “environmental” conditions that favor the accumulation of specific mutations.

Publications Insinga A, Cicalese A, Faretta M, Gallo B, Albano L, Ronzoni S, Furia L, Viale A, Pelicci PG. DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions. Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):3931-6. doi: 10.1073/ pnas.1213394110. Dellino GI, Cittaro D, Piccioni R, Luzi L, Banfi S, Segalla S, Cesaroni M, Mendoza-Maldonado R, Giacca M, Pelicci PG. Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing. Genome Res. 2013 Jan;23(1):1-11. doi: 10.1101/gr.142331.112. Gambino V, De Michele G, Venezia O, Migliaccio P, Dall’olio V, Bernard L, Minardi SP, Fazia MA, Bartoli D, Servillo G, Alcalay M, Luzi L, Giorgio M, Scrable H, Pelicci PG, Migliaccio E. Oxidative stress activates a specific p53 transcriptional-response that regulates cellular senescence and aging. Aging Cell. 2013 Feb 28. doi: 10.1111/acel.12060. 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 Breccia M, Mazzarella L, Bagnardi V, Disalvatore D, Loglisci G, Cimino G, Testi AM, Avvisati G, Petti MC, Minotti C, Latagliata R, Foà R, Pelicci PG, Lo-Coco F. Increased BMI correlates with higher risk of disease relapse and differentiation syndrome in patients with acute promyelocytic leukemia treated with the AIDA protocols. Blood. 2012 Jan 5;119(1):49-54. doi: 10.1182/ blood-2011-07-369595.

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Basic Research

Immunobiology of Dendritic Cells and Immunotherapy Maria RESCIGNO, PhD Director

STAFF Staff Scientist: Giuseppe Penna Post-doctoral Fellows: Giulia Fornasa, PhD, Gerhard Antonie Floris Fransen, PhD, Silvia Guglietta, PhD, Chiara Pozzi, PhD, Fabiana Saccheri, PhD (until April 2012), Tiziana Schioppa, PhD, Dario Brunelli, PhD PhD Students: Elisa Mazzini, Ilaria Spadoni, Katerina Tsilingiri, Elena Zagato Technicians: Erika Mileti Temporary Fellows: Maria Rosa Ciranna Undergraduate students: Lapo Morelli

Activities 2012. 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 bacteria both in vitro and in vivo with the aim to study the interaction between the host and the intestinal flora (microbiota) and to establish new protocols for cancer immunotherapy. It is becoming increasingly clear that the interaction with the microbiota can control several essential functions of our body, such as the development of the immune system, the digestion of complex macromolecules, the control of intestinal homeostasis and the detoxification of carcinogens.

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Hence, understanding how our mucosal immune system copes with the millions of microorganisms that inhabit our gut is fundamental to unravel important physiological functions of our body and how deregulations can lead to tumor development. In the laboratory we have two major lines of research, one is aimed at studying how microorganisms influence tumor development and how we can exploit them for the generation of new immunotherapy approaches of cancer. The other line of research deals in understanding basic mechanisms of host-microbiota interactions with particular focus on inflammatory bowel disease. Within cancer, we try to assess 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. We are generating recombinant bacteria that express on their cell surface antibodies that target the bacteria specifically to tumor cells. These bacteria also carry payloads of enzymes important for the catabolization of prodrugs into drugs for the local delivery of active antitumor compounds. 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 with 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 have evaluated whether beneficial microbes like probiotics can reestablish 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. To study the anti-inflammatory properties of bacteria we have established a new organ culture model system that allows to grow gut tissue specimens and to apply a stimulus in a polarized fashion by the addition of a cave cylinder on the apical face of the tissue. This method allows to carry out stimulations that resemble the in vivo route of bacterial interaction with the host with great relevance for a rapid translation of the findings into the clinical practice.

Publications Spadoni I, Iliev ID, Rossi G, Rescigno M. Dendritic cells produce TSLP that limits the differentiation of Th17 cells, fosters Treg development, and protects against colitis. Mucosal Immunol. 2012 Jan 11. doi: 10.1038/mi.2011.64. [Epub ahead of print] Tsilingiri K, Barbosa T, Penna G, Caprioli F, Sonzogni A, Viale G, Rescigno M. Probiotic and postbiotic activity in health and disease: comparison on a novel polarised exvivo organ culture model. Gut. 2012 Feb 1. De Pas T, Giovannini M, Rescigno M*, Catania C, Toffalorio F, Spitaleri G, Delmonte A, Barberis M, Spaggiari L, Solli P, Veronesi G, De Braud F. Vaccines in non-small cell lung cancer: Rationale, combination strategies and update on clinical trials. Crit Rev Oncol Hematol. 2012 Feb 24. [Epub ahead of print] * Corresponding author Enterotypes of the human gut microbiome. Arumugam M, et al. Nature. 2011 May 12;473(7346):174-80. Epub 2011 Apr 20. Klaenhammer TR, Kleerebezem M, Kopp MV, Rescigno M.The impact of probiotics and prebiotics on the immune system. Nat Rev Immunol. 2012 Oct;12(10):728-34. doi: 10.1038/nri3312.

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Basic Research

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

STAFF Post-doctoral Fellows: Antonio Adamo, Silvia Cristofanon, Pasquale Laise, Elena Signaroldi, Prem Tripathi PhD Students: Sina Atashpaz, Giulia Barbagiovanni, Serena Buontempo, Giuseppe D’Agostino, Vivek Das, Giulia Fragola, Pierre Luc Germain, Pietro Lo Riso, Jacopo Sgualdino Temporary Fellows: Matteo Zanella

Activities 2012.

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 selfrenewal and 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

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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, our lab pursue three complementary lines of research in epigenetics: i) the physiology of genome programming, focusing on the role of selected chromatin modifiers in the acquisition and maintenance of neuronal fate during corticogenesis; ii) the pathologic counterpart of neural cell fate determination, through the investigation of silencing chromatin in gliomagenesis; iii) cell reprogramming, both as tool to dissect the epigenetic basis of fate plasticity and as novel experimental system to model neurodevelopmental disorders and cancer. 1. The physiology of genome programming: chromatin regulation of neural fate 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 regulate distinct phases of neurogenesis as well as the maintenance of the differentiated state in neural cells. To this end we harnessed the power of both constitutive and conditional mutagenesis to establish murine strains and embryonic stem cell (ESC) in which to modulate the activity of key effectors of the epigenetic axis centered on H3K27me3 and define its the role in neurodevelopmental transitions. We discovered that Jmjd3 is required for survival, since Jmjd3 mutants die of perinatal lethality due to respiratory failure (Burgold et al. Cell Reports 2012).

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Basic Research ??? This is due to the complete and selective disruption of the preBötzinger complex (PBC), the pacemaker of the respiratory rhythm generator (RRG), and represents the most severe respiratory phenotype described to date. Importantly, a combination of genetic and electrophysiological approaches allowed us to establish that the enzymatic activity of Jmjd3 is selectively required for the maintenance of the PBC through the activation of critical regulators of PBC function, uncovering an unanticipated role of this enzyme in the late structuring of neuronal networks. 2. Aberrant genome programming 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 pre-marked 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 counter- part of the neurogenic and gliogenic program, namely the development of malignant gliomas, with the aim of elucidating the epigenetic basis of the lineage

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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 functional dissection in primary cells isolated from human tumors. 3. Cell reprogramming a) Epigenetic mechanisms of cell fate reassignment Here we took 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 has been to dissect functionally their relative contribution to cell fate reassignment, defining what are the epigenetic windows of sensitivity during reprogramming. To this send we have been using both experimental paradigms of induced pluripotent stem cells (iPSC) and induced neuronal cells (iNCs). Specifically, we defined the role of Polycomb Repressive Complex 2 (PRC2) in reprogramming somatic cells to pluripotency through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming (Fragola et al. PLoS Genetics 2013). We found that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, an innovative combination of mass-spec analysis and chromatin immunoprecipitation coupled to next generation sequencing (ChIPseq) in Ezh2-mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for

developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. We thus established that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TFdependent cell reprogramming, setting stage for defining the functional relevance of this core gene subset in other physiopathological paradigms of cell reprogramming, including cancer. b) Modeling disease through cell reprogramming One of the most tangible outputs of somatic cell reprogramming has been a paradigm shift in our ability to model human diseases. Fundamental limitations in the study of human diseases have been so far: i) the scarce availability of primary diseased tissues, which is particularly salient for disorders of the nervous system; and ii) the difficulty of reconstructing disease history, which is salient also for cancer pathogenesis. 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 the latest approaches in somatic cell reprogramming, based on synthetic mRNA delivery, to develop physiopathologically meaningful in vitro models of both neurodevelopmental disorders and cancer, thereby aiming at the dissection of the genomic versus epigenomic components of their pathogenesis.

Publications G. Fragola, P.L. Germain, P. Laise, A. Cuomo, A. Blasimme, F. Gross, E. Signaroldi, G. Bucci, C. Sommer, G. Pruneri, G. Mazzarol, T. Bonaldi, G. Mostoslavsky, S. Casola and G. Testa Cell reprogramming requires silencing of a core subset of Polycomb targets PLoS Genetics 2013 Feb;9(2):e1003292. doi: 10.1371/journal. pgen.1003292 Corresponding author T. Burgold, N. Voituron, M. Caganova, P.P. Tripathi, C Menuet, B.K. Tusi, F. Spreafico, M. Bévengut, C. Gestreau, S. Buontempo, A. Simeone, L. Kruidenier, G. Natoli, S. Casola, G. Hilaire and G. Testa The H3K27 demethylase JMJD3 is required for maintenance of the embryonic respiratory neuronal network, neonatal breathing and survival, Cell Reports 2(5), 2012: 1244-58 Corresponding author L. Austenaa, I. Barozzi, A. Chronowska, A. Termanini, R. Ostuni, F. Stewart, G. Testa and G. Natoli The histone methyltransferase Wbp7 (Mll4) controls macrophage function through GPI anchor synthesis, Immunity 36(4), 2012: 572-85 Blasimme, A., Schmietow, B. and Testa, G. Reprogramming potentiality: the co-production of stem cell policy and democracy. Am J Bioeth. 2013;13(1):30-2. doi: 10.1080/15265161.2012.747032 Corresponding author M. Curnutte and G. Testa Consuming genomes: scientific and social innovation in direct-to-consumer genetic testing (2012) New Genetics and Society, 31:2, 159-181 Corresponding author

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Basic Research

Mechanisms Controlling Chromosome Segregation Rosella VISINTIN, PhD Director

STAFF Post-doctoral Fellows: Sara Busnelli PhD Students: Michela Roccuzzo, Federico Tili Technician: Clara Visintin Undergraduate students: Cecilia Claudi

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Activities 2012. 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 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 chromatid segregation. However, mounting evidence suggests that cohesin-independent 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. 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 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 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. Cohesin is cleaved by separase whose activity is restrained by securin. 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. 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

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Basic Research ??? 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).

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. Visintin C., Tomson B.N., Rahal R., Paulson J., Cohen M., Taunton J., Amon A. and Visintin R. (2008). Apc/C-Cdh1mediated degradation of the Polo kinase Cdc5 promotes the return of Cdc14 into the nucleolus. Genes Dev, 22: 79-90

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. Publications Visintin R. (2011). Cdc14B: When a good kid turns bad. Cell Cycle 10: 2416-17 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

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Educational Programs

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Educational Programs

Educational Programs

SEMM - European School of Molecular Medicine Pier Giuseppe PELICCI MD, PhD Scientific Director

STAFF Foundation Assistant: Annalisa Ariesi Graduate Office: Francesca Fiore (coordinator), Veronica Viscardi (Students’ administrator) Events coordinator: Sabrina Frata

Domenico TRIARICO Administrative Director

The European School of Molecular Medicine The mission of the European School of Molecular Medicine (SEMM) is to promote the training and research of young scientists in the emerging sectors of biomedicine with a special focus on Molecular Oncology and Human Genetics. SEMM collaborates with two Italian Universities, University of Milan and University of Naples “Federico II” to create its training programs and operates within research centers of excellence bridging together higher education and front-line research training. SEMM is currently running five PhD programs: Molecular Oncology, Human Genetics, Computational Biology, Medical Nanotechnology, Foundations of Life Sciences and Their Ethical Consequences and a post doc program. The faculty of the school includes 34 scientists, eight of which are also professors at the University of Milan. PhD programs: the characterizing traits of the PhD programs are their strong interdisciplinary traits, the international dimension and the training platform. Training includes intensive laboratory work for the development of technical skills and attendance to specifically designed courses for the acquisition of new theoretical tools. 166 were the students enrolled at the PhD programs in 2012, 28 of them are foreigners coming from 16 different countries from Europe (Albania, Austria, Bulgaria, Georgia, Germany, Greece, Lithuania, Poland, Serbia), and other countries (Canada, China, Kenya, India, Iran, Nigeria, Turkey). Since SEMM started its activity, 110 students got graduated and 90% of them found a new position within one year from the graduation. The vast majority of them (75%) continued their scientific career with a post doc position.

aims to support “International Mobility for non-resident Italians and foreigners. It is designed to boost the career of post-docs and to encourage them to become successful and independent scientists. During 2012, 18 were the post docs enrolled in the program, 12 were foreigners coming from Egypt, France, Germany, India, Japan, Netherland, Russia, Spain, Turkey, USA. Training courses and seminars During 2012, 23 training courses and 60 scientific seminars were organized. The SEMM faculty with the support of 10 visiting professors held the training courses for PhD students. Courses covered both basic knowledge such as Scientific Methodology, Molecular Oncology, Biochemistry, specialized advanced courses specific per each educational program. Seminars are held by internationally recognized scientists from around the world and cover a wide range of topics related to the subjects of the educational programs, such as Molecular Oncology, Immunology, Structural Biology, Genomics & Proteomics, Network Biology, Bioinformatics, Nanotechnology, Bioethics.

