Final program Auspices
Under the auspices of:
Supported by:
1 Committees
SCIENTIFIC COORDINATOR Gabriella Sozzi (IRCCS National Cancer Institute, Milan, Italy)
LOCAL SCIENTIFIC AND ORGANIZING COMMITTEE Giovanni Apolone (Scientific Director, IRCCS National Cancer Institute, Milan, Italy) Andrea Anichini (IRCCS National Cancer Institute, Milan, Italy) Mario Colombo (IRCCS National Cancer Institute, Milan, Italy) Filippo De Braud (IRCCS National Cancer Institute, Milan, Italy) Massimo Di Nicola (IRCCS National Cancer Institute, Milan, Italy) Andrea Ferrari (IRCCS National Cancer Institute, Milan, Italy) Marina Garassino (IRCCS National Cancer Institute, Milan, Italy) Marilena Iorio (IRCCS National Cancer Institute, Milan, Italy) Delia Mezzanzanica (IRCCS National Cancer Institute, Milan, Italy) Ugo Pastorino (IRCCS National Cancer Institute, Milan, Italy) Filippo Pietrantonio (IRCCS National Cancer Institute, Milan, Italy) Luca Roz (IRCCS National Cancer Institute, Milan, Italy) Elda Tagliabue (IRCCS National Cancer Institute, Milan, Italy)
SIC SCIENTIFIC BOARD President Gabriella Sozzi (IRCCS National Cancer Institute, Milan, Italy)
President Elect Nicola Normanno (National Cancer Institute “G. Pascale”, Naples, Italy)
Board Paola Chiarugi (University of Florence, Italy) Amedeo Columbano (University of Cagliari, Italy) Rita Falcioni (Regina Elena National Cancer Institute, Rome, Italy) Davide Melisi (University of Verona, Italy) Katia Scotlandi (IRCCS Orthopaedic Rizzoli Institute, Bologna, Italy) Elda Tagliabue (IRCCS National Cancer Institute, Milan, Italy) SIC Secretariat Giulia Taraboletti (“Mario Negri” Institute for Pharmacological Research, Bergamo, Italy) Società Italiana di Cancerologia Via G. Venezian, 1 20133 Milano MI Members under 35 E-mail: [email protected] Antonino Bruno (MultiMedica, Milan, Italy) www.cancerologia.it Marco Macagno (University of Turin, Italy) Organizing Secretariat
Past President Silvia Giordano (University of Turin, Italy) Via Sassonia, 30 47922 Rimini RN E-mail: [email protected] www.adriacongrex.it
1 Faculty
Adriana Albini MultiMedica, Milan, Italy Andrea Anichini IRCCS National Cancer Institute, Milan, Italy Alberto Bardelli University of Turin, Italy Tommaso Balestra IRCCS Orthopaedic Rizzoli Institute, Bologna, Italy Anne-Lise Børresen-Dale University Hospital The Norwegian, Oslo, Norway Gerard Brady Cancer Research UK Manchester Institute, Manchester, United Kingdom Antonino Bruno MultiMedica, Milan, Italy Paola Chiarugi University of Florence, Italy Mario Paolo Colombo IRCCS National Cancer Institute, Milan, Italy Amedeo Columbano University of Cagliari, Italy Filippo De Braud IRCCS National Cancer Institute, Milan, Italy Giannino Del Sal University of Trieste, Italy Serena Di Cosimo IRCCS National Cancer Institute, Milan, Italy Rodrigo Dienstmann Vall d’Hebron Institute of Oncology, Barcelona, Spain Massimo Di Nicola IRCCS National Cancer Institute, Milan, Italy Rita Falcioni Regina Elena National Cancer Institute, Rome, Italy Federica Fusella University of Turin, Italy Marina Garassino IRCCS National Cancer Institute, Milan, Italy Cyrus Ghajar Fred Hutchinson Cancer Research Center, Seattle WA, USA Luca Gianni IRCCS San Raffaele Hospital, Milan, Italy Silvia Giordano University of Turin, Italy Douglas Hanahan EPFL, École Polytechnique Fédérale de Lausanne, Switzerland Marilena Valeria Iorio IRCCS National Cancer Institute, Milan, Italy Franco Locatelli Bambino Gesù Hospital, Rome, Italy Domenica Lorusso IRCCS National Cancer Institute, Milan, Italy Marco Macagno Candiolo Cancer Institute, Turin, Italy Alberto Mantovani University of Milan, Italy Enzo Medico University of Turin Ignacio Melero Bermejo University of Navarra, Pamplona, Spain Davide Melisi University of Verona, Italy Delia Mezzanzanica IRCCS National Cancer Institute, Milan, Italy Andrea Necchi IRCCS National Cancer Institute Milan, Italy Paola Nisticò Regina Elena National Cancer Institute, Rome, Italy Nicola Normanno National Cancer Institute “G. Pascale”, Naples, Italy Ugo Pastorino IRCCS National Cancer Institute, Milan, Italy Solange Peters Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Stefan Pfister German Cancer Research Center, Heidelberg, Germany Filippo Pietrantonio IRCCS National Cancer Institute, Milan, Italy Paolo Porporato University of Turin, Italy Maria Paula Roberti Institute Gustave Roussy, Villejuif, France Maurizio Scaltriti Memorial Sloan Kettering Cancer Center, New York, USA Katia Scotlandi IRCCS Orthopaedic Rizzoli Institute, Bologna, Italy Gabriella Sozzi IRCCS National Cancer Institute, Milan, Italy Andrea Speciale University of Genoa, Italy Lorenzo Stramucci Regina Elena National Cancer Institute, Rome, Italy Giulia Taraboletti “Mario Negri” Institute for Pharmacological Research, Bergamo, Italy Alice Turdo University of Palermo, Italy
2 3 Program at a Glance
Wednesday, September 19th Plenary Room
10.00 - 12.30 Annual Meeting of SIC Young Investigators 13.30 Registration 14.00 - 15.00 Opening Address 15.00 - 16.00 Opening Lecture 16.00 - 16.30 Coffee break 16.30 - 18.30 LUNG CANCER 19.00 - 20.30 Welcome Cocktail
Thursday, September 20th Plenary Room
08.30 - 10.00 BREAST CANCER 10.00 - 10.30 Coffee break 10.30 - 12.00 IMMUNOTHERAPY: PRECLINICAL & CLINICAL 12.00 - 13.00 Poster viewing with authors 13.00 - 14.00 Lunch 14.00 - 17.00 POSTER DISCUSSION (4 PARALLEL SESSIONS) 14.00 - 15.30 Poster Discussion 1A (Plenary Room) 14.00 - 15.30 Poster Discussion 1B (Workshop Room) 15.30 - 17.00 Poster Discussion 2A (Plenary Room) 15.30 - 17.00 Poster Discussion 2B (Workshop Room) 17.00 - 17.30 Coffee break 17.30 - 19.00 CHILDHOOD TUMORS
Friday, September 21st Plenary Room
08.30 - 10.00 COLON CANCER 10.00 - 10.30 Coffee break 10.30 - 12.00 MICROBIOME, METABOLISM & CANCER 12.00 - 13.00 Poster viewing with authors 13.00 - 14.00 Lunch 14.00 - 16.00 SIC meets Pharma Industry 16.00 - 16.30 Coffee break 16.30 - 17.30 Award ceremony 17.30 - 19.00 BIOMARKERS, NEW MOLECULAR TARGETS & TARGETED THERAPIES 19.00 - 20.00 SIC General Assembly 20.30 Social Event
Saturday, September 22nd Plenary Room
08.45 - 10.15 TUMOR MICROENVIRONMENT & METASTASIS 10.15 - 11. 15 “Giorgio Prodi” lecture 11. 15 - 11.45 Coffee break 11.45 - 12.30 Award ceremony 12.30 - 13.30 Closing lecture 13.30 Closing remarks
2 3 4 5 Scientific Program
Wednesday, September 19th
Plenary Room
10.00 - 12.30 Annual Meeting of SIC Young Investigators
13.30 Registration
Plenary Room
14.00 - 15.00 Opening Address Greetings from the Authority
15.00 - 16.00 Opening Lecture Hallmarks of Cancer: Applications for Cancer Medicine? Douglas Hanahan, Lausanne (Switzerland)
16.00 - 16.30 Coffee Break
16.30 - 18.30 Lung Cancer Chairs: Filippo De Braud, Milan (Italy) - Gabriella Sozzi, Milan (Italy)
CTC-Single cell analysis provides informative genetic profiles for lung cancer Gerard Brady, Manchester (UK)
E13 Chemotherapy can promote metastasis outgrowth through recruitmente at distant sites of both metastasis initiating cells and myeloid-derived suppressor cells via the CXCL12/CXCR4 axis G. Bertolini (IRCCS National Cancer Istitute, Milan, Italy), V. Cancila, M. Tortoreto, G. Centonze, C. D’ Alterio, S. Scala, C. Tripodo, G. Sozzi, L.Roz
Personalized prevention and screening of lung cancer Ugo Pastorino, Milan (Italy)
C18 Investigating an aggressive molecular subtype of Stage I lung adenocarcinoma: evidences for the acquirement of a stem-like phenotype and immunoevasion V. Melocchi, E. Dama, R. Cuttano, T. Colangelo, F. Bianchi (IRCCS-Casa Sollievo della Sofferenza - San Giovanni Rotondo, Italy)
Defining and optimizing the use of immunotherapy in NSCLC Solange Peters, Lausanne (Switzerland)
19.00 Welcome Cocktail
4 5 Thursday, September 20th
Plenary Room
8.30 - 10.00 Breast Cancer Chairs: Massimo Di Nicola, Milan (Italy) - Serena Di Cosimo, Milan (Italy)
Multilevel molecular analyses in breast cancer: gauging inter- and intra-tumor heterogeneity and elucidating its role in treatment response Anne-Lise Børresen-Dale, Oslo (Norway)
B5 Insight into genetic susceptibility to BRCA-negative male breast cancer by multigene panel testing: results from a multicenter study in Italy V. Zelli (Sapienza University of Rome, Italy), P. Rizzolo, V. Silvestri, V. Valentini, A. Spinelli, M.G. Tibiletti, A. Russo,L. Varesco, G. Giannini, D. Calistri, L. Cortesi, A. Viel, M. Montagna, P. Peterlongo, P. Radice, D. Palli, L. Ottini
Translational research and neoadjuvant therapy of breast cancer: lessons learned and new perspectives Luca Gianni, Milan (Italy)
P1 Bone marrow hematopoietic adaptation as a sensor of early, pre-invasive, mammary carcinoma C. Chiodoni (IRCCS National Cancer Istitute, Milan, Italy), V. Cancila, T.A. Renzi, A.M. Tomirotti, M. Perrone, M. Dugo, M. Milani, C. Tripodo, M.P. Colombo
10.00 - 10.30 Coffee break
10.30 - 12.00 Immunotherapy: preclinical & clinical Chairs: Mario Paolo Colombo, Milan (Italy) - Marina Garassino, Milan (Italy)
Innate Immunity, Inflammation and cancer: a double edged sword Alberto Mantovani, Milan (Italy)
U5 Galectin-3 contributes to prostate cancer malignancy and immunosuppression in the early phases of disease development and metastasis S. Caputo (San Raffaele Scientific Institute, Milan, Italy) M. Grioni, C. Brambillasca, E. Jachetti, M. Freschi, C. Doglioni, R. Galli, M. Bellone
Two pieces in the immunotherapy puzzle CD137 and IL-8 Ignacio Melero Bermejo, Pamplona (Spain)
U10 Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-Small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade M. Moro (IRCCS National Cancer Institute, Milan, Italy), G. Lo Russo, M. Sommariva, V. Cancila, M. Boeri, G. Centonze, M. Ganzinelli, M. Milione, G. Pruneri, C. Proto, S. Sangaletti, P. Gasparini, V. Torri, A. Anichini, L. Rivoltini, C. Tripodo, M.P. Colombo, A. Balsari, G. Sozzi, M.C. Garassino
12.00 - 13.00 Poster viewing with authors Poster Area
13.00 - 14.00 Lunch
6 7 Thursday, September 20th
14.00-17.00 POSTER DISCUSSION (4 PARALLEL SESSIONS)
Plenary Room
14.00-15.30 Poster Discussion 1A
Biomarkers and Therapy Chairs: Marilena Iorio, Milan (Italy) - Alice Turdo, Palermo (Italy)
E2 CD73: a new driver and a therapeutic target in ovarian cancer stem cells M. Lupia, K. Sachsenmeier, N. Colombo, F. Bianchi, U. Cavallaro (European Institute of Oncology, Milan, Italy)
F9 Identification of a novel miR-200b-c/ETAR/ZEB1 network involved in ovarian cancer progression R. Sestito (IRCCS Regina Elena National Cancer Institute, Rome, Italy), L. Rosanò, R. Cianfrocca, P. Tocci, V. Di Castro, A. Sacconi, G. Blandino, A. Bagnato
F11 Plasma membrane-associated platforms to control tumor cell motility and invasion K. Sala (Università Vita-Salute San Raffaele, Milan, Italy), A. Corbetta, D. Tonoli, C. Minici, M. Degano, E. Cammarota, D. Mazza, I. De Curtis
K2 Detection and monitoring of EWS-FLI1 fusion transcripts by liquid biopsy in ewing sarcoma patients M. Allegretti, B. Casini (IRCSS Regina Elena National Cancer Institute, Rome, Italy) C. Mandoj, S. Benini, L. Alberti, M. Novello, L. Conti, R. Covello, E. Pescarmona, G.M. Milano, A. Annovazzi, V. Anelli, V. Ferraresi, R. Biagini, P. Giacomini
L2 Coated Cationic Liposomes entrapping mir-660 inhibits tumor growth in Patients Derived Xenografts lung cancer models O. Fortunato (IRCCS National Cancer Institute, Milan, Italy), M. Moro, D. Di Paolo, M. Milione, G. Centonze, V. Bornaghi, C. Borzi, P. Perri, U. Pastorino, M. Ponzoni G. Sozzi
L11 Targeting the pro-neoplastic role played by the chaperone TRAP1 in tumor cell mitochondria C. Sanchez-Martin, M. Ferraro, E. Moroni, I. Masgras, M. Serra, A. Columbano, G. Colombo, A. Rasola (University of Padua, Italy)ova - Padova)
Q9 Identification of a 3-miRNAs signature in human osteosarcoma discriminating patient prognosis V. Chiadini (IRCCS Orthopaedic Rizzoli Institute, Bologna, Italy) G. Romano, D. Veneziano, G. Nigita, P. Fadda, C.M. Croce, K. Scotlandi
T1 Analysis of cfDNA reveals high levels of tumor heterogeneity in NSCLC R. Pasquale (National Cancer Institute “G. Pascale” Foundation, Mercogliano, Italy) F. Bergantino, F. Fenizia, L. Forgione, C. Roma, A. De Luca, R. Azzaro, V. Iervolino, G. Rocco, A. Morabito, N. Normanno
6 7 Thursday, September 20th
Worshop Room
14.00-15.30 Poster Discussion 1B
Cancer genetics, epigenetics and control of gene expression Chairs: Tommaso Balestra, Bologna (Italy) - Marco Macagno, Turin (Italy)
A2 Genetic inactivation of Nrf2 prevents clonal expansion of initiated cells in a nutritional model of rat hepatocarcinogenesis C. Orrù (University of Cagliari, Italy), M. Szydlowska, K. Taguchi, P. Zavattari, A. Perra, M. Yamamoto, A. Columbano
C12 Whole exome sequencing to discover lung tumor predisposition in women with previous breast cancer S. Coco (“San Martino” Hospital, Genoa, Italy), S. Bonfiglio, D. Cittaro, C. Genova, I. Vanni, S. Boccardo, M. Mora, M.G. Dal Bello, E. Rijavec, A. Alama, A. Truini, V. Rossella, D. Lazarevic, A. Ballestrero, F. Grossi
C20 Radiological and genomic evolution of individual metastasis during HER2 blockade in colorectal cancer G. Crisafulli (University of Turin, Candiolo Cancer Institute, Candiolo, Italy), G. Siravegna, L. Lazzari, A. Sartore-Bianchi, B. Mussolin, A. Cassingena, C. Martino, R.B. Lanman, R.J. Nagy, S. Fairclough, G. Rospo, G. Corti, A. Bartolini, P. Arcella, M. Montone, F. Lodi, A. Lorenzato, A. Vanzati, E. Valtorta, G. Cappello, A. Bertotti, S. Lonardi, V. Zagonel, F. Leone, M. Russo, A. Balsamo, M. Truini, F. Di Nicolantonio, A. Amatu, E. Bonazzina, S. Ghezzi, D. Regge, A. Vanzulli, L. Trusolino, S. Siena, S. Marsoni, A. Bardelli
C21 Identification of actionable cancer genes and treatment options for metastatic ovarian carcinomas using patient derived xenografts (PDXs) and PDX derived tumor cells (PDTCs) C. D’ Ambrosio (Candiolo Cancer Institute FPO-IRCCS, Candiolo, Italy), M. Olivero J. Erriquez, M. Arigoni, S. Capellero, G. Mittica, F. Borella, D. Katsaros, S. Privitera, E. Berrino, T. Venesio, G. Valabrega, R. Calogero, M.F. Di Renzo
C23 Genetic Profiling of Malignant Pleural mesothelioma F. Torricelli (IRCCS Reggio Emilia, Italy), F. Lococo, M. Pagano, R. Gnoni, G. Veronesi, O. Rena, C. Casadio, P. Novellis, S. Piana, B. Donati, T. Di Stefano, C. Pinto, A. Ciarrocchi
H1 MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN amplified neuroblastoma M. Petroni (Italian Technological Institute, Rome, Italy), F. Sardina, P. Infante, A. Bartolazzi, E. Locatelli, F. Fabretti, C. Capalbo, B. Cardinali, A. Coppa, V. Colicchia, M. Sahun Roncero, F. Belardinilli, L. Di Marcotullio, S. Soddu, M. Comes Franchini, E. Petricci, G. Giannini
K1 MYC-related micrornas signatures in non-hodgkin B-cell lymphomas and their relationships with core cellular pathways G. Malpeli (University of Verona, Italy), S. Barbi, G. Tosadori, S. Greco, S. Zupo, S. Pedron, M. Brunelli, A. Bertolaso, M.T. Scupoli, P. Takam Kamga, M. Krampera, C. M. Croce, G.A. Calin, A. Scarpa, A. Zamò
M3 A novel VEGFR2 mutation has oncogenic potential in braf wild-type melanoma E. Grillo (University of the Study of Brescia, Italy), M. Di Somma, M. Corsini, C. Ravelli, L. Zammataro, M. Presta, S. Mitola
8 9 Thursday, September 20th
Plenary Room
15.30-17.00 Poster Discussion 2A
Drug resistance, targeted therapy, cell metabolism Chairs: Andrea Speciale, Genoa (Italy) - Lorenzo Stramucci, Rome (Italy)
D2 Combined positron emission tomography imaging approach for identification of new potential biomarker for treatment response in glioma models S. Valtorta, I. Raccagni, A. Lo Dico, B. Zinnhardt, S. Belloli, C. Martelli, L.S. Politi, S. Todde, C. Monterisi, V. Vaira, A.H. Jacobs, L. Ottobrini, R.M. Moresco (University of Milan Bicocca, Monza, Italy)
F15 Targeting semaphorin 4A-expressing myeloid cells to impair cancer progression F. Maione (Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy) Y. Qiu, E. Garibaldi, A. Miranti, P. Gabriele, S. Brundu, E. Giraudo
G1 Pik3c2g loss promotes pancreatic cancer through Mtor regulation and metabolic rewiring M. Martini (University of Turin, Italy), M.C. De Santis, E. Ratto, L. Gozzelino, A. Derle, P.E. Porporato, E. Hirsch
G8 Metastatic and metabolic features of patient-derived ovarian cancer xenografts C. Ghilardi (IRCCS-Mario Negri Institute for Pharmacological Research, Milan, Italy) A. Decio, L. Brunelli, M. Galimberti, R. Pastorelli, M.R. Bani, R. Giavazzi
I4 A proteomic approach identified HSP90 as a central hub in CDDP-resistant ovarian cancer cells: role of HSP90 inhibitors in overcoming drug resistance R. Lombardi (CROM - National Cancer Institute “G. Pascale”, Mercogliano, Italy) M. Sonego, L. Addi, M.R. Milone, A. Costa, F. Bruzzese, B. Pucci, G. Baldassarre, A. Budillon
I7 Genome scale CRISPR/Cas9 screen identifies Hippo pathway as key determinant for susceptibility to BET inhibitors in lung cancer G. Gobbi, B. Donati, I.F. Do Valle, F. Reggiani, F. Torricelli, G. Castellani, D.C. Ambrosetti, A. Ciarrocchi, V. Sancisi (IRCCS Reggio Emilia, Italy)
I11 MIR146A regulates melanoma sensitivity to BRAF/MEK inhibitors E. Vergani, M. Dugo, C. Gargiuli, M. Cossa, F. Gallino, L. Di Guardo, F. Ferrarini, F. Ferrero, M. Sensi, L. Rivoltini, M. Rodolfo, V. Vallacchi (IRCCS National Cancer Institute, Milan, Italy)
O3 MIR-506-RAD17 regulatory axis is a key node for synthetic lethal approaches targeting DNA repair pathway R. Nicoletti, L. De Cecco, P. Alberti, F. Raspagliesi, E. Cecchin, D. Califano, S. Pignata, M. Bagnoli (IRCCS National Cancer Institute, Milan, Italy), D. Mezzanzanica
8 9 Thursday, September 20th
Workshop Room
15.30-17.00 Poster Discussion 2B
Cancer stem cells, microenvironment, tumor immunology and microbiome Chairs: Antonino Bruno, Milan (Italy) - Federica Fusella, Turin (Italy)
E1 WNT signaling modulates PD-L1 expression in stem cell compartment enhancing immune- evasion of triple negative breast cancer S. Faraci (IRCCS, National Cancer Institute, Milan, Italy), L. Castagnoli, S. Romero Cordoba, V. Cancila, C. Chiodoni, G. Talarico, M. Milani, E. Fasano, T. Volpari, M.V. Iorio, E.Tagliabue, C. Tripodo, S. Sangalett, M. Di Nicola and S.M. Pupa
E4 Chronic myeloid leukemia stem cells are sensitive to the pharmacological inhibition of ERK5 pathway E. Rovida (University of Florence, Italy), I. Tusa, G. Cheloni, A. Gozzini, X. Deng, N.S. Gray, S. Li, P. Dello Sbarba
E5 Comparative transcriptomics of triple negative breast cancer stem cells and differentiated tumor cells identifies Teneurin-4 as a novel CSC-Associated molecule R. Ruiu (University of Turin, Italy), M. Arigoni, F. Riccardo, L. Conti, S. Lanzardo, R.A. Calogero, F. Cavallo, E. Quaglino
J2 Antibiotics or probiotic aerosolization modulate pulmonary microbiota and promotes immunosurveillance against melanoma lung metastases V. Le Noci (IRCCS National Cancer Institute, Milan, Italy), S. Guglielmetti, S. Arioli C. Camisaschi, M. Sommariva, C. Storti, F. Bianchi, T. Triulzi, C. Castelli, A. Balsari, E. Tagliabue, L. Sfondrini
U7 Newly developed CXCR4 antagonist potentiates T-cell trafficking and enhances PD-1 blockade efficacy in poor/high immunoresponsive mouse models C. D’ Alterio (National Cancer Institute ”G. Pascale”, Naples, Italy), M. Napolitano, L. Portella, G. Rea, A. Barbieri, A. Luciano, C. Arra, G. Scognamiglio, F. Tatangelo, A. Anniciello, M. Monaco, L. Gabriele, M. Buoncervello, G. Romagnoli, E. Cavalcanti, G. Botti, S. Scala, C. Ieranò
U3 Maternal immunization against ALK hinders tumor progression in neuroblastoma-prone offspring G. Barutello (University of Turin, Italy) , F. Riccardo, C. Voena, E. Bolli, E. Quaglino, R. Chiarle, F. Cavallo
V6 Breast cancer-adipocytes crosstalk as promoter of cancer progression I. Rybinska (IRCCS National Cancer Institute, Milan, Italy), V. Regondi, C. Ghirelli, E. Tagliabue, T. Triulzi
V16 Notch Signaling Promotes Bone Marrow-Induced Drug Resistance in Multiple Myeloma Through the Regulation of the CXCR4/CXCL12 System M. Colombo (University of the Study of Milan, Italy) M. Mazzola, M. Barbieri, N. Platonova, M.T. Palano, S. Garavelli, D. Giannandrea, E. Lazzari, A. Basile, A. Pistocchi, A. Neri, R. Chiaramonte
17.00 - 17.30 Coffee break
17.30 - 19.00 Childhood Tumors Chairs: Rita Falcioni, Rome (Italy) - Katia Scotlandi, Bologna (Italy)
New concepts for the diagnosis and treatment of childhood malignancies Stefan Pfister, Heidelberg (Germany)
N4 Zyxin in ewing sarcoma: a novel role as mediator between CD99 and EWS-FLI1 T. Balestra (IRCCS Orthopaedic Rizzoli Institute, Bologna, Italy), M.C. Manara, C. Garofalo, S. Santi, K. Scotlandi
Innovative approches of immunotherapy to treat acute leukemias refractory to conventional therapies Franco Locatelli, Rome (Italy)
N6 mRNA expression level of SRCIN1/p140CAP is an independent prognostic marker of outcome in neuroblastoma S. Grasso (University of Turin, Italy), M. Alzona, D. Cangelosi, J. Chapelle, V. Salemme, A. Lamolinara, F. Di Cunto, M. Iezzi, C. Riganti, A. Pezzolo, L. Varesio, A. Eva, E. Turco, P. Defilippi
10 11 Friday, September 21st
Plenary Room
8.30 - 10.00 Colon Cancer Chairs: Davide Melisi, Verona (Italy) - Nicola Normanno, Naples (Italy)
Precision medicine in colorectal cancer Rodrigo Dienstmann, Barcelona (Spain)
Q25 Outcome prediction of metastatic colorectal cancer patients undergoing liver resection by analyzing serum metabolomics S. Costantini, A. Sorice, F. Capone, E. Di Gennaro, C. Vitagliano, A. De Stefano, P. Delrio, F. Bianco, F. Izzo, A. Avallone, A. Budillon (National Cancer Institute “G. Pascale”, Naples, Italy)
Evolving landscape of immunotherapy in colorectal cancer Filippo Pietrantonio, Milan (Italy)
T3 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies G. Corti, M. Russo (Candiolo Cancer Institute - FPO - Candiolo, Italy), S. Lamba, A. Lorenzato, A. Sogari, G. Rospo, M. Montone, L. Lazzari, S. Arena, D. Oddo, M. Linnebacher, A. Sartore-Bianchi, F. Pietrantonio, S. Siena, F. Di Nicolantonio, A. Bardelli
10.00 - 10.30 Coffee break
10.30 - 12.00 Microbiome, metabolism & cancer Chairs: Paola Chiarugi, Florence (Italy) - Silvia Giordano, Turin (Italy)
Ileal Apoptosis and Microbiome Shape the Immunosurveillance and Prognosis of Proximal Colon Cancer Maria Paula Roberti, Villejuif (France)
G7 Stromal contact regulates prostate cancer epigenetic and metabolic reprogramming L. Ippolito (University of Florence, Italy) L. Ferrone, G. Comito, A. Morandi, E. Giannoni, P. Chiarugi
Metabolic control of tumor progression and cachexia Paolo Porporato, Turin (Italy)
J1 Microbiota-driven interleukin-17-producing cells and eosinophils synergize to accelerate multiple myeloma progression A. Calcinotto, A. Brevi (San Raffaele Scientific Institute, Milan, Italy) M. Chesi, R. Ferrarese, L. Garcia Perez, S. Kumar, K.J. Henderson, G. Tonon, M. Tomura, Y. Miwa, R.A. Flavell, S. Huber, F. Canducci, V.S. Rajkumar, P.L. Bergsagel, M. Bellone
12.00 - 13.00 Poster viewing with authors Poster Area
13.00 - 14.00 Lunch
10 11 Friday, September 21st
Plenary Room
SIC meets Pharma Industry
14.00 - 14.30 Use of PI3K (fosfoinositide 3-chinasi) inhibitors in the clinic: ready for prime time? Maurizio Scaltriti, New York (USA)
14.30 - 15.00 Comprehensive Genomic Profile and F One to better select patients for clinical practice and research* Andrea Necchi, Milan (Italy)
15.00 - 15.30 The value of research for AstraZeneca’s Oncology* Constanza Oliveros, Milan (Italy) * Not accredited CME lecture
15.30 - 16.00 The EurOPDX EDIReX project: towards a European Research Infrastructure on patient-derived cancer models* Enzo Medico, Turin (Italy) * Not accredited CME lecture
16.00 - 16.30 Coffee break
16.30 - 17.30 Award Ceremony “Elena Cappannini” award for the best 2017 publication “Pezcoller Foundation” Fellowship awards “Pezcoller Foundation - Ferruccio ed Elena Bernardi” Fellowship 2017-2018 Exploring new therapeutic approaches for pediatric Acute Myeloid Leukemia (AML): preclinical evaluation of a combined treatment with DOT1L and FLT3 inhibitors Annalisa Lonetti (University of Bologna, Italy) “Pezcoller Foundation - Alice Triangi” Fellowship 2017-2018 Prospective validation of a circulating microRNA signature classifier for early lung cancer detection: the BioMILD trial Mattia Boeri (IRCCS National Cancer Institute, Milan, Italy) “Pezcoller Foundation - Ferruccio ed Elena Bernardi” Fellowship award 2019-2020 “Pezcoller Foundation - Alice Triangi” Fellowship award 2019-2020
17.30 - 19.00 Biomarkers, new molecular targets & targeted therapies Chairs: Amedeo Columbano, Cagliari (Italy) - Delia Mezzanzanica, Milan (Italy)
Mutant p53 at the crossroads of mechano-signalling and metabolic pathways in cancer Giannino Del Sal, Trieste (Italy)
C1 Quantative mapping of epigenetic marks by MS-proteomics in breast cancer samples, for patient stratification and discovery of new therapeutic targets T. Bonaldi (European Insitute of Oncology, Milan, Italy), R. Noberini
Role of parp inhibitor in the ovarian cancer treatment Domenica Lorusso, Milan (Italy)
L21 Palbociclib, a CDK4/6 inhibitor, as a new strategy of treatment in triple negative breast cancer, malignant pleural mesothelioma, and hepatocellular carcinoma D. Cretella (University of Parma, Italy), M. Bonelli, G. Digiacomo, C. Fumarola, A. Ravelli, P.G. Petronini
19.00 - 20.00 SIC General Assembly
12 13 Saturday, September 22nd
Plenary Room
8.45 - 10.15 Tumor microenvironment & metastasis Chairs: Andrea Anichini, Milan (Italy) - Giulia Taraboletti, Bergamo (Italy)
HMena splicing, a key mediator of pro-invasive stroma: a useful candidate for deciphering the tumor microenvironment? Paola Nisticò, Rome (Italy)
V14 Inhibition of EXPORTIN-1 (XPO1) activity reverses pro-tumorigenic secretory phenotype of lung cancer fibroblasts A. Bruccoleri, F. Andriani, F. Facchinetti,O. Rondinone, U. Pastorino, G. Sozzi, L. Roz (IRCCS, National Cancer Institute, Milan, Italy)
Perivascular regulation of tumor dormancy and drug resistance supported by Cyrus Ghajar, Seattle (USA)
I12 Increased lactate secretion by cancer cells sustains non-cell-autonomous adaptive resistance to met and EGFR targeted therapies M. Apicella, E. Giannoni, S. Fiore, C. Isella, C. Granchi, F. Minutolo, A. Sottile, P. Comoglio, E. Medico, F. Pietrantonio, M. Volante, P. Chiarugi, S. Corso, S. Giordano (University of Turin, Italy)
10.15 - 11.15 “Giorgio Prodi” Lecture Targeting invasion, angiogenesis and the tumor microenvironment: from therapy to cancer prevention Adriana Albini, Milan (Italy)
11.15 - 11.45 Coffee break
11.45 - 12.30 Award Ceremony “Piero Trivella” Award for the Best Posters
12.30 - 13.30 Closing Lecture Inactivation of DNA repair to improve cancer immune surveillance Alberto Bardelli, Turin (Italy)
13.30 Closing remarks
12 13 Scientific Information
CME CREDITS No. 5,4 Italian Ministry of Health for the following Professions and Disciplines: Medical Surgeon (Disciplines: Haematology, General (Family Doctors) Medicine, Medical Genetics, Oncology, Pathological Anatomy, Pharmacology and Clinical Toxicology, Clinical Pathology, Internal Medicine); Biologist, Pharmacist, Chemist (Discipline: Analytical Chemistry); Physicist (Discipline: Health Physics); Veterinary Surgeon.
SLIDES Slides must be in English. Computer videoprojection will be available in Power Point. Please communicate to the Organizing Secretariat your different requirements as soon as possible. Slides have to be handed to the slide centre one hour before the session beginning.
PRIZES Friday, September 21st “Pezcoller Foundation” Fellowship 2017-2018: Winner award - Annalisa Lonetti, Bologna (Italy) Winner award - Mattia Boeri, Milan (Italy) “Pezcoller Foundation” Fellowship 2019-2020 “Elena Cappannini” award for the best 2017 publication
Saturday, September 22nd “Piero Trivella” Award for the Best Posters
General Information
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14 15 General Information
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14 15 Thanks to
16 17 ABSTRACT BOOK
Abstract Book
16 17 18 19 ABSTRACT BOOK
CONTENTS
A .....Animal models of cancer...... Pag. 20
B .....Cancer epidemiology ...... Pag. 21
C .....Cancer genetics, genomics and epigenetics ...... Pag. 23
D...... Cancer modelling and imaging...... Pag. 32
E...... Cancer stem cells...... Pag. 34
F...... Cell adhesion, migration, invasion and metastasis...... Pag. 38
G...... Cell metabolism...... Pag. 43
H...... DNA damage and molecular responses to damage...... Pag. 46
I...... Drug resistance...... Pag. 47
J...... Microbiome...... pag. 51
K...... Molecular pathology...... pag. 52
L...... Novel therapeutic targets and experimental therapeutics...... pag. 55
M...... Oncogenes and tumor suppressor genes...... Pag. 65
N...... Pediatric cancer...... Pag. 67
O...... Personalized medicine...... Pag. 70
P...... Prevention and early detection...... Pag. 72
Q...... Prognostic and predictive biomarkers...... Pag. 74
R...... Signal transduction and intracellular trafficking...... Pag. 82
S...... System biology and emerging technologies...... Pag. 83
T...... Tumor heterogeneity...... Pag. 84
U...... Tumour immunology and immunotherapy...... Pag. 86
V...... Tumour microenvironment: inflammation, hypoxia and angiogenesis...... Pag. 92
18 19 ABSTRACT BOOK
fatty liver and steatohepatitis with fibrosis and allows to identify early stages of ANIMAL MODELS OF CANCER hepatocarcinogenesis. Results: We found that Nrf2 activation takes place already in early preneoplastic A1 lesions identified by the marker glutathione S-transferase placental form (GSTP). Loss of hepatic RuvBL1 causes chronic liver damage and Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in regeneration, promoting hepatocellular carcinoma 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether 1)Mello T. 2)Materozzi M. 3)Simeone I. 4)Zanieri F. 5)Ceni E. 6)Polvani S. 7)Tarocchi Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. M. 8) Bereshchenko O. 9)GalliA. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to 1)Dept. of Experimental and Clinical Biomedical Sciences “Mario Serio”, University increased liver injury and chronic compensatory hepatocyte regeneration when rats of Florence, Florence, Italy 2)Dept. of Medicine, Surgery and Neurosciences, were fed CMD diet. However, in spite of such a permissive environment, the livers University of Siena, Siena, Italy 3)Dept. of Experimental and Clinical Biomedical of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. Sciences “Mario Serio”, University of Florence, Florence, Italy 4)Dept. of Conclusions: These results demonstrate that, in a model of hepatocarcinogenesis Experimental and Clinical Biomedical Sciences “Mario Serio”, University of resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps Florence, Florence, Italy 5)Dept. of Experimental and Clinical Biomedical Sciences of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of “Mario Serio”, University of Florence, Florence, Italy 6)Dept. of Experimental and initiated cells, indicating that Nrf2 is critical in the onset of HCC. Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 7) Dept. of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 8)Faculty of Medicine, University of Perugia, Perugia, Italy A3 9)Dept. of Experimental and Clinical Biomedical Sciences “Mario Serio”, University ABROGATION OF EMILIN1-a4ß1 INTEGRIN INTERACTION AFFECTS of Florence, Florence, Italy EXPERIMENTAL COLITIS AND COLON CARCINOGENESIS ENHANCING LYMPHATIC DYSREGULATION AND INCREASING INFLAMMATORY CASCADE Background and Aim 1)Capuano A. 2)Bosisio G. 3) Pivetta E. 4)Scanziani E. 5)Minoli L. 6)Bortoluzzi S. 7) Overexpression of the AAA+ ATPase RuvBL1 correlates with a poor prognosis in Gaffo E. 8)Bucciotti F. 9)Doliana R. 10)Colombatti A. 11)Spessotto P. several human cancers, including Hepatocellular Carcinoma (HCC). A growing 1)Translational research, Cro national cancer institute, Aviano, Italy 2)Translational body of data from in vitro models shows that RuvBL1 supports cancer cell growth research, Cro national cancer institute, Aviano, Italy 3)Translational research, Cro through multiple mechanisms and thus it is receiving increasing consideration as a national cancer institute, Aviano, Italy 4)Medicina veterinaria, University of Milan, potential target for anti-cancer therapies. However, whether RuvBL1 participates Milan, Italy 5)Medicina veterinaria, University of Milan, Milan, Italy 6)Molecular in the oncogenic transformation towards hepatocellular carcinoma remains open medicine, University of Padova, Padova, Italy 7)Molecular medicine, University to speculation. To address this question, we realized a hepatocyte-conditional hep-/- of Padova, Padova, Italy 8)Translational research, Cro national cancer institute, RuvBL1 knock-out mouse model (RuvBL1 ) and evaluated the development of Aviano, Italy 9)Translational research, Cro national cancer institute, Aviano, Italy 10) chemically-induced HCC. Translational research, Cro national cancer institute, Aviano, Italy 11)Translational Methods hep-/- research, Cro national cancer institute, Aviano, Italy RuvBL1 mice were generated by crossing RuvBL1-floxed with Albumin-Cre mice. HCC was induced by i.p injection of DEN (5mg/kg) at 14 days of age. HCC Colon cancer is one of the principal cancer types where a functional link between progression was monitored after 3, 6, 9 and 12 months. Lineage-tracing experiments hep-/- mT/mG chronic inflammation, tumor microenvironment and progression had been noted; were performed by crossing RuvBL1 mice with with B6 mice to obtain a + -/- + - wt/ inflammation driving the colitis-associated cancer is also related to striking changes progeny in which mature (Alb , RuvBL1 , EGFP ) and non-mature (Alb , RuvBL1 wt + in the lymphatic vasculature and dysfunction in the intestinal lymphatic network is , mTRed ) hepatocytes could be traced in vivo. a well-established feature of human and experimental inflammatory bowel disease. Results EMILIN1 is expressed, among several other tissues, in the normal colonic mucosa; Contrary to our expectation, DEN-induced carcinogenesis was strikingly increased hep-/- hep-/- it controls elastogenesis and blood pressure homeostasis and is a key structural in RuvBL1 vs floxed mice. Moreover, RuvBL1 mice developed spontaneous element in the maintenance of the integrity of lymphatic vessels. It is an important HCC with aging. We found that RuvBL1 deletion causes hepatocellular damage, ECM adhesive ligand of a4b1 integrin via its gC1q domain and, in contrast with apoptosis and compensatory proliferation. The liver damage induced by loss large body of evidence that signals generated by ligand-activated integrins are pro- of RuvBL1 peaked around 2 weeks of age. Starting at 3 weeks of age, massive proliferative, this interaction down-regulates proliferation. The impact of intestinal proliferation of non-mature hepatocytes and partial recovery of RuvBL1 protein levels inflammation was analysed in Emilin1-/ - (KO) and in E933A EMILIN1 transgenic were observed. Further characterization revealed that non-mature hepatocytes b origin from CK19+CD133+HNF4+ cells. We established that cycles of RuvBL1 mice (E933A-TG), in which a mutant EMILIN1 unable to be engaged by a4 1 is deletion, hepatocytes loss and compensatory proliferation occurs throughout the deposited. Mice were chronically treated with DSS 2% in the drinking water; KO hep+/- and E933A-TG models presented higher colitis scores and more severe mucosal lifetime of RuvBL1 mice. injury, fibrosis and inflammatory infiltrates (CD3, CD45/B220, MPO and Iba1 Conclusion positive cells) than WT animals. RNAseq analysis confirmed the up-regulation of This is the first report highlighting the essential role of RuvBL1 for hepatocyte survival several inflammatory response genes (i.e. IL1A, IL1B) and the down-regulation of and the impact of its loss on liver carcinogenesis. The increased HCC incidence cell-cell adhesion molecules in E933A-TG animals. To determine if KO and E933A- does not reflect a tumor-suppressor role of RuvBL1, rather, it is the outcome of a TG backgrounds influence also the extent of carcinogenesis, colon tumours were chronic regenerative process involving staminal precursors. This model may prove induced with a single injection of AOM followed by 1-week exposures to 2% DSS. useful to investigate the contribution of the hepatic progenitor compartment to liver Tumour growth was observed over time by endoscopic and clinical scoring of regeneration and carcinogenesis in the context of chronic hepatic damage. colitis severity was performed applying the MEICS and DAI indexes. In line with experimental colitis results, in colon carcinogenesis KO and E933A-TG mice had A2 higher incidence, bigger tumours and less survival. A possible explanation comes Genetic inactivation of Nrf2 prevents clonal expansion if initiated from preliminary analyses on whole mount colon specimens from untreated animals: cells in a nutritional model of rat hepatocarcinogenesis immunostaining for podoplanin, showed that both KO and E933A-TG lymphatic 1)Orrù C. 2)Szydlowska M. 3)Taguchi K. 4)Zavattari P. 5)Perra A. 6)Yamamoto M. vessels are irregular, dilated and characterized by dysmorphic structures and wide 7)Columbano A. lacunae. The abnormal architecture of lymphatic network could enhance lymphatic 1)Biomedical sciences, University of Cagliari, Cagliari, Italy 2)Biomedical sciences, dysregulation, decreased lymph flow and inflammatory cell drainage impairing University of Cagliari, Cagliari, Italy 3)Medical biochemistry, University of Tohoku, inflammatory resolution. We are presently completing LV analysis on treated colon Sendai, Japan 4)Biomedical sciences, University of Cagliari, Cagliari, Italy 5) samples. Biomedical sciences, University of Cagliari, Cagliari, Italy 6)Medical biochemistry, University of Tohoku, Sendai, Japan 7)Biomedical sciences, University of Tohoku, A4 Sendai, Japan Patient-derived tumor xenografts as pharmacological model of human pancreatic ductal adenocarcinoma Background & Aims: Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in 1)Anastasia A. 2)Resovi A. 3)Ghilardi C.4)Micotti E. 5)Minoli L. 6)Morandi E. 7) cancer development. Here we examined whether Nrf2 activation occurs at early Ostano P. 8)Chiorino G. 9)Belotti D. 10) Giavazzi R. 11)Bani M.R. steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the 1)Department of Oncology, Mario Negri Institute for Pharmacological Research, onset of hepatocellular carcinoma (HCC). Milano, Italy 2)Department of Oncology, Mario Negri Institute for Pharmacological Methods: We used wildtype (WT) and Nrf2 knockout (Nrf2KO) rats treated Research, Milano, Italy 3)Department of Oncology, Mario Negri Institute for with a single injection of diethylnitrosamine (DENA) followed by choline-devoid Pharmacological Research, Milano, Italy 4)Department of Neuroscience, Mario methionine-deficient (CMD) diet. This experimental model causes massive Negri Institute for Pharmacological Research, Milano, Italy 5)Department of Veterinary Medicine, University of Milan, Milano, Italy 6)Ospedale di Rho, ASST-
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Rhodense, Rho (MI), Italy 7)Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia, Biella Italy 8)Laboratory of Cancer Genomics, Fondazione Edo CANCER EPIDEMIOLOGY ed Elvo Tempia, Biella Italy 9)Department of Oncology, Mario Negri Institute for B1 Pharmacological Research, Milano, Italy 10)Department of Oncology, Mario Negri THYROID CANCER INCIDENCE IN BELARUSIAN RESIDENTS EXPOSED TO Institute for Pharmacological Research, Milano, Italy 11)Department of Oncology, CHERNOBYL FALLOUT Mario Negri Institute for Pharmacological Research, Milano, Italy 1)Veyalkin I. 2)Cheshyk A. 3)Zaitsau A. 4)Razhko A. Introduction and aim 1)Laboratory of epidemiology, Center for radiation medicine and human ecology, Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with an overall survival Gomel, Belarus 2)Chernobyl registry, Center for radiation medicine and human rate of 5% and a median survival time of less than 6 months. The poor prognosis ecology, Gomel, Belarus 3)Directorate, Institute of radiology, Gomel, Belarus 4) is due to late diagnosis and high refractoriness to treatment. Current systemic Directorate, Center for radiation medicine and human ecology, Gomel, Belarus treatments offer only modest benefits in symptom control and surgery remains the most effective treatment. Thus, the exploitation of new therapeutics is an urgent Introduction. The accident at the Chernobyl nuclear power plant was the largest need, but the research is braked by the limited value of the current preclinical radiation-ecological catastrophe, which was accompanied by a massive release models as predictors of patient’s response. of the radioactive isotope of iodine. The aim of this work was to investigate the The aim of our study is to develop patient-derived xenografts (PDA-PDXs) that incidence of thyroid cancer in Belarus and assess the risk of developing this disease mimic the biology of the patient’s tumor and retain the capability to provoke a robust in affected cohorts. stroma reaction, considered one of the main causes for the resistance to systemic Material and Methods. The data from the State Register of persons affected by therapy. the Chernobyl accident (from 1987 to 2015) and the data of the Belarusian Cancer Methods and results Registry (from 1978 to 2015) were used in the study. The analysis was conducted To reproduce an experimental setting reflecting the human disease, cancer using crude rates (CR), age-specific rates (AsR) and age-standardized rates (ASR) specimens from patients undergoing cephalic duodenopancreatectomy were and standardized incidence ratio (SIR). Affected population was divided into four xenografted orthotopically in the pancreas of immunodeficient mice. Histologically groups: liquidators (99 498 persons), evacuees (13 096 persons), residents of confirmed PDA, engrafted successfully in 5 cases (out of 11) and were propagated contaminated areas (139 811 persons) and their offspring (30 475 persons), which by their re-implantation into the mouse pancreas. were analyzed by sex, age at time of the disaster, year of being in contaminated Gene expression data identify all the PDA-PDXs as “Classical” according to area, duration of staying in contaminated area, density of radiation contamination Collisson [Collisson et al, Nat Med. 2011] or “Pancreatic Progenitor” according to and individualized doze in thyroid gland. Bailey [Bailey et al, Nature 2016]. Results and Discussions. It was shown the intensive increase in the incidence Mutational landscape of the PDA-PDXs reflect the clinical scenario: four out of rates of thyroid cancer in the Belorussian population after the Chernobyl accident, which finished after 2001 (CR1986=1.5±0.24, CR2001=10.3±0.63, CR2015=12.5±0,71). five PDA-PDXs are mutated in KRAS (G12D, G12R) and/or TP53 (I162F, R213*, Thyroid cancer growth was most significant in most contaminated Gomel, Mogilev Y220C, R248Q), one out of five shows the deletion of SMAD4 and one PDA-PDX and Brest areas. It was shown that the maximum increase in incidence was carries a BRCA1 mutation. observed in persons aged 0-4 years at the time of the accident. In the affected When grown in the mouse pancreas, PDA-PDXs maintain the histological features population, significantly high risk of thyroid cancer was noted in all groups. The of the human disease and exhibit a robust amount of host derived stroma with risk was highest in the evacuees in 1995-1999 (SIR=13.0 (9.21-17.70)) and was collagen deposition, constant through generations. significantly higher than in the liquidators (SIR=4.4 (3.63-5.25)) and in the cohort of PDA-PDXs were marginally responsive to gemcitabine treatment. Better response living in contaminated areas (SIR=2,4 (1.88-2.98)). The risk was higher in men than (stable disease and regression) was observed with the combination regimen in women, for those who were children at the time of the accident and who were gemcitabine and nab-paclitaxel. in the most contaminated areas. The dose-response relationship was shown for Conclusion the evacuees (rSpearman=0.9, p=0.037) and for persons living in contaminated areas PDA-PDXs growing in the pancreas of mice retained the molecular, morphological (rSpearman=1.0, p<0.001). For the group of liquidators, the dose-effect relationship was and biological features of the patient tumors, thus they are valuable tools for the not noted (rSpearman=-0.48, p=0.329). preclinical exploitation of novel therapeutics. Conclusion. In our work we have shown that Chernobyl disaster increased thyroid Supported by AIRC 5 per mille n. 12182 and Fondazione “Eugenio Morandi” ONLUS cancer incidence in both whole population and most affected groups. Especially in per lo studio e la cura dei tumori del pancreas. people who were children at the time of disaster.