Post doc program: the program is an international program co-funded by the European Commission under the FP7-Marie Curie Actions – People program, which

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Educational Programs

IEO Education Pier Giuseppe PELLICCI, MD, PhD Scientific Coordinator

STAFF Scientific Secretary: Nicoletta Tradati Executive 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 2012 saw the implementation of several activities of IEO Education, which was set up to coordinate all IEO educational and training activities related to patient management and clinical research, integrate them in an innovative manner, and thereby promote, both internally and externally the Institute’s knowledge. The main areas of actions are a) Clinical Science Seminars in Oncology with at least one meeting per month 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), providing education and training courses online and on demand with a considerable scientific impact; d) the monthly publication of the IEO Edu newsletter designed to circulate and promote the main scientific and training events, e) the launch of the new catalogue of IEO Web Education with seven courses on: Early Glottic Cancer, Management of Clinical Studies, Counseling in Medicine, Clinical Risk Management, Breast Reconstructive Surgery, Patients Radioprotection, Primary Nursing, two of them are in English. The English version of the Italian courses will be available by the end of 2013. Other three online courses (on urology, gynaecology, and pain treatment) will be released by June 2013. IEO Education has divided its internal and external activities into ad hoc scientific programs handled by especially appointed program coordinators. Internal education and training activities The hospital education and training activities in 2012 were: • 48 accredited CME courses. Some courses were repeated, bringing the total number to 66. • 21 non-accredited CME courses. Some courses were repeated, bringing the total number to 24.

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• 7 behavioral courses linked to needs arising from career development plans, during the annual staff evaluation. Some courses were repeated, bringing the total course number to 132. The accredited courses produced a total of 1473 CME credits (1147,5 credits in 2011). The number of hours totalled 640 for CME-accredited courses, 523 for non-accredited CME courses, and 238 for behavioral courses. The number of participants totalled 1187 for CMEaccredited courses, 339 for non-accredited CME courses, and 238 for behavioral courses. Regarding participation in scientific congresses, a total of 1066 participants were registered, of whom 532 attended national events and 534 attended international events.

Following are the main educational activities organized in 2012: • Milan Breast Cancer Conference • Breast Cancer: Oncologic and Reconstructive Surgery. Interactive course with live surgery - 3D during live surgery as technological innovation • Esagon Biennial Course: a 2-week residential and online course with live surgery and theory sessions, course on animal models. Due to technical problems, the courses of the School of Robotic Surgery did not take place. The activities, temporarily suspended in 2012, will start again in 2013.

External education and training activities In 2012, 42 educational activities (courses, meetings, and congresses) were organized by the European Institute of Oncology. Some of these were repeated bringing the total number to 47. Of these: • 19 events had both Italian and European CME accreditation,, • 23 events had no CME accreditation (some were repeated and totalled 28). The total number of hours invested in external educational activities was 585. The total number of attendees (i.e., general practitioners, specialists, and other health care professionals/providers) amounted to 2031 with over 90% coming from national and international institutions. Based on evaluation questionnaires, the satisfaction rate was 88,14%. Participants in IEO education and training activities gained 5793,4 Italian CME credits and 504 European CME credits.

IEO — Scientific Report 2012 — Ongoing Research 2013

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4

Seminars 2012 Basic science seminars (SEMM)

April 05 – Philipp Kaldis (Singapore): “In vivo functions of Cdk1 in proliferation, liver regeneration, and cancer”

1

February 28 – Luca Magnani (Lebanon, USA): “PBX1 significance as a novel therapeutic target in ERa positive breast cancer”

January 20 – David Komander (Cambridge, UK): “Specificity in the ubiquitin system”

3

2

February 02 – Laura Soucek (Barcelona, Spain): “Pre-clinical validation of Myc as a target in cancer therapy” February 10 – Stefano Piccolo (Padua, Italy): “How soluble cues and mechanical forces drive tumor progression” February 14 – Alberto Puliafito (Candiolo, Italy): “Collective and single cell behavior in epithelial contact inhibition” February 20 – Daniel Durocher (Toronto, Canada): “The interplay between the response to DNA damage and ubiquitin function”

5

March 09 – Luigi Naldini (Milan, Italy): “Recent Advances in Hematopoietic Stem Cell Gene Therapy: from microRNA Regulation to Targeted Gene Transfer”

May 07 – Maria Carmo-Fonseca (Lisbon, Portugal): “Splicing alive”

March 12 – Sara Rubinelli (Nottwil, Switzerland): “The paradox of information and patient empowerment” March 16 – Alessio Maiolica (Zurich, Switzerland): “Investigation of the phosphosphorylation-mediated signaling in mitosis by mass spectrometry” March 16 – Alessio Zippo (Milan, Italy): “A Myc-driven epigenetic switch controls cell fate decision” March 23 – Maarten van Lohuizen (Amsterdam, The Netherlands): Role of Polycomb repressors in stem cells, cancer, DNA repair and iPS cell reprogramming March 27 – John O’Shea (Bethesda, USA): “Mechanisms of Stability and Plasticity of CD4 Helper T Cells”

February 21 – Licia Selleri (New York, USA): “Making Faces: a Short TALE of Noses, Lips and Clefts”

March 30 – Josef Jiricny (Zurich, Switzerland): “Mammalian mismatch repair: error-free or error-prone?”

IEO — Scientific Report 2012 — Ongoing Research 2013

April 20 –Adriana Albini (Milan, Italy): “The tumor microenvironment as target for cancer therapy and prevention”

March 09 – Laxman Gangwani (El Paso, USA): “Deficiency of the Zinc Finger Protein ZPR1 Causes Defect in Cell Proliferation”

February 21 – Kelly A. Frazer (La Jolla, USA): “Genomics in Oncology: Insights into Cancer Biology and Personalized Medicine”

February 22 – Massimo Tommasino (Lyon, France): “Transforming properies of Beta cutaneous human papillomaviruses and their possible role in carcinogenesis”

April 12 – Christine Nardini (Shangai, China): “Multiomic integration: application to Rheumatoid Arthritis” April 18 – Andreas Ernst (Toronto, Canada): “A new strategy to develop inhibitors of the Ubiquitin Proteasome System”

January 19 – Christophe Lancrin (Monterotondo, Italy): “Understanding the birth of blood cells in the mouse embryo”

January 25 – Alberto Cambrosio (Paris, France): “Hybrid platforms, disruptive technologies and the reassembling of oncology”

256

April 02 – Utz Herbig (Newark, USA): “Telomeres in aging and cancer: good things can have bad ends”

March 30 – Ewa Paluch (Dresden, Germany): “Mechanics and regulation of blebs and lamellipodia formation during cell migration”

May 07 – Yijun Qi (Beijing, China): “Mechanism and biology of small RNAs: what we have learned from plants”

May 10 – David Bacon (London, UK): “The problem of image integrity and photo editing software” May 11 – Haruhiko Koseki (Yokohama, Japan): “Polycomb repression during mammalian development” May 18 – Federico Calegari (Dresden, Germany): “Conditional expansion of neural stem cells in the mammalian brain” May 25 – Joshua Z. Rappoport (Birmingham, UK): “Imaging receptor trafficking and directed cell motility” May 31 – Valter Tucci (Genoa, Italy): “Genetics and epigenetics of sleep and brain development”

6

June 01 – Genevieve Almouzni (Paris, France): “(Hetero)chromatin assembly and nuclear organization” June 05 – Dónal O’Carroll (Monterotondo Scalo, Italy): “Establishment and maintenance of transposon silencing in the male germ line”

June 08 – Guido Serini (Candiolo, Italy): “Regulation of cell adhesion by integrin conformation and traffic: an emerging indissoluble partnership”

Educational Programs

Educational Programs

June 12 – Alberto R. Kornblihtt (Buenos Aires, Argentina): “Chromatin and transcription regulate alternative splicing” June 13 – Manuel J. Muñoz (Buenos Aires, Argentina): “UV irradiation, DNA damage and alternative splicing” June 18 – Walter Marcotti (Sheffield, UK): “Control of hair cell development: genetic program and sensoryindependent electrical activity” June 18 – Douglas R. Higgs (Oxford, UK): “Understanding how genes are switched on and off in haematopoiesis” June 19 – Rutledge G. Ellis-Behnke (Cambridge, USA): “The Intersection of Nanotechnology and Healthcare: using self-assembled nanomaterials to delay the formation of human metastatic prostate cancer stem cell colonies” June 21 – Paola Picotti (Zurich, Switzerland): “Targeted proteomics to study protein networks in health and disease”

7

July 02 – Mauro Giacca (Trieste, Italy): “How can you mend a broken heart (Bee Gees, 1971): Searching for novel genes and microRNAs inducing myocardial protection and re generation” July 05 – Giorgio Ascoli (Fairfax, USA): “Neuronal Morphology goes Digital: A Research Hub for Cellular and System Neuroscience” July 10 – Eros Lazzerini Denchi (La Jolla, USA): “A two step mechanism for TRF2-mediated end protection” July 27 – Jennifer Gamble (Newtown, Australia): “The Ageing Endothelium”

9

September 07 – Ronald McKay (Baltimore, USA): “Stem cell technologies and human functional genomics” September 14 – Jacco Van Rheenen (Utrecht, The Netherlands): “Intravital imaging of metastasis through imaging windows”

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Educational Programs

Educational Programs

Seminars 2012 Clinical - science seminars (IEO Education)

September 24 – Daniel Gerlich (Vienna, Austria): “Image-based screening for cell division regulators” September 28 – David Dominguez-Sola (New York, USA): “MYC, germinal center homeostasis and B cell lymphomagenesis”

10

October 15 – Nils Hoppe (Hannover, Germany): “Legal and Ethical Issues of Property Rights in the Human Body” October 18 – Giulia Rancati (Singapore): “Aneuploidy in cellular adaptation, drug resistance and cancer” October 19 – Ana-Maria Lennon-Duménil (Paris, France): “Coupling Cell function to Cell Migration: the example of Dendritic Cells” October 22 – David Sassoon (Paris, France): “Adult stem cells: towards a common regulatory stress pathway?” October 23 – Shamil R. Sunyaev (Boston, USA): “Human germ line and somatic mutation rates: evolution, biology and statistical genetics” October 29 – Antonio Iavarone (New York, USA): “Master regulators of stem cells and drivers of oncogenesis in the brain”

11

November 05 – Oliver Pabst (Hannover, Germany): “A complex microbiota drives diverse IgA repertoires in the intestine”

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November 12 – Anna Bigas (Barcelona, Spain): “Regulation of Hematopoietic Stem Cell development in the mouse embryo” November 23 – Wieland Huttner (Dresden, Germany): “Neural stem and progenitor cells and the evolution of the cerebral cortex”

12

December 04 – Luciano Di Croce (Barcelona, Spain): “From protein structure to control of embryonic stem cell differentiation: the role of Polycomb proteins” December 13 – Henk Stunnenberg (Nijmegen, The Netherlands): “The transcriptome, epigenome and methylome profiles define two distinct mouse pluripotent embryonal stem cell states” December 14 – Francois Schweisguth (Paris, France): “A live imaging analysis of Notch signaling in Drosophila” December 18 – Chiara Francavilla (Copenhagen, Denmark): “Functional proteomics defines the molecular switch underlying FGFR2b trafficking and response”

1

6

2

7

January 18 – Jürg Bernhard (Bern, Switzerland): “Research in quality of life issues in cancer patients: challenges and future directions”

February 01 – Eva Szabo (Bethesda, USA): “New opportunities for chemoprevention trials in lung and oral cancer” February 29 – Marie E. Wood (Burlington, USA): “Breast Cancer Prevention: What’s new”

3

March 07 – Daniel Lenihan (Nashville, USA): “How Cardiotoxicity with Cancer Therapy can lead to Promising Heart Failure Treatment: The Story of Neuregulin” March 20 – Robert Kerbel (Toronto, Canada): “Anti-angiogenic and metronomic therapies, where to go next?”

4

April 04 – Peter Goldstraw (London, UK): “New TNM classification for lung cancer and its implications April 18 – Agnes Lee (Vancouver, Canada): “Cancer associated thrombosis: have we made any progress since Trousseau?”

5

May 15 – Angelita Habr-Gama (São Paolo, Brazil): “Complete pathological response after chemo/ radiotherapy in rectal cancer: non operative strategy”

June 21 – Charles M. Perou (Chapel Hill, USA): “Tumor subtypes and personalized genetics for breast cancer patients”

July 04 – Jan B. Vermorken (Edegem, Belgium): “Recent Advances in the Non-Surgical Treatment of Squamous Cell Carcinoma of the Head and Neck (SCCHN)”

9

September 19 – Yasuhiro Ito (Kobe, Japan): “Early thyroid cancer management in 2012: the Japanese experience”

10

October 24 – Alberto Sobrero (Genoa, Italy): “Management of advanced CRC”

11

November 07 – Hirohiko Tsujii (Chiba, Japan): “Experience in Carbon Ion Therapy for Head and Neck Tumors” November 30 – Riccardo Fodde (Rotterdam, The Netherlands): “Wnt signaling, methylation and (triple-negative) breast cancer: dangerous liaisons and therapeutic opportunities

12

December 06 – George Hanna (London, UK): “Oncological Quality of Minimally Invasive Gastroesophaegeal Surgery”

IEO — Scientific Report 2012 — Ongoing Research 2013

259


Publications, Clinical Trials, Ongoing Grants, and IEO Foundation

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Publications

Full Papers 2012

AUTHORS

TITLE

JOURNAL

VOLUME

PAGE

I.F.

ABED EL RAHMAN D., MATEI V. D., MUSI G., BOTTERO D., BRESCIA A., GALASSO G., MAZZOLENI F., DE COBELLI O.

Neoadjuvant chemotherapy for invasive bladder cancer: an interesting case report.

ARCH ITAL UROL ANDROL

84

167 - 170

0

ACETO N., SAUSGRUBER N., BRINKHAUS H., GAIDATZIS D., MARTINY-BARON G., MAZZAROL G., CONFALONIERI S., QUARTO M., HU G., BALWIERZ P. J., PACHKOV M., ELLEDGE S. J., VAN NIMWEGEN E., STADLER M. B., BENTIRESALJ M.

Tyrosine phosphatase SHP2 promotes breast cancer progression and maintains tumor-initiating cells via activation of key transcription factors and a positive feedback signaling loop

NAT MED

18

529 - 537

22,462

ADAMS D., ALTUCCI L., ANTONARAKIS S. E., BALLESTEROS J., BECK S., BIRD A., BOCK C., BOEHM B., CAMPO E., CARICASOLE A., DAHL F., DERMITZAKIS E. T., ENVER T., ESTELLER M., ESTIVILL X., FERGUSON-SMITH A., FITZGIBBON J., FLICEK P., GIEHL C., GRAF T., GROSVELD F., GUIGO R., GUT I., HELIN K., JARVIUS J., KUPPERS R., LEHRACH H., LENGAUER T., LERNMARK A., LESLIE D., LOEFFLER M., MACINTYRE E., MAI A., MARTENS J. H., MINUCCI S., OUWEHAND W. H., PELICCI P. G., PENDEVILLE H., PORSE B., RAKYAN V., REIK W., SCHRAPPE M., SCHUBELER D., SEIFERT M., SIEBERT R., SIMMONS D., SORANZO N., SPICUGLIA S., STRATTON M., STUNNENBERG H. G., TANAY A., TORRENTS D., VALENCIA A., VELLENGA E., VINGRON M., WALTER J., WILLCOCKS S.

BLUEPRINT to decode the epigenetic signature written in blood.

NAT BIOTECHNOL

30

224 - 226

23,268

ALCAZAR J. L, GUERRIERO S., AJOSSA S., PARODO G., PIRAS B., PEIRETTI M., JURADO M., IDOATE M. A

Extragenital endometrial stromal sarcoma arising in endometriosis.

GYNECOL OBSTET INVES

73

265 - 271

1,276

ALGAITHY Z. K., PETIT J. Y., LOHSIRIWAT V., MAISONNEUVE P., REY P., BAROS N., LAI H. W., MULAS P., MEIRELLES BARBALHO D., VERONESI P., RIETJENS M.

Nipple sparing mastectomy: Can we predict the factors predisposing to necrosis?

EJSO-EUR J SURG ONC

38

125 - 129

2,499

ALIMOGLU E., ALIMOGLU M. K., CEKEN K., KABAALIOGLU A., APAYDIN A., CASSANO E., SINDEL T.

Bi-RADS category 3 nonpalpable breast masses on sonography: long-term results of a prospective cohort study.

J CLIN ULTRASOUND

40

125 - 134

0,806

AL-TAMEEMI M., CHAPLAIN M., D'ONOFRIO A.

Evasion of tumors from the control of the immune system: consequences of brief encounters.

BIOL DIRECT

7

31 - 31

4,017

AMSON R., PECE S., LESPAGNOL A., VYAS R., MAZZAROL G., TOSONI D., COLALUCA I., VIALE G., RODRIGUEZ 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 - 100

22,462

ANDREOLA G., BABIC A., RABASCIO C., NEGRI M., MARTINELLI G., LASZLO' D.

Plerixafor and Filgrastim XM02 (Tevagastrim®) s a first line peripheral blood stem cell mobilisation strategy in patients with multiple myeloma and lymphoma candidated to autologous bone marrow transplantation.

EUR J HAEMATOL

88

154 - 158

2,614

IEO — Scientific Report 2012 — Ongoing Research 2013

263


264

Who should be really considered as a poor mobilizer in the plerixafor era?

TRANSFUS APHER SCI

47

27 - 32

1,25

BACCHIANI G., CARDINALE D.

Using biomarkers and early prophylactic treatment to prevent cardiotoxicity in cancer patients on chemotherapy

SA HEART

9

244 - 256

0

ANDRIULLI A., FESTA V., BOTTERI E., VALVANO M. R., KOCH M., BASSI C., MAISONNEUVE P., SEBASTIANO P. D.

Neoadjuvant/preoperative gemcitabine for patients with localized pancreatic cancer: a meta-analysis of prospective studies.

ANN SURG ONCOL

19

1644 1662

4,166

BACHEM A., HARTUNG E., GUTTLER S., MORA A., ZHOU X., HEGEMANN A., PLANTINGA M., MAZZINI E., STOITZNER P., GURKA S., HENN V., MAGES H. W., KROCZEK R. A.

Expression of XCR1 Characterizes the Batf3-Dependent Lineage of Dendritic Cells Capable of Antigen Cross-Presentation.

IMMUNOLOGY

3

214 - 214

3,321

ANNONI M., SCHIAVONE G., CHIAPPERINO L., BONIOLO G.

Constructing the Medical Humanities gaze.

CRIT REV ONCOL HEMAT

84 Suppl 2

S5 - S10

4,411

379

633 - 640

38,278

Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers.

BREAST CANCER RES

14

R33

5,245

Lapatinib with trastuzumab for HER2positive early breast cancer (NeoALTTO): a randomised, open-label, multicenter, phase 3 trial.

LANCET

ANTONIOU A. C., KUCHENBAECKER K. B., et al.

ARBYN M., DE SANJOSE S., SARAIYA M., SIDERI M., PALEFSKY J., LACEY C., GILLISON M., BRUNI L., RONCO G., WENTZENSEN N., BROTHERTON J., QIAO Y. L., DENNY L., BORNSTEIN J., ABRAMOWITZ L., GIULIANO A., TOMMASINO M., MONSONEGO J.

EUROGIN 2011 roadmap on prevention and treatment of HPV-related disease.

INT J CANCER

131

1969 1982

5,444

BASELGA J., BRADBURY I., EIDTMANN H., DI COSIMO S., DE AZAMBUJA E., AURA C., GOMEZ H., DINH P., FAURIA K., VAN DOOREN V., AKTAR G., GOLDHIRSCH A., CHANG T. W, HORVATH Z., COCCIA PORTUGAL M., DOMONT J., TSENG L. M, KUNZ G., SOHN J. H, SEMIGLAZOV V., NEOALTTO STUDY TEAM, COLLEONI M. BELLOCCO R., PASQUALI E., ROTA M., BAGNARDI V., TRAMACERE I., SCOTTI L., PELUCCHI C., BOFFETTA P., CORRAO G., LA VECCHIA C.

Alcohol drinking and risk of renal cell carcinoma: results of a meta-analysis.

ANN ONCOL

23

2235 2244

6,425

BELLOMI M.

260

0

Assessment of colorectal hepatic metastases by quantitative T2 relaxation time

EUR J RADIOL

ECANCERMEDICALSCIENCE

6

ARSALAN RAZA S., FUNICELLI L., SOHAIB S. A., COLLINS D. J., SCURR E., LEACH M. O., KOH D. M.

A classification of pulmonary nodules by CT scan

BELLOMI M., DE PAS T., TESSITORE A., PREDA L.

Lung cancer

7

ASCIERTO P. A., KIRKWOOD J. M., GROB J. J., SIMEONE E., GRIMALDI A. M., MAIO M., PALMIERI G., TESTORI A., MARINCOLA F. M., MOZZILLO N.

The role of BRAF V600 mutation in melanoma.

J TRANSL MED

Springer-Verlag Italia 2012 - AGLIETTA M., REGGE D. Imaging Tumor response to therapy

BELSEY J., CROSTA C., EPSTEIN O., FISCHBACH W., LAYER P., PARENTE F., HALPHEN M.

CURR MED RES OPIN

28

1883 1890

2,38

ASIOLI S., MALETTA F., VERDUN DI CANTOGNO L., SATOLLI M. A., SCHENA M., PECCHIONI C., BOTTA C., CHIUSA L., MOLINARO L., CONTI L., VIALE G., INGRAVALLO G., MAIORANO E., SAPINO A.

Approaching heterogeneity of human epidermal growth factor receptor 2 in surgical specimens of gastric cancer.

HUM PATHOL

Meta-analysis: efficacy of small bowel preparation for small bowel video capsule endoscopy.

BELSEY J., CROSTA C., EPSTEIN O., FISCHBACH W., LAYER P., PARENTE F., HALPHEN M.

Meta-analysis: the relative efficacy of oral bowel preparations for colonoscopy 1985-2010.

ALIMENT PHARM THER

35

222 - 237

3,769

2249 2259

5,176

BREAST J

Sustained hippocampal neurogenesis in females is amplified in P66(Shc-/-) mice: An animal model of healthy aging.

22

Oral Metronomic Cyclophosphamide and Methotrexate Plus Fulvestrant in Advanced Breast Cancer Patients: A Mono-Institutional Case-Cohort Report

BERRY A., AMREIN I., NOETZLI S., LAZIC S.E., BELLISARIO V., GIORGIO M., PELICCI P. G., ALLEVA E., LIPP H.P., CIRULLI F.

HIPPOCAMPUS

AURILIO G., MUNZONE E., BOTTERI E., SCIANDIVASCI A. S., ADAMOLI L., MINCHELLA I., ESPOSITO A., CULLURA' D., CURIGLIANO G., COLLEONI M. A., GOLDHIRSCH A., NOLE' F.

Role of robotic surgery in colorectal resections for cancer.

MINERVA GASTROENTEROL DIETOL

58

191 - 200

0

AURILIO G., SCIANDIVASCI A. S., MUNZONE E., SANDRI M. T., ZORZINO L., CASSATELLA M. C., VERRI E., COSSU ROCCA M., NOLE' F.

Prognostic value of circulating tumor cells in primary and metastatic breast cancer.

EXPERT REV ANTICANC

BERTANI E., CHIAPPA A., UBIALI P., FIORE B., CORBELLINI C., COSSU M. L., MINICOZZI A., ANDREONI B.

10

184

1,12

The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis.

IMMUNITY

Recurrence and prognostic factors in patients with aggressive fibromatosis. The role of radical surgery and its limitations.

WORLD J SURG ONCOL

AUSTENAA L. M., BAROZZI I. G., CHRONOWSKA A., TERMANINI A., OSTUNI R., PROSPERINI E., STEWART A. F., TESTA G., NATOLI G.

BERTANI E., TESTORI A., CHIAPPA A., MISITANO P., BIFFI R., VIALE G., MAZZAROL G., DE PAS T., BOTTERI E., CONTINO G., VERRECCHIA F., BAZOLLI B., ANDREONI B. BERTOLINI F.