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B2 respectively. MCPyV DNA sequences were studied by PCR methods in MCCs RISK OF MALIGNANT NEOPLASMS OF BLOOD AND LYMPHATIC SYSTEM OF and in PBMCs. Immunohistochemical (IHC) analyses were carried out in MCCs to LIQUIDATORS OF THE CHERNOBYL ACCIDENT IN BELARUS reveal MCPyV LT. 1)Veyalkin I. 2)Cheshyk A. Three MCC were detected in two patients affected by rheumatoid arthritis and one 1)Laboratory of epidemiology, Center for radiation medicine and human ecology, patient with ankylosing spondylitis, respectively. Patient 1 developed MCC after 5 Gomel, Belarus 2)Chernobyl registry, Center for radiation medicine and human months of abatacept therapy. Patient 2 developed MCC after 5 yrs of infliximab ecology, Gomel, Belarus therapy; MCC growth arrest occurred after the withdrawal of the biological drug. Introduction Patient 3 developed MCC after 42 months of etanercept therapy. MCPyV DNA The accident at the Chernobyl nuclear power plant was the most huge radiation and sequences identified in in the three MCCs showed 100% homology with MCPyV ecological catastrophe. About 100,000 Belarusian citizens took part in the liquidation MKL-1, which is the European strain. PBMCs from patients tested MCPyV-negative. of the Chernobyl accident. During the first years after the accident, the growth of Viral DNA loads in MCCs were in the range of 0.1-30 copy/cell. Anti-LT/ST IgG Abs incidence of malignant neoplasms of blood of the affected population was predicted. were detected in patients 1 and 3, whereas patient 2 did not react with MCPyV LT/ But till now, there is no clear conclusion about the contribution of the radiation factor to ST antigen. Sera of the three MCC patients contained anti-MCPyV VP1 IgG Abs. the incidence of leucosis and lymphomas. The purpose of the study was to analyze the MCCs tested positive for the MCPyV LT antigen in IHC, with a strong, nuclear LT features of forming the incidence of malignant neoplasms of the blood and lymphatic expression. Normal tissues, employed as control, tested MCPyV LT negative. system of the liquidators of the Chernobyl accident in the Republic of Belarus. Pharmacologic immunosuppressive therapy with biologics in managing Material and Methods rheumatologic disorders could modify the immune response against MCPyV as well The data of the Chernobyl State Register of persons exposed to radiation as reducing host anti-cancer immune response, which in turn it may increase the following the Chernobyl catastrophe for the period from 1987 to 2015 were used risk of MCC onset/progression. It is striking to note that in our patients the incidence (99 498 liquidators). The epidemiological analysis of the incidence of leukemias, of MCC increased from 1/>106, detected in the general population, to 1/< 103. lymphomas and multiple myeloma was made using the standardized incidence ratio (SIR). Liquidators were analysed by sex, age at time of the disaster, year of work, duration of staying in contaminated area, density of radiation contamination and B4 individualized doze in bone marrow. DianaWeb: a community-based participatory research to Results and Discussions improve breast cancer prognosis through lifestyles The excess of incidence of leukemias in the cohort of liquidators of the Chernobyl 1)Villarini M. 2)Nucci D. 3)Gianfredi V. 4)Berrino F. 5)Gargano G. 6)Acito M. 7) accident was noted. The excess fraction of leukemias was about 20–40 % (SIR Moretti M. 8)Villarini A. = 1.3 (1.2–1.46)). It was shown that the high risk of leukemia was formed due 1)Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy to chronic lymphocytic (SIR = 1.3 (1.14–1.53)) and myelocytic (SIR = 1.7 (1.35– 2)Digestive Endoscopy Unit, Veneto Institute of Oncology IOV-IRCCS, Padova, 2.03)) leukemias regardless of the status of a liquidator. There was also no clear Italy 3)School of Specialization in Hygiene and Preventive Medicine, Department dependence between the risk of hemoblastosis and the density of contamination, as of Experimental Medicine, University of Perugia, Perugia, Italy 4)Associazione “La well as the individualized absorbed dose on the blood marrow. However, it can be Grande Via”, Brescia, Italy 5)Department of Research, Fondazione IRCCS Istituto stated that a large proportion of cases of multiple myeloma (32 %) was found in the Nazionale dei Tumori, Milano, Italy 6)Department of Pharmaceutical Sciences, liquidators who performed their work in the territory with the Cs137 pollution density University of Perugia, Perugia, Italy 7)Department of Pharmaceutical Sciences, of more than 40 Ku/km2 (SIR = 1.8 (1.16–2.8)). University of Perugia, Perugia, Italy 8)Department of Research, Fondazione IRCCS Conclusion Istituto Nazionale dei Tumori, Milano, Italy The conducted research allowed to characterize the risks of development of various forms of hemoblastosis in liquidators and to show trends in the distribution of DianaWeb is an ongoing community-based participatory research offered to Italian incidence of certain their forms. breast cancer (BC) patients with or without metastasis, local recurrences or second cancers, with in situ or invasive cancer; patients are eligible for enrolment whatever the disease stage at diagnosis, the histological diagnosis, and the time elapsed B3 since diagnosis. MERKEL CELL CARCINOMAS ARISING IN AUTOIMMUNE DISEASE The aim of the study is the evaluation of effectiveness of a lifestyle intervention AFFECTED PATIENTS TREATED WITH BIOLOGICAL DRUGS, INCLUDING (inspired to the traditional Mediterranean Diet (MD) and to the World Cancer ANTI-TNF ALPHA Research Fund (WCRF) recommendations) to improve the prognosis through the 1)Rotondo J.C. 2)Bononi I. 3)Puozzo A. 4)Govoni M. 5)Foschi V. 6)Lanza G. 7)Gafà reduction BC risk factors such as abdominal obesity, metabolic syndrome (MetS), R. 8)Gaboriaud P. 9)Touzé F.A. 10)Selvatici R. 11)Martini F. 12)Tognon M. sedentary lifestyle, subclinical chronic inflammation, high plasma insulin and 1)Department of Morphology, Surgery and Experimental Medicine, School of testosterone. Medicine, University of Ferrara, Ferrara, Italy 2)Department of Morphology, With the support of the IT’s Soluzioni Informatiche (Brescia, Italy), a dedicated website Surgery and Experimental Medicine, School of Medicine, University of Ferrara, (www.dianaweb.org) was developed to collect clinical and anthropometric data, to Ferrara, Italy 3)Department of Morphology, Surgery and Experimental Medicine, monitor patient’s lifestyle, and to provide realistic and practical recommendations for School of Medicine, University of Ferrara, Ferrara, Italy 4)Department of Medical healthy lifestyle based on the best available scientific evidence. We obtained written Sciences, School of Medicine, University of Ferrara, Ferrara, Italy 5)Department informed consent from all participants. of Medical Sciences, School of Medicine, University of Ferrara, Ferrara, Italy 6) Recruitment started in September 2016 and over 1000 women enrolled. 223 Department of Medical Sciences, School of Medicine, University of Ferrara, Ferrara, participants were assessed at baseline and 1-year follow-up. A 24-point food-items Italy 7)Department of Morphology, Surgery and Experimental Medicine, School of questionnaire was used to assess adherence to the traditional MD and a 24-hours Medicine, University of Ferrara, Ferrara, Italy 8)Université Francois Rabelais, UMR recall sheet was used to calculate a score of adherence to WCRF recommendations. INRA 1282 ISP, Faculté des Sciences Pharmaceutiques, Tours, France 9)Université The mean age of participants was 51.0 ± 8.8 years. Participants showed to have a Francois Rabelais, UMR INRA 1282 ISP, Faculté des Sciences Pharmaceutiques, high education level (92.4% with >12 years education) and predominantly married Tours, France 10)Department of Medical Sciences, School of Medicine, University (67.7%). At baseline 3.6% of the participants were current smokers, and 40% of of Ferrara, Ferrara, Italy 11)Department of Morphology, Surgery and Experimental the participants were sedentary. At follow-up we observed a significant increase in Medicine, School of Medicine, University of Ferrara, Ferrara, Italy 12)Department consumption of whole grains and a significant decrease in intake of refined foods. of Morphology, Surgery and Experimental Medicine, School of Medicine, University The relationships between adherence to MD and BC risk factors and between of Ferrara, Ferrara, Italy adherence to the WCRF recommendations and BC risk factors were evaluated using Spearman’s correlation. Merkel cell carcinoma (MCC) is a rare but aggressive tumor with an incidence We observed that greater adherence to a MD was associated with decreasing of 1/~3 million/yr in Europe. Merkel cell polyomavirus (MCPyV) is the causative levels of MetS (significantly reduced), BMI, insulin and testosterone. We report also agent of this tumor. In immunocompromised host the anti-viral/cancer response is a significant reduction in MetS and BMI with an increasing score for adherence to an important adverse event, which may occur due to biologic disease–modifying WCRF recommendations. anti-rheumatic drugs, including anti-TNFs, therapies that are employed in the management of autoimmune diseases. We aimed to characterize MCCs arisen in three patients affected by auto-immune diseases, treated with biologics, belonging to a 700-patients cohort. We studied in detail: (i) the clinical features of patients; (ii) MCPyV sequences and proteins in their PBMCs and MCCs; (iii) anti-MCPyV antibodies (Abs) and titers in serum samples. Serum IgG Abs against the viral oncoproteins Large T (LT) and small t (ST) antigens and the viral capsid protein 1 were analyzed by indirect E.L.I.S.As. Viral antigens were recombinant LT/ST and virus-like particles (VLPs),
22 23 ABSTRACT BOOK
B5 Insight into genetic susceptibility to BRCA-negative male CANCER GENETICS, GENOMICS AND breast cancer by multigene panel testing: Results from a EPIGENETICS multicenter study in Italy 1)Zelli V. 2)Rizzolo P. 3)Silvestri V. 4)Valentini V. 5)Spinelli A. 6)Tibiletti M.G. 7) C1 Russo A. 8)Varesco L. 9)Giannini G. 10)Calistri D. 11)Cortesi L. 12)Viel A. 13) QUANTITATIVE MAPPING of EPIGENETIC MARKS BY MS-PROTEOMICS Montagna M. 14)Peterlongo P. 15)Radice P. 16)Palli D. 17)Ottini L. IN BREAST CANCER SAMPLES, FOR PATIENT STRATIFICATION AND 1)Department of Molecular Medicine, Sapienza University of Rome, Italy 2) DISCOVERY OF NEW THERAPEUTIC TARGETS Department of Molecular Medicine, Sapienza University of Rome, Italy 3) 1)Bonaldi T. 2)Noberini R. Department of Molecular Medicine, Sapienza University of Rome, Italy 4) 1)Experimental oncology, European insitute of oncology, Milano, Italy 2)Experimental Department of Molecular Medicine, Sapienza University of Rome, Italy 5)S.C. oncology, European insitute of oncology, Milano, Italy Genetica Medica, IRCCS, Burlo Garofolo, Trieste, Italy 6)Unit of Pathology, Ospedale di Circolo, Varese, Italy 7)Section of Medical Oncology, Department of Background Surgical and Oncological Sciences, University of Palermo, Italy 5)Unit of Hereditary Triple-negative breast cancers (TNBCs) comprise a highly diverse group of tumors, Cancers, IRCCS AOU San Martino – IST, Genoa, Italy 8)Department of Molecular for which well-defined molecular targets have not yet been identified and targeted Medicine, Sapienza University of Rome, Italy 9)Istituto Scientifico Romagnolo per lo therapies do not exist. A better understanding of the molecular mechanisms Studio e la Cura dei Tumori (IRST), Meldola (FC), Italy 10)Department of Oncology underlying this type of disease is therefore needed in order to develop effective and Haematology, University of Modena and Reggio Emilia, Modena, Italy 11)Unit of targeted therapies as well as discover biomarkers to be used as prognostic and Experimental Oncology 1, CRO Aviano, National Cancer Institute, Aviano (PN), Italy predictive tools. Building on the increasing evidence that links aberration in histone 12)Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV - post-translational modifications (hPTMs) and cancer, in this study we employed a IRCCS, Padua, Italy 13)IFOM, The FIRC (Italian Foundation for Cancer Research) quantitative MS-based analysis of hPTMs to profile epigenetic patterns in different Institute of Molecular Oncology, Milan, Italy 14)Unit of Molecular Bases of Genetic breast cancer subtypes, focusing on TNBCs in particular. Risk and Genetic Testing, Department of Research, Fondazione IRCCS Istituto Methods Nazionale Tumori (INT), Milan, Italy 15)Molecular and Nutritional Epidemiology Unit, We profiled breast cancer patient samples using our recently developed PAT-H-MS ISPRO, Florence, Italy 16)Department of Molecular Medicine, Sapienza University (pathology tissue analysis of histones by mass spectrometry) method, which allows of Rome, Italy the MS-analysis of hPTM patterns from formalin fixed paraffin embedded tissues, in combination with a histone-focused super-SILAC approach. MS data were acquired Background: Male breast cancer (MBC) is a rare disease, representing less than on a high-resolution HF Q Exactive instrument with methods/protocols adapted for 1% of all breast cancers (BC). Inherited mutations in BRCA1 and, more frequently, in depth analysis of histones. BRCA2 genes predispose to MBC and account for up to 13% of all cases. In order to Results gain more insight into the genetic susceptibility of MBC, we performed an extensive The MS-analysis of >100 breast cancer patient samples belonging to different genomic screening of a large series of BRCA1/2-negative MBCs by multigene panel subtypes (Luminal A- and B-like, Her positive and TNBCs) revealed epigenetic testing. signatures, which included the H3 K27me3 and H3K9me3 marks, that distinguish Methods: A well-characterized series of 503 BRCA1/2 mutation negative MBC different subtypes. Furthermore, we defined TNBC subgroups characterized by cases, recruited in the frame of the ongoing Italian multicenter study on MBC, was different epigenetic signature, as well as epigenetic marks that differentiate TNBC analyzed by a multi-gene custom panel of 50 cancer-related genes, using MiniSeq patients with and without relapse after chemotherapy. Histone PTMs profiles were platform (Illumina). The main clinical-pathologic characteristics of MBCs were then intersected with global proteomics and RNA-seq patient data, identifying compared between pathogenic variant carriers and non-carriers. aberrantly expressed histone-modifying enzymes, which may serve as targets for Results: Overall, 27 of the 503 (5.4%) BRCA1/2 mutation negative MBC cases were epigenetic therapy for breast cancer subtypes or TNBC subgroups. carriers of pathogenic variants in 14 of the 50 genes analyzed. PALB2 and ATM Conclusions were the most frequently altered genes (1.2% and 0.6%, respectively). Pathogenic The hPTM marks and signatures revealed by our work offer insights into epigenetic variants in known/proposed BC genes, including BARD1, BLM, CASP8, CHEK2, mechanisms that underlie breast cancer, and particularly TN, not only providing FANCM, NF1, RAD50, RAD51C and RAD51D, as well as in genes not closely biomarkers useful for patient stratification, but also suggesting novel epigenetic related to BC predisposition, including APC, EPCAM and MUTYH, were also pathways targetable for therapy. identified. The majority of carriers had one pathogenic variant. Only two carriers Keywords had two pathogenic variants: one had biallelic MUTYH variants and the other had Quantiative MS, histone post-translational modifications, epigenetics, breast RAD51C and MUTYH variants. Pathogenic variant carriers were more likely to cancer have a personal history of cancer in addition to BC (p=0.0001) and family history of breast/ovarian cancer (BC/OC) and/or other cancer (p=0.004) rather than family history of BC/OC only (p=0.3). C2 Conclusions: Our results support the central role of PALB2 in MBC susceptibility, COMPARISON BETWEEN MODELS OF MYXOID/ROUND CELL LIPOSARCOMA point to a relevant role of ATM and confirm a low impact of CHEK2 pathogenic SENSITIVE AND RESISTANT TO TRABECTEDIN THROUGH NEXT GENERATION variants on MBC predisposition in the Italian population. Considering that pathogenic SEQUENCING AND MICROARRAY APPROACH variants were found only in a fraction of the genes analysed, our data indicate that 1)Craparotta I. 2)Mannarino L. 3)Ballabio S. 4)Frapolli R. 5)Bello E. 6)Ponzo M. 7) the identification of the more appropriate genes for the genomic screening of MBC Barisella M. 8)Sanfilippo R. 9)Pilotti S. 10)Brich S. 11)Casali P. 12)Markowetz F. 13) cases is essential in order to develop a comprehensive and specific BC susceptibility Marchini S. 14)D’incalci M. panel. Moreover, our results indicate that hereditary MBC does not necessarily 1)Oncology, Irccs Mario Negri, Milan, Italy 2)Oncology, Irccs Mario Negri, Milan, appear in BC/OC-families but also in families characterized by the presence of Italy 3)Oncology, Irccs Mario Negri, Milan, Italy 4)Oncology, Irccs Mario Negri, cancers other than BC/OC, with important implications for clinical management of Milan, Italy 5)Oncology, Irccs Mario Negri, Milan, Italy 6)Oncology, Irccs Mario MBC patients and their relatives. Study supported by AIRC (IG 16933) to L.O. Negri, Milan, Italy 7)Diagnostic pathology, Irccs istituto nazionale dei tumori, Milan, Italy 8)Medical oncology, Irccs istituto nazionale dei tumori, Milan, Italy 9)Diagnostic pathology, Irccs istituto nazionale dei tumori, Milan, Italy 10)Diagnostic pathology, Irccs istituto nazionale dei tumori, Milan, Italy 11)Medical oncology, Irccs istituto nazionale dei tumori, Milan, Italy 12)Cancer research uk Cambridge, University of Cambridge, Cambridge, Uk 13)Oncology, Irccs Mario Negri, Milan, Italy 14) Oncology, Irccs Mario Negri, Milan, Italy Myxoid/round cell liposarcoma (MLPS) is a rare tumor within the soft tissue sarcomas. Its main genetic lesion is the FUS-CHOP chimera, due to t(12;16) (q13;p11). One of the biological effects of the aberrant FUS-CHOP activity is the impairing of the adipocyte differentiation. Trabectedin, a marine compound, has demonstrated a great efficacy on MLPS by promoting adipogenesis. Unfortunately, most patients become progressively resistant. In this study, differences between sensitive and resistant patients derived xenograft (PDX) models and the effects of trabectedin on both were investigated. PDX resistant to trabectedin (ML017/ET) has been previously generated by prolonged in vivo treatments of the ML017 sensitive model. Genomic analysis
22 23 ABSTRACT BOOK through next generation sequencing technology and microarray experiments were C4 performed in order to investigate differences between models at baseline and after Identification of novel transcripts related to platinum different time of trabectedin exposure. resistance in high grade serous epithelial ovarian cancer Although transcriptional data reveals no changes in the expression profile between patients by RNA sequencing approaches ML017 and ML017/ET at basal level, genomic analysis of the tumor architecture 1)Paracchini L. 2)Calura E. 3)Beltrame L. 4)Federica Donati F. 5)Ceppi L. 6)Fruscio shows that ML017/ET is characterized by a more marked chromosome instability R. 7)Todeschini P. 8)Bignotti E. 9)D’Incalci M. 10)Romuladi C. 11)Marchini S. than the ML017 with several deletions, amplifications and translocations. 1)Laboratory of Cancer Pharmacology, Department of Oncology, IRCCS “Mario In ML017, already after a single dose, trabectedin increases chromosome instability Negri” Institute for Pharmacological Research, Milan, Italy 2)Department of with many deletions; lower rearrangements were found on the resistant type. Biology, University of Padova, Padova, Italy 3)Laboratory of Cancer Pharmacology, Prolonged exposure to the drug causes a worsening of chromosomes alterations Department of Oncology, IRCCS “Mario Negri” Institute for Pharmacological in ML017, while the genomic landscape of ML017/ET mostly overlaps the untreated Research, Milan, Italy 4)Laboratory of Cancer Pharmacology, Department of control. Oncology, IRCCS “Mario Negri” Institute for Pharmacological Research, Milan, Italy As far as transcriptional effects, single dose trabectedin affects several pathways 5)Division of Obstetrics and Gynecology, San Gerardo Hospital, Monza, Italy 6) related to cell cycle, mitotic spindle transition and apoptosis in ML017 and not in Division of Obstetrics and Gynecology, San Gerardo Hospital, Monza, Italy 7)Division ML017/ET. After prolonged treatment, adipogenesis is the main activated pathway of Gynecologic Oncology, Angelo Nocivelli Institute of Molecular Medicine, University in ML017 while only few lipid metabolic networks are up-regulated in ML017/ET. of Brescia, Brescia, Italy 8)Division of Gynecologic Oncology, Angelo Nocivelli Preliminary data suggests that: Institute of Molecular Medicine, University of Brescia, Brescia, Italy 9)Laboratory (1) no major differences in transcriptional profile were detected comparing ML017 of Cancer Pharmacology, Department of Oncology, IRCCS “Mario Negri” Institute and ML017/ET, despite profound discrepancies in their genomic structure; for Pharmacological Research, Milan, Italy 10)Department of Biology, University (2) trabectedin exposure modifies the genomic architecture of ML017 but notof of Padova, Padova, Italy 11)Laboratory of Cancer Pharmacology, Department of ML017/ET. Oncology, IRCCS “Mario Negri” Institute for Pharmacological Research, Milan, Italy In ML017 the presence of chromosome instability could be due to the transcriptional changes, possibly related to the drug early cytotoxic effect, which is not present on Background the ML017/ET. High-grade serous epithelial ovarian cancer (HGS-EOC) has a 5-years survival Furthermore, resistance to trabectedin appears to be associated to the lack of the rate less than 40%. This poor outcome is due to Platinum (Pt) resistance: in fact, activation of adipocyte differentiation. although most of patients initially respond to Pt-based therapy, they generally relapse with a disease that becomes progressively resistant. Nowadays there are no clinical parameters or molecular biomarkers able to predict Pt response. C3 To elucidate the molecular mechanisms underlying Pt-resistance, we exploited Molecular stratification of HPV-related head and neck RNA sequencing approach to profile the entire transcriptome searching for novel squamous cell carcinoma reveals subtype with biological transcripts that are differentially expressed between Pt-sensitive (relapsing after 12 and clinical relevance months from the end of therapy) and Pt-resistant patients (relapsing within 6 month 1)Serafini M.S. 2)De Cecco L. 3)Canevari S. 4)Bossi P. 5)Licitra L. 6)Locati L.D. from the end of therapy). 1)Platform of Integrated Biology, Department of Applied Research and Technology Methods Development, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 2) Two independent cohorts of HGS-EOC (Cohort A= 28 and Cohort B = 55), balanced Platform of Integrated Biology, Department of Applied Research and Technology in terms of Pt-sensitive and Pt resistant patients, were selected. TruSeq Stranded Development, Fondazione IRCCS Istituto Nazionale Tumori Milan, Italy 3)Platform of RNA kit (Illumina) was used to analyze the entire transcriptome in cohort A. Reads Integrated Biology, Department of Applied Research and Technology Development, were mapped to a reference genome using bowtie and RABT assembler, and Fondazione IRCCS Istituto Nazionale Tumori Milan, Italy 4)Head and Neck Medical novel transcripts reconstructed with StringTie. RSEM was used for transcripts Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5) quantification. Differentially expressed transcripts (DETs) were validated, using Head and Neck Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei targeted RNA sequencing (Capture Seq, Roche) on cohorts A and B. Libraries were Tumori, Milan, Italy 6)Head and Neck Medical Oncology Unit, Fondazione IRCCS run on Nextseq 500 system (Illumina). Istituto Nazionale dei Tumori, Milan, Italy Results RNAseq experiments on Cohort A identified 1371 DETs. Only 10% of them were Background: HPV-related head and neck squamous cell carcinoma (HNSCC) reported as known transcripts. Targeted RNA sequencing was exploited to validate patients have different epidemiologic, clinical and outcome behaviors in comparison the 1371 DETs first on Cohort A (technical validation) and further on CohortB with HPV-negative, having HPV-related patients a 70% of reduction in their risk of (clinical validation). Analysis confirmed 1313 and 820 DETs in Cohort A andin death. Smoking exposure and bulky neck nodes are negative prognostic factors, Cohort B, respectively. Considering the intersection of DETs with concordant fold however the reasons of treatment failure are almost unknown in HPV-related change between the two cohorts, 77 DETs (FDR 15%) were validated. Known cases. In the last years high-throughput gene expression assays allowed to transcripts represent the smallest portion of DETs validated (5 out of 77). identify predictive signatures in several tumor types. In the present study, we aim at Conclusion disclosing molecular subtypes in HPV-related HNSCC with potential biological and Technical and clinical validations of the RNA sequencing data, highlight different clinical relevance. transcriptional programs between Pt-sensitive and Pt-resistant HGS-EOC patients. Of Methods: through a literature revision, publicly gene-expression data for HPV- these, less than 7% of transcriptional changes can be ascribed to coding transcripts, positive HNSCC were retrieved from the following databases: ArrayExpress, GEO, exploiting why previous attempts to identify transcriptional signatures associated to and TCGA. Only studies with HPV status confirmed by DNA sequencing or qPCR Pt response and patients’ prognosis were not successful. The 93% of transcriptional were considered eligible. A total of 346 available cases from 11 studies were merged alterations were due to unknown or partially unknown transcripts. Studies are ongoing through a meta-analysis approach. The biological pathways related to each subtype to define the functional and prognostic roles of DETs identified in the present study. were investigated using gene set enrichment analysis (GSEA). Results: unsupervised consensus clustering analysis provided evidence of the presence of three main clusters with significant stability confirmed by Silhouette plot. C5 The clinical relevance of this classification was tested for its association to overall- AN INTEGRATED COMPUTATIONAL APPROACH TO DEFINE TRANSCRIPTOME survival. Kaplan-Meier analysis demonstrated that Cl3 has the worst behavior, ALTERATIONS IN PLATINUM RESISTANCE while Cl1 has the better one, differing for the enrichment of a number of pathways 1)Benvenuto G. 2)Calura E. 3)Todeschini P. 4)Paracchini L. 5)Ravaggi A. 6)Odicino including epithelial mesenchymal transition (EMT), cell motility, and immune-related F. 7)D’Incalci M. 8)Bignotti E. 9)Marchini S. 10)Romualdi C. gene-sets. In addition Cl3 shows lower immune-score assessed by a deconvolution 1)Department of Biology, University of Padova, Padova, Italy 2)Department approach compared to the other clusters. of Biology, University of Padova, Padova, Italy 3)”Angelo Nocivelli” Institute of Conclusions: At present, our analysis is the largest meta-analysis in HPV-related Molecular Medicine, Division of Obstetrics and Gynecology, University of Brescia, HNSCC and demonstrated the existence of molecular heterogeneity in this type Brescia, Italy 4)Department of Oncology, IRCCS, “Mario Negri” Institute for of tumors. We report a molecular subtype classification that can improve patient Pharmacological Research, Milan, Italy 5)”Angelo Nocivelli” Institute of Molecular selection and pave the way to the development of appropriate therapeutic strategies. Medicine, Division of Obstetrics and Gynecology, University of Brescia, Brescia, Validation in an independent series provided within the H2020 “BD2decide” project Italy 6)”Angelo Nocivelli” Institute of Molecular Medicine, Division of Obstetrics is ongoing. and Gynecology, University of Brescia, Brescia, Italy 7)Department of Oncology, This work was supported by Associazione Italiana Ricerca Cancro (AIRC IG 18519 IRCCS, “Mario Negri” Institute for Pharmacological Research, Milan, Italy 8)”Angelo to LDC) and by the European Union’s Horizon 2020 research and innovation Nocivelli” Institute of Molecular Medicine, Division of Obstetrics and Gynecology, programme under grant agreement No 689715. University of Brescia, Brescia, Italy 9)Department of Oncology, IRCCS, “Mario Negri” Institute for Pharmacological Research, Milan, Italy 10)Department of Biology, University of Padova, Padova, Italy
24 25 ABSTRACT BOOK
METHODS: 639 mCRC samples were sequenced by IT-PGM platform using a panel Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy of hotspots and targeted regions of 22 genes (including RAS) commonly involved in due to its diagnosis at advanced stages. EOC is generally sensitive to first line CRCs. MSI analyses on 89 patients have been performed with a single fluorescent chemotherapy, and the vast majority of patients respond to platinum (Pt)-based system comprising BAT25 and BAT26 mononucleotide repeats. therapy after debulking surgery. Unfortunately, the median progression free survival RESULTS: We identified recurrent mutations (≥1%) in 12/22 genes, being KRAS, (PFS) lasts only 18 months, and more than 80% of Pt-responsive patients relapse TP53 and PIK3CA the most frequently mutated ones. Statistical analysis, indicated with a disease that progressively becomes Pt-resistant. The process by which that the mutation associations follow a non-random distribution. Categorization disease relapses is still poorly understood, so the aim is to identify biomarkers of of the cases on the base of KRAS and p53 mutation status led us the definition sensitivity to chemotherapy and therapeutic targets in HGS-EOC by integrating of 8 Mutation Association Patterns (MAPs) characterized by specific mutation transcriptomic data, coding and non-coding RNAs. associations. Analysis of the clinicopathological data available for 89 out of 639 To this end, we used matched miRNA and mRNA expression data obtained by cases indicates interesting trends for the associations of MAPs with specific both microarray and sequencing technologies of HGS-EOC biopsies (at the time parameters, some of which reached statistical significance. of diagnosis). HGS-EOC tumour samples are divided as platinum-resistant (Pt- CONCLUSIONS: Application of gene panel NGS as a routine for the characterization r), platinum-sensitive (Pt-s) and platinum-partially sensitive, having a complete of RAS/BRAF status required for predictive purposes in CRC patients, may provide follow up of at least five years. After data normalization, we performed differential additional prognostic/predictive information, with no significant extra-costs. expression analysis on coding and non-coding elements, using the two more Keywords: Colorectal cancer, Next Generation Sequencing, RAS extreme categories of patients (Pt-r and Pt-s), and we applied an integrated pathway approach to identify circuits associated to the therapy response. In addition, we used a set of strand specific paired-end RNA-seq experiments, C7 selected from tumour tissue collection, to perform transcriptome reconstruction in Exosomal shuttle of tumoral miR-19b and miR-92a enhances order to identify new aberrant splicing such as circular RNAs and to estimate their lung epithelial cell proliferation expressions. Borzi C., Fortunato O., Sozzi G. Our findings suggest that the molecular landscape between Pt-s and Pt-r patients at Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto the time of diagnosis is highly complex and heterogeneous. This variability seems Nazionale dei Tumori, Milan, Italy to involve mainly novel transcripts, including specific non conding RNAs. In this perspective a two-step approach using classical array-based gene expression Background: Lung cancer causes around a fifth of all cancer deaths. Tumor cells and recent NGS technology is the best way to approach the problem. Here we actively communicate with the surrounding microenvironment to support malignant identify a regulatory circuit composed of 127 genes and 5 miRNAs wiring mainly progression. Exosomes can mediate cell-to-cell communication and modulate ATP syntesis, calciumchannel, HLAs and CREB proteins, and also a list of novel recipient cells by the delivery of their contents, including microRNAs (miRNAs). transcripts potentially involved in Pt resistance mechanism. The aim of this study was to characterize miRNA content of lung cancer derived- Specifically using RNA-Seq we report a large number of circRNAs differentially exosomes and to evaluate their pro-tumorigenic potential on epithelial cells. expressed between tumor resistance types. The consistency of circular RNA Methods: Exosomes were isolated from conditioned medium of human lung cancer expression, in conjunction with the regulatory circuit, may offer new candidates for (A549, LT73) and non-tumorigenic bronchial epithelial (HBECs) cell lines using cancer treatment and prognosis, revealing that the circRNA-miRNA-mRNA network ultracentrifugation method. Exosomes purification was evaluated by sizing (Nanosight) may shed light on the biological functions of circRNAs in ovarian cancer. and expression of tetraspanins such as CD9, CD63, CD81 and Alix (FACS, Western Blot). Exosomes uptake was detected by FACS and confocal microscopy. Proliferation was assessed by cell count and cell metabolic assay, while migration by scratch C6 assay. miRNAs and mRNA expression level were analyzed by digital PCR and Real- NGS-based approach for a better molecular stratification of Time PCR, respectively. In silico analysis was performed by DIANA-miRPath tool. colorectal cancer patients Results: Purity of exosomes isolated from A549, LT73 and HBECs was confirmed 1)Belardinilli F. 2)Capalbo C. 3)Pisapia P. 4)Malapelle U. 5)Raimondo D. 6)Milanetti by sizing and expression of exosome-enriched proteins. HBEC-KRASV12highcells E. 7)Colella M. 8)Magri V. 9)Mezi S. 10)Coppa A. 11)Nicolussi A. 12)Valentini V. 13) treated with exo-A549 and exo-LT73 clearly showed exosomes uptake. Of note, we Petroni M. 14)Colicchia V. 15)Prete A. 16)Cortesi E. 17)Troncone G. 18)Giannini G. observed a significant proliferation increase in treated cells (fold increase: 2.3±0.4 1)Department of Molecular Medicine, University La Sapienza, Rome, Italy 2) and 1.5±0.04 for exo-A549 and exo-LT73 compared to control, respectively) that Department of Molecular Medicine, University La Sapienza, Rome, Italy 3) was confirmed by cell cycle analysis. Conversely, migration was not affected by Department Department of Public Health, “Federico II” University of Naples, Italy exosomal treatment. Analysis of cellular and exosomal-miRNAs levels revealed 4)Department Department of Public Health, “Federico II” University of Naples, miR-19b and -92a enrichment in cancer-derived exosomes. Up-regulation of the Italy 5)Department of Molecular Medicine, University La Sapienza, Rome, Italy same miRNAs was detected in treated HBECs (miR-19b fold increase: 8.6 and 6)Department of Physics, University La Sapienza, Rome, Italy 7)Department 7.6, miR-19b fold increase: 2.4 and 2.5 for exo-A549 and exo-LT3, respectively) of Molecular Medicine, University La Sapienza, Rome, Italy 8)Department of without alteration in pri-miRNAs transcription suggesting an exogenous transfer of Medical Oncology, Policlinico Umberto I “Sapienza” University of Rome, Italy 9) miR-19b and -92a. Over-expression of the two miRNAs by miRNA mimics in HBECs Department of Medical Oncology, Policlinico Umberto I “Sapienza” University of confirmed their ability to promote proliferation. These findings was supported by in Rome, Italy 10)Department of Experimental Medicine, University La Sapienza, silico analysis data which showed that miR-19b and -92a target genes are involved Rome, Italy 11)Department of Experimental Medicine, University La Sapienza, in cell cycle and cancer-related pathways. Rome, Italy 12)Department of Molecular Medicine, University La Sapienza, Rome, Conclusions: Our data demonstrate lung cancer exosomes ability to promote Italy 13)Department of Molecular Medicine, University La Sapienza, Rome, Italy HBECs proliferation and involvement of exosomal onco-miR-19b and -92a in pro- 14)Department of Molecular Medicine, University La Sapienza, Rome, Italy 15) tumorigenic features. Department of Medical Oncology, Policlinico Umberto I “Sapienza” University of Rome, Italy 16)Department of Medical Oncology, Policlinico Umberto I “Sapienza” University of Rome, Italy 17)Department Department of Public Health, “Federico II” University of Naples, Italy 18)Department of Molecular Medicine, University La Sapienza, Rome, Italy INTRODUCTION: Colorectal carcinoma (CRC) is one of the most commonly diagnosed cancers worldwide. The metastatic disease contributes to the high mortality rate reported for such tumors. Significant benefit on overall survival was brought about the introduction of monoclonal antibodies anti-EGFR and anti-VEGF used in combination with chemotherapy in metastatic CRC (mCRC). While anti- VEGF treatment does not require biomarker-based selection criteria, the potential efficacy of anti-EGFR antibodies is neglected to patients with activating mutations in KRAS and NRAS (RAS) genes, whose molecular analysis became a clinical routine. The advent of Next Generation Sequencing (NGS) instruments, able to reach quick testing of multiple clinically-relevant hotspots, yet maintaining precision and low costs, allows the simultaneous determination of the mutation status of an expanding number of genes. Despite only few of these molecular biomarkers have gained clinical utility in the routine oncological practice, the acquisition of more complex cancer mutational patterns may provide more efficient tumor characterization for prognostic and predictive purposes and highlight actionable targets.
24 25 ABSTRACT BOOK
C8 by a high incidence of relapse, which is the common cause of death in HNSCC RNA SEQUENCING APPROACH TO INVESTIGATE THE ROLE OF NEW patients. The identification of biomarkers supporting the management of HNSCC TRANSCRIPTS INVOLVED IN PLATINUM RESISTANCE IN HIGH-GRADE disease is still an unmet need in clinical oncology. SEROUS OVARIAN CANCER Meterials and methods: To this end, we designed a mutational chip for next 1)Calura E. 2)Todeschini P. 3)Paracchini L. 4)Romani C. 5)Tognon G. 6)Sartori E. 7) generation sequencing (NGS) analysis to detect mutations in tumor tissues and Odicino F. 8)Ceppi L. 9)Fruscio R. 10)Ravaggi A. 11)D’incalci M. 12)Bignotti E. 13) matched plasma of HNSCC patients. The analysis of the HNSCC TCGA cohort Marchini S. 14)Romualdi C. revealed that TP53 (72%), CDKN2A (22%) and FAT1 (24%) are the most frequently 1)Department of biology, University of Padova, Padova, Italy 2)Department of mutated genes in HNSCCs. Notably TP53 mutations associate with poor outcome. obstetrics and gynecology, “Aangelo Nocivelli” institute of molecular medicine, A cohort of 250 fresh frozen tissues from HNSCC patients has been collected. Brescia, Italy 3)Department of oncology, Irccs “Mario Negri” institute for Specifically, for each patient three biopsies representing non-tumoral (resection pharmacological, Milano, Italy 4)Department of obstetrics and gynecology, “Angelo margin), peritumor (histologically tumor-free tissue at ≥1cm from the tumor) and Nocivelli” institute of molecular medicine, Brescia, Italy 5)Division of obstetrics and tumor tissues were collected. Results:This cohort has been challenged with a gynecology, Asst spedali civili di Brescia, Brescia, Italy 6)Department of obstetrics customized mutational chip for NGS analysis of mutations that includes the entire and gynecology, University of Brescia, Brescia, Italy 7)Department of obstetrics and CDS of the TP53, CDKN2A and FAT1 genes. We found that the TP53 (743%) and gynecology, University of Brescia, Brescia, Italy 8)Clinic of obstetrics and gynecology, CDKN2A (243%) mutation frequency in tumors of our cohort was similar to that of University of Milan Bicocca, San Gerardo hospital, Monza, Italy 9)Clinic of obstetrics TGCA. While the frequency of FAT1 (38,6%) mutations was higher in our dataset. and gynecology, University of Milan Bicocca, San Gerardo hospital, Monza, Italy 10) Many of the identified FAT1 mutations have not been annotated yet. Since we have Department of obstetrics and gynecology, “Angelo Nocivelli” institute of molecular also collected matched plasma at the surgery and during follow-up, our analysis medicine, Brescia, Italy 11)Department of oncology, Irccs “Mario Negri” institute for is progressing toward the identification of mutations in ccf-DNA from patients. pharmacological, Milano, Italy 12)Department of obstetrics and gynecology, “Angelo Conclusions: This analysis is ongoing and the related data will be presented. Nocivelli” institute of molecular medicine, Brescia, Italy 13)Department of oncology, Irccs “Mario Negri” institute for pharmacological, Milano, Italy 14)Department of biology, University of Padova, Padova, Italy C10 Multigene panel testing in selected BRCA-mutation negative The most common histological subtype of epithelial ovarian cancer (EOC), the breast and/or ovarian cancer families high-grade serous ovarian carcinoma (HGSOC), is generally diagnosed late, 1)Coppa A. 2)Nicolussi A. 3)Valentini V. 4)Fratini B. 5)Capalbo C. 6)Belardinilli F. 7) when multiple synchronous tumor lesions are localized in the ovary, as well as in Colicchia V. 8)D’ Inzeo S. 9)Ottini L. 10)Giannini G. other anatomical sites within the peritoneum cavity. The five-years survival rate is 1)Experimental medicine, Sapienza university of Rome, Rome, Italy 2)Experimental less than 30%, and most patients, despite an initial response to platinum agents, medicine, Sapienza university of Rome, Rome, Italy 3)Molecular medicine, Sapienza become progressively resistant and die from the tumor that becomes incurable. The university of Rome, Rome, Italy 4)Molecular medicine, Sapienza university of conventional array-based approach studies, drawn on known transcript structures, Rome, Rome, Italy 5)Molecular medicine, Sapienza university of Rome, Rome, failed to identify biomarker transcripts for platinum resistance. Thus, with the aim to Italy 6)Molecular medicine, Sapienza university of Rome, Rome, Italy 7)Molecular discover new coding and non-coding variants of known transcripts or totally novel medicine, Sapienza university of Rome, Rome, Italy 8)Experimental medicine, transcripts, associated with the mechanism of resistance, we have sequenced the Sapienza university of Rome, Rome, Italy 9)Molecular medicine, Sapienza entire transcriptome of tumor biopsies from 14 platinum-sensitive and 14 platinum- university of Rome, Rome, Italy 10)Molecular medicine, Sapienza university of resistant patients, deriving from the Spedali Civili cohort in Brescia. Rome, Rome, Italy The transcriptome reconstruction of the 28 sequencing experiments allowed us to identify 1371 transcripts differentially expressed between pt-resistant and pt- Background: About 5-10% of the hereditary breast and/or ovarian cancer (BC/ sensitive samples. Among them, 125 transcripts showed a complete match of intron BOC) is associated with an autosomal dominant genetic susceptibility due to highly chain with known transcripts, 686 are potentially novel isoforms of known transcripts penetrant mutations of the BRCA1/2 genes. In particular, BRCA1/2 gene mutations and the remaining are novel intergenic transcripts or anti-sense transcripts with are found in 25-30% of the BC/BOC families subjected to genetic testing. These an exonic overlap with a known transcript. Interestingly, a very small part of the numbers suggest the possible involvement of other genes in BC/BOC genetic collected transcriptional alterations can be ascribed to coding-genes, suggesting a predisposition and a fraction of these cases remains to be assigned to specific prominent non-coding role in HGSEOC platinum resistance. genetic factors. Here, we report on the application of the NGS multigene panel to a Capture Sequencing of the 1371 transcripts has been performed in the same cohort group of BRCA1/2 mutation negative BC/BOC cases, in order to identify germline of 28 patients as well as in an external and independent cohort of 55 samples, mutations that could further explain BC/BOC genetic susceptibility. collected at San Gerardo Hospital, Monza and stored at Mario Negri Institute in Materials and methods: We selected a group of 26 BRCA1/2 negative BC and BOC Milan. The further analysis resulted in a list of 77 transcripts validated in both families on the basis of a clear dominant inheritance pattern and/or a moderate/ cohorts, 6 up- and 71 down-regulated in platinum-resistant compared to platinum- high BRCAPro score. We performed a genomic screening by a comprehensive sensitive. Among the 71 we found 5 known transcripts, 27 new isoforms of known multigene custom panel of 29 cancer-related genes, using Ion Torrent platform transcripts, 45 unknown transcripts with 28 that can be antisense transcripts. (Thermo Fisher Scientific). Further functional validations are ongoing to define the functional and predictive/ Results and discussion: In three cases (11,5%) we found mutations described as prognostic role of the detected transcriptional alterations. pathogenic in ATM, MUTYH and PALB2 genes. In the series analyzed, the most frequently altered gene was ATM (22,2%) but were also identified mutations in MSH6, APC and TP53 (16,7%), MUTYH (11,1%), RAD51B, EPCAM, PALB2, C9 BRIP1, CHEK2, RAD50, STK11 and BARD1 (5,6%). In particular, we found nine A MUTATIONAL CHIP TO DETECT TP53, CDKN2A AND FAT1 MUTATIONS genomic variants of conflicting interpretations of pathogenicity in TP53, MSH6, IN CIRCULATING CELL FREE-DNA OF HEAD AND NECK SQUAMOUS CELL APC, CHEK2, ATM, EPCAM and MUTYH genes; six genomic variants of uncertain CARCINOMA PATIENTS: A PILOT STUDY significance (VUS) inATM , APC, MSH6, BRIP1 and RAD50 genes; and six genomic 1)Spinella F. 2)Ganci F. 3)Cottone G. 4)Cotroneo E. 5)Nuccitelli A. 6)Biroccio A. 7) variants not reported in ClinVar in ATM, TP53, APC, RAD51B, STK11 and BARD1 Sacconi A. 8)Manciocco V. 9)Pallocca M. 10)Rollo F. 11)Covello R. 12)Benevolo M. genes, predicted deleterious by in silico analysis. Their biological significance and 13)Spriano G. 14)Fiorentino F. 15)Blandino G. involvement in the development of the pathology is still unknown today. Only eight 1)Genoma Group, Rome, Italy 2)Oncogenomic and Epigenetic Unit, Italian National patients were negative for the presence of mutations in the 29 genes analyzed. Cancer Institute “Regina Elena”, Rome, Italy 3)Genoma Group, Rome, Italy 4) Conclusions: Preliminary results of this study suggest that NGS could offer a great Genoma Group, Rome, Italy 5)Genoma Group, Rome, Italy 6)Oncogenomic and contribution to identify the genetic component of susceptibility to BC/BOC and could Epigenetic Unit, Italian National Cancer Institute “Regina Elena”, Rome, Italy 7) potentially be used with implications for clinical management and counselling of Oncogenomic and Epigenetic Unit, Italian National Cancer Institute “Regina patients and their families. Moreover, our results suggest that multigene panel Elena”, Rome, Italy 8)Otolaryngology Department, Italian National Cancer Institute testing approach may benefit appropriately selected patients, especially those with “Regina Elena”, Rome, Italy 9)Otolaryngology Department, Italian National Cancer increased risk of BC/BOC development. Institute “Regina Elena”, Rome, Italy 10)Pathology Department, Italian National Cancer Institute “Regina Elena”, Rome, Italy 11)Pathology Department, Italian National Cancer Institute “Regina Elena”, Rome, Italy 12)Pathology Department, Italian National Cancer Institute “Regina Elena”, Rome, Italy 13)Otolaryngology Department, Italian National Cancer Institute “Regina Elena”, Rome, Italy 14) Genoma Group, Rome, Italy 15)Oncogenomic and Epigenetic Unit, Italian National Cancer Institute “Regina Elena”, Rome, Italy Introduction: Head and neck squamous cell carcinoma (HNSCC) is characterized
26 27 ABSTRACT BOOK
C11 C13 SGK1 affects RAN/RANBP1/RANGAP1 via SP1 to play a critical role in pre- CAMPARISON OF TWO IN SILICO NGS BASED METHODS CHARACTERIZING miRNA nuclear export: a new route of epigenomic regulation GENOMIC INSTABILITY OF GYNAECOLOGIC TUMORS Dattilo V., D’Antona L., Catalogna G., Iuliano R., Perrotti N., Amato R. 1)López-Reig R. 2)Fernández-Serra A. 3)Romero I. 4)Garcia-Casado Z. 5)Illueca Dipartimento di Scienze della Salute, Università degli Studi “Magna Graecia” di C. 6)Poveda A. 7)López-Gguerrero J.A. Catanzaro, Catanzaro, Italia 1)Laboratory of molecular biology, Fundación instituto valenciano de oncología, Valence, Spain 2)Laboratory of molecular biology, Fundación instituto valenciano The serum- and glucocorticoid-regulated kinase (SGK1) controls cell transformation de oncología, Valence, Spain 3)Clinical area of gynocologic oncology, Fundación and tumor progression. SGK1 affects mitotic stability by regulating the expression instituto valenciano de oncología, Valence, Spain 4)Laboratory of molecular of RANBP1/RAN. Here, we demonstrate that SGK1 fluctuations indirectly modify biology, Fundación instituto valenciano de oncología, Valence, Spain 5)Pathology the maturation of pre-miRNAs, by modulating the equilibrium of the RAN/RANBP1/ department, Fundación instituto valenciano de oncología, Valence, Spain 6) RANGAP1 axis, the main regulator of nucleo-cytoplasmic transport. The levels of Clinical area of gynocologic oncology, Fundación instituto valenciano de oncología, pre-miRNAs and mature miRNAs were assessed by qRT-PCR, in total extracts Valence, Spain 7)Laboratory of molecular biology, Fundación instituto valenciano de and after differential nuclear/cytoplasmic extraction. RANBP1 expression is the oncología, Valence, Spain limiting step in the regulation of SGK1-SP1 dependent nuclear export. These results were validated in unrelated tumor models and primary human fibroblasts and Tumors with Homologous Recombination Deficiency (HRD) are more sensitive corroborated in tumor-engrafted nude mice. The levels of pri-miRNAs, DROSHA, to DNA damaging agents. Currently, mutational analysis of BRCA1/2 is the DICER and the compartmental distribution of XPO5 were documented. Experiments standard surrogate of HRD, however, it is insufficient. The introduction of targeted using RANGTP conformational antibodies confirmed that SGK1, through RANBP1, therapies in clinical management of cancer has promoted the hunt for reliable decreases the level of the GTP-bound state of RAN. This novel mechanism may molecular techniques of genomic profiling. Due to the nature of the HRD scars, play a role in the epigenomic regulation of cell physiology and fate. a comprehensive method useful for simultaneously interrogate single nucleotide and copy number variation events (CNV) is highly desirable. For that reason, the aim of this study is to develop a reliable strategy applicable in the clinical lab C12 setting. Whole exome sequencing to discover a lung cancer Mutational and CNV analysis were performed on FFPE blocks, following the predisposition in women with a previous breast cancer SureSelectXT HRD 35-genes custom panel (Agilent) plus OneSeq backbone 1)Coco S. 2)Bonfiglio S. 3)Cittaro D. 4)Genova C. 5)Vanni I. 6)Boccardo S.7) protocol, interrogating 147.000 single nucleotide polymorphism. For data analysis, Mora M. 8)Dal Bello M.G. 9)Rijavec E. 10)Alama A. 11)Truini A. 12)Rossella V. 13) two different pipelines were implemented in parallel. The first method relies on Lazarevic D. 14)Ballestrero A. 15)Grossi F. the CNVkit that uses both target and non-covered regions in order to infer the 1)Lung Cancer Unit, Ospedale Policlinico San Martino, Genova, Italy 2)Centre for normalized read counts. For the CNV calling, comparison median read counts of Translational Genomics and Bioinformatics, IRCCS San Raffaele Scientific Institute, the series was used. The second method consists on the alignment and variant Milano, Italy 3)Centre for Translational Genomics and Bioinformatics, IRCCS San recalibration with GATK pipeline. Normalization was perform based on a control Raffaele Scientific Institute, Milano, Italy 4)Lung Cancer Unit, Ospedale Policlinico sample (healthy tissue). For visualization of the results and CNV calling, saasCNV San Martino, Genova, Italy 5)Lung Cancer Unit, Ospedale Policlinico San Martino, R library was used. Genova, Italy 6)Lung Cancer Unit, Ospedale Policlinico San Martino, Genova, Italy The combination of both methods allow us to obtain a CNVs profile of each sample, 7)Department of Pathology, Ospedale Policlinico San Martino, Genova, Italy 8)Lung defining regions with statistically significant CNV or loss of heterozygosity events Cancer Unit, Ospedale Policlinico San Martino, Genova, Italy 9)Lung Cancer Unit, and the genes involved in each alteration. saasCNV method detects lower total Ospedale Policlinico San Martino, Genova, Italy 10)Lung Cancer Unit, Ospedale number of regions (n=48) compared with CNVkit method (n=78). However, the Policlinico San Martino, Genova, Italy 11)Lung Cancer Unit, Ospedale Policlinico median length of these regions is larger in the saasCNV method, 39.3 Mb [3.82- San Martino, Genova, Italy 12)Swiss Stem Cell Biotech, Vacallo, Switzerland 13) 242.55], compared with CNVkit, 25.3 Mb [0.97-147.39], being this difference Lung Cancer Unit, Ospedale Policlinico San Martino, Genova, Italy 14)Department significant (p=0.04). Although both methodologies showed very similar CNV of Internal Medicine (Di.M.I.), University of Genova, Genova, Italy 15)Lung Cancer patterns, CNVkit shows a more fragmented one due to the recalibration step used Unit, Ospedale Policlinico San Martino, Genova, Italy in saasCNV pipeline. Even so, the recalibration step is required to compensate both methodological and biological biases despite subtracting resolution. The use of Approximately 10% of women with breast cancer (BC) develop a second unrelated two different approaches serves us as validation process of the method, reinforcing tumor, and lung cancer (LC) is one of the most diagnosed across them. For decades, the reliability of the results. Although prelaminar these results are promising, need the BC radiotherapy-related effects were linked to LC occurrence in BC survivors. further analysis. However, the current radiotherapy and fractionation schedules do not apparently CNAs results, together with mutational analysis, open new possibilities in the support this assumption. Rare germline variants in highly penetrant genes such as establishment of an HRD score (profile) for treatment guidance. TP53, BRCA1/2 have been linked to cancer susceptibility. We hypothesize that BC (This research is supported by a grant from La Generalitat Valenciana; “Ayudas survivors who develop primary LC may inherit cancer predisposition by carrying rare para la contratación de personal investigador en formación de carácter predoctoral, germline variants in cancer predisposition genes. convocatoría 2015, expediente ACIF/2016/008”) Twenty-eight women with LC subsequent to BC (study patients: SP) and thirty- two females with BC and no secondary neoplasms after at least 10 years from BC diagnosis (control patients: CP) were enrolled for Whole Exome Sequencing (WES). C14 For each patient, the genomic DNA was isolated by GeneRead DNA FFPE kit from MYXOID LIPOSARCOMAS TYPE I AND III ARE CHARACTERISED BY matched BC and LC of SP and from the unaffected tissue of SP and CP. All exons DIFFERENT GENOMIC FEATURES WHICH COULD DETERMINE DIFFERENT were captured using Agilent SureSelect XT v6 and sequenced on Illumina HiSeq RESPONSE TO TRABECTEDIN TREATMENT 2500. Variant calling was performed with FreeBayes software. Somatic signatures 1)Mannarino L. 2)Craparotta I. 3)Ballabio S. 4)Frapolli R. 5)Bello E. 6)Barisella M. were calculated on all nucleotidic substitutions and burden tests were computed 7)Casali PG. 8)Pilotti S. 9)Markowetz F. 10)Marchini S. 11)D’ Incalci M. using WSS and C-a statistics. 1)Oncology, Irccs Mario Negri, Milan, Italy 2)Oncology, Irccs Mario Negri, Milan, The average age of SP at BC diagnosis was 62 years with LC occurring nearly 5 Italy 3)Oncology, Irccs Mario Negri, Milan, Italy 4)Oncology, Irccs Mario Negri, years later; 25% of patients did not receive any adjuvant radio-treatment and 46% Milan, Italy 5)Oncology, Irccs Mario Negri, Milan, Italy 6)Diagnostic pathology and were no smokers. Two main mutational signatures (S) were extracted from our tumor laboratory medicine, Fondazione Irccs Istituto Nazionale dei tumori, Milan, Italy 7) cohort and then compared to the COSMIC cancer signatures. The S1, that included Medical oncology department, Fondazione Irccs Istituto Nazionale tumori, Milan, all SP-BCs and 16/28 LCs, was found to be most similar to COSMIC-S30 (cosine Italy 8)Diagnostic pathology and laboratory medicine, Fondazione Irccs Istituto distance d = 0.53) which has been observed in a small subset of BC but unknown Nazionale tumori, Milan, Italy 9)Cancer research uk Cambridge Institute, University etiology. The S2, enriched in LC (12/28) only, positively correlated with smoking of Cambridge, Cambridge, United Kingdom 10)Oncology, Irccs Mario Negri, Milan, (10/12 patients were current/former smokers) and matched with COSMIC-S4, Italy 11)Oncology, Irccs Mario Negri, Milan, Italy linked to tobacco use. These signatures may reflect two distinct mutagenic processes underlying LC development: smoking could have played a main role in Background S2-LC subgroup while genetic predisposition could enhance LC development in S1- Myxoid/round cell liposarcoma (MLPS) is a soft tissue sarcoma whose pathogenesis LC patients. Therefore we performed a gene-based burden test over rare germline is related to the expression of FUS-CHOP which is formed by CHOP, exons 2-4, variants in S1-LC of SP versus CP identifying 249 candidate genes (FDR<0.05). and FUS, exons 1-7 in type I, or exons 1-8 in type III, and prevents adipocytic Our results report two mutational signatures underlying LC development. A validation differentiation. Trabectedin is a natural compound which has demonstrated a great step of germline data is ongoing to confirm them in an independent cohort of SP. efficacy on MLPS by restoring differentiation. To investigate the genomic features of these types and their response to drug treatment, we used patient-derived MLPS xenograft (PDX).
26 27 ABSTRACT BOOK
Methods C16 The transcriptional regulation of PDX at basal or under treatment conditions TRANSCRIPTIONAL CLASSIFICATION OF CUTANEOUS MELANOMA was analysed through microarray. The mutational profile and chromosomal IDENTIFIES THREE CONSENSUS SUBTYPES WITH DISTINCT PHENOTYPES instability were investigated through high throughput sequencing approaches at AND CLINICAL IMPLICATIONS basal conditions, under treatment with the standard drug, doxorubicin, and with Dugo M., Canevari S., Sensi M. trabectedin, at 24 and 72 hours after the first dose and 15 days after the third weekly Platform of Integrated Biology, Department of Applied Research and Technological dose. Data were processed on a high performance computing platform through a Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy public available pipeline, bcbionextgen. Results were then post-processed by integrating different bioinformatics approaches and tools for data visualisation and Cutaneous melanoma (SKCM) is a highly aggressive disease resistant to comparison. conventional treatments and characterized by poor prognosis. Targeted therapies Results against MAPK pathway and immune checkpoint inhibitors have dramatically At transcriptional level, the activation of adipogenesis in response to trabectedin improved survival of metastatic melanoma patients but the extent and duration of treatment was present in type I and not in type III in which changes in P53 and response are variable. Classification systems based on gene expression profiling apoptosis pathways were found. From the genomic point of view, type I and III have have so far allowed stratification of SKCM patients and cell lines in distinct molecular a common deletion on chromosome 19, however the general chromosomal portrait subtypes with relevant clinical implications. However clinical translation of molecular is different, since type I has two big inversions on chromosomes 1 and 12, while type subtypes is hampered by inconsistencies among the different gene expression- III has chromosome 8 completely amplified. On both models, doxorubicin does not based classification schemes. Here, through meta-analysis of public gene expression induce great rearrangements, instead, trabectedin causes many losses in almost all datasets we compare and combine eight reported classification schemes to derive chromosomes, and gains mainly on chromosomes 3, 10 and 14. Together with the the consensus transcriptional subtypes of SKCM. We show an extensive agreement canonical FUS-CHOP, there are other fusion genes characterising both typesand between the different classification systems that together define three consensus others were found induced by the treatment. transcriptional subtypes (Invasive-A, Invasive-B and Proliferative). Exploiting TCGA Conclusions multi-omics data, we demonstrate that these entities have distinctive features at Even if the two MLPS models represent the same tumour type, their transcriptional the transcriptional, epigenomic, proteomic and microenvironmental level but do not regulation program and their genomic arrangement show marked differences differ in terms of mutational load, frequency of mutated genes and copy number at basal condition. Moreover, their response to trabectedin at subsequent time aberrations. The Invasive-A subtype comprises tumors with high immune and points affects distinctive chromosomal regions and does not seem to be due to stromal infiltration and displays high expression of genes involved in epithelial to mutations on DNA repair-related genes. Further investigation on these models and mesenchymal transition and low expression of melanocyte differentiation genes. a larger cohort of MLPS is needed in order to determine whether or not the genomic Invasive-B group has a decreased level of immune infiltration and it shows heavy differences are correlated to different isoforms of FUS-CHOP. down-regulation of cell cycle genes. The Proliferative subtype is characterized by higher tumor purity, high expression of melanocyte differentiation, pigmentation and proliferation-related genes. In terms of prognosis, the Invasive-B subtype has C15 longer overall survival than Invasive-A and Proliferative groups. In each subtype, A NEW INSIGHT INTO MYC ACTION: CONTROL OF RNA POLYMERASE II patients with highly infiltrated tumors have better prognosis compared to poorly METHYLATION AND TRANSCRIPTION TERMINATION infiltrated ones, highlighting an independent role of tumor-intrinsic and immune- 1)Scagnoli F. 2)Favia A. 3)Scuoppo C. 4)Salvatori L. 5)Greenblatt J. 6)Yanling zhao related factors. Overall, combining different gene expression-based classification D. 7)Illi B. 8)Nasi S. systems we are able to identify three robust SKCM subtypes, determined at the 1)Department of biology and biotechnology, Sapienza university, Rome, Italy 2) transcriptional and epigenomic level. These consensus subtypes are supported by Institute of molecular biology and pathology, Ibpm-cnr, Rome, Italy 3)Instiute for marked biological and clinical differences and, after further refinement, could have cancer genetics, Columbia university, New york, Usa 4)Institute of molecular biology strong potential for stratification and management of SKCM patients. and pathology, Ibpm-cnr, Rome, Italy 5)Department of molecular genetics, University of Toronto, Toronto, Canada 6)Department of molecular genetics, University of toronto, Toronto, Canada 7)Institute of molecular biology and pathology, Ibpm-cnr, C17 Rome, Italy 8)Institute of molecular biology and pathology, Ibpm-cnr, Rome, Italy GENOMICS AND TRANSCRIPTOMICS OF DESMOPLASTIC SMALL ROUND CELL TUMOR (DSRCT), A RARE, AGGRESSIVE AND POORLY RESPONSIVE MYC regulates many aspects of transcription by RNA Polymerase II (RNAP II) – DISEASE activation, pause release, elongation – but its role on termination is unknown. That 1)Devecchi A. 2)De Cecco L. 3)Sensi M. 4)Pilotti S. 5)Dugo M. MYC may specifically affect termination is suggested by our finding of a functional 1)Applied Research and Technology Development, Fondazione IRCCS Istituto interaction between MYC and PRMT5, an arginine methyltransferase controlling Nazionale dei Tumori, Milan, Italy 2)Applied Research and Technology Development, gene expression and cell signaling. PRMT5 monomethylates and symmetrically di- Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Applied Research methylates arginine residues in a number of targets like histones 3 and 4, P53, and Technology Development, Fondazione IRCCS Istituto Nazionale dei Tumori, MAPK signaling components. Notably, PRMT5 symmetrically di-methylates RNAP Milan, Italy 4)Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS II at R1810, a modification that recruits proteins like SMN necessary for resolving Istituto Nazionale dei Tumori, Milan, Italy 5)Applied Research and Technology R-loops in transcription termination regions, thus allowing proper termination and Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy splicing of transcripts. Here we report that MYC overexpression in 293T cells strongly increases symmetrical RNAP II arginine di-methylation (R1810me2s) and DSRCT is a rare form of soft tissue sarcoma occurring prevalently in young men SMN recruitment. Interfering with MYC or PRMT5 expression restrains the RNAP and characterized by poor prognosis and resistance to conventional therapies. II di-methylation increase. The same result was obtained by expressing Omomyc, Beside the diagnostic t(11;22)(p13:q12) chromosomal translocation that results in a MYC inhibitory peptide that affects MYC DNA binding and protein interactions. the EWS/WT1 fusion transcript, little is known about the molecular alterations of To assess the relevance of RNAP II di-methylation control for MYC oncogenic this tumor. To gain insights into the genomics of this rare disease we performed, on functions, we investigated cancer stem cells from glioblastoma multiforme (GBM) Illumina NextSeq500, exome and transcriptome sequencing of six primary DSRCTs, harboring a doxycycline-inducible Omomyc. Glioblastoma stem cells (GSCs), which surgically removed after combined chemotherapy. are responsible for GBM resistance to therapy and relapse, overexpress both MYC Somatic mutations were identified in 137 genes of which 135 were patient-specific and PRMT5. We analyzed by ChIP-seq the RNAP II occupancy at transcription start and only two recurred in more than one case. The 30% of somatically mutated genes sites (TSSs) and termination sites (TTS) in the presence or absence of Omomyc. were also expressed whereas the remaining could not be validated due to coverage Omomyc influences RNAPII di-methylation and strongly affects RNAP II ratio of failure in RNA-sequencing data (RNA-seq). Despite high mutational heterogeneity at TSS vs TTS in most genes. This suggests that MYC may control occupancy at several mutations affected genes related to DNA damage-response network and initiation and termination regions of target genes by influencing RNAP II arginine epithelial to mesenchymal transition. Analysis of COSMIC mutational signatures di-methylation. While Omomyc affects gene expression, the relationship between ruled out that the identified mutations could be linked to pre-treatment with alkylating gene expression changes measured by RNA-seq and RNAP II occupancy changes agents. The pattern of copy number aberrations revealed a large amplification of is not a simple one. In conclusion, MYC regulates – via PRMT5 – the RNAP II the long arm of chromosome 1 (chr. 1) and complete or partial loss of chr. 6 each R1810me2s modification that influences transcription termination. This maybe co-occurring in at least 50% of cases together with several regions altered in a case- important for the balance of RNAP II occupancy at initiation and termination regions specific fashion. Analysis of RNA-seq through single sample gene set enrichment and the transcriptional output, and may contribute to the oncogenic properties of analysis showed that the samples clustered into two groups associated to chr. 6 overexpressed MYC. By affecting this pathway, Omomyc may fine-tune expression deletion and chr. 1 amplification. Samples positive for these aberrations had lower of a variety of genes, including the master transcription factors responsible for expression of inflammatory response and immune systems gene sets, and up- glioblastoma stem-like cell identity. regulation of cell cycle gene sets. This is concordant with the fact that the short arm of chr. 6 carries the major histocompatibility complex which encodes HLA class I antigens, mandatory for tumor cells to be recognized by cytotoxic T cells. Immune
28 29 ABSTRACT BOOK
deconvolution with CIBERSORT confirmed the absence of immune infiltration in were generated using Affymetrix Clariom D assays. Samples were assigned to DSRCTs, especially for samples with loss of chr. 6. claudin-low (CL), basal (B) or luminal (L) subtypes combining the BASE47 and Taken together, this study represents the first attempt to comprehensively define the BCL40 classification systems. Sample classification was further refined using the molecular features of DSRCTs to date and shows a high inter-tumor heterogeneity GSC predictor and consensus samples were identified. Pts were divided in 11 L that favors genomic instability and explains the refractory DSRCT profile. and 6 CL. Differentially expressed genes between CL-NR or R and L were analyzed using Linear Model for Microarray data Analysis (LIMMA). Gene Set Enrichment Analysis focusing on the contribution of hallmark and immune gene sets from the C18 Molecular Signatures Database were also performed in these groups. Investigating an aggressive molecular subtype of Stage I lung Results: adenocarcinoma: evidences for the acquirement of a stem-like Association between subtypes and therapeutic response was observed with all L phenotype and immunoevasion samples falling in the R group while CL were equally split between R and NR (3 Melocchi V., Dama E., Cuttano R., Colangelo T., Bianchi F. CL-R vs 3 CL-NR). ISBREMIT - Institute for Stem-cell Biology, Regenerative Medicine and Innovative In order to assess whether immune components, known to be upregulated in the Therapies, IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy CL compared to the L subtype, could further distinguish between CL-R and CL- NR, we tested differential expression of genesets corresponding to defined immune Lung cancer early diagnosis is effective in reducing the risk of metastatic disease subpopulations (FDR<0.05, FC>=2). We found a specific downregulation of immune and improve overall survival. However, a significant fraction of patients (~20%) components and, in particular, of T cells (both CD8 and CD4), NK, monocytes, with early stage disease (Stage I) experience tumor recurrence and metastatic dendritic cells and macrophages in CL-NR that render them more similar to L (all disease. Recent advances in high-throughput genomic screening allowed more in- R) than to CL-R. CL-NR were however significantly different from both CL-R and L depth analyses of molecular features of lung cancer, with a particular emphasis for the activation of several hallmark gene sets, including those related to cell cycle for the identification of prognostic gene expression signatures. Indeed, we recently and metabolism. described the existence of an aggressive lung cancer molecular subtype (Stage I), Similar results have been confirmed by Ingenuity Pathway Analysis (IPA) comparing namely C1-subtype, with a peculiar expression and mutational profile resembling the same groups. metastatic cancer (Dama et al., 2017). Conclusions: Using an integrative approach relying on computational biology and lung cancer Altogether, the results indicate that, beside subtypes classification, the combination experimental models, we now discovered that this C1-subtype is characterized by of immune landscape and metabolic pathways are relevant for the response to SGC stem-like and immune-evading phenotype. In particular, we surveyed a total of 3766 chemotherapy and such pathways will be more deeply investigated. In addition, gene-signatures representing virtually all known molecular pathways and shown that DNA copy number alterations will be studied to extend the identification of potential lung cancer C1-subtype is enriched in stem-cells expression signatures (FDR<5%). genomic response predictors to neoadjuvant therapy in MIBC. The gene expression network of C1-subtype can be activated by upstream modulators known being involved in cancer stem cells biology such as MYB, LIN28A, YAP1, TP63, FOXO1, FOXM1. In line with this, we found several iPS/Stem and EMT C20 biomarkers overexpressed in lung cancer C1-subtype and in a panel of lung cancer Radiologic and genomic evolution of individual metastases cells which we classified as ‘C1-like’ by using anad hoc developed classifier. Notably, during HER2 blockade in colorectal cancer forced overexpression of HOXB7 gene, which is a determinant of lung cancer C1- 1)Crisafulli G. 2)Siravegna G. 3) Lazzari L 4)Sartore-Bianchi A. 5) Mussolin B. 6) subtype and a member of the Antp homeobox family, promotes the expansion of Cassingena A. 7)Martino C. 8)Lanman R.B. 9)Nagy R.J. 10)Fairclough S. 11) lung sphere-forming cells and reprogramming into iPS cells (Monterisi et al., 2018). Rospo G. 12)Corti G. 13) Bartolini A. 14)Arcella P., 15)Montone M., 16)Lodi F. 17) Lastly, we estimated the abundances of immune cell types in lung tumors using Lorenzato A. 18)Vanzati A. 19)Valtorta E. 20)Cappello G. 21)Bertotti A. 22)Lonardi CIBERSORT (Newman et al., 2015), and found that lung cancer C1-subtype is S. 23)Zagonel V. 24)Leone F. 25)Russo M. 26) Balsamo A. 27)Truini M. 28)Di enriched in neutrophils (TAN), uncommited macrophages (M0) and activated mast Nicolantonio F. 29)Amatu A. 30)Bonazzina E. 31)Ghezzi S. 32)Regge D. 33)Vanzulli cells, which are all hallmark of cancer immunoediting toward immunosurveillance A. 34)Trusolino L. 35)Siena S. 36)Marsoni S. 37)Bardelli A. escaping and tumor progression. 1)Department of Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), In conclusion, we described here a novel molecular subtype characterized by a Italy 2) Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 3)Department stem-like and immunoevasion phenotype. C1 lung tumors can be identified by of Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 4) qPCR analysis using a 10-gene signature previously described (Dama et al., 2017). Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy, Our findings improve the understanding of lung cancer molecular features which 5)Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 6)Niguarda Cancer can aid the identification of more effective therapeutic strategies. Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy 7)Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 8)Guardant Health, Redwood City, CA, USA 9)Guardant Health, Redwood City, CA, USA 10)Guardant Health, Redwood C19 City, CA, USA 11) Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 12) Response to neoadjuvant sorafenib, gemcitabine and cisplatin Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 13) Candiolo Cancer (SGC) in an open-label, single-arm, phase 2 study in muscle Institute-FPO, IRCCS, Candiolo (TO), Italy 14) Candiolo Cancer Institute-FPO, invasive urothelial bladder cancer: role of molecular IRCCS, Candiolo (TO), Italy 15) Candiolo Cancer Institute-FPO, IRCCS, Candiolo subtypes, immune landscape and metabolic pathways (TO), Italy 16)Department of Oncology, University of Toni, Candiolo (TO), Italy 1)Gargiuli C. 2) Raggi D. 3)Dugo M. 4)Giannatempo P. 5)Paoli A. 6)De Cecco L. 7) 17)Department of Oncology, University of Toni, Candiolo (TO), Italy 18)Niguarda Colecchia M. 8)Daidone M.G. 9)Necchi A. 10)Sensi M. Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy 19)Niguarda 1)Department of applied research and technical development, National Institute Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy 20)Candiolo of Tumors, Milan, Italy 2)Department of Medical Oncology, National Institute of Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 21)Department of Oncology, Tumors, Milan, Italy 3)Department of Applied Research and Technical Development, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 22)Oncologia Medica National Institute of Tumors, Milan, Italy 4)Department of Medical Oncology, 1, Istituto Oncologico Veneto - IRCCS, Padova, Italy 23)Oncologia Medica 1, Istituto National Institute of Tumors, Milan, Italy 5)Department of Applied Research and Oncologico Veneto - IRCCS, Padova, Italy 24)Department of Oncology, Candiolo Technical Development, National Institute of Tumors, Milan, Italy 6)Department of Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 25)Candiolo Cancer Institute- Applied Research and Technical Development, National Institute of Tumors, Milan, FPO, IRCCS, Candiolo (TO), Italy 26)Candiolo Cancer Institute-FPO, IRCCS, Italy 7)Department of Pathology and Laboratory Medicine, National Institute of Candiolo (TO), Italy 27)Niguarda Cancer Center, Grande Ospedale Metropolitano Tumors, Milan, Italy 8)Department of Applied Research and Technical Development, Niguarda, Milan, Italy 28)Department of Oncology, Candiolo Cancer Institute- National Institute of Tumors, Milan, Italy 9)Department of Medical Oncology, FPO, IRCCS, Candiolo (TO), Italy 29)Niguarda Cancer Center, Grande Ospedale National Institute of Tumors, Milan, Italy 10)Department of Applied Research and Metropolitano Niguarda, Milan, Italy 30)Niguarda Cancer Center, Grande Ospedale Technical Development, National Institute of Tumors, Milan, Italy Metropolitano Niguarda, Milan, Italy 31)Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy 32)Candiolo Cancer Institute-FPO, IRCCS, Background: Candiolo (TO), Italy 33)Department of Oncology and Hemato-Oncology, Università Genomic analyses demonstrated that MIBC can be grouped into molecular subtypes degli Studi di Milano, Milan, Italy 34)Department of Oncology, Candiolo Cancer that have different prognosis and responses to systemic and neoadjuvant treatments. Institute-FPO, IRCCS, Candiolo (TO), Italy 35)Department of Oncology and Hemato- Neoadjuvant SGC was active in MIBC, showing a response rate (downstaging to Oncology, Università degli Studi di Milano, Milan, Italy 36)Department of Oncology, pT<2) of 54.3% in 46 patients (pts) in a phase 2 trial (NCT01222676, Necchi et al, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy 37)Department of Urol Oncol 2018). We analyzed gene expression profiles (GEP) of transurethral Oncology, Candiolo Cancer Institute-FPO, IRCCS, Candiolo (TO), Italy resections (TURB) from available pts. Methods: Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal We analyzed 25 pts, 18 responders (R) and 7 non-responders (NR). GEP profiles cancer patients, however, secondary resistance occurs in most of the cases. How
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ERBB2-amplified tumors that initially respond to HER2 blockade evolve during PIK3R1W624R PDX line. Buparlisib, a pan-class I PI3K inhibitor, showed the ability to therapy and become resistant is largely unknown. Further knowledge is crucial to block proliferation of PDTCs and the growth in vivo of PDXs in regression preclinical devise additional lines of treatment or mount combinatorial preventive strategies. trial. These data proofed-the-concept that a PDX-based pipeline is able to unveil To address this, we exploited longitudinal radiologic profiles and a unique resource actionable pathways for the treatment of advanced/metastatic ovarian cancer. of plasma samples serially collected for each patient from treatment start to progression. We also collected tissue samples prior to therapy and at resistance through a warm autopsy trial, we purposely designed to interrogate the impact of C22 drug resistance on the genomic evolution of mCRCs. The evolution of individual Methylation alteration of SHANK1 as a diagnostic and metastases during treatment in combination with cell-free circulating tumor DNA prognostic biomarker for chronic lymphocytic leukemia (ctDNA) analysis enable the identification of alterations associated with resistance 1)Loi E. 2)Moi L. 3)Fadda A. 4)Satta G. 5)Zucca M. 6)Amini Nia S. 7)Padoan M. 8) in most of the patients clinically refractory to HER2 blockade. We revealed organ Magnani C. 9)Miligi L. 10)Gentilini D. 11)Ennas M.G. 12) Cocco P. 13)Zavattari P. and metastases private-evolutionary patterns as well as a high level of intra-patient 1)Unit of Biology and Genetics, Department of Biomedical Sciences, University of molecular heterogeneity. Genomic and functional analyses on samples from eight Cagliari, Cagliari, Italy 2)Unit of Biology and Genetics, Department of Biomedical metastases of a patient co-recruited to the post-mortem study unveiled lesion- Sciences, University of Cagliari, Cagliari, Italy 3)Unit of Biology and Genetics, specific evolutionary trees and pharmacologic vulnerabilities. Using a patient- Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy 4) and lesion-specific NGS panel approach, clonal and sub-clonal alterations were Department of Medical Sciences and Public Health, Occupational Health Unit, monitored identifing not only the overall disease burden but also the contribution University of Cagliari, Cagliari, Italy 5)Department of Biomedical Sciences, of individual lesions (and their sub-clonal components) in ctDNA. We found a Cytomorphology Unit, University of Cagliari, Cagliari, Italy 6)Department of Medical remarkable correlation between the size of each lesion and its contribution to the Sciences and Public Health, Occupational Health Unit, University of Cagliari, pool of ctDNA in the blood, and patterns of expansion and reduction of the individual Cagliari, Italy 7)Department of Medical Sciences, Unit of Medical Statistics and metastases were manifest in blood and could be correlated with clinical course. We Cancer Epidemiology, University of Eastern Piedmont, Novara, Italy 8)Department asked whether and to what extent the repertoire of TCR clonotypes might reflect of Medical Sciences, Unit of Medical Statistics and Cancer Epidemiology, University tumor evolution during the emergence of therapeutic resistance. We revealed a of Eastern Piedmont, Novara, Italy 9)Institute of Oncology Studies and Prevention, high level of heterogeneity in TCR distribution and T-cell infiltration intra- and inter- Florence, Italy 10)Department of Brain and Behavioral Sciences, University of lesions, however, when metastases-specific TCR rearrangements were profiled in Pavia, Pavia, Italy 11)Department of Biomedical Sciences, Cytomorphology Unit, the blood there was no direct correlation but rather an anti-correlation with ctDNA University of Cagliari, Cagliari, Italy 12)Department of Medical Sciences and Public patterns of the corresponding lesions. Health, Occupational Health Unit, University of Cagliari, Cagliari, Italy 13)Unit of Biology and Genetics, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy C21 Identification of actionable cancer genes and treatment Introduction options for metastatic ovarian carcinomas using Patient Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. In fact, Derived Xenografts (PDXs) and PDX Derived Tumor Cells (PDTCs) while some patients have a slow progression of the disease, others experience 1)D’Ambrosio C. 2)Olivero M. 3)Erriquez J. 4)Arigoni M. 5) Capellero S. 6)Mittica G. a more aggressive form of leukemia and require adequate therapies sooner after 7)Borella F. 8)Katsaros D. 9)Privitera S. 10)Berrino E. 11)Venesio T. 12)Valabrega diagnosis. Thus, the identification of diagnostic biomarkers is needed in order to G. 13)Calogero R. 14)Di Renzo M.F. characterize patients showing different clinical courses. Aim of this work was to 1)Oncology, Irccs, Candiolo, Italy 2)Oncology, Irccs, Candiolo, Italy 3)Oncology, identify specific-CLL methylation patterns by conducting a genome-wide methylation Irccs, Candiolo, Italy 4)Oncology, Molecular biotechnology center, Torino, Italy analysis on 18 CLL cases and six non-tumoral controls, and to evaluate whether 5)Oncology, Irccs, Candiolo, Italy 6)Oncology, University of turin, Torino, Italy 7) methylation aberrations in selected genes are associated with changes in gene Ginecologia ostetricia, Sant’anna hospital, Torino, Italy 8)Ginecologia ostetricia, expression. Sant’anna hospital, Torino, Italy 9)Anatomia e istologia patologica e citodiagnostica, Materials and methods Sant’anna hospital, Torino, Italy 10)Oncology, Irccs, Candiolo, Italy 11)Pathology, The global methylation profiling was performed using Illumina HumanMethylation450 Irccs, Candiolo, Italy 12)Medical oncology ovarian cancer, Irccs, Candiolo, Italy 13) BeadChips. Methylation raw data were analyzed using RnBeads by conducting Dipartimento di biotecnologie molecolari e scienze, Molecular biotechnology center, a differential methylation analysis between CLL and non-tumoral samples. We Torino, Italy 14)Oncology, Irccs, Candiolo, Italy selected the 100-top ranked differentially methylated CpG islands and annotated them by referring to the 450k manifest. An additional differential methylation analysis Patients with advanced ovarian cancers have experienced little improvement was performed by adding the absolute lymphocyte count as a covariate. We tested in overall survival with standard treatments even after the incorporation of anti- the levels of expression of selected differentially methylated genes in blood cell angiogenic therapies. Besides anti-PARP inhibitors, matching individual critical samples of 27 CLL cases and 16 non-tumoral subjects by qRT-PCR. genomic alterations with the best available drugs has not advanced as in other Results cancers, likely because a handful of cancer-related genes are mutated at high We detected cancer specific methylation patterns characterizing CLL samples. frequency, while many more are found mutated at much lower frequencies. This so We also found a positive correlation between methylation levels and absolute called “mutation tail” is not only long but also mostly unexplored. lymphocyte count in CLL patients. Interestingly, among the tested genes, we found We used Patient Derived Xenografts (PDXs) to identify actionable cancer genes that SHANK1 showed a significant gene body hypermethylation and downregulation and PDX Derived Tumor Cells (PDTCs) to accelerate the discovery of treatment in CLL. Consistent with our findings, recent publications suggested that gene body options. We envisioned that the alleged weakness of PDX models, i.e. lack of methylation may be correlated with alternative transcript isoforms expression, and human stromal and immune cells, might be instrumental to identify mutations in it may regulate cell context-specific alternative promoters. Our preliminary findings cancer and to test approved or experimental targeted drugs as monotherapy or in suggest that there is an alteration in the pattern of expression of the different different combinations to link biomarkers to treatments. transcript isoforms of this gene in CLL. Fourty-nine PDX lines from metastatic epithelial ovarian carcinomas have been Conclusion propagated and fully characterized as far as histology, immunohistochemistry of Our study identified specific alterations in the DNA methylation of CLL cells, and epithelial and high-grade serous-specific markers and presence of TP53 and highlighted the association between gene-body methylation alteration and transcript BRCA1/2 mutations. isoform regulation in CLL. In addition, the positive correlation between methylation Copy number variations (CNV) analysis and Whole Exome Sequencing (WES) levels and the absolute lymphocyte count in CLL patients would suggest a role were carried out of 12 PDX lines derived from naïve metastatic high-grade serous of methylation markers, not only in characterizing the tumor, but also in the epithelial ovarian carcinomas. We studied non-synonymous mutations with personalized quantification of tumor aggression. allele frequencies ≥0.1. Only mutations in cancer genes listed in databases were further analyzed. SNPdb allowed ruling out polymorphisms. SIFT and PROVEAN softwares predicted deleterious or damaging effects onto the protein sequences. DGIdb helped selecting actionable genes. We identified mutations in 1-4 cancer genes in 8/12 PDX lines. In one PDX line, a possibly loss-of-function mutation of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) had an allele frequency=0.9 in early and late passages. Moreover, in two micro-dissected FFPE samples of the source tumor this mutation had an allele frequency nearly identical to that of the mutated TP53. Hence, PIK3R1W624R could be a trunk mutation in the PDX line and possibly in the human counterpart. Treatment options were assayed ex-vivo, on short-term cultures of PDTCs of the
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C23 neoplastic lesions, the level of protein oxidation, as expected, is consistently and Genetic Profiling of Malignant Pleural mesothelioma significantly higher than that of non –photoexposed areas. Unexpectedly, proteins 1)Torricelli F. 2)Lococo F. 3)Pagano M. 4)Gnoni R. 5)Veronesi G. 6)Rena O. 7) form perilesional areas were also, significantly more oxidised than those for lesinal Casadio C. 8)Novellis P. 9)Piana S. 10) Donati B. 11)Di Stefano T. 12)Pinto C. 13) areas lesional areas! Ciarrocchi A. Through Mass spectromentry analysis, over 40 differentially oxidised proteins were 1)Laboratory of translational research, Ausl_irccs, Reggio Emilia, Italy 2)Unit of identified. thoracic surgery, Ausl_irccs, Reggio Emilia, Italy 3)Oncology unit, Ausl_irccs, In lesional areas the GRP78 (78 kDa glucose-regulated protein) and the 14-3-3 Reggio Emilia, Italy 4)Oncology unit, Ausl_irccs, Reggio Emilia, Italy 5)Unit of isophorm sigma were sharply less oxidised compared with non–phoptoexposed thoracic surgery, Humanitas research hospital, Milano, Italy 6)Unit of thoracic tissues, while comparted with the perilesional tissues the proteins Nucleoline, surgery, University of Novara, Novara, Italy 7)Unit of thoracic surgery, University UV excision repair RAD23 omolog B; Perossiredoxin-2, Serine/threonin-protein of Novara, Novara, Italy 8)Unit of thoracic surgery, Humanitas research hospital, chinasi SIK3, were found more oxidised. Comparing the perilesional tissues with Milano, Italy 9)Unit of pathology, Ausl_irccs, Reggio Emilia, Italy 10)Laboratory of non-photoexposed tissues the protein Protein Disulfide Isomerase A3, Calreticulin; translational research, Ausl_irccs, Reggio Emilia, Italy 11)Unit of thoracic surgery, HSP90B1; HSP72; HSP90AB1; Tumor Protein D52; Tumor protein D54; Ubiquitin Ausl_irccs, Reggio Emilia, Italy 12)Oncology unit, Ausl_irccs, Reggio Emilia, Italy carboxyl-terminal hydrolase 5; Junction plakoglobin; Vinculin and Collagen 1A1 13)Laboratory of translational research, Ausl_irccs, Reggio Emilia, Italy were found differentially oxidised. Discussion. Introduction: Malignant Pleural mesothelioma (MPM) is a very aggressive and Reported data as a whole suggest that protein oxidation represent a further fatal form of cancer. The relative rarity of these lesions, have prevented the underexplered level of deregulation in cancer initiation and progression. identification of MPM-driver molecules, as well as the identification of specific drug to target this cancer. Morphologically, MPM can be divided in epithelioid and sarcomatoid histotype. These subtypes have different aggressive potential being the sarcomatoid phenotype more aggressive. 30% of MPM, called biphasic, present both epithelial and sarcomatoid component. This intermediate phenotype of MPM is currently poorly characterized. To improve our knowledge on the genetic events that underline MPM and in particular to define genetic alterations associated with biphasic form of MPM, we evaluated by NGS the genetic profile of a large cohort of MPM. Methods: A population of 100 MPM was available. About 70% of these lesions displayed biphasic histotype, while the remaining 30% were epithelioid. Based on the TCGA data we designed a MPM-specific gene panel, covering 33 genes frequently altered in these tumors. These panel was analyzed by NGS. Results: To date 58 MPM (30 epithelioid and 28 biphasic) were sequenced. A total of 4352 genetic alterations were found but focusing only on potentially functional mutations 251 were considered: 166 were missense, 50 led to a frameshift, 23 were splice variants and 12 caused a start loss/stop gain. All patients displayed at least one mutation and the average number of mutations per sample was 4.3 (range 1- 14). PIK3CA, RDX, MXRA5 and NF2 were the most frequently alterated genes being mutated in 72%, 43%, 32% and 19% of samples respectively. Interestingly, PIK3CA and MXRA5 showed a higher frequency of mutations in the biphasic as compared with the epithelioid subgroup. PIK3CA was mutated in 86% of biphasic and only in 60% of epithelial tumors while MXRA5 was altered in 43% of biphasic and in 23% of epithelioid tumors. Finally we focus on the biphasic group to investigate the effect of these gene mutations on overall survival. Mutations in NF2 and MXRA5 showed a trend of association with reduced median survival time (NF2, 4 vs 16 months, p=0.016) (MXRA5, 9 vs 16 months, p=0.165). Conclusion: Our data provide additional insights into the genetic of MPM suggesting a crucial role for genes that were not previously linked to this cancer. Furthermore, studying the largest set of biphasic MPM ever analyzed,we identify mutations which occurrence likely characterizes this subtype.
C24 The contribution of protein oxidative damage to UV related human skin carcinogenesis De Marco F. Ridait, Regina Eelena National Cancer Institute IRCCS, Rome, Italy Introduction The Ultra Violet (UV) component of solar radiation is the cause of Human skin cancer, a group of diseases whose incidence is steadily increasing all over the world. UV radiation has the potential to damage any molecular structure of the cell. Nonetheless the vast majority of experimental studies are focused on DNA damage while the contribution of protein oxidative damage remains largely underexplored. Yet, the alteration of protein structure and functions is a crucial event for genetic damage accumulation and cancer development. In a previous work on in vitro Human Diploid Skin Keratinocytes we showed that the UV radiation specifically induced oxidative damage on a number of proteins involved in regulatory mechanisms thus providing a causative link between UV radiation, damage repair impairing and skin cancer. Aims In this work we report about the protein oxidative damage found in solar induced skin neoplastic lesions and discuss the present results whitin the context of in vitro and ex vivo , already reported data. Results: The study was approved by the local ethical committee. 20 patients with solar neoplastic lesions gave their full informed consent to participate to the study. The total amount of protein oxidation showed that, in the areas surrounding the
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Methods CANCER MODELING AND IMAGING In vivo glioma models obtained with cells with different sensitivity to TMZ were divided in treatment groups (control, TMZ alone, metformin alone and combination D1 therapy). Mice were monitored before therapy and after 7 days using MRI and ET-1 receptor blockade in engineered 3D high-grade serous 18 18 18 ovarian cancer tumoroids PET with [ F]FLT (cell proliferation), [ F]FAZA (hypoxia) and [ F]VC701 (TSPO receptor, inflammation). At sacrifice, brains were collected and processed for 1)Rosanò L. 2)Pape J. 3)Cheema U. 4)Loizidou M. 5)Bagnato A. immunohistochemistry (IHC) analysis. 1)Unit of Preclinical Models and New Therapeutic Agents, Regina Elena National Results Cancer Institute, Rome, Italy 2)UCL Institute of Orthopaedics and Musculoskeletal In vitro metformin treatment both improved sensitivity of TMZ and overcame Sciences, UCL Division of Surgery and Interventional Science, Stanmore Campus resistance to TMZ. In vivo, combined treatment with TMZ and MET influenced 3)UCL Institute of Orthopaedics and Musculoskeletal Sciences, UCL Division of tumour growth and survival in a cell-dependent manner. Metformin alone didn’t Surgery and Interventional Science, Stanmore Campus 4)UCL Division of Surgery increase survival independently from TMZ sensitivity. Treated TMZ-sensitive tumour and Interventional Science, Royal Free Campus, London, UK 5)Unit of Preclinical displayed a significant decrease in [18F]FLT and [18F]FAZA uptake compared to Models and New Therapeutic Agents, Regina Elena National Cancer Institute, control. In TMZ-sensitive group, the addition of metformin significantly increased Rome, Italy survival and decreased [18F]VC701 uptake. Markers for ki67, HIF-1α, CD133 High-grade serous ovarian cancer (HG-SOC), accounting for 70-80% of ovarian showed variable changes in different treatment groups. cancer deaths, is characterized by widespred and rapid metastatic nature, Conclusions suggesting that unraveling determinants of metastasis may implement new In conclusion, metformin is able to enhance the efficacy of TMZ both in TMZ- therapeutic strategies on clinical care. In this tumor, autocrine or paracrine sensitive and in TMZ-resistant cell lines. The use of combined PET radiotracers activation of the endothelin-1 receptors (ET-1R) are recognized drivers of allows to evaluate changes of several cancer-related features induced by treatment. tumor progression, promoting EMT, invasion, and metastasis, as well as tumor Acknowledgement/References angiogenesis and lymphangiogenesis. However, since the metastatic spread and This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC), response to therapy are influenced by the extracellular matrix (ECM) density and project number IG15771. composition of the surrounding tumour microenvironment (TME), the development of a three-dimensional in vitro model mimicking the in vivo tumors is necessary D3 to better understand the dynamic interactions between tumor cells and TME 68Gallium-NOTA-Ahx-Peptide R54, a novel Peptide CXCR4 antagonist elements, including fibroblasts, endothelial cells and the ECM, regulated byET- for imaging of CXCR4 overexpressing solid tumors 1R. With this in mind, we have developed HG-SOC tumoroids by engineering a 1)Rea G. 2)Trotta A.M. 3)Aurilio M. 4)D’Alterio C. 5)Di Martino D. 6)Barbieri A. 7) dense central artificial cancer mass (ACM) containing HG-SOC cells, nested within Luciano A. 8)Arra C. 9)Gaballo P. 10)Di Maro S. 11)Lastoria S. 12)Scala S. an compressed hydrogel representing the stromal compartment comprising type 1)Functional Genomics, Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, I collagen, laminin and fibronectin, and stromal cells (fibroblasts and endothelial Naples, Italy 2)Functional Genomics, Istituto Nazionale Tumori, Fondazione cells). After 21 days, ET-1-stimulated HG-SOC cells showed an altered migration “G.Pascale”-IRCCS, Naples, Italy 3)Nuclear Medicine, Istituto Nazionale Tumori, pattern and formed cellular aggregates, mimicking micrometastases that invaded the Fondazione “G.Pascale”-IRCCS, Naples, Italy 4)Functional Genomics, Istituto stroma. As compared to control cells, ET-1 treated cells showed a higher number of Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, Naples, Italy 5)Nuclear cellular aggregates, which were reduced by treatment with dual receptor antagonist Medicine, Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, Naples, Italy 6) macitentan. In addition, ET-1 increased the size of the invading aggregates Animal Facility, Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, Naples, compared to control cells. These findings suggest that HG-SOC tumoroids might Italy 7)Animal Facility, Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, be useful to investigate the cross-talk between HG-SOC cells and TME in cancer Naples, Italy 8)Animal Facility, Istituto Nazionale Tumori, Fondazione “G.Pascale”- progression and in the response to ET-1R antagonist. IRCCS, Naples, Italy 9)Nuclear Medicine, Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, Naples, Italy 10)Department of Pharmacy, Università degli studi di Napoli “Federico II”, Naples, Italy 11)Nuclear Medicine, Istituto Nazionale D2 Tumori, Fondazione “G.Pascale”-IRCCS, Naples, Italy 12)Functional Genomics, Combined Positron Emission Tomography imaging approach Istituto Nazionale Tumori, Fondazione “G.Pascale”-IRCCS, Naples, Italy for identification of new potential biomarker for treatment response in glioma models CXCR4 is a G-protein-coupled receptor widely expressed on the membranes of 1)Valtorta S. 2)Raccagni I. 3)Lo Dico A. 4)Zinnhardt B. 5)Belloli S. 6)Martelli C. 7) neutrophils, lymphocytes and monocytes. In cancer, CXCR4 is overexpressed on Politi L.S. 8)Todde S. 9)Monterisi C. 10)Vaira V. 11)Jacobs A.H. 12)Ottobrini L. 13) the majority of haematological and solid cancers and its expression correlated with Moresco R.M. the metastatic capability. Recently, a direct correlation between microenvironment 1)Medicine and Surgery dept. and Tecnomed foundation, University of Milano- receptor upregulation and tumor progression, neovascularization, invasion Bicocca, Monza, Italy 2)Institute of Molecular Bioimaging and Physiology, CNR, and metastasis was demonstrated. CXCR4 is a potential imaging target for the Segrate, Italy 3)Department of Pathophysiology and Transplantation, University of diagnosis, staging and therapeutic monitoring of metastases. 68Ga-Pentixafor, a Milan, Milan, Italy 4)European Institute for Molecular Imaging (EIMI), Westfälische radiolabeled cyclic pentapeptide with high affinity to CXCR4, was recently developed Wilhelms-University Münster, Münster, Germany 5)Institute of Molecular in hematological and solid tumors. In comparison to the conventional PET with Bioimaging and Physiology, CNR, Segrate, Italy 6)Department of Pathophysiology 18F-FDG, 68Ga-Pentixafor-PET proved superior in multiple myeloma but less accurate and Transplantation, University of Milan, Milan, Italy 7)Hematology/oncology div. in solid tumors. With the intent to design a PET probe ideal for CXCR4 expressing and radiology dept., Boston Children’s Hospital, Boston (MA), USA 8)Medicine solid tumors the efficacy of recently reported CXCR4 antagonist peptide, R54 and Surgery dept. and Tecnomed foundation, University of Milano-Bicocca, (PCT/IB2011/000120/ EP2528936B1/US2013/007 9292A1) was evaluated. R54 Monza, Italy 9)Experimental Imaging Center- Nuclear Medicine div., IRCCS San selectively binds the human receptor CXCR4 (hCXCR4) with an IC50 of 20±2 nM Raffaele Scientific Institute, Milan, Italy 10)Division of Pathology, Fond. IRCCS Ca’ and 68Ga-labeled-R54, 68Ga-DOTA-Ahx-R54 and 68Ga-NOTA-Ahx-R54 as potential Granda Osp. Maggiore Policlinico, Milan, Italy 11)European Institute for Molecular PET tracer for targeting hCXCR4 in vivo were developed. DOTA and NOTA-Ahx-R54 Imaging (EIMI), Westfälische Wilhelms-University Münster, Münster, Germany 12) were synthesized by combining solid and solution-phase peptide synthesis. DOTA- Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Ahx-R54 and NOTA-Ahx-R54 derivatives bind CXCR4 with high affinity (IC50 values 13)Medicine and Surgery dept. and Tecnomed foundation, University of Milano- of 107±39.6 and 35.8±11.1 nM respectively) in 125I-CXCL12 competitive binding Bicocca, Milan, Italy assay on CCRF-CEM cells overexpressing CXCR4. Biodistribution studies and PET imaging were carried out in nude mice bearing subcutaneous Chinese Hamster Introduction Ovary cells (CHO) transfected with hCXCR4 (CHO-hCXCR4) vs non transfected Glioblastoma multiforme (GBM), the most aggressive brain tumour, carries a cells (wt-CHO). Biodistribution studies revealed high and specific tumor uptake in poor prognosis with a 27% 2-years survival rate and a median survival of 10–11 CHO-hCXCR4 xenograft model at 45min p.i. of 68Ga-NOTA-Ahx-R54 (4.29%ID/g) months with standard treatment (cytoreduction associated to radiotherapy, RT, plus compared to 68Ga-DOTA-Ahx-R54 (1.5%ID/g). Competition with cold R54 reduced chemotherapy with Temozolomide, TMZ). Despite the low rate of survival, alternative by approximately 80% and 88% respectively. Both tracers showed rapid clearance, treatment does not exist. The combined use of chemotherapeutic agents with drugs predominant renal excretion and considerable accumulation in the bladder. The PET/ targeting cell metabolism is becoming an interesting therapeutic option for cancer. CT imaging revealed that both 68Ga-NOTA-Ahx-R54 and 68Ga-DOTA-Ahx-R54 uptake Metformin, a biguanide currently used for diabetes II patients, displays anticancer in CHO-hCXCR4 xenograft was CXCR4 specific. 68Ga-NOTA-Ahx-R54 is a suitable effects probably mediated by Adenosine Monophosphate-Activated Protein Kinase tracer for targeting and imaging of human CXCR4 expression in vivo. Studies in (AMPK) activation and mTOR signaling inhibition. Here, we evaluated the response PES43, human melanoma xenograft, are ongoing to demonstrate the efficacy of new of GBM mouse models to metformin and TMZ using combined Positron Emission probes in detecting CXCR4 melanoma primary and secondary lesions. Tomography (PET) imaging.
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D4 hyaluronidase that selectively degrades stromal hyaluronan (HA), in combination IN VIVO OPTICAL IMAGING: A RELIABLE TECHNIQUE TO EVALUATE with paclitaxel (PTX) in ovarian cancer xenografts. MULTIPLE ASPECTS OF BREAST CANCER Parental SKOV3 or HA overexpressing SKOV3-HAS3 cells were injected s.c. in 1)Diceglie C. 2)Martelli C. 3)Lo Dico A. 4)Bertoli G. 5)Cava C. 6)Maffey A. 7) nude mice. After randomization mice were treated with PTX 20 mg/kg i.v. weekly Lucignani G. 8)Bianco F. 9)Castiglioni I. 10)Ottobrini L. for three times alone or with PEGPH2 0 0.1 mg/kg, given 24 h before each PTX 1)Department of Pathophysiology and Transplantion, Univerisity of Milan, Segrate dose. Antitumor activity was expressed as T/C% where T and C are the mean tumor (MI), Italy 2)Department of Pathophysiology and Transplantion, Univerisity of volume of treated and control mice, respectively. To evaluate the pharmacokinetics Milan, Segrate (MI), Italy 3)Department of Pathophysiology and Transplantion, of PTX, samples were collected at 1, 3, 6, 24 and 48 h after treatment. Mass Univerisity of Milan, Segrate (MI), Italy 4)Institute of Molecular Bioimaging and spectrometry imaging (MSI) was performed to visualize PTX distribution within Physiology, National Research Council (IBFM-CNR), Segrate (MI), Italy 5) Institute tumor tissue. A threshold analysis was performed to calculate the percentage of of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), pixels positive to PTX signal in each tumor slide. Segrate (MI), Italy 6)Neuro-Zone srl, OpenZone, Bresso (MI), Italy 7)Department In the HA rich SKOV3-HAS3 model the combination with PEGPH20 improved of Health Sciences, University of Milan, Milan, Italy 8)Fondazione Fernando PTX activity. Best T/C were 28% and 57% for the combination and PTX alone, Santarelli, Neuroinflammation Lab, Milano, Italy 9)Institute of Molecular Bioimaging respectively. Instead, PEGPH20 did not enhance treatment efficacy in the parental and Physiology, National Research Council (IBFM-CNR), Segrate (MI), Italy 10) SKOV3 xenograft, which has low levels of HA. PTX concentrations in plasma and Department of Pathophysiology and Transplantion, Univerisity of Milan, Segrate tumor after administration of PTX alone or in combination were similar in both (MI), Italy models. MSI showed a more homogeneous PTX distribution in SKOV3 tumors (86% of positive pixels) than in SKOV3-HAS3 tumors (38% of positive pixels). In the Introduction: Breast cancer is one of the most common cancers and the most frequent SKOV3-HAS3 model, PEGPH20 improved PTX distribution, that became similar to in women. Two important nodes in breast cancer knowledge are the understanding that of the parental one (74% of positive pixels). of the role of microenvironment (TME) and the identification of molecular features In conclusion, PEGPH20 pre-treatment increases PTX antitumor activity and useful for precise grading. Bioluminescence (BLI) and Fluorescence (FLI) non- influences PTX tumor distribution in HA-rich SKOV3-HAS3. Drug distribution is invasive imaging can allow fast, easy and reliable procedures to in vivo assess more homogeneous when PTX is given in combination with PEGPH20, suggesting various aspects of cancerogenesis, using both specific reporter genes and injectable that a remodeling of the extracellular matrix may improve drug penetration. This probes. In fact, it is possible to monitor tumour growth, the response to treatments, effect explains the increased efficacy observed with the combination. the expression and activity of specific proteins important in tumour progression. Methods: The effect of Mesenchymal Stem Cells (MSCs) and miRNA-567 modulation were studied in vitro and in vivo. For in vivo experiments, female mice were orthotopically injected in mammary fat pad with breast cancer cells (SUM159 and MDA-MB-231), previously engineered for the constitutive expression of luciferase gene. Moreover, MDA-MB-231 cells were also engineered to stably overexpress miR-567. A fluorescent probe was used in SUM-159 bearing mice for angiogenesis study. The IVIS Spectrum CT system was used to perform in vivo BLI and FLI 2D and 3D acquisitions on small animals. Results: The MSCs, a crucial component of TME, exert a pro-tumoral effect on breast cancer cells. In vitro assay showed that, by co-culturing MSCs and breast cancers cells, there was an increase in breast cancer cell number compared to control. Pharmacological blockage of the crosstalk between MSCs and breast cancer cells allowed to prevent this phenomenon. Optical imaging in SUM-159 bearing mice allowed the assessment of MSCs influence on breast cancer tumours, showing that MSC presence in TME exert a pro-tumoral effect by increasing tumour growth and angiogenesis. In the second model, optical imaging can also be used to non-invasively monitor the effect of a specific miR upregolation in MDA-MB-231 tumour growth. In fact, breast cancer cells overexpressing miR567 (whose importance has been previously identified by bioinformatics study), induced a lower growth rate compared to MDA control cells as well as in silico and in vitro studies suggested Conclusions: These data demonstrate the reliability of bioluminescence imaging in evaluating breast cancer response to different stimuli and growth. This approach allows in vivo longitudinal monitoring of tumour growth and features, confirming its usefulness, versatility and reliability.
D5 PEGylated hyaluronidase (PEGPH20) improves the antitumor activity of paclitaxel in a hyaluronan overexpressing ovarian carcinoma xenograft by influencing its tumor distribution 1)Ponzo M. 2)Morosi L. 3)Falcetta F. 4)Bello E. 5)Fuso Nerini I. 6)Zucchetti M. 7) Kang D. 8)D’Incalci M. 9)Frapolli R. 1)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 2)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 3)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 4)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 5)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 6)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 7) Halozyme, San Diego, CA 8)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 9)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy Ovarian cancer is the most lethal gynecologic malignancy. Most patients respond to the first-line therapy based on platinum and taxane but the majority relapse within 2 years. A fact that can contribute to resistance is the poor drug penetration in tumor tissue. The extracellular matrix, together with anomalous blood and lymphatic vessels, may cause the increase of the interstitial fluid pressure within the tumor impairing the drug distribution. Modification of the desmoplastic stroma could be a strategy to improve drug tumor penetration and to increase chemotherapeutic efficacy. Here we have tested PEGPH20, a PEGylated recombinant human
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to generate the entire progeny of short-lived, differentiated and heterogeneous CANCER STEM CELLS cells that compose the tumor bulk. Ovarian cancer stem cells (OCSC), in particular, E1 have been proposed to drive and sustain tumor dissemination, recurrence and WNT signaling modulates PD-L1 expression in stem cell chemoresistance, most likely due to the slow cycling rate and the detoxifying compartment enhancing immune-evasion of triple negative molecular machineries. This provides the rationale to characterize OCSC as ideal breast cancer targets for OC eradication. 1)Faraci S. 2)Castagnoli L. 3)Romero Cordoba S. 4)Cancila V. 5)Chiodoni C. 6) We have established a repository of patient-derived primary cells isolated either Talarico G. 7)Milani M. 8)Fasano E. 9)Volpari T. 10)Iorio M.V. 11)Tagliabue E. 12) from OC or from its tissues of origin. The OCSC subpopulation and their normal counterpart were then derived from primary cultures and used to profile their specific Tripodo C. 13)Sangaletti S. 14)Di Nicola M. 15)Pupa S.M. transcriptome. Among the genes differentially expressed in OCSC, we focused on 1)Molecular Targeting Unit, Department of Research, Fondazione IRCCS Istituto CD73, which encodes a membrane-associated 5’-ectonucleotidase and has been Nazionale dei Tumori, Milan, Italy 2)Molecular Targeting Unit, Department of implicated in tumor immune escape. Genetic ablation experiments revealed that Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3) CD73 acts as a driver of OC cell stemness and tumor initiation. Furthermore, the Molecular Targeting Unit, Department of Research, Fondazione IRCCS Istituto pharmacological inhibition of CD73 reduced OCSC self-renewal and tumorigenesis, Nazionale dei Tumori, Milan, Italy 4)Tumor Immunology Unit, Department of Health highlighting the druggability of CD73 in the context of OCSC-directed therapies. The Science, Human Pathology Section, University of Palermo School of Medicine, biological function of CD73 in OCSC required its enzymatic activity and involved Palermo, Italy 5)Molecular Immunology Unit, Department of Research, Fondazione adenosine signaling. Mechanistically, CD73 promotes the expression of stemness IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Molecular Immunology Unit, and epithelial-mesenchymal transition-associated genes, implying a regulation of Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, OCSC function at the transcriptional level. Italy 7)Molecular Immunology Unit, Department of Research, Fondazione IRCCS In summary, CD73 is involved in OCSC biology and may represent a therapeutic Istituto Nazionale dei Tumori, Milan, Italy 8)Molecular Targeting Unit, Department target for innovative treatments aimed at OC eradication. In this context, CD73- of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9) targeted therapies may combine the benefit of OCSC neutralization with Immunotherapy and Innovative Therapeutics Unit, Department of Medical Oncology, overcoming CD73-driven immune escape, resulting in a synergistic effect Fondazione IRCCS Istituto Nazionale, Milan, Italy 10)Molecular Targeting Unit, towards OC eradication. Furthermore, CD73 neutralization might help preventing Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, OCSC-dependent chemoresistance, thus enhancing the therapeutic efficacy of Italy 11)Molecular Targeting Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 12)Tumor Immunology Unit, Department conventional chemotherapy. The therapeutic potential of CD73-based treatments of Health Science, Human Pathology Section, University of Palermo School of and the molecular function of CD73 in OCSC are currently under investigation. Medicine, Palermo, Italy 13)Molecular Immunology Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 14)Immunotherapy E3 and Innovative Therapeutics Unit, Department of Medical Oncology, Fondazione 4SC-202 (DOMATINOSTAT), A NOVEL HISTONE DEACETYLASE INHIBITOR, IRCCS Istituto Nazionale, Milan, Italy 15)Molecular Targeting Unit, Department of IMPROVES CHEMOTHERAPY EFFICACY AND OVERCOMES DRUG Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy RESISTANCE IN PANCREATIC CANCER MODELS Triple negative breast cancers (TNBCs) are characterized by a poor prognosis and Roca M.S., Leone A., De Rienzo S., Moccia T., Di Gennaro E., Budillon A. lack of specific-targeted agents, thus new therapeutic strategies represent a clinical Experimental Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. challenge. Anti-PD-1 and anti-PD-L1 inhibitors have shown significant efficacy Pascale - Napoli, Italy against various solid tumors, however the susceptibility of TNBCs to immunotherapy remain uncertain. In this study we deeply investigated cancer stem cell (CSC) Pancreatic cancer is the fourth leading cause of cancer-related death in developed programs and the expression of PD-L1 in TNBCs. Human TNBCs molecularly countries. Although new standard first line regimens, such as FOLFIRINOX and stratified for high levels of PD-L1 (PD-L1High) resulted significantly enriched in the gemcitabine combined with nab-paclitaxel, have improved overall survival, the expression of immune and cancer stemness gene pathways compared to cases prognosis of this disease is still very poor with 5-year survival rate of 8%. Thus new with low PD-L1 expression (PD-L1Low), and PD-L1High cases were also positively treatment options in pancreatic cancer represent a critical medical need. Epigenetic associated with high stemness score (SSHigh). TNBC cell lines gated for ALDH and alterations play an important role in initiation and progression of several cancers, CD44 expression, two canonical cancer stemness biomarkers, exhibited higher including pancreatic cancer and epigenetic manipulation, particularly by histone levels of PD-L1 than the ALDH-negative and CD44Low counterparts, and PD-L1High deacetylase inhibitors (HDACi), has emerged as an attractive novel anticancer TNBC cells generated significantly more mammospheres (TNBCSCs) than those treatment. In order to improve therapeutic efficacy of conventional treatment, in this PD-L1Low. Moreover, human TNBC samples contained morphologically athypical study we test potential combination strategies of 4SC-202 (domatinostat), a new tumor elements co-expressing PD-L1 with both ALDH1A1 and/or CD44v6 cancer class I HDACi currently in clinical development, plus chemotherapy (CT) regimens stemness biomarkers, and 104 murine mammary tumor cells sorted for PD-L1High such as gemcitabine/paclitaxel (GP) or 5’DFUR/SN38 (capecitabine and irinotecan expression grafted in vivo in 75% of injected syngeneic immune-competent mice, metabolite, respectively) (CAPIRI), in pancreatic cancer models. First, we evaluated whereas PD-L1Low cells administered at the same cell multiplicity did not graft. the antitumoral activity of 4SC-202, as single agent, demonstrating inhibition of Furthermore, the treatment of TNBC cells with a selective WNT signaling inhibitor tumor growth, evaluated as antiproliferative and anticlonogenic effects, as well or activator respectively down- or up-regulated PD-L1 expression supporting that as G2 phase cell cycle block and induction of apoptosis and necrosis in Panc28, PD-L1 expression in the SSHigh TNBC cells is driven by Wnt genes. Notably, we Panc1 and AspC1 cell lines. Then, by combination indexes (CI) evaluated by the also observed an in vivo intra-tumor close contact of PD-L1-positive murine and Chou-Talalay method, we showed a synergistic inhibition of cell growth induced by human mammary morphologically athypical neoplastic elements and CD3/CD8/ 4SC-202 in combination with GP or CAPIRI, compared to 4SC-202 or CT alone, PD-1-positive T-cells. Overall, our study implies that the expression of PD-L1 in in all three cell lines. Synergistic antitumor effect was confirmed by apoptosis and TNBCSCs orchestrated by Wnt signaling could represent a still unknown and necrosis induction as well as by clonogenic and sphere formation assays. Notably, targetable mechanism of immune-evasion. the best synergistic antitumor effect was obtained in sequential treatment with the HDACi administered 24h before CT, compared to concomitant treatment. Since we found that 4SC-202 alone reduced stem features evaluated by sphere formation E2 assay and by RT-PCR of stem cell markers, we hypothesize that by targeting CSC- CD73: a new driver and a therapeutic target in ovarian cancer enriched population and reducing tumor cells apoptotic threshold, this HDACi can stem cells potentiate CT. Additional studies are ongoing to test this combination strategy 3D co- 1)Lupia M. 2)Sachsenmeier K.F. 3)Colombo N. 4)Fabrizio Bianchi F. 5)Cavallaro U. culture models of tumor and stroma cells and in vivo in pancreatic cancer xenograft 1)Unit of Gynecological Oncology Research, European Institute of Oncology, model in nude mice. Overall our preliminary data suggests a novel combinatorial Milan, Italy 2)MedImmune, Gaithersburg, MD, USA 3)Division of Gynecologic therapeutic strategies aimed to improve efficacy and to overcame resistance of Oncology, European Institute of Oncology, Milan, Italy 4)Institute for Stem Cell commonly used chemotherapeutics in advanced pancreatic cancer. Biology, Regenerative Medicine and Innovative Therapies, IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy 5)Unit of Gynecological Oncology Research, European Institute of Oncology, Milan, Italy Epithelial ovarian carcinoma (OC) is the most lethal gynaecological tumor in developed countries, with ~4500 new cases and 3000 deaths occurring annually in Italy. The high mortality of OC is essentially due to the frequency of tumor relapse and to acquired chemoresistance. Many biological and clinical features of OC support the notion that the disease is driven by a subpopulation of self-renewing cancer stem cells (CSC), that are able
34 35 ABSTRACT BOOK
E4 a shorter relapse-free survival. CHRONIC MYELOID LEUKEMIA STEM CELLS ARE SENSITIVE TO THE 4T1 tumorspheres transfected with a siRNA specific to TENM4 showed a decrease PHARMACOLOGICAL INHIBITION OF ERK5 PATHWAY in TENM4 mRNA and protein levels, which was reflected by a significant impairment 1)Rovida E. 2)Tusa I. 3)Cheloni G. 4)Gozzini A. 5)Deng X. 6)Gray N.S. 7)Li S. 8) of tumorsphere-forming ability. TENM4 silencing also led to a decrease in Focal Dello Sbarba P. Adhesion Kinase (FAK) phosphorylation, which has been linked to CSC biology, 1)Department of Experimental and Clinical Biomedical Sciences, University of thus strengthening the possible link between TENM4 and a CSC-like phenotype. Florence and Istituto Toscano Tumori, Florence, Italy 2)Department of Experimental Overall, our results indicate that the stem-like status of TNBC cells is accompanied and Clinical Biomedical Sciences, University of Florence and Istituto Toscano Tumori, by altered regulation of apoptosis, cell cycle and lipid metabolism pathways. Florence, Italy 3)Department of Experimental and Clinical Biomedical Sciences, Furthermore, we identified TENM4 as a potential novel player in CSC biology, and its University of Florence and Istituto Toscano Tumori, Florence, Italy 4)Hematology potential role as a novel target to improve the outcome of TNBC patients in the future. Unit, AOU Careggi, Florence, Italy 5)Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 6)Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 7)Department of Medicine, University of Massachusetts Medical E6 School, Worcester, MA, USA 8)Department of Experimental and Clinical Biomedical Sam68 modulates the response to DNA repair inhibitors in Sciences, University of Florence and Istituto Toscano Tumori, Florence, Italy breast cancer stem cells 1)Turdo A. 2)Gaggianesi M. 3)Chinnici A. 4)Apuzzo T. 5)Veschi V. 6)Scavo E. 7) Tyrosine kinase inhibitors (TKi) targeting BCR/ABL are very effective for the Nicotra A. 8)Mangiapane L.R. 9)Bianca P. 10)Di Franco S. 11)Stassi G. 12)Todaro M. treatment of Chronic Myeloid Leukemia (CML). However, discontinuation and/ 1)Department of Surgical and Oncological Sciences, University of Palermo, Italy 2) or inefficacy on CML leukemia stem cells (LSC) may lead to relapse. To identify Department of Surgical and Oncological Sciences, University of Palermo, Italy 3) new druggable targets alternative to BCR/ABL, we investigated the role of the Department of Surgical and Oncological Sciences, University of Palermo, Italy 4) Extracellular signal-Regulated Kinase 5 (ERK5) pathway in CML LSC maintenance. Department of Surgical and Oncological Sciences, University of Palermo, Italy 5) KCL22 and K562 CML cell lines, patient-derived CML cells or CD34+ peripheral Department of Surgical and Oncological Sciences, University of Palermo, Italy 6) blood cells from healthy donors (informed consent) were incubated in normoxic Department of Surgical and Oncological Sciences, University of Palermo, Italy 7) or hypoxic (0.1% O2) primary cultures (LC1) in the presence or the absence of Department of Surgical and Oncological Sciences, University of Palermo, Italy 8) drugs. At the end of incubation (day 7), cells were analyzed on a flow cytometer to Department of Surgical and Oncological Sciences, University of Palermo, Italy 9) determine the expression of stem cell markers or transferred to drug-free normoxic Department of Surgical and Oncological Sciences, University of Palermo, Italy 10) secondary cultures (LC2) to measure LC2 repopulation as a read-out of progenitor/ Department of Surgical and Oncological Sciences, University of Palermo, Italy 11) stem cell potential (CRA assay). In the serial Colony Formation Ability (CFA) assay Department of Surgical and Oncological Sciences, University of Palermo, Italy 12) colonies were scored on day 7 of each passage (III passages). In the Long-Term Department of DiBiMIS, University of Palermo, Italy Culture-Initiating Cells (LTC-IC) assay the number of colonies was scored after 14 days. Compounds: XMD8-92 (ERK5 inhibitor) and BIX02189 (MEK5 inhibitor); Background: Breast cancer (BC) is the most common cancer among women imatinib and dasatinib (BCR/ABL inhibitors). worldwide. Although considerable improvements have been made in BC survival In CML patient-derived cells and cell lines we found that the MEK5/ERK5 pathway rates, inter- and intra-tumor heterogeneity impedes the efficacy of current standard is active and necessary for optimal proliferation in low oxygen, a condition typical therapies. Recurrence and relapse are commonly caused by the emergence of of normal hematopoietic and leukemic stem cell niches. Treatment of primary a remnant cancer stem cell (CSCs) population, following the administration of CML cells with XMD8-92 or BIX02189, but not with TKi, strikingly reduced Culture standard treatments. CSCs are intrinsically resistant to anti-cancer compounds due Repopulation Ability (CRA), serial Colony Formation Ability and Long-Term Culture- to higher expression of ABC transporters, anti-apoptotic factors and a proficient Initiating Cells (LTC-IC). Importantly, inhibition of MEK5/ERK5 was effective on CML DNA damage response (DDR). Hence, CSCs are optimal candidates for DNA repair cells regardless of the presence or absence of imatinib (IM), and did not reduce targeted therapeutics. CRA or LTC-IC of normal CD34+ cells. Interestingly, in hypoxia, combined treatment Sam68 is an RNA/DNA binding protein involved in a plethora of biological processes XMD8-92/IM decreased the expression of genes relevant for stem cell maintenance and plays a crucial role in BC onset and progression. Most recently, a novel function such as c-MYC, SOX2 and NANOG and the expression of CD26, a CML LSC of Sam68 in DDR has been unveiled. Sam68 is recruited to the DNA and, together marker. with PARP1, orchestrates DNA repair and modulates the expression of anti-apoptotic The combination of ERK5 pathway inhibition with TKi appears capable to target CML genes. Of note, BCs might benefit from treatment with PARP-inhibitor based therapy stem cells as well as the overall CML cell population, thus appearing a promising regardless of the BRCA mutational status. Hence, we propose that targeting the strategy to prevent relapse of disease and at the same to contribute to the TKi- molecular effectors sustaining an efficient DDR, such as Sam68, PARP and Rad51, driven induction of remission. could serve as a powerful therapeutic strategy for advanced BCs. Material and methods: Breast CSC were obtained from mechanical and enzymatic digestion of human BC specimens. Sam68 knockdown (shSam68) was generated E5 by cells lentiviral transduction followed by puromycin selection. BC cells (3 x 105) COMPARATIVE TRANSCRIPTOMICS OF TRIPLE NEGATIVE BREAST CANCER were suspended in 1:6 matrigel and injected in the mammary fat pad of NOD/SCID STEM CELLS AND DIFFERENTIATED TUMOR CELLS IDENTIFIES TENEURIN-4 mice. AS A NOVEL CSC-ASSOCIATED MOLECULE. Results: Tissue microarray (TMAs) analysis showed that high expression levels Ruiu R., Arigoni M., Riccardo F., Conti L., Lanzardo S., Calogero R.A., Cavallo F., of Sam68 are associated with a reduced distant relapse free survival in the most Quaglino E. aggressive subtypes of BCs (p=0.008). Stable knockdown of Sam68 reduced Molecular biotechnology and health sciences, University of Turin, Turin, Italy the proliferation, invasiveness and in vivo tumor initiating capabilities of triple negative BC (TNBC) cell lines. Moreover, shSam68 TNBC cells are more sensitive Triple-negative breast cancer (TNBC) is insensitive to the most effective therapies to chemotherapeutic agents as compared to control. However, BCSCs resulted for other breast cancers, including endocrine and Her2-directed therapies. The lack inherently resistant to DNA damage agents due to the high expression levels of of specific treatments prompted us to search for TNBC-associated molecules to Rad51 and Sam68, thus underlying the importance of interfering with DNA repair be used as targets for cancer therapy. As patients with TNBC usually experience mechanism as a promising strategy to counteract CSCs survival. a quicker relapse and metastatic progression compared to other breast cancer Conclusions: Herein, we show that Sam68 fosters proliferation and invasive subtypes, we hypothesized that cancer stem cells (CSC) could play a central role in behavior of BC cell lines as well as patient-derived breast CSCs and mediates the TNBC. We thus directed our focus on genes differentially expressed between CSC recovery from DNA damaging agents. and differentiated cancer cells of TNBC cell lines. We established tumorsphere cultures from mouse and human mammary cancer cell lines to enrich the CSC population. RNA-Seq was used to identify differences in gene expression between tumorspheres and their monolayer counterparts. Seventy-four transcripts were found up-regulated in the tumorspheres, while forty- two genes were down-regulated. Enrichment analysis of biological processes showed an up-regulation in genes involved in regulation of apoptosis, and a down-regulation in genes involved in lipid metabolism and cell cycle regulation in tumorspheres as compared to their monolayer counterparts. By focusing on up-regulated genes coding for cell membrane-associated proteins, we selected Teneurin-4 (TENM4) as a candidate for further studies. Meta-analysis of publicly available datasets revealed that TENM4 mRNA is up-regulated in both lobular and ductal invasive carcinoma specimens compared to normal breast, and that high expression of TENM4 in TNBC patients shows a trend of correlation with
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E7 weeks. CSC propagation, GSH levels (HPLC analysis) and metabolic parameters Disrupting glutamine metabolism: an opportunity to defeat (biochemical analyses) were analyzed. breast cancer stem cells In the CSCs originating from HTLA-ER, 5-week treatment with sulfasalazine or 1)Gaggianesi M. 2)Chinnici A. 3)Turdo A. 4)Apuzzo T. 5)Stassi G. 6)Todaro M. PKC-alpha inhibitor plus etoposide reduced cell propagation and GSH levels, 1)Laboratory of Cellular and Molecular Oncology, Department of Surgical and also promoting anaerobic glycolytic metabolism whereas in the CSCs originating Oncological Sciences, University of Palermo, Palermo, Italy 2)Laboratory of from parental HTLA cells, similar effects were obtained already after 2 weeks of Cellular and Molecular Oncology, Department of Surgical and Oncological etoposide but co-treatments with sulfasalazine or PKC-alpha inhibitor did not modify Sciences, University of Palermo, Palermo, Italy 3)Laboratory of Cellular and these results. Collectively, our data suggests that targeting CD44/xCT by reducing Molecular Oncology, Department of Surgical and Oncological Sciences, University GSH levels and favoring the anaerobic metabolism, could be an effective strategy to of Palermo, Palermo, Italy 4)Laboratory of Cellular and Molecular Oncology, sensitize neuroblastoma stem cells to etoposide-induced cytotoxic effects (Grants Department of Surgical and Oncological Sciences, University of Palermo, Palermo, from Genoa University). Italy 5)Laboratory of Cellular and Molecular Oncology, Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy 6)Department of Internal and Specialist Medicine (DIBIMIS), University of Palermo, Palermo, Italy E9 Differentiation of colorectal cancer stem cells affects the Background content and the effects of released exosomes Notwithstanding improvement in early diagnosis and treatment ameliorated 1)Lucchetti D. 2)Colella F. 3)Calapà F. 4)Perelli L. 5)Vincenzoni F. 6)Marzano V. 7) life expectancy of BC patients, metastatic disease lacks effective therapeutic Ricciardi tenore C. 8)Guttieri L. 9)Fiori ME. 10)Castagnola M. 11)Crucitti A. 12)De approaches. Resistance to anti-cancer therapy results from the refractoriness of a maria R. 13)Sgambato A. subpopulation of cancer stem cells (CSCs). 1)Institute of general pathology, Università cattolica del sacro cuore, Rome, Italy Breast cancer is the most common malignancy in women worldwide and the second- 2)Institute of general pathology, Università cattolica del sacro cuore, Rome, Italy leading cause of cancer-related death worldwide. The onset and progression of 3)Institute of general pathology, Università cattolica del sacro cuore, Rome, Italy breast cancer depends on the ability of a small population of cancer cells, called 4)Institute of general pathology, Università cattolica del sacro cuore, Rome, Italy CSCs, which are endowed with tumor-initiating and metastasis formation capabilities 5)Institute of biochemistry and clinical biochemistr, Università cattolica del sacro and chemoresistance. Tumor metabolism is heterogeneous and cancer cells could cuore, Rome, Italy 6)Institute of biochemistry and clinical biochemistr, Università replace glucose with glutamine or fatty acids as sources of energy. Despite being cattolica del sacro cuore, Rome, Italy 7)Institute of general pathology, Università a non essential amino acid, glutamine becomes ‘conditionally essential’ since its cattolica del sacro cuore, Rome, Italy 8)Institute of general pathology, Università synthesis is not sufficient in cancer cells, becoming reliant on its exogenous supply. cattolica del sacro cuore, Rome, Italy 9)Department of oncology and molecular Results medicine, Istituto superiore di sanità, Rome, Italy 10)Institute of biochemistry and To understand the importance of glutamine in cancer metabolism, we explore clinical biochemistr, Università cattolica del sacro cuore, Rome, Italy 11)Department whether glutamine starvation or glutaminase inhibition may affect breast CSCs of surgery, Università cattolica del sacro cuore, Rome, Italy 12)Institute of general growth. pathology, Università cattolica del sacro cuore, Rome, Italy 13)Institute of general We inhibit glutamine metabolism by using CB-839, a glutaminase inhibitor (GLSi) pathology, Università cattolica del sacro cuore, Rome, Italy under clinical evaluation. GLSi hampers the proliferation rate of glutamine- dependent breast cancer cell lines, whereas glutamine-independent cells show Introduction: exosomes are extracellular vesicles involved in inter-cellular a behavior similar to breast CSCs, reflecting the presence of a tumor metabolic communication and largely mirroring the molecular profile of the originating cells. heterogeneity. Moreover, glutaminase inhibition sensitizes glutamine-dependent We previously reported that NaBu-induced differentiation of HT29 colon cancer cells breast cancer cell lines, but not breast CSCs, to standard chemotherapy. In order to is associated with a reduced expression of CD133 and with an increased release of identify new vulnerability targets that could be coupled with glutamine consumption exosomes positive for CD133. This study aimed to analyze the role and content of inhibition for eradicating breast CSCs, we performe transcriptomic and biochemical exosomes in the differentiation process of colorectal cancer cells. assays. Methods: exosomes were isolated using differential centrifugations from cell Conclusions supernatant of HT29 and from two colorectal cancer stem cell lines (CSC). The lack of effective therapeutic regimens, especially for metastatic breast cancer, Differentiation was induced in HT29 cells by NaBu administration and in CSC by points up the need of studying alternative strategies able to reduce side effects and adhesion on plates and NaBu treatment. Differentiation process was monitored to increase patient response. Our preliminary results show that targeting metabolic evaluating phosphatase alkaline activity and confirmed assessing CSC markers demands of breast CSCs could be exploited as a promising tool to counteract CD133 and CD44 by flow cytometry. Proteomic analysis was performed by high- cancer progression. resolution nano-HPLC-ESI-MS/MS of proteins extracted from exosomes released by undifferentiated and differentiated HT29 cells. mRNA expression levels were evaluated by RT-PCR. E8 Results: These preliminary data showed that exosomes released by HT29 cells are TARGETING CD44/XCT ENHANCES THE SENSITIVITY OF NEUROBLASTOMA enriched in protein related to cancer progression, metastasis and metabolism such STEM CELLS TO ETOPOSIDE. as PKM2, HMGB1, LAMP-1, EZR, CD44, Integrin, CD147 and others. PKM2 and 1)Speciale A. 2)Marengo B. 3)Traverso N. 4)Monteleone L. 5)Cantoni C. 6)Ravera CD147 expression increased in exosomes released by differentiating cells as well S. 7)Garbarino O. 8)Pronzato M.A. 9)Domenicotti C. as in HT29 and CSC cells induced to differentiate. 1)Department of Experimental Medicine, University of Genoa, Italy 2)Department HT29 and CSC cells differentiation and the increase of cellular CD147 expression of Experimental Medicine, University of Genoa, Italy 3)Department of Experimental levels were prevented by blocking multivescicular body maturation using NH4Cl Medicine, University of Genoa, Italy 4)Department of Experimental Medicine, or GW4869. Differentiation of HT29 and CSC was associated with an increased University of Genoa, Italy 5)Department of Experimental Medicine, University of release of CD9 and CD63 positive exosomes. Genoa, Italy 6)Department of Pharmacy, University of Genoa, Italy 7)Department Exposure to exosomes released by differentiating HT29 and colorectal CSC of Experimental Medicine, University of Genoa, Italy 8)Department of Experimental induced an increased expression of the CD147 and MMP2 proteins and modified Medicine, University of Genoa, Italy 9)Department of Experimental Medicine, the expression of IL6, TGF-beta and VEGF in HCT116 colorectal cancer cells and University of Genoa, Italy in cancer-associated fibroblasts. Conclusions: release of exosomes is affected by differentiation of colon cancer cells. Neuroblastoma (NB) is the most frequent extracranial childhood tumor and Exosomes might be used by differentiating cells to get rid of cellular components its clinical treatment with etoposide, exerting a pro-oxidant action, is initially that continue to exert their effects on recipient cells through protein related to cancer effective but subsequently it is able to induce chemoresistance. This condition is progression, metastasis and metab. characterized by an increase in glutathione (GSH) levels and by the presence of cancer stem cells (CSCs) which, having a low proliferative rate, are less sensitive to therapies acting on highly-proliferating cells. CSCs express CD44, a PKC-alpha- modulated staminality marker, which is able to influence GSH levels by stabilizing xCT, a cystine-glutamate antiporter promoting GSH synthesis by catalyzing cystine uptake and glutamate release. Based on this evidence, our aim was to investigate whether targeting CD44/xCT might be a useful strategy to increase neuroblastoma sensitivity to therapy. Therefore, CSCs selected from HTLA-230, an high-risk NB cell line, and from etoposide-resistant HTLA (HTLA-ER) cells were treated once a week with 1.25 µM etoposide alone or in combination with 0.5 µM PKC-alpha inhibitor or 5 µM sulfasalazine, an xCT inhibitor. These treatments were carried out for 2 and 5
36 37 ABSTRACT BOOK
E10 used: i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment Microenviroment and metastatic spread: 3D-decellularized with PTX to select for resistant cells (RC); and combined the two, by treating RC matrix as a tool to dissect the main features of the colorectal with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) cancer peritoneal metastatic niche activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar 1)Varinelli L. 2)Belfiore A. 3)Bruccoleri A. 4)Guaglio M. 5)Kusamura S. 6)Baratti D. approach was followed for in vivo studies, with NSG female injected through intra- 7)Milione M. 8)Sozzi G. 9)Pierotti M.A. 10)Deraco M. 11)Roz L. 12)Gariboldi M. nipple injection of 100,000 SUM159/pLUC/pSlik34a cells, and separated into: not 1)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto treated (NT), 34a alone, PTX alone, and PTX+34a. Nazionale dei Tumori, Milan, Italy 2)Department of Pathology and Laboratory Results: As observed for aggressive breast cancer, treatment of SUM159 with Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Tumor PTX generated resistant cells (RC) with increased CSC content (ALDH activity Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale and spheres forming ability). When miR-34a was induced in the RC, cells changed dei Tumori, Milan, Italy 4)Peritoneal Surface Malignancies Unit, Colon and Rectal morphology, slowed proliferation rate, and decreased CSC content (ALDH activity Surgery, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 5)Peritoneal and spheres). Moreover, a second PTX treatment almost completely abolished Surface Malignancies Unit, Colon and Rectal Surgery, Fondazione IRCCS Istituto the ALDH activity and SFE. The tumor growth was evaluated in vivo, following by Nazionale Tumori, Milan, Italy 6)Peritoneal Surface Malignancies Unit, Colon luminescence the primary tumor and lungs for metastasis. As expected, PTX alone and Rectal Surgery, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 7) showed an increase of lung metastasis but, interestingly, PTX+34a mice showed Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto reduced lung metastasis. Nazionale dei Tumori, Milan, Italy 8)Tumor Genomics Unit, Department of Research, Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9)IFOM (FIRC cells reducing cancer stem cell capacities, and limited the aggressive phenotype Institute of Molecular Oncology) Foundation/Cogentech, Milan, Italy 10)Peritoneal of breast cancer, including proliferation, drug resistance, relapse and metastasis. Surface Malignancies Unit, Colon and Rectal Surgery, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 11)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 12)Tumor Genomics E12 Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, BCL6: a novel target of triple negative breast cancer stem Milan, Italy cells 1)Volpari T. 2)Fucà G. 3)Romero Cordoba S. 4)De Santis F. 5)Rondinone O. 6) Peritoneal metastases (PM) are one of the most common routes of dissemination Faraci S. 7)Farris F. 8)Puricelli C. 9)Castagnoli L. 10)Marullo R. 11)Cerchietti L. 12) for colorectal cancer (CRC). PM development is caused by a cross-talk between M. Pupa S.M. 13)Di Nicola M. cancer cells and the microenvironment involving several passages, known as 1)Immunotherapy and Innovative Therapeutics Unit, Department of Medical peritoneal metastatic cascade, which is governed and modulated by refined Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2) interactions between biochemical factors and biomechanical events, allowing the Immunotherapy and Innovative Therapeutics Unit, Department of Medical Oncology, formation of the metastatic niche. The mechanisms involved in PM spread are still Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Molecular Targeting obscure and could be elucidated by an in vitro 3D-culture system that integrates Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, all the elements involved in PM development. The 3D-organoid cultures better Milan, Italy 4)Immunotherapy and Innovative Therapeutics Unit, Department of reflect the physio/pathological characteristics of the organs than the traditional cell Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5) culture models, but still fail to represent the heterogeneity of the microenvironment. Immunotherapy and Innovative Therapeutics Unit, Department of Medical Oncology, Methodologies have been recently established to remove cells from tissues and Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Molecular Targeting obtain 3D models in which extra cellular matrix (ECM) and tissue architecture are Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, maintained (3D-dECM models), which could be used as the most representative Milan, Italy 7)Immunotherapy and Innovative Therapeutics Unit, Department scaffold for 3D cultures. of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, We aimed to obtain a 3D model that closely recapitulates the microenvironment Italy 8)Immunotherapy and Innovative Therapeutics Unit, Department of Medical where the CRC-PM develops and includes d-ECM repopulated with organoids Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9)Molecular of epithelial origin and stromal cells derived from the same PM. We purified PM- Targeting Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei derived ECMs from mucosa obtained from PM samples and from the matched Tumori, Milan, Italy 10)Hematology and Oncology Division, Weill Cornell Medicine, normal mucosa using hypotonic solutions containing ionic and nonionic detergents, Cornell University, New York, NY, USA 11)Hematology and Oncology Division, Weill hypertonic solution and endonuclease without denaturing agents. dECMs analyses Cornell Medicine, Cornell University, New York, NY, USA 12)Molecular Targeting demonstrated that the procedure is able to maintain the specific characteristics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, of their tissue of origin also in terms of distribution, localization and architectural Milan, Italy 13)Immunotherapy and Innovative Therapeutics Unit, Department of organization of ECM-related proteins. The obtained dECMs showed a different Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy spatial rearrangement among normal versus PM-derived stroma, suggesting that 3D-dECMs scaffolds more closely recapitulate the native PM microenvironment. Introduction: Despite improvements in breast cancer treatment in recent years, triple From the same tissues we have also isolated the subpopulation of cells belonging to negative breast cancer (TNBC) is still characterized by a poor prognosis and a lack the stromal counterpart, such as fibroblast and mesothelial cells that were positive of specific actionable alterations. Thus, the identification of new molecular targets for αSMA, Calreticulin and WT1. Finally, organoids have been generated from represents a clinical priority. BCL6 is a well-known transcriptional modulator that PM tissue. Further analyses will focus on the repopulation of 3D-ECMs with their acts as a repressor of DNA-damage sensing and cell cycle progression pathways corresponding organoids and stromal cells in order to mimic the main characteristics in the context of B-cell lymphomas, while its implication in controlling breast of the peritoneal lesion, thus providing a robust model for recapitulating the main cancer cell growth, survival, and invasiveness has been recently described. This features of the metastatic niche and a valid tool for ex vivo drug screening. study investigated the role of BCL6 expression in TNBC initiation and stemness (TNBCSCs). Methods: The correlation between BCL6 expression and the overall survival (OS) E11 of TNBC patients, as well as the association with stemness-related pathways MIRNA-34A SENSITIZES TRIPLE-NEGATIVE BREAST CANCER CELLS TO were assessed in silico using the METABRIC dataset. Following BCL6 silencing PACLITAXEL, REDUCING CANCER STEM CELL POPULATION AND LUNG or pharmacological inhibition by the small molecule FX1, mammosphere-forming METASTASIS efficiency (MFE) and ALDH and CD44 CSC markers expression were evaluated in Climent MC., Bonetti PB., Nicassio FN. SUM149 and SUM159 TNBC cell lines. Center for genomic science of iit@semm, Istituto italiano di tecnologia, Milan, Italy Results: In silico analysis of the METABRIC dataset has showed that BCL6 overexpression is associated with poor overall survival and enrichment in stemness Background: Cancer stem cells (CSCs) have inherent or developed drug resistance signatures in TNBC patients. In vitro, BCL6 silencing resulted in a significantly for which chemotherapy fails to eliminate all tumor cells, a property that is critically reduced number of mammospheres compared to internal control, as well as a contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known simultaneous decreased percentage of ALDH+ and CD44+ cells in all the TNBC tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, cell lines tested. Moreover, blocking the transcriptional repressor activity of BCL6, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on using its selective inhibitor FX1, confirmed the impairment of the MFE. Of note, our preliminary observations (Bonetti et al., under revision), we hypothesized a following BCL6 silencing, we observed a global reduction in the repressive marker combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing H3K27me3, suggesting a cooperation between BCL6 and the breast EMT- and CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as stemness-related enzyme EZH2 in inducing specific gene transcription repression a model. in TNBC. Methodology: We exploited SUM159pt cells (TNBC mesenchymal-like, with CSC Conclusion: Downregulation and pharmacological inhibition of BCL6 significantly features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which impaired TNBC stem-like phenotype, suggesting a critical role of BCL6 in regulating is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we TNBCSC self-renewal. This still unknown BCL6 biological feature could be mediated
36 37 ABSTRACT BOOK by BCL6-dependent epigenetic silencing of stemness-related genes, even though further ongoing studies will be necessary to precisely elucidate such mechanism. CELL ADHESION, MIGRATION, Overall, we identified BCL6 as a novel target to counteract TNBCSCs paving the INVASION AND METASTASIS way to a novel “tailored” cure for TNBC. F1 THE IKK/ NF-kB SIGNALLING PATHWAY REQUIRES MORGANA TO DRIVE E13 BREAST CANCER METASTASIS CHEMOTHERAPY CAN PROMOTE METASTASIS OUTGROWTH THROUGH 1)Fusella F. 2)Seclì L. 3)Moiso E. 4)Rocca S. 5)Conti L. 6)Annaratone L. 7)Turco E. RECRUITMENT AT DISTANT SITES OF BOTH METASTASIS INITIATING CELLS 8)Morotti A. 9)Castellano I. 10)Cavallo F. 11)Provero P. 12)Brancaccio M. AND MYELOID-DERIVED SUPPRESSOR CELLS VIA THE CXCL12/CXCR4 1)Department of Molecular Biotechnology and Health Sciences, University of AXIS Torino, Torino, Italy 2)Department of Molecular Biotechnology and Health Sciences, 1)Bertolini G. 2)Cancila V. 3)Tortoreto M. 4)Centonze G. 5)D’Alterio C. 6)Scala S. 7) University of Torino, Torino, Italy 3)Department of Molecular Biotechnology and Tripodo C. 8)Sozzi G. 9)Roz L. Health Sciences, University of Torino, Torino, Italy 4)Department of Molecular 1)Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Biotechnology and Health Sciences, University of Torino, Torino, Italy 5)Department Italy 2)Tumor Immunology Unit, Department of Health Sciences, University of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy of Palermo, Palermo, Italy 3)Molecular Pharmacology, Fondazione IRCCS 6)Department of Medical Sciences, University of Torino, Torino, Italy 7)Department Istituto Nazionale dei Tumori, Milan, Italy 4)Tumor Genomics Unit, Fondazione of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Functional Genomics, Istituto 8)Department of Clinical and Biological Sciences, University of Torino, Orbassano, Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”-IRCCS. Italy 9)Department of Medical Sciences, University of Torino, Torino, Italy 10) Naples, Italy 6)Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Department of Molecular Biotechnology and Health Sciences, University of Torino, Tumori, Fondazione “G. Pascale”-IRCCS, Naples, Italy 7)Tumor Immunology Unit, Torino, Italy 11)Department of Molecular Biotechnology and Health Sciences, Department of Health Sciences, University of Palermo, Palermo, Italy 8)Tumor University of Torino, Torino, Italy 12)Department of Molecular Biotechnology and Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9) Health Sciences, University of Torino, Torino, Italy Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Triple Negative Breast Cancers (TNBCs) are very aggressive tumors associated with Standard chemotherapy regimens have limited long-term efficacy in lung cancer metastases, high recurrence after chemotherapy and high mortality rates. Aberrant patients due to chemoresistance and inefficacy in controlling metastatic disease. activation of NF-κB signaling is frequently found in TNBC cells, but unfortunately, the In pre-clinical models we have shown that cisplatin treatment enriches for the cause of this deregulation is unclear. NF-kB is a transcription factor involved in the chemoresistant fraction of CD133+CXCR4+ lung cancer metastasis initiating cells regulation of multiple physiological and pathological cellular processes, including (MICs), increasing distant metastasis development that can be prevented by CXCR4 inflammation, cell survival, proliferation and cancer cell metastasis. Here we found blockade. Therefore, we hypothesize that the CXCL12/CXCR4 axis, implied in MICs that NF-kB activation in triple negative breast cancer cells depends on the presence maintenance/migration and in immune and stromal cells trafficking, could play a of the CHORDC1 gene product Morgana, a new component of the IKK complex critical role in cisplatin-induced pro-metastatic effects. essential for IkBa substrate recognition and phosphorylation. Morgana silencing To study the effects of cisplatin in promoting a pre-metastatic niche, naïve SCID blocks metastasis formation in breast cancer mouse models and this phenotype mice were treated with cisplatin plus/minus peptide R, a novel inhibitor of CXCR4. is reverted by IkBa downregulation. Moreover, morgana overexpression in cancer Mouse lungs were evaluated through IHC and FACS analyses 72h post-treatment, cells can shape tumor microenvironment and immune system cells recruitment revealing increased expression of CXCL12 in PDGFRβ+ stromal cells linked to to further support tumor aggressiveness and progression. In fact, high Morgana recruitment of CXCR4+CD11b+ myeloid cells and in particular to the subset of expression levels inhibit accumulation of natural killer cells in the first phases of inflammatory monocytes (CD11b+Ly6-Chigh), known as myeloid-derived suppressor tumor growth and, by activating NF-kB, induce the expression of cytokines able cells (MDSCs). Peptide R partially prevented these effects. to attract neutrophils in the primary tumor as well as in the pre-metastatic lungs, Tail-vein injection of H460 human lung cancer cells 72h after cisplatin administration fuelling cancer metastasis. In accordance, high Morgana levels positively correlate resulted in augmented number of lung metastases (p=0.003), that showed a 3.5-fold with NF-kB target gene expression and poor prognosis in human patients. Taken enrichment in CD133+CXCR4+ MICs (p=0.005) and increase of monocytic-MDSCs, together all these results highlighted a previously underscribed role for morgana suggesting a role for MDSCs in MICs expansion/overgrowth. Pre-treatment with overexpression in breast tumorigenesis as a prognostic marker of metastasis peptide R abolished these effects. formation through NF-kB activation. CXCL12 was also increased in cisplatin-treated subcutaneous H460 xenografts and was associated to a greater number of tumor-associated inflammatory monocytes and CXCR4+F480high macrophages, directly correlated with enrichment F2 in the chemoresistant MICs subset (R=0.75 p=0.0001). CXCR4 inhibition partially Overexpression of the dsRNA-dependent kinase PKR in impaired these effects, also by preventing myeloid cells extravasation. osteosarcoma cells promotes attachment independent Moreover cisplatin treatment of H460 xenografts caused, at the lung metastatic growth and migration but not invasion site, an increase in stromal CXCL12 and recruitment of both CXCR4+ inflammatory 1)Focaccia EF. 2)Piazzi MP. 3)Greco SG. 4)Orsini AO. 5)Cocco LC. 6)Faenza IF. 7) monocytes/macrophages (1.6-fold change p=0.01) and MICs subset (1.8- Bavelloni AB. 8)Blalock WB. fold change p=0,04), overall resulting in a boost in micrometastases partially 1)Institute of molecular genetics, National research council of italy, Bologna, Italy counteracted by CXCR4 blocking. 2)Institute of molecular genetics, National research council of italy, Bologna, Italy Our data reveal a paradoxical pro-metastatic effect of cisplatin that fosters MIC- 3)Department of biomedical and neuromotor sciences, University of bologna, MDSCs recruitment and cross-talk via CXCL12/CXCR4 axis activation. A new Bologna, Italy 4)Department of biomedical and neuromotor sciences, University combination strategy based on CXCR4 inhibition may disrupt these interactions, of bologna, Bologna, Italy 5)Department of biomedical and neuromotor sciences, providing more effective results for lung cancer treatment. University of bologna, Bologna, Italy 6)Department of biomedical and neuromotor sciences, University of bologna, Bologna, Italy 7)Department of musculoskeletal cell biology, Rizzoli orthopedic institute, Bologna, Italy 8)Institute of molecular genetics, National research council of italy, Bologna, Italy Osteosarcoma, which mainly occurs in children and young adults, is the most common primary tumor of the bone; typically affecting the proximal tibia and distal femur. While surgical removal of the primary tumor is often the initial course of action, this tumor has remained extremely hard to treat, due to the high level of post- operative relapse and pulmonary metastasis. Chromosomal abnormalities affecting tumor suppressor genes, such as Rb and p53, remain the most common etiology of this tumor, but alterations in other signaling pathways associated with growth (Wnt/beta catenin, AKT and MAPK), chemokine signaling (CXCR4), apoptosis (Fas and Bcl-2), transcription, (RUNX2), chemoresistance (MDR1, CYP3A4/5) and angiogenesis (VEGF) have each been shown to play a significant role. As in other tumors, inflammatory/stress signaling has also been shown to play a fundamental role in osteosarcoma. Among the most relevant inflammatory /stress proteins shown to have a role in tumorgenesis, is the double-strand RNA activated protein kinase PKR. PKR, which is best known for its ability to phosphorylate the eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) leading to the inhibition of
38 39 ABSTRACT BOOK