Lenalidomide for Multiple Myeloma

NEW ENGL J MED

367

573 - 575

53,298

AUTIER P., GANDINI S., MULLIE P

A Systematic Review: Influence of Vitamin D Supplementation on Serum 25-Hydroxyvitamin D Concentration.

J CLIN ENDOCR METAB

5,967

BERTOLINI F., LOHSIRIWAT V., PETIT J. Y., KOLONIN M. G

Adipose tissue cells, lipotransfer and cancer: A challenge for scientists, oncologists and surgeons

BBA-REV CANCER

1826

209 - 214

9,38

AWADELKARIM K. D, MARIANI COSTANTINI R., OSMAN I., BARBERIS M.

Ki-67 Labeling Index in Primary Invasive Breast Cancer from Sudanese Patients: A Pilot Study

PATHOLOGY

2,378

BERTOLINI F., MANCUSO P., BENAYOUN L., GINGIS-VELITSKI S., SHAKED Y.

Evaluation of circulating endothelial precursor cells in cancer patients.

904

165 - 172

0

AZIM H. A., SANTORO L., RUSSELL-EDU W., PENTHEROUDAKIS G., PAVLIDIS N., PECCATORI F. A.

Prognosis of pregnancy-associated breast cancer: a meta-analysis of 30 studies.

CANCER TREAT REV

38

834 - 842

6,054

Mikhail G. Kolonin and Paul J. Simmons (eds.), Stem Cell Mobilization: Methods and Protocols, Methods in Molecular Biology

279 - 290

32,403

ACTA ONCOL

51

653 - 661

3,33

Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.

150

The biological features and prognosis of breast cancer diagnosed during pregnancy: a case-control study.

BHATT D. M., PANDYA-JONES A., TONG A. J., BAROZZI I., LISSNER M. M., NATOLI G., BLACK D. L., SMALE S. T.

CELL

AZIM H., BOTTERI E., RENNE G., DELL'ORTO P., ROTMENSZ N., GENTILINI O. D., SANGALLI C. A., PRUNERI G., DI NUBILA B., LOCATELLI M. A., SOTIRIOU C., PICCART M., GOLDHIRSCH A., VIALE G., PECCATORI F. A.

BIANCHI F., NICASSIO F., VERONESI G., DI FIORE P. P.

Circulating microRNAs: next-generation biomarkers for early lung cancer detection.

ECANCERMEDICALSCIENCE

6

246

0

AZIM JR H. A, KROMAN N., PAESMANS M., GELBER S., ROTMENSZ N., AMEYE L., DE MATTOS-ARRUDA L., PISTILLI B., PINTO A., JENSEN M. B, CORDOBA O., DE AZAMBUJA E., GOLDHIRSCH A., PICCART M. J, PECCATORI F. A

Prognostic Impact of Pregnancy After Breast Cancer According to Estrogen Receptor Status: A Multicenter Retrospective Study.

J CLIN ONCOL

BIANCHI S., BENDINELLI B., CASTELLANO I., PIUBELLO Q., RENNE G., CATTANI M. G., DI STEFANO D., CARRILLO G., LAURINO L., BERSIGA A., GIARDINA C., DANTE S., DI LORETO C., QUERO C., ANTONACCI C. M., PALLI D., VANCB STUDY GROUP

Morphological parameters of flat epithelial atypia (FEA) in stereotactic vacuum-assisted needle core biopsies do not predict the presence of malignancy on subsequent surgical excision.

VIRCHOWS ARCH

461

405 - 417

2,491

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10

43

18

12

36

97

38

e536 e540

2,606

85 - 85

3,474

2070 2079

2,876

470 - 474

1,643

203 - 214

572 - 585

2606 2613

834 - 842

2,652

21,637

18,372

Publications

ANDREOLA G., VANAZZI A., RADICE D., BABIC A., RABASCIO C., NEGRI M., MARTINELLI G., LASZLO' D.

0

IEO — Scientific Report 2012 — Ongoing Research 2013

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266

Power technology: how to safely use ports and central catheters to deliver contrast medium in radiology procedures.

Totally Implantable Venous Access Devices -Management in Mid.and Long.term Clinical Setting

34

239-246

0

BIFFI R.

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Totally Implantable Venous Access Devices -Management in Mid.and Long.term Clinical Setting

11

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BORNSTEIN J., SIDERI M. G., TATTI S., WALKER P., PRENDIVILLE W., HAEFNER H. K, FOR THE NOMENCLATURE COMMITTEE OF THE INTERNATIONAL FEDERATION FOR CERVICAL PATHOLOGY AND COLPOSCOPY

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Cigarette smoking and pancreatic cancer: an analysis from the International Pancreatic Cancer Case-Control Consortium (Panc4).

ANN ONCOL

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BOSOTTI R., CARPINELLI P., HEALY S., LOCATELLI G., CAPPELLA P., LANFRANCONE L. M., CALOGERO R., MOLL J., ISACCHI A.

Transcriptional analysis of the Aurora inhibitor Danusertib leading to biomarker identification in TP53 wild type cells.

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Choice of venous sites. Percutaneous implant/technique/US guidance.

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Surgical site infections following colorectal cancer surgery: a randomized prospective trial comparing common and advanced antimicrobial dressing containing ionic silver.

WORLD J SURG ONCOL

10

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BOTRUGNO O. A., ROBERT T., VANOLI F., FOIANI M., MINUCCI S.

Molecular pathways: old drugs define new pathways: non-histone acetylation at the crossroads of the DNA damage response and autophagy.

CLIN CANCER RES

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2,491

Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore

NAT CELL BIOL

19,488

Biopsy of liver metastasis for women with breast cancer: impact on survival.

BREAST

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. A.

BOTTERI E., DISALVATORE D., CURIGLIANO G., BROLLO J., BAGNARDI V., VIALE G., ORSI F., GOLDHIRSCH A., ROTMENSZ N.

Mastectomy without radiotherapy: outcome analysis after 10 years of follow-up in a single institution.

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EUR J NUCL MED MOL I

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BOHLEN T. T., BRONS S., DOSANJH M., FERRARI A., FOSSATI P., HABERER T., PATERA V., MAIRANI A.

Investigating the robustness of ion beam therapy treatment plans to uncertainties in biological treatment parameters.

PHYS MED BIOL

57

7983 8004

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BOTTERI E., MAISONNEUVE P.

How strong is the association between smoking and colorectal cancer?

COLORECTAL CANCER

1

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BOYLE P., BONIOL M., KOECHLIN A., ROBERTSON C., VALENTINI F., COPPENS K., FAIRLEY L. L., BONIOL M., ZHENG T., ZHANG Y., PASTERK M., SMANS M., CURADO M. P., MULLIE P., GANDINI S., BOTA M., BOLLI G. B., ROSENSTOCK J., AUTIER P.

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Dual effect of metformin on breast cancer proliferation in a randomized presurgical trial.

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BOZZETTI F., MARIANI L., LO VULLO S., THE SCRINIO WORKING GROUP, AMERIO M. L, BIFFI R., CACCIALANZA R., CAPUANO G., CORREJA I., COZZAGLIO L., DE LEO A., DI COSMO L., FINOCCHIARO C., GAVAZZI C., GIANNONI A., MAGNANINI P., MANTOVANI G., PELLEGRINI M., ROVERA G. M, ROVERA L., SANDRI G., TINIVELLA M., VIGEVANI E.

The nutritional risk in oncology: a study of 1,453 cancer outpatients

SUPPORT CARE CANCER

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BONIOL M., AUTIER P., BOYLE P., GANDINI S.

Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis.

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BIOETHICS

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Increased BMI correlates with higher risk of disease relapse and differentiation syndrome in patients with acute promyelocytic leukemia treated with the AIDA protocols.

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BRECCIA M., MAZZARELLA L., BAGNARDI V., DISALVATORE D., LOGLISCI G., CIMINO G., TESTI A. M, AVVISATI G., PETTI M. C., MINOTTI C., LATAGLIATA R., FOA' R., PELICCI P. G., LO COCO F.

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Alarming shift away from sharing results

NATURE

488

Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.

HUM MUTAT

BONIOLO G., VACCARI T. BONIOLO G., VALENTINI S.

Objects. A Study in Kantian Formal Epistemology

NOTRE DAME J FORM L

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The emerging role of p53 in stem cells.

TRENDS MOL MED

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BREWSTER B.L., ROSSIELLO F., FRENCH J.D., EDWARDS S.L., WONG M., WRONSKI A., WHILEY P., WADDELL N., CHEN X., BOVE B., KCONFAB, HOPPER J.L., JOHN E.M., ANDRULIS I., DALY M., VOLORIO S., BERNARD L., PEISSEL B., MANOUKIAN S., BARILE M., PIZZAMIGLIO S., VERDERIO P., SPURDLE A.B., RADICE P., GODWIN A.K., SOUTHEY M.C., BROWN M.A., PETERLONGO P.

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2011 colposcopic terminology of the international Federation for Cervical Pathology and Colposcopy.

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Globally stable endemicity for infectious diseases with information-related changes in contact patterns.

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The H3K27 Demethylase JMJD3 Is Required for Maintenance of the Embryonic Respiratory Neuronal Network, Neonatal Breathing, and Survival.

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Has color Doppler a role in the evaluation of mammary lesions?

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CALABRESE L., OSTUNI A., ANSARIN M., GIUGLIANO G., MAFFINI F., ALTERIO D., ROCCA M. C., PETRALIA G., BRUSCHINI R., CHIESA F., ASSOCIATION OF RADIOTHERAPY AND ONCOLOGY OF THE MEDITERRANEAN AREA (AROME)

Future challenges in head and neck cancer: from the bench to the bedside?

CRIT REV ONCOL HEMAT

84 Suppl 1

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CAMBIAGHI V., GIULIANI V., LOMBARDI S., MARINELLI C., TOFFALORIO F., PELICCI P. G

Trim proteins in cancer

Trim/RBCC Proteins - Meroni G. -Editor Springer

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77-83

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Two sides of the Myc-induced DNA damage response: from tumor suppression to tumor maintenance.

CELL DIV

7

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Immunotherapy against the radial glia marker GLAST effectively triggers specific antitumor effectors without autoimmunity.

ONCOIMMUNITY

CAPITANI N., PATRUSSI L., TRENTIN L., LUCHERINI O. M., CANNIZZARO E., MIGLIACCIO E., FREZZATO F., GATTAZZO C., FORCONI F., PELICCI P., SEMENZATO G., BALDARI C. T.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis.

BLOOD

120

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CARAVAGNA G., BARBUTI R., D'ONOFRIO A.

Fine-tuning anti-tumor immunotherapies via stochastic simulations.

BMC BIOINFORMATICS

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CARDILLO G., GALETTA D., VAN SCHIL P., ZUIN A., FILOSSO P., CERFOLIO R. J., FORCIONE A. R., CARLEO F.

Completion pneumonectomy: a multicentre international study on 165 patients.

EUR J CARDIO-THORAC

42

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La donna, le cure oncologiche e la protezione del cuore

AAOHN J

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Cardiovascular Complications of Cancer Therapeutic Agents

Braunwald's Heart Disease, A textbook of Cardiovascular Madicine (Ninth Edition) Bonow, Mann, Zipes, Libby

CARDINALE D. M., COLOMBO A., BACCHIANI G., CIPOLLA C.

La cardioprotezione nelle donne con patologia oncologica

G ITAL CARDIOL

13

461 - 468

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Role of biomarkers in cardioncology.

CLIN CHEM LAB MED

49

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CARDOSO F., COSTA A., NORTON L., CAMERON D., CUFER T., FALLOWFIELD L., FRANCIS P., GLIGOROV J., KYRIAKIDES S., LIN N., PAGANI O., SENKUS E., THOMSSEN C., AAPRO M., BERGH J., DI LEO A., EL SAGHIR N., GANZ P. A., GELMON K., GOLDHIRSCH A., HARBECK N., HOUSSAMI N., HUDIS C., KAUFMAN B., LEADBEATER M., MAYER M., RODGER A., RUGO H., SACCHINI V., SLEDGE G., VAN'T VEER L., VIALE G., KROP I., WINER E.

1st International consensus guidelines for advanced breast cancer (ABC 1).

BREAST

21

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2,491

CARDOSO F., LOIBL S., PAGANI O., GRAZIOTTIN A., PANIZZA P., MARTINCICH L., GENTILINI O., PECCATORI F., FOURQUET A., DELALOGE S., MAROTTI L., PENAULT-LLORCA F., KOTTIKITROMILIDOU A. M., RODGER A., HARBECK N.

The European Society of Breast Cancer Specialists recommendations for the management of young women with breast cancer.