general protein synthesis, acts as a general sentinel of external and internal cellular expressing specifically the CD44v8-10 isoform and displayed a more stem-like and stresses, and its activation results in the modification of signaling complexes invasive phenotype in vitro that is lost by silencing the CD44 variant. The gene involved in ribosome biogenesis, RNA processing, chromatin organization and DNA for epithelial splicing regulatory protein 1 (ESRP-1) regulates alternative splicing repair. of CD44 and ZEB-1 is a ESRP-1 repressor. High level of ESRP-1 is significantly To determine whether PKR has a significant role in the development and progression associated with a lower rate of overall survival, suggesting that increased ESRP- of osteosarcoma, osteosarcoma cell lines were stably transfected with wild-type 1 expression is related to the malignant behaviour of many human cancer. We and dominant-negative (DN) forms of PKR; and then the rate of proliferation, demonstrated the abundance of ESRP-1 mRNA and low levels of ZEB-1 mRNA sensibility to chemotherapeutic drugs (methotrexate, doxorubicin and vincristin), in CD44v8-10+ cells. When the two sorted subpopulations were cultured in attachment independent growth, migration and invasion were all assayed. While stem medium, also CD44v8-10- population acquired high levels of CD44v8-10 overexpression of wild-type PKR had no clear effect on the rate of proliferation, it and in this conditions both PC3 populations show the same invasion capability, had a significant effect on the response to doxorubicin treatment, resulting in a more suggesting that stem conditions selected cells expressing CD44v8-10 with high rapid and potent activation of caspase 3. In addition, PKR overexpression resulted invasive potential. We investigated the basal reactive oxygen species (ROS) in attachment independent growth as well as enhanced migration. In contrast, level in both subpopulations and we observed that CD44v8-10+ population shows overexpression of DN-PKR inhibited attachment independent growth, while it had higher basal ROS levels than the negative population with a better response to no significant effect on migration or invasion. oxidative stress. To investigate the differences of two PC3 subpopulations in in vivo These results seem to support a role for PKR in primary tumor establishment, while tumour growth ability, we generated subcutaneous xenografts in male nude mice subsequent loss of PKR activity may contribute to metastasis. by inoculation of CD44v8-10+ PC3 cells compared to CD44v8-10- PC3 cells. We did not observe statistically significant difference in in vivo cell growth between the two PC3 subpopulations. To verify whether primitive rate of CD44v8-10 expression F3 was maintained after in vivo growth, we performed a flow cytometry analysis for DISSECTING THE SIGNALING AND ASSEMBLY PATHWAYS OF INVADOPODIA CD44v8-10 protein from xenografts for each PC3 subpopulation. CD44v8-10 TRIGGERED BY ENDOTHELIN-1 RECEPTOR/ß-ARRESTIN 1 AXIS expression, migratory and invasive capabilities in xenografts analyzed mirrored that 1)Chellini L. 2)Caprara V. 3)Spadaro F. 4)Bagnato A. 5)Rosanò L. of two injected cell populations. CD44 variant isoforms are known to be specifically 1)Preclinical Models and New Therapeutic Agents Unit, Regina Elena National expressed in cancer stem cells and metastatic cells than standard CD44 (CD44s). Cancer Institute, Rome, Italy 2)Preclinical Models and New Therapeutic Agents Unit, The goal of this work will be to study the role of CD44v8-10 in the prostate cancer Regina Elena National Cancer Institute, Rome, Italy 3)Confocal Microscopy Unit, development and progression by investigating the molecular mechanisms that Istituto Superiore di Sanità, Rome, Italy 4)Preclinical Models and New Therapeutic make the CD44v8-10+ population more stem-like and invasive. Agents Unit, Regina Elena National Cancer Institute, Rome, Italy 5)Preclinical Models and New Therapeutic Agents Unit, Regina Elena National Cancer Institute, Rome, Italy F5 Targeting of melanosomal Two-Pore Channel 2 (TPC2) as a novel The “invasive phenotype” of serous ovarian cancer (SOC) cells is characterized strategy to impair melanoma progression and metastasis by the formation of actin-based structures, invadopodia, operating extracellular 1)D’Amore A. 2)Hanbashi A. 3)Papacci F. 4)Parrington J. 5)Palombi F. 6)Filippini A. matrix (ECM) degradation and cell invasion. Environmental cues, as signals from 1)Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit growth factor receptors and stiffness, might cause the assembly of a F-actin core of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy and the engagement of integrins and associated proteins, like IQGAP1, to form an 2)Department of Pharmacology, University of Oxford, Oxford, United Kingdom 3) “adhesion ring”, helping to invadopodia function. Endothelin-1 (ET-1)/ETA receptor Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of (ETAR) axis is a regulator of invadopodia by the scaffolding function of β-arrestin1 Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy 4) (β-arr1) driving well defined pathways, as RhoC and Mena, which are coordinately Department of Pharmacology, University of Oxford, Oxford, United Kingdom 5) regulated in invasive SOC cells; however, a deeper understanding of how ETAR Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of promotes the integration of adhesive components in the invadopodia lifecycle needs Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy 6) to be reached. Here, we assessed whether, in SOC cells, ETAR/β-arr1 axis functions Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, Unit of as signal-integrating module for adhesion signaling components in invadopodia. Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy By using public gene data sets, we found that IQGAP1 mRNA is overexpressed in SOC compared to normal tissues and that its expression is enhanced from Melanoma is one of the most aggressive and treatment-resistant human cancers. low to high grade, and Kaplan–Meier analysis and the log-ranked test showed It has been shown that the ion channel TPC2, activated by the second messenger that high expression level IQGAP1 is predictive of worse overall survival and NAADP and typically found on the membrane of acidic organelles, is present progression-free survival in patients diagnosed with SOC, implying its pathological in melanosomes and involved in pigmentation. In addition, our laboratory has significance in SOC progression. In SOC cells expressing IQGAP1, ET-1 promotes demonstrated NAADP/TPC2/Ca2+signalling is involved in melanoma progression the upregulation of IQGAP1 and its interaction with β-arr1, which can be inhibited and metastasis. To date, it is well known that the microenvironment has a key by macitentan, the dual ET-1R antagonist, as shown by imaging and biochemical role in cancer progression. It was shown very recently that melanosomes release analyses. This molecular complex is involved in the fine regulation of ET-1-induced pro-metastatic factors. It is therefore important to understand the mechanism RhoA/RhoC activation, and Rac1 inhibition, through a mechanism involving Rac of melanosome release. We have observed that melanin release in the B16-F0 GTPase-activating protein (RacGAP)1. Moreover, IQGAP1/b-arr1 interaction melanoma cell line is markedly reduced by the TPC2 inhibitor Naringenin. is involved in the localization of F-actin and adhesion proteins, as vinculin, with TPC2 inhibition increased CREB (cAMP response element-binding protein) invadopodia markers (cortactin, TKS5), and invadopodia maturation, indicating phosphorylation, via increased p-38 activity and decreased p-ERK activity. These its involvement in invadopodia lifecycle. Both macitentan and IQGAP1 or β-arr1 data suggest that TPC2 might be a novel therapeutic target in melanoma. silencing inhibited ETAR-induced protease secretion, ECM degradation, and cell invasion. In vivo, macitentan inhibited SOC metastatic dissemination and IQGAP1 protein expression. Collectively, these data establish an unpredicted role for the F6 β-arr1/IQGAP1 complex as a novel network of protein driven by ETAR fostering HMGA1 AND FOXM1 SYNERGISTICALLY REGULATE A COMMON GENE invadopodia formation and the metastatic process in SOC, which might be impaired NETWORK MODULATING ANGIOGENESIS IN BREAST CANCER by macitentan treatment 1)Pegoraro S. 2)Zanin R. 3)Ros G. 4)Ciani Y. 5)Piazza S. 6)Agostinis C. 7)Bossi F. 8)Bulla R. 9)Tonon F. 10)Zennaro C. 11)Sgarra R. 12)Manfioletti G. 1)Dept. of life sciences, University of Trieste, Trieste, Italy 2)Dept. of life sciences, F4 University of Trieste, Trieste, Italy 3)Dept. of life sciences, University of Trieste, Role of CD44v8-10 splicing variant in the androgen-independent Trieste, Italy 4)Lncib, Area science park, Trieste, Italy 5)Cibio, University of Trento, prostate cancer cells PC3 Trento, Italy 6), Institute for maternal and child health, burlo gar, Trieste, Italy 7)Dept. Fontanella R.A., Di Stefano C., Marampon F., De Cesaris P., Padula F., D’Amore A., of medical, surgical and health sciences, University of Trieste, Trieste, Italy 8)Dept. Campese A.F., Ziparo E., Filippini A., Riccioli A. of life sciences, University of Trieste, Trieste, Italy 9)Dept. of medical, surgical and D.A.H.F.M.O.- Department of Anatomy, Histology, Forensic Medicine and health sciences, University of Trieste, Trieste, Italy 10)Dept. of medical, surgical Orthopaedics, Section of Histology and Medical Embryology, “Sapienza” University and health sciences, University of Trieste, Trieste, Italy 11)Dept. of life sciences, of Rome, Italy University of Trieste, Trieste, Italy 12)Dept. of life sciences, University of Trieste, Trieste, Italy The cell surface protein CD44 is a known as a marker for stem-like and metastatic cancer cells in prostate cancer (PCa). We detected and isolated a minor The triple negative breast cancer represents an open challenge to oncologists subpopulation of CD44-negative highly aggressive prostate cancer (PCa) cell lines and researchers, as it does not respond to hormonal or targeted treatments. PC3. In culture, these sorted cells rapidly converted to a CD44-positive phenotype Many aspects of this highly aggressive cancer have not been clarified yet, so it is
38 39 ABSTRACT BOOK extremely important to keep on looking for the factors involved. One of the actors CTMs will provide important insight into early steps of metastatic progression. accounting for breast cancer progression is the High mobility group A1 (HMGA1) protein, an architectural transcription factor that has a causal role in the malignant transformation of normal breast cells by controlling the transcription of genes F8 involved in stemness, cell motility, cell adhesion and invasion. In order to further cIAP1 is a novel determinant of Snai2 expression by promoting explore the molecular networks HMGA1 orchestrates, we subjected a triple negative the EGFR-dependent signaling pathways in triple negative breast cancer cell line, MDA-MB-231, silenced for HMGA1 expression, to deep breast cancer cells RNA sequencing. By performing a bioinformatic analysis aimed to unravel possible 1)Majorini M.T. 2)Manenti G. 3)Mano M. 4)De Cecco L. 5)Pinciroli P. 6)Tagliabue E. HMGA1 molecular partners in governing downstream pathways, we focused on 7)Chiodoni C. 8)Colombo M.P. 9)Delia D. 10)Lecis D. the Forkhead box protein M1 (FOXM1), a transcription factor known to control 1)Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, several malignant features of breast cancer. In particular, we found that HMGA1 Milano, Italy 2)DRAST, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, collaborates with FOXM1 in regulating the transcription of a common target gene, Italy 3)Functional Genomics and RNA-Based Therapeutics Laboratory, University the Vascular endothelial growth factor (VEGF). Moreover, we studied how HMGA1, of Coimbra, Coimbra, Portugal 4)DRAST, Fondazione IRCCS Istituto Nazionale dei by modulating FOXM1 network, could regulate the formation of new blood vessels Tumori, Milano, Italy 5)DRAST, Fondazione IRCCS Istituto Nazionale dei Tumori, within the tumour context by acting on the proliferation and migration of endothelial Milano, Italy 6)Department of Research, Fondazione IRCCS Istituto Nazionale cells. Finally we validated in vivo that these two factors synergistically regulate neo- dei Tumori, Milano, Italy 7)Department of Research, Fondazione IRCCS Istituto angiogenesis in Zebrafish larvae. This study shed light on a new molecular partner Nazionale dei Tumori, Milano, Italy 8)Department of Research, Fondazione IRCCS of HMGA1 and how HMGA1 and FOXM1 collaborate to regulate a critical aspect of Istituto Nazionale dei Tumori, Milano, Italy 9)Department of Research, Fondazione breast cancer: the angiogenetic process. IRCCS Istituto Nazionale dei Tumori, Milano, Italy 10)Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy F7 Inhibitor of apoptosis (IAP) proteins contribute to cancer aggressiveness by EARLY DISSEMINATION IN NON-METASTATIC BREAST CANCER PATIENTS: controlling several pro-survival pathways and signaling cascades. They are often DETECTION OF CIRCULATING TUMOR MICROEMBOLI deregulated in cancer cells where they promote anchorage-independent growth, 1)Reduzzi C. 2)Motta R. 3)Schamberger T. 4)Zannini L. 5)Silvestri M. 6)Di Cosimo motility and eventually dissemination by metastasis. Hence, the compounds that S. 7)Martinetti A. 8)Klein C.A. 9)Daidone M.G. 10)Cappelletti V. inhibit the activity of IAPs could be potentially useful in cancer therapy. Accordingly, 1)Department of Applied Research and Technological Development, Fondazione we have recently synthesized and characterized a library of small molecules IRCCS Istituto Nazionale dei Tumori, Milano, Italy 2)Department of Applied directed against three members of the IAP family, cIAP1, cIAP2 and XIAP, and Research and Technological Development, Fondazione IRCCS Istituto Nazionale evaluated their in vitro cytotoxicity. Moreover, the in vivo activity of SM83, a novel dei Tumori, Milano, Italy 3)Experimental Medicine and Therapy Research, molecule with nanomolar affinities for IAPs, was tested by employing NOD/SCID University of Regensburg, Regensburg, Germany 4)Istituto di genetica molecolare, mice subcutaneously engrafted with the human triple-negative breast cancer Consiglio Nazionale delle Ricerche, Pavia, Italy 5)Department of Applied Research (TNBC) MDA-MB231 cell line. In this way, we found that the inhibition of IAPs and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, reduced the growth of primary tumors and hindered their capacity to metastasize Milano, Italy 6)Department of Applied Research and Technological Development, to lungs. Moreover, we profiled the primary tumors for gene expression and found Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 7)Department that the treatment affected the expression of several genes. The majority of the of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, modulated genes were targets of the NF-kB pathway, as expected, since cIAP1 Italy 8)Experimental Medicine and Therapy Research, University of Regensburg, and cIAP2 are apical regulators of this pathway, but we demonstrated that SM83 Regensburg, Germany 9)Department of Applied Research and Technological treatment prevented also the expression of Snai2. This regulator of the epithelial- Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 10) to-mesenchymal transition (EMT) process favors the stem-like properties and Department of Applied Research and Technological Development, Fondazione metastatic potential of cancer cells, and it promotes the aggressiveness in particular IRCCS Istituto Nazionale dei Tumori, Milano, Italy of breast cancers. Through the employment of a panel of breast cancer cells, we proved that cIAP1 is a determinant of Snai2 levels and provided evidence that this Background: IAP is a novel regulator of the epidermal growth factor receptor (EGFR). Accordingly, Tumor cells can migrate from solid tumors either as single cells (circulating tumor the genetic and pharmacological inhibition of cIAP1 prevented the EGFR-mediated cells, CTCs) or as clusters (circulating tumor microemboli, CTMs). In a literature activation of the mitogen-activated protein kinase (MAPK) pathway upon EGF study using a breast cancer (BC) mouse model, CTMs showed a 23-50-fold higher and TGFα stimulation, and reduced the expression of Snai2. In a number of cell metastatic capacity than CTCs. In few studies, the presence of CTMs was also lines, cIAP1 depletion caused the reduction even of EGFR itself, but this event was associated with worse survival in metastatic (M1) BC patients. Here, we investigate dispensable for the SM83-dependent block of its signaling, and Snai2 transcription. the presence of CTMs, in both M1 and M0 BC patients and define an innovative In conclusion, our work provides evidence that IAP-directed therapy could result in workflow for CTMs molecular characterization. the inhibition of the downstream pathways of EGFR and be particularly effective Methods: in tumors characterized by high expression of EGFR and Snai2, as in the case of Nine ml of blood were collected at baseline or during treatment from 37 M0 and TNBCs. 24 M1 BC patients and processed for CTM-enrichment with SceenCell filters (4 healthy donors were also analyzed). After a cytological staining of filters, CTMs were counted based on morphological criteria. The expression of epithelial and F9 leukocyte markers (pan-cytokeratin (CK) and CD45, respectively) was assessed by Identification of a novel miR-200b-c/ETAR/ZEB1 network involved immunofluorescence (IF) on 26 filters from 19 patients. in ovarian cancer progression CTMs and single cells from CTMs, picked from the filters with a micromanipulator, 1)Sestito R. 2)Rosanò L. 3)Cianfrocca R. 4)Tocci P. 5)Di castro V. 6)Sacconi A. 7) underwent whole genome amplification and lowpass whole genome sequencing for Blandino G. 8)Bagnato A. copy number alteration (CNA) analysis. 1)Preclinical model and new therapeutic agents unit, Regina Elena national cancer Results: institute, Rome, Italy 2)Preclinical model and new therapeutic agents unit, Regina At baseline, CTM positivity rates were 26/37 (70%) vs. 5/24 (28%) in M0 and M1 Elena national cancer institute, Rome, Italy 3)Preclinical model and new therapeutic patients, respectively (P=0.0002). Similar positivity rates were observed in serial agents unit, Regina Elena national cancer institute, Rome, Italy 4)Preclinical model blood samples obtained during treatment: 30/42 (71%) vs. 5/23 (22%) in M0 and M1 and new therapeutic agents unit, Regina Elena national cancer institute, Rome, patients, respectively (P=0.0002). CTMs evaluated by IF were negative for CD45, Italy 5)Preclinical model and new therapeutic agents unit, Regina Elena national but had a heterogeneous expression of CK. cancer institute, Rome, Italy 6)Oncogenomic and epigenetic unit, Regina Elena CNA profiles obtained from 25 CTMs (from 4 M0 patients) showed the presence national cancer institute, Rome, Italy 7)Oncogenomic and epigenetic unit, Regina of aberrations only in few CTMs, while single cells isolated from CTM were clearly Elena national cancer institute, Rome, Italy 8)Preclinical model and new therapeutic aberrant. This suggests that a mixture with normal cells could occur, possibly agents unit, Regina Elena national cancer institute, Rome, Italy blurring the aberrations. Conclusions: Identifying key mediators of epithelial ovarian cancer (EOC) invasion and metastasis Surprisingly, CTMs were detected 2.5-times more frequently in M0 than in M1 is crucial for early detection and development of more effective therapies. miR- patients, whereas control samples were free of clusters. In addition, clusters were 200/ZEB1 negative feedback loop represents a driving force for EOC progression negative for CD45 staining, suggesting that CTM are not composed of differentiated towards metastasis. Emerging novel mechanistic aspects of endothelin (ET)- blood cells. However, the observation that most clusters –in contrast to individual 1 receptor signaling in EOC progression provide a strong rationale to target it. cells isolated from clusters – did not display CNAs by bulk analysis indicates that Despite the important contribute of ET-1/ET-1 A receptor (ETAR) and ZEB1/miR- the majority of CTM-forming cells are either genomically balanced or are genetically 200 networks in EOC, their possible cross-talk has never been investigated. In very heterogeneous. Therefore, deep sequencing or cell-by-cell analysis of M0 this study, we demonstrated the existence of a complex network involving ETAR,
40 41 ABSTRACT BOOK
miR-200b/-c and ZEB1 in EOC. In particular, we found that aggressive EOC cells phase separation of cytosolic proteins. Fluorescence recovery after photobleaching expressed low levels of miR-200b/-c and high levels of ETAR and ZEB1. Importantly, (FRAP) shows that PMAP-like ERC1-positive assemblies are dynamic structures the ectopic introduction of miR-200b/-c in these cells reduced both ZEB1 and showing fusion and fission events, and rapidly exchange their molecular components ETAR at mRNA and protein levels. Besides targeting ZEB1 3’UTR, these miRNA with the surrounding cytosolic phase. Number and brightness experiments in living specifically bond the ETAR 3’UTR and inhibited its transcription. As a consequence cells have demonstrated the dimeric nature of ERC1 in the diluted cytosolic phase, of the ETAR down-regulation, the ectopic expression of miR-200b/-c impaired the as confirmed by single molecule analysis by rotary shadowing electron microscopy ET-1-driven proliferation, apoptosis resistance, epithelial-to-mesenchymal transition of purified full length proteins ERC1 and liprin-a1. Experiments with deletion mutants (EMT), cell invasion and migration. Of note, ETAR and ZEB1 reciprocally control highlight the influence of structurally disordered regions of ERC1 in the assembly their expression. ET-1 induced ZEB1 expression by regulating its transcription and, of these newly identified dynamic membrane-less organelles. Inclusion of other viceversa, a reduction of ETAR expression was observed upon ZEB1silencing. This molecular components of PMAPs in ERC1-induced assemblies alters their dynamic latter was accompanied by a miR-200b/-c up-regulation, revealing the existence properties, and suggest the formation of multiphase organelles, reflecting the of a miR-200/ETAR/ZEB1 crass-talk in these cells. Of translational interest, the function of these structures in promoting tumor cell migration. Our results support dual ET-1 receptor antagonist macitentan, reduced metastatic dissemination and the hypothesis that liquid-liquid phase separation of proteins at specific sites of ZEB1 expression in xenograft models of EOC. We validated our findings in the the cell promotes disassembly of focal adhesions and invadosomes in support of cohort of high-grade serous ovarian cancer (HG-SOC) patients from The Cancer tumor cell invasion, identifying these structures as novel targets to interfere with the Genome Atlas (TCGA), reporting that ETAR and ZEB1 gene expression inversely formation of metastasis. correlated with miR-200b/-c and that ETAR and ZEB1 expression was associated with an unfavorable prognosis. Collectively, these findings establish a double-circuit controlling ETAR-ZEB1 and miR-200 family members that, representing a driving F12 force for cancer progression, regulates the EOC metastatic process. Antimetastatic activity of trabectedin in cutaneous melanoma 1)Carminati L. 2)Pinessi D. 3)Borsotti P. 4)Minoli L. 5)Giavazzi R. 6)D’Incalci M. 7) Taraboletti G. F10 1)Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche TRF2 in breast cancer aggressiveness and metastasis Farmacologiche Mario Negri, Bergamo 2)Tumor Angiogenesis Unit, Department 1)Petti E. 2)Salvati E. 3)Porru M. 4)Dinami R. 5)Zizza P. 6)Sacconi A. 7)Leonetti C. of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo 8)Biroccio A. 3)Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche 1)Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Farmacologiche Mario Negri, Bergamo 4)Mouse and Animal Pathology Lab Rome, Italy 2)Oncogenomic and Epigenetic Unit, Regina Elena National Cancer (MAPLab), Fondazione Filarete and Department of Veterinary Pathology, University Institute, Rome, Italy 3)SAFU, Regina Elena National Cancer Institute, Rome, of Milan, Italy 5)Tumor Angiogenesis Unit, Department of Oncology, IRCCS- Italy 4)Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Istituto di Ricerche Farmacologiche Mario Negri, Bergamo 6)Laboratory of Cancer Rome, Italy 5)Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Pharmacology, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Institute, Rome, Italy 6)Oncogenomic and Epigenetic Unit, Regina Elena National Mario Negri, Milano 7)Tumor Angiogenesis Unit, Department of Oncology, IRCCS- Cancer Institute, Rome, Italy 7)SAFU, Regina Elena National Cancer Institute, Istituto di Ricerche Farmacologiche Mario Negri, Bergamo Rome, Italy 8)Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Rome, Italy Introduction. Trabectedin is a marine-derived anti-neoplastic drug, approved for the treatment of soft tissue sarcoma and relapsed ovarian cancer. The drug targets Breast Cancer (BC) is the leading cause of cancer-related mortality in women in both cancer cells and the tumor microenvironment, with marked effects on the developed countries and this is due to cancer recurrence as distant metastasis vasculature and the immune response. Since a favorable microenvironment is a (bone, brain, liver, lung, and distant lymph nodes) or locoregional relapse. Among key factor in the progression of cutaneous melanoma, the aim of this study was to molecular subtypes, Triple Negative Breast Cancers (TNBCs), defined by lack of investigate the activity of trabectedin on the growth and metastasis of this highly expression of estrogen, progesterone, or HER2 receptors are characterized by the aggressive cancer. most aggressive clinical course, early relapse and poor outcome. For this reason, Material and Methods. The activity of trabectedin was studied on the murine treatment of TNBC cannot be limited to conventional chemotherapy and requires melanoma B16-BL6 and K1735-M2, implanted in syngeneic mice. Its peculiar dual the identification of novel molecular targets. activity on tumor and microenvironment was studied on tumor cell proliferation and Our study focuses on the role of the Telomeric Repeat binding factor 2 (TRF2) in invasion in vitro, and on vasculature and TAM in vivo. The anti-metastatic effect was aggressiveness and metastasis of triple negative breast cancer. Although TRF2 is a investigated in a lung colonization assay, following i.v. injection of tumor cells, and in well-known regulator of telomere integrity, recent evidences demonstrated that TRF2 a spontaneous metastasis model, after primary tumor surgical removal. exerts extra-telomeric functions during tumor progression and is overexpressed in Results. Trabectedin inhibited the subcutaneous growth of the murine melanoma different human malignancies including BC. B16-BL6 and K1735-M2, and caused a significant reduction of tumor blood vessels Here, we found that high levels of TRF2 correlate with a worse clinical outcome of density and tumor associated macrophages. Trabectedin had a remarkable anti- BC patients and that TRF2 expression is significantly higher in TNBCs compared metastatic activity, inhibiting the formation of B16-BL6 and K1735-M2 lung colonies to other breast cancer molecular subtypes. Moreover, by using an orthotopic model in vivo. The drug was also active in the clinical-relevant spontaneous metastasis of metastatic TNBC, we found that TRF2 knockdown impairs the formation of assay, given before (neo-adjuvant) and after (adjuvant) surgical removal of the spontaneous metastasis. In order to reveal the molecular mechanism(s) that link primary tumor. In line with its anti-metastatic activity, trabectedin inhibited melanoma TRF2 depletion with a less aggressive TNBC phenotype, we found that knockdown cell invasiveness in vitro, associated with increased TIMP-1 production and of TRF2 impacts on nuclear and cytoskeletal organization and reduces in vitro alteration in cell shape and cytoskeleton organization. The anti-metastatic activity migration and invasion ability of TNBC cells. of trabectedin was further increased by combination with the immune checkpoint inhibitor anti-PD1 antibody. Conclusions. This study shows that trabectedin affects melanoma growth and F11 metastasis, by targeting both the tumor cells and the vascular and inflammatory Plasma membrane-associated platforms to control tumor tumor microenvironment. These findings open a new prospect for the use of cell motility and invasion trabectedin for the treatment of melanoma, also in combination with other stroma- Sala K., Corbetta A., Tonoli D., Minici C., Degano M., Cammarota E., Mazza D., modifying agents, including immunotherapy. Curtis I. San Raffaele Scientific Institute, Milan, Italy We have identified plasma-membrane associated platforms (PMAPs) assembling specifically at the front of migrating invasive tumor cells, and near extracellular matrix–degrading invadosomes. PMAPs include the core scaffold proteins liprin-a1, ERC/ELKS and LL5, which contain both coiled coils as well as regions of predicted structural disorder. Silencing of components of PMAPs inhibit tumor cell migration in vitro and the formation of metastasis by invasive breast cancer cells in vivo. By combining biochemical and electron microscopy analysis, with high resolution confocal imaging in living cells, we have characterized the properties of PMAPs and their role in promoting the protrusive activity of breast cancer tumor cells, defining a role of their protein components in the turnover of integrin-mediated focal adhesions and in the dynamics of matrix-degrading invadosomes in transformed cells. Interestingly, recent results indicate that PMAPs formation is driven by liquid-liquid
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F13 F15 Assessing the molecular and clinical impact of RNASET2 in TARGETING SEMAPHORIN 4A-EXPRESSING MYELOID CELLS TO IMPAIR epithelial ovarian cancer CANCER PROGRESSION 1)Roggiani F. 2)Riva C. 3)Taramelli R. 4)Acquati F. 5) Mezzanzanica D. 6)Tomassetti A. 1)Maione F. 2)Qiu Y. 3)Garibaldi E. 4)Miranti A. 5)Gabriele P. 6)Brundu S. 7)Giraudo E. 1)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS 1)Transgenic mouse models laboratory, Candiolo cancer institute-fpo,irccs, Istituto Nazionale dei Tumori, Milan, Italy 2)Department of Medicine and Surgery, Candiolo (To), Italy 2)Transgenic mouse models laboratory, Candiolo cancer Università degli Studi dell’Insubria, Varese, Italy 3)Department of Theoretical and institute-fpo,irccs, Candiolo (To), Italy 3)Radiation therapy laboratory, Candiolo Applied Sciences, Università degli Studi dell’Insubria, Varese, Italy 4)Department of cancer institute-fpo,irccs, Candiolo (To), Italy 4)Radiation therapy laboratory, Theoretical and Applied Sciences, Università degli Studi dell’Insubria, Varese, Italy Candiolo cancer institute-fpo,irccs, Candiolo (To), Italy 5)Radiation therapy 5)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto laboratory, Candiolo cancer institute-fpo,irccs, Candiolo (To), Italy 6)Transgenic Nazionale dei Tumori, Milan, Italy 6)Unit of Molecular Therapies, Department of mouse models laboratory, Candiolo cancer institute-fpo,irccs, Candiolo (To), Italy 7) Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Ransgenic mouse models laboratory, Candiolo cancer institute-fpo,irccs, Candiolo (To), Italy Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related deaths among women, and the leading cause of death from gynecological cancer. High In solid cancers the heterogeneous population of tumor cells is enclosed in a grade serous EOCs, the most frequent and aggressive tumors, are diagnosed in particular microenvironment characterized by high levels of hypoxia and acidosis about 80% of cases when patients present peritoneal solid primary and secondary responsible of the reduction in the efficacy of chemotherapy, radiotherapy and lesions associated to malignant ascites. RNASET2 is a cytoplasmic and secreted immunotherapy. These conditions favor the recruitment of bone marrow derived- ribonuclease with anti-tumorigenic capability independently from its catalytic activity cells (BMDCs) which differentiate into tumor-associated macrophages (TAMs). This in several carcinomas. Recently, we have assessed that RNASET2 causes actin subpopulation of myeloid cells is classified as M2-like and promotes tumor growth cytoskeleton remodelling and affects cell interaction with the extracellular matrix directly by favoring tumor cell proliferation and survival. Semaphorin(s) are a family (ECM), suggesting a role of this protein in the regulation of integrin pathway. of cell-surface and soluble proteins that play a role in regulating both physiological With the aim to deeply assess RNASET2 role during EOC progression, we first and pathological angiogenesis. A growing body of evidences indicate that several performed molecular and immunohistochemical evaluation of RNASET2 expression Semaphorin(s) and their receptors, Neuropilin(s) and Plexin(s), are critically involved in EOC samples. At molecular level, analyzing two datasets of gene expression in various phases of the immune response during tumor inflammation. In particular, from EOC samples, we found that patients showing the highest RNASET2 transcript we previously demonstrated that Semaphorin 4A (Sema4A) is overexpressed in levels displayed longer overall survival. In an EOC cohort representative of the myeloid cells under inflammatory conditions and exerts a pro-angiogenic effect EOC incidence, RNASET2 protein was widely expressed among EOC samples enhancing the production of VEGF-A in macrophages. However, its function in independently from their grading and staging, being also detected in 70% of high tumor inflammation and angiogenesis is still poorly investigated. In the present grade serous EOC. Interestingly, when expressed, RNASET2 was mainly found at study, by confocal microscope and RT-Real-Time RT-PCR analysis we observed the boundaries between tumor cells and ECM. We therefore decided to silence or increased levels of Sema4A and its receptors Plexin D1 and Plexin B2 in tumors of transfect RNASET2 in two different EOC cellular models representative of low and HPV16/E2 cervical and pancreatic ductal adenocarcinoma (PDAC) mouse models high aggressive EOC. Silencing of RNASET2 conferred high proliferation rate and compared with normal tissues. Notably, we observed that in these two models, increased src kinase activation with marked changes in the actin cytoskeleton with Sema4A is mainly expressed by myeloid cells and particularly by pro-tumoral M2 ticker stress fibers and loss of cell-cell contacts. Treatment of RNASET2-silenced macrophages and to a lesser extent by T cells. Interestingly, Sema4A is present in cells with the human recombinant RNASET2 was able to revert the cytoskeleton bone marrow (BM) myeloid cells of wild type mice and its expression significantly assembly of these cells. Conversely, ectopic RNASET2 transfection negatively increased during tumor progression. Stemming from these findings, in order better affect proliferation and displayed decreased integrin-dependent src activation thus to clarify the role of Sema4A in tumor inflammation, by means of lentiviral shRNA leading to impaired migration and growth rate when grow on Matrigel. Therefore, technology we silenced Sema4A in the whole BM isolated from a group of normal RNASET2 appears to exert an inhibition of tumor aggressiveness by modulating mice. Then, a cohort of tumor bearing HPV16/E2 and PDAC mice were irradiated integrin-depentent src kinase activation thus leading to cytoskeleton changes and at 7Gy to completely abolish myeloid cells and repopulated with Sema4A-depleted impaired proliferation rate. Mimicking RNASET2 function might also represent a cells. Remarkably, we observed a significant reduction in tumor volume both in possible inhibitory approach of EOC progression. HPV16/E2 and PDAC mice lacking Sema4A compared with controls. In addition, Partially supported by AIRC and Cariplo Foundation. Sema4A depletion was able to re-program the phenotype of resident macrophages from M2-like to anti-tumor macrophages (M1-like) functional state, suggesting a crucial role of Sema4A on tumor-associated inflammation, in tumor progression. F14 AN ACTIONABLE AXIS CONNECTING NFATC2 TO FOXM1 AND EZH2 CONTROLS THE EMT-LIKE PROFILE OF MELANOMA CELLS Perotti V., Baldassari P., Molla A., Nicolini G., Bersani I., Vegetti C., Maurichi A., Santinami M., Anichini A., Mortarini R. Department of research, Fondazione IRCCS istituto nazionale dei tumori, Milan, Italy Melanoma is one of the most aggressive human tumors with the high capacity to metastatize. This type of tumor takes advantages from an EMT-like phenotype to gain migratory and invasive properties and resistance to therapy. The determination of regulators of EMT process would lead to the improvement of current therapies. We found that NFATc2, a member of a family of transcription factors, has a key role in promoting the EMT-like, dedifferentiation process in melanoma. Gene targeting experiments allowed to define the functional axis that control the EMT-like process. This axis is regulated by NFATc2 and involves c-myc, FOXM1 and EZH2. In a large panel of human melanoma cell lines NFATc2 expression correlated with main EMT markers, such as ZEB1 and CDH2. Immunohistochemistry analysis of tissue sections of primary and metastatic lesions corroborated this finding. Silencing or pharmacological inhibition of NFATc2, and of other components of the identified axis, down-regulated the expression of the EMT-related genes and suppressed the migration and invasion of melanoma cells. NFATc2 inhibition reduced melanoma cell proliferation in vitro, but also in vivo in SCID mice. Co-targeting of different components of this axis by specific inhibitors promoted melanoma apoptosis, independently of the mutational profile (BRAF, NRAS or wt/wt) and was effective even in BRAF-inhibitor resistant melanoma cells. These results indicate that NFATc2 regulates an actionable axis that controls the EMT-like phenotype of melanoma. Acknowledgments: This work was supported by AIRC grant 15860 to RM.
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health promoting activities; δ- and g-TT, in particular, were reported to be endowed CELL METABOLISM with anticancer properties in different tumors, based on the modulation of different intracellular molecular pathways. G1 In preliminary experiments we could demonstrate that δ-TT exerts a proapoptotic Pik3c2g Loss Promotes Pancreatic Cancer through mTOR activity in cells derived from two aggressive tumors, such as human castration- Regulation and Metabolic Rewiring resistant prostate cancer (DU145 and PC3 cells) and melanoma (A375 and BLM Martini M., De Santis M.C., Ratto E., Gozzelino L., Derle A., Porporato P.E., Hirsch E. cells), while sparing human normal prostate epithelial cells and melanocytes. Aim Molecular Biotechnology Center, Dip of Mol Biotechnology and Health Science, of this study was to investigate the effects of δ-TT on cancer cells metabolism, in Torino, Italy particular on mitochondrial activity. First, we demonstrated that the expression levels of respiratory chain complexes are increased in prostate cancer and melanoma Introduction cells when compared with non-tumoral cells, thus supporting a reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) is the most lethal cancer across the the OXPHOS mechanism. We then observed that δ-TT: • decreases the expression world, with incidence equaling mortality. The lack of progress in early diagnosis and levels of the proteins of the respiratory chain complexes, specifically of complex I, the effective therapies are the main reasons why improvements in PDAC death rates major entry point of respiratory chain substrates; • induces mitochondrial swelling; have been so scarce. A wealth of studies have identified the PI3K/mTOR axis as an • reduces mitochondrial activity without affecting mitochondrial mass; • decreases important player in PDACs, impacting on tumor growth and metabolism. Whereas both basal oxygen consumption and ATP production • induces ROS production. the majority of efforts have so far focused on class I PI3K, increasing evidence is These results demonstrate that δ-TT exerts a selective antitumor activity in cells pointing to the importance of class II enzymes in cell proliferation and survival. In derived from different aggressive tumors (prostate cancer and melanoma) through particular, PI3K-C2γ, differently from other class II members, is mainly expressed the reprogramming of mitochondrial metabolism. in the pancreatic and survival. In particular, PI3K-C2γ, differently from other class II (Supported by PRIN 2015 and Comitato Emme Rouge onlus) members, is mainly expressed in the pancreatic tissue where it plays a pivotal role in insulin signaling. Material and Methods G3 Mouse model of PDAC (K-RASG12D/Trp53R172H/CrePdx1) was crossed with DEVELOPMENT OF A PANEL OF MARKERS TO STUDY METABOLIC mouse strain lacking PI3K-C2γ expression. Mice were weekly followed for survival, HETEROGENEITY OF OVARIAN CANCER XENOGRAFTS tumor appearance and growth. Tumor lesions were evaluated by histopathological Verza M., Pinazza M., Nardo G., Zulato E., Esposito G., Indraccolo S. and immunofluorescence analysis. Functional in vitro and in vivo experiments were Istituto Oncologico Veneto, IRCCS, Padova, Italy performed. Results and Discussions Previous studies suggested that acquired resistance to anti-VEGF drugs in tumor We modeled PI3K-C2γ loss in PDAC by targeting PIK3C2G gene in a mouse model models is associated with a metabolic rewiring marked by increased expression of of pancreatic cancer (KPC) and found that its deletion both initiates and promotes MCT4, a lactate transporter considered a reliable marker of glycolysis. As corollary pancreatic tumor development. Loss of PI3K-C2γ in KPC mice strongly reduces of this observation it might be argued that high level expression of MCT4 might mice mean survival rate (18 vs 36 w) and drives rapid progression to PDAC. Low correlate with primary resistance to anti-VEGF drugs, although this hypothesis has PI3K-C2γ expression was significantly associated with poor survival and increased not been adequately validated in the literature. These considerations prompted us to resistance to chemotherapeutic agents in pancreatic tumors. We observed that investigate MCT4 expression in a panel of patient-derived ovarian cancer xenografts PI3K-C2γ can control autophagy in tumor cells through the mTORC1 pathway. (PDX). Expression of this glycolytic marker was analyzed by immunohistochemistry In addition, we showed that PI3K-C2γ promotes the metabolic rewiring of PDAC, (IHC) in n=21 out of n=32 PDX available in our laboratory that formed a solid mass regulating several prominent metabolic factors, including glycolytic enzymes (PKM2, after intraperitoneal (i.p.) injection in NOD/SCID mice. MCT4 expression was very HK2 and LDH), glucose (GLUT1) and monocarboxylate (MCT4) transporters. -/- heterogeneous among the PDX analyzed and allowed us to classify them based on Furthermore, pharmacological inhibition of autophagy in PIK3C2G KPC leads to our scoring system into highly glycolytic (intensity score ≥6) and poorly glycolytic tumor regression (intensity score <6). We previously demonstrated that tumors formed by highly Conclusion glycolytic cancer cell lines grow faster than poorly glycolytic tumors in subcutaneous Overall, these findings establish PI3K-C2γ as a PDAC tumor suppressor and models and confirmed here this correlation also in PDX models grown i.p. In order to suggest that the metabolic phenotype of PI3K-C2γ-deficient tumors can be exploited understand if faster in vivo growth of highly glycolytic PDX was related to increased by specific therapeutic strategies. cell proliferation, we performed IHC analysis for a nuclear proliferation marker (phospho-histone-H3), but we observed no differences between highly and poorly glycolytic PDX. We thus conclude that increased cell proliferation did not account for G2 the phenomenon observed. In addition, we extended metabolic characterization of Targeting mitochondrial metabolism in prostate cancer and the PDXs by including markers related to other metabolic pathways like glutamine melanoma cells with δ-tocotrienol metabolism, serine/glycine pathway and fatty acid metabolism. Concerning 1)Raimondi M. 2)Fontana F. 3)Marzagalli M. 4)Longo R. 5)Crestani M. 6)Procacci P. glutamine metabolism, we analyzed expression of the aminoacid transporter SLC1A5 7)Sartori P. 8)Moretti R.M. 9)Montagnani Marelli M. 10)Limonta P. and the enzyme glutaminase (GLS). Serine/Glycine metabolism was investigated 1)Department of Pharmacological and Biomolecular Sciences, Università degli by analysis of serine hydroxymethyltranferase1 (SHMT1) and phosphoglycerate- Studi di Milano, Milano, Italy 2)Department of Pharmacological and Biomolecular dehydrogenase (PHGDH). Finally, we monitored lipids metabolism by analyzing Sciences, Università degli Studi di Milano, Milano, Italy 3)Department of Experimental expression of fatty-acid-synthase (FAS), acetylcoenzyme-A-carboxylase (ACC) and Therapeutics, Beckman Research Institute, City of Hope National Medical Center, carnitine-palmitoyl-transferase1A (CPT1A). We first validated anti-GLS, PHGDH Duarte, CA, USA 4)Department of Pharmacological and Biomolecular Sciences, and SHMT1 antibodies by using specific siRNAs and performed IHC analysis using Università degli Studi di Milano, Milano, Italy 5)Department of Pharmacological and both cell-blocks and tumor masses derived from two ovarian cell lines in order to Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy 6)Department assess intensity of staining in interspersed single cells. of Biomedical Sciences for Health, Università degli Studi di Milano, Milano, Italy 7) Our aim will be to correlate tumor growth rates of ovarian xenografts in vivo with Department of Biomedical Sciences for Health, Università degli Studi di Milano, expression of the various metabolic markers included in the panel. To do this, Milano, Italy 8)Department of Pharmacological and Biomolecular Sciences, we are now trying to identify appropriate cut-off values for IHC markers in order Università degli Studi di Milano, Milano, Italy 9)Department of Pharmacological to classify PDX into high-metabolism and low-metabolism. In future studies, our and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italy 10) metabolic panel will be used to characterize general metabolic features of PDX and Department of Pharmacological and Biomolecular Sciences, Università degli Studi primary tumors, with the final aim of exploiting it a prognostic tool. di Milano, Milano, Italy Metabolic reprogramming is recognized as a key feature of cancer cells. In addition to high glycolysis, upregulated mitochondrial metabolism sustains the uncontrolled growth rate in tumor cells, that require good supply of macromolecules for survival. Thus, mitochondria constitute promising targets for novel cancer treatment strategies, based on both synthetic or natural compounds. Vitamin E is composed of two groups of hydrophobic compounds, tocopherols (TPs) and tocotrienols (TTs) and each group consists of four isomers: a, β, g and δ. The chemical structure of TPs and TTs is composed of a chromanol ring linked to a isoprenoid side chain; this chain is saturated in TPs and unsaturated in TTs. The four isoforms, both TPs and TTs, differ on the number and position of methyl groups. In the last decades, TTs (but not TPs) have attracted great interest for their
42 43 ABSTRACT BOOK
G4 ALL cells are known to express low-to-absent levels of Asparagine Synthetase TRAP1 favor glycolytic metabolism and resistance to EGFR (ASNS), the enzyme that synthetizes asparagine (Asn) from glutamine (Gln) and inhibitors in human colorectal carcinoma through regulation aspartate. This phenotype renders ALL cell growth strictly dependent on extracellular of PFK Asn availability. It has been proposed that MSCs support ALL cells with Asn, during 1)Maddalena F. 2)Condelli V. 3)Li Bergolis V. 4)Sisinni L. 5)Pacelli C. 6)Pietrafesa M. the treatment with Asparaginase, which depletes blood Asn and Gln. However, the 7)Lettini G. 8)Lepore S. 9)Storto G. 10)Capitanio N. 11)Esposito F.12)Landriscina M. mechanism involved in this nutritional support has been unknown thus far. 1)Laboratory of Pre-Clinical and Translational Research, IRCCS-CROB, Rionero MSCs, isolated from bone marrow aspirates of healthy donors (HD-MSCs) and ALL in Vulture (PZ), Italy 2)Laboratory of Pre-Clinical and Translational Research, patients (ALL-MSC), have been cultured in asparagine-free DMEM, with 2 mM Gln IRCCS-CROB, Rionero in Vulture (PZ), Italy 3)Clinical Oncology Unit, Department and 10% FBS. MSCs were maintained as monoculture or co-cultured with freshly of Medical and Surgical Sciences, University of Foggia, Italy 4)Laboratory of Pre- isolated BPC-ALL blasts or with ALL cell lines. Asparaginase (ASNase) treatment Clinical and Translational Research,, IRCCS-CROB, Rionero in Vulture (PZ), Italy was performed with the enzyme from Erwinia chrysanthemi (1U/ml). 5)Biochemistry Unit, Department of Clinical and Experimental Medicine, University Compared with HD-MSCs, ALL-MSCs exert an higher protection on ALL cell lines of Foggia, Italy 6)Laboratory of Pre-Clinical and Translational Research, IRCCS- during ASNase treatment. Both HD-MSCs and ALL-MSCs express comparable CROB, Rionero in Vulture (PZ), Italy 7)Laboratory of Pre-Clinical and Translational levels of ASNS and GS. However, compared to HD-MSCs, ALL-MSCs express Research, IRCCS-CROB, Rionero in Vulture (PZ), Italy 8)Laboratory of Pre-Clinical higher levels and activity of the transporter SNAT5, a transporter that mediates the and Translational Research, IRCCS-CROB, Rionero in Vulture (PZ), Italy 9)Nuclear efflux of Asn and Gln. Consistently, 3H-Gln efflux was higher in ALL-MSCs than Medicine Unit, IRCCS-CROB, Rionero in Vulture (PZ), Italy 10)Biochemistry Unit, HD-MSCs. When co-cultured with freshly purified ALL blasts or lines, an increase Department of Clinical and Experimental Medicine, University of Foggia, Italy 11) of SNAT5 mRNA was detected in HD-MSCs, which was associated with a decrease Department of Molecular Medicine and Medical Biotechnology, University of Naples of ASNS mRNA in ALL blasts. Federico II, Italy 12)Laboratory of Pre-Clinical and Translational Research and These data suggest that ALL blasts drive MSCs to increase the secretion of 4Nuclear Medicine Unit, IRCCS-CROB, Rionero in Vulture (PZ), Italy asparagine via the SNAT5 transporter, and disclose a new possible target to overcome resistance to ASNase. Introduction. Metabolic rewiring is necessary to support the increased need for energy production and synthesis of nucleic acids intermediates in rapidly proliferating cancer cells. Thus, tumor cells growth and cellular metabolism are G6 closely interrelated and this may be relevant in modulating the clinical activity of AMINO ACIDS METABOLIC REPROGRAMMING SUSTAINS ENDOCRINE cytostatic agents, such as EGFR inhibitors. TRAP1 is a member of the HSP90 THERAPY RESISTANCE OF ER+ BREAST CANCER. family, involved in control of a variety of cellular functions, including intracellular 1)Bacci M. 2)Lorito N. 3)Ferracin M. 4)Ramazzotti M. 5)Ippolito L. 6)Luti S. 7) signaling, cell cycle progression, apoptosis and drug resistance, stemness and Chiarugi P. 8)Morandi A. bioenergetics through co-traslational regulation of a network of client proteins. 1)Department of Experimental and Clinical Biomedical Sciences, University of Consequently, the activation of TRAP1 network is responsible for poor outcome in Florence, Florence, Italy 2)Department of Experimental and Clinical Biomedical human colorectal carcinomas (CRC). Thus, the role of this molecular chaperone in Sciences, University of Florence, Florence, Italy 3)Department of Specialised, controlling the metabolic rewiring of human CRC cells and its relationship with the Experimental, and Diagnostic Medicine, University of Bologna, Bologna, Italy 4) cytostatic activity of cetuximab were evaluated. Department of Experimental and Clinical Biomedical Sciences, University of Materials and Methods. These issues were assessed by standard molecular and Florence, Florence, Italy 5)Department of Experimental and Clinical Biomedical cell biology methods and by using in vitro CRC cell models and human CRCs. Sciences, University of Florence, Florence, Italy 6)Department of Experimental and Results. The analysis of a series of human CRCs showed that TRAP1 is co- Clinical Biomedical Sciences, University of Florence, Florence, Italy 7)Department expressed with the glucose transporter GLUT1 and correlates with high 18F-FDG of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, PET uptake. In addition, TRAP1 is responsible for favoring the Warburg metabolism Italy 8)Department of Experimental and Clinical Biomedical Sciences, University of in CRC cell lines and this occurs upon interaction with phosphofructokinase-1 Florence, Florence, Italy (PFK1), a key glycolytic enzyme, and through regulation of its activity. Interestingly, this TRAP1 regulation on PFK1 activity is impaired by intermediates of TCA cycle. Endocrine therapy is the first choice of treatment for oestrogen receptor positive Functionally, TRAP1 silencing results in decreased glucose uptake, PFK1 activity, (ER+) breast cancers. Unfortunately, the clinical benefit is still limited due to lactate production and increased mitochondrial oxygen consumption. Finally, the resistance. We have previously published that endocrine therapy resistant (ETR) inhibition glycolytic metabolism by the glucose analog 2-deoxy-D-glucose (2-DG) cancer cells are characterised by enhanced central carbon metabolic plasticity. In or the PFK1 inhibitor 3PO, as well as TRAP1 silencing by siRNA induce cell cycle the current study, we performed a global gene expression profiling of ETR cells arrest in G0-G1 transition and greatly increase the cytostatic activity of cetuximab in compared to parental cells. Interestingly, Gene Set Enrichment Analysis revealed RAS wild type CRC cell lines. a global decreased expression of amino acids transporters that correlates with Conclusions. These data identify TRAP1 as central regulator of Warburg metabolism an impairment of amino acids uptake in the ETR cells compared to the parental in human CRC and suggest a molecular link between glycolytic metabolism and cell counterpart. Particularly, the expression of the amino acid transporter SLC6A14 cycle progression, making TRAP1 an attractive therapeutic target to potentiate the is strongly decreased in ETR cells and its deregulation correlated with autophagy cytostatic activity of EGFR inhibitors. activation in ETR cells. Interestingly, analysis of publicly available clinical datasets shows that lower SLC6A14 expression correlates with poor survival in ER+ breast cancer. HPLC analysis of the amino acids intracellular content of ETR cells showed G5 a selective increase in aspartate and glutamate levels when compared to parental THE SNAT5 TRANSPORTER IS INVOLVED IN THE ASPARAGINE SUPPLY OF cells. This enhancement is not dependent by the increased autophagy activation that ACUTE LYMPHOBLASTIC LEUKEMIA BLASTS BY MESENCHYMAL STROMAL characterised ETR cells but is mediated by increased expression of the aspartate/ CELLS glutamate transporter SLC1A2, the only amino acid transporter found significantly 1)Chiu M. 2)Taurino G. 3)Dander E. 4)Bardelli D. 5)Mirandola P. 6)Carubbi C. 7) upregulated in ETR cells, which leads to an increase of exogenous aspartate and Andreoli R. 8)Rizzari C. 9)D’Amico G. 10)Bussolati O. glutamate uptake in ETR cells. Therefore, we analysed aspartate and glutamate 1)Laboratory of General Pathology, Department of Medicine and Surgery, University destiny by radioactive tracing assays and Seahorse analysis. Notably, aspartate of Parma, Parma Italy 2)Laboratory of General Pathology, Department of Medicine fuels mitochondria-dependent metabolism and the associated-anabolic pathways and Surgery, University of Parma, Parma, Italy 3)“M. Tettamanti” Research Center, (e.g. lipids, proteins and nucleotides synthesis) whereas glutamate seems to be Department of Pediatrics, University of Milano Bicocca, MBBM Foundation, Maria preferentially involved in DNA synthesis but can rescue mitochondrial metabolic Letizia Verga Center, Monza, Italy 4)“M. Tettamanti” Research Center, Department potential when aspartate is not available. Aspartate and glutamate deprivation of Pediatrics, University of Milano Bicocca, MBBM Foundation, Maria Letizia Verga impairs colony formation ability of ETR cells and interfering with SLC1A2 expression Center, Monza, Italy 5)Human Anatomy, Department of Medicine and Surgery, leads to reduction of cell proliferation and invasion in ETR cells, suggesting that University of Parma, Parma, Italy 6)Human Anatomy, Department of Medicine and negatively charged amino acids are involved in the survival and aggressiveness Surgery, University of Parma, Parma, Italy 7)Industrial Toxicology, Department of of ETR cells. In conclusion, these results suggest that amino acid metabolic Medicine and Surgery, University of Parma, Parma, Italy 8)Pediatric Hematology reprogramming could represent a strategic adaptive mechanism during ETR. The Oncology Unit, Department of Pediatrics, San Gerardo Hospital, University of molecular mechanisms involved in this metabolic reprogramming may be targeted Milano Bicocca, Monza, Italy 9)“M. Tettamanti” Research Center, Department of to combat or delay endocrine therapy resistance in ER+ breast cancers. Pediatrics, University of Milano Bicocca, MBBM Foundation, Maria Letizia Verga Cente, Monza, Italy10)Laboratory of General Pathology, Department of Medicine and Surgery, University of Parma, Parma, Italy
Mesenchymal stromal cells (MSCs) are in close contact with acute lymphoblastic leukemia (ALL) blasts in a confined niche in the bone marrow microenvironment.
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G7 We found that OC-PDX are heterogeneous in term of metabolic features (the STROMAL CONTACT REGULATES PROSTATE CANCER EPIGENETIC AND different approaches allowed to categorize OC-PDX as highly or poorly glycolytic) METABOLIC REPROGRAMMING and malignant behavior. Ippolito L., Ferrone L., Comito G., Morandi A., Giannoni E., Chiarugi P. Furthermore we established that: Department of Experimental and Clinical Biomedical Sciences, University of - the low glycolytic OC-PDXs developed more abdominal metastases than the Florence, Florence, Italy highly glycolytic ones - OC-PDX capable to adapt their metabolism are more malignant: the expression of Background: Stromal and tumor cells orchestrate the establishment of a peculiar glycolysis-related genes increased during the progression of the disease in those microenvironment shaped by reciprocal metabolic interactions, leading to dramatic OC-PDX exhibiting shorter survival time (more aggressive), while it remained changes of malignant phenotype of tumor cells. Our prior research efforts unchanged in the less aggressive one (longer survival time). demonstrate that cancer-associated fibroblasts (CAFs) exert a supporting role in Altogether our data indicate that ovarian cancer cells modulates their metabolism prostate cancer (PCa) progression by promoting a TCA cycle reprogramming of to best fit the needs favoring metastatic spread and suggest that perturbing the PCa cells primed by stromal lactate. However, the altered metabolism induced by tumor’s metabolic re-programming is exploitable to affect metastatic dissemination. stromal contact is likely intimately connected to specific epigenetic changes. Indeed, Further studies are needed to fully understand this phenomenon. our aim was to clarify the interplay between epigenetic and metabolic rewiring in Supported by AIRC IG2016 18853 to RG. and Cariplo n. 2017-0896 to AD CAF-conditioned PCa cells. Methods: Human PC3 and DU145 PCa cells were co-cultured with or treated by conditioned medium from human prostate fibroblasts (HPFs) or CAFs derived from G9 PCa patients. Epigenetic signature was analyzed by WB, ChIP and specific kits. Aberrant choline metabolism in epithelial ovarian cancer: Results: Our data support a clear CAF-induced TCA cycle rearrangement in using a cancer hallmark as therapeutic target PCa cells, resulting in the accumulation of succinate and fumarate. Accordingly, 1)Rizzo A. 2)Alberti P. 3)Satta A. 4)Figini M. 5)Raspagliesi F. 6)Iorio E. 7)Bagnoli M. by administrating them to cancer cells, we were able to mimic the pro-invasive 8)Mezzanzanica D. CAF effect. We also address the histone methylation pattern in PCa cells and, 1)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto accordingly with succinate and fumarate amount, we found a hypermethylator Nazionale dei Tumori, Milan, Italy 2)Unit of Molecular Therapies, Department of pattern (H3K27me3 and H3K9me2) in CAF-exposed PCa cells. Furthermore, by Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Unit of using an inhibitor of lactate inward transporter MCT1, we impaired the methylation Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale status of conditioned cancer cells, suggesting that CAF-derived lactate could act dei Tumori, Milan, Italy 4)Unit of Molecular Therapies, Department of Research, as an epigenetic regulator for PCa cells. Also, we found that CAF conditioning Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Department of is crucial for the repression of CDH1 promoter in PCa cells via both histone and Gynecologic Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, DNA methylation, thereby boosting the aggressive phenotype of PCa cells. Finally, Italy 6)Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, we found a peculiar expression of succinate receptor SUCNR1 and transporter Rome, Italy 7)Unit of Molecular Therapies, Department of Research, Fondazione SLC13A3 in CAFs and CAF-reprogrammed PCa cells, compared to their respective IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8)Unit of Molecular Therapies, controls, suggesting a hypothetical role of extracellular succinate in shaping tumor- Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, stroma interplay. Italy These data show a particular epigenetic mark – driven by stroma and orchestrated not only by lactate but also (exogenous and endogenous) succinate – underlying Epithelial ovarian cancer (EOC), the most lethal gynecological disease, is PCa cell malignancy. characterized by the “cholinic phenotype”, a metabolic alteration sustained by choline kinase-α (CHKA) overexpression and hyper-activation that reflects deep interactions between oncogenic signaling and cell metabolism. We have shown G8 that CHKA down-modulation in different EOC cellular models affected cancer cells Metastatic and metabolic features of patient-derived ovarian aggressiveness by reducing typical tumor features such as cell proliferation, motility cancer xenografts and invasion capabilities. Recently, we observed that CHKA knock-down sensitized 1)Ghilardi C..2)Decio A. 3)Brunelli L. 4)Galimberti M. 5)Pastorelli R. 6)Bani M.R. 7) cells to receptor-mediated apoptotic stimuli. Giavazzi R. Upon CHKA silencing both in-vitro and in ex-vivo derived EOC cells, we observed 1)Laboratory of Biology and Treatment of Metastasis, Department of Oncology, an enhanced expression of several TNF receptor superfamily members, known IRCCS- Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy 2)Laboratory death receptors involved in the apoptotic process once engaged by their ligands. of Biology and Treatment of Metastasis, Department of Oncology, IRCCS- Istituto In particular, our data indicate an increased cell surface expression of TRAIL-R2 di Ricerche Farmacologiche “Mario Negri”, Milan, Italy 3)Laboratory of Mass which sensitized siCHKA cells to TRAIL-mediated cell death or to the redirection Spectrometry, Department of Environmental Health Sciences; IRCCS- Istituto di of lymphocytes cytotoxic activity by a bi-specific anti-TRAIL-R2/anti-CD3 antibody. Ricerche Farmacologiche “Mario Negri”, Milan, Italy 4)Laboratory of Biology and With the aim to dissect the apoptotic pathway triggered by the expression of Treatment of Metastasis, Department of Oncology, IRCCS- Istituto di Ricerche TRAIL-R2 following CHKA silencing, we also evaluated changes at the level of Farmacologiche “Mario Negri”, Milan, Italy 5)Laboratory of Mass Spectrometry, mitochondrial damages. Our results indicate a loss of mitochondrial membrane Department of Environmental Health Sciences; IRCCS- Istituto di Ricerche potential, thus weakening CHKA silenced cells. Farmacologiche “Mario Negri”, Milan, Italy 6)Laboratory of Biology and Treatment of Since further metabolomic and mass spectrometry analysis showed variations in Metastasis, Department of Oncology, IRCCS- Istituto di Ricerche Farmacologiche intracellular contents of different metabolites as side effect of CHKA knock-down, “Mario Negri”, Milan, Italy 7)Laboratory of Biology and Treatment of Metastasis, we are now evaluating metabolites and related pathways whose alterations may Department of Oncology, IRCCS- Istituto di Ricerche Farmacologiche “Mario Negri”, be responsible for the observed EOC sensitization and immunological recognition. Milan, Italy Considering that EOC is characterized by ascites development, a microenvironment where tumor aggregates float together with immune effector cells, increasing the Ovarian adenocarcinoma (OC) is the most lethal gynecologic malignancy in sensitivity of cancer cells to receptor-mediated apoptosis could be considered an the developed countries, with a 5-yr survival rate of <30% that has shown little interesting therapeutic strategy in combination with drugs or with immunomodulators. improvement in the last 20 years. Partially supported by CARIPLO Foundation, AIRC and 5x1000 Institutional More than 70% of cases are diagnosed after tumors have widely spread within Research Funding. the peritoneal cavity, limiting the effectiveness of surgery and chemotherapy. Understanding the mechanisms facilitating metastatic spread is needed to develop therapeutic strategies. Metabolic re-programming is an emerging hallmark of cancer. Alterations of glycolysis as well as oxidative phoshorylation (OxPhos) are frequently observed in cancer cells and have effects on gene expression, cellular differentiation and tumor behavior. Our aim is the comprehension of the metabolic perturbations associated to metastatic dissemination of OC. To this purpose we characterized a panel of OC-patient derived xenografts (OC- PDX) for their malignant behavior (ascites production, abdominal metastasis formation and survival time) and metabolic features (glucose and lactate levels in ascites fluid, gene expression of glycolysis- and OxPhos- related genes and metabolomic analysis in cell aggregates from abdominal effusion and in metastases of peritoneal organs (tumor masses from pancreas, liver, lymph nodes).
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a slower cell growth rate and a lower clonogenic ability if compared to the wild DNA DAMAGE AND type (WT) cells, and a double DNA content. CDK12 KO clones display a higher MOLECULAR RESPONSES TO DAMAGE basal induction of apoptosis than the WT cells, while no differences in autophagy or senescence were observed. The mRNAs expression of some DNA repair genes, H1 measured by real time PCR, were decreased in CDK12 KO clones as compared to MRE11 inhibition highlights a replication stress-dependent WT cells (BRCA1, CHK1, WEE1 and FANCD2). Western blot analysis corroborated vulnerability of MYCN amplified neuroblastoma the decreased expression protein level of BRCA1. Cytotoxic effect of anticancer 1)Petroni M. 2)Sardina F. 3)Infante P. 4)Bartolazzi A. 5)Locatelli E. 6)Fabretti F. 7) agents with different mechanisms of action were evaluated on CDK12 KO clones Capalbo C. 8)Cardinali B. 9)Coppa A. 10)Colicchia V. 11)Sahùn Roncero M. 12) and WT cells by MTS assays. CDK12 KO clones are less sensitive to ATR, CHK1 Belardinilli F. 13)Di Marcotullio L. 14)Soddu S. 15)Comes Franchini M. 16)Petricci and WEE1 inhibitors treatment as compared to WT cells, while platinum, paclitaxel, E. 17)Giannini G. trabectedin, olaparib, ATM and CDK7/12 inhibitors have similar cytotoxic activity 1)Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy in KO and WT cells. When CDK12 KO clones and WT cells were subcutaneously 2)Dept. of Molecular Medicine, University La Sapienza, Rome, Italy 3)Center for Life transplanted in nude mice, tumor take, tumor lag appearance and tumor growth Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy 4)Pathology were impaired in KO clones as compared to WT ones. In conclusion, we were able Research Laboratory, St. Andrea University Hospital, Rome, Italy 5)Department of to obtain CDK12 KO. We think that these models could help in disclosing new Industrial Chemistry “Toso Montanari”, Bologna, Italy 6)Dept. of Molecular Medicine, roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new University La Sapienza, Rome, Italy 7)Dept. of Molecular Medicine, University combination therapies for tumors with CDK12 impairments. La Sapienza, Rome, Italy 8)Institute of Cell Biology and Neurobiology, National Research Council, Campus A. Buzzati-Traverso, Monterotondo, Rome, Italy 9)Dept. Experimental Medicine, University La Sapienza, Rome, Italy 10)Dept. of Molecular H3 Medicine, University La Sapienza, Rome, Italy 11)Dept. of Molecular Medicine, ERCC1-XPF complex as a read out of nucleotide excision repair University La Sapienza, Rome, Italy 12)Dept. of Molecular Medicine, University La activity in ovarian patient derived xenografts with different Sapienza, Rome, Italy 13)9Istituto Pasteur-Fondazione Cenci Bolognetti,, Rome, response to cisplatin treatment Italy 14)Unit of Cellular Networks and Molecular Therapeutic Targets, Regina 1)Guffanti F. 2)Caiola E. 3)Ganzinelli M. 4)Ricci F. 5)Affatato R. 6)Damia G. Elena National Cancer Institite _IRCCS, Rome, Italy 15)Department of Industrial 1)Laboratory of molecular pharmacology, Irccs- Istituto Mario Negri, Milan, Italy 2) Chemistry “Toso Montanari”, Bologna, Italy 16)Dept.of Biotechnology, Chemistry Laboratory of molecular pharmacology, Irccs- Istituto Mario Negri, Milan, Italy 3) and Pharmacy, University of Siena, Siena, Italy 17)Istituto Pasteur-Fondazione Medical oncology department, Fondazione Irccs istituto nazionale dei tumori, Milan, Cenci Bolognetti, Rome, Italy Italy 4)Laboratory of molecular pharmacology, Irccs- Istituto Mario Negri, Milan, Italy 5)Laboratory of molecular pharmacology, Irccs- Istituto Mario Negri, Milan, Italy 6) High risk neuroblastoma patients are frequently characterized by MYCN Laboratory of molecular pharmacology, Irccs- Istituto Mario Negri, Milan, Italy amplification. Despite aggressive treatments, these children very often experience consecutive recurrences and fatal outcome, making the search for effective After an initial response to a platinum (DDP) based therapy, almost 70% of ovarian therapies an absolute priority. Since targeting MYCN in the clinical settings cannot cancer patients will develop resistance to such treatment. Indirect evidence suggest be achieved, yet, discovering novel MYCN-associated vulnerabilities might provide that cellular DNA repair activity is inversely correlated with DDP sensitivity. Few alternative strategies for the therapy of MYCN-driven neuroblastoma. functional repair assays are available to directly address the relevance of DNA The consolidated notion that MYCN induces replication stress prompted us to repair cellular capacity in DDP sensitivity/resistance. With this aim, we set up a explore the possibility to trigger intolerable levels of replication stress-dependent proximity ligation assay (PLA) to quantify the activity of nucleotide excision repair DNA damage to treat MYCN-amplified neuroblastoma. (NER), one of the pathway involved in the repair of DDP induced DNA damage. Previously, we reported that the MRE11 complex, a crucial player in the DNA damage An ovarian PDX platform was recently established in our laboratory, whose response response (DDR) and in the maintenance of replication fork stability, is essential for to a DDP based therapy is known. In addition, we have available PDXs resistant to MYCN-dependent proliferation and survival of neuronal precursor cells. DDP (MNHOC124R, MNHOC124LPR and MNHOC239R) obtained after several Now we addressed MRE11 role in human neuroblastoma. Interestingly low MRE11 in vivo DDP treatments of sensitive PDXs (MNHOC124S, MNHOC124LPS and expression characterizes primary human MYCN-single copy neuroblastomas with MNHOC239S). Tumours grown in mice both as subcutaneous masses or ascitic bad prognosis, consistent with its established oncosuppressive function. In sharp fluid, were obtained in their exponential growth, fixed in formalin and paraffin contrast, high MRE11 expression is associated with bad prognosis and occurs in embedded (FFPE) at the time of sacrifice. The protein complexes ERCC1-XPF, that MYCN-amplified tumors, suggesting it might be required for tumor growth, in this represent a biomarker of the NER activity, was quantified by PLA as foci per nucleus subset. Consistently, either MRE11 knock-down or its pharmacological inhibitor in FFPE tumour sections. mirin, induced accumulation of replication stress and DNA damage biomarkers Statistically significant higher level of ERCC1-XPF foci could be observed in (i.e. p53BP1 foci, H2AX phosphorylation), specifically in MYCN-amplified cells. MNHOC124R and -124LPR as compared to their sensitive counterparts. No The consequent DDR recruited p53 and resulted in the expression of pro-apoptotic differences were observed between MNHOC239S and R, even if the number of p53 target genes and in p53-dependent cell death, as revealed by p53 loss- and ERCC1-XPF foci in MNHOC239S were statistically higher than the ones observed gain-of-function experiments. Encapsulation of mirin in water-dispersible polymeric in MNHOC124S and in -124LPS. mRNA and protein levels of the different ERCC1 nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts. Mirin isoforms and of XPF were similar between the pairs of sensitive and resistant PDXs. treatment resulted in a sharp impairment of tumor growth, associated with DDR PDXs highly sensitive to DDP (MNHOC266, -230) showed a low number of ERCC1- activation, p53 accumulation and cell death. XPF complexes similar to MNHOC124S and -124LPS. In conclusion, our findings show that MRE11 inhibition might be an effective strategy These preliminary results highlight a possible link between DDP resistance and a to treat MYCN-amplified and p53 wild-type neuroblastoma and support the idea higher NER activity in ovarian cancer that need to be confirmed in a wider panel of that targeting the replication stress response to induce DNA damage-dependent tumours. This study corroborates the importance to develop new functional assays apoptosis should be further exploited for the cure of these tumors. to directly evaluate the activity of different DNA repair pathways that could predict the DDP activity in patients.
H2 Characterization of CDK12 knocked out ovarian cancer cell H4 lines Role of DNA Polymerase β in platinum resistance in non-small Chilà RC., Panini NP., Guffanti FG., Damia GD. cell lung cancer (NSCLC) Oncology, Mario Negri Institute, Milan, Italy Tomanelli M., Tumiatti F., Caiola E., Marabese M., Broggini M. Laboratory of Cancer Pharmacology, Department of Oncology, IRCCS - Istituto di Cyclin-dependent kinases (CDKs) have a key role both in controlling transition Ricerche Farmacologiche “Mario Negri”, Milan, Italy between the different phases of the cell cycle (i.e CDK4/6) and in the regulation of transcription (CDK7, 9 and 12). Specifically, CDK12 has been shown to regulate Background the expression of genes involved in DNA damage and to maintain genomic stability. Lung Cancer is the first cause of cancer-related death in the world. The alterations CDK12 mutations, interfering with its catalytic activity, have been reported in 3% in KRAS oncogene are very frequent (25%), but, this protein is at present of high grade serous ovarian carcinomas. Impairment of its activity has been undruggable. The gold standard therapy for KRAS-mutated NSCLC is platinum- demonstrated to sensitize to PARP inhibitor and cisplatin treatments in different based, but the emerging of drug-resistance limits its effectiveness. Previous studies, cellular systems. To better understand its role in ovarian cancer and in response to performed in NCI-H1299-derived isogenic cell line system that over-express the chemotherapy, we generate ovarian cancer cell lines knocked out (KO) for CDK12 most common NSCLC KRAS mutations (G12C,G12V,G12D), showed a different using CRISPR/Cas9 technology. We obtained 2 CDK12 KO clones, A2780 KO sensitivity to platinum-based treatment and the resistance could be mediated by and SKOV3 KO, out of more than 300 clones screened. CDK12 KO clones have the deregulation of base excision repair (BER) through the over-expression of the
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DNA polymerase beta (POLB). Here we cloned and analyzed the POLB promoter and we explored strategies that could potentially be used to treat tumor cells with DRUG RESISTANCE high POLB expression. I1 Methods PPARγ AGONIST PROMOTES ADIPOCYTIC DIFFERENTIATION AND POTENTIATES Two regions of POLB promoter (about 600 bp and 1500 bp), containing core THE ANTITUMOR ACTIVITY OF TRABECTEDIN IN MYXOID LIPOSARCOMA promoter, was cloned and analyzed through luciferase assays to characterize the PATIENT-DERIVED XENOGRAFTS molecular mechanism of POLB regulation in NSCLC. 1)Ballabio S. 2)Craparotta I. 3)Mannarino L. 4)Frapolli R. 5)Bello E. 6)Ponzo M. 7) The isogenic system and a panel of NSCLC cell lines both expressing different Carrassa L. 8)Pilotti S. 9)Brich S. 10)Sanfilippo R. 11)Casali P.G. 12)Marchini S. 13) levels of POLB were treated with carboplatin as alternative strategy to overcome D’Incalci M. the cisplatin resistance. 1)Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Results Milano, Italy 2)Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche The cloned regions of POLB promoter did not seem to be responsible for the “Mario Negri”, Milano, Italy 3)Department of Oncology, IRCCS Istituto di Ricerche different POLB expression observed in both the isogenic system and the cell lines Farmacologiche “Mario Negri”, Milano, Italy 4)Department of Oncology, IRCCS panel. Carboplatin treatment was able to overcome, in the isogenic system, the Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 5)Department of resistance to cisplatin, being its adducts less susceptible to repair by BER. This Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 6) result was not confirmed when the whole NSCLC cell lines panel was considered. Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Conclusion Milano, Italy 7)Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche In cells in which the overall DNA repair system is similar (as in the isogenic system “Mario Negri”, Milano, Italy 8)Department of Diagnostic Pathology and Laboratory, used here), changes in BER activity could impact on cisplatin sensitivity. However Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 9)Department of when other DNA repair systems (such as NER and HR) differ among the different Diagnostic Pathology and Laboratory, Fondazione IRCCS Istituto Nazionale cells, the role of BER in cisplatin response seems quantitatively poorly relevant. Tumori, Milan, Italy 10)Department of Medical Oncology Unit II, Fondazione IRCCS In the future we will perform ad hoc assays to better characterize the relative Istituto Nazionale dei Tumori, Milan, Italy 11)Department of Medical Oncology Unit contribution of various DNA repair systems, compared to BER activity, in platinum II, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 12)Department of treatment response. Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy 13)Department of Oncology, IRCCS Istituto di Ricerche Farmacologiche “Mario H5 Negri”, Milano, Italy Ganoderma lucidum extracts restrain the oncogenic potential Myxoid/round cell liposarcoma (MLPS) is the second most common type of of triple negative breast cancer and increase the therapeutic liposarcoma. It is characterized by the chromosomal translocation t(12;16)(q13;p11), efficacy of doxorubicin. which produces FUS-CHOP fusion gene. It is known to promote malignant 1)Romero Cordoba S. 2)Cosentino G. 3)Peña-Luna M. 4)Hidalgo A. 5)Tagliabue E. transformation by deregulating RNA transcription and thereby blocking adipocyte 6)Iorio M.V. 7)Martínez Carrera D.C. differentiation. Trabectedin is able to block the FUS-CHOP activity and promotes 1)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, adipogenesis. Since pioglitazone is a PPARγ agonist with pro-differentiation effects, Milan, Italy 2)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei the aim of our work is to evaluate the efficacy of combined treatment of trabectedin Tumori, Milan, Italy 3)Cancer Genomics Laboratory, National Institute of Genomic with pioglitazone in different types of MRCL patient-derived xenografts (PDX). Medicine, Mexico 4)Cancer Genomics Laboratory, National Institute of Genomic Three different MLPS xenografts model, namely ML017 (type I), ML017/ET (type Medicine, Mexico 5)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale I resistant) and ML006 (type III), have been established our laboratory. Mice were dei Tumori, Milan, Italy 6)Molecular Targeting Unit, Fondazione IRCCS Istituto sacrificed 15 days after the last dose of trabectedin and 3 hours after the last dose Nazionale dei Tumori, Milan, Italy 7)Biotecnología de Hongos Comestibles, of pioglitazone. Funcionales y Medicinales, Colegio de Postgraduados (CP), Campus Puebla, Comparing gene expression data, we performed Gene Set Enrichment Analysis Puebla, Puebla, Mexico (GSEA) which classified genes into pathway. qRT-PCR was used for orthogonal Breast cancer heterogeneity has been characterized by the differential expression validation. Molecular results were integrated with antitumor activity and histological of estrogen and progesterone receptors (ER and PgR) and the epidermal growth data. factor receptor 2 (HER2), which are therapeutic targets. Tumors lacking the Gene expression analysis from treated and untreated models revealed that: 1) on expression of ER, PgR and HER2 are defined as triple negative breast cancer ML006 PDX model, less sensitive to trabectedin, adipogenic process is activated (TNBC), which has an aggressive phenotype and for which treatment is limited by both pioglitazone and combined treatment; 2) trabectedin exposure activates to standard chemotherapy. Identifying alternative therapeutic options is an urgent adipogenesis pathway in the PDX ML017 but not in the PDX ML017/ET resistant challenge, thus, the properties of natural products are also under evaluation. one. Pioglitazone exposure activates adipogenic differentiation in both models, as a Ganoderma lucidum (Gl) is a medicinal mushroom containing bioactive compounds single treatment or in combination with trabectedin. with well-documented anti-oncogenic properties. Thus, the aim of the present To support this evidence, gene expression data confirmed that the main genes study was to analyze the effects of two standardized hydroalcoholic extracts well- implicated in adipogenesis process (PLIN1, FABP4 and ADIPOQ) are activated in characterized in terms of biochemical composition of Gl strain, whose efficacy has the ML017 after trabectedin treatment and in the ML006 and ML017/ET models in not been evaluated on TNBC. Gl extracts significantly reduced the proliferation combination with pioglitazone. ADIPOQ seems to be the most up-regulated one, rate of triple negative cell line models (MDAMB468, MDAMB231, BT545, therefore it was validated by qRT-PCR and then selected as a protein marker for SUM149, HCC1937), induced apoptosis and decreased migration capability. western blot studies. Notably, nontumorigenic mammary epithelial cells MCF10A showed an attenuated The study provides evidence that drug resistance can be counteracted by PPARγ impairment of the proliferation rate after treatment, compared to MDAMB468 cells, agonist in MLPS patients. The combination with pioglitazone reduces tumor growth suggesting a reduced cytotoxicity of the natural extracts on a normal epithelium. and increases mice survival in all three xenograft models, in particular on ML006 Additionally, the combinatory treatment of Gl extracts and doxorubicin increased the and ML017/ET models. Pioglitazone potentiates trabectedin activity by inducing anti-tumor effect of the chemotherapeutic agent on MDAMB468 and BT549 cells. adipocytic maturation. A whole transcriptome evaluation of mRNAs, lncRNAs and microRNA after fungal The data provides a rational for clinical investigations on the combination of treatments on MDAMB468 revealed the alteration of cell viability, cell proliferation, trabectedin with PPARγ agonist. cell migration, cell differentiation and DNA repair signaling as actionable anti- oncogenic mechanisms. We then functionally validated the impairment of DNA repair by Gl extracts through the down-modulation of BRCA1, ATM and PARP protein expression and the increased of DNA fragmentation after UV-induced DNA damage. Ongoing experiments are testing the responsiveness of different TNBC cell lines to the combined action of the mushroom extracts and Olaparib, a PARP inhibitor, to confirm that Gl extracts enhance a BRcaness-like phenotype, which may sensitize tumors to target therapies directly inhibiting proteins related to DNA repair mechanisms. Gl extracts represent a new source of bioactive compounds showing anti-oncogenic properties in TNBC. Further evaluations on in vivo models would validate and strengthen our previous results.
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I2 treated with increasing etoposide concentrations and an etoposide-resistant cell Modulating TAK1 expression through the inhibition of GSK3 line (HTLA-ER) was obtained. HTLA-ER cells acquired a resistant phenotype impairs YAP/TAZ oncogenic functions in pancreatic cancer and, following acute etoposide exposure, evaded apoptosis by increasing the 1)Santoro R. 2)Zanotto M. 3)Piro G. 4)Carbone C. 5)Tortora G. 6)Melisi D. anti-apoptotic protein Bcl2 and decreasing the pro-apoptotic Bax. Since both cell 1)Digestive Molecular Clinical Oncology Research Unit, Department of Medicine, populations shared a homozygous TP53 mutation encoding a partially-functioning Università degli studi di Verona, Verona, Italy 2)Digestive Molecular Clinical protein, other components of the P53 network were investigated. In HTLA-ER cells, Oncology Research Unit, Department of Medicine, Università degli studi di Verona, but not in the parental cell line, a mono-allelic deletion of the 13q14.3 locus, where Verona, Italy 3)Laboratory of Oncology and Molecular Therapy, Department of the P53 inducible miRNA-15a/16-1 are located, was detected. Consequently, a Medicine, Università degli studi di Verona, Verona, Italy 4)Digestive Molecular down-regulation of miRNA-15a/16-1 levels was observed exclusively in HTLA-ER Clinical Oncology Research Unit, Department of Medicine, Università degli studi di cells. This event correlated with an up-regulation of the oncoprotein BMI-1 with Verona, Verona, Italy 5)Laboratory of Oncology and Molecular Therapy, Department pleiotropic effects including the decrease of the tumor suppressor protein p16 and of Medicine, Università degli studi di Verona, Verona, Italy 6)Medical Oncology Unit, the activation of glutathione (GSH)-dependent responses which could be involved Azienda Ospedaliera Universitaria Integrata, Verona, Italy in the metabolic adaptation of the drug-resistant NB cell line. Taken collectively, these results highlight the potential role of miRNA-15a/16-1 as markers of Background: Resistance to chemotherapeutic drugs poses one of the greatest chemoresistance and suggest them as therapeutic targets in order to modulate the challenges in pancreatic cancer (PC) treatment. TGFb-activated kinase 1 (TAK1) has epigenetic changes supporting NB chemoresistance (Grants from Genoa University been demonstrated to drive chemoresistance in PC through the phosphorylation- and AIRC: G#5506 to G.F). dependent activation of the NF-kB transcription factor. The transcriptional regulators Yes Associated Protein (YAP) and transcriptional coactivator with a PDZ binding domain (TAZ) are central determinants of malignancy and have been identified as a I4 critical oncogenic effectors of KRAS in PC. We hypothesized that TAK1 could drive A proteomic approach identified HSP90 as a central hub in CDDP- PC aggressiveness by sustaining YAP and TAZ oncogenic activities. resistant ovarian cancer cells: role of HSP90 inhibitors in Methods: TAK1 expression was silenced by shRNA in AsPC1, Panc1, and MDA- overcoming drug resistance Panc28 cell lines. GSK3 was targeted by using the small molecule inhibitor 1)Lombardi R. 2)Sonego M. 3)Addi L. 4)Milone M.R. 5)Costa A. 6)Bruzzese F. 7) LY2090314. The expression of TAK1, YAP/TAZ and their target genes in PC cell Pucci B. 8)Baldassarre G. 9)Budillon A. lines was studied by both Western blot and qPCR. Cell migration was assessed by 1)CROM-Centro Ricerche Oncologiche di Mercogliano (AV), Istituto transwell assays, and stemness by both spheroid formation and percentage of cancer Nazionale Tumori, IRCCS Fondazione G. Pascale, Napoli, Italy 2)Division of stem cells (CSC). SRB assays were used to assess the in vitro chemopotentiation Molecular Oncology, CRO National Cancer Institute, Aviano, Italy 3)CROM-Centro of nab-paclitaxel, gemcitabine, oxaliplatin, and SN-38. In vivo activity of LY2090314 Ricerche Oncologiche di Mercogliano (AV), Istituto Nazionale Tumori, IRCCS alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude Fondazione G. Pascale, Napoli, Italy 4)CROM-Centro Ricerche Oncologiche di mouse model with luciferase-expressing AsPC1 tumors. Expression of YAP/TAZ Mercogliano (AV), Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale, Napoli, target genes in tumor samples was measured by both qPCR and IHC. Italy 5)Division of Molecular Oncology, CRO National Cancer Institute, Aviano, Italy Results: knockdown of TAK1 resulted in inhibition of K48-linked ubiquitination 6)Experimental Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione and proteasomal degradation of YAP and TAZ, which was independent on TAK1 G. Pascale Napoli, Italy 7)CROM-Centro Ricerche Oncologiche di Mercogliano kinase activity. Significant reduction of proliferation, migration and chemoresistance (AV), Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale, Napoli, Italy 8) of shTAK1 PC cell lines was also observed. Treatment with LY2090314 induced Division of Molecular Oncology, CRO National Cancer Institute, Aviano, Italy 9) silencing of TAK1, as well as a remarkable reduction of the expression of both YAP CROM-Centro Ricerche Oncologiche di Mercogliano (AV), Istituto Nazionale and TAZ and their targets. In combination, the GSK3 inhibitor strongly potentiated Tumori, IRCCS Fondazione G. Pascale, Napoli, Italy the cytotoxic activities of chemotherapeutic agents in all three PC cell lines. In nude mice, i.p. admnistration of LY2090314 plus nab-paclitaxel significantly reduced Epithelial ovarian cancer (EOC) remains one of the leading causes of cancer-related tumor burden and increased median OS. deaths among women worldwide. Standard treatments for newly diagnosed cancer Conclusions: Our study identified GSK3 and TAK1 as major modulators ofYAP consist of cytoreductive surgery and platinum-based chemotherapy. However, and TAZ stability and oncogenic activities, thus indicating that inhibition of TAK1 acquired resistance to platinum occurs frequently and predicts poor prognosis. expression by modulating GSK3 activity could represent a valid approach to revert Therefore, understanding the mechanisms underlying platinum resistance and in vivo the intrinsic chemoresistance of PC. finding ways to overcome them are active areas of study in ovarian cancer.On this regard, we have generated and characterized three cisplatin (CDDP)-resistant isogenic high grade EOC cell lines (TOV-112D Mi-res, OVSAHO Mi-res and MDAH I3 Mi-res) from their parental counterpart. To further investigate the mechanism by ETOPOSIDE-RESISTANCE IN HIGH-RISK NEUROBLASTOMA IS ASSOCIATED which cells acquire CDDP-resistance, we took advantage of the 2-D DIGE followed WITH MIRNA-15A/16-1 DOWN-REGULATION by LC-MS/MS to compare the protein expression profile of the three CDDP-resistant 1)Marengo B. 2)Monti P. 3)Miele M. 4)Menichini P. 5)Ottaggio L. 6)Foggetti G. 7) models compared to parental cells. We identified several differentially expressed Pulliero A. 8)Izzotti A. 9)Speciale A. 10)Garbarino O. 11)Traverso N. 12)Fronza G. proteins and specifically 23, 24 and 20 for TOV-112D, OVSAHO and MDAH 13)Domenicotti C. respectively. The IPA analysis showed a relevant relationship between almost all the 1)Department of Experimental Medicine, General Pathology Section, University of identified proteins in the three models. Interestingly, ten of the identified proteins, Genova, Italy 2)UOC Mutagenesis and Oncologic Prevention, Ospedale Policlinico HSP7C, HNRPL, ATPA, EFTU, PHB, HNRPK, LMNA, PHGDH, ACO2 and GRP75, San Martino, Genova, Italy 3)UOC Mutagenesis and Oncologic Prevention, shared at least within two cell lines, are related in one main network. Multiple Ospedale Policlinico San Martino, Genova, Italy 4)UOC Mutagenesis and Oncologic central nodes, namely GRB2, p85, p110, AKT and HSP90 were identified in the Prevention, Ospedale Policlinico San Martino, Genova, Italy 5)UOC Mutagenesis protein-protein networks related with cancer. In particular the protein chaperone and Oncologic Prevention, Ospedale Policlinico San Martino, Genova, Italy 6)Yale HSP90 emerged as a central hub and its activation, evaluated in terms of increased Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA phosphorylation levels, was observed in the CDDP-resistant cells. Given that 7)Department of Health Sciences, University of Genova, Italy 8)UOC Mutagenesis numerous oncoproteins have been identified as HSP90 clients, HSP90 inhibitors and Oncologic Prevention, Ospedale Policlinico San Martino, Genova, Italy 9) have the potential to block multiple oncogenic pathways as well as drug resistance Department of Experimental Medicine, General Pathology Section, University of in several cancer types. Initial molecular characterization of this pathway pointed Genova, Italy 10)Department of Experimental Medicine, General Pathology Section, to the transcription factor HSF1 as a relevant HSP90 client in the acquisition of the University of Genova, Italy 11)Department of Experimental Medicine, General resistant phenotype. On this regard, we evaluated the effect of CDDP in combination Pathology Section, University of Genova, Italy 12)UOC Mutagenesis and Oncologic with two HSP90 inhibitors: 17-AAG and ganetespib, both of them in phase II/III Prevention, Ospedale Policlinico San Martino, Genova, Italy 13)Department of in cancers patients, demonstrating synergistic anti-cancer activity by combination Experimental Medicine, General Pathology Section, University of Genova, Italy indexes (CI) evaluated by the Chou-Talalay method. Overall, our data demonstrated a potential role of HSP90 inhibitors as an innovative antitumor strategy able to re- Neuroblastoma (NB), the most common paediatric tumor, is initially sensitive to sensitize to CCDP the CDDP-resistant EOC cells and that warrants further clinical etoposide, but following intense treatment several patients become chemoresistant evaluation. and the molecular mechanisms underlying this phenomenon are not yet fully understood. In several cancers, TP53 mutations confer resistance to therapy- induced apoptosis. In NB, TP53 mutations are rare at diagnosis but the inactivation of TP53 occurs in 50% of the cases after therapy due to mutation-dependent or independent events. The aim of this study was to investigate the basis of etoposide resistance in NB, focusing the attention on the dysregulation of P53-dependent pathways. To this end, a MYCN-amplified NB cell line (HTLA-230) was chronically
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I5 metabolomics approaches. LMW-PTP targeting to improve the effectiveness of chemo- and Both strategies converged to define that the major metabolomic changes radiotherapy associated with BEZ235 resistance were found in both membrane lipid species 1)Paoli P. 2)Lori G. 3)Pranzini E. 4)Pardella E. 5)Caselli A. 6)Cirri P. 7)Marzocchini (lysophosphatidilcholines, phosphatidylcholines, and sphingomyelins) and in R. 8)Caderni G. 9)Raugei G. their degree of saturation. This membrane lipid alteration was reflected in a slight 1)Department of Experimental and Clinical Biomedical Sciences “Mario Serio”; increase in membrane fluidity in the BEZ235 resistant cells compared to parental University of Florence, Firenze, Italy 2)Department of Experimental and Clinical cells and was not associated to a general cellular lipid storage (lipid droplets). The Biomedical Sciences “Mario Serio”; University of Florence, Firenze, Italy 3) striking difference, observed for both the resistant clones, in the membrane lipid Department of Experimental and Clinical Biomedical Sciences “Mario Serio”; portion, could open new possibilities for proposing new intervention to overcome University of Florence, Firenze, Italy 4)Department of Experimental and Clinical resistance. Biomedical Sciences “Mario Serio”; University of Florence, Firenze, Italy 5) Department of Experimental and Clinical Biomedical Sciences “Mario Serio”; University of Florence, Firenze, Italy 6)Department of Experimental and Clinical I7 Biomedical Sciences “Mario Serio”; University of Florence, Firenze, Italy 7) Genome scale CRISPR/Cas9 screen identifies Hippo pathway as Department of Experimental and Clinical Biomedical Sciences “Mario Serio”; key determinant for susceptibility to BET inhibitors in lung University of Florence, Firenze, Italy 8)NEUROFARBA Department, Section of cancer Pharmacology and Toxicology, University of Florence, Firenze, Italy 9)Department 1)Gobbi G. 2)Donati B. 3)Faria Do Valle I. 4)Reggiani F. 5)Torricelli F. 6)Castellani of Experimental and Clinical Biomedical Sciences “Mario Serio”; University of G. 7)Ambrosetti D. 8)Ciarrocchi A. 9)Sancisi V. Florence, Firenze, Italy 1)Laboratory of Translational Research, Azienda USL di Reggio Emilia – IRCCS, Reggio Emilia, Italy 2)Laboratory of Translational Research, Azienda USL di Reggio The set point of new effective anticancer strategies to overcome cancer resistance Emilia – IRCCS, Reggio Emilia, Italy 3)“L. Galvani” Center for Bioinformatics, is one the major goals of pharmaceutical companies worldwide. We demonstrated Biophysics and Biocomplexity, University of Bologna, Bologna, Italy 4)Laboratory that Low Molecular Weight Protein Tyrosine Phospatase (LMW‐PTP) is a new of Translational Research, Azienda USL di Reggio Emilia – IRCCS, Reggio Emilia, interesting target to revert intrinsic resistance of tumors toward chemo- and Italy 5)Laboratory of Translational Research, Azienda USL di Reggio Emilia – radiotherapy. This enzyme is generally overexpressed in aggressive and therapy- IRCCS, Reggio Emilia, Italy 6)“L. Galvani” Center for Bioinformatics, Biophysics resistant tumors and its expression is related to poor prognosis. We demonstrated and Biocomplexity, University of Bologna, Bologna, Italy 7)Dipartimento di Farmacia that LMW-PTP silencing, in many different cell lines, allows the reprogramming e Biotecnologie (FaBit), University of Bologna, Bologna, Italy 8)Laboratory of of resistant cancer cells toward a phenotype more sensitive to various anticancer Translational Research, Azienda USL di Reggio Emilia – IRCCS, Reggio Emilia, drugs, including dacarbazine, docetaxel and 5-FU, or radiotherapy. Furthermore, Italy 9)Laboratory of Translational Research, Azienda USL di Reggio Emilia – LMW-PTP silencing strongly impairs self-renewal ability of different types of IRCCS, Reggio Emilia, Italy cancer cells, thereby suggesting that this enzyme sustains survival of staminal cancer cells, promoting relapse. Treating cancer cells with a LMW-PTP inhibitors Lung cancer is the first cause of human cancer-related mortality. Despite the recent leads to the same effects of silencing, improving the effectiveness of chemo- and advances in lung cancer therapy, drug resistance can lead to poor survival, making radiotherapy. Interestingly, we observed that LMW-PTP inhibitors are not effective urgent the development of new therapeutic options. on non-cancerous cells, which express low LMW-PTP levels. This finding reinforce Inhibitors of the Bromodomain and Extraterminal domain proteins (BETi) are a class the hypothesis that expression of LMW-PTP contribute to make cancer cells more of anti-cancer epigenetic drugs that showed efficacy in pre-clinical settings on many resistant toward cytotoxic stimuli triggered by traditional anticancer therapies. cancer types and are currently included in more than 20 clinical trials for different Studies conducted on PIRC rats, which spontaneously develop multiple tumours in tumors, including lung cancer. However, the definition of the mechanisms leading the colon (a model for both familial adenomatous polyposis and sporadic colorectal to susceptibility or resistance to these drugs is needed to stratify patients that may cancer) confirmed that treatment with LMW-PTP inhibitors reduces LMW-PTP benefit from this treatment. expression in rat tumors, increasing sensitivity of cancer cells toward chemotherapy. To uncover new mechanisms responsible of susceptibility and/or resistance to BETi, In conclusion, LMW-PTP is a promising target, for improving the outcome of patients we performed a genome-scale CRISPR/Cas9 screening in lung cancer cells (A549), affected by cancers refractory to treatments. using the GeCKOv2 library, targeting over 19000 genes in the human genome. We identified three genes belonging to the Hippo pathway, LATS2, TAOK1 and NF2, as key determinants for lung cancer cells susceptibility to BETi. The Hippo pathway I6 is a cascade of kinases phosphorylating YAP and TAZ oncogenic transcriptional RESISTANCE TO PI3K/akt/mTOR INHIBITORS IS ASSOCIATED WITH LIPID regulators. Phosphorylated YAP/TAZ are sequestered into cytosol and/or directed ALTERATION IN NSCLC CELLS to degradation, restraining their pro-oncogenic activity. We showed that the 1)Caiola E. 2)Brunelli L. 3)Iezzi A. 4)Marabese M. 5)Sestito G. 6)Pastorelli R. 7) knockout of LATS2, TAOK1 or NF2 sustains TAZ activity by increasing its nuclear Broggini M. localization. Furthermore, we demonstrated that TAZ is a direct target of BRD4 and 1)Laboratory of Molecular Pharmacology, IRCCS – Istituto ‘Mario Negri’, Milano, that treatment of A549 cells with BETi downregulates TAZ expression. Finally, we Italy 2)Unit of Protein and Metabolite Biomarker, IRCCS – Istituto ‘Mario Negri’, showed that knockout of TAZ increases susceptibility to BETi. Milano, Italy 3)Laboratory of Molecular Pharmacology, IRCCS – Istituto ‘Mario Our data uncover a new function of the Hippo pathway in modulating the response Negri’, Milano, Italy 4)Laboratory of Molecular Pharmacology, IRCCS – Istituto ‘Mario to BETi in lung cancer. Notably, molecular alterations in Hippo pathway genes Negri’, Milano, Italy 5)Unit of Protein and Metabolite Biomarker, IRCCS – Istituto are present in 33% of NSCLC patients, whereas about 12% of them display an ‘Mario Negri’, Milano, Italy 6)Unit of Protein and Metabolite Biomarker, IRCCS – amplification of YAP or TAZ, suggesting a relevant role for this pathway in modulating Istituto ‘Mario Negri’, Milano, Italy 7)Laboratory of Molecular Pharmacology, IRCCS BETi response in patients. Furthermore, showing that TAZ is downregulated by BETi – Istituto ‘Mario Negri’, Milano, Italy treatment, we laid the basis for an innovative pharmacological strategy to counter- act TAZ pro-oncogenic activity in lung cancer patients. The PI3K/akt/mTOR pathway is an important signaling pathway regulating several essential cellular functions and is one of the most deregulated pathways in human cancers. Alterations in the signaling cascade lead to aberrant growth, proliferation and metabolic rewiring. Several inhibitors of PI3K, akt and mTOR have been extensively studied at preclinical level where very promising results have been obtained. The translation into the clinic, however, resulted in benefits below the expectancies. Among the possible reasons for the disappointing clinical results there is resistance to treatments. To study the mechanisms underlying the acquired resistance to PI3K/akt/mTOR inhibitors, H1299 human NSCLC cells, overexpressing wild-type (wt) or G12C mutant KRAS, were used to generate BEZ235 (dual pan-PI3K/mTOR inhibitor) resistant clones by repeated exposures to the drug. The IC50 ratio between the resistant cell line and its sensitive counterpart was about 5 for both wt and G12C KRAS cells. Pharmacological and molecular analyses suggested that the resistance was mainly associated with the activity of mTOR, independently from KRAS status. Considering the role played by mTOR in controlling cell metabolism, we determined the metabolic asset of BEZ235 resistant clones comparing them with their respective parental cells using targeted and untargeted mass spectrometry-based
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I8 expression profiling of A498 and SN12C -drug sensitive and resistant cell lines Axl downstream targeting unravels synergistic drug (A498 -WT vs RAD 1-5-10 uM resistant cells and SN12C-WT vs RAD1-5-20 uM). combinations in ovarian carcinoma cells Through comparative analysis of gene expression data in sensitive and resistant 1)Corno C. 2)Alampi D.M. 3)Ciusani E. 4)Carenini N. 5)Corna E. 6)Zaffaroni N. 7) lines we identified among 20 top differentially expressed genes including CXCR4, Gatti L. 8)Perego P. CXCR7 and CD274, down-regulated, and Serpin B2, PBRM1, up-regulated, in 1)Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei each one of the three resistance phenotypes, as validated by qRT-PCR and western Tumori, Milan, Italy 2)Molecular Pharmacology Unit, Fondazione IRCCS Istituto blot analysis. Ingenuity Pathway Analysis (IPA®) is ongoing to identify the principal Nazionale dei Tumori, Milan, Italy 3)Laboratory of Clinical Pathology and Medical molecular pathways responsible for resistance in renal cancer, thus suggesting new Genetics, Fondazione IRCCS Istituto Neurologico C. Besta, Milan, Italy 4)Molecular therapeutic opportunities as potential strategy to modulate Everolimus resistance. Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Molecular Pharmacology Unit, Fondazione IRCCS Istituto Nazionale I10 dei Tumori, Milan, Italy 7)Molecular Pharmacology Unit, Fondazione IRCCS Istituto A FUNCTIONAL GENOMICS APPROACH TO THE IDENTIFICATION OF GENES Nazionale dei Tumori, Milan, Italy 8)Molecular Pharmacology Unit, Fondazione INVOLVED IN RESISTANCE TO OXALIPLATIN IN COLORECTAL CANCER IRCCS Istituto Nazionale dei Tumori, Milan, Italy 1)Invrea F. 2)Cavicchioli F. 3)Petti C. 4)Cantarella D. 5)Russo R. 6)Isella C. 7) Medico E. Ovarian cancer is a disease characterized by relatively low incidence, but it is a 1)Department of oncology, University of Turin, Turin, Italy 2), Istituto nazionale frequent cause of cancerrelated deaths in women. Although trends in relative biostrutture e biosistemi, Rome, Italy 3)Department of oncology, Candiolo cancer survival and population mortality have shown improvements over time, ovarian institute - Irccs, Turin, Italy 4)Department of oncology, Candiolo cancer institute cancer, particularly ovarian carcinoma, is usually diagnosed as advanced disease. - Irccs, Turin, Italy 5), Istituto nazionale biostrutture e biosistemi, Rome, Italy 6) The standard of care is surgery followed by systemic platinum-based chemotherapy. Department of oncology, Candiolo cancer institute - Irccs, Turin, Italy 7)Department Despite a good clinical response of ovarian carcinoma to therapy, drug resistance of oncology, Candiolo cancer institute - Irccs, Turin, Italy often develops. The knowledge gained on the molecular features of ovarian carcinoma has not been fully translated to the therapeutic setting. Therefore, further efforts are needed to define rational pharmacological approaches to hit aggressive Currently, the first line therapy for metastatic colorectal cancer (CRC) isthe features of this tumor. Thus, since molecular targeting of Receptor Tyrosine FOLFOX regimen, which includes Oxaliplatin in combination with Fluorouracil Kinases (RTK) sustaining tumor cell survival impairs factors that may modulate and Leucovorin. Responses to FOLFOX range from overt regressions (35%) to drug response, the aim of this study was to examine if the RTK Axl downstream full resistance (25%), and eventually all patients become resistant. Indeed, the targeting could be exploited to improve cell response to cisplatin. We previously molecular mechanisms of Oxaliplatin resistance remain mostly to be understood. reported that Axl is over-expressed in cisplatin-resistant ovarian carcinoma cells To identify genes involved in sensitivity/resistance to Oxaliplatin, we designed and is involved in sustaining increased invasive ability through activation of the an integrated approach including forward genetic screens and computational p38 Mitogen Activatel Protein Kinase (MAPK) and Stat3. Therefore, we employed analyses. The pipeline includes: (i) stable transduction of CRC cells with pooled small molecules inhibitors of p38 and of Stat3 in combination with cisplatin in drug- shRNA libraries; (ii) Oxaliplatin treatment for six weeks to select a drug-resistant sensitive and -resistant cells and we carried out growth inhibition assays. The subpopulation; (iii) identification of shRNA constructs enriched in the Oxaliplatin- possible drug interaction was analyzed using the Chou and Talalay method. The resistant subpopulation by next-generation sequencing analysis. We focused on the p38 inhibitor SB203580 produced a synergistic effect in combination with cisplatin shRNA libraries targeting phosphatases and ubiquitin conjugation pathway genes. in the cisplatin-resistant IGROV-1/Pt1 cells, but not in the other cell lines. When the The first are involved in several biological processes, reversing the biochemical combination of cisplatin with the Stat 3 inhibitor stattic was tested in IGROV-1 and activity of kinases and typically acting as feedback loop regulators. The second IGROV-1/Pt 1 cells no synergistic interaction was found. Differently, a favorable direct proteins to degradation by the proteasome regulating homeostasis, cell cycle, drug interaction was observed in A2780 cells when pre-incubated with cisplatin and DNA repair pathways. HCT116 CRC cells, markedly sensitive to Oxaliplatin, prior to stattic exposure. The analysis of cell response after treatment indicated were transduced in duplicate with both shRNA libraries. After Oxaliplatin treatment that exposure of IGROV-1/Pt1 cells to the p38 inhibitor-cisplatin combination for six weeks, resistant populations emerged from the library-transduced cells, resulted in increased apoptosis with respect to that observed with cisplatin alone. but not from control-transduced cells. To reveal shRNA constructs responsible In A2780 cells, cisplatin alone induced high levels of apoptosis. Our results support of such phenotype, we extracted genomic DNA from transduced cells and, upon that downstream targets of Axl in ovarian carcinoma cells are valuable to modulate amplification of shRNA constructs by targeted PCR and next-generation sequencing, cisplatin activity in ovarian carcinoma models. we compared the repertoire of shRNA sequences in HCT116 cells selected with Oxaliplatin with that of cells grown in standard medium. Screening hits were then prioritized for functional characterization, resulting in a phosphatase (CDC25C) and I9 an ubiquitin ligase candidate (DCAF17). In conclusion, we successfully setup a The CXCR4-mTOR pathway is involved in Everolimus resistance in functional genomic screening for acquired resistance to Oxaliplatin in CRC. Specific human renal cancer cell lines. Therapeutic approaches to re- shRNA constructs from both the phosphatase and ubiquitin ligase libraries were induce everolimus sensitivity enriched after Oxaliplatin treatment, highlighting candidate genes whose loss of 1)Galdiero F. 2)Ieranò C. 3)Di Martino M.T. 4)Napolitano M. 5)Portella L. 6)Scionti function could potentially drive resistances to Oxaliplatin. F. 7)Vuttariello E. 8)Scala S. 1)Functional Genomics Unit, Istituto Nazionale Tumori -IRCCS -Fondazione G. Pascale, Napoli, Italy 2)Functional Genomics Unit, Istituto Nazionale Tumori I11 -IRCCS -Fondazione G. Pascale, Napoli, Italy 3)Department of Experimental MIR146A REGULATES MELANOMA SENSITIVITY TO BRAF/MEK INHIBITORS and Clinical Medicine, Magna Graecia University, Salvatore Venuta University 1)Vergani E. 2)Dugo M. 3)Gargiuli C. 4)Cossa M. 5)Gallino F. 6)Di Guardo L. 7) Campus, Catanzaro, Italy 4)Functional Genomics Unit, Istituto Nazionale Tumori Ferrarini M. 8)Ferrero E. 9)Sensi M. 10)Rivoltini L. 11)Rodolfo M. 12)Vallacchi V. -IRCCS -Fondazione G. Pascale, Napoli, Italia 5)Functional Genomics Unit, Istituto 1)Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Nazionale Tumori -IRCCS -Fondazione G. Pascale, Napoli, Italy 6)Department of Milan, Italy 2)Department of Applied Research and Technological Development, Experimental and Clinical Medicine, Magna Graecia University, Salvatore Venuta Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Department of University Campus, Catanzaro, Italy 7)Functional Genomics Unit, Istituto Nazionale Applied Research and Technological Development, Fondazione IRCCS Istituto Tumori -IRCCS -Fondazione G. Pascale, Napoli, Italy 8)Functional Genomics Unit, Nazionale dei Tumori, Milan, Italy 4)Department of Pathology, Fondazione IRCCS Istituto Nazionale Tumori -IRCCS -Fondazione G. Pascale, Napoli, Italy Istituto Nazionale dei Tumori, Milan, Italy 5)Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Department of Medical The chemokine receptor CXCR4 is a seven-membrane-spanning G-protein coupled Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7)Division receptor (GPCR). The crosstalk between the mTOR pathway, activated in several of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy 8)Division of human malignancies, and the CXCR4–CXCL12–CXCR7 chemokine receptor Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy 9)Department axis has been investigated in human renal cancer cells. Treatment of metastatic of Applied Research and Technological Development, Fondazione IRCCS Istituto renal cell carcinoma (mRCC) has significantly improved with the advent of agents Nazionale dei Tumori, Milan, Italy 10)Department of Research, Fondazione IRCCS targeting the mTOR pathway, such as Temsirolimus and Everolimus (RAD001). Istituto Nazionale dei Tumori, Milan, Italy 11)Department of Research, Fondazione However, their efficacy is thought to be limited by feedback loops and crosstalk IRCCS Istituto Nazionale dei Tumori, Milan, Italy 12)Department of Research, with other pathways leading to the development of drug resistance. Previous results Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy have shown that targeting CXCR4–CXCL12–CXCR7 axis with CXCR4 antagonists re-induced Everolimus sensitivity in SN12C- and A498- RAD001 resistant renal Targeted therapy with BRAF and MEK inhibitors (BRAF/MEKi) has improved survival cancer cells. With the intent to identify other genes and pathways crucial to the for patients with metastatic melanoma but most patients eventually progress due to development of drug resistance in renal cancer, we performed microarray gene resistance mechanisms, which include genetic and epigenetic tumor alterations.
50 51 ABSTRACT BOOK
We and others have shown that miRNA dysregulation contributes to melanoma resistance by regulating genes involved in prosurvival pathways. MICROBIOME miRNA expression analysis by Nanostring technology in six melanoma cell lines J1 with acquired resistance induced by chronic drug treatment in comparison to their Microbiota-driven interleukin-17-producing cells and sensitive parental lines revealed miR146a was decreased and inversely correlated eosinophils synergize to accelerate multiple myeloma with IC50 values of BRAF/MEKi, thus indicating the association between its progression expression and acquisition of resistance. miR146a has been reported to promote 1)Calcinotto A. 2)Brevi A. 3)Ches M. 4)Ferrarese R. 5)Garcia Perez L. 6)Grioni M. melanoma cell growth and progression but its role in resistance was not investigated. 7)Kumar S. 8)Henderson K.J. 9)Tonon G. 10)Tomura M. 11)Miwa Y. 12)Flavell R.A. Overexpression of miR146a in resistant cell lines significant reduced cell viability, and increased apoptotic cells and LDH release upon BRAF/MEKi treatment. 13)Huber S. 14)Canducci F. 15)Rajkumar V.S. 16)Leif Bergsagel P. 17)Bellone M. Conversely, knock-down of miR146a in sensitive cells decreased sensitivity to drug 1)San Raffaele Scientific Institute, Division of Immunology, Transplantation and treatment. Upon miR146a overexpression in resistant cells, drug treatment induced Infectious Diseases, Milan, Italy - Institute of Oncology Research, Oncology deactivation of PI3K/AKT/NFkB signaling cascades and the downregulation of Institute of Southern Switzerland, Bellinzona, Switzerland 2)San Raffaele Scientific antiapoptotic factors MCL1 and BCL2L1. In addition, several protumoral factors Institute, Division of Immunology, Transplantation and Infectious Diseases, Milan, showed reduction, including IL8 and CCL5, which are direct targets of miR146a, Italy 3)Mayo Clinic Arizona, Comprehensive Cancer Center, Scottsdale, United and CCL2, IL6 and VEGFA, regulated via NFkB activity. Similar to what observed in States 4)San Raffaele Scientific Institute, Laboratory of Microbiology, Milan, cell lines, a lower expression level of miR146a was detected in melanoma biopsies Italy 5)Universitätsklinikum Hamburg-Eppendorf, Molekulare Immunologie und from treated patients when compared to pretreatment tumors. GSEA showed that Gastroenterologie, Hamburg, Germany 6)San Raffaele Scientific Institute, Division gene sets significantly correlated to miR146a are shared between melanoma cell of Immunology, Transplantation and Infectious Diseases, Milan, Italy 7)Mayo Clinic lines (GSE67638) and melanoma tumor TCGA public datasets. Moreover, enforced Rochester, Division of Hematology, Rochester, United States 8)Mayo Clinic Arizona, expression of miR146a in melanoma tissue explants upon drug treatment in short- Comprehensive Cancer Center, Scottsdale, United States 9)San Raffaele Scientific term dynamic culture system in bioreactor reduced proliferation, as shown by Institute, Division of Experimental Oncology, Milan, Italy 10)Osaka Ohtani University, the decrease of cell positive for Ki67 staining, and decreased the expression of Faculty of Pharmacy, Osaka, Japan 11)University of Tsukuba, Tsukuba, Japan 12) antiapoptotic and cyto-chemokine genes. Yale University, Department of Immunobiology, School of Medicine, and Howard Taken together, these results showed that miR146a downregulation contributes Hughes Medical Institute, Yale, United States 13)Universitätsklinikum Hamburg- Eppendorf, Molekulare Immunologie und Gastroenterologie, Hamburg, Germany to melanoma cell proliferation and survival in melanoma resistance to BRAF/ 14)University of Insubria, Department of Biotechnology and Life Sciences, Varese, MEKi treatment. The elucidation of the complex molecular mechanisms involved Italy 15)Mayo Clinic Rochester, Division of Hematology, Rochester, United States in miR146a expression regulation may disclose novel targets for more effective 16)Mayo Clinic Arizona, Comprehensive Cancer Center, Scottsdale, United States combination treatments. 17)San Raffaele Scientific Institute, Division of Immunology, Transplantation and Supported by CARIPLO Foundation (Grant 2015-0911). Infectious Diseases, Milan, Italy
I12 The gut microbiota has been causally linked to cancer, yet the mechanisms by Increased lactate secretion by cancer cells sustains which intestinal microbes influence progression of extramucosal tumors are non-cell-autonomous adaptive resistance to MET and EGFR poorly understood. Here we show that the commensal microbiota and Prevotella targeted therapies heparinolytica in particular, promotes the differentiation of Th17 cells colonizing the 1)Apicella M. 2)Giannoni E. 3)Fiore S. 4)Isella C. 5)Granchi C. 6)Minutolo F. 7) Peyer’s patches and migrating to the bone marrow (BM) of transgenic Vk*MYC Sottile A. 8)Comoglio P.M. 9)Medico E. 10)Pietrantonio F. 11)Volante M. 12)Chiarugi mice, where they favor progression of multiple myeloma (MM). Lack of IL-17 in P. 13)Corso S. 14) Giordano S. Vk*MYC mice, or disturbance of their microbiome imposed either by sterility of 1)University of Torino, Dept. Of Oncology, Candiolo, Italy 2)Dept. of Experimental the housing conditions or by antibiotics led to decreased BM levels of IL-17 and and Clinical Biomedical Sciences, University of Florence, Florence, Italy 3) delayed disease appearance. Similarly, in patients with smoldering MM a higher University of Torino, Dept. Of Oncology, Candiolo, Italy 4)Candiolo Cancer Institute level of BM IL-17 predicted faster disease progression. Mechanistically, IL-17 - FPO, IRCCS, Candiolo, Italy 5)Dept. of Pharmacy, University of Pisa, Pisa, Italy induced STAT3 phosphorylation in plasma cells, and activated eosinophils. Indeed, 6)Dept. of Pharmacy, University of Pisa, Pisa, Italy 7)Candiolo Cancer Institute - treatment of Vk*MYC mice with antibodies blocking IL-17, IL-17RA and IL-5, the FPO, IRCCS, Candiolo, Italy 8)Candiolo Cancer Institute - FPO, IRCCS, Candiolo, latter activating and recruiting eosinophils, reduced BM accumulation of Th17 cells Italy 9)University of Torino, Dept. Of Oncology, Candiolo, Italy 10)Medical Oncology and eosinophils and delayed disease progression. Thus, in Vk*MYC mice, the gut Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 11) microbiota precociously unleashes a paracrine signaling network between adaptive Pathology Unit, San Luigi Hospital, Orbassano, Italy 12)Tuscany Tumor Institute and innate immunity that accelerates progression to MM, and can be targeted by and “Center for Research, Transfer and High Education DenoTHE”, Florence, Italy already available therapies. 13)Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Italy 14)Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Italy J2 Microenvironment is known to influence cancer drug response and sustain Antibiotics or probiotic aerosolization modulate pulmonary resistance to therapies targeting receptor-tyrosine kinases. However if and how microbiota and promotes immunosurveillance against tumor microenvironment can be altered during treatment, contributing to resistance melanoma lung metastases onset is not known. In our work we show that, under prolonged treatment with 1)Le Noci V. 2)Guglielmetti S. 3)Arioli S. 4)Camisaschi C. 5)Sommariva M. 6)Storti tyrosine kinase inhibitors (TKIs), EGFR- or MET-addicted cancer cells displayed a C. 7)Bianchi F. 8)Triulzi T. 9)Castelli C. 10)Balsari A. 11)Tagliabue E. 12)Sfondrini L. metabolic shift towards increased glycolysis and lactate production. We identified 1)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, secreted lactate as the key molecule able to instruct Cancer Associated Fibroblasts Milan, Italy 2)Dipartimento di Scienze degli Alimenti, Nutrizione e Ambiente (CAFs) to produce Hepatocyte Growth Factor (HGF) in a NF-KB dependent (DeFENS), Università degli Studi di Milano, Milan, Italy 3)Dipartimento di Scienze manner. Increased HGF, activating MET-dependent signaling in cancer cells, degli Alimenti, Nutrizione e Ambiente (DeFENS), Università degli Studi di Milano, sustained resistance to TKIs. Functional targeting or pharmacological inhibition of Milan, Italy 4)Immunotherapy Unit, Fondazione IRCCS Istituto Nazionale dei lactate dehydrogenase prevented and overcame in vivo resistance, demonstrating Tumori, Milan, Italy, 5)Dipartimento di Scienze Biomediche per la Salute, Università the crucial role of this metabolite in the adaptive process. degli Studi di Milano, Milan, Italy 6)Molecular Targeting Unit, Fondazione IRCCS This non-cell-autonomous, adaptive resistance mechanism was observed in Istituto Nazionale dei Tumori, Milan, Italy 7)Molecular Targeting Unit, Fondazione NSCLC patients progressed on EGFR TKIs, demonstrating the clinical relevance IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8)Molecular Targeting Unit, of our findings and opening novel scenarios in the challenge to drug resistance. Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9)Immunotherapy To our knowledge, this is the first report identifying a metabolism-driven, non-cell- Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 10)Molecular autonomous mechanism of acquired resistance to TKIs, opening exciting clinical Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 11) opportunities to bypass a major hurdle in cancer treatment. Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 12)Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy Immunological tolerance in the lung microenvironment is essential to control inflammation in response to inhaled particulates, but it also creates a permissive milieu for the setting of lung metastasis. Lung microbiota is implicated in establishing pulmonary tolerance. Thus, we explored whether lung microbiota manipulation via antibiotics or probiotics aerosolization in C57BL/6 mice limits melanoma metastasis
50 51 ABSTRACT BOOK implantation and growth, by subverting local immune suppression and boosting immune responses. MOLECULAR PATHOLOGY Our results indicate that vancomycin/neomycin aerosolization induced a decrease in bacterial load in bronchoalveolar lavage of mice was associated to a significant K1 reduction in regulatory T cells in the lung (p = 0.005). Moreover, an enhanced MYC-RELATED MICRORNAS SIGNATURES IN NON-HODGKIN B-CELL activation of lung T and NK cells, assessed by cytofluorimetric analysis, paralleled LYMPHOMAS AND THEIR RELATIONSHIPS WITH CORE CELLULAR the significant reduction of lung metastases in mice aerosolized with antibiotics and PATHWAYS intravenously injected with melanoma B16 cells (Mean ± SEM: 3.3 ± 0.4 antibiotics 1)Malpeli G. 2)Barbi S. 3)Tosadori G. 4)Greco S. 5)Zupo S. 6)Pedron S. 7)Brunelli versus 47.8 ± 7.1 saline-treated mice; p < 0.0001). Reduction of lung metastases M. 8)Bertolaso A. 9)Scupoli MT. 10)Takam kamga P. 11)Krampera M. 12)Croce CM. also occurred in lung transplanted with bacteria isolated from the bronchoalveolar 13)Calin GA. 14)Scarpa A. 15)Zamò A. lavage of antibiotic-treated mice; likewise, transplantation of bacteria isolated from 1)Diagnostics and public health, University of Verona, Verona, Italy 2)Diagnostics untreated mice attenuated the anti-metastatic effects of antibiotics. Aerosolized and public health, University of Verona, Verona, Italy 3)Center for biomedical probiotic Lactobacillus rhamnosus, a human commensal bacterium, strongly limited computing, University of Verona, Verona, Italy 4)Department of medicine, University B16 metastases implantation (Mean ± SEM: 6.5 ± 1.8 probiotic versus 26.4 ± of Verona, Verona, Italy 5)Laboratory of molecular diagnostics, Irccs-aou san 8.2 saline-treated mice; p = 0.02), and this effect was associated to effector cells martino-ist, Genoa, Italy 6)Diagnostics and public health, University of Verona, activation, maturation of resident APCs, assessed by cytofluorimetric analysis, and Verona, Italy 7)Diagnostics and public health, University of Verona, Verona, Italy 8) a reduced expression of M2 genes (Il10, Ido and Irf4) as revealed by real-time Diagnostics and public health, University of Verona, Verona, Italy 9)Department of PCR analysis performed on mRNA extracted from adherent cell fraction of digested medicine, University of Verona, Verona, Italy 10)Department of medicine, University lungs. Furthermore, probiotics or antibiotics improved the therapeutic effects of of Verona, Verona, Italy 11)Department of medicine, University of Verona, Verona, dacarbazine, a chemotherapeutic agent used in metastatic melanoma patients, Italy 12)Department of molecular virology, The Ohio State University, Columbus, in advanced B16 metastases bearing mice (Mean ± SEM: 10.8 ± 1.8 probiotic/ Oh, Usa 13)Department of experimental therapeutics and the ce, The University of DTIC versus 31.8 ± 2.8 DTIC treated mice p < 0.0001; Mean ± SEM: 3.6 ± 0.9 Texas md Anderson Cancer Center, Houston, Tx, Usa 14)Diagnostics and public antibiotics/DTIC versus 10.1 ± 2.0 DTIC treated mice p = 0.01). Our results reveal health, University of Verona, Verona, Italy 15)Department of oncology, University that commensal bacteria in the lungs are relevant in creating an immunological of Turin, Torino, Italy milieu permissive for metastatization. Thus, targeting lung microbiota via probiotic or In order to investigate the role of microRNAs in the pathogenesis of different B-cell antibiotic aerosolization could represent a new therapy to prevent metastases and lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed enhance the response to chemotherapy. non-Hodgkin B-cell lymphomas, including Burkitt’s lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC-translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC- related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.
K2 DETECTION AND MONITORING OF EWS-FLI1 FUSION TRANSCRIPTS BY LIQUID BIOPSY IN EWING SARCOMA PATIENTS 1)Allegretti M. 2)Casini B. 3)Mandoj C. 4)Benini S. 5)Alberti L. 6)Novello M. 7)Conti L. 8)Covello R. 9)Pescarmona E. 10)Milano G.M. 11)Annovazzi A. 12)Anelli V. 13) Ferraresi V. 14)Biagini R. 15)Giacomini P. 1)Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Rome, Italy 2)Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 3)Clinical Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 4)Department of Pathology, The Rizzoli Institute, Bologna, Italy 5)Department of Pathology, Centre Léon Bérard, Lyon, France 6)Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 7)Clinical Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 8)Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 9)Pathology, IRCCS Regina Elena National Cancer Institute, Rome, Italy 10)Department of Hematology/Oncology, Bambino Gesù Children’s Hospital, Rome, Italy 11)Nuclear Medicine, IRCCS Regina Elena National Cancer Institute, Rome, Italy 12)Radiology and Diagnostic Imaging, IRCCS Regina Elena National Cancer Institute, Rome, Italy 13)Medical Oncology 1, IRCCS Regina Elena National Cancer Institute, Rome, Italy 14)Orthopedics, IRCCS Regina Elena National Cancer Institute, Rome, Italy 15)Oncogenomics and Epigenetics, IRCCS Regina Elena National Cancer Institute, Rome, Italy Background Ewing Sarcomas (ES) are extremely rare tumors of bone and soft tissues driven by defined chromosomal translocations. Diagnostic offer for ES is limited, with conventional solid tumor biomarkers usually not applicable to long-term monitoring and relapse/response prediction. Oncogenic fusion transcripts are hallmarks of ES and are not down-regulated or lost during tumor progression, thus representing an appealing source of circulating ES-specific analytes. Detection of these markers
52 53 ABSTRACT BOOK
directly in the bloodstream would greatly improve the molecular diagnostic silencing significantly decreased the intracellular ATP level and led to the activation standards in ES. of caspase 3 and 7. Moreover, we observed increased sub G0/G1 phase and Material and methods reduction of the S phase of cell cycle after silencing. FFPE tissue blocks and blood samples were collected from six and four ES patients, Overall, our preliminary data demand further investigation in NEAT1 transcriptional respectively. Plasma was obtained by two successive rounds of centrifugation and configuration and suggest its potential role in the regulation of MM proliferation. stored frozen until RNA extraction by the QIAmp CNA kit (Qiagen). RNA was retro- transcribed by the SuperScript VILO kit (ThermoFisher) and subjected RT-qPCR and dPCR. Reactions were set up using two custom primer sets identifying types 1 K4 and 2 EWS-FLI1 fusion transcripts. UVEAL MELANOMA WITH A MUTATION IN THE Gα-PROTEIN GNA11 SHOW Results A MORE AGGRESSIVE CLINICAL COURSE THAN THOSE WITH A GNAQ Types 1 and 2 EWS-FLI1 rearrangements could be identified using only two sets of MUTATION PCR primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR 1)Piaggio F. 2)Dogrusöz M. 3)Bordo D. 4)Puzone R. 5)Viaggi S. 6)Coviello D. 7) and dPCR unequivocally discriminated the two variants in all (tissues and plasma) Lanza F. 8)Liguori P. 9)Mosci C. 10)Van der Velden P. 11)Jager M.J. 12)Queirolo P. tested samples. Of note, EWS-FLI1 molecular diagnosis was possible using blood 13)Pfeffer U. 14)Amaro A. samples even when the corresponding tumor tissue was not available. Circulating 1)Ospedale Policlinico San Martino, Genova, Italy 2)Department of Ophthalmology, tumor RNAs (ctRNAs) levels in plasma significantly (p<0.05) correlated with Leiden University Medical Center, Leiden, The Netherlands 3)Ospedale Policlinico volume-based Positron Emission Tomography (PET) parameters (MTV and TLG) San Martino, Genova, Italy 4)Ospedale Policlinico San Martino, Genova, Italy 5) and allowed the fine tracking of disease course after surgery, during adjuvant as Department of Earth Sciences, Environment, and Life, Università Degli Studi well as neo-adjuvant chemotherapy. Importantly, non-invasive long-term monitoring di Genova, Genova, Italy 6)Ente Ospedaliero Galliera3, Genova, Italy 7)Ente of ES was also possible by LB in one patient. Ospedaliero Galliera, Genova, Italy 8)Ente Ospedaliero Galliera, Genova, Italy Conclusions 9)Ente Ospedaliero Galliera, Genova, Italy 10)Department of Ophthalmology, LB is clinically applicable to ES for diagnosis, confirmation of lesions upon progression Leiden University Medical Center, Leiden, The Netherlands 11)Department of and rapid assessment of response to treatments. Nevertheless, optimization efforts Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands 12) are warranted to yield a widely applicable and reliable assay detecting circulating Ospedale Policlinico San Martino, Genova, Italy 13)Ospedale Policlinico San tumor nucleic acids (DNAs and RNAs) for highly personalized ES management. Martino, Genova, Italy 14)Ospedale Policlinico San Martino, Genova, Italy This work was supported by EU commission (grant #633937 – ULTRAPLACAD), AIRC (IG #19052 to PG, Nuvenia Fellowship to MA), and Regina Elena intramural Mutations of the Gα-proteins GNAQ and GNA11 are found in 85% cases of uveal funding. melanomas (UM) and are considered driver mutations. Metastatic UM shows additional mutations among which, prominently, mutations in the BRCA1 Associated Protein 1 gene, BAP1, monosomy of chromosome 3 and amplification of chr8q. K3 Several reports found a tendency towards more frequent GNA11 mutations in MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF NEAT1 IN metastatic UM. GNAQ and GNA11 show extended homology (90% amino acid MULTIPLE MYELOMA identity) and are believed to exert redundant functions in GPCR-signaling. 1)Taiana E. 2)Favasuli V. 3)Ronchetti D. 4)Agnelli L. 5)Todoerti K. 6)Amodio N. Here we show protein structure modeling indicating that all the mutated residues, 7)Cantafio M.E.G. 8)Platonova N. 9)Manzoni M. 10)Vinci C. 11)Pelizzoni F.12) except Glu95 -> Asp, are located at positions not involved in the quaternary Chiaramonte R. 13)Tassone P. 14)Neri A. structure formation with the β and γ subunits and are likely to affect other protein- 1)Department of Oncology and Hemato-oncology, University of Milan, Italy 2) protein interactions consistent with different physical interaction networks. The Department of Oncology and Hemato-oncology, University of Milan, Italy 3) expression of GNAQ and GNA11 did not show consistent differences in the tumors. Department of Oncology and Hemato-oncology, University of Milan, Italy 4) We analyzed the association of GNAQ and GNA11 mutations with disease-specific Department of Oncology and Hemato-oncology, University of Milan, Italy 5) survival, gene expression profiles, and cytogenetic alterations in 221 UMs. Our Department of Oncology and Hemato-oncology, University of Milan, Italy 6) analysis showed a shorter disease specific survival of GNA11 mutated cases as Department of Experimental and Clinical Medicine, Magna Graecia University of compared to GNAQ mutated ones (median months: 63.4, 95%CI 14.4-112, vs not Catanzaro, Catanzaro, Italy 7)Department of Experimental and Clinical Medicine, reached, p=0.011; HR=1.78, 95%CI 1.13-2.81, p=0.012). The GNA11 mutation Magna Graecia University of Catanzaro, Catanzaro, Italy 8)Department of Health was associated with tumor thickness (p=0.032), expression alterations of the Sciences, University of Milan, Italy 9)Department of Oncology and Hemato- BRCA1-associated protein 1 (BAP1; p=0.001), Chr3 monosomy (p=0.0003), chr8q oncology, University of Milan, Italy 10)Department of Oncology and Hemato- amplification (p=0.040), the combination of the latter two (p=0.0004) and inversely oncology, University of Milan, Italy 11)Hematology Unit, Fondazione IRCCS Cà with chr6p amplification (p=0.006). The effect of GNA11 mutations is likely due to the Granda Ospedale Maggiore Policlinico, Milan, Italy 12)Department of Health different interaction networks the two G-proteins belong to and might be mediated Sciences, University of Milan, Italy 13)Department of Experimental and Clinical by DDEF1/ASAP1 and RHOQ, two genes already implicated in UM progression, Medicine, Magna Graecia University of Catanzaro, Catanzaro, Italy 14)Department that are overexpressed in GNA11- as compared to GNAQ- mutated cases. of Oncology and Hemato-oncology, University of Milan, Italy
Multiple myeloma (MM) is a malignant proliferation of antibody-secreting bone K5 marrow plasma cells (PCs) characterized by genomic instability. The discovery of Correlation between LKB1 gene status and expression in non- lncRNA has added a further layer of complexity to the pathobiology of the disease. small cell lung cancer (NSCLC) patients NEAT1 is a lncRNA, mapped at 11q13, transcribed in two different isoforms (3.7Kb 1)Centonze G. 2)Paolino C. 3)Belfiore A. 4)Busico A. 5)Conca E. 6)Ganzinelli M. 7) and 22.3Kb), retained in the nucleus where it forms the core structural component Caiola E. 8)Biganzoli E. 9)Fornili M. 10)De Bortoli D. 11)Pastorino U. 12)Fabbri A. of the paraspeckle sub-organelles. It may act as transcriptional regulator for several 13)Massimo M. 14)Pruneri G. 15)Sozzi G. 16)Perrone F. 17)Tamborini E. genes, including cancer related ones. 1)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto This study is aimed at the molecular and functional characterization of NEAT1 in MM. Nazionale dei Tumori, Milan, Italy 2)Thoracic Surgery Unit, Department of Surgery, We generated lncRNA transcriptional profiles of purified bone marrow PCs from 50 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Clinical Research MM, 15 plasma cell leukemia and 4 normal donors by GeneChip® Human Gene Lab (CRAB), Department of Pathology and Laboratory Medicine, Fondazione 2.0 ST microarray and by RNA-seq on HiSeq 2000 (Illumina) in 30 MM of the same IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Department of Pathology and cohort. qRT-PCR has been used for validation. We designed LNA-gapmeRs to Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy silence NEAT1 in MM cell lines by the use of Exiqon algorithm and optimized the 5)Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto condition for NEAT1 silencing by gymnotic delivery. Biological effect upon silencing Nazionale dei Tumori, Milan, Italy 6)Medical Oncology Department, Fondazione was evaluated by viable count and ATP measurement, and by caspase 3 and IRCCS Istituto Nazionale di Tumori, Milan, Italy 7)Department of Oncology, IRCCS caspase 7 cleavage assessment. Cell cycle upon NEAT1 silencing was analyzed Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy 8)Unit of Medical by FACS acquisition. Statistics, Biometry and Bioinformatics “Giulio A. Maccacaro,” Department of Transcriptional analysis identified the upregulation of both NEAT1 isoforms Clinical Sciences and Community Health, University of Milan, Campus Cascina in pathological samples compared to healthy controls. qRT-PCR confirmed Rosa, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 9)Unit of Medical available literature data about the more abundant expression of the short isoform. Statistics, Biometry and Bioinformatics “Giulio A. Maccacaro,” Department of Furthermore, RNA-seq profile highlighted the existence of two still uninvestigated Clinical Sciences and Community Health, University of Milan, Campus Cascina non-overlapping shorter isoforms at the 5’ end. We confirmed the expression of Rosa, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 10)Unit of Medical both isoforms by specific qRT-PCR assays able to selectively detect the different Statistics, Biometry and Bioinformatics “Giulio A. Maccacaro,” Department of Clinical variants. Sciences and Community Health, University of Milan, Campus Cascina Rosa, Finally, we tested six in-house designed LNA-gapmeRs and chose the one with Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 11)Thoracic Surgery the highest silencing efficiency by gymnotic delivery of the oligonucleotide. NEAT1 Unit, Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori,
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Milan, Italy 12) Department of Pathology and Laboratory Medicine, Fondazione K7 IRCCS Istituto Nazionale dei Tumori, Milan, Italy 13)Tumor Genomics Unit, Implications of TERT promoter mutations and telomerase Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, expression in head and neck cancer and cervical carcinoma Italy 14)Department of Pathology and Laboratory Medicine, Fondazione IRCCS 1)Tornesello M.L. 2)Annunziata C. 3)Pezzuto F. 4)Botti G. 5)Buonaguro L. 6) Istituto Nazionale dei Tumori, Milan, Italy 15)Tumor Genomics Unit, Department Buonaguro F.M. of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 16) 1)Molecular Biology and Viral Oncology Unit, Istituto Nazionale Tumori IRCCS Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Fondazione Pascale, Napoli, Italy 2)Molecular Biology and Viral Oncology Unit, Nazionale dei Tumori, Milan, Italy 17)Department of Pathology and Laboratory Istituto Nazionale Tumori IRCCS Fondazione Pascale, Napoli, Italy 3)Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Biology and Viral Oncology Unit, Istituto Nazionale Tumori IRCCS Fondazione Pascale, Napoli, Italy 4)Department of Pathology, Istituto Nazionale Tumori IRCCS Background: The liver kinase B1 (LKB1) tumor suppressor is a key sensor of Fondazione Pascale, Napoli, Italy 5)Molecular Biology and Viral Oncology Unit, metabolic stress. Its major direct target is the 5’-AMP activated protein kinase Istituto Nazionale Tumori IRCCS Fondazione Pascale, Napoli, Italy 6)Molecular (AMPK), a master regulator of growth, metabolism and cell survival. LKB1 gene Biology and Viral Oncology Unit, Istituto Nazionale Tumori IRCCS Fondazione mutations occours in about 25% of NSCLC patients and LKB1 loss-of-activity is Pascale, Napoli, Italy related to sporadic lung cancer development. LKB1 mutated NSCLC models showed, both in vitro and in vivo, a high sensitivity to metabolic stress-inducing INTRODUCTION: The telomerase activity is regulated by reversible mechanisms treatments. Loss of LKB1 expression may represent a possible predictive marker such as TERT gene transcription control, TERT pre-mRNA alternate splicing and for therapies affecting cell metabolism. In this study, we investigated the correlation post-translational modifications, as well as by irreversible mechanisms such as between LKB1 protein expression (phenotype) and its mutational status (genotype) genetic alterations and recurrent mutations in TERT promoter region. In the present in a set of NSCLC patients. study we analysed TERT promoter mutations and TERT mRNA levels in head and Experimental design: LKB1 protein expression was performed on H1299 (LKB1wt), neck cancer and cervical neoplasia as well as in cervical carcinoma-derived cell H1299 KO (LKB1 KO by Crispr/Cas9 technology) and H460 (LKB1pQ37*) cell lines to lines. Moreover, we investigated the effect of the increased telomerase expression validate the efficacy and specificity of immunohistochemistry (IHC) and evaluated on the p53 pathway in cervical neoplasia. with semi-quantitative H-score method (H score = 0-300). To confirm the experimental METHODS: Genomic DNA and total RNA were extracted from cancer biopsies and IHC conditions, we analyzed LKB1 expression on 38 NSCLC cases, retrospectively autologous peri-tumor tissues. TERT promoter mutations were searched by DNA analyzed by Next Generation Sequencing (NGS) (Thermofisher custom panel), and amplification and nucleotide sequence analysis. RNA was reverse transcribed and comprising 28 wild type and 10 mutated cases. LKB1 mutations were classified as cDNA used to perform gene expression analysis of HPV16 E6, TERT gene and of benign or damaging/pathogenic by ClinVar, Cosmic and Poliphen. Consequently, 84 p53-related genes by real time PCR. two different group of cases were recorded: wild type/benign mutations with likely RESULTS: Nucleotide sequence analysis of TERT promoter region showed protein expression and damaging mutations with likely loss of function and no that 33.3% of oral SCC and 16.8% of cervical squamous cell carcinoma (SCC) protein expression. harboured mutually exclusive G to A transitions at nucleotide position -124 or -146. Results: IHC analysis for LKB1 on NSCLC cell lines showed strong positivity in TERT promoter was mutated at nucleotide -146 (G>A) in SiHa cell line. Other H1299 and no expression in H1299 KO and H460. nucleotide changes creating in some cases putative ETS binding sites were more Wild type and benign mutation (32 cases) showed variable and heterogeneous frequent in oral SCC (26.7%) than in cervical carcinoma (4.8%). Such hot spot LKB1 IHC expression (Average H score = 94), whereas damaging mutation (6 changes were not detected in oropharyngeal SCC, cervical adenocarcinoma and cases) revealed negative or low expression (Average H score = 5). Interestingly, CIN lesions. Expression of TERT gene was significantly higher in TERT promoter we found a significant association between LKB1 IHC expression and gene status mutated (-124 G>A or -146 G>A) cervical SCC compared to not mutated SCC with Fisher exact test (P= 0.0078) and ROC curve (AUC = 0.90, CI: 0.82-0.99). irrespective of HPV16 E6 and E7 levels. The expression profile of 84 p53-related Conclusions: We showed the potential role of IHC to predict LKB1 pathogenic genes showed among TERT promoter mutated SCC a significant increase in the mutations in order to identify tumors with particular sensitivity to metabolic stress- expression level of genes involved in the apoptosis and proliferation. inducing treatments. CONCLUSIONS: Recurrent activating mutations in TERT promoter are frequent and heterogeneously distributed in different types of head and neck as well as in cervical carcinoma. The increased telomerase levels in TERT promoter mutated K6 cervical SCC activates telomerase noncanonical functions affecting the expression The microRNA profile of multiple primary cutaneous melanoma of several p53-related genes. Dika E., Riefolo M., Baraldi C., Porcellini E., Broseghini E., Lambertini M., Fanti P.A., Ferracin M. Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy It is estimated that a personal history of melanoma is a risk factor for the development of a subsequent primary melanoma. This risk is higher in the first year after the first diagnosis and remains increased for at least 20 years. These patients may represent a high-risk model of melanoma occurrence. Though patients affected by Multiple Primary Cutaneous Melanoma (MPCM) may have a higher genetic susceptibility, the frequency of germline mutations is much lower than expected. We hypothesized a role for microRNAs (miRNAs) in the pathogenesis of the disease. To study the miRNA profile of multiple primary melanomas with and without family history of cutaneous melanomas, we collected 24 formalin fixed and paraffin embedded samples from 3 benign nevi, 4 single cutaneous melanomas, 17 multiple primary or familial melanomas from 8 different patients. We analyzed the global smallRNA expression using Illumina smallRNA protocol. The miRNA profile was found to be statistically different between multiple primary and single primary melanoma. No significant difference was observed between multiple primary melanomas with family history and multiple primary melanomas without family history. In addition, confronting the first and the second tumor of multiple melanomas, we noticed that the miRNA profile also changes in a statistically significant manner. In conclusion, our results suggest that multiple melanomas have a peculiar miRNA deregulation that could reflect their specific biology and allow the identification of new biomarkers.