CARRARA L., GADDUCCI A., LANDONI F., MAGGINO T., SCAMBIA G., GALLETTO L., LISSONI A. A., FUSO L., ZOLA P., SARTORI E.

Could Different Follow-Up Modalities Play a Role in the Diagnosis of Asymptomatic Endometrial Cancer Relapses?: An Italian Multicentric Retrospective Analysis.

INT J GYNECOL CANCER

CASSATELLA M., ZORZINO L., SANDRI M.

Single circulating tumor cell profiling: a new perspective for targeted therapy?

FUTURE ONCOL

IEO — Scientific Report 2012 — Ongoing Research 2013

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CATANIA C. M., SPITALERI G., DELMONTE A., GIOVANNINI M., TOFFALORIO F., NOBERASCO C., BRESOLIN N., COMI G., DE PAS T.

Safety of systematic chemotherapy in patient with mitochondrial myopathy and non-small cell lung cancer

J CLIN ONCOL

30

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CATUCCI I., COLOMBO M., VERDERIO P., BERNARD L., FICARAZZI F., MARIETTE F., BARILE M., PEISSEL B., CATTANEO E., MANOUKIAN S., RADICE P., PETERLONGO P.

Sequencing analysis of SLX4/FANCP gene in Italian familial breast cancer cases.

PLOS ONE

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CATUCCI I., MILGROM R., KUSHNIR A., LAITMAN Y., PALUCH-SHIMON S., VOLORIO S., FICAZZI F., BERNARD L., RADICE P., FRIEDMAN E., PETERLONGO P.

Germline mutations in BRIP1 and PALB2 in Jewish high cancer risk families.

FAM CANCER

11

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CATUCCI I., VERDERIO P., PIZZAMIGLIO S., BERNARD L., DALL'OLIO V., SARDELLA D., RAVAGNANI F., GALASTRI L., BARILE M., PEISSEL B., ZAFFARONI D., MANOUKIAN S., RADICE P., PETERLONGO P.

The SNP rs895819 in miR-27a is not associated with familial breast cancer risk in Italians.

BREAST CANCER RES TR

133

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CAZZANIGA M., BONANNI B.

Prevention of ER-negative breast cancer: where do we stand?

EUR J CANCER PREV

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CECCARONI M., ROVIGLIONE G., SPAGNOLO E., CASADIO P., CLARIZIA R., PEIRETTI M., BRUNI F., PETERS I., ALETTI G.

Pelvic dysfunctions and quality of life after nerve-sparing radical hysterectomy: a multicenter comparative study.

ANTICANCER RES

32

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CHEN X., BAROZZI I., TERMANINI A., PROSPERINI E., RECCHIUTI A., DALLI J., MIETTON F., MATTEOLI G., HIEBERT S., NATOLI G.

Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages.

P NATL ACAD SCI USA

CHINOL M., MANSI L.

Blaine T. Smith and Kara D. Weatherman (eds): Diagnostic Imaging for Pharmacists

EUR J NUCL MED MOL I

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Lifting the Threshold between Life and Death: SUMO and HDAC Fine-Tune HIPK2 to Sense Redox Status.

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CHIRGWIN J., SUN Z., SMITH I., PRICE K. N., THURLIMANN B., EJLERTSEN B., BONNEFOI H., REGAN M. M., GOLDHIRSCH A., COATES A. S., FOR THE BIG 1-98 COLLABORATIVE AND INTERNATIONAL BREAST CANCER STUDY GROUPS, COLLEONI M. A., VERONESI P., PERUZZOTTI G., CORSETTO L. A., GHISINI R., RENNE G., LUINI A., DE PAS T., MUNZONE E., GALIMBERTI V. E., ZURRIDA S., INTRA M., NOLE' F., ORECCHIA R., MARTINELLI G., DE BRAUD F., VIALE G., DELL'ORTO P., MASTROPASQUA M. G., DEL CURTO B.

The advantage of letrozole over tamoxifen in the BIG 1-98 trial is consistent in younger postmenopausal women and in those with chemotherapy-induced menopause.

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CIOCCA M., CANTONE M. C., VERONESE I., CATTANI F., PEDROLI G., MOLINELLI S., VITOLO V., ORECCHIA R.

Application of failure mode and effects analysis to intraoperative radiation therapy using mobile electron linear accelerators.

INT J RADIAT ONCOL

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CLIBY W., ALETTI G., CHI D., BRISTOW R.

Re: defining the limits of radical cytoreductive surgery in ovarian cancer.

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Is adjuvant chemotherapy useful for women with luminal a breast cancer?

J CLIN ONCOL

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COATES A. S., MILLAR E. K., O'TOOLE S. A., MOLLOY T. J., VIALE G., GOLDHIRSCH A., REGAN M. M., GELBER R. D., SUN Z., CASTIGLIONE-GERTSCH M., GUSTERSON B., MUSGROVE E. A., SUTHERLAND R. L.

Prognostic interaction between expression of p53 and estrogen receptor in patients with node-negative breast cancer: results from IBCSG Trials VIII and IX.

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Neoadjuvant therapy for ER-positive breast cancers.

ANN ONCOL

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ANN ONCOL

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COLOMBO N., CARINELLI S., COLOMBO A., MARINI C., ROLLO D., SESSA C., ESMO GUIDELINES WORKING GROUP

Cervical cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.

ANN ONCOL

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COLOMBO N., KUTARSKA E., DIMOPOULOS M., BAE D. S, RZEPKA GORSKA I., BIDZINSKI M., SCAMBIA G., ENGELHOLM S. A, JOLY F., WEBER D., EL-HASHIMY M., LI J., SOUAMI F., WING P., ENGELHOLM S, BAMIAS A., SCHWARTZ P.

Randomized, open-label, phase III study comparing patupilone (EPO906) with pegylated liposomal doxorubicin in platinum-refractory or -resistant patients with recurrent epithelial ovarian, primary fallopian tube, or primary peritoneal cancer.

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COLOMBO N., MANGILI G., MAMMOLITI S., KALLING M., THOLANDER B., STERNAS L., BUZENET G., CHAMBERLAIN D.

A phase II study of aflibercept in patients with advainced epithelial ovarian cancer and symptomatic malignant ascites.

GYNECOL ONCOL

COLOMBO N., PEIRETTI M., GARBI A., CARINELLI S., MARINI C., SESSA C., ESMO GUIDELINES WORKING GROUP

Non-epithelial ovarian cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up.

ANN ONCOL

COLUCCIA P., CROVETTI G., DEL FANTE C., DALLAVALLE F. M., LASZLO D., FERREMI P., MARENCHINO D., SANTOLERI L., DE FILIPPO C., MATTANA F., MARIANI L., PERSEGHIN P., RAVAGNANI F.

Screening of related donors and peripheral blood stem cell collection practices at different Italian apheresis centers.

BLOOD TRANSFUS-ITALY

COMBS S. E., KIESER M., HABERMEHL D., WEITZ J., JAGER D., FOSSATI P., ORECCHIA R., ENGENHART-CABILLIC R., POTTER R., DOSANJH M., JAKEL O., BUCHLER M. W., DEBUS J.

Phase I/II trial evaluating carbon ion radiotherapy for the treatment of recurrent rectal cancer: the PANDORA-01 trial.

BMC CANCER

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CORBELLINI C., VINGIANI A., MAFFINI F. A., CHIAPPA A., BERTANI E., ANDREONI B.

Retroperitoneal pararenal isolated neurofibroma: report of a case and review of literature.

ECANCERMEDICALSCIENCE

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CORTES-DERICKS L., GALETTA D., SPAGGIARI L., SCHMID R. A., KAROUBI G.

High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter disease-free intervals.

EUR J CARDIO-THORAC

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COSTA S., VENTUROLI S., NEGRI G., SIDERI M. G., PRETI M., PESARESI M., FALASCA A., BARBIERI D., ZERBINI M., SANTINI D., SANDRI M. T, GHIRINGHELLO B., CAROPPO VENTURINI N., SYRJANEN S., SYRJANEN K.

Factors predicting the outcome of conservatively treated adenocarcinoma in situ of the uterine cervix: an analysis of 166 cases.

GYNECOL ONCOL

COUCH F. J., GAUDET M. M., ANTONIOU A. C., et al.

Common Variants at the 19p13.1 and ZNF365 Loci Are Associated with ER Subtypes of Breast Cancer and Ovarian Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

CANCER EPIDEM BIOMAR

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Dosimetry for Peptide Receptor Radionuclide Therapy.

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Tumor-Associated Antigens in Breast Cancer

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New drugs for breast cancer subtypes: targeting driver pathways to overcome resistance.

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Cardiovascular toxicity induced by chemotherapy, targeted agents and radiotherapy: ESMO Clinical Practice Guidelines.

ANN ONCOL

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CURIGLIANO G., LOCATELLI M. A., FUMAGALLI L., BROLLO J., MUNZONE E., NOLE' F., CRISCITIELLO C., GOLDHIRSCH A.

Targeting the subtype s of breast cancer: rethinking investigational drugs.

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Consuming genomes: scientific and social innovation in direct-to-consumer genetic testing

NEW GENET SOC

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DANIELLI R., RIDOLFI R., CHIARION-SILENI V., QUEIROLO P., TESTORI A., PLUMMER R., BOITANO M., CALABRO' L., DE ROSSI C., DI GIACOMO A. M., FERRUCCI P. F., RIDOLFI L., ALTOMONTE M., MIRACCO C., BALESTRAZZI A., MAIO M.

Ipilimumab in pretreated patients with metastatic uveal melanoma: safety and clinical efficacy

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61

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D'ANTONIO M., PENDINO V., SINHA S., CICCARELLI F.

Network of Cancer Genes (NCG 3.0): integration and analysis of genetic and network properties of cancer genes.

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A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B.

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DE BACCO F., CASANOVA E., MEDICO E., PELLEGATTA S., ORZAN F., ALBANO R., LURAGHI P., REATO G., D'AMBROSIO A., PORRATI P., PATANE M., MADERNA E., POLLO B., COMOGLIO P. M., FINOCCHIARO G., BOCCACCIO C.

The MET oncogene is a functional marker of a glioblastoma stem cell subtype.

CANCER RES

72

4537 4550

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DE CENSI A., DUNN B. K, PUNTONI M., GENNARI A., FORD L. G

Exemestane for Breast Cancer Prevention: A Critical Shift?

CANCER DISCOV

2

25 - 40

0

DE CENSI A., PRUNERI G., GUERRIERI GONZAGA A.

Estrogen receptor in breast ductal carcinoma in situ: good cop, bad cop?

J CANCER ONCOLOGY

30

1384 1386

18

DE FRANCISCIS S., D'ONOFRIO A.

Spatiotemporal bounded noises and transitions induced by them in solutions of the real Ginzburg-Landau model.

PHYS REV E

86

21118

2,255

DE GIORGI V., GANDINI S., GRAZZINI M., BENEMEI S., MARCHIONNI N., GEPPETTI P.

[ß-blockers: a new and emerging treatment for melanoma].

RECENTI PROG MED

103

11-16'

0

DE GIORGI V., GORI A., GRAZZINI M., ROSSARI S., ORANGES T., LONGO A. S., LOTTI T., GANDINI S.

Epidemiology of melanoma: is it still epidemic? What is the role of the sun, sunbeds, Vit D, betablocks, and others?

DERMATOL THER

25

392 - 396

1,687

DE GIORGI V., GRAZZINI M., GANDINI S., BENEMEI S., ASBURY C. D, MARCHIONNI N., GEPPETTI P.

ß-adrenergic-blocking drugs and melanoma: current state of the art.

EXPERT REV ANTICANC

12

1461 1467

2,652

DE GIORGI V., GRAZZINI M., ROSSARI S., GORI A., PAPI F., SCARFI F., SAVARESE I., GANDINI S.

Is skin self-examination for cutaneous melanoma detection still adequate? A retrospective study.

DERMATOLOGY

225

31 - 36

2,053

DE LORENZI F., LOHSIRIWAT V., BARBIERI B., RODRIGUEZ PEREZ S., GARUSI C., PETIT J. Y., GALIMBERTI V. E., RIETJENS M.

Immediate breast reconstruction with prostheses after conservative treatment plus intraoperative radiotherapy. Long term esthetic and oncological outcomes.

BREAST

21

374 - 379

2,491

DE PAS T., GIOVANNINI M., RESCIGNO M., CATANIA C. M., TOFFALORIO F., SPITALERI G., DELMONTE A., BARBERIS M., SPAGGIARI L., SOLLI P., VERONESI G., DE BRAUD F.

Vaccines in non-small cell lung cancer: Rationale, combination strategies and update on clinical trials.