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K8 ONCOGENIC ROLE OF A SET OF MIRNAS IN THYROID CANCER CELLS NOVEL THERAPEUTIC TARGETS 1)Orlandella F.M. 2)Mariniello R.M. 3)Iervolino P.L.C. 4)De Stefano A.E. 5) AND EXPERIMENTAL THERAPEUTICS Munciguerra F. 6)Verde A. 7)Salvatore G. 1)IRCCS SDN, Naples, Italy 2)Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy 3)IRCCS SDN, Naples, Italy 4)CEINGE - L1 Biotecnologie Avanzate s.c. a r.l., Naples, Italy 5)CEINGE - Biotecnologie Avanzate THE TRANSERI STUDY: EFFECT OF ERIBULIN ON CIRCULATING TGFβ AND s.c. a r.l., Naples, Italy 6)CEINGE - Biotecnologie Avanzate s.c. a r.l., Naples, Italy 7) TNFα IN METASTATIC BREAST CANCER PATIENTS. RELATIONSHIP WITH Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, OUTCOME Italy 1)Garrone O. 2)Lo Nigro C. 3)Michelotti A. 4)Vandone A.M. 5)Abbona A. 6)De Angelis C. 7)Tonissi F. 8)Vanella P. 9)Denaro N. 10)Merlano M.C. Thyroid carcinoma is one of the malignancies with the highest increase in incidence 1)Medical Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy 2)Medical in the last years. In general the most frequent form, papillary thyroid carcinoma, has Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy 3)Medical Oncology, an overall favorable prognosis, however a fraction of patients develop metastasis AOU Pisana, Ospedale S. Chiara, ITT, Pisa, Italy 4)Medical Oncology, S. Croce and resistance to therapy. Anaplastic or undifferentiated thyroid cancer is a rare & Carle Teaching Hospital, Cuneo, Italy 5)Medical Oncology, S. Croce & Carle and lethal form of thyroid cancer in which cells have lost thyroid differentiation Teaching Hospital, Cuneo, Italy 6)Medical Oncology, AOU Pisana, Ospedale S. markers and have acquired highly proliferative and motile phenotype. Thus, there is Chiara, ITT, Pisa, Italy 7)Medical Oncology, S. Croce & Carle Teaching Hospital, a compelling need to identified novel molecular biomarkers associated with thyroid Cuneo, Italy 8)Medical Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy carcinoma cells aggressiveness. 9)Medical Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy 10)Medical MicroRNAs (miRNAs) are a small class of negative regulators that play a key role Oncology, S. Croce & Carle Teaching Hospital, Cuneo, Italy in the control of many biologic processes and in many human disease including thyroid cancers. miRNAs can acts by modulating both cancer initiation and cancer **Note: The appearance of your abstract here is an approximation of how the progression by regulating the expression of target mRNAs . abstract would appear in print, if accepted. Through a miRnome profile, we have identified several miRNAs deregulated in Background papillary thyroid cancer cells. We have previously showed that miR-584 mediates Eribulin (E) is approved for the treatment of metastatic breast cancer (mBC) thyroid cancer cell resistance to apoptosis by regulating the tumor suppressor patients (pts) after previous exposure to antracyclines and taxanes. E interferes with TUSC2. microtubule leading to apoptosis and cell cycle arrest in G2/M. E reverses epithelial Here, we studied the role of miR-650 in thyroid carcinoma tissues and cell lines. mesenchimal transition (EMT) in human cancer cell lines and in mice. Moreover, We demonstrated that miR-650 is over-expressed in papillary and anaplastic thyroid reduces metastatization in mice. TGFβ is an immunosuppressive cytokine, is a carcinoma compared to normal thyroids tissues. Further, we obtained evidences growth factor for cancer-associated fibroblasts (CAFs) and promotes EMT. TNFα that in thyroid cancer cells, miR-650 increases cell migration and invasion. Finally, synergizes with TGFβ to promote EMT. EMT permits cancer cells trafficking thus we identified, the serine-threonine protein phosphatase 2 catalytic subunit alpha, as driving metastatization. Our study investigates the interference among E and TGFβ a direct target of miR-650. and TNFα levels in pts and the correlation with the outcome and the metastatic In conclusions, we have identified novel molecular players of thyroid carcinogenesis spread. that can be used as novel biomarkers of thyroid cancer cells aggressiveness. Methods Blood levels of TGFβ and TNFα were determined at baseline, before cycle (C) 3, 5 and at disease progression in mBC pts treated with E at 1.23 mg/m2, d 1–8 every 21 d. Results We report data on 24 pts who completed 5 C or progressed before C 5. We did not observe changes of TNFα during treatment. The median (M) basal TGFβ value was higher in pts than in 4 healthy volunteers (M concentrations: 232 pg/ml vs 114 pg/ml respectively). We divided our population according to basal TGFβ levels upper or lower the M value. M PFS was similar in the two groups (112 vs 100 d). TGFβ increased in 9 pts from basal to C 5, and decreased in 15 pts. We observed a numerical difference in M PFS between the pts with decreased and increased values (107 vs 82 d respectively). In 8 pts TGFβ decreased at each subsequent point. M PFS in these pts was 167 vs 84 d in the remaining. Notwithstanding the continuous decline of TGFβ, 2 of these pts progressed at C 5 (both at CNS). In pts with continuous TGFβ decline the M value approaches healthy controls (160 pg/ml [range 303-90] vs 114 pg/ml [range 120-85] respectively). At C 5 9 pts did not progress: 3 PR and 6 SD (M PFS 175 d). TGFβ decreased in all but 2 pts (1PR+1SD). In these 2 pts PD occurred as early as 21 and 30 d respectively from C 5. The behaviour of pts with SD suggests that there is no correlation between tumor burden and TGFβ level. Conclusions Basal TGFβ does not predict outcome. TGFβ changed during treatment with E regardless of tumor load. Our results suggest that TGFβ changes might correlate with outcome in mBC.
L2 Coated Cationic Liposomes entrapping mir-660 inhibits tumor growth in Patients Derived Xenografts lung cancer models 1)Fortunato O.1, 2)Moro M.1, 3)Di Paolo D.2, 4)Milione M.3, 5)Centonze G.1, 6) Bornaghi V.1, 7)Borzi C.1, 8)Perri P. 2, 9)Pastorino U.4, 10)Ponzoni M.2 11)Sozzi G.1 1)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Laboratory of Experimental Therapies in Oncology, Istituto Giannina Gaslini, Genoa, Italy 4) Anatomic Pathology Unit, Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8) Laboratory of Experimental Therapies in Oncology, Istituto Giannina Gaslini, Genoa, Italy 9)Thoracic Surgery Unit, Fondazione IRCCS Istituto Nazionale dei
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Tumori, Milan, Italy 10)Laboratory of Experimental Therapies in Oncology, Istituto Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Giannina Gaslini, Genoa, Italy 11)Tumor Genomics Unit, Department of Research, Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase INTRODUCTION Lung cancer is the leading cause of cancer-related deaths. and cell growth and in inducing apoptotic cell death in colon carcinoma cells. Late diagnosis and inadequate therapies contribute to poor outcomes. MicroRNA (miRNA) are small non coding RNAs involved in lung cancer development. Since miRNAs can simultaneously regulate different cancer-related genes, they represent L4 an interesting therapeutic approach for cancer treatment. We previously reported HMGA1/E2F1 axis and NFkB pathways regulate LPS progression that mir-660 inhibits lung cancer growth through the inhibition of the MDM2-p53 and trabectedin resistance axis. 1)Loria R. 2)Laquitana V. 3)Bon G. 4)Trisciuoglio D. 5)Frapolli R. 6)Covello R. 7) MATERIAL AND METHODS We have developed Coated Cationic Liposomes Amoreo C.A. 8)Ferraresi V. 9)Zoccali C. 10)Novello M. 11)Del Bufalo D. 12)Milella entrapping mir-660 (CCL660) and administered (1,5mg/Kg) i.p. twice a week for M. 13)Biagini R. 14)D’Incalci M. 15)Falcioni R. four weeks into SCID mice subcutaneously carrying lung cancer Patients Derived 1)Cellular Network and Molecular Therapeutic Target Unit, IRCCS Regina Elena Xenografts (PDXs). National Cancer Institute, Roma, Italy 2)Cellular Network and Molecular Therapeutic RESULTS Systemic delivery of liposomal mir-660 increased miRNA levels in Target Unit, IRCCS Regina Elena National Cancer Institute, Roma, Italy 3)Cellular tumors (20-fold compared to controls) and caused a significant tumor growth Network and Molecular Therapeutic Target Unit, IRCCS Regina Elena National reduction (25% volume reduction in CCL660 treated tumors compared to controls) Cancer Institute, Roma, Italy 4)Preclinical Models and New Therapeutic Agents in PDX305, a P53 wild type model. Effects of CCL660 were visible in terms of tumor Unit, IRCCS Regina Elena National Cancer Institute, Roma, Italy 5)Department of growth delay as shown by Kaplan-Meier curves where percent of survival indicating Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy the percent of tumors that reached 1000mm3 volume (0% in vehicle-treated and 6)Pathology Unit, Department of Research Advanced Diagnostic and Technological 80% in CCL660-treated tumors) Innovation, IRCCS Regina Elena National Cancer Institute, Roma, Italy 7)Pathology Mir-660 administration reduced the number of proliferating cancer cells (32% Unit, Department of Research Advanced Diagnostic and Technological Innovation, reduction of Ki-67 positive cells in PDX305) through inhibition of MDM2 and IRCCS Regina Elena National Cancer Institute, Roma, Italy 8)Medical Oncology A, restoration of p53 and its downstream effector, p21. Moreover, the effects of mir- IRCCS Regina Elena National Cancer Institute, Roma, Italy 9)Orthopedic Surgery, 660 over-expression on tumor growth were lost, although MDM2 down-modulation Department of Experimental Clinical Oncology, IRCCS Regina Elena National was still appreciable, in a P53 mutated PDX model, PDX111. Cancer Institute, Roma, Italy 10)Pathology Unit, Department of Research Advanced Furthermore, CCL660 long lasting treatment (8 weeks) inhibited tumor growth (50% Diagnostic and Technological Innovation IRCCS Regina Elena National Cancer reduction compared to controls) in another P53wt model, PDX302. Institute, Roma, Italy 11)Preclinical Models and New Therapeutic Agents Unit, Interestingly, we reported that liposome delivery of synthetic mir-660 had no IRCCS Regina Elena National Cancer Institute, Roma, Italy 12)Medical Oncology immunological off-target and acute toxicity effects in terms of blood biochemistry, A, IRCCS Regina Elena National Cancer Institute, Roma, Italy 13)Orthopedic organ weights, immune cells infiltrates in organ tissues, chemokines, cytokines and Surgery, Department of Experimental Clinical Oncology, IRCCS Regina Elena growth factors production. National Cancer Institute, Roma, Italy 14)Department of Oncology, IRCCS-Istituto Furthermore, stable mir-660 expression inhibited the capacity of metastatic lung di Ricerche Farmacologiche Mario Negri, Milan, Italy 15)Cellular Network and cancer H460 cells to form lung nodules when injected i.v. in immunocompromised Molecular Therapeutic Target Unit, IRCCS Regina Elena National Cancer Institute, mice. The reduction of lung nodules growth was observed by animal PET analysis Roma, Italy and confirmed by H&E and pan-cytokeratin staining of the lungs. CONCLUSION Altogether, our results highlight the potential role of coated cationic Although the medical treatments of sarcoma have evolved in the last years, a liposomes entrapping mir-660 in lung cancer treatment without inducing immune- significant portion of patients develops recurrence after therapies suggesting the related toxic effects. need to identify novel targets to improve the treatments. By the use of patient- derived and established cell lines from liposarcoma, as well as specimens from patient biopsies, we found that HMGA1 is involved in the progression of L3 dedifferentiated and myxoid liposarcoma. The immunohistochemical and RT-PCR New TRAP1 and Hsp90 chaperone inhibitors with cationic analyses of 68 liposarcoma specimens revealed a significant high expression components: preliminary studies on mitochondrial targeting of HMGA1, at the protein and RNA levels, both in myxoid and dedifferentiated 1)Rondanin R. 2)Lettini G. 3)Oliva P. 4)Baruchello R. 5)Costantini C. 6)Trapella C. liposarcoma subtypes compared to differentiated ones. Loss- and gain-of-function 7)Simoni D. 8)Bernardi T. 9)Sisinni L. 10)Pietrafesa M. 11)Maddalena F. 12)Lepore experiments by HMGA1-specific depletion and overexpression in dedifferentiated S. 13)Ponterini G. 14)Costi M.P. 15)Vignudelli T. 16)Luciani R. 17)Matassa D.S. 18) and myxoid liposarcoma cells showed the contribution of this oncogenic factor Esposito F. 19)Landriscina M. in cell proliferation, motility, invasion and drug resistance. The in vitro and in 1)Dep. of Chemical and Pharmaceutical Sciences, University of Ferrara, Italy vivo treatment of myxoid liposarcoma with trabectedin, a drug with a potent anti- 2)Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral tumor activity, revealed down-regulation of HMGA1, E2F1 and its-downstream Cancer Center of Basilicata, Rionero in Vulture, Italy 3)Dep. of Chemical and targets, vimentin and ZEB1, indicating a critical role of trabectedin in inhibiting the Pharmaceutical Sciences, University of Ferrara, Italy 4)Dep. of Chemical and mesenchymal markers of these tumors through the HMGA1/E2F1 axis. These data Pharmaceutical Sciences, University of Ferrara, Italy 5)Dep. of Chemical and were also confirmed in patients’ tumor biopsies being HMGA1, E2F1, and vimentin Pharmaceutical Sciences, University of Ferrara, Italy 6)Dep. of Chemical and expression significantly reduced upon trabectedin therapy, administered as neo- Pharmaceutical Sciences, University of Ferrara, Italy 7)Dep. of Chemical and adjuvant chemotherapy. Furthermore, trabectedin treatment inhibits in vitro NFkB Pharmaceutical Sciences, University of Ferrara, Italy 8)Dep. of Chemical and pathway in mixoyd liposarcoma sensitive but not in resistant counterparts, and the Pharmaceutical Sciences, University of Ferrara, Italy 9)Laboratory of Pre-Clinical inhibition of NFkB pathway re-sensitizes the resistant cells to trabectedin treatment. and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero These data support the rational for combining NFkB inhibitors with trabectedin in in Vulture, Italy 10)Laboratory of Pre-Clinical and Translational Research, IRCCS, liposarcoma patients, who have become resistant to the drug. Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 11)Laboratory of Pre- Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, L5 Rionero in Vulture, Italy 12)Laboratory of Pre-Clinical and Translational Research, cxcr4 role for new therapeutic strategy in osteosarcoma IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 13)Dep. 1)Bientinesi E. 2)Pollino S. 3)Pazzaglia L. 4)Dozza B. 5)Palmerini E. 6)Lucarelli E. of Life Sciences, University of Modena and Reggio Emilia, Italy 14)Dep. of Life 7)Benassi M.S. Sciences, University of Modena and Reggio Emilia, Italy 15)Dep. of Life Sciences, 1)Rizzoli Orthopaedic Institute -IRCCS, Experimental Oncology Laboratory, University of Modena and Reggio Emilia, Italy 16)Dep. of Life Sciences, University Bologna, Italy 2)Rizzoli Orthopaedic Institute -IRCCS, Experimental Oncology of Modena and Reggio Emilia, Italy 17)Dep. of Molecular Medicine and Medical Laboratory, Bologna, Italy 3)Rizzoli Orthopaedic Institute -IRCCS, Experimental Biotechnology, University of Napoli Federico II, Italy 18)Dep. of Molecular Medicine Oncology Laboratory, Bologna, Italy 4)Rizzoli Orthopaedic Institute -IRCCS, and Medical Biotechnology, University of Napoli Federico II, Italy 19)Dep. of Medical Osteoarticolar Regeneration Laboratory, Bologna, Italy 5)Rizzoli Orthopaedic and Surgical Sciences, University of Foggia, Italy Institute - IRCCS, Chemotherapy Unit, Bologna, Italy 6)Rizzoli Orthopaedic Institute -IRCCS, Osteoarticolar Regeneration Laboratory, Bologna, Italy 7)Rizzoli TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone Orthopaedic Institute -IRCCS, Experimental Oncology Laboratory, Bologna, Italy family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been Osteosarcoma (OS), a highly malignant bone tumor, predominantly affects young envisaged and a series of compounds has been developed by binding the simple adults and pediatric patients. The low survival rate for patients with metastatic OS, pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a due to the development of drug-resistance and high toxicity in treatment, has led us permanent cationic head, or a basic group highly ionizable at physiologic pH. to focus on CXCL12 / CXCR4 pathway, considered responsible for the development
56 57 ABSTRACT BOOK
and progression of many malignant neoplasms and still little studied in OS. Università degli Studi di Milano, Milano, Italia 5)Experimental therapeutics, In this study CXCR4 gene expression was analyzed by RT-PCR in 52 OS patients Beckman Research Institute, City of Hope National Medical Center, Duerte, CA, and 13 normal tissues and a significant overexpression in tumors compared to Usa 6)Department of Biomedical Sciences for Health, Università degli Studi di normal samples (p<0.05) was found. Milano, Milano, Italia 7)Department of Biomedical Sciences for Health, Università The IHC analysis showed that 45.5% of high-grade OS analyzed presented a degli Studi di Milano, Milano, Italia 8)Department of Pharmaceutical Sciences, moderate to strong positivity for CXCR4 protein and statistical analysis revealed Università degli studi di milano, Milano, Italia 9)Pharmacological and biomolecular that the risk of developing metastasis increased 2-fold (95% CI = 1.2-3.4; p = 0.008) sciences, Università degli Studi di Milano, Milano, Italia for each increase in the level of receptor expression. Moreover the patients with a low and focal CXCR4 protein expression had a higher probability of disease-free Tocotrienols are members of the vitamin E family compounds known as α-, β-, γ-, survival than patients with a strong and homogeneous distribution. δ-tocopherols (TPs) or tocotrienols (TTs). Recent findings have identified TTs as Afterwards the effect of two CXCR4 inhibitors (MDX1338 and AMD3100) on having more potent anticancer potential than TPs. Among TTs, δ-TT shows the most proliferation, apoptosis and migration was analyzed in U2OS, SAOS2 and 143B cell potent antiproliferative activities against various cancers. Prostate cancer represents lines. The cells were treated with MDX1338 at the dose of 0.005 μg/mL and with the second major cause of cancer death among men in developed countries. At AMD3100 at the dose of 30 μg/mL A reduction of cell proliferation associated to an the early stages, prostate cancer responds to androgen deprivation therapy but increased apoptotic fraction was found after 48h of both treatments. Concomitantly, in the later stages it becomes aggressive with severe chemoresistence, referred a slowing down of cell migration was found after 24 with 0.001 μg/mL of MDX1338 to as castration-resistant prostate cancer (CRPC). Despite recent advances in the and 20 μg/mL of AMD3100. treatment of CRPC, the outcome of patients remains poor, so new therapeutical Further studies are ongoing to validate these results and in order to better approaches are needed. The aim of this study was to obtain insights into the understand the role of CXCL12 / CXCR4 pathway in OS progression and to focus mechanisms that underlie the δ-TT cytotoxicity in DU145 and PC3 CRPC cells. on the planning of tailored therapies that should be more effective and less toxic. In this study we observed that δ-TT induces cytotoxicity and apoptosis (canonical cell death) in both cell lines with an extensive cytoplasmic vacuolation. To analyze the nature of vacuoles, the morphological changes were evaluated by TEM that L6 highlighted mitochondria swelling and endoplasmic reticulum (ER) dilatation. These ERAP1 promotes the Hedgehog signaling and medulloblastoma observations suggest that δ-TT, beside the well known apoptotic death, might development by controlling βTrCP/USP47 axis induce in these cell line paraptosis, a non-canonical cell death, characterized by 1)Lospinoso Severini L. 2)Bufalieri F. 3)Infante P. 4)Bernardi F. 5)Caimano M. 6) ER- or mitochondria-derived vacuoles. To confirm this hypothesis, we observed Romania P. 7)Peschiaroli A. 8)Pazzaglia S. 9)Locatelli F. 10)Gulino A. 11)Ayrault O. that pretreatment of the cells with ER stress inhibitors prevents δ-TT-induced 12)Fruci D. 13)Di Marcotullio L. vacuolation. 1)Department of Molecular Medicine, University La Sapienza, Rome, Italy 2) Chronic ER stress and autophagy are two processes that might be involved in δ-TT Department of Molecular Medicine, University La Sapienza, Rome, Italy 3) cytotoxicity. We found that δ-TT induces an increased expression of ER stress Centre of Life NanoScience@Sapienza, Istituto Italiano di Tecnologia, Rome, markers (Bip, PERK, EIF2α, ATF4 and CHOP) in both cell lines, while it activates Italy 4)Department of Molecular Medicine, University La Sapienza, Rome, Italy 5) autophagy only in PC3 cells. To demonstrate the interplay between the ER stress/ Department of Molecular Medicine, University La Sapienza, Rome, Italy 6)Pediatric autophagy axis and apoptosis, we performed MTT and Western blot assays in Haematology/Oncology Department, Bambino Gesù Children’s Hospital IRCCS, the presence or absence of ER stress or autophagy inhibitors, highlighting that Rome, Italy 7)National Research Council of Italy CNR, Institute of Translational the cytotoxic effect of δ-TT as well as the expression of apoptotic markers were Pharmacology, Rome, Italy 8)Biotechnology Unit, ENEA CR Casaccia, Rome, Italy significantly reduced. In conclusion, our results demonstrated the complexity in 9)Pediatric Haematology/Oncology Department, Bambino Gesù Children’s Hospital δ-TT-mediated cytotoxicity, involving both paraptosis-like and apoptosis cell death IRCCS, Rome, Italy 10)Department of Molecular Medicine, University La Sapienza, in CRPC cells. Moreover, δ-TT activity is mediated, at least partially, by the induction Rome, Italy 11)Institut Curie, PSL Research University, CNRS UMR, INSERM, of the ER stress/autophagy axis in PC3 cells and only by the induction of ER stress Orsay, France 12)Pediatric Haematology/Oncology Department, Bambino Gesù in DU145 cells. Children’s Hospital IRCCS, Rome, Italy 13)Department of Molecular Medicine, (Supported by PRIN 2015) University La Sapienza, Rome, Italy Hedgehog pathway is essential for embryonal development and tissues homeostasis. Inappropriate Hedgehog signaling causes several disparate human cancers, including medulloblastoma, the most common brain malignancy in childhood. Since Hedgehog signaling interacts with multiple pathways during tumorigenesis, its better knowledge is mandatory for developing innovative anti-cancer approaches. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1) as a previously unknown player of Hedgehog signaling. ERAP1 is crucial for the maturation of a wide spectrum of substrates and is involved in several biological functions such as antigen processing, cytokine receptor shedding and neo-angiogenesis. ERAP1 enzymatic activity contributes to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. Recently, inhibition of ERAP1 peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses. Here, we show a new biological role for ERAP1 in a not-immune mediated control of cancer and identify ERAP1 as a novel activator of the Hedgehog signaling. Our data demonstrate that genetic or pharmacological inhibition of ERAP1 suppresses Hedgehog-dependent tumor growth in vitro and in vivo by modulating the stability of Gli transcription factors, the final effectors of the Hedgehog pathway. Mechanistically, ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated ubiquitin ligase βTrCP and promotes βTrCP degradation, thus leading to the modulation of Gli transcription factors. These data identify a novel molecular mechanism in the regulation of Hedgehog signaling and strongly indicate ERAP1 as a promising therapeutic target for Hedgehog-dependent tumors.
L7 CANONICAL AND NON-CANONICAL CELL DEATHS INDUCED BY δ-TOCO- TRIENOL IN HUMAN PROSTATE CANCER CELLS 1)Montagnani Marelli M. 2)Moretti R.M. 3)Fontana F. 4)Raimondi M. 5)Marzagalli M. 6)Procacci P. 7)Sartori P. 8)Beretta G. 9)Limonta P. 1)Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italia 2)Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italia 3) Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milano, Italia 4)Department of Pharmacological and Biomolecular Sciences,
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L8 various proteins and transcription factors involved in colorectal carcinogenesis, Synergistic antitumor interaction of valproic acid and such as c-Myc. simvastatin sensitizes prostate cancer to docetaxel by Materials and Methods: CRC cell lines (HCT116, DLD1 and SW48) were treated targeting cancer stem cells compartment via YAP-pathway with increasing doses of iPA for 24h or 48h and cell proliferation was evaluated by modulation BrdU incorporation assay. Total lysates from CRC cells treated with 10 µM iPA at 1)Iannelli F. 2)Roca M.S. 3)Ciardiello C. 4)De Rienzo S. 5)Lombardi R. 6)Sorice A. different time points were analyzed by Western blot. The isolation and enrichment 7)Costantini S. 8)Moccia T. 9)Milone M. 10)Pucci B. 11)Budillon A. 12)Bruzzese F. of ubiquitinylated c-Myc was carried out using UBI-QAPTURE-Q® kit (Enzo Life 1)Experimental Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione Science). Interaction between FBXW7 and c-Myc was analyzed through IP assay. G. Pascale Napoli, Italy 2)Experimental Pharmacology Unit, Istituto Nazionale To assess the direct effect of iPA on c-Myc promoter activity, luciferase assay was Tumori, IRCCS Fondazione G. Pascale Napoli, Italy 3)Experimental Pharmacology performed in HCT116 and DLD1 cell lines. Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale Napoli, Italy 4) Results: iPA significantly inhibited CRC cells proliferation in a dose- and time- Experimental Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. dependent manner starting from a concentration of 5 µM. iPA treatment was able to Pascale Napoli, Italy 5)CROM-Centro Ricerche Oncologiche di Mercogliano (AV), up-regulate the expression of FBXW7 and the ubiquitination levels of its oncogenic Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale; Italy 6)CROM-Centro substrate c-Myc in CRC cell lines. Accordingly, iPA induced the interaction between Ricerche Oncologiche di Mercogliano (AV), Istituto Nazionale Tumori, IRCCS FBXW7 and c-Myc in HCT116 and DLD1 cells but not in SW48 cells harbouring Fondazione G. Pascale; Italy 7)CROM-Centro Ricerche Oncologiche di Mercogliano heterozygous FBXW7 mutation. Moreover, the effect observed in FBXW7 wild-type (AV), Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale; Italy 8)Experimental cells, such as HCT116 and DLD1, was attended by a significant reduction of Myc- Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale Max promoter activity. Napoli, Italy 9)CROM-Centro Ricerche Oncologiche di Mercogliano (AV), Istituto Conclusions: Our findings confirm the antiproliferative effect of iPA in CRC. The Nazionale Tumori, IRCCS Fondazione G. Pascale; Italy 10)CROM-Centro Ricerche obtained results suggested that in HCT116 and DLD1 cells iPA could be able to Oncologiche di Mercogliano (AV), Istituto Nazionale Tumori, IRCCS Fondazione regulate c-Myc turnover or degradation in FBXW7-dependent manner, pointing to a G. Pascale; Italy 11)Experimental Pharmacology Unit, Istituto Nazionale Tumori, novel potential biochemical target of iPA for the treatment of malignant CRC. IRCCS Fondazione G. Pascale Napoli, Italy 12)Experimental Pharmacology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale Napoli, Italy L10 Docetaxel (DTX) represents a standard of care for castration-resistant prostate YAP/mutant p53 protein complex orchestrates endothelin A cancer (PCa) patients. However, the onset of side effects hampers patient’s receptor/β-arrestin1 signaling during metastatization and compliance and DTX resistance invariably emerges leading to disease relapse. platinum response of high-grade serous ovarian cancer Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be 1)Tocci P. 2)Cianfrocca R. 3)Di Castro V. 4)Rosanò L. 5)Sacconi A. 6)Donzelli S. 7) responsible for the development of resistance to DTX. The mevalonate pathway Ferrandina G. 8)Tonon G. 9)Blandino G. 10)Bagnato A. (MVP) plays a critical role in PCa progression and is implicated in cell stemness, 1)Preclinical Models and New Therapeutic Agents Unit, IRCCS, Regina Elena proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we National Cancer Institute, Rome, Italy 2)Preclinical Models and New Therapeutic evaluate the combination treatment of valproic acid (VPA), an histone deacetylase Agents Unit, IRCCS, Regina Elena National Cancer Institute, Rome, Italy 3) (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor Preclinical Models and New Therapeutic Agents Unit, IRCCS, Regina Elena of the rate limiting enzyme in MVP HMGCoA reductase (HMGCR), alone and plus National Cancer Institute, Rome, Italy 4)Preclinical Models and New Therapeutic DTX, in PCa models. Synergistic antiproliferative and proapoptotic effect of VPA-SIM Agents Unit, IRCCS, Regina Elena National Cancer Institute, Rome, Italy 5) was observed in four PCa cell lines and was confirmed in PCa spheroids. Mevalonic Oncogenomic and Epigenetic Unit; IRCCS, Regina Elena National Cancer Institute, acid (MEV), the product of HMGCR activity, reverts all the VPA-SIM combined Rome, Italy 6)Oncogenomic and Epigenetic Unit; IRCCS, Regina Elena National antitumor effects, suggesting the involvement of MVP in the synergistic interaction. Cancer Institute, Rome, Italy 7)Gynecologic Unit, Catholic University of Rome, Italy Mechanistically, the VPA/SIM combination was able to induce a reduction of 8)Functional Genomics of Cancer Unit, IRCCS, San Raffaele Scientific Institute, HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP. Milan, Italy 9)Oncogenomic and Epigenetic Unit; IRCCS, Regina Elena National Furthermore, YAP cytoplasmatic inactive form, increased upon VPA/SIM treatment, Cancer Institute, Rome, Italy 10)Preclinical Models and New Therapeutic Agents and this effect was antagonized by both MEV, confirming an MVP-dependent effect. Unit, IRCCS, Regina Elena National Cancer Institute, Rome, Italy In addition, in 22Rv1 cells transfected with the constitutively active YAP (YP5SA) mutated form, VPA/SIM treatment was unable to inhibit YAP nuclear translocation The limited clinical response to platinum-based therapeutics observed in high-grade compared with control cells. In line with all these observations, we demonstrated serous ovarian cancer (HG-SOC), characterized by high frequency of recurrence strong reduction of YAP transcriptional targets, CTGF and Cyr61, induced by VPA/ and TP53 mutations, may be related to the connections between hyper-activated SIM combination. As a functional read-out of these observations, we performed a pathways. The aberrant activation of endothelin-1 (ET-1)/endothelin A receptor limiting dilution assay (LD) in 22Rv1 control and mutant YAP transfected (22Rv1- (ETAR) signaling, through the ability of the scaffold protein β-arrestin1 (β-arr1) to YAP5SA) cells, demonstrating a strong reduction of stem cell frequency induced by interact with a broad repertoire of signaling proteins, integrates several pathways VPA/SIM combination in control cells, partially loss in 22Rv1-YAP5SA cells. Finally, involved in a plethora of tumor functions. Here, we show that the Hippo effector YAP the VPA/SIM combination, sensitizes PCa cells to DTX treatment in vitro and in vivo works as crucial signal transducer to mediate ETAR/β-arr1 signaling during HG-SOC in two PCa xenograft models. Overall, this study suggests that the combination of growth, metastasis and platinum response. In patient-derived HG-SOC cells and two safe generic drugs such as VPA and SIM, may improve the therapeutic index of platinum sensitive and resistant OC cell lines, we demonstrate that ET-1R activation DTX representing an innovative and feasible antitumor strategy for the treatment of promotes YAP and TAZ de-phosphorylation and to a greater extent in platinum- prostate cancer that warrants further clinical evaluation. resistant OC cells. Of note, ET-1R through β-arr1, engaging a physical interaction with the novel partner YAP, triggers its cytoplasmic-nuclear shuttling. This effect is further improved in platinum resistant OC cells and is abrogated by the mutant β-arr1, L9 unable of nuclear distribution. In parallel, β-arr1 interacting with Trio, mediates the N6-ISOPENTENYLADENOSINE INHIBITS PROLIFERATION OF COLORECTAL ET-1R-induced Trio/RhoA/actin cytoskeleton-dependent YAP nuclear accumulation. CANCER CELLS THROUGH UP-REGULATION OF TUMOR SUPPRESSOR Mechanistically, downstream of ETAR signaling, nuclear β-arr1 and YAP bind mutant FBXW7. p53 (mutp53), forming a competent transcriptional complex consisting of β-arr1/ 1)Piscopo C. 2)Fiore D. 3)Proto M.C. 4)Fusco B.M. 5)Bifulco M. 6)Gazzerro P. YAP/mutp53/TEAD. This leads to the aberrant transcriptional activation of target 1)Department of Pharmacy, University of Salerno, Fisciano (SA), Italy 2)Department genes, including ET-1, thereby fueling a feed-forward loop that sustains a persistent of Pharmacy, University of Salerno, Fisciano (SA), Italy 3)Department of Pharmacy, YAP activity and ensures cell survival and invasion. ET-1R inactivation, by using University of Salerno, Fisciano (SA), Italy 4)Department of Pharmacy, University the dual ET-1R antagonist macitentan, shuts-down β-arr1-mediated YAP/mutp53/ of Salerno, Fisciano (SA), Italy 5)Department of Molecular Medicine and Medical TEAD transcriptional program, reduces tumor growth, metastatic dissemination and Biotechnologies, University of Naples “Federico II”, Naples (NA), Italy 6)Department enhances the sensitivity to chemotherapy in patient-derived HG-SOC xenografts. of Pharmacy, University of Salerno, Fisciano (SA), Italy Of clinical relevance, the combined expression of ETAR/β-arr1/YAP gene signature correlates with a worst prognosis in HG-SOC patients carrying TP53 mutations. Introduction: N6-isopentenyladenosine (iPA), the only known cytokinin found in Altogether, these findings identify YAP as a central mediatorA ofET R/β-arr1 animal cells, has been shown to exert potent in vitro anti-tumor activity on different signaling, thereby providing mechanistic and therapeutic insights to the targeting of types of human cancers, including colorectal cancer (CRC). Although some the ETAR/β-arr1/YAP/mutp53 axis using an ET-1R antagonist that impairs malignant potential iPA biochemical targets have been identified, its precise mechanism of progression and sensitizes to platinum-based therapy in HG-SOC. (Supported by action remains to be elucidated. AIRC). Here we found that iPA affects CRC cells proliferation by increasing the expression of a well-established tumor suppressor, FBXW7, a component of the SCF (SKP1, CUL1, F-box protein) E3 ubiquitin ligase complex that promotes degradation of
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L11 therapies. Targeting the pro-neoplastic role played by the chaperone Functional analysis of MKK3 knockdown-mediated effects in microsatellite stable TRAP1 in tumor cell mitochondria (MSS)-CRC revealed that MKK3 targeting was able to induce autophagy and 1)Sanchez Martin C. 2)Ferraro M. 3)Moroni E. 4)Masgras I. 5)Serra M. 6)Columbano apoptosis only in some MSS-CRC cell lines, while it was able to boost 5-FU efficacy, A. 7)Colombo G. 8)Rasola A. likely by inducing accumulation of DNA-damage. Moreover, we found that MKK3 1)Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy 2) levels are rapidly (6-24h) increased in MSS-CRC cell lines upon non-lethal 5-FU Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle treatment: strikingly, while increased MKK3 activity has been previously reported Ricerche, Milano, Italy 3)IRCCS Multimedica, Milano, Italy 4)Dipartimento di to be responsible for 5-FU induced apoptosis via p38alfa activation in CRC, we Scienze Biomediche, Università di Padova, Padova, Italy 5)Dipartimento di observed reduced cell proliferation and increased cell death upon combination of Scienze Biomediche, Università di Cagliari, Cagliari, Italy 6)Dipartimento di 5-FU treatment and MKK3 knockdown. Analysis of p38 isoforms expression revealed Scienze Biomediche, Università di Cagliari, Cagliari, Italy 7)Istituto di Chimica del that 5-FU exposure consistently caused increase in p38delta but not p38alfa levels Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Milano, Italy 8) in MSS-CRC, suggesting that pro-survival signaling could be mediated by p38delta Dipartimento di Scienze Biomediche, Università di Padova, Padova, Italy and that this overcomes the previously reported p38alfa-transduced apoptotic signaling in this cell-context. Mitochondria take actively part in the process of tumor onset and growth by allowing Altogether, results from this study indicate MKK3 as a therapeutic target in MSS- to escape death signals, to cope with oxidative stress and to meet energy demands CRC: correct MKK3 targeting may contribute to efficient MSS-CRC killing by for neoplastic transformation in an environment where nutrients and oxygen can both inducing autophagic cell death (in some contexts) and DNA damage, hence be spatially and temporally heterogeneous. In this scenario, the mitochondrial potentiating 5-FU efficacy. chaperone TRAP1, a member of the Hsp90 family, was identified as a master regulator of the mitochondrial bioenergetics in several tumor cell types [1]. We have previously shown that TRAP1 inhibits oxidative phosphorylation by down- L13 regulating the enzymatic activity of succinate dehydrogenase (SDH), the complex A pre-clinical assessment of the HSP90 inhibitor AUY922 II of the respiratory chain, thus leading to a succinate-dependent stabilization of (Luminespib) in hepatocellular carcinoma the transcription factor HIF1α that is crucial for tumor progression [2]. Moreover, 1)Augello G. 2)Cusimano A. 3)Emma M.R. 4)Mongiovì S. 5)Azzolina A. 6)Puleio R. SDH inhibition decreases ROS levels and shields neoplastic cells from oxidative 7)Cicero L. 8)Gulino A. 9)Belmonte B. 10)Gramignoli R. 11)Strom S. 12)Montalto insults [3]. We have also found that TRAP1 is involved in a mitochondrial signaling G. 13)Cervello M. cascade, as it forms a multimeric complex with SDH and the mitochondrial fraction 1)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy” – CNR – of the kinase ERK1/2, which phosphorylates TRAP1 thus enhancing its inhibitory Palermo, Italy 2)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy” effect on complex II activity. In tumor models endowed with deregulated activation – CNR – Palermo, Italy 3)Istituto di Biomedicina e Immunologia Molecolare “Alberto of the Ras/ERK pathway, such as cells lacking the Ras-GAP neurofibromin, this Monroy” – CNR – Palermo, Italy 4)Istituto di Biomedicina e Immunologia Molecolare mitochondrial signalling axis contributes to neoplastic growth [4]. “Alberto Monroy” – CNR – Palermo, Italy 5)Istituto di Biomedicina e Immunologia Taken together, these observations suggest that TRAP1 could be a good target to Molecolare “Alberto Monroy” – CNR – Palermo, Italy 6)Istituto Zooprofilattico develop innovative therapeutic approaches for cancer treatment. In this context, Sperimentale della Sicilia “A. Mirri”, Histopathology and Immunohistochemistry we have exploited computational approaches based on the characterization of Laboratory, Palermo, Italy 7)Istituto Zooprofilattico Sperimentale della Sicilia “A. the chaperone internal dynamics in order to identify TRAP1 regions that can be Mirri”, Histopathology and Immunohistochemistry Laboratory, Palermo, Italy 8) potentially targeted by selective TRAP1 inhibitors in an allosteric way. Tumor Immunology Unit, Department of Health Science, University of Palermo, By analyzing long-range coordination propensities, which are defined as functions Italy 9)Tumor Immunology Unit, Department of Health Science, University of of the distance fluctuation of two given amino acid residues, of residue-pairs in Palermo, Italy 10)Division of Pathology, Department of Laboratory Medicine, Cell the whole structure, we have pinpointed the residues that provide the highest Transplantation and Regenerative Medicine, Karolinska Institutet, Stockholm, contribution in controlling functional motions. This analysis was carried out in the Sweden 11)Division of Pathology, Department of Laboratory Medicine, Cell presence/absence of a ligand, thus allowing detection of druggable allosteric sites Transplantation and Regenerative Medicine, Karolinska Institutet, Stockholm, and providing the chemical information necessary to design molecules optimized to Sweden 12)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy” – efficiently bind such sites. CNR – Palermo, Italy 13)Istituto di Biomedicina e Immunologia Molecolare “Alberto Based on this information, we have fished out and tested a set of putative TRAP1 Monroy” – CNR – Palermo, Italy allosteric inhibitors, finding that all these compounds inhibit TRAP1 ATPase activity in a dose-dependent way, with a potency very similar to that displayed by classical Hepatocellular carcinoma (HCC) is a highly aggressive liver tumor, and the survival Hsp90-family inhibitors, such as the geldanamycin derivative 17-AAG and radicicol. rate for HCC patients remains dismal. Therefore, new therapeutic strategies need Additionally, none of these compounds exerted an inhibitory effect on Hsp90α to be developed. Heat shock protein 90 (HSP90), a molecular chaperone, is highly activity, suggesting that they are highly selective for TRAP1. At a cellular level, these expressed in different types of tumors, though, its role in hepatocarcinogenesis lead compounds, at a concentration of 50 µM, were able to completely reverse the remains unclear. The potent third-generation HSP90 inhibitor AUY922 (luminespib) inhibition of the SDH activity exerted by TRAP1 in both mouse malignant peripheral has antitumor activity in a wide range of human cancer cells, and it is currently being nerve sheath tumors and human cervix carcinoma cell models, while cells where tested in several phase II clinical studies in patients with different cancer types. TRAP1 expression had been knocked out were completely insensitive to the However, to date, there have been no studies of AUY922 administration in HCC. effects of these molecules. Our studies provide evidence that novel computational This study aimed to investigate the intracellular events leading to AUY922-induced approaches can be used to design highly selective TRAP1 inhibitors. These antitumor effects in HCC. molecules are useful tools for the comprehension of the role played by TRAP1 in We studied the expression of HSP90 in primary human HCC tissues by the regulation of signaling and metabolic pathways in tumor cell mitochondria and immunohistochemistry. HSP90 expression was significantly higher in HCC tissues can be further developed in order to find novel antineoplastic strategies. compared to peritumoral liver cirrhotic tissues (p < 0.01), suggesting that the overexpression of HSP90 might be involved in hepatocarcinogenesis. Therefore, the antitumor effects of AUY922 were investigated in the HepG2, Hep3B, Huh7, PLC/ L12 PRF/5 and SNU475 HCC cell lines. AUY922 treatment reduced cell proliferation, MKK3 TARGETING AS A PROMISING TOOL IN MICROSATELLITE-STABLE cell viability and the migration of HCC cells, and triggered apoptosis. Notably, COLORECTAL CARCINOMA AUY922 was not toxic in primary normal human hepatocytes. AUY922 treatment 1)Stramucci L. 2)Amoreo C.A. 3)Diodioro M.G. 4)Bossi G. of HCC cells led to the upregulation of HSP70, a hallmark of HSP90 inhibition, and 1)Ricerca, diagnostica avanzata e innovazione tecnol, Istituto nazionale tumori the simultaneous depletion of several HSP90 client proteins known to be involved in Regina Elena, Roma, Italia 2)Dipartimento di patologia, Istituto nazionale tumori hepatocarcinogenesis, such as EGFR, ERK1/2, and AKT. In addition, the treatment- Regina Elena, Roma, Italia 3)Dipartimento di patologia, Istituto nazionale tumori induced caspase-dependent fragmentation of β-catenin resulted in inhibition of Regina Elena, Roma, Italia 4)Ricerca, diagnostica avanzata e innovazione tecnol, β-catenin-mediated transcriptional activity. Moreover, AUY922 activated ER stress Istituto nazionale tumori Regina Elena, Roma, Italia genes and increased the expression of the stress inducible gene nuclear protein 1 (NUPR1). Furthermore, NUPR1 knockdown sensitized Huh7 cells to AUY922 We previously reported the mitogen activated protein kinase (MAPK) kinase 3 (MKK3) treatment, suggesting that NUPR1 expression contributes to AUY922 resistance. to be able, upon genetic depletion, to induce autophagy and boost 5-Fuorouracyl Notably, the combination of AUY922 with sorafenib, the only FDA approved drug for (5-FU) efficacy in vitro and in vivo. Query of publicly available datasets revealed treating advanced HCC, synergistically inhibited HCC cell viability. Finally, in vivo poor prognosis in colorectal cancer (CRC) patients displaying high MKK3 levels, AUY922 inhibited the growth of HCC tumor cells in a xenograft animal model. In and tissue micro-array (TMA) analysis of 185 CRC patients revealed a correlation conclusion, these results demonstrate that HSP90 is a promising therapeutic target of high MKK3 levels with advanced tumor stages, indicating that MKK3 could serve in HCC, and AUY922 could be a drug candidate for its treatment. as a prognostic marker and therapeutic target in CRC. Among CRC patients, those Supported in part by the Italian Association for Cancer Research (AIRC) displaying microsatellite stability have worse prognosis, and urgently need new
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L14 addition, apoptosis increase was also evaluated by measuring positive annexin the transcription regulator nupr1 regulates sorafenib V staining, by flow cytometry technique, and apoptosis markers, such as PARP and resistance IN hepatocellular carcinoma cells by inducing cleaved caspase 3, by immunoblot assay. At the same time, ongoing results show apoptosis and inhibiting autophagy that pharmacologic inhibitors of autophagy, such as hydroxichloroquine, are able to 1)Emma M.R. 2)Augello G. 3)Azzolina A. 4)Cusimano A. 5)Montalto G. 6)Cervello M. reduce cell viability in cell lines exposed to Ipatasertib and Taselisib, proving a new 1)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, CNR, therapeutic combinatorial approach potentially translatable to patients. Palermo, Italy 2)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, CNR, Palermo, Italy 3)Istituto di Biomedicina e Immunologia Molecolare “Alberto Monroy”, CNR, Palermo, Italy 4)Istituto di Biomedicina e Immunologia Molecolare L16 “Alberto Monroy”, CNR, Palermo, Italy 5)Dipartimento Biomedico di Medicina Interna p65BTK is a novel therapeutic target in ovarian cancer e Specialistica, Università di Palermo, Italy 6)Istituto di Biomedicina e Immunologia 1)Conconi D. 2)Fruscio R. 3)Cialdella A. 4)Buda A. 5)Leone B.E. 6)Bonomo S. 7) Molecolare “Alberto Monroy”, CNR, Palermo, Italy Decio A. 8)Giavazzi R. 9)Damia G. 9)Giovannoni R. 10)Dalprà L. 11)Grassilli E. 12) Lavitrano M. Hepatocellular carcinoma (HCC) is a highly aggressive tumor with a low response to 1)Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy conventional chemotherapy as well as molecularly targeted treatments. Sorafenib, 2)Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy a multikinase inhibitor, is the only approved systemic therapy able to improve 3)Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy HCC patient survival. However, resistance to sorafenib treatment remains a major 4)Gynecologic Oncology Unit, Department of Obstetrics and Gynaecology, San obstacle to the effective treatment of HCC. Increasing evidence supports the Gerardo Hospital, University of Milano-Bicocca, Monza, Italy 5)Department of notion that autophagy plays a pivotal role in resistance to cancer therapy. Recent Medicine and Surgery, University of Milano-Bicocca, Monza, Italy 6)Department of findings have demonstrated that sorafenib activates a protective form of autophagy Medicine and Surgery, University of Milano-Bicocca, Monza, Italy 7)Department of in different tumor types, including HCC. In addition, we recently identified several Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy 8) genes involved in the ER stress response modulated after the treatment of HCC Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, cells with sorafenib, including the stress-inducible gene nuclear protein 1 (NUPR1). Milan, Italy 9)Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Interestingly, several studies have shown that NUPR1 regulates autophagy, Mario Negri, Milan, Italy 10)Department of Medicine and Surgery, University of apoptosis and chemo-resistance. Based on these observations, the present Milano-Bicocca, Monza, Italy 11)Department of Medicine and Surgery, University of study aimed to characterize the role of NUPR1 in HCC resistance to sorafenib, Milano-Bicocca, Monza, Italy 12)Department of Medicine and Surgery, University of focusing on its possible role in modulating autophagy and apoptosis. Treatment Milano-Bicocca, Monza, Italy 13)Department of Medicine and Surgery, University of of HCC cells with sorafenib resulted in the activation of autophagic flux, as shown Milano-Bicocca, Monza, Italy by an increase in LC3II expression and a decrease in p62 protein expression, two well-known autophagic markers. Furthermore, in basal condition, the silencing of Ovarian cancer is the seventh most common cancer worldwide accounting for more NUPR1 induced an accumulation of p62, suggesting it may block autophagic flux. than 150000 death/years. The absence of early distinctive symptoms and accurate Next, chloroquine (CQ, an autophagy inhibitor) or a small interfering RNA (siRNA) screening tests cause a late stage diagnosis in more than 70% ovarian cancer targeting NUPR1 was used to investigate whether autophagy protects against patients, making this cancer the most lethal gynaecological malignancy in western sorafenib-induced cytotoxicity in HCC cells, and therefore its inhibition could enhance countries. For this reason, possible new treatment options are emerging from recent HCC cell sensitivity s to the drug. CQ treatment or NUPR1 knockdown significantly clinical trials, but these approaches have not yet proven to cure ovarian cancer. An increased tumor cell sensitivity to sorafenib, and this effect was associated with an unmet need is therefore the finding of targets amenable to therapeutic inhibition. increase of p62, suggesting an impairment of autophagic flux. In addition, after CQ We recently identified p65BTK, a novel isoform of the Bruton’s kinase expressed in treatment or NUPR1 KD, apoptosis was also induced, as demonstrated by PARP colorectal carcinoma (CRC) acting as a powerful oncogene. In addition, p65BTK re- cleavage, a well-known caspase 3/7 substrate. In conclusion, all together, these sensitize to chemotherapy p53 null drug-resistant CRC cell lines and patient derived results suggest that sorafenib administration induces protective autophagy through organoids in vitro, and xenografts in vivo. We investigated whether p65BTK might NUPR1 up-regulation, which confers a survival advantage to HCC cells and that, be a therapeutic target also in ovarian cancer. therefore, the inhibition of NUPR1 expression may be an attractive strategy for We detected p65BTK expression in a panel of 10 ovarian carcinoma cell lines unlocking the antitumor potential of sorafenib in HCC. with different genetic background and in cancer tissues derived from 45 patients. Supported by the Italian Association for Cancer Research (AIRC) Using a homemade specific anti-p65BTK antibody in ELISA, we found significantly higher levels of p65BTK expression in biopsies from patients undergoing an early relapse (within 6 months). L15 In vitro experiments on Caov-3, OVCAR3 and OV-90 cell lines showed that p65BTK Role played by Autophagy in Breast Cancer models exposed to inhibition significantly decreased both short and long term cell proliferation. These new PI3K/AKT inhibitors, GDC-0068 and GDC-0032 results were also confirmed on primary cultures obtained from patient’s derived 1)Cocco S. 2)Leone A. 3)Buonfanti G. 4)Sisalli MJ. 5)Costantini S. 6)Budillon A. 7) xenograft. De laurentiis M. Testing ex vivo cells isolated from 59 ovarian cancer biopsies for sensitivity/ 1)U.O.C. Oncologia Medica Senologica, INT Fondazione G .Pascale, Napoli, Italia resistance to standard of care drugs (Paclitaxel, Carboplatin and Bevacizumab) and 2)Unità di Farmacologia Sperimentale, INT Fondazione G .Pascale, Napoli, Italia Ibrutinib as BTK inhibitor, we showed that inhibition of BTK strongly affected cell 3)U.O.C. Oncologia Medica Senologica, INT Fondazione G .Pascale, Napoli, Italia survival in high grade serous histotype tumors. In addition, we found a statistically 4)Dipartimento di Neuroscienze, Scienze riproduttive, Università degli studi di significant difference in treatment efficacy, measured as cell viability, between Napoli “Federico II, Napoli, Italia 5)Lab Bioinformatica e biologia dei sistemi, Centro standard of care drugs (both single and combined) and Ibrutinib. These results Ricerche Oncologiche di Mercogliano, Avellino, Italia 6)Unità di Farmacologia confirmed that ex-vivo inhibition of p65BTK is more potent than standard of care Sperimentale, INT Fondazione G. Pascale, Napoli, Italia 7)U.O.C. Oncologia therapeutics. Moreover, Ibrutinib treatment caused a decrease in short term survival Medica Senologica, INT Fondazione G .Pascale, Napoli, Italia especially in cells from patients that subsequently relapsed. Our data suggest that p65BTK could be a novel therapeutic target in ovarian cancer. Abundant preclinical evidences indicate that stress-induced autophagy in tumor cells is predominantly cytoprotective and that inhibition of autophagy can enhance tumor cell death by diverse anticancer therapies. A major negative regulator of L17 autophagy is the mammalian target of rapamycin (mTOR), activated downstream Interactions between CXCR4 pathway and Homologous of PI3KAKT pathway. mTOR inhibitors, including rapamycin, have been shown Recombinant Deficiency (HRD) targeted agents PARP inhibitors to induce autophagy in tumor cells (Takeuchi H. 2005), while the combination of in human ovarian cancer cell lines PI3K-AKT/mTOR and autophagy inhibitors shown synergistic effect on increased Russo D., Spina A., Ieranò C., Bello A.M., Califano D., Scala S. apoptosis and reduced autophagy. This project aimed to characterize the role of Istituto Nazionale Tumori “Fondazione G. Pascale”, Napoli, Italia autophagy in Breast Cancer models exposed to new potent Genentech PI3K/ AKT inhibitors GDC0068 (Ipatasertib) and GDC0032 (Taselisib) currently in phase Introduction. Epithelial Ovarian Cancer (EOC) is the 7th most common cause of III clinical trials on TNBC and ER+ patients. However, the efficacy of PI3K/AKT cancer death for women worldwide. Despite improvements in the treatment of ovarian inhibitors may be limited by resistance mechanisms that result in minimal cell death cancer, the majority of patients who achieve a complete response (CR) with front- in tumor cells. In order to investigate the role of autophagy as possible mechanisms line platinum-based chemotherapy ultimately develop recurrent disease, with over of resistance, Ipatasertib and Taselisib have been evaluated in three breast cancer 50% of women diagnosed with EOC eventually dying from their disease. Poly(ADP- cell lines, characterized by different receptors profile: the TNBC MDMB231, ribose) polymerase (PARP) inhibitors olaparib and niraparib, showed interesting the HER2/c-erb-2 SKBR3, and the luminal A MCF-7 cell line. Our results showed results in clinical trials in the setting of maintenance treatment in BRCA1/2 EOC. that both drugs are able to induce G1/S cell cycle block and increase of autophagy Ovarian tumor microenvironment is a critical target to impair cancer progression. signaling measured by p62 level and LC3 II/LC3 I ratio, by immunoblot assay. In CXC chemokine ligand 12 (CXCL12) activates the chemokine receptors CXCR4
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and CXCR7 regulating tumor growth, cell migration and aggressiveness through via L19 activation pathways such as PI-3K/Akt/mTOR and MAPKinases. BENEFICIAL EFFECTS OF DASATINIB ADDITION TO STANDARD It has been recently shown that dual blockade of PI3K and PARP in OC cell lines CHEMOTHERAPY IN PRECLINICAL MODELS OF PERIPHERAL T CELL resulted in attenuated PI3K/AKT/mTOR signaling. Aim of this project is to evaluate LYMPHOMA the efficacy of the combined treatment of PARP inhibitors, olaparib and niraparib, 1)Magni M. 2)Biancon G. 3)Carniti C. 4)Corradini P. with the newly developed CXCR4 antagonist, Peptide R54, in human ovarian 1)Dept. of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale cancer cell lines. dei Tumori, Milan, Italy 2)Dept. of Medical Oncology and Hematology, Fondazione Methods. OVCAR5, OVCAR8, SKOV3 and IGROV1 human ovarian cancer cell IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Dept. of Medical Oncology and lines were characterized for CXCR4, CXCR7 and CXCL12 expression by Western Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Dept. blot and Real Time PCR. Growth curves and cytotoxicity assays were conducted in of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei the presence of R54. CXCR4 signalling was also evaluated. Tumori, Milan, Italy; Dept. of Oncology and Hemato-oncology, Università degli Studi Results. OVCAR5, OVCAR8, SKOV3 and IGROV1 express CXCR4. CXCR7 di Milano, Milan, Italy. mRNA was found in OVCAR8, OVCAR5 and IGROV1 cell lines, whereas CXCL12 mRNA was detected in all cell lines except OVCAR8. R54 inhibited the CXCL12 Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of dependent/independent growth of OVCAR8, SKOV3 and OVCAR5. aggressive non-Hodgkin’s lymphomas comprising different entities. Anthracycline- Conclusions. Preliminary evidence demonstrate that OVCAR8 and SKOV3 human based regimens (usually CHOP, cyclophosphamide/doxorubicin/vincristine/ ovarian cancer cells are growth inhibited by the CXCR4 antagonist Peptide R54. prednisone or CHOEP, CHOP+Etoposide) are considered the standard of care Ongoing experiments are investigating on the effect of CXCR4 antagonism on in the front-line treatment. However, in contrast to diffuse large B-cell lymphomas PARP inhibitors, olaparib and niraparib, efficacy in human ovarian cancer cells. (DLBCLs), responses to these approaches have been neither adequate nor durable. Thus, new treatment strategies are needed to improve survival. In this study we investigated the responses to CHOEP in in vitro and in vivo models of PTCL to L18 uncover novel pathways to be targeted to improve CHOEP effects. A novel bispecific antibody to harness the hERG1-β1 macro- To this aim we employed six T-cell lymphoma and leukemia cell lines (OCI-Ly12, molecular complex for cancer therapy SUP-T1, KARPAS-299, HH, HDMAR-2 and Jurkat) to represent the heterogeneity 1)Duranti C. 2)Lottini T. 3)Iorio J. 4)Stefanini M. 5)Petroni G. 6)Giustetto P. 7) and complexity of PTCLs. Cells were incubated with escalating doses of CHOEP to Coppola S. 8)Lastraioli E. 9)Arcangeli A. calculate IC50. We found that CHOEP treatment induces different responses, with 1)University of Florence, Experimental and Clinical Medicine, Florence, Italy 2) the most sensitive cells being HH and the least sensitive being HDMAR-2. To gain University of Florence, Experimental and Clinical Medicine, Florence, Italy 3) insights into the molecular mechanisms of action of CHOEP, we performed a gene University of Florence, Experimental and Clinical Medicine, Florence, Italy 4) expression profiling, revealing that the treatment induces a significant upregulation University of Florence, Experimental and Clinical Medicine, Florence, Italy 5) of genes encoding for tyrosine kinases belonging to the Src family (SFKs), which University of Florence, Experimental and Clinical Medicine, Florence, Italy 6) are involved in T cell receptor (TCR) signalling. Subsequent western blot analyses Fujifilm Visualsonics Inc., Toronto, Canada 7)University of Leiden, Physics of Life confirmed that SFKs are activated upon CHOEP treatment, suggesting the rationale Processes- Huygens-Kamerlingh Onnes Laboratory, Leiden, The Netherlands 8) of combining a tyrosine kinase inhibitor to CHOEP, to potentiate its activity. For University of Florence, Experimental and Clinical Medicine, Florence, Italy 9) this, the pan-tyrosine kinase inhibitor dasatinib was titrated and suboptimal University of Florence, Experimental and Clinical Medicine, Florence, Italy concentrations (IC20) of CHOEP and dasatinib were used to assess the efficacy of the combination. Analyses performed revealed that addition of dasatinib to CHOEP Introduction significantly inhibits cell proliferation and the antiproliferative effect of the drug Among hindrances in cancer treatment, the lack of appropriate markers to be combination was supported by a significant increase in necrotic/apoptotic cell death exploited for targeted therapy, and the need of new potential drugs are two big associated with severe mitochondrial depolarization. The effects of the combination challenges.hERG1 potassium channels area novel class of oncological targets were further assessed in vivo, subcutaneously inoculating HDMAR-2 and OCI-Ly12 and one of the most intriguing aspects of their involvement in tumor establishment cell lines in NOD/SCID mice. Treatment with CHOEP+dasatinib strongly reduced and progression is the interaction with adhesion molecules, such as integrins. It tumor growth when compared to CHOEP and dasatinib administered alone. has been recently demonstrated that macromolecular complexes formed between Data presented provide the rationale for the addition of dasatinib to CHOEP and hERG1 and β1 integrins selectively occurs in many types of cancer (Becchetti A demonstrate that dasatinib could potentiate CHOEP efficacy in PTCLs irrespective et al., 2017). In this scenario, hERG1 could be exploited as a therapeutic target of histologies and of CHOEP sensitivity. providing non cardiotoxic strategies aimed at blocking hERG1. Materials and Methods A scDb, a bifunctional single-chain diabody, directed against hERG1/β1 L20 complex, was developed via SOE-PCR methodology. Such antibody was tested PLK1: A NEW THERAPEUTIC TARGET IN MUCINOUS OVARIAN CARCINOMA on HCT116 cells in lateral motility and western blotting experiments. Moreover Affatato R., Lupi M., Restelli V., Carrassa L., Damia G. immunohistochemistry (IHC) was performed on metastatic colorectal cancer Oncology, Istituto Mario Negri, Milan, Italy (mCRC) paraffin embedded samples using the scDb, an anti-hERG1 and an anti-β1 Ovarian mucinous carcinomas (mEOCs) represent a group of rare neoplasms. integrin. Moreover, we are investigating the antibody biodistribution and toxicity in Although these tumors are different from the other histological subtypes of epithelial vivo in atimic nude mice using optical imaging (PhotoImager, Biospace Lab) and ovarian neoplasms, they are still treated with a similar chemotherapeutic approach a novel developed photoacustic station, Ultrasound Imaging VevoLazr-x available (cis-platinum-paclitaxel combination),to which however they are generally poor in our laboratory. responsive. To find new therapeutic options for mEOC, we have performed a Results and Discussion high-throughput screening using a siRNA library directed against 719 human Performing IHC on sequential sections of mCRC confirmed the specificity of the scDb protein kinases in the mEOC cell line MCAS, resistant to cis-platinum. After 2 for both hERG1 and β1 integrin. In vitro data provide evidences that the administering independent screenings and validation procedures using specific esiRNAs, PLK1 of the bispecific antibody has an impact on lateral motility. Moreover, signaling (Polo-like kinase1), belonging to the family of mitotic serine/threonine kinases pathways are also affected by the antibody treatment, as AKT phosphorylation was identified as the most significant kinase whose downregulation interfered with and HIF1α levels are decreased when the molecule is administered. Such findings both proliferation and survival. Both PLK1 siRNA and nanomolar concentrations might suggest a possible effect of the bispecific antibody on the VEGF-A signaling of commercially available inhibitors against PLK1 inhibited cell growth in other two pathway, which are consistent with our previous hypothesis (Becchetti A et al., additional mEOC cell lines (EFO-27 and JHOM-1). In addition, in all three mEOC 2017) of a possible cross-talk leading to a deep impact on VEGF expression and, cell lines PLK1 inhibition was able to induce apoptosis and to block cells in the G2-M thus, on neoangiogenesis. phase of the cell cycle. We then evaluated in vitro combinations between the PLK1 Conclusions inhibitors and standard chemotherapeutic drugs used in mEOC, i.e. cis-platinum scDb-hERG1/β1 could be used as a potential new treatment for cancer patients and and paclitaxel. We found that the antimitotic drug paclitaxel, but not cis-platinum as an early molecular diagnostic marker. In fact, the selective expression of hERG1/ synergized with PLK1 inhibitors in MCAS cell line. A similar synergistic effect was β1 complex in cancer cells and its role in angiogenesis and cancer progression observed combining with another antimitotic drug, eribulin, which acts by blocking suggests that a molecule selectively targeting the complex will be an invaluable tool microtubule polymerization. These drug combinations are being evaluated in the for cancer treatment. other two mEOC cell lines. Further investigation aiming at better understanding the molecular mechanisms at the basis of these drug combinations is undergoing and will hopefully provide new and effective drug combinations to test in clinical setting in mEOC patients whose outcome is still very poor.
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L21 molecular patterns (DAMPs) such as: HMGB-1, Calreticulin, IL1β, IL6 (by WB), Palbociclib, a CDK4/6 inhibitor, as a new strategy of treatment PGE2 (by ELISA), ATP (ATP assay). in Triple Negative Breast Cancer, Malignant Pleural In conclusion, we found that COPZ1 depletion boosts a complex intracellular Mesothelioma, and Hepatocellular Carcinoma program, that finally converges in the production of several inflammatory molecules Cretella D., Bonelli M., Digiacomo G., Fumarola C., Ravelli A., Petronini P.G. that could modulate the microenvironment response. Dept. of Medicine and Surgery, Lab. of Experimental Oncology, University of Parma, Italy L23 Background: Palbociclib is a reversible CDK4/6 inhibitor that acts by inhibiting the Citron kinase inactivation inhibits medulloblastoma transition from G1 to S phase of the cell cycle. Cell sensitivity to palbociclib can progression by inducing apoptosis and cell senescence be predicted by the presence of a functional Rb pathway. Given the successes 1)Pallavicini G. 2)Sgrò F. 3)Garello F. 4)Falcone M. 5)Bitonto V. 6)Berto G.E. 7) obtained with palbocicib in treating ER-positive breast cancer, we evaluated its Bianchi F.T. 8)Gai M. 9)Chiotto A.M.A. 10)Filippi M. 11)Cutrin J.C. 12)Terreno E. 13) efficacy in aggressive tumors with poor prognosis that require the development of Turco E. 14)Di Cunto F. novel therapeutic strategies. 1)Department of Molecular Biotechnology and Health Sciences, University of Turin, Methods: Cell proliferation/viability assays, RT-PCR, Western blotting, glucose Italy 2)Department of Molecular Biotechnology and Health Sciences, University uptake, glycolysis and lactate production were performed in a panel of Triple of Turin, Italy 3)Department of Molecular Biotechnology and Health Sciences, Negative Breast Cancer (TNBC), Malignant Pleural Mesothelioma (MPM), and University of Turin, Italy 4)Division of Stem Cells and Cancer, German Cancer Hepatocellular Carcinoma (HCC) cell lines exposed to palbociclib and PI3K Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany 5) inhibitors alone or combined with different schedules. Department of Molecular Biotechnology and Health Sciences, University of Turin, Results: Palbociclib inhibited cell proliferation only in TNBC, MPM, and HCC cell Italy 6)Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy 7) lines with a functional Rb signaling. In particular, palbociclib induced cell cycle Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy 8)Department arrest in G0/G1 phase and consequently down-regulated p-Rb, Rb and p-CDK6, of Molecular Biotechnology and Health Sciences, University of Turin, Italy 9) while up-regulating cyclin-D1, both in dose- and time-dependent manner. Then, Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy 10)Department we evaluated the effects of palbociclib on cell energy metabolism both in normoxic of Molecular Biotechnology and Health Sciences, University of Turin, Italy 11) and hypoxic conditions. Palbociclib was able to down-regulate hypoxia-mediated Department of Molecular Biotechnology and Health Sciences, University of Turin, induction of HIF-1α and GLUT-1 expression as well as glucose uptake and glucose Italy 12)Department of Molecular Biotechnology and Health Sciences, University consumption. These inhibitory effects were presumably associated with palbociclib- of Turin, Italy 13)Department of Molecular Biotechnology and Health Sciences, mediated down-regulation of c-myc protein. In addition, considering that treatment University of Turin, Italy 14)Neuroscience Institute Cavalieri Ottolenghi, University with palbociclib induced AKT activation in all the cell models, the combination of Turin, Italy of palbociclib with PI3K/AKT inhibitors (BYL719 or BEZ235) showed additive or synergistic effects depending on the cell line and the schedule of treatment. Medulloblastoma (MB) is the most common malignant brain tumor in children. Such combinations were more effective in impairing glucose metabolism than the Current treatment for MB, consisting of surgery followed by irradiation of the whole individual treatments under both normoxic and hypoxic conditions. neuraxis and high-dose multi-agent chemotherapy, is only partially effective and Conclusions: Impairment of glucose metabolism may contribute to the anti-cancer is associated with highly invalidating side effects. Therefore, the identification and activity of palbociclib; combining palbociclib with PI3K inhibitors enhanced this effect validation of novel target molecules, capable of contrasting MB growth without and may represent a promising strategy for the treatment of TNBC, MPM, and HCC disturbing brain development, is needed. The Citron kinase protein (CITK), encoded cancers. by primary microcephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability and apoptosis in developing brain, but has limited effects L22 on other tissues. On this basis, we hypothesized that CITK could be an effective Dissection of the paracrine effects of cell death induced by target for MB treatment. We tested this hypothesis using the MB cell lines DAOY COPZ1 targeting in thyroid tumor cells and ONS-76. In these cells CITK knockdown increases both cytokinesis failure and 1)Di Marco T. 2)Todoerti K. 3)Pagliardini S. 4)Neri A. 5)Anania M.C. 6)Greco A. DNA damage, impairing proliferation and inducing cell senescence and apoptosis 1)Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Fondazione via TP53 or TP73. Similar effects were obtained in MB arising in the NeuroD-SmoA1 IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, and Department of Oncology transgenic mouse model, in which CITK deletion increases apoptotic cells and and Hemato-oncology, University of Milan, Milan, Italy 3)Fondazione IRCCS Istituto senescence markers, such as P21CIP1, P27KIP1 and P16INK4A. Most importantly, Nazionale dei Tumori, Milan, Italy 4)Fondazione IRCCS Ca’ Granda, Ospedale CITK deletion decreases tumor growth and increases overall survival in these mice, Maggiore Policlinico, and Department of Oncology and Hemato-oncology, with no apparent side effects. These results suggest that CITK can be a useful University of Milan, Milan, Italy 5)Fondazione IRCCS Istituto Nazionale dei Tumori, therapeutic molecular target for MB treatment. Milan, Italy 6)Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Thyroid carcinoma (TC) is generally associated with good prognosis, nevertheless L24 no effective treatments are available for aggressive forms refractory to current Resveratrol acts as a SGK1-Kinase inhibitor in vitro and in vivo, therapies. We previously identified the coatomer protein complex ζ1 (COPZ1) leading an anticancer effect in hepatocarcinoma cell lines (involved in ER-Golgi secretory pathway, endosome maturation, autophagy and 1)Catalogna G. 2)D’antona L. 3)Muraca F. 4)Dattilo V. 5)Perrotti G. 6)Ortuso F. 7) lipid homeostasis) as a new putative therapeutic target, since: i) it is required for the Iuliano R. 8)Trapasso F. 9)Alcaro S. 10)Amato R. 11)Perrotti N. growth of different TC cell lines but not for that of normal thyrocytes; ii) its depletion 1)Department of “Health Sciences”, University “Magna Graecia” of Catanzaro, leads to abortive autophagy, ER stress, unfolded protein response and apoptosis; Catanzaro, Italy 2)Department of “Health Sciences”, University “Magna Graecia” of iii) the local treatment with siRNA oligos targeting COPZ1 reduces the tumor growth Catanzaro, Catanzaro, Italy 3)Department of “Health Sciences”, University “Magna of TC xenograft models. Graecia” of Catanzaro,, Catanzaro, Italy 4)Department of “Health Sciences”, To identify other mediators that are activated upon COPZ1 silencing and that University “Magna Graecia” of Catanzaro, Catanzaro, Italy 5)Department of “Health contribute to cell death, we performed a gene expression profiling of COPZ1- Sciences”, University “Magna Graecia” of Catanzaro, Catanzaro, Italy 6)Department depleted TPC-1 and 8505C cells (papillary and anaplastic thyroid tumor cells, of “Health Sciences”, University “Magna Graecia” of Catanzaro, Catanzaro, Italy respectively), 72h after siRNA transfection, by using Affymetrix Gene ST 2.0 array. 7)Department of “Health Sciences”, University “Magna Graecia” of Catanzaro, Functional annotation analyses by means of DAVID 6.8 bioinformatic tool and Gene Catanzaro, Italy8)Department of “Experimental and Clinical Medicine”, University Set Enrichment Analysis were performed. We found 227 and 321 genes specifically “Magna Graecia” of Catanzaro, Catanzaro, Italy 9)Department of “Health Sciences”, modulated in COPZ1-depleted TPC-1 and 8505C cells, respectively. The main University “Magna Graecia” of Catanzaro, Catanzaro, Italy 10)Department of altered functional categories dealt with cell cycle and cell death, protein transport “Health Sciences”, University “Magna Graecia” of Catanzaro,Catanzaro, Italy 11) and metabolism. Specifically, we found the up regulation of gene sets associated Department of “Health Sciences”, University “Magna Graecia” of Catanzaro, with RNA metabolism (involved in spliceosome and RNA processing) and interferon Catanzaro, Italy pathway (viral mimicry response). Real time PCR confirmed the up regulation of genes involved in the Interferon I pathway and viral mimicry response (ISG15, IRF7, The SGK1 kinase plays important roles in insulin and steroid signal transduction IFIT1, MX1, USP18, DDX58, STAT1, OAS1), and in inflammation (COX-2, IDO1, pathways operating in sodium retention, metabolism and cell transformation. IL1α, IL1β, IL8). Moreover, by western blot (WB) analysis, we found that some We recently described the biologic effects of a SGK1 kinase inhibitor, SI113, of the corresponding proteins were overexpressed in total protein extracts or in characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold. SI113 was able the conditioned media derived from COPZ-1 depleted cells. We then investigated to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. whether COPZ1 depletion is associated with immunogenic cell death. Interestingly, Insulin resistance, obesity and type 2 diabetes are associated with and incresed we found that COPZ1-depleted cells overexpress some damage-associated risk of cancer, in different circumstances caloric restriction protects from the risk of
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cancer. mesva, University of L’Aquila, L’Aquila, Italy 14), Mediapharma srl, Chieti, Italy 15), Resveratrol is a naturally occurring polyphenol that mimics caloric restriction with Centro de investigación del cáncer, Salamanca, Salamanca, Spain possible beneficial effects in cancer prevention. Based on a series of docking experiments carried out on both the wild-type and The HER-3 receptor is emerging as an attractive therapeutic target because of its phosphorylated forms of SGK1 we demonstrated the molecular recognition of trans overexpression across many cancers and its role in several compensatory processes resveratrol by SGK1. Moreover we demonstrated that resveratrol inhibits SGK1 leading to resistance to anticancer drugs. In this study, we present evidence of the kinase activity in vitro and in intact cells, affecting proliferation and survival of HUH7 preclinical efficacy of novel Antibody-Drug Conjugates (ADC) s targeting HER-3. human hepatoma cancer cells. EV20 is a humanized anti-HER-3 monoclonal antibody developed by our group, The scientific interest in the pharmacology of plant-derived natural product has been which is rapidly and efficiently internalized by HER3+ cancer cells. We have increasing in recent years. generated a panel of EV20-based ADCs using different payloads, including tubulin Our findings demonstrate a novel molecular mechanism for resveratrol to function polymerization inhibitors (maytansine derivative DM3 and monomethyl auristatin F) as a SGK1 inhibitor, suggesting possible applications in the prevention sodium and DNA minor groove binding alkylating agents (duocarmycin derivative). Cytotoxic retention and cancer. agents were coupled to EV20 using both random and site-specific conjugations by means of cleavable or not cleavable linkers. We describe here the potent and specific in vitro and in vivo therapeutic activity of L25 these EV20-based ADCs in several HER-3+ tumor models, including BRAF V600E NUCLEAR SPHINGOSINE KINASE 1 (SPHK1): A MULTIFACETED ROLE IN melanoma, trastuzumab and T-DM1 resistant breast cancer and hepatocellular LUNG CANCER carcinoma. Also, the results of pharmacokinetic, biodistribution and safety profile Napolitano F., Rosa R., D’Amato V., Di Mauro C., Formisano L., Orsini R.C, Servetto studies are reported. A., Santaniello A., Ciciola P., Marciano R., De Placido S., Bianco R. In summary, our study validates HER-3 as an attractive therapeutic target in multiple Dipartimento ad Attività Integrata di Ematologia, Oncologia, Anatomia Patologica e solid tumors and supports further clinical development and application of EV20- Diagnostica per Immagini, Università degli Studi di Napoli “Federico II”, Napoli, Italy based ADC for anti-HER-3 therapy. Background: Sphingosine kinases (SphKs) are enzymes catalyzing the formation of sphingosine- L27 1-phosphate (S1P), a bioactive lipid messenger, through phosphorylation of NOTCH-TARGETED THERAPEUTIC STRATEGY IN MULTIPLE MYELOMA sphingosine. SphK exists in two isoforms: SphK1, with a cytosolic distribution, and BASED ON SMALL MOLECULES HAMPERING RECEPTOR-LIGAND SphK2, known to locate predominantly in the nucleus. Hyperactivation of SphKs INTERACTION is involved in tumor growth and progression through regulation of cell proliferation 1)Platonova N. 2)Parravicini C. 3)Palazzolo L. 4)Saporiti S. 5)Colombo M. 6) and apoptosis. Vallelonga V. 7)Giannandrea D. 8)Giana D. 9)Neri A. 10)Eberini I. 11)Chiaramonte R. Methods: 1)Department of Health Sciences, University of Milano, Milan, Italy 2)Department We studied the expression, the intracellular localization and the activity of SphK1 of Pharmacological and Biomolecular Sciences, University of Milano, Milan, Italy 3) and SphK2 in non-small cell lung cancer (NSCLC) cell lines and patients’ samples. Department of Pharmacological and Biomolecular Sciences, University of Milano, Results: Milan, Italy 4)Department of Pharmacological and Biomolecular Sciences, University In NSCLC cells, SphK1 was found in the cytosol, while SphK2 showed a nuclear of Milano, Milan, Italy 5)Department of Health Sciences, University of Milano, localization. Surprisingly, we observed SphK1 expression also in the nuclear Milan, Italy 6)Department of Health Sciences, University of Milano, Milan, Italy 7) compartment. In the majority of the tested cell lines, SphK1 expression was even Department of Health Sciences, University of Milano, Milan, Italy 8)Department of higher in the nucleus compared to the cytosol. Consistently, in a NSCLC patients’ Health Sciences, University of Milano, Milan, Italy 9)Department of Oncology and derived tissues microarray, SphK1 nuclear expression was detected in a wide Hemato-oncology, University of Milano; Unit of Hematology, Fondazione Cà Granda number of samples. Based on these data, we hypothesized the presence of a IRCCS Policlinico, Milano 10)Department of Pharmacological and Biomolecular nuclear import signal in the SphK1 sequence, probably recognized by Karyopherin Sciences, University of Milano, Milan, Italy 11)Department of Health Sciences, beta2. We found, in the aminoacidic sequence of SphK1, a R/K/H-X(5)-P-Y University of Milano, Milan, Italy C-terminal motif within a structurally disordered and positively charged regions of about 30 amino acids, compatible with a nuclear import signal. In order to dissect Multiple myeloma (MM) is an incurable hematological cancer characterized by MM the role of nuclear SphK1, specific inhibitors of SphKs were tested. Among them, cells accumulation in the bone marrow (BM) where they interact and shape the fingolimod (FTY720), an oral drug initially defined as a S1P receptors antagonist nearby BM milieu, resulting in tumor progression, acquisition of drug resistance but also acting as a SphK1 inhibitor, is effective in NSCLC cell lines: a reduction and consequent patient’s relapse. Despite recent advances, poor clinical response of cell density of about 50% is obtained with a drug concentration of about 2.5 and relapse remain major problems. The oncogenic Notch signaling consists of 4 µM. Interestingly, fingolimod seems to be more effective in cells with a nuclear/ receptors (Notch1-4) and 5 ligands (Jag1,2 and Dll1,3,4) and plays a crucial role cytosolic ratio of SphK1 expression > 1. Moreover, at a 5-years follow up, we found in MM. In particular, aberrant Notch2 activation and Jag1 and 2 overexpression that NSCLC patients with higher expression of nuclear SphK1 had a better overall stimulate MM cells to establish pathological interactions with BM that trigger MM survival. Lastly, we are now evaluating the epigenetic regulation of several genes progression. Our previous data showed that these effects can be interfered by mediated by SphKs: this is a matter under investigation. knocking down of Jag1 and 2 expression. Conclusions: Currently, indirect approaches to inhibit Notch signaling are mainly based on inhibition We demonstrate for the first time that SphK1 has a nuclear localization in NSCLC of g-Secretase that catalyzes Notch activation along with other several g-Secretase and we are now investigating its biological and epigenetic role in this context. substrates. Moreover, inhibition of all four Notch receptors is associated with gut Moreover, we propose nuclear SphK1 as a novel druggable target in human NSCLC toxicity that might be avoided by selectively blocking Notch signaling triggered by cells. Since fingolimod is a clinically available drug, our results may suggest SphK1 only one family of ligands, Jag or Dll ligands. targeting as a therapeutic opportunity to be tested in NSCLC patients. This evidence prompts us to develop a therapeutic tool to selectively inhibit Notch2 signaling triggered by Jag1 and 2. We applied in silico protein-protein docking and virtual high-throughput screening L26 (HTS) of a chemoteque to select druglike small molecules. The biological activity EV20-based Antibody-Drug Conjugates display potent and was validated by a Notch responsive reporter assay and co-culture assay that allow target-dependent therapeutic activity in HER-3+ human cancers to measure Notch2 transcriptional activity induced either by Jag or Dll ligands. 1)Capone EC. 2)Gandullo LG. 3)D’agostino DD. 4)Diaz-Rodriguez ER. 5)Lamolinara Based on previous setup of integrated in silico pipeline, we applied a strategy to AL. 6)Iezzi MI. 7)Ponziani SP. 8)Gentile RG. 9)Giansanti FG. 10)Dituri FD. 11) exclusively uncouple Notch2::Jag1/2, leaving unaltered the interaction with Dll. 100 Giannelli GG. 12)De Laurenzi VD. 13)Ippoliti RI. 14)Iacobelli SI. 15)Pandiella AP. top-scoring compounds directed exclusively to Notch2::Jag2 surface were selected 1)Medical, oral and biotechnological sciences, University of Chieti-Pescara, Chieti, by HTS of the chemoteque. 2 of 100 compounds were validated in vitro by the Italy 2), Centro de investigación del cáncer, Salamanca, Salamanca, Spain 3) Notch responsive reporter assay on HEK293T cells and showed a significant reduce Medical, oral and biotechnological sciences, University of Chieti-Pescara, Chieti, in Notch transcriptional activity. The co-culture assay of Hela cells overexpressing Italy 4), Centro de investigación del cáncer, salamanca, Salamanca, Spain 5) Notch2 with NIH3T3 overexpressing Jag1 or Dll4 ligands identified one promising Medicine and aging cesi-met, University of Chieti-Pescara, Chieti, Italy 6)Medicine compound that specifically inhibited Notch2::Jag1 and not Notch2::Dll4 interactions. and aging cesi-met, University of Chieti-Pescara, Chieti, Italy 7)department mesva, Overall, our results showed that the identified compounds can selectively antagonize University of L’Aquila, L’Aquila, Italy 8)4department mesva, University of L’Aquila, Notch activation. This lays a basis for an effective and safe Notch-directed anti- L’Aquila, Italy 9)4department mesva, University of L’Aquila, L’Aquila, Italy 10)S. De tumor therapy in MM and other Notch-dependent tumors. Bellis” research hospital,, S. De Bellis” research hospital,, Bari, Italy 11)S. De Bellis” research hospital,, S. De Bellis” Research hospital,, Bari, Italy 12)Medical, oral and biotechnological sciences, University of Chieti-Pescara, Chieti, Italy 13)4department
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L28 L30 miR-199a-3p Modulates MTOR and PAK4 Pathways and Inhibits Development of novel FGF trap small molecules for the Tumor Growth in a Hepatocellular Carcinoma Transgenic treatment of FGF-dependent cancers Mouse Model 1)Taranto S. 2)Giacomini A. 3)Castelli R. 4)Spadoni G. 5)Matarazzo S. 6)Foglio E. 1)Callegari E. 2)D’Abundo L. 3)Guerriero P. 4)Simioni C. 5)Bassi C. 6)Gramantieri 7)Maccarinelli F. 8)Ronca R. 9)Mor M. 10)Presta M. L., 7)Neri L.M. 8)Sabbioni S. 9)Negrini M. 1)Department of Molecular and Translational Medicine, University of Brescia, 1)Morphology, surgery and experimental medicine, University of Ferrara, Ferrara, Brescia, Italy 2)Department of Molecular and Translational Medicine, University Italy 2)Morphology, surgery and experimental medicine, University of Ferrara, of Brescia, Brescia, Italy 3)Department of Food and Drug, University of Parma, Ferrara, Italy 3)Morphology, surgery and experimental medicine, University of Italy 4)Department of Biomolecular Sciences, University of Urbino, Urbino, Italy 5) Ferrara, Ferrara, Italy 4)Morphology, surgery and experimental medicine, University Department of Molecular and Translational Medicine, University of Brescia, Brescia, of Ferrara, Ferrara, Italy 5)Morphology, surgery and experimental medicine, Italy 6)Department of Molecular and Translational Medicine, University of Brescia, University of Ferrara, Ferrara, Italy 6)Center for applied biomedical research, St. Brescia, Italy 7)Department of Molecular and Translational Medicine, University Orsola-Malpighi university hospital, Bologna, Italy 7)Morphology, surgery and of Brescia, Brescia, Italy 8)Department of Molecular and Translational Medicine, experimental medicine, University of Ferrara, Ferrara, Italy 8)Life sciences and University of Brescia, Brescia, Italy 9)Department of Food and Drug, University of biotechnology, University of Ferrara, Ferrara, Italy 9)Morphology, surgery and Parma, Italy 10)Department of Molecular and Translational Medicine, University of experimental medicine, University of Ferrara, Ferrara, Italy Brescia, Brescia, Italy Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related The FGF/FGFR system plays a key role in tumor/stroma compartments of different death worldwide. Prognosis is poor, and therapeutic options are limited. MicroRNAs tumors, thus representing a major target for the development of novel anti- (miRNAs) have emerged as potential therapeutic molecules against cancer. Here, tumor drugs. The soluble pattern recognition receptor long pentraxin-3 (PTX3) is we investigated the therapeutic efficacy of miR-199a-3p, an miRNA highly expressed a natural FGF antagonist able to inhibit the growth and vascularization of FGF- in normal liver and downregulated in virtually all HCCs. The therapeutic value dependent cancers. Molecular characterization of FGF2/PTX3 interaction allowed ofmiR-199a-3p mimic molecules was assayed in the TG221 mouse, a transgenic the identification of the first orally active low molecular weight FGF trap (NSC12) model highly predisposed to the development of liver cancer. Administration of endowed with a significant anti-tumor activity in various FGF-dependent tumor miR-199a-3p mimics in the TG221 transgenic mouse showing liver cancer led to models. a significant reduction of number and size of tumor nodules compared to control NSC12 structure consists of a steroid backbone linked to a hexafluoroisopropanol animals. In vivo delivery confirmed protein downregulation of the miR-199a-3p direct group. In order to better characterize the structure/function relationship of NSC12 targets, mechanistic target of rapamycin (MTOR) and p21 activated kinase 4 (PAK4), and to improve its potency and physicochemical properties, chemical derivatives ultimately leading to the repression of FOXM1. Remarkably, the anti-tumor activity of were generated and tested on the FGFR3-overexpressing human multiple myeloma miR-199a-3p mimics was comparable to that obtained with sorafenib. These results cell line KMS-11. suggested that miR-199a-3p may be considered a promising HCC therapeutic option. Cell proliferation and FGFR phosphorylation assays identified four active derivatives out of the 27 molecules tested, modification of the hexafluoroisopropanol group causing the loss of antitumor activity of the compound. Similar to NSC12, active L29 derivatives blocked the proliferation of KMS-11 cells with an IC50 @ 3 µM, whereas Blockage of beta adrenergic receptors as a new strategy in they appeared to be more effective in inhibiting FGFR phosphorylation in KMS-11 combined treatment with Trabectedin cells. Notably, only one of them prevented the formation of the bioactive HSPG/ 1)Di Fonte R. 2)Garofoli M. 3)Mazzotta A. 4)Iacobazzi R. M. 5)Guida M. 6)Strippoli FGF2/FGFR ternary complex in a FGF2-dependent “cell-cell adhesion assay” better S. 7)Azzariti A. 8)Porcelli L. than NSC12, thus representing our lead compound. Accordingly, when compared 1)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy to NSC12, intraperitoneal delivery of the lead compound appeared to be more 2)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy effective in reducing the growth of KMS-11 xenografts in immunodeficient mice. 3)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy Altogether, these results define the pivotal role of the hexafluoroisopropanol group 4)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, in mediating the FGF trap activity of NSC12 and identify a new FGF trap lead Italy 5)Medical Oncology Unit, Istituto Tumori “Giovanni Paolo II”, Bari, Italy 6)Medical compound with improved efficacy in vivo. Oncology Unit, Ospedale Mons. R. Dimiccoli, Barletta, Bat, Italy 7)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy 8)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy L31 FGF/PTX3 cross talk in bladder cancer: novel prognostic and The investigation on enhancing the response to chemotherapy, through the selective therapeutic implications inhibition of β-adrenergic receptors (β-ARs) relies on recent reports demonstrating Matarazzo S., Maccarinelli F., Ghedini G.C., Melocchi L., Bugatti M., Vermi W., that adrenergic stimulation impairs the response to several chemotherapeutics; Presta M., Ronca R. whereas therapeutic targeting of β-ARs has recently shown remarkable efficacy in Department of Molecular and Translational Medicine, University of Brescia, Brescia, combination with vinblastine in the treatment of angiosarcoma patients. β-adrenergic Italy receptors are expressed on tumor and stromal cells in diverse cancers. Upon activation by catecholamines from local sympathetic nerve fibers (norepinephrine) Bladder cancer is one of the most common cancers in the world. Although most adrenergic receptors trigger signaling pathways that regulate multiple cellular of urothelial carcinomas (UCs) are low-grade papillary tumors, their propensity to processes that contribute to the initiation and progression of cancer. The aim of recur and to progress to become invasive with a poor survival rate represents a our study was to evaluate whether β-blokade with propranolol in combination with major challenge. trabectedin (Yondelis - PharmaMar) resulted in increased effectiveness of the Different members of the Fibroblast growth factor (FGF) family are expressed by chemotherapeutic agent in the treatment of ovarian cancer, leyomiosarcoma and human bladder cancer cells and aberrant expression of FGF receptors (FGFRs) is liposarcoma, all currently treated with trabectedin as second line treatment option. a typical feature of bladder cancer compared to normal urothelium. High expression To this purpose 2D and 3D models obtained by ovarian cancer cells A2780 CP, levels of FGFR1 are associated with poor survival and FGFR3 is frequently Caov-3 and SK-OV-3, SK-LMS-1 vulva leiomyosarcoma and SW872 connective dysregulated in UC. Thus, the FGF/FGFR system may represent a promising liposarcoma were treated with trabectedin and propranolol alone or in combination therapeutic target in invasive and non-invasive bladder cancer. and cytotoxicity was assayed by MTT and CCK-8 assays. Apoptosis induction was Long-pentraxin 3 (PTX3) acts as a natural FGF-trap by binding FGFs and hampering determined by staining cells with Annexin V/PI, followed by flow cytometry analysis. their biological activity in various tumor models. Cell targets modulation, was evaluated by western blotting and real time PCR. Preliminary observations have shown that low grade UCs express PTX3 while Protein localization was evaluated by ICC and IF. All analysis were carried out with high grade/invasive UCs lose PTX3 expression. Accordingly, bladder cancer cell or without 10 µM norepinephrine (NE). Results demonstrated that β-adrenergic lines share an overall presence of FGFRs and FGFs but express different levels activation by NE reduced the effectiveness of trabectedin in all cells; whereas of PTX3 in vitro and in vivo. Of note, high grade/invasive cells express very low or blockage by propranolol resulted in the recovery of cytotoxicity and apoptogenic undetectable levels of PTX3, whereas low grade, papilloma-like cells express high potential of trabectedin. The evaluation of underlying mechanisms of combination levels of PTX3. Indeed, invasive bladder cancer cell xenografts grow faster in nude effectiveness in ovarian cancer, pointed out the induction of COX-2 expression, that mice and express much lower levels of PTX3 than low grade-derived lesions, thus localized into the nucleus of the cells and the simultaneous increase of p-erk1/2 and confirming an inverse correlation between UC grade and PTX3 expression. p-p53(Ser15) expression level, regarded as mechanisms of apoptosis induction. In This hypothesis is reinforced by a preliminary case study performed on a small conclusion, blockage of β-adrenergic receptors enhances trabectedin effectiveness cohort of biopsies from UC patients confirming that the presence of PTX3 isa in ovarian cancer, liposarcoma and leyomiosarcoma cell models. Further evaluation common feature of low grade samples while PTX3 is poorly expressed or absent in of underlying mechanisms of combination effectiveness are warranted. aggressive/invasive cases. These data point to PTX3 as a natural FGF-trap that could play a role in modulating
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bladder cancer progression and invasiveness and indicate that inhibition of the FGF/FGFR system by natural and synthetic FGF traps may represent a promising ONCOGENES AND target for the therapy of UCs. TUMOR SUPPRESSOR GENES L32 M1 IMPLICATION FOR BREAST CANCER TREATMENT OF CDK4/6 INHIBITION mir-140-5p functions as a tumor suppressor in HER2+ breast AND MODULATION OF THE CROSSTALK BETWEEN THE ESTROGEN cancer RECEPTOR AND ERBB RECEPTORS 1)Pala V. 2)Di Cosimo S. 3)Zaffaroni N. 4)Daidone M.G. 5)Appierto V. 1)Viganò L. 2)Locatelli A. 3)Zambetti M. 4)Zambelli S. 5)Zucchinelli P. 6)Bianchini 1)Biomarker Unit, Department of Applied Research and Technological Development, G. 7)Ulisse A. 8)Sica L. 9)Daniele T. 10)Gianni L. Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Biomarker Unit, 1)Medical oncology, Irccs ospedale San Raffaele, Milano, Italy 2)Medical oncology, Department of Applied Research and Technological Development, Fondazione Irccs ospedale San Raffaele, Milano, Italy 3)Medical oncology, Irccs ospedale San IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Molecular Pharmacology Unit, Raffaele, Milano, Italy 4)Medical oncology, Irccs ospedale San Raffaele, Milano, Italy Department of Applied Research and Technological Development, Fondazione 5)Medical oncology, Irccs ospedale San Raffaele, Milano, Italy 6)Medical oncology, IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Biomarker Unit, Department Irccs ospedale San Raffaele, Milano, Italy 7)Medical oncology, Irccs ospedale San of Applied Research and Technological Development, Fondazione IRCCS Istituto Raffaele, Milano, Italy 8)Medical oncology, Rccs ospedale San Raffaele, Milano, Nazionale dei Tumori, Milan, Italy 5)Biomarker Unit, Department of Applied Italy 9)Centro imaging sperimentale, Rccs ospedale San Raffaele, Milano, Italy 10) Research and Technological Development, Fondazione IRCCS Istituto Nazionale Medical oncology, Rccs ospedale San Raffaele, Milano, Italy dei Tumori, Milan, Italy In many subtypes of breast cancer (BC) growth and progression are governed by MicroRNAs (miRNAs) are small non coding RNAs that regulate gene expression a complex interaction between estrogen, progesterone receptors (ER, PR) and at post transcriptional level. They play a significant role in biological processes and tyrosine kinase receptors (RTKs) of the erbB family. ER+/HER2+ BC has relatively dysregulation of miRNAs is involved in many diseases including cancer. Several low sensitivity to ER-directed therapies, and the combination of endocrine therapy studies have proved the presence of miRNAs in plasma and serum samples and and anti-HER2 drugs only leads to modest improvement of therapeutic results have highlighted the potential of circulating miRNAs as biomarkers for cancer (Kaufman JCO 2009), suggesting that the disruption of the HER2/ERα cross-talk diagnosis and prognosis. In our laboratory the role of circulating miRNAs as non- is insufficient or is bypassed by alternative pathways of tumor survival. Mitogenic invasive biomarkers to predict the efficacy of neoadjuvant anti-HER2 therapy was signals driven by RTKs and hormonal receptors (HR) converge to the Cyclin-D1/ explored in HER2+ breast cancer patients. Specifically, we analyzed in terms of CDK4/6 complex, which is a key regulator of the G1-S transition and cell cycle circulating miRNAs plasma samples collected from patients entering the NeoALTTO progression. In 5 BC cell lines characterized by different level of expression of HR trial, a randomized, multicenter phase III protocol that compared the efficacy of and HER2 we investigated a triple targeting through combination of an anti-ER drug neoadjuvant lapatinib and trastuzumab alone and in combination after 2 weeks (fulvestrant, F), a dual block of HER2 (trastuzumab and pertuzumab; H, Pert) and of therapy in HER2+ breast cancer patients. Through a RT-PCR-based high- a CDK4/6 inhibitor (palbociclib, P). The 3 ER+ cell lines with HER2 amplification throughput miRNA-profiling we identified a signature of 5 miRNAs able to predict (MDA-MB-361, BT474, ZR-75-30) had strongly heterogeneous drug sensitivity, pathological complete response in patients treated with trastuzumab. Notably, one of but in all of them the 4-drug PFHPert combination showed a more than additive the 5 miRNAs, mir-140-5p, also showed prognostic relevance on 7 years event-free cytotoxic effect (MTT assay). In the HER2- cell lines MCF7 and T47D PFHPert survival. Considering our findings and published studies suggesting an antitumor showed clear synergism according to the Bliss test (24 and 10% respectively). In activity of mir-140-5p in many malignancies, we decided to investigate the role of the above HER2low cells, 72h treatment with P led to a senescence-like morphology mir-140-5p in SKBR3 and BT474 cell lines, two cellular models of HER2+ breast (B-galactosidase test) that was reversible after drug removal. However, when P was cancer. By Real-time PCR mir-140-5p expression was analyzed upon trastuzumab combined with F and anti-HER2 antibodies the senescence-like phenotype became treatment and an increase was observed in treated compared to control cells. In irreversible and led to cells degeneration. No association with an increment of G0- addition, the effects on gene perturbation caused by miR-140-5p overexpression, G1 block, apoptosis or p27 protein was demonstrated. However treatment with F, obtained by miRNA transfection, were evaluated by gene set enrichment analysis. and even more P plus F, led to an increment of p-EGFR Tyr 845 (>100 fold) and A negative association was observed between miR-140-5p upregulation and three p-HER3 Tyr1289 (>2.5fold) suggesting that the block of HR induced an adaptive gene-sets: E2F targets, DNA repair and MYC targets, which are pathways involved in response with the activation of RTKs normally unused but inducible in MCF7 and DNA replication and proliferation. Accordingly, we observed that ectopic expression T47D cells. The findings are in line with the clinical results in the companion clinical of mir-140-5p caused a reduction of cell viability and an increase of trastuzumab NA-PHER2 study in ER+/HER2 amplified BC (Gianni L, Lancet Oncol 2018), in efficacy. Interestingly, preliminary results also indicated that mir-140-5p induces a which the no-chemotherapy regimen led to high pathological (pCR 27%) and clinical G0-G1 cell cycle-arrest and reduces cell migration and invasion. Collectively, our (OR 97%) responses. The results also suggest that addition of the anti-HER2 to P results support the hypothesis that mir-140-5p is endowed with oncosuppressor and F might also be useful in HER2low /ER+ BC. The hypothesis is being investigated activity in HER2+ breast cancer cells. in an extension of the NA-PHER2 study.
M2 INHIBITION OF BRAFV600E INDUCES NIS EXPRESSION IN THYROID CANCER CELL LINES Bonaldi E., Minna E., Rizzetti M.G., Mazzoni M., Greco A., Borrello M.G. Molecular Mechanisms Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Introduction. The incidence of thyroid cancer has continuously increased in last years. Most newly diagnosed thyroid tumors are papillary thyroid carcinomas (PTC), and BRAFV600E mutation represents the most frequently identified genetic lesion (62%). Most PTCs are effectively treated with surgical removal followed by radioactive iodine (RAI) therapy; nevertheless, a small fraction of patients display RAI resistance and have poor prognosis. These patients represent a clinical challenge as effective therapies are still lacking. The aberrant expression, localization and/or function of sodium iodide symporter (NIS), a membrane glycoprotein physiologically involved in the iodine uptake and thyroid hormones synthesis, have been identified as important factors for RAI resistance. Several studies have already reported the possibility of restoring NIS activity, and therefore RAI uptake, as novel therapeutic strategy for RAI-resistant tumors. Recent clinical trials with these so called “redifferentiation therapies” followed by RAI treatment showed promising results. In this study we tested the possibility of restoring NIS expression by pharmacological inhibition of BRAFV600E in a panel of thyroid cancer cell lines. Methods. Fourteen thyroid derived cell lines were subjected to quality control by STR profiling; BRAFV600E mutation was assessed by PCR followed by direct sequencing and by mutant allele-specific PCR amplification (MASA). The basal
64 65 ABSTRACT BOOK expression of NIS was evaluated by quantitative RT-PCR. Four thyroid cancer cell M4 lines harboring BRAFV600E, two with homozygous (BCPAP and 8505C) and two with PROLINE DEHYDROGENASE EXPRESSION, REGULATION AND FUNCTION heterozygous mutation (K1 and NIM), were selected and treated with increasing IN NON-SMALL CELL LUNG CARCINOMA doses of the BRAFV600E inhibitor Vemurafenib ranging from 1 to 15µM. Cell growth 1)Parnigoni A. 2)Grossi S. 3)D’Antona P., 4)Corbetta E. 5)Sanvito F. 6)Chiaravalli and NIS expression were evaluated in control and treated cells by crystal violet A.M. 7)Sessa F. 8)Campomenosi P. assay and qRT-PCR, respectively. 1)Department of Biotechnology and Life Sciences, DBSV, University of Insubria, Results. The tested cell lines have unique genetic profile and 7/14 harbor BRAFV600E Varese, Italy 2)Department of Biotechnology and Life Sciences, DBSV, University (2/7 homozygous and 5/7 heterozygous mutation). In all the treated cell lines of Insubria, Varese, Italy 3)Department of Biotechnology and Life Sciences, DBSV, pharmacological inhibition of BRAFV600E impairs cell growth at 48 hours after University of Insubria, Varese, Italy 4)Department of Biotechnology and Life treatment with greater extent in K1 and NIM1 cells, both expressing heterozygous Sciences, DBSV, University of Insubria, Varese, Italy 5)Department of Biotechnology BRAFV600E. In all treated cell lines NIS expression is induced in a dose-dependent and Life Sciences, DBSV, University of Insubria, Varese, Italy 6)Pathology Unit, manner at 24 and 48 hours after treatment. Ospedale di Circolo e Fondazione Macchi, Varese, Italy 7)Department of Medicine Conclusions. The in vitro inhibition of the BRAFV600E oncoprotein is associated with and Surgery, DMS, University of Insubria, Varese, Italy 8)The Protein Factory, restored expression of NIS. Centro Interuniversitario di Ricerca in Biotecnologie Proteiche, Politecnico di Milano, ICRM-CNR Milano and University of Insubria, Varese, Italy M3 Introduction. Non-Small Cell Lung Cancer (NSCLC) is one of the most frequent A NOVEL VEGFR2 MUTATION HAS ONCOGENIC POTENTIAL IN BRAF WILD- and deadliest cancers and comprises two main histotypes, adenocarcinoma (ADC) TYPE MELANOMA and squamocellular carcinoma (SCC). Identification of markers to better define the 1)Grillo E. 2)Di Somma M. 3)Corsini M. 4)Ravelli C. 5)Zammataro L. 6)Presta M. diagnosis, prognosis and therapeutic options of NSCLC is needed. We investigated 7)Mitola S. if proline dehydrogenase (PRODH), a mitochondrial flavoenzyme catalyzing the key 1)Dmmt, Università degli studi di Brescia, Brescia, Italy 2)Dmmt, Università degli step in proline degradation, and involved in the regulation of cell survival, autophagy studi di Brescia, Brescia, Italy 3)Dmmt, Università degli studi di Brescia, Brescia, and apoptosis, may be one such marker. Italy 4)Dmmt, Università degli studi di Brescia, Brescia, Italy 5)Dep of obstetrics, Materials and methods. We characterized PRODH expression in NSCLC by gynecology and reproductive sc., Yale university, New Haven, Usa 6)Dmmt, immunohistochemistry and qPCR and tested if there was correlation between Università degli studi di Brescia, Brescia, Italy 7)Dmmt, Università degli studi di expression of PRODH and clinical features of the tumors or expression of other Brescia, Brescia, Italy markers. Phenotypic assays were performed to investigate the cellular influenced by PRODH in lung ADC cell lines. We also tested the regulation of the PRODH gene Tyrosine kinase receptors (RTKs) play central roles in tumor progression and by TTF-1 by means of transfection experiments, expression analyses and luciferase represent an important class of drug targets. Nevertheless, clinical use of RTK reporter assays. inhibitors shows limited response and rapid resistance, which may be due to the Results and discussion. We found PRODH immunostaining in the majority (70%) of presence of cancer cells expressing mutant forms of RTKs. lung ADCs. Patients with PRODH positive tumors had better overall survival than Melanoma is an aggressive cancer which partially responds to existing therapies. those with negative tumors. Protein staining was paralleled by high transcript levels. Recently identified driver mutations (BRAFV600E) have allowed the development of Clonogenic assays showed that PRODH favors survival of ADC cells. Moreover, effective targeted inhibitors. However, 30% of melanoma tumors lack activating TTF-1, a transcription factor essential for thyroid and lung development and mutations in BRAF or other drivers. physiology, that shows similar expression pattern as PRODH was found to regulate VEGFR2 is a pro-angiogenic RTK which is also expressed in melanoma cells where PRODH expression. This prompted us to investigate if PRODH could be a target it is implicated in tumor progression. Nevertheless, the possibility that activating of TTF-1. Transfection of a TTF-1 expression construct into ADC cell lines led to an vegfr2 gene mutations drive BRAFWT melanoma progression and response to TK increase in PRODH transcript and luciferase reporter assays showed that a RE was inhibitors remains unexplored. indeed transactivated by TTF-1. In this study we used a new computational tool for non-synonymous low frequency Conclusion. Our data support a possible application of PRODH immunostaining mutation analysis (LowMACA) to simultaneously study the pattern of low frequency as a prognostic marker and warrant further research aimed to investigate PRODH mutations conserved in the TK domain of various RTKs (PFAM: 07714) in human transactivation by TTF-1 and the role of PRODH in the biology of NSCLC. cancer samples. Heterozygous mutation of position 236, located in the catalytic loop of the TK domain, was found in VEGFR2, FGFR4, FLT4, PDGFRA and TIE1 in lung, breast and skin cancer. Of note, mutation of position 236 of VEGFR2 showed to be mutually exclusive with BRAFV600E in melanoma patients. Functional characterization demonstrated that VEGFR2236 cannot be phosphorylated in Y1175 residue and has a reduced membrane lateral diffusion when compared to VEGFR2WT as it is expected for signaling receptors. In BRAFWT melanoma, when co-expressed with endogenous VEGFR2WT to mimic heterozygosity, mutated VEGFR2236 modulates multiple phospho-kinases involved in cell metabolism. Accordingly, VEGFR2236 makes melanoma cells metabolically more active when compared to cells expressing only wild-type receptor, which also reflects in a higher rate of ATP production. In vitro and in vivo analysis revealed that mutated VEGFR2236 confers to melanoma cells a strong pro-oncogenic phenotype and a reduced sensitivity to the multi-target TK inhibitor Linifanib. Here we demonstrate that mutated VEGFR2236 drives a pro-oncogenic phenotype in a BRAFWT melanoma. We speculate that VEGFR2236 modulates the activity of co- expressed VEGFR2WT to induce such phenotype. Our study suggests that screening for somatic VEGFR2 mutations could help to predict inter-individual differences in terms of tumor growth and response to TK therapy.
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PEDIATRIC CANCER Background. Patients with diffuse pontine gliomas (DIPG), a disease primarily of early school-aged children, show a brief history characterized by cranial nerve N1 deficits, pyramidal and cerebellar signs and symptoms. To date, radiotherapy is the Biological differences among pediatric and adolescent/young only treatment of choice for DIPG. With the aim of exploring add-on strategies, an adult (AYA) rhabdomyosarcomas observational pilot study was conducted at Fondazione IRCCS Istituto Nazionale 1)Gasparini P. 2)De Cecco L. 3)Orazio F. 4)Dugo M. 5)Boeri M. 6)Bergamaschi L. Tumori (Milan) to assess the efficacy in terms of objective response rate according 7)Bornaghi V. 8)Sensi ML. 9)Collini P. 10)Massimino M. 11)Casanova M. 12)Sozzi to the RECIST criteria of combining nimotuzumab and vinorelbine with radiation in G. 13)Ferrari A. newly-diagnosed DIPG. Twenty-five consecutive children (median age 7.4 years) 1)Department of Research, Tumor Genomics Unit, Fondazione IRCCS Istituto were enrolled and median PFS and OS were 8.5 and 15 months, respectively. Nazionale dei Tumori, Milano, Italy 2)Applied Research Department and Methods. With the aim to identify novel non-invasive biomarkers able to improve the Technological Development, Integrated Biology Platform, Fondazione IRCCS outcome prediction, serum specimens for 25 patients were collected at baseline, Istituto Nazionale dei Tumori, Milano, Italy 3)Department of Research, Tumor at each therapy administration and during follow-up. Hemolysis quality check was Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 4) performed by spectrophotometic methods and RNA was extracted using miRCURY Applied Research Department and Technological Development,Integrated Platform, RNA isolation kit (Exiqon). We used a high-throughput approach using Agilent Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 5)Department of platform and Human miRNA SureSelect 8x60K containing 2006 miRNAs annotated Research, Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumor, on miRBase19.0 to assess circulating miRNA expression. Milano, Italy 6)Department of Pediatric Oncology, Fondazione IRCCS Istituto Results. After quality check, only one patient was excluded for high hemolysis levels Nazionale dei Tumori, Italy 7)Department of Research, Tumor Genomics Unit, proving the feasibility of using blood specimens in DIPG patients. Primary data Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 8)Applied Research analysis at baseline yielded a matrix containing 330 detectable miRNA. Association Department and Technological Development, Integrated Biology Platform, with PFS allowed us to disclose a signature of 10 miRNAs able to stratify patients Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 9)Soft tissues and with high and low risk of progression (HR=4.33, 95%CI 1.49-12.54; p=4.27E-05) bone, and pediatric pathology unit, Fondazione IRCCS Istituto Nazionale dei Tumori, and independent of confounding factors (i.e. age at diagnosis and gender). Italy 10) Department of Pediatric Oncology, Fondazione IRCCS Istituto Nazionale Subsequently, miRNA expression was evaluated in specimens collected at different dei Tumori, Italy 11) Department of Pediatric Oncology, Fondazione IRCCS Istituto time points until tumor progression or for two years after tumor diagnosis. A model Nazionale dei Tumori, Italy 12) Department of Research, Tumor Genomics Unit, reporting miRNA expression and risk of progression related to the longitudinal time Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 13)Department of points was build and underlies the incremental boosting strategy of radiotherapy Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Italy along with the maintenance frame of targeted agents. Conclusion. We report a circulating miRNA signature independent on analytical Background. Adolescents and young adults with rhabdomyosarcoma (RMS) form confounding variables suggesting an appropriate performance in assessing the a subgroup of patients whose optimal clinical management and best possible outcome status of DIPG patients. Due to the small sample size in the discovery access to care remain a challenge and survival lacks behind that of children while phase, a validation step is ongoing. Further studies are warranted to investigate the diagnosed with histologically similar tumors. A better understanding of tumor biology benefit of conventional treatment backbone with local radiotherapy versus multiple that differentiate children from AYA RMS could provide critical information and drive elective radiotherapy courses and concurrent treatment with targeted agents. new initiatives to improve their final outcome. MiRNA and gene expression analyses This work was supported by Fondazione G. Celeghin Onlus, Fondo di Giò Onlus, have the potential to lead to discovery of new targets pathways for a more tailored and AIRC. treatment in these age groups of young RMS patients. Material and Methods. Good quality RNA was isolated from tumor tissues of 49 primary FFPE RMS, 27 pediatric (Peds; 0-14 years) and 22 AYA (15-30), as well as non- N3 neoplastic tissue (from 9 RMS from each group). A fine analysis of miRNA profile ITCH/β-ARRESTIN2-DEPENDENT NON-PROTEOLYTIC UBIQUITYLATION utilizing SurePrint G3 Human miRNA r21 with chips on miRBAse 21.0 (miRNA) Agilent OF SuFu CONTROLS HEDGEHOG SIGNALLING AND MEDULLOBLASTOMA was performed. In vitro effects on RMS proliferation (metabolic cell proliferation assays), TUMORIGENESIS migration (transwell assay) and apoptosis (annexinV FACS assay) were assessed. 1)Infante P. 2)Faedda R., 3)Bernardi F. 4)Bufalieri F. 5)Lospinoso Severini L. 6)Kool Results. MiRNA profile analysis identified 10 miRNAs differentially expressed considering age, 30 miRNA differentially expressed by examining survival and 15 M. 7)Pfister S.M. 8)Guardavaccaro D. 9)Gulino A. 10)Di Marcotullio L. different miRNA between the two embryonal and alveolar histotypes (p<0.01 in 1)Istituto Italiano di Tecnologia CLNS@Sapienza, Rome, Italy 2)Department tumoral and p>0.5 in non neoplastic tissue). Real Time PCR confirmed differences of Molecular Medicine, University La Sapienza, Rome, Italy 3)Department of in expression for the following miRNAs: miR-223 and miR-431 (RMS and age), Molecular Medicine, University La Sapienza, Rome, Italy 4)Department of Molecular the “family” of miR-500 (miRNA and survival) and miR-362 (miRNA and histology), Medicine, University La Sapienza, Rome, Italy 5)Department of Molecular Medicine, all reported to be deregulated in important pathways for tumorigenesis, including University La Sapienza, Rome, Italy 6)German Cancer Research Center (DKFZ), pathways involving FOXO1, metabolism, cell cycle and transcriptional misregulation. Division of Pediatric Neurooncology, Heidelberg, Germany 7)Heidelberg University Functional studies revealed that transient over-expression of mir-223 using miRNA Hospital, Department of Pediatric Hematology and Oncology, Heidelberg, Germany mimics reduced migration and proliferation properties, while increasing apoptosis 8)Hubrecht Institute-KNAW and University, Medical Center Utrecht, Utrecht, The in RH30 alveolar AYA-RMS cells. Anti-tumoral effects of mir-223 were absent in Netherlands 9)Department of Molecular Medicine, University La Sapienza, Rome, embryonal RMS Peds-RD cells, suggesting a peculiar mechanism of this miRNA in Italy 10)Department of Molecular Medicine, University La Sapienza, Rome, Italy RH30. Ongoing studies to identify the potential role of mir-223 in the development of Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is RMS utilizing primary cell lines, are in progress. Gene expression profiling will also a central player of Hh signalling, a pathway crucial for development and deregulated be correlated with miRNAs results. in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh Conclusion. Improving knowledge on the biological profile and identifying distinct signalling, our understanding of the mechanism regulating this key event remains pathways of pediatric compared to AYA cancers may provide an alternative way to limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces overcome the limits of the current treatment approach. its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into N2 a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises Liquid biopsies in DIPG patients: discovery of predictors of the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations response to targeted therapy occurring in medulloblastoma patients are insensitive to Itch activity, thus leading 1)Ianno’ M.F. 2)De Cecco L. 3)Schiavello E. 4)Anichini A. 5)Biassoni V. 6)Gandola to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our find- L.4 7)Massimino M.2 ings uncover mechanisms controlling the tumour suppressive functions of SuFu 1)Platform of Integrated Biology, Department of Applied Research and Technology and reveal that their alterations are implicated in medulloblastoma tumorigenesis. Development, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 2) Platform of Integrated Biology, Department of Applied Research and Technology Development, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 3)Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 4)Human Tumors Immunobiology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 6)Radiology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7)Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy
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N4 protein involved in the regulation of gene transcription. Che-1 is required for ZYXIN IN EWING SARCOMA: A NOVEL ROLE AS MEDIATOR BETWEEN CD99 proliferation in early embryogenesis, and exhibits an anti-apoptotic activity in different AND EWS-FLI1 tumour contests but its role in haematological malignancies has not been deeply 1)Balestra T. 2)Manara MC. 3)Garofalo C. 4)Santi S. 5)Scotlandi K. explored. We investigated the role of c-Myc and Che-1 in BCP-ALL. In this study, 1)Experimental oncology laboratory, Orthopedic Rizzoli institute, Bologna, Italy 2) we demonstrated that these two proteins are highly expressed in a cohort of BCP- Experimental oncology laboratory, Orthopedic Rizzoli institute, Bologna, Italy 3) ALL patients at the onset of disease, irrespective of genetic heterogeneity, but were Advanced translational research laboratory, Veneto institute of oncology, Padova, barely detectable when patients obtained leukemia remission. Notably, elevated Italy 4) Institute of Molecular Genetics (CNR), Bologna, Italy 5)Experimental levels of Che-1 and c-Myc were also found at time of relapse. Taken together, these oncology laboratory, Orthopedic Rizzoli institute, Bologna, Italy data provide the first evidence of co-expression of the oncogene c-Myc and Che-1 in the most common pediatric hematological malignancy. Importantly, analysis of Ewing sarcoma (EWS) is a rare and aggressive tumor of the bones, affecting several databases confirmed a positive correlation between c-Myc and Che-1 in children and adolescents. EWS cells typically express a disease–specific oncogenic different hematological malignancies. Addressing the correlation between these two chimeric transcription factor, usually EWS–FLI1. Another relevant feature of EWS proteins, the experiments we conducted showed the presence of c-Myc on both cells is the high expression of the glycoprotein of membrane CD99, necessary to human and murine Che-1 promoter, and its capability to activate the transcription maintain the tumor’s malignancy. Despite various studies, the relation between of this gene. Consistent with these results, either up- or down-modulation of c-Myc EWS-FLI1 and CD99 remain obscure for the most part. In this context, we decide expression strongly affected Che-1 expression. RNA-seq experiments conducted to focus our attention on the protein zyxin. This protein normally localizes in the cell in primary BCP-ALL cells showed a significant correlation between the genes membrane and cytosol, where it regulates the organization of actin microfilaments. modulated by these two proteins, indicating how the two factors are involved in the After specific stimulations, zyxin can shuttle in the nucleus and modulate gene same processes that regulate cell proliferation. Notably, Che-1 depletion strongly expression. In EWS cells zyxin acts as a tumor suppressor and undergoes silencing affected growth of ALL cells by inhibiting the expression of several c-Myc target by EWS-FLI1. In addition, zyxin mediates EWS cell death induced by engagement genes, and sensitized ALL cells to Adriamycin, a drug widely used in the treatment of CD99 with a specific antibody (0662 MAb). Here we analyze a cohort of EWS of BCP-ALL. Finally, further evidence of the close connection existing between patients, confirming the tumor suppressor role of this protein in EWS. In fact, we c-Myc and Che-1 arises from the demonstration of their physical interaction on the report a global low expression of zyxin and find out a positive prognostic role. promoters of different c-Myc target genes, and from the demonstration that Che-1 is Moreover, we record that EWS cell lines poorly express zyxin, which inhibits key required for recruitment of c-Myc on these promoters. Silencing of Che-1 expression processes of cancer progression, such as cellular migration, proliferation and in an immortalized cell line generated from lymph nodes of the Burkitt’s Lymphoma anchorage-independent growth. Then we analyze the potential interactions of zyxin mouse model, Em-Myc, allowed us to confirme, in ex-vivo, that Che-1 is strongly with CD99 and EWS-FLI1. We prove that inhibition of CD99 activity, through either required for c-Myc-driven blast-cell proliferation. Moreover, Che-1 overexpression is its engagement with 0662 MAb or its silencing, causes an increase of zyxin level in a shared feature of different possible avenues leading to the ultimate tumorigenesis term of mRNA and protein. Furthermore, we observe that the treatment with 0662 of BCP-ALL, which makes Che-1 especially attractive as a drug target for this MAb induces physical interaction between CD99 and zyxin, which then shuttles into genetically heterogeneous disease. the nucleus. Intrigued by these effects, we verify if the nuclear presence of zyxin could interfere with EWS-FLI1 activity. We show that the treatment with 0662 MAb is able to reduce EWS-FLI1 binding of its target genes promoters. Simultaneously N6 zyxin increases its occupancy on the same promoters analyzed for EWS-FlI1. From mRNA EXPRESSION LEVEL OF SRCIN1/p140CAP IS AN INDEPENDENT these evidences, we hypothesize a novel role for zyxin in EWS. EWS-FLI1 and PROGNOSTIC MARKER OF OUTCOME IN NEUROBLASTOMA CD99 repress zyxin’s activity, but upon inhibition of CD99 zyxin shifts in the nucleus 1)Grasso S. 2)Alzona M. 3)Cangelosi D. 4)Chapelle J. 5)Salemme V. 6)Lamolinara and regulates negatively EWS–FLI1 functions. A. 7)Di Cunto F. 8)Iezzi M. 9)Riganti C. 10)Pezzolo A. 11)Varesio L. 12)Eva A. 13) Grants: AIRC_IG18451 to KS. Turco E. 14)Defilippi P. 1)Dept of molecular biotechnology and health science, University of Torino, Torino, N5 Italy 2)Dept of molecular biotechnology and health science, University of Torino, Che-1/AATF is a new actor in the c-Myc-driven cell proliferation Torino, Italy 3)Laboratory of molecular biology, Giannina Gaslini institute, Genova, in pediatric B-cell precursor acute lymphoblastic leukemia Italy 4)Dept of molecular biotechnology and health science, University of Torino, KEYWORDS: BCP-ALL, Che-1, c-Myc, lymphomagenesis Torino, Italy 5)Dept of molecular biotechnology and health science, University of 1)Folgiero V.1 2)Sorino C.2 3)Pallocca M.2 4)Caforio M.1 5)De Nicola F.2 6)Goeman Torino, Torino, Italy 6)Dept of medicine and aging science, G. D’Annunzio university, F. 3 7)Bertaina V.1 8)Pitisci A.1 9)Strocchio L.1 10)Fanciulli M.2 11)Locatelli F.1,4 Chieti, Italy 7)Dept of molecular biotechnology and health science, University of 1)Department of Pediatric Hematology/Oncology and of Cell and Gene Therapy, Torino, Torino, Italy 8)Dept of medicine and aging science, G. D’Annunzio university, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy 2)SAFU, Chieti, Italy 9)Dept of oncology, University of Torino, Torino, Italy 10)Laboratorio Department of Research, Advanced Diagnostics, and Technological Innovation, cellule staminali post natali e terapi, Giannina Gaslini institute, Genova, Italy 11) Translational Research Area, Regina Elena National Cancer Institute, Rome, Laboratory of molecular biology, Giannina Gaslini institute, Genova, Italy 12) Italy 3)SAFU, Department of Research, Advanced Diagnostics, and Technological Laboratory of molecular biology, Giannina Gaslini institute, Genova, Italy 13)Dept Innovation, Translational Research Area, Regina Elena National Cancer Institute, of molecular biotechnology and health science, University of Torino, Torino, Italy 14) Rome, Italy 4)Department of Pediatric Hematology/Oncology and of Cell and Gene Dept of molecular biotechnology and health science, University of Torino, Torino, Therapy, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy 5)SAFU, Italy Department of Research, Advanced Diagnostics, and Technological Innovation, Translational Research Area, Regina Elena National Cancer Institute, Rome, Italy Background. Neuroblastoma (NB), the most common extra-cranial pediatric solid 6)Epigenetic, Department of Research, Advanced Diagnostics, and Technological tumor, responsible for 13-15% of pediatric cancer death, is a complex disease, Innovation, Translational Research Area, Regina Elena National Cancer Institute, characterized by genetic, biological, clinical, and morphological heterogeneity. Rome, Italy 7)Department of Pediatric Hematology/Oncology and of Cell and Despite chemotherapy, surgery and radiotherapy, it is difficult to target it for Gene Therapy, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy 8) successful therapy. Thus, there is an urgent need for understanding better its Department of Pediatric Hematology/Oncology and of Cell and Gene Therapy, progression, improving treatment and increasing survival rate. The adaptor protein Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy 9)Department of p140Cap, encoded by the SRCIN1 gene, is highly expressed in neurons and in some Pediatric Hematology/Oncology and of Cell and Gene Therapy, Bambino Gesù epithelial tissues. We have recently showed that p140Cap negatively regulates Children’s Hospital, IRCCS, 00146 Rome, Italy 10)SAFU, Department of Research, tumor cell features and limits ERBB2 breast cancer progression, by interfering with Advanced Diagnostics, and Technological Innovation, Translational Research Area, specific signaling pathways. The aim of this study was to determine whether and Regina Elena National Cancer Institute, Rome, Italy 11)Department of Pediatric how SRCIN1/p140Cap is also a key determinant of NB outcome. Science, University of Pavia, Italy Methods. Bioinformatic analysis of RNAseq expression profiles obtained from 498 primary NBs. CGH+SNP microarray platform for detecting genome-wide copy Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood number aberrations and copy-neutral loss of heterozygosity in primary tumors from accounting for almost 30% of pediatric cancer and around 80-85% have a disease 124 patients with high-risk NB and in 8 NB cell lines. Gain of function experiments originating from B-cell precursors that is BCP-ALL. Despite the overall progress in in human neuroblastoma cells. In vitro and in vivo functional assays: proliferation, treatment of B-ALL, relapse occurs across the whole spectrum of all subtypes and migration, tumor growth, metastasis formation, regulation of signaling pathways; has a dismal prognosis with an overall survival of 30%. This disease is typically sensitivity to chemotherapeutic drugs. characterized by genetic lesions resulting in oncogene activation, loss of tumor Results. Here we show that, in NB patients, expression of SRCIN1 mRNA correlates suppressor gene function and concurrent activation of pro-survival signaling with outcome as an independent prognostic marker. In primary NB and in cell lines pathways. The oncogene c-Myc is a master transcription factor governing many SRCIN1 gene was hemizygously deleted, or subjected to copy-neutral loss of critical cell functions such as metabolism, proliferation and survival and it is involved heterozygosity, or disrupted because located at breakpoint of 17q segment involved in up to 70% of cancers. Che-1/AATF is an important RNA polymerase II binding in 17q gain formation. De novo expression of p140Cap protein in the ACN human NB
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cells leads to down-modulation of the Src and STAT3 signaling pathways, impairs in vitro anchorage-independent growth and migration, and significantly reducesin vivo Introduction. PEG-ASP is a cornerstone of the chemotherapy protocols for the tumor growth and spontaneous lung metastasis formation. p140Cap also increases treatment of Acute Lymphoblastic Leukemia (ALL) in children. It acts depleting at sensitivity of ACN cells to chemotherapy drugs. systemic level, asparagine, an essential amino acid for leukemic cell growth. PEG- Conclusions: Overall, these results indicate that SRCIN1/p140Cap is a new ASP has been used in the AIEOP-BFM-ALL-2009 protocol as a first-line therapeutic independent prognostic marker in NB patients. In NB tumors, translocations, option. The activity of the drug was subjected to serum pharmacological monitoring. deletions or cn(LOH) could affect SRCIN1 expression. In NB cells, p140Cap The pharmacokinetic profile of PEG-ASP is described here limited to the induction negatively regulates signaling pathways, impairing tumor growth and metastasis phase of the protocol. formation, and enhancing sensitivity to chemotherapy. Methods. The study involved several Italian hospitals which collected and delivered to our laboratory serum samples withdrawn at different days during the patient’s treatment in the induction phase of the protocol. PEG-ASP was twice administered N7 as 2-hours intravenous infusion at a dose of 2500 U/m2 on days 12 and 26 in TAILORING EPIGENETIC PRECISION MEDICINE IN NEUROBLASTOMA: induction phase. Samples were taken on days 19, 26, 33, 40 and 45 and then PHARMACOLOGICAL INHIBITION OF SETD8 RESCUES P53 CANONICAL analyzed for PEG-ASP activity. The activity was measured by a quantitative FUNCTIONS enzymatic test (MAAT, MEDAC). Pharmacokinetic analysis was conducted using 1)Veschi V. 2)Liu Z. 3)Yan C. 4)Blum G. 5)Luo M. 6)Ma A. 7)Jin J. 8)Thiele C. the NCPKAv2.7 software developed in MAT-LAB. 1)Surgical and oncological sciences, University of Palermo, Palermo, Italy Results. 2660 samples from 440 patients were available for our analysis. The 2)Pediatric oncology branch, National cancer institute, Bethesda, Usa 3) pharmacokinetics of PEG-ASP are described according to a biphasic model with an Oncogenomics section, genetics branch, National cancer institute, Bethesda, Usa average estimate of the clearance in the population of 0.138 ± 0.047 L/day/m2, with 4)Chemical biology program, Memorial sloan kettering cancer center, New york, an inter-individual variability of 34.3%. The decrease of PEG-ASP activity correlated Usa 5)Chemical biology program, Memorial sloan kettering cancer center, New with the time elapsed post-dose. york, Usa 6)Structural and chemical biology, Icahn school of medicine at mount We assessed two different constant of elimination (Ke) for PEG-ASP: Ke1=0.14 (1/ sinai, New York, Usa 7)Structural and chemical biology, Icahn school of medicine at day) determined from day 7 to 19 after the second dose administered and Ke2=0.27 mount sinai, New York, Usa 8)Pediatric oncology branch, National cancer institute, (1/day) determined from day 22 to 28. The activity values (mean ± SD) were Bethesda, Usa 1386±394, 777±317, 1893±712, 934±405 and 387±266 IU/L on days 19, 26, 33, 40 and 45, respectively. The adequate value of activity (≥100 IU/L) was found in 97% of High-risk neuroblastoma (NB) is one of the most aggressive pediatric tumors cases 14 days after each drug administration and in 90% on day 45 (19 days after accounting for 15% of all pediatric oncology deaths, and less than 50% of patients the second dose). experience long-term survival, despite intense multimodal treatment. The tumor Conclusions. The clearance of PEG-ASP is characterized by a biphasic model with suppressor p53 is rarely (2%) mutated in NB but its functions are diminished. a slow initial phase, due to the conjugated structure, followed by a rapid phase Multiple mechanisms have been identified that attenuate the activity of p53 in of decay of activity probably due to the release of the native form of ASP. This is MYCN-amplified NB cells, but few affect MYCN-WT NBs. Thus, a major challenge is reflected by the increase in the Ke and consequently by the simultaneous reduction to identify novel targeted therapies for high-risk NB (HR-NB) patients, specifically for of the half-life of PEG-ASP over time. In the phase of therapy here considered, the large fraction (70%) that present with MYCN-WT. Recently we identified SETD8, PEG-ASP activity levels 14 days after the drug administration were found to be the H4K20me1 methyltransferase, as a crucial epigenetic regulator of growth and adequate in over 97% of the population examined. differentiation in NB. In addition to targeting other non-histone proteins, SETD8 monomethylates p53 on lysine 382 (p53K382me1), attenuating its pro-apoptotic and growth arrest functions. Genetic and pharmacological (UNC0379) inhibition of N9 SETD8 impairs NB growth in vivo. Moreover, SETD8 levels are associated with Monitoring of the asparaginase (ASP) activity during the poor prognosis only in MYCN-WT Stage 4 NBs. In order to identify targeted therapy treatment with Erwinia C. ASP (ERW-ASP) in children affected by less toxic for HR-NB, we evaluated a more specific SETD8 inhibitor with enhanced Acute Lymphoblastic leukemia (ALL), after allergic reaction activity and selectivity, SGSS05-NS3 (SG3). Our results indicated that in NB cells in or silent inactivation to E. Coli PEG ASP (PEG-ASP) vitro treatment with SG3 rescues the canonical p53 functions leading to increases 1)Ceruti T. 2)Matteo C. 3)Colombini A. 4)Silvestri D. 5)Conter V.6)Falcetta F. 7)Lebiu in p53 protein levels and of its target p21 by decreasing p53K382me1, impairing M. 8)Brivio E. 9)Barisone E. 10)Bettini L. 11)Lo Nigro L. 12)Micalizzi C. 13)Mura R.