CRIT REV ONCOL HEMAT

83

432 - 443

4,411

DE ROBERTO G., DE LEONE A., TAMAYO D., FIORI G., RAVIZZA D., TROVATO C. M., DE LISI S., CROSTA C.

Stent migration after stent-in-stent technique using a biodegradable stent

ENDOSCOPY

44

E51 - E52

5,21

DE ROSA A., PELLEGATTA S., ROSSI M., TUNICI P., MAGNONI L., SPERANZA M. C., MALUSA F., MIRAGLIOTTA V., MORI E., FINOCCHIARO G., BAKKER A.

A Radial Glia Gene Marker, Fatty Acid Binding Protein 7 (FABP7), Is Involved in Proliferation and Invasion of Glioblastoma Cells.

PLOS ONE

7

e52113 e52113

4,092

DEL PUP L., PECCATORI F. A., AZIM H. A., MICHIELI M., MOIOLI M., GIORDA G., TIRELLI U., BERRETTA M.

Obstetrical, fetal and postnatal effects of gestational antiblastic chemotherapy: how to counsel cancer patients.

INT J IMMUNOPATH PH

25

33S - 46S

2,991

3,888

23 Suppl 7

e173 e181

CURIGLIANO G., CARDINALE D., SUTER T., PLATANIOTIS G., DE AZAMBUJA E., SANDRI M. T., CRISCITIELLO C., GOLDHIRSCH A., CIPOLLA C., ROILA F., ESMO GUIDELINES WORKING GROUP

IEO — Scientific Report 2012 — Ongoing Research 2013

Publications

COLLET T. H., GUSSEKLOO J., BAUER D. C., DEN ELZEN W. P., CAPPOLA A. R., BALMER P., IERVASI G., ASVOLD B. O., SGARBI J. A., VOLZKE H., GENCER B., MACIEL R. M., MOLINARO S., BREMNER A., LUBEN R. N., MAISONNEUVE P., CORNUZ J., NEWMAN A. B., KHAW K. T., WESTENDORP R. G., FRANKLYN J. A., VITTINGHOFF E., WALSH J. P., RODONDI N., FOR THE THYROID STUDIES COLLABORATION

271


272

Systematic lymphadenectomy in ovarian cancer at second-look surgery: a randomised clinical trial

DELLAPASQUA S., BAGNARDI V., BERTOLINI F., SANDRI M. T., PASTRELLO D., CANCELLO G., MONTAGNA E., BALDUZZI A., MANCUSO P., LUINI A., GOLDHIRSCH A., COLLEONI M. A.

Increased mean corpuscular volume of red blood cells predicts response to metronomic capecitabine and cyclophosphamide in combination with bevacizumab.

BREAST

DELLAPASQUA S., BAGNARDI V., REGAN M. M., ROTMENSZ N., MASTROPASQUA M. G., VIALE G., MAIORANO E., PRICE K. N., GELBER R. D., CASTIGLIONE-GERTSCH M., GOLDHIRSCH A., COLLEONI M. A.

A risk score based on histopathological features predicts higher risk of distant recurrence in premenopausal patients with lymph node-negative endocrineresponsive breast cancer.

BREAST

21

621 - 628

2,491

DELUCA J. G., MUSACCHIO A.

Structural organization of the kinetochore-microtubule interface.

CURR OPIN CELL BIOL

24

48 - 56

12,897

DEMEESTERE I., BASSO O., MOFFA F., PECCATORI F., POIROT C., SHALOM-PAZ E.

Fertility preservation in female cancer patients.

OBSTET GYNECOL

2012

695041 695041

4,73

DEMEESTERE I., MOFFA F., PECCATORI F., POIROT C., SHALOM-PAZ E.

Multiple approaches for individualized fertility protective therapy in cancer patients.

OBSTET GYNECOL

2012

961232 961232

4,73

DEPUYDT T., PENNE R., VERELLEN D., HRBACEK J., LANG S., LEYSEN K., VANDEVONDEL I., POELS K., REYNDERS T., GEVAERT T., DUCHATEAU M., TOURNEL K., BOUSSAER M., COSENTINO D., GARIBALDI C., SOLBERG T., DE RIDDER M.

Computer-aided analysis of star shot films for high-accuracy radiation therapy treatment units.

PHYS MED BIOL

57

2997 3011

2,829

DI GIACOMO A. M, ASCIERTO P. A, PILLA L., SANTINAMI M., FERRUCCI P. F, GIANNARELLI D., MARASCO A., RIVOLTINI L., SIMEONE E., NICOLETTI S. V, FONSATTI E., ANNESI D., QUEIROLO P., TESTORI A., RIDOLFI R., PARMIANI G., MAIO M.

Ipilimumab and fotemustine in patients with advanced melanoma (NIBIT-M1): an open-label, single-arm phase 2 trial.

LANCET ONCOL

13

879 - 886

22,589

DI LEGGE A., TESTA A. C., AMEYE L., VAN CALSTER B., LISSONI A. A., LEONE F. P., SAVELLI L., FRANCHI D., CZEKIERDOWSKI A., TRIO D., VAN HOLSBEKE C., FERRAZZI E., SCAMBIA G., TIMMERMAN D., VALENTIN L.

Lesion size affects diagnostic performance of IOTA logistic regression models, IOTA simple rules and risk of malignancy index in discriminating between benign and malignant adnexal masses.

ULTRASOUND OBST GYN

40

345 - 354

3,007

DI MAIO M., LEIGHL N. B., GALLO C., FELD R., CIARDIELLO F., BUTTS C., MAIONE P., GEBBIA V., MORGILLO F., WIERZBICKI R., FAVARETTO A., ALAM Y., CINIERI S., SIENA S., BIANCO R., RICCARDI F., SPATAFORA M., RAVAIOLI A., FELLETTI R., FREGONI V., GENESTRETI G., ROSSI A., MANCUSO G., FASANO M., MORABITO A., TSAO M. S., SIGNORIELLO S., PERRONE F., GRIDELLI C.

Quality of Life Analysis of TORCH, a Randomized Trial Testing First-Line Erlotinib Followed by Second-Line Cisplatin/ Gemcitabine Chemotherapy in Advanced Non-Small-Cell Lung Cancer.

J THORAC ONCOL

7

1830 1844

3,661

DIDIER F., ARNABOLDI P., GANDINI S., MALDIFASSI A., GOLDHIRSCH A., RADICE D., MINOTTI I., BALLARDINI B., LUINI A., SANTILLO B., RIETJENS M., PETIT J. Y.

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 RES TR

132

1177 - 1184

4,431

DIDIER F., RADICE D., MALDIFASSI A., GATTI G., LUINI A., LEONARDI M. C., LUPO F., ROTMENSZ N., SANTILLO B., GALIMBERTI V. E., GOLDHIRSCH A.

Do early breast cancer patients with only sentinel lymph node biopsy experience lower psychological morbidity over time?

BREAST J

18

622 - 624

1,643

D'ONOFRIO A, CERRAI P., GANDOLFI A.

New challenges for cancer systems biomedicine

D'onofrio A., Cerrai P., Gandolfi A Editori - Springer Series

D'ONOFRIO A.

Spatiotemporal effects of a possible chemorepulsion of tumor cells by immune system effectors.

J THEOR BIOL

296

41 - 41

2,208

D'ONOFRIO A., GANDOLFI A., GATTONI S.

The Norton-Simon hypothesis and the onset of non genetic resistance to chemiotherapy induced by stochastic fluctuations

PHYSICA A

391

6484 6496

1,373

IEO — Scientific Report 2012 — Ongoing Research 2013

BRIT J CANCER

107

21

785 - 792

309 - 313

5,042

D'ONOFRIO A., MANFREDI P., POLETTI P.

The interplay of public intervention and private choices in determining the outcome of vaccination programmes.

PLOS ONE

7

e45653 e45653

4,092

DUBSKY P. C., CURIGLIANO G.

Immunotherapy in Breast Cancer Towards a New Understanding of Both Tumor and Host

BREAST CARE

7

258 - 260

0,446

DUELL E. J., LUCENTEFORTE E., OLSON S. H., BRACCI P. M., LI D., RISCH H. A., SILVERMAN D. T., JI B. T., GALLINGER S., HOLLY E. A., FONTHAM E. H., MAISONNEUVE P., BUENODE-MESQUITA H. B., GHADIRIAN P., KURTZ R. C., LUDWIG E., YU H., LOWENFELS A. B., SEMINARA D., PETERSEN G. M., LA VECCHIA C., BOFFETTA P.

Pancreatitis and pancreatic cancer risk: a pooled analysis in the International Pancreatic Cancer Case-Control Consortium (PanC4).

ANN ONCOL

23

2964 2970

6,425

EARLY BREAST CANCER TRIALISTS' COLLABORATIVE GROUP (EBCTCG), PETO R., DAVIES C., GODWIN J., GRAY R., PAN H. C, CLARKE M., CUTTER D., DARBY S., MCGALE P., TAYLOR C., WANG Y. C, BERGH J., DI LEO A., ALBAIN K., SWAIN S., PICCART M., PRITCHARD K., VIALE G., PRUNERI G.

Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100,000 women in 123 randomised trials.

LANCET

EGGERMONT A. M. M., SUCIU S., TESTORI A., KRUIT W. H., MARSDEN J., PUNT C. J., SANTINAMI M., SALES F., SCHADENDORF D., PATEL P., DUMMER R., ROBERT C., KEILHOLZ U., YVER A., SPAZ A.

Ulceration and stage are predictive of interferon efficacy in melanomamelanoma: Results of the phase III adjuvant trials EORTC 18952 and EORTC 18991

EUR J CANCER

48

218 - 225

5,536

EGGERMONT A. M. M., SUCIU S., TESTORI A., SANTINAMI M., KRUIT W. H. J., MARSDEN J., PUNT C. J. A., SALES F., DUMMER R., ROBERT C., SCHADENDORF D., PATEL P., DE SCHAETZEN G., SPATZ A., KEILHOLZ U.

Long-term Results of the Randomized Phase III Trial EORTC 18991 of Adjuvant Therapy With Pegylated Interferon ?-2b Versus Observation in Resected Stage III Melanoma

J CLIN ONCOL

30

3810 3818

18,372

EIERMANN W., BERGH J., CARDOSO F., CONTE P., CROWN J., CURTIN N. J., GLIGOROV J., GUSTERSON B., JOENSUU H., LINDERHOLM B. K., MARTIN M., PENAULT-LLORCA F., PESTALOZZI B. C., RAZIS E., SOTIRIOU C., TJULANDIN S., VIALE G.

Triple negative breast cancer: proposals for a pragmatic definition and implications for patient management and trial design.

BREAST

21

20 - 26

2,491

EJLERTSEN B., ALDRIDGE J., NIELSEN K. V., REGAN M. M., HENRIKSEN K. L., LYKKESFELDT A. E., MULLER S., GELBER R. D., PRICE K. N., RASMUSSEN B. B., VIALE G., MOURIDSEN H., DANISH BREAST CANCER COOPERATIVE GROUP, BIG 1-98 COLLABORATIVE GROUP, INTERNATIONAL BREAST CANCER STUDY GROUP

Prognostic and predictive role of ESR1 status for postmenopausal patients with endocrine-responsive early breast cancer in the Danish cohort of the BIG 1-98 trial.

ANN ONCOL

23

1138 1144

6,425

ELKINS J. M, SANTAGUIDA S., MUSACCHIO A., KNAPP S.

Crystal structure of human aurora B in complex with INCENP and VX-680.

J MED CHEM

7841 7848

0

ERBA P. A., SOLLINI M., ORCIUOLO E., TRAINO C., PETRINI M., PAGANELLI G., BOMBARDIERI E., GRANA C. M., GIOVANNONI L., NERI D., MENSSEN H. D., MARIANI G.

Radioimmunotherapy with Radretumab in Patients with Relapsed Hematologic Malignancies

J NUCL MED

53

922 - 927

6,381

ERIKSEN J. G., BEAVIS A. W., COFFEY M. A., LEER J. W., MAGRINI S. M., BENSTEAD K., BOELLING T., HJALM-ERIKSSON M., KANTOR G., MACIEJEWSKI B., MEZECKIS M., OLIVEIRA A., THIRION P., VITEK P., OLSEN D. R., EUDALDO T., ENGHARDT W., FRANCOIS P., GARIBALDI C., HEIJMEN B., JOSIPOVIC M., MAJOR T., NIKOLETOPOULOS S., RIJNDERS A., WALIGORSKI M., WASILEWSKA-RADWANSKA M., MULLANEY L., BOEJEN A., VAANDERING A., VANDEVELDE G., VERFAILLIE C., POTTER R.

The updated ESTRO core curricula 2011 for clinicians, medical physicists and RTTs in radiotherapy/radiation oncology.

RADIOTHER ONCOL

103

103 - 108

5,58

ESPOSITO A., MUNZONE E., BAGNARDI V., ADAMOLI L., SCIANDIVASCI A. S., CULLURA' D., GOLDHIRSCH A., NOLE' F.

Are there benefits in routine clinical practice of continuing trastuzumab after progression for metastatic breast cancer patients?

ANTICANCER DRUGS

23

1089 1098

0

2,491

0

Publications

DELL'ANNA T., SIGNORELLI M., BENEDETTI PANICI P., MAGGIONI A., FOSSATI R., FRUSCIO R., MILANI R., BOCCIOLONE L., BUDA A., MANGIONI C., SCAMBIA G., ANGIOLI R., CAMPAGNUTTA E., GRASSI R., LANDONI F.

38,278

IEO — Scientific Report 2012 — Ongoing Research 2013

273


274

Obesity and Risk of Recurrence or Death After Adjuvant Endocrine Therapy With Letrozole or Tamoxifen in the Breast International Group 1-98 Trial.

FAN T. Y, ZHANG L., CHEN W., LIU Y., HE M., HUANG X., ORSI F., WANG Z.

Feasibility of MRI-guided high intensity focused ultrasound treatment for adenomyosis

EUR J RADIOL

FASCHING P. A., PHAROAH P. D., et al.

The role of genetic breast cancer susceptibility variants as prognostic factors.

HUM MOL GENET

FATTORI G., RIBOLDI M., DESPLANQUES M., TAGASTE B., PELLA A., ORECCHIA R., BARONI G.

Automated fiducial localization in CT images based on surface processing and geometrical prior knowledge for radiotherapy applications.

FERNANDEZ-CUESTA L., OAKMAN C., FALAGAN-LOTSCH P., SMOTH K. S., QUINAUX E., BUYSE M., DOLCI M. S., AZAMBUJA E. D., HAINAUT P., DELL'ORTO P., LARSIMONT D., FRANCIS P. A., CROWN J., PICCART-GEBHART M., VIALE G., LEO A. D., OLIVIER M.

J CLIN ONCOL

30

18,372

GADDUCCI A., COSIO S., LANDONI F., MAGGINO T., ZOLA P., FUSO L., SARTORI E.

Analysis of Treatment Failures and Survival of Patients With Uterine Papillary Serous Carcinoma: A Cooperation Task Force (CTF) Study.

INT J GYNECOL CANCER

22

1355 1360

1,646

GALETTA D., SOLLI P., BORRI A., GASPARRI R., PETRELLA F., PARDOLESI A., SPAGGIARI L.

Bronchovascular Reconstruction for Lung Cancer: Does Induction Chemotherapy Influence the Outcomes?

ANN THORAC SURG

94

907 - 913

3,741

GALETTA D., SOLLI P., BORRI A., PETRELLA F., GASPARRI R., BRAMBILLA D., SPAGGIARI L.

Bilobectomy for Lung Cancer: Analysis of Indications, Postoperative Results, and Long-Term Outcomes.

ANN THORAC SURG

93

251 - 258

3,741

GALIMBERTI V. E., BOTTERI E., CHIFU C., GENTILINI O. D., LUINI A., INTRA M., BARATELLA P., SARGENTI M., ZURRIDA S., VERONESI P., ROTMENSZ N., VIALE G., SONZOGNI A. M., COLLEONI M. A., VERONESI U.

Can we avoid axillary dissection in the micrometastatic sentinel node in breast cancer?

BREAST CANCER RES TR

131

819 - 825

4,431

GANDINI S., NEGRI E., BOFFETTA P., LA VECCHIA C., BOYLE P.

Mouthwash and Oral Cancer Risk Quantitative Meta-analysis of Epidemiologic Studies.

ANN AGR ENV MED

2

173 - 180

2,311

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

130

665 - 670

5,444

GARUSI C., DI CAGNO A.

A seno nudo. 14 donne raccontano come la nuova chirurgia plastio-ricostruttiva ha restituito loro il sorriso.

Tecniche Nuove Ed. Garusi C., Di Cagno A.

3624 3630

2,606

21

3926 3939

7,636

IEEE T BIOMED CIRC S

59

2191 2199

2,032

Prognostic and predictive value of TP53 mutations in node-positive breast cancer patients treated with anthracycline- or anthracycline/taxane-based adjuvant therapy: results from the BIG 02-98 phase III trial.

BREAST CANCER RES

14

R70 - R70

5,245

FERRARI D., CODECA' C., BERTUZZI C., BROGGIO F., CREPALDI F., LUCIANI A., FLORIANI I, ANSARIN M., CHIESA F., ALTERIO D., FOA P.

3D dosimetry in patients with early breast cancer undergoing Intraoperative Avidination for Radionuclide Therapy (IART) combined with external beam radiation therapy.

BMC CANCER

12

208

3,011

12

208

3,011

3771 3776

4,166

BMC CANCER

Repeating Conservative Surgery after Ipsilateral Breast Tumor Reappearance: Criteria for Selecting the Best Candidates.

19

Role of plasma EBV DNA levels in predicting recurrence of nasopharyngeal carcinoma in a Western population.

GENTILINI O. D., BOTTERI E., VERONESI P., SANGALLI C. A., DEL CASTILLO A. P., BALLARDINI B., GALIMBERTI V. E., RIETJENS M., COLLEONI M. A., LUINI A., VERONESI U.

ANN SURG ONCOL

FERRARI D., CODECA' C., BERTUZZI C., BROGGIO F., CREPALDI F., LUCIANI A., FLORIANI I., ANSARIN M., CHIESA F. G., ALTERIO D., FOA P. FERRARI M. E., CREMONESI M., DI DIA G. A., BOTTA F., DE CICCO C., SARNELLI A., PEDICINI P., CALABRESE M., ORECCHIA R., PEDROLI G., PAGANELLI G.

3D dosimetry in patients with early breast cancer undergoing Intraoperative Avidination for Radionuclide Therapy (IART(®)) combined with external beam radiation therapy.

EUR J NUCL MED MOL I

39

1702 - 1711

4,991

GENTILINI O. D., VERONESI U.

Abandoning sentinel lymph node biopsy in early breast cancer: A new trial in progress at the European Institute of Oncology of Milan (SOUND: Sentinel node vs Observation after axillary UltraSouND).

BREAST

21

678 - 681

2,491

FERRUCCI P. F., TOSTI G., DI PIETRO A., PASSONI C., PARI C., TEDESCHI I., CATALDO F., MARTINOLI C., TESTORI A.

Newly Identified Tumor Antigens as Promising Cancer Vaccine Targets for Malignant Melanoma treatment.

CURR TOP MED CHEM

12

11'-31

4,174

GERMAIN P. L

Cancer cells and adaptive explanations

BIOL PHILOS

27

785 - 810

1,203

7

120 - 120

2,321

Chemo-conization for early staged cervical cancer

Textbook of gynaecological oncology - Editors: Ayhan A., Reed N., Gultekin M., Dursun P.

88

530 - 536

0

Implementation of HybridArc treatment technique in preoperative radiotherapy of rectal cancer: dose patterns in target lesions and organs at risk as compared to helical Tomotherapy and RapidArc.

RADIAT ONCOL

FESTI A., LANDONI F.

GEVAERT T., ENGELS B., GARIBALDI C., VERELLEN D., DECONINCK P., DUCHATEAU M., REYNDERS T., TOURNEL K., DE RIDDER M.

FIELD J. K., SMITH R. A., ABERLE D. R., OUDKERK M., BALDWIN D. R., YANKELEVITZ D., PEDERSEN J. H., SWANSON S. J., TRAVIS W. D., WISBUBA I. I., NOGUCHI M., MULSHINE J. L., IASLC CT SCREENING WORKSHOP 2011 PARTICIPANTS, BARBERIS M., VERONESI G.

International Association for the Study of Lung Cancer Computed Tomography Screening Workshop 2011 report.

J THORAC ONCOL

7

10-19'

3,661

GHIORZO P., BONELLI L., PASTORINO L., BRUNO W., BARILE M., ANDREOTTI V., NASTI S., BATTISTUZZI L., GROSSO M., BIANCHI-SCARRA G., QUEIROLO P.

MC1R variation and melanoma risk in relation to host/clinical and environmental factors in CDKN2A positive and negative melanoma patients.

EXP DERMATOL

21

718 - 720

3,543

11

41 - 47

1,302

Dose prescription in carbon ion radiotherapy: a planning study to compare NIRS and LEM approaches with a clinicallyoriented strategy.

PHYS MED BIOL

57

7543 7554

2,829

Contribution of germline mutations in the BRCA and PALB2 genes to pancreatic cancer in Italy.

FAM CANCER

FOSSATI P., MOLINELLI S., MATSUFUJI N., CIOCCA M., MIRANDOLA A., MAIRANI A., MIZOE J., HASEGAWA A., IMAI R., KAMADA T., ORECCHIA R., TSUJII H. FRAGA GUEDES C., GOBBI H., MASTROPASQUA M. G, BOTTERI E., LUINI A., VIALE G.

Primary and secondary angiosarcomas of the breast: a single institution experience.

BREAST CANCER RES TR

132

1081 1088

4,431

GHIORZO P., PENSOTTI V., FORNARINI G., SCIALLERO S., BATTISTUZZI L., BELLI F., BONELLI L., BORGONOVO G., BRUNO W., GOZZA A., GARGIULO S., MASTRACCI L., NASTI S., PALMIERI G., PAPADIA F., PASTORINO L., RUSSO A., SAVARINO V., VARESCO L., BERNARD L., BIANCHI SCARRA G., GENOA PANCREATIC CANCER STUDY GROUP GHOUSSAINI M., FLETCHER O., et al.

NAT GENET

44

312 - 318

35,532

FUMAGALLI C., PRUNERI G., POSSANZINI P., MANZOTTI M., BARILE M., FEROCE I., COLLEONI M. A., BONANNI B., MAISONNEUVE P., RADICE P., VIALE G., BARBERIS M.

Methylation of O (6)-methylguanine-DNA methyltransferase (MGMT) promoter gene in triple-negative breast cancer patients.

BREAST CANCER RES TR

131 - 137

4,431

Genome-wide association analysis identifies three new breast cancer susceptibility loci.

GIBELLI B., ZAMPERINI P., PROH M., GIUGLIANO G.

Management and follow-up of thyroid cancer in pregnant women.

ACTA OTORHINOLARYNGO

31

358 - 365

0,863

FUMAGALLI M., ROSSIELLO F., CLERICI M., BAROZZI S., CITTARO D., KAPLUNOV J. M., BUCCI G., DOBREVA M., MATTI V., BEAUSEJOUR C. M., HERBIG U., LONGHESE M. P., D'ADDA DI FAGAGNA F.

Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation.

NAT CELL BIOL

GIGLI F., GARDELLINI A., BERTAZZONI P., MARTINELLI G.

Re: secondary haematological malignancies after radioimmunotherapy.

ANN HEMATOL

91

969 969

2,615

GIGLI F., GARDELLINI A., BERTAZZONI P., MARTINELLI G.

Secondary haematological malignancies after radioimmunotherapy

ANN HEMATOL

91

969

2,615

IEO — Scientific Report 2012 — Ongoing Research 2013

81

3967 3975

134

14

355 - 365

19,488

Publications

EWERTZ M., GRAY K. P., REGAN M. M., EJLERTSEN B., PRICE K. N., THURLIMANN B., BONNEFOI H., FORBES J. F., PARIDAENS R. J., RABAGLIO M., GELBER R. D., COLLEONI M., LANG I., SMITH I. E., COATES A. S., GOLDHIRSCH A., MOURIDSEN H. T.

0

IEO — Scientific Report 2012 — Ongoing Research 2013

275


Preoperative FDG PET/CT in breast cancer patients: where are we going?

EUR J NUCL MED MOL I

39

1667 1669

4,991

GIORDANO A., FERRARI G., RADICE D., RANDI G., BISANTI L., SOLARI A., ON BEHALF OF THE POSMOS STUDY

Health-related quality of life and depressive symptoms in significant others of people with multiple sclerosis: a community study.

EUR J NEUROL

19

847 - 854

GIORGI ROSSI P., SIDERI M., CAROZZI F. M., VOCATURO A., BUONAGURO F. M., TORNESELLO M. L., BURRONI E., MARIANI L., BOVERI S., ZAFFINA L. M., CHINI F., THE HPV PREVALENCE ITALIAN WORKING GROUP

HPV type distribution in invasive cervical cancers in Italy: pooled analysis of three large studies.

INF AGENTS CAN

GIORGI ROSSO P., RONCO G., CONFORTINI M., MACCALLINI V., NALDONI C., SEGNAN N., SIDERI M., ZAPPA M., ZORZI M., CALVIA M., GIORGI ROSSO P.

[Health technology assessment report. Use of liquid-based cytology for cervical cancer precursors screening].

EPIDEMIOL PREV

GIORGIO M., BERRY A., BERNIAKOVICH I., POLETAEVA I., TRINEI M., STENDARDO M., HAGOPIAN K., RAMSEY J. J., CORTOPASSI G., MIGLIACCIO E., NOTZLI S., AMREIN I., LIPP H. P, CIRULLI F., PELICCI P. G.

The p66(Shc) knocked out mice are short lived under natural condition.

AGING CELL

GIOVANNETTI E., TOFFALORIO F., DE PAS T., PETERS G. J.

Pharmacogenetics of conventional chemotherapy in non-small-cell lung cancer: a changing landscape?

PHARMACOGENOMICS

13

1073 1086

3,974

GJERDE J., GANDINI S., GUERRIERI GONZAGA A., HAUGAN MOI L. L, ARISTARCO V., MELLGREN G., DE CENSI A., LIEN E. A

Tissue distribution of 4-hydroxy-N-desmethyltamoxifen and tamoxifen-N-oxide.

BREAST CANCER RES TR

134

693 - 700

4,431

GODDI A., BONARDI M., ALESSI S.

Breast elastography: A literature review.

J ULTRAS MED

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1,245

GOLDHIRSCH A., GELBER R. D.

Breast cancer: Anastrozole and fulvestrant-combination to unlock efficacy.

NAT REV CLIN ONCOL

9

556 - 557

11,963

GORI S., GRECO M. T, CATANIA C. M., COLOMBO C., APOLONE G., ZAGONEL V., AIOM GROUP FOR THE INFORMED CONSENT IN MEDICAL ONCOLOGY

A new informed consent form model for cancer patients: Preliminary results of a prospective study by the Italian Association of Medical Oncology (AIOM).

PATIENT EDUC COUNS

87

243 - 249

2,305

GRANA C. M., CHINOL M., DE CICCO C., BARTOLOMEI M., CREMONESI M., BODEI L., ROCCA P. A., PACIFICI M., TIBERINI S., BAIO S. M., BROGGI G., SEVERI S., PAGANELLI G.

Eleven-Year Experience with the AvidinBiotin Pretargeting System in Glioblastoma: Toxicity, Efficacy and Survival

O NUCL MED J

4

14 - 20

0

GRUSZKA A. M, ALCALAY M.

Clinical and Biological Relevance of Gene Expression Profiling in Acute Myeloid Leukemia

Myeloid Leukemia – Clinical Diagnosis and Treatment

10

197 - 210

0

GRUSZKA A. M., MARTINELLI C., SPARACIO E., PELICCI P. G., DE MARCO A.

The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients.

LEUKEMIA

26

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9,561

GUIU S., MICHIELS S., ANDRE F., CORTES J., DENKERT C., DI LEO A., HENNESSY B. T., SORLIE T., SOTIRIOU C., TURNER N., VAN DE VIJVER M., VIALE G., LOI S., REIS-FILHO J. S.

Molecular subclasses of breast cancer: how do we define them? The IMPAKT 2012 Working Group Statement.

ANN ONCOL

23

HEIN R., MARANIAN M., et al.

Correction: Comparison of 6q25 Breast Cancer Hits from Asian and European Genome Wide Association Studies in the Breast Cancer Association Consortium (BCAC).

PLOS ONE

7

Rare variants in XRCC2 as breast cancer susceptibility alleles.

J MED GENET

HILBERS F. S., WIJNEN J. T., HOOGERBRUGGE N., OOSTERWIJK J. C., COLLEE M. J., PETERLONGO P., RADICE P., MANOUKIAN S., FEROCE I., CAPRA F., COUCH F. J., WANG X., GUIDUGLI L., OFFIT K., SHAH S., CAMPBELL I. G., THOMPSON E. R., JAMES P. A., TRAINER A. H., GRACIA J., BENITEZ J., VAN ASPEREN C. J., DEVILEE P.

276

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7

36

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2997 3006

Prognosis of medullary breast cancer: analysis of 13 International Breast Cancer Study Group (IBCSG) trials.

ANN ONCOL

23

2843 2851

6,425

3,692

HUOBER J., GELBER S., GOLDHIRSCH A., COATES A. S., VIALE G., OHLSCHLEGEL C., PRICE K. N., GELBER R. D., REGAN M. M., THURLIMANN B.

HER2-negative (1+) breast cancer with unfavorable prognostic features: to FISH or not to FISH?

ANN ONCOL

23

1371 1372

6,425

0

IORFIDA M., DELLAPASQUA S., BAGNARDI V., CARDILLO A., ROTMENSZ N., MASTROPASQUA M. G., BOTTIGLIERI L., GOLDHIRSCH A., VIALE G., COLLEONI M. A.

Invasive lobular breast cancer: subtypes and outcome.

BREAST CANCER RES TR

133

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4,431

0,651

IORFIDA M., MAIORANO E., ORVIETO E., MAISONNEUVE P., BOTTIGLIERI L., ROTMENSZ N., MONTAGNA E., DELLAPASQUA S., VERONESI P., GALIMBERTI V. E., LUINI A., GOLDHIRSCH A., COLLEONI M. A., VIALE G. JERECZEK B. A., BELTRAMO G., FARISELLI L., FODOR C., SANTORO L., VAVASSORI A., ZERINI D., GHERARDI F., ASCIONE C., BOSSI ZANETTI I., MAURO R., BREGANTIN A., BIANCHI L. C., DE COBELLI O., ORECCHIA R.

Robotic Image-Guided Stereotactic Radiotherapy, for Isolated Recurrent Primary, Lymph Node or Metastatic Prostate Cancer.

INT J RADIAT ONCOL

82

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4,105

JERECZEK B. A., KAANDERS J. H., DUBRAY B., COTTRILL C., LEER J. W., ON BEHALF OF THE FACULTY OF THE TEACHING COURSE ON EVIDENCE-BASED RADIATION ONCOLOGY OF THE EUROPEAN SOCIETY OF THERAPEUTIC RADIOLOGY AND ONCOLOGY

Focal HIFU for prostate cancer.

LANCET ONCOL

13

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22,589

JOHNSTON K., LEVY A. R, LORIGAN P., MAIO M., LEBBE C., MIDDLETON M., TESTORI A., BEDANE C., KONTO C., DUEYMES A., SBARIGIA U., VAN BAARDEWIJK M.

Economic impact of healthcare resource utilization patterns among patients diagnosed with advanced melanoma in the United Kingdom, Italy, and France: Results from a retrospective, longitudinal survey (MELODY study)

EUR J CANCER

48

2175 2182

5,536

KALOGERA E., DOWDY S. C., MARIANI A., ALETTI G., BAKKUM-GAMEZ J. N., CLIBY W. A.

Utility of closed suction pelvic drains at time of large bowel resection for ovarian cancer.

GYNECOL ONCOL

126

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3,888

KARLSSON P., COLE B. F., CHUA B. H., PRICE K. N., LINDTNER J., COLLINS J. P., KOVACS A., THURLIMANN B., CRIVELLARI D., CASTIGLIONEGERTSCH M., FORBES J. F., GELBER R. D., GOLDHIRSCH A., GRUBER G., FOR THE INTERNATIONAL BREAST CANCER STUDY GROUP

Patterns and risk factors for locoregional failures after mastectomy for breast cancer: an International Breast Cancer Study Group report.

ANN ONCOL

23

2852 2858

6,425

KESSEL K. A, BOUGATF N., BOHN C., HABERMEHL D., OETZEL D., BENDL R., ENGELMANN U., ORECCHIA R., FOSSATI P., POTTER R., DOSANJH M., DEBUS J., COMBS S. E

Connection of European particle therapy centers and generation of a common particle database system within the European ULICE-framework.

RADIAT ONCOL

7

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KLAENHAMMER T. R., KLEEREBEZEM M., KOPP M. V., RESCIGNO M.

The impact of probiotics and prebiotics on the immune system.

NAT REV IMMUNOL

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AUTOPHAGY

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KNEUBIL M. C, LOHSIRIWAT V., CURIGLIANO G., BROLLO J., BOTTERI E., ROTMENSZ N., MARTELLA S., MASTROPASQUA M. G., IERA M., COELHO M. B, INTRA M., ORECCHIA R., REY P., RIETJENS M., PETIT J. Y.

Risk of Locoregional Recurrence in Patients With False-Negative Frozen Section or Close Margins of Retroareolar Specimen in Nipple-Sparing Mastectomy

ANN SURG ONCOL

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4117 4123

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KRENGLI M., TERRONE C., JERECZEK B. A., BELDI' D., ORECCHIA R.

May intra-operative radiotherapy have a role in the treatment of prostate cancer?

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Structural analysis reveals features of the spindle checkpoint kinase Bub1kinetochore subunit Knl1 interaction.

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KWIATKOWSKI N., DENG X., WANG J., TAN L., VILLA F., SANTAGUIDA S., HUANG H. C., MITCHISON T., MUSACCHIO A., GRAY N.

Selective aurora kinase inhibitors identified using a taxol-induced checkpoint sensitivity screen.

ACS CHEM BIOL

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6,446

LAMBRECHTS D., TRUONG T., et al.

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5,686

6,265

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GILARDI L., DE CICCO C., PAGANELLI G.

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LOCOCO F., CESARIO A., CARDILLO G., FILOSSO P., GALETTA D., CARBONE L., OLIARO A., SPAGGIARI L., CUSUMANO G., MARGARITORA S., GRAZIANO P., GRANONE P.

Malignant Solitary Fibrous Tumors of the Pleura: Retrospective Review of a Multicenter Series.

J THORAC ONCOL

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1698 1706

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LOHSIRIWAT V., MARTELLA S., RIETJENS M., BOTTERI E., ROTMENSZ N., MASTROPASQUA M. G., GARUSI C., DE LORENZI F., MANCONI A., SOMMARIO M., BARBIERI B., KNEUBIL M. C., MINOTTI I., PETIT J. Y.

Paget's Disease as a Local Recurrence after Nipple-Sparing Mastectomy: Clinical Presentation, Treatment, Outcome, and Risk Factor Analysis.

ANN SURG ONCOL

19

1850 1855

4,166

LANCONELLI N., PACILIO M., LO MEO S., BOTTA F., DI DIA G. A., TORREAS AROCHE L. A, COCA PEREZ M. A, CREMONESI M.

A free database of radionuclide voxel S values for the dosimetry of nonuniform activity distributions

PHYS MED BIOL

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LANDONI F., MANEO A., ZAPARDIEL I., ZANAGNOLO V., MANGIONI C.

Class I versus class III radical hysterectomy in stage IB1-IIA cervical cancer. A prospective randomized study.

EUR J SURG ONCOL

38

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0

LONSDALE M. N., PEDROLI G.

Technological innovations in nuclear medicine imaging.

Q J NUCL MED MOL IM

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2,256

LOPERA SIERRA M., CREMONESI M., GRANA C. M

Tratamiento del linfoma en medicina nuclear.

Medicina Nuclear en La Practica Clinica. Ed A. Soriano, J. Martín Comín, A. García. 2012.

LANKISCH P. G., WEBER-DANY B., MAISONNEUVE P., LOWENFELS A. B.

Pancreatic pseudocysts: prognostic factors for their development and their spontaneous resolution in the setting of acute pancreatitis.

PANCREATOLOGY

Alcohol consumption and pancreatic cancer: a pooled analysis in the International Pancreatic Cancer Case-Control Consortium (PanC4).

ANN ONCOL

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6,425

0,446

LUCENTEFORTE E., LA VECCHIA C., SILVERMAN D., PETERSEN G. M., BRACCI P. M., JI B. T., BOSETTI C., LI D., GALLINGER S., MILLER A. B., BUENO DE MESQUITA H. B., TALAMINI R., POLESEL J., GHADIRIAN P., BAGHURST P. A., ZATONSKI W., FONTHAM E., BAMLET W. R., HOLLY E. A., GAO Y. T., NEGRI E., HASSAN M., COTTERCHIO M., SU J., MAISONNEUVE P., BOFFETTA P., DUELL E. J.

LAZZERONI M., GUERRIERI GONZAGA A., SERRANO D., CAZZANIGA M., MORA S., CASADIO C., JEMOS C., PIZZAMIGLIO M., CORTESI L., RADICE D., BONANNI B.

Breast ductal lavage for biomarker assessment in high risk women: rationale, design and methodology of a randomized phase II clinical trial with nimesulide, simvastatin and placebo.

BMC CANCER

12

575 - 575

3,011

LAZZERONI M., SERRANO D.

Potential Use of Vaccines in the Primary Prevention of Breast Cancer in High-Risk Patients

BREAST CARE

7

281 - 287

LAZZERO