NB cell viability and inducing caspase-dependent cell death at lower IC50 compared 14)Petruzziello F. 15)Rabusin M. 16)Vinti L. 17)Uggeri M. 18)Valsecchi M.G. 19) with UNC0379 (1µM). Gene expression profile (RNA-seq analysis) confirmed that Zucchetti M. 20)Rizzari C. the most significantly upregulated genes upon SG3 treatment were among the 1)Dipartimento di Oncologia, Istituto di Ricerche Farmacologiche M. Negri – IRCCS, p53 pathway targets. In pre-clinical xenograft NB models, pharmacological SETD8 Milan, Italy 2Dipartimento di Oncologia, Istituto di Ricerche Farmacologiche M. Negri inhibition by SG3 conferred a significant survival advantage in MYCN WT NB. Our – IRCCS, Milan, Italy 3)Clinica Pediatrica, Università di Milano-Bicocca, Ospedale S. study provides further evidence for targeting SETD8 as a therapeutic strategy in NB, Gerardo, Monza, Italy 4)Dipartimento di Medicina Clinica e Prevenzione, Università particularly in MYCN-WT subgroup. di Milano-Bicocca, Monza, Italy 5)Clinica Pediatrica, Università di Milano-Bicocca, Ospedale S. Gerardo, Monza, Italy 6)Dipartimento di Oncologia, Istituto di Ricerche Farmacologiche M. Negri – IRCCS, Milan, Italy 7)Clinica Pediatrica, Università di N8 Milano-Bicocca, Ospedale S. Gerardo, Monza, Italy 8)Clinica Pediatrica, Università Pharmacokinetic profile of PEG-ASP in children with Acute di Milano-Bicocca, Ospedale S. Gerardo, Monza, Italy 9)Centro AIEOP di Torino, Lymphoblastic Leukemia treated in the induction phase of the Turin, Italy 10)Clinica Pediatrica, Università di Milano-Bicocca, Ospedale S. Gerardo, AIEOP-BFM-ALL 2009 protocol Monza, Italy 11)Centro AIEOP di Catania, Italy 12)Centro AIEOP di Genova, Genoa, 1)Matteo C. 2)Colombini A. 3)Silvestri D. 4)Conter V. 5)Falcetta F. 6)Ceruti T. 7) Italy 13)Centro AIEOP di Cagliari, Italy 14)Centro AIEOP di Napoli Pausilipon, Zanta D. 8) Barisone E. 9)Casini T. 10)Lo Nigro L. 11)Micalizzi C 12)Parasole R. 13) Naples, Italy 15)Centro AIEOP diTrieste, Italy 16)Centro AIEOP di Roma Bambino Provenzi M. 14)Zecca M. 15)Vinti L. 16)D’Incalci M. 17)Valsecchi M.G. 18)Rizzari Gesù, Rome, Italy 17)Dipartimento di Medicina Clinica e Prevenzione, Università di C. 19)Zucchetti M. Milano-Bicocca, Monza, Italy 18)Dipartimento di Medicina Clinica e Prevenzione, 1)Department of Oncology, IRCCS-Istituto di Ricerche farmacologiche Mario Negri, Università di Milano-Bicocca, Monza, Italy 19)Dipartimento di Oncologia, Istituto Milan, Italy 2)Pediatric Clinics, University of Milano-Bicocca, S. Gerardo Hospital, di Ricerche Farmacologiche M. Negri – IRCCS, Milan, Italy 20)Clinica Pediatrica, Monza, Italy 3)Department of Clinical Medicine and Prevention, University of Milan- Università di Milano-Bicocca, Ospedale S. Gerardo, Monza, Italy Bicocca, Monza, Italy 4)Pediatric Clinics, University of Milano-Bicocca, S. Gerardo Hospital, Monza, Italy 5)Department of Oncology, IRCCS-Istituto di Ricerche Introduction. A pharmacological monitoring study of the ERW-ASP activity has farmacologiche Mario Negri, Milan, Italy 6)Department of Oncology, IRCCS- been conducted in the recent AIEOP-BFM ALL 2009 protocol. ERW-ASP is an Istituto di Ricerche farmacologiche Mario Negri, Milan, Italy 7)Pediatric Clinics, asparaginase formulation had to be used in case of allergic reaction or silent University of Milano-Bicocca, S. Gerardo Hospital, Monza, Italy 8)AIEOP Center inactivation (enzymatic activity <100 IU/L in the absence of clinical allergy), of Torino, Turin, Italy 9)AIEOP Center of Firenze, Florence, Italy 10)AIEOP Center observed during the first line treatment with PEG-ASP. The preliminary results of of Catania, Italy 11)AIEOP Center of Genova, Genoa, Italy 12)AIEOP Center of this monitoring study (ASP activity levels in serum) are here presented. Napoli Pausilipon, Naples, Italy 13)AIEOP Center of AIEOP Center of Bergamo, Methods. Seven doses of ERW-ASP (20,000 IU/m2 i.v. in 2 hours) were administered Italy 14) AIEOP Center of Pavia, Italy 15)AIEOP Center of Roma, Bambino Gesù, at intervals of 48/72 hours to replace one dose of PEG-ASP (2500 IU/m2). The serum Rome, Italy 16) Department of Clinical Medicine and Prevention, University of activity of ERW-ASP was measured by an enzymatic test (MAAT test, MEDAC). We Milan-Bicocca, Monza, Italy 17)Department of Clinical Medicine and Prevention, have defined three classes of asparaginase activity: optimal ≥100 IU/L, border-line University of Milan-Bicocca, Monza, Italy 18)Pediatric Clinics, University of Milano- ≥50 <100 IU/L and inadequate <50 IU/L. Bicocca, S. Gerardo Hospital, Monza, Italy 19)Department of Oncology, IRCCS- Results. The asparaginase activity levels analyzed in 307 samples of 43 patients Istituto di Ricerche farmacologiche Mario Negri, Milan, Italy showed a wide inter-patient and intra-patient variability and were optimal 48 hour
68 69 ABSTRACT BOOK post-dose in 62% (116/187; mean 343±289 IU/L), border-line in 24.6% (79±14 IU/L) and inadequate in 13.4% (30±15 IU/L) of the samples, respectively. The ERW-ASP PERSONALIZED MEDICINE activity measured in the samples obtained 72 hours after its administration was O1 adequate only in 15.6% of the samples. LIQUID BIOPSY: TRACKING MUTATIONAL TRAJECTORIES IN ERBB2 Conclusions. ERW-ASP, used instead of PEG-ASP in cases with allergic reaction BREAST CANCER PATIENTS UNDERGOING T-DM1 TREATMENT or silent inactivation, and administered as suggested in the treatment protocol, 1)Allegretti M. 2)Giordani E. 3)Casini B. 4)Romania P. 5)Nisticò C. 6)Gasparro S. 7) determines levels of adequate or border-line enzymatic activity in 86.6 % of the Ferretti G. 8)Malaguti P. 9)Russillo M. 10)Gallo E. 11)Buglioni S. 12)Pescarmona E. samples 48-hour post dose. Conversely, the inadequate activity levels detected 72 13)Cognetti F. 14)Ciliberto G. 15)Giacomini P. 16)Fabi A. hour post-dose in more than 60% of the samples highlight the need to stringently 1)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, follow the clinical protocol indication to administer the drug every 48 hours. Rome, Italy 2)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 3)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 4)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 5)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 6)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 7)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 8)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 9)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 10)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 11)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 12)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 13)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy 14)Scientific Direction, IRCSS Regina Elena National Cancer Institute, Rome, Italy 15)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 16)Medical Oncology 1, IRCSS Regina Elena National Cancer Institute, Rome, Italy Background. The antibody-drug conjugate Ado-Trastuzumab emtansine (T-DM1) is standard of care in ERBB2 breast cancer progressing upon Trastuzumab and/or Pertuzumab and/or taxanes. Despite considerable clinical benefit, some patients rapidly develop progressive disease, but the molecular mechanisms remain largely unknown. Analysis of mutational trajectories by liquid biopsy (LB) may help to uncover molecular patterns linked to T-DM1 resistance/responsiveness. Materials and methods. Tumor tissues (n=11) and plasma samples (n=80) were collected, following informed consent, from 6 breast cancer patients undergoing T-DM1 administration. Tissue (tDNA) and circulating tumor DNAs (ctDNA) were extracted by the QIAmp DNA FFPE and CNA kits (Qiagen), and analyzed by ultra- deep sequencing and dPCR (IonTorrent S5 and QuantStudio 3D, LifeTechnologies). Genomic data were correlated with conventional clinical imaging (CT/PET). Results. Four out 6 (67%) patients experienced progression on T-DM1 within 1 year of treatment (mean 221±105 days). In 3/4 of these patients, LB revealed both blood increases in pre-existing mutations (TP53 p.R273H) and de novo appearance of aberrations (PIK3CA p.H1047R, ESR1 p.Y537C plus CCND1 amplification) not seen at baseline. LB anticipated clinical progression (as routinely assessed by imaging) by an average lead time of 1.9 months (range 0.7-2.8). Surprisingly, the fourth patient underwent rapid progression (3 months) in spite of decreased PIK3CA p.E545K in blood, suggesting heterogeneous response to T-DM1 across multiple cancer cell populations. Finally, we observed a progressive accumulation of ERBB2 p.L755S (mechanistically associated with Lapatinib resistance) in multiple serial metastatic foci from a single patient over several years of multimodal ERBB2 blockade. However, a single administration of T-DM1 resulted in ultra-fast (within days) ctDNA clearance, two distinct ‘bystander’ TP53 ctDNAs (p.R273H and p.S241T) remaining stable throughout. Conclusions. Non-invasive LB monitoring of a small cohort of T-DM1-treated patients provides proof of principle of intersecting mutational trajectories, anticipation of resistance, and de novo onset/clearance of resistance mutations. Thus, LB may provide clues about disease evolution and successive lines of medical treatment. This work was supported by AIRC (Nuvenia Fellowship to MA, IG 19052 to PG), EU commission (grant #633937 – ULTRAPLACAD), and Regina Elena intramural funding.
O2 Case report: preclinical testing of NEDD8 and proteasome inhibitors for a treatment-refractory, metastatic high-grade mucinous colorectal cancer patient 1)Torchiaro E. 2)Petti C. 3)Isella C. 4)Corti G. 5)Montone M. 6)Mussolin B. 7)Bardelli A. 8)Medico E. 1)Oncogenomics, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 2) Oncogenomics, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 3) Oncogenomics, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 4)Molecular Oncology, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 5)Molecular Oncology, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 6)Molecular Oncology, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 7)Molecular Oncology, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy 8)Oncogenomics, Candiolo Cancer Institute - FPO IRCCS, Turin, Italy
Colorectal cancer (CRC) is the third leading cause of death in the world. CRC shows variable phenotypic make-ups; among them, a particularly aggressive histological
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subtype is the “high grade mucinous” (HGM) adenocarcinoma, highly mucinous, O4 prone to metastasis and typically refractory to treatments. In some cases, HGM Unique true predicted neoantigens (TPNAs) correlates with is accompanied by a peculiar “signet ring” phenotype of cancer cells. Early stage anti-tumor immune control in HCC patients diagnosis of signet ring HGM is rare, since clinical symptoms tend to occur late 1)Tagliamonte M. 2)Petrizzo A. 3)Mauriello A. 4)Costa V. 5)Caporale A. 6)Arra C. 7) and most cases are usually detected at an advanced stage, with a poor overall Tornesello M.L. 8)Buonaguro F.M. 9)Buonaguro L. survival. We previously demonstrated that a transcriptional signature of HGM 1)Laboratory of Cancer Immunoregulation, Istituto Nazionale per lo Studio e displays negative prognosis and sensitivity to the NEDD-8 inhibitor pevonedistat, la Cura dei Tumori, “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 2) suggesting the involvement of neddylation- and ubiquitination-based mechanisms Laboratory of Cancer Immunoregulation, Istituto Nazionale per lo Studio e la Cura in these cases (Picco et al., JNCI J, 2017). dei Tumori, “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 3)Laboratory of To assess the clinical potential of colorectal cancer HGM identification and targeting Cancer Immunoregulation, Istituto Nazionale per lo Studio e la Cura dei Tumori, by pevonedistat or other inhibitor of proteasome pathway, we recently propagated “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 4)Institute of Genetics and cells and PDXs from a case of early onset, metastatic CRC in a Lynch syndrome Biophysics “A. Buzzati-Traverso” (IGB), National Research Council, 80131 Naples, patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, Italy 5)Institute of Biostructures and Biomaging (IBB), National Research Council, surprisingly, also to nivolumab. The tumor was highly mucinous, with signet ring 80134, Naples, Italy 6)Animal Facility, Istituto Nazionale per lo Studio e la Cura undifferentiated cells. dei Tumori, “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 7)Laboratory of Mutational analysis on tumor tissue from the first surgery highlighted PIK3CA Molecular Biology and Viral Oncology, Istituto Nazionale per lo Studio e la Cura H1047R mutation and no mutations in KRAS, NRAS or BRAF. Surprisingly, exome dei Tumori, “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 8)Laboratory of analysis on two lesions from a subsequent surgery displayed a different scenario: Molecular Biology and Viral Oncology, Istituto Nazionale per lo Studio e la Cura KRAS G13D but not PIK3CA mutation. Probably these discording results suggest dei Tumori, “Fondazione Pascale” - IRCCS, 80131 Naples, Italy 9)Laboratory of a strong tumor heterogeneity and evolution. Considering the failure of all previous Cancer Immunoregulation, Istituto Nazionale per lo Studio e la Cura dei Tumori, therapies, the lack of actionable genetic alterations and the peculiarity of the “Fondazione Pascale” - IRCCS, 80131 Naples, Italy phenotypic features, patient-derived models (organoids and 2D cell cultures) were derived from the two latter lesions and tested for sensitivity to the NEDD8 pathway A novel prediction algorithm is needed for the identification of effective tumor inhibitor pevonedistat and the proteasome inhibitors bortezomib and carfilzomib. associated mutated neoantigens. Only those with no homology to self wild type Interestingly, all models showed a strong sensitivity to both drugs, in particular to antigens are true predicted neoantigens bortezomib. (TPNAs) and can elicit an antitumor T cell response, not attenuated by central This is an example of a rare case of HGM colorectal cancer where the integration tolerance. To this aim, the mutational landscape was evaluated in HCV-associated of several approaches proves useful to identify possible therapeutic strategies for a hepatocellular carcinoma. patient without further standard treatment options. Liver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted O3 by comparison with corresponding wild-type sequences, non-related self and MiR-506-RAD17 regulatory axis is a key node for synthetic pathogen-related antigens. Immunological confirmation was obtained in preclinical lethal approaches targeting DNA repair pathway as well as clinical setting. 1)Nicoletti R. 2)De Cecco L. 3)Alberti P. 4)Raspagliesi F. 5)Cecchin E. 6)Califano D. The development of such a novel algorithm resulted in a handful of TPNAs despite 7)Pignata S. 8)Bagnoli M. 9)Mezzanzanica D. the large number of predicted neoantigens. Furthermore, TPNAs may share 1)Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Fondazione homology to pathogen’s antigens and be targeted by a pre-existing T cell immunity. IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Fondazione IRCCS Istituto Cross-reactivity between such antigens was confirmed in an experimental pre- Nazionale dei Tumori, Milan, Italy 4)Fondazione IRCCS Istituto Nazionale dei clinical setting. Finally, TPNAs homologous to pathogen’s antigens were found Tumori, Milan, Italy 5)Centro di Riferimento Oncologico, Aviano, Italy 6)Fondazione in the only HCC long-term survival patient, suggesting a correlation between the G. Pascale Istituto Nazionale Tumori, Naples, Italy 7)Fondazione G. Pascale Istituto pre-existing T cell immunity specific for these TPNAs and the favourable clinical Nazionale Tumori, Naples, Italy 8)Fondazione IRCCS Istituto Nazionale dei Tumori, outcome. Milan, Italy 9)Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy The new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term Introduction. Overcoming chemo-resistance remains a major challenge in Epithelial survival. Only such TPNAs represent the optimal candidates for immunotherapy Ovarian Cancer (EOC). We previously identified the ChrXq27.3 miRNA cluster strategies. independently associated with early relapse in advanced stage EOC patients. MiR- 506, belonging to this cluster, was shown to regulate several genes of the DNA damage repair (DDR) pathway thus supporting its involvement in response to drug O5 treatments. In EOC, inactivation of DDR genes (BRCA1/2) is associated with better EFFICACY OF COMBINED MEK AND NOTCH INHIBITION IN A UVEAL prognosis, due to sensitization to DNA damaging drugs. Deciphering miR-506 role MELANOMA CELL LINE in regulating signaling networks related to DDR and drug resistance is expected to 1)Mazzotta A. 2)Porcelli L. 3)Iacobazzi R.M. 4)Di Fonte R. 5)Garofoli M. 6)Guida G. improve the management of the disease. 7)Guida M. 8)Azzariti A. Experimental Design. The involvement of miR-506 in regulating DDR machinery was 1)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, investigated in in-vitro EOC models with different p53 mutational status, assessing Italy 2)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo the potential of miR-506 expression to mimic the effects of BRCA1/2 mutation in II”, Bari, Italy 3)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni sensitizing tumor cells to platinum (Pt), PARP, CHK1 and WEE1 inhibitors (PARPi, Paolo II”, Bari, Italy 4)Experimental Pharmacology Laboratory, Istituto Tumori CHK1i, WEE1i). We investigated the correlation of miR-506 expression with Pt- “Giovanni Paolo II”, Bari, Italy 5)Experimental Pharmacology Laboratory, Istituto sensitivity in clinical setting including only optimally debulked patients. Tumori “Giovanni Paolo II”, Bari, Italy 6)Department of Basic Medical Sciences, Results. In this clinical setting we found a significant association of miR-506 Neurosciences and Sense Organs, University of Bari, Italy 7)Medical Oncology expression with Pt-sensitivity in two independent cohorts, thus supporting its pivotal Unit, Istituto Tumori “Giovanni Paolo II”, Bari, Italy 8)Experimental Pharmacology role as chemosensitizing miRNA. Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy We identified and validated the direct targeting of miR-506 on the DNA damage early sensor RAD17, whose activation culminates in BRCA1/2 recruitment to DNA Although considered a rare tumour, uveal melanoma (UM) is the most common damaged sites with the stabilization of an active signalling for damage repair. MiR- primary ocular malignancy in adults. Primary tumours can be treated effectively, 506 expression was shown to impair the G2/M checkpoint activation upon DNA but approximately 50% of the patients develop metastasis; so, novel strategies are damage leading to mitotic defects and micronuclei formation and allowing for urgently needed. Activating mutations in genes encoding the G-protein-α subunits mitotic entry with increased defects and therefore boosting the effectiveness of DNA GNAQ or GNA11 are reported in more than 80% of UMs. Currently there are no damaging treatments. Interestingly, we observed that miR-506-RAD17 regulatory known GNAQ/GNA11 targeted drugs and an alternative approach involves the axis is synthetically lethal with Pt, PARPi, CHK1i and WEE1i treatments in EOC selective inhibition of critical downstream effectors, including MAPK signalling. cells. Moreover the combination treatments of Pt with CHK1i and WEE1i is more Recent clinical data has demonstrated modest efficacy in UM patients treated with effective than mono treatments and synergistic in the context of miR-506 expression. the MEK1/2 inhibitor. It seems that mutant GNAQ promotes UM cell viability and Conclusion. We propose miR-506 as a key node in the regulation of DDR pathway. migration even through the activation of Notch signalling. Thus, in this study, we Its expression, recapitulating a BRCAness phenotype, sensitizes EOC cells to explored combined MAPK and NOTCH pathways inhibition with the allosteric MEK chemotherapy and may help in selecting patients susceptible to DNA damaging inhibitor, cobimetinib, and the small-molecule inhibitor of the γ-secretase complex drugs in combination with new small molecules targeting DDR pathway. (GS), nirogacestat, in a UM 92.1 cell line carrying activating GNAQ mutation. To this Partially supported by AIRC and CARIPLO Foundation. purpose UM 92.1 cells were treated with cobimetinib and GS inhibitor (GSI) alone or in combination and cell proliferation was assayed after 3 days by MTT assay.
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The effects on cell cycle progression and apoptosis induction were determined after 24/48h of treatment, after staining cells with propidium iodide and Annexin PREVENTION AND EARLY DETECTION V/PI, respectively, followed by flow cytometry analysis. Cell targets modulation, P1 related to apoptosis induction and cell cycle arrest, was evaluated by western Bone marrow hematopoietic adaptation as a sensor of early, blotting. The combination of cobimetinib and GSI resulted in further 50% inhibition pre-invasive, mammary carcinoma of cell proliferation compared to each drug alone. Cell cycle analysis after 24h 1)Chiodoni C. 2)Cancila V. 3)Renzi T.A. 4)Tomirotti A.M. 5)Perrone M. 6)Dugo M. 7) evidenced a significant arrest induced by the combination in the G0/G1 phase. At Milani M. 8)Tripodo C. 9)Colombo M.P. 48h, GSI strongly accumulated cells in G0/G1, while the combination resulted in 1)Molecular Immunology Unit, Department of Research, Fondazione IRCCS Istituto the slowdown of proliferation rate evidenced by the reduction of S and G2/M-phase Nazionale dei Tumori, Milan, Italy 2)Department of Human Pathology, University of cells population. However the mitotic arrest, triggered by the combination, didn’t Palermo, Italy 3)Molecular Immunology Unit, Department of Research, Fondazione increase apoptosis induced by cobimetinib alone. At the protein level, according IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Molecular Immunology Unit, to G0/G1 arrest, the level of cyclin D1, the allosteric regulator of CDK4/CDK6, was Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, strongly decreased; whereas p27 level increased according to the inhibition of entry Milan, Italy 5)Molecular Immunology Unit, Department of Research, Fondazione into S-phase. In conclusion, the simultaneous inhibition of MEK and GS resulted IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Functional Genomics and in induced apoptosis and slow down of cell proliferation, thus showing to be a Bioinformatics, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7) promising innovative approach for the treatment of mutant GNAQ uveal melanoma. Molecular Immunology Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8)Department of Human Pathology, University of O6 Palermo, Italy 9)Molecular Immunology Unit, Department of Research, Fondazione TARGETING WNT10B*R-MEDIATED TRANCRIPTOME AND INTERACTOME IN IRCCS Istituto Nazionale dei Tumori, Milan, Italy ACUTE LYMPHOBLASTIC LEUKEMIA: SINGLE-CELL ANALYSES 1)Lazzaroni F. 2)Cassaro A. 3)Esposito I. 4)Reda G. 5)Cairoli R. 6)Beghini A. Tumor development and progression is in part dependent on the ability of 1)Department of Biochemistry, University of Lausanne, Epalinges, Switzerland 2) bystander cells, mostly of bone marrow (BM) origin, to establish a pro-tumorigenic Hematology Unit, Niguarda Cancer Center, Milan, Italy 3)Department of Health microenvironment. We hypothesized that signs of the cross-talk between the BM Sciences, University of Milan, Italy 4)Hematology Unit, Fondazione IRCCS Ca’ and elements located in the tumor microenvironment, either neoplastic or stroma/ immune cells, can be identified in the very early phases of cancer development, and Granda Ospedale Maggiore Policlinico, Milan, Italy 5)Hematology Unit, Niguarda lead to the instruction of a tumor-promoting hematopoiesis. Cancer Center, Milan, Italy 6)Department of Health Sciences, University of Milan, By integrating flow cytometry analysis and gene expression profiling of hematopoietic Italy populations with in situ BM histopathological and immunophenotypical analyses, Acute lymphoblastic leukemia (ALL) is a malignant disease characterized we investigated, in a spontaneous mouse model of breast carcinogenesis (MMTV/ by uncontrolled proliferation of immature lymphocytes and according to the NeuT), the modifications in the hematopoietic and stromal BM compartments and cell type that is affected it can be divided into two subtypes: B- and T-cell ALL. their correlation with the pathological stage of the mammary lesions. At the invasive Deregulated signaling is considered a major contributing factor in leukomogenesis carcinoma stage, the analysis of the BM hematopoietic compartments revealed a of T-ALL, and in particular Wnt signaling has been implicated in the regulation of clear-cut switch towards a preferential granulo-monocytic fate, which was paralleled hematopoietic stem cells and plays an important role during T-cell development by a significant contraction in B-cell lymphoid populations and associated with the in thymus. The analysis of WNT10B locus revealed the presence of a recurrent remodeling of the BM mesenchymal niches. At this stage, gene expression profiling rearrangement (WNT10B*R) recently described in acute myeloid leukemia, of BM cells identified a differential gene signature that allowed, on unsupervised leading to expression of the transcript variant WNT10B*IVS1 in an Italian cohort analysis, distinguishing between transgenic and control mice on the basis of of 34 primary acute lymphoblastic leukemia (5 T-ALL and 29 B-ALL) patients. inflammatory and innate immune response programs. The investigation at earlier We detected the WNT10B*R rearrangement in 19/34 ALL patients analyzed, stages of cancer development of such modifications in BM microarchitecture, supporting our hypothesis of its broad involvement in the acute leukemogenesis. hematopoietic composition, and gene profile demonstrated that their induction In order to determine the FZD receptor/s involved in WNT10B-mediated interaction, was already detectable at the high-grade dysplasia/in situ cancer stage, which we performed specific proximity ligation assay (PLA) for FZD4/5/6 in the MOLT4 provided the first evidence of the BM acting as a very early sensor of peripheral T-ALL cell model, characterized by the monoallelic expression of the WNT10B*R. transformation. Hypothesizing that the variations of the BM transcriptional profile Interestingly, we detected high intensity signals only for WNT10B/FZD6 ligand/ could be paralleled by a reprogramming of the miRNA circulating in the peripheral receptor-interaction. To assess the functional significance of WNT10B*R autocrine blood, we profiled plasmatic miRNAs during tumor progression and found, beginning signaling, we infected the MOLT4 cells with WNT10B*R-silencing lentiviruses versus at early stage, differentially expressed miRNAs, which could potentially be used as empty vector control and we evaluated the transcriptomic and proteomic effects at early biomarkers of cancerogenesis. a single cell level. Moreover, we down-modulated the autocrine ligand-dependent In conclusion, our data demonstrate that BM hematopoietic adaptation to peripheral Wnt signaling by porcupine acyltransferase inhibitor-mediated interference of tissue transformation represents an early process co-evolving with malignant WNTs secretion.. We showed that in MOLT4 cell line both WNT10B*R silencing or progression. These results, besides casting light on the identification of potential porcupine inhibition resulted in a decreasing proliferation and increased senescence, new biomarkers for early cancer detection, offer a new prospect on investigating the paralleled by decreased levels of Wnt effector genes (b-catenin, PYGO2) and by a significance of early cancer-adapted hematopoiesis. strong reduction of WNT10B-FZD6 interaction events. Treatment with increasing concentration of porcupine inhibitor decreased the MOLT4 cell viability in a dose- P2 dependent manner, highlighting the WNT10B*R involvement in the autocrine Wnt Cross-sectional analysis of tissue and circulating tumor DNA signaling activation also in ALL. These overall findings establish the relevance of the in primary colorectal cancer patients at surgery WNT10B*R-mediated autocrine WNT signalling activation and identified FZD6 as a 1)Giordani E. 2)Allegretti M. 3)Cottone G. 4)Cotroneo E. 5)Casini B. 6)Buglioni S. 7) potential receptor functionally involved in acute lymphoblastic leukemia, suggesting Carboni F. 8)Valle M. 9)Garofalo A. 10)Cigliana G. 11)Conti L. 12)Pescarmona E. 13) new molecular targeting strategies for an improved clinical outcome in ALL. Giacomini P. 14)Spinella F. 1)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 2)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 3)Oncogenomics Division, Genoma Group, Rome, Italy 4)Oncogenomics Division, Genoma Group, Rome, Italy 5)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 6)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 7)Digestive Surgery, IRCSS Regina Elena National Cancer Institute, Rome, Italy 8)Digestive Surgery, IRCSS Regina Elena National Cancer Institute, Rome, Italy 9)Digestive Surgery, IRCSS Regina Elena National Cancer Institute, Rome, Italy 10)Clinical Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 11)Clinical Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 12)Pathology, IRCSS Regina Elena National Cancer Institute, Rome, Italy 13)Oncogenomics and Epigenetics, IRCSS Regina Elena National Cancer Institute, Rome, Italy 14)Oncogenomics Division, Genoma Group, Rome, Italy
Background. Liquid biopsy approaches are being applied to progressively earlier surgical and clinical oncology settings, and show considerable promise. Circulating
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tumor DNA (ctDNA) monitoring in advanced cancers is quite easy due to elevated P4 variant allele frequencies (VAFs), but difficult in early cancer settings since few Circulating microRNAs combined with PSA for accurate and non- copies per mL have to be detected. We performed a cross-sectional analysis of invasive prostate cancer detection matched tissue and blood samples from 32 colorectal cancer (CRC) patients at 1)Gregnanin I. 2)Mello-Grand M. 3)Sacchetto L. 4)Ostano P. 5)Zitella A. 6)Bottoni G. surgery to evaluate whether LB can detect small primary cancers. 7)Oderda M. 8)Marra G. 9)Munegato S. 10)Gasparini M. 11)Pardini B. 12)Naccarati Materials and methods. Matched tissues and blood samples were collected from A. 13)Gontero P. 14)Chiorino G. 32 CRC patients at surgery, and from two metastatic patients as controls. Genomic 1)Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia, Biella, Italy analysis was performed by combining two NGS platforms (Ion S5 and NextSeq) and 2)Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia, Biella, Italy the QuantStudio 3D dPCR. Clinical pathological data (stage, grade, tumor size), 3)Department of Mathematical Sciences of Politecnico di Torino, Turin, Italy 4) and plasma biomarkers (CEA and Ca19.9) were correlated with ctDNA levels. Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia, Biella, Italy Results. A total of 52 hits covering 40 unique aberrations were concordantly 5)University of Torino, Turin, Italy 6)Department of Biochemistry, University of detected by the two NGS platforms in tissues from 29/32 (90.6%) CRC patients, Lausanne, Epalinges, Switzerland 7)Department of Urology, Ospedale San with negligible differences in VAFs (1.3%±1.2; range 0.0-4.1%). Twenty-eight out Lazzaro, Alba, Italy 8)Department of Urology, Ospedale San Lazzaro, Alba, Italy 52 mutational hits were tested by dPCR and all of them were validated. Only 15/28 9)Department of Urology, Ospedale San Lazzaro, Alba, Italy 10)Department of (53.6%) ‘index’ mutations found in tissues were identified in the bloodstream in Mathematical Sciences of Politecnico di Torino, Turin, Italy 11)Italian Institute for 12/29 (41.4%) CRC patients. As expected, dPCR showed higher sensitivity than Genomic Medicine (IIGM), Turin, Italy 12)Italian Institute for Genomic Medicine NGS, but the latter identified 18 aberrations not detected in matched tumor tissues. (IIGM), Turin, Italy 13)Department of Urology, Ospedale San Lazzaro, Alba, Italy No relationship was apparent between the abundance of a mutation in tissue 14)Laboratory of Cancer Genomics, Fondazione Edo ed Elvo Tempia, Biella, Italy and its representation in blood. Likewise, no significant correlations were found between ctDNA and stage, grade or tumor size. Enumeration of ctDNA, CEA and Background. Early detection of prostate cancer (PCa) is limited by the lack of Ca19.9 values in a scoring matrix revealed that ctDNA outperformed conventional accurate non-invasive biomarkers easily evaluable in men within a screening biomarkers since it detected tumor presence in 10/18 patients whereas biomarkers context. Prostate-specific antigene (PSA) measurement leads to high percentages were above threshold only in 5/18 patients. of both false positives and false negatives. In the era of liquid biopsies, we propose Conclusions. Detection of ctDNAs in CRC patients is feasible even in non- to combine PSA dosage with the evaluation of circulating microRNAs (miRs), in metastatic disease. A combined use of NGS and dPCR helps to correctly assign order to discriminate between men harbouring or not PCa. WT/MUT status. No obvious correlation could be observed between ctDNA levels Methods. miR profiling of plasma samples from 60 men with PCa, 51 menwith and the clinical pathological features of primary CRC. Notably, ctDNA behaves as benign prostatic hyperplasia (BPH) and 27 healthy donors (HD) was performed an independent biomarker, and provides incremental information over clinically using microarrays. Univariate analysis (linear models with an empirical Bayes approved biomarkers. approach) and multivariate penalized logistic regression models were applied to This study was supported by EU (grant #633937 – ULTRAPLACAD) and AIRC (IG highlight miRs and clinical variables associated with the presence of malignant 19052, Nuvenia Fellowship to MA). disease, and were combined in a classification score. An independent dataset of 242 plasma samples (68 PCa, 8 premalignant lesions, 93 BPH, 73 HD), analyzed by RT-qPCR, was used to validate the developed score. P3 Results. Out of 2006 miRNAs analyzed, a linear combination of miR-103a-3p and SERUM MICRORNAS AS BIOMARKERS FOR EARLY DIAGNOSIS OF NON let-7a-5p with PSA defined a score. A classification rule based on this score allowed SMALL CELL LUNG CANCER to reclassify samples with accuracy (0.61), sensitivity (0.87), specificity (0.35) 1)D’Antona P. 2)Moretti F. 3)Finardi E. 4)Barbetta M. 5)Cattoni M. 6)Dominioni L. 7) and AUC (0.68) higher than those of PSA alone. On the validation set, the same Noonan D.M. 8)Poli A.. 9)Campomenosi P. classification rule still performed better than PSA alone in terms of specificity (0.57 1)Department of Biotechnology and Life Science, DBSV, University of Insubria, versus 0.55) and AUC (0.76 versus 0.74), allowing to correctly reclassify all but one Varese, Italy 2)Department of Diagnostic and Public Health, University of Verona, tumors not detected by PSA and 33% of BPH with PSA in the 4-16 ng/ml range. Verona, Italy 3)Department of Diagnostic and Public Health, University of Verona, Conclusions. The combination of two circulating miRs with PSA could be an Verona, Italy 4)Department of Diagnostic and Public Health, University of Verona, interesting biomarker to detect the presence of PCa (even when PSA levels are Verona, Italy 5)Department of Medicine and Surgery, DMS, Center for Thoracic below the standard cut-off of 4 ng/ml) and the absence of PCa in an important Surgery, University of Insubria, Itali 6)Department of Medicine and Surgery, fraction of men with high PSA, who may avoid unnecessary biopsies. These results DMS, Center for Thoracic Surgery, University of Insubria, Itali 7)Scientific and warrant further investigation in large multicentre studies. Technological Pole, IRCCS MultiMedica, Milan, Italy 8)Department of Diagnostic and Public Health, University of Verona, Verona, Italy 9)The Protein Factory, Centro Interuniversitario di Ricerca in Biotecnologie Proteiche, Politecnico di Milano, ICRM- CNR Milano and University of Insubria, Varese, Italy Introduction. Lung cancer is the main cause of cancer-related mortality worldwide. Patients with early stage (I-II) non-small cell lung cancer (NSCLC) have a much better prognosis than those diagnosed at late stage. Thus, the development of sensitive and non-invasive methods for screening individuals at high risk for NSCLC is needed. microRNAs (miRNAs) have been suggested as a novel class of tumor biomarkers; their stability in biofluids and their change in levels in disease suggest their potential application as circulating biomarkers. Experimental model. Upon a critical review of the literature, we selected 8 miRNAs for a two-step screening of early lung cancer, based on their reported sensitivity and specificity and the fact that are not influenced by hemolysisa. Since smoking habit or inflammatory conditions may influence miRNA levels in serum, we quantified miRNAs of our panels in three groups of controls (non-smokers, smokers and COPD patients) and in stage I-II NSCLC patients. Droplet digital PCR was applied for quantification of miRNAs. Results. The two-step screening is composed of a panel of 4 miRNAs endowed with high sensitivity and a second panel with high specificity. The chosen miRNAs should allow to identify true positive patients, that would undergo CT scan. For 3 of the 6 miRNAs analyzed there was no significant difference among control subgroups (non-smokers, smokers, and subjects affected by Chronic Obstructive Pulmonary Disease), whereas miRNA levels were expressed at significantly different levels in tumor and control groups, confirming their possible role as biomarkers previously described in the literature. Conclusions. The selected miRNAs may help to identify high risk subjects who need further investigation for the presence of early NSCLC; in particular, miR-223 showed a good accuracy in distinguishing tumor samples from controls.
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Nuclear and cytoplasmic Sox7 staining was also negatively associated with poorly PROGNOSTIC AND PREDICTIVE BIOMARKERS differentiated grades of prostate cancer. The Bath cohort did not have enough Q1 benign tissues to confirm the relationship between cancer and benign tissue, but BROMODOMAIN-CONTAINING PROTEIN 7 (BRD7) IS A CANDIDATE TUMOUR showed a negative association with grade and biochemical relapse. Changes in SUPPRESSOR IN BREAST CARCINOMA AND PROGNOSTIC BIOMARKER Sox7 staining in tissues from both sets of samples was not associated with prostate 1)Lo Nigro C. 2)Garrone O. 3)Vivenza D. 4)Brizio R. 5)Mantovani F. 6)Del Sal G. 7) cancer stages. A second independent antibody confirmed these results in both Crook T. 8)Merlano M.C. cohorts of patient tissue samples. 1)Oncology Department, S. Croce & Carle Teaching Hospital, Cuneo, Italy 2) It can be seen from this preliminary data that Sox7 might play an important role in Oncology Department, S. Croce & Carle Teaching Hospital, Cuneo, Italy 3)Oncology prostate tumour formation and/or aggressiveness and warrants further investigation Department, S. Croce & Carle Teaching Hospital, Cuneo, Italy 4)Pathology to understand its function and establish if it could represent a potential diagnostic Department, S. Croce & Carle Teaching Hospital, Cuneo, Italy 5)Laboratorio or prognostic biomarker. Nazionale CIB (LNCIB), Area Science Park, Trieste, Italy 6)Laboratorio Nazionale CIB (LNCIB), Area Science Park, Trieste, Italy 7)Department of Oncology, Royal Q3 Surrey County Hospital, Guildford, UK 8)Oncology Department, S. Croce & Carle LIQUID BIOPSY AS TOOL TO MONITOR ADVANCED NON-SMALL CELL LUNG Teaching Hospital, Cuneo, Italy CANCER (NSCLC) DURING SYSTEMIC TREATMENT 1)Zulato E. 2)Bonanno L. 3)Nardo G. 4)Del Bianco P. 5)Verza M. 6)Pasqualini L. Background. Multiple genetic and epigenetic factors drive tumour initiation and 7)Attili I. 8)Pavan A. 9)Alberti G. 10)Pasello G. 11)Zago G.12)Frega S. 13)Fassan progression in breast cancer (BC). Bromodomain-containing protein 7 (BRD7), M. 14)Rugge M. 15)De Salvo G.L. 16)Calabrese F. 17)Amadori A. 18)Conte P. 19) member of the bromodomain-containing protein family, is mainly localized in the cell Indraccolo S. nucleus, is a transcriptional co-activator for p53, modulates histone acetylation and 1)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV – promotes p53 target genes. Previous studies show that BRD7 is down-regulated in IRCCS, Padua, Italy 2)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, cancer and this may correlate with clinical outcomes. However, the involvement of Padua, Italy 3)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto BRD7 in BC and its prognostic importance are poorly understood. IOV – IRCCS, Padua, Italy 4)Clinical Purpose of the study. We have investigated the expression, CpG island methylation Trials and Biostatistics Unit, Istituto Oncologico Veneto IOV – IRCCS, Padua, Italy and sub-cellular localization of BRD7 in BC cell lines and clinical cases and thereby assessed its prognostic significance by correlating with clinicopathologic features 5)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV – and time-dependent clinical outcomes. IRCCS, Padua, Italy Materials and methods. BRD7 expression was analysed by qRT-PCR and IHC 6)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV – in 13 BC cancer cell lines. Expression was also assessed by IHC in 50 cases of IRCCS, Padua, Italy 7)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, primary BC and 14 paired metastatic lesions. CpG island methylation was quantified Padua, Italy 8)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Padua, by pyrosequencing. BC cases were divided for nuclear and non-nuclear BRD7 Italy 9)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Padua, Italy localization and then stratified for high and low BRD7 expression. The effects of 10)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Padua, Italy 11) BRD7 expression and clinical data on OS and PFS were evaluated using univariate Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Padua, Italy 12) and multivariate Cox regression analyses. Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Padua, Italy 13) Results. BRD7 was expressed at levels similar to normal breast epithelium and the Department of Medicine, Surgical Pathology Unit, University of Padua, Italy 14) CpG island unmethylated in all 13 cell lines. Methylation analysis was successful in Department of Medicine, Surgical Pathology Unit, University of Padua, Italy 15) 42 FFPE collected, with 26,2% of them methylated (11/42). IHC on primary tissues Clinical Trials and Biostatistics Unit, Istituto Oncologico Veneto IOV – IRCCS, showed that BDR7 is mainly localized Padua, Italy 16)Department of Cardiothoracic and Vascular Sciences, University of in the nucleus (34/50: 68%). Patients with BRD7 nuclear localization (N=34) Padua, Italy 17)Department of Surgery, Oncology and Gastroenterology, University showed smaller tumours (p=0.012), a longer OS (p=0.002) and a lower relapse rate of Padua, Italy 18)Department of Surgery, Oncology and Gastroenterology, (p=0.03). Moreover, nuclear BRD7 expression is associated with wild-type TP53 University of Padua, Italy 19)Immunology and Molecular Oncology Unit, Istituto (p=0.04) and unmethylated BRD7 (p=0.04), compared to the non-nuclear cases Oncologico Veneto IOV – IRCCS, Padua, Italy (N=16). No difference was seen in nodal status, grading, clinical stage, histological Background. Detecting tumor-specific genetic alterations in plasma has the type, ER, PR, HER2 and Ki-67 expression. Patients (N=12) showing both nuclear potential to monitor biological effects of treatment without invasive procedures. localization and high BRD7 expression presented the smallest tumours (10/12 = Aim of this study is to validate the method in advanced EGFR-ALK-ROS1 wild-type 83%) and both a longer OS (p=0,005) and PFS (p=0,001). Among the 4/14 patients NSCLC and explore the correlation of dynamic variation in plasma of tumor-specific with nuclear localization in their primary tumours, 3 (75%) presented a diffuse or mutations with outcome. negative BRD7 in the paired metastasis. Methods. Advanced NSCLCs undergoing systemic anti-cancer treatment were Conclusions. BRD7 is a strong candidate for breast cancer tumor suppressor and prospectively enrolled in this mono-institutional trial (MAGIC-1). Tissue genetic prognostic biomarker. alterations were screened through Mass Array (Sequenom) or Next-generation sequencing. Plasma samples were collected at baseline (T1), after the first cycle of treatment (T2), at first radiological restaging (T3) and at radiological progression Q2 (T4). The first cohort of patients, bearingKRAS mutations in tumor DNA (KRAS-m), Sox7 expression is reduced in prostate cancer and negatively was analyzed by using digital droplet PCR (ddPCR). Semiquantitative index of associated with increasing Gleason grade fractional abundancy (FA) of mutated allele is used. 1)Alghezi D. A. 2)Whitley P. 3)Beresford M. 4)Bowen R. 5)Mitchard J. 6)Chalmers Results. Between January and December 2017, we prospectively enrolled 161 A.D. pts, including 55 (33%) treated with immunotherapy. Here, we depict the KRAS-m 1)Biology and biochemistry, Bath university, Bath, Uk 2)Biology and biochemistry, cohort (n= 45). At baseline, KRAS mutations in cfDNA were detected in 21 out Bath university, Bath, Uk 3)Oncology, Royal united hospital, Bath, Uk 4)Oncology, of 43 samples analyzed (49% sensitivity). Predictive value of KRAS mutation in Royal united hospital, Bath, Uk 5)Histopathology, Royal united hospital, Bath, Uk 6) plasma was statistically significant at T3 with an AUC 0.73 (95%CI: 0.55-0.91, Biology and biochemistry, Royal united, Bath, Uk p-value=0.0132); sensitivity and specificity for non-PD at a cut-off value of 0.013 FA were 58% (95% CI 28-85) and 89% (95% CI 67-99), respectively. The reduction of The aim of our study is to investigate the hypothesis that proteins that have been FA (T1-T3) showed an overall ability to discriminate between groups (PD vs non- linked to stem cells might play an important role in prostate cancer progression PD) of 85% (95% CI: 72-97, p-value<0.0001) with a sensitivity for detection of non- and could be potential diagnostic or prognostic biomarkers. One of the potential PD of 67% (95% CI 35-90) and a specificity of 89% (95% CI 67-99). In the subgroup biomarkers that is being investigated is Sox7, a zinc finger transcriptional factor of patients undergoing immunotherapy (n=11), the predictive value of reduction of which has a role in stem cell development and oncogeneses, including a possible FA (T1-T3) was 86% (95% CI: 58-100, p-value 0.0124), with a sensitivity of 100% role in prostate cancer progression and relapse, making it an important protein for (95% CI 16-100) and a specificity of 86% (95% CI 42-100). further analysis. Conclusions. KRAS mutations were found at baseline in 49% (95% CI 33.3- Nuclear and cytoplasmic Sox7 staining was evaluated by immunohistochemistry 64.5) of the KRAS-m cohort and in 45.5% (95% CI 16.7-76.6) of pts treated with using two sources of patient samples, a tissue microarray group and a Bath cohort. immunotherapy. Dynamic variations of KRAS mutation in plasma significantly The tissue microarray group consists of 96 cases including normal prostate, correlate with radiological response, indicating the feasibility of detecting KRAS adjacent normal prostate, and prostate cancer samples. The Bath cohort consists of mutation in clinical practice and the potential usefulness of tracking biological 27 samples, including samples from patients that had recurrent disease and those changes in plasma. Prospective trial in pts treated with immunotherapy is ongoing. who remained disease free. Nuclear and cytoplasmic Sox7 staining was decreased significantly in prostate cancer samples compared to benign tissues from the tissue microarray group.
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Q4 Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 10)Biomarkers Unit, MONITORING TUMOR PROGRESSION USING CIRCULATING TUMOR DNA IN Department of Applied Research and Technological Development, Fondazione EARLY AND LOCALIZED TRIPLE NEGATIVE BREAST CANCER PATIENTS IRCCS Istituto Nazionale dei Tumori, Milano, Italy 1)Ortolan E. 2)Appierto V. 3)Di Cosimo S. 4)Veneroni S. 5)De Marco C. 6)Cavadini E. 7)Vismara M. 8)Cascone F. 9)Pruneri G. 10)Bianchi G. 11)Daidone M. G. Characterization of circulating tumor cells (CTCs) could enable the monitoring of 1)Biomarkers Unit, Department of Applied Research and Technological tumor progression and of adaption occurring during treatment. Unfortunately CTCs Development, Fondazione IRCCS “Istituto Nazionale dei Tumori, Milan, Italy 2) are very rare and enrichments from blood samples and subsequent identification of Biomarkers Unit, Department of Applied Research and Technological Development, these cells is technically very challenging. Fondazione IRCCS “Istituto Nazionale dei Tumori, Milan, Italy 3)Biomarkers Unit, We have developed a CTC-characterization worflow including Parsortix enrichment Department of Applied Research and Technological Development, Fondazione and visualization, enumeration and recovery of single CTCs and analysis with IRCCS “Istituto Nazionale dei Tumori, Milan, Italy 4)Biomarkers Unit, Department DEPArray™platform. Cells were stained for epithelial markers (EpCAM, CK, of Applied Research and Technological Development, Fondazione IRCCS “Istituto EGFR) and white blood cells markers (CD45, CD14, CD16) and harvested as Nazionale dei Tumori, Milan, Italy 5)Biomarkers Unit, Department of Applied putative CTCs classified as epithelial cells (positive only for epithelial markers) or Research and Technological Development, Fondazione IRCCS “Istituto Nazionale non-conventional cells (negative for all markers). Cells were processed to obtain dei Tumori, Milan, Italy 6)Biomarkers Unit, Department of Applied Research and Whole Genome Amplification products (Ampli1 WGA kit, Silicon Biosystems), then Technological Development, Fondazione IRCCS “Istituto Nazionale dei Tumori, multiplexed sequencing-ready libraries were generated (Ampli1™ Low pass kit, Milan, Italy 7)Biomarkers Unit, Department of Applied Research and Technological Silicon Biosystems) for Whole Genome Sequencing to evaluate Copy Number Development, Fondazione IRCCS “Istituto Nazionale dei Tumori, Milan, Italy 8) Alteration (CNA) (Ion Torrent Platforms). Biomarkers Unit, Department of Applied Research and Technological Development, This workflow allowed to select eCTCs and/or ncCTCs in samples obtained from Fondazione IRCCS “Istituto Nazionale dei Tumori, Milan, Italy 9)Department of patients of different types of solid tumors, as breast, kidney, gynecological and Pathological Anatomy, Fondazione IRCCS “Istituto Nazionale dei Tumori”, Milan, biliary tract cancer (BTC) and to confirm their cancer nature at genomic level. In Italy 10)Department of Medical Oncology, Fondazione IRCCS “Istituto Nazionale particular, in BTC we observed a difference between CTC-phenotype in patients dei Tumori”, Milan, Italy 11)Biomarkers Unit, Department of Applied Research and with intrahepatic and extrahepatic disease, showing a lower presence of eCTC Technological Development, Fondazione IRCCS “Istituto Nazionale dei Tumori, alone in extrahepatic patients compared to the intrahepatic ones. In BTC patients Milan, Italy we detected also a difference in CTC subpopulations between baseline and treated patients, observing CTCs in 100% of patients before treatment dropping to 80% Triple negative breast cancers (TNBC) are characterized by lack of treatment after therapy. Furthermore, in a few gynecological patient samples we detected a targets, aggressive behavior and frequent DNA mutations. Tissue biopsies subpopulation of putative CTCs expressing a new marker for cancer mesenchymal represent the gold standard to profile tumor genomic changes, but are invasive and cells (cell surface vimentin, CSV) that could be useful to better discriminate CTC only partially reflect tumor heterogeneity. Circulating biomarkers such as circulating subpopulations also in other solid cancers. tumor DNA (ctDNA) and circulating tumor cells (CTCs) could track disease Globally with this new workflow in all clinical samples, we detected ncCTCs in progression, treatment response and identify new potential treatment targets. We comparable number with respect to eCTCs, hence isolating a CTC population that previously demonstrated the feasibility of anticipating new disease manifestations would have been lost using positive selection based only on epithelial markers. by ctDNA tracking in serial plasma samples collected during post-surgery follow-up Such CTC might be relevant to better understand the role of epithelial-mesenchymal from patients with stage I breast cancer undergoing surgery more than 20 years transition (EMT) in cancer spread. Our results support the importance of using ago in our Institute. Based on these results, a prospective observational study was an unbiased method for CTC phenotypical selection in order to maximize the activated in patients with early/localized TNBC to 1) confirm the potential of ctDNA informative content of liquid biopsies. in tracing minimal residual disease and in the early detection of recurrences; 2) characterize progressive disease through direct ctDNA sequencing and CTCs analysis. As of May 2018, blood of 105 patients has been longitudinally collected Q6 every 4-6 months during follow-up, with a total of 284 plasma samples. So far tumor MONITORING ADVANCED NSCLC THROUGH PLASMA GENOTYPING DURING tissue mutational analysis has been successfully performed in 75% of cases by IMMUNOTHERAPY: PRELIMINARY RESULTS targeted next generations sequencing. Until now the identified somatic mutations 1)Boscolo A. 2)Zulato E. 3)Bonanno L. 4)Nardo G. 5)Verza M. 6)Pasqualini L. 7)Del were analyzed in plasma from 81% of patients by digital PCR assays. Samples Bianco P. 8)Attili I. 9)Pavan A. 10)Alberti G. 11)Pasello G. 12)Zago G. 13)Frega S. 14) collected at surgery were positive for ctDNA in 43% of cases in the adjuvant setting Fassan M. 15)Rugge M. 16)De Salvo G.L. 17)Calabrese F. 18)Amadori A. 19)Conte while ctDNA became undetectable after tumor resection. In the neoadjuvant setting P. 20)Indraccolo S. 70% of patients were positive for ctDNA before starting chemotherapy. ctDNA 1)Department of Surgery, Oncology and Gastroenterology, University of Padua, analysis anticipated of 7 months the metastatic recurrence of a neoadjuvant patient. Italy 2)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV– The TP53 mutation (H179Q), characterizing the patient primary tumor and the IRCCS, Italy 3)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Italy residual disease after chemotherapy, was undetectable at surgery but started to 4)Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV– increase 5 months after tumor resection (64 copies/ml of plasma) and continued to IRCCS, Italy 5)Immunology and Molecular Oncology Unit, Istituto Oncologico dramatically increase in the post-surgery plasma samples until clinical diagnosis Veneto IOV– IRCCS, Italy 6)Immunology and Molecular Oncology Unit, Istituto of liver metastasis (22.000 copies/ml of plasma). The mutation was also found in a Oncologico Veneto IOV– IRCCS, Italy 7)Clinical Trials and Biostatistics Unit, Istituto CTC collected at relapse. Currently, direct sequencing of ctDNA is ongoing to define Oncologico Veneto IOV – IRCCS, Italy 8)Medical Oncology 2, Istituto Oncologico the genetic events of metastatic disease. Preliminarily, these results indicate that Veneto IOV – IRCCS, Italy 9)Medical Oncology 2, Istituto Oncologico Veneto IOV – ctDNA monitoring can identify breast cancer patients at high risk of relapse and IRCCS, Italy 10)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Italy might be included as a part of follow-up management. 11)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Italy 12)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Italy 13)Medical Oncology 2, Istituto Oncologico Veneto IOV – IRCCS, Italy 14)Department of Medicine, Surgical Q5 Pathology Unit, University of Padua, Italy 15)Department of Medicine, Surgical FEASIBILITY OF A PROTOCOL FOR SINGLE-CELL ANALYSIS OF Pathology Unit, University of Padua, Italy 16)Clinical Trials and Biostatistics Unit, CIRCULATING TUMOR CELLS IN PATIENTS WITH SOLID TUMORS Istituto Oncologico Veneto IOV – IRCCS, Italy 17)Pathology Unit, Department of 1)Vismara M. 2)Reduzzi C. 3)Silvestri M. 4)Motta R. 5)Miodini P. 6)Martinetti A. 7) Cardiothoracic Sciences, University of Padua, Italy 18)Department of Surgery, Sottotetti E. 8)Cascone F. 9)Daidone M.G. 10)Cappelletti V. Oncology and Gastroenterology, University of Padua, Italy 19)Medical Oncology 1)Biomarkers Unit, Department of Applied Research and Technological Development, 2, Istituto Oncologico Veneto IOV – IRCCS, Italy 20)Immunology and Molecular Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 2)Biomarkers Unit, Oncology Unit, Istituto Oncologico Veneto IOV– IRCCS, Italy Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 3)Biomarkers Unit, Department Background. Detecting genetic alterations in plasma has the potentiality to monitor of Applied Research and Technological Development, Fondazione IRCCS Istituto biological effects of treatment without invasive procedures. We are going to perform Nazionale dei Tumori, Milano 4)Biomarkers Unit, Department of Applied Research a prospective trial (MAGIC-1) in order to validate this method in advanced EGFR- and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, ALK-ROS1 wild-type NSCLC and explore the correlation of dynamic variation of Milano, Italy 5)Biomarkers Unit, Department of Applied Research and Technological tumor-specific mutations in plasma with outcome. Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 6) Methods. Advanced NSCLC patients undergoing anti-cancer systemic treatment Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, are prospectively enrolled in this trial. Tissue genetic alterations are screened Italy 7)Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, through Sequenom massarray system and Next-generation sequencing panel. Milano, Italy 8)Biomarkers Unit, Department of Applied Research and Technological Plasma samples are collected at baseline (T1), after the first cycle of treatment (T2), Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 9) at the moment of first radiological restaging (T3) and radiological progression (T4). Biomarkers Unit, Department of Applied Research and Technological Development, The first cohort of patients (KRAS-m) is analyzed in plasma by using digital droplet
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PCR (ddPCR). Semiquantitative index of fractional abundancy (FA) of mutated Q8 allele is used. Primary end-point of MAGIC-1 study is to assess the sensitivity of TLR3 expression has opposing role and prognostic significance technologies to detect genetic alterations in plasma. in tumor and immune cells in human NSCLC smokers Results. Since January 2017 we prospectively enrolled 156 patients, including 1)Bianchi F. 2)Milione M. 3)Casalini P. 4)Camisaschi C. 5)Centonze G. 6)Le Noci 51 (33%) treated with immunotherapy. 72 tissue samples have been analyzed V.M. 7)Storti C. 8)Alexiadis S. 9)Truini M. 10)Sozzi G. 11)Pastorino U. 12)Balsari A. by Sequenom or by NGS analysis: 43 (27%) patients were positive for KRAS 13)Tagliabue E. 14)Sfondrini L. mutations, including 11 who underwent immunotherapy. Here we depicted four 1)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, representative cases analyzed by ddPCR. Two out of four patients showed partial Italy 2)Pathological Anatomy Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, response (PR) whereas two experienced progression disease (PD). One patient Milan, Italy 3)Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei experiencing PD was negative at baseline and showed an increase of FA of KRAS Tumori, Milan, Italy 4)Immunotherapy of Human Tumors Unit, Fondazione IRCCS mutation at T2 when clinical progression was observed. The other patients were all Istituto Nazionale dei Tumori, Milan, Italy 5)Pathological Anatomy Unit, Fondazione positive at T1. One patient presented an increase of FA at T2 and T3 and showed IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Molecular Targeting Unit, radiological progression. A decrease of FA was observed in the other two patients at Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7)Università degli T3. Consistent with molecular data, these two patients showed partial response at Studi di Milano, Dipartimento di Scienze Biomediche per la Salute, Milan, Italy the first radiological restaging (T3). 8)Pathological Anatomy Unit, ASST Grande Ospedale Metropolitano Niguarda, Conclusions. Our preliminary results are promising and indicate the feasibility of Piazza dell’Ospedale Maggiore, Milan 9)Pathological Anatomy Unit, ASST Grande detecting KRAS mutation in clinical practice and the potential usefulness of tracking Ospedale Metropolitano Niguarda, Piazza dell’Ospedale Maggiore, Milan 10) biological changes in plasma. We are going to complete analysis in the cohort of Tumor Genomics Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, KRAS patients to further validate this method. Italy 11)Thoracic Surgery Unit, Department of Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan 12)Università degli Studi di Milano, Dipartimento di Scienze Biomediche per la Salute, Milan, Italy 13)Molecular Targeting Unit, Q7 Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 14)Università degli A Comprehensive genomic profiling of KRAS/NRAS/BRAF/PIK3CA Studi di Milano, Dipartimento di Scienze Biomediche per la Salute, Milan, Italy WILD-TYPE METASTATIC COLORECTAL CANCER patients from the CAPRI CLINICAL trial TLR3 is expressed on immune and epithelial cells, responding to microbial signals 1)Rachiglio A.M. 2)Lambiase M. 3)Fenizia F. 4)Iannaccone A. 5)Roma C. 6)Cardone to activate the innate immune response. The prognostic value of TLR3 is debated in C. 7)De Luca A. 8)Martinelli E. 9)Maiello E. 10)Ciardiello F. 11)Normanno N. cancer and no data are available on TLR3 expression in NSCLC, for which therapeutic 1)Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. approaches that target the immune system are currently under investigation, Pascale”-IRCCS, Naples, Italy 2)Cell Biology and Biotherapy Unit, Istituto Nazionale including TLR3 agonists. 80% of NSCLC patients are smokers and cigarette smoke Tumori “Fondazione G. exposure, in addition to inducing dysplastic epithelial changes, has been reported Pascale”-IRCCS, Naples, Italy 3)Cell Biology and Biotherapy Unit, Istituto to affect immune microenvironment by hyperactivating or attenuating local innate Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy 4)Cell Biology and and adaptive immunity. The aim of this study was to determine the expression and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, prognostic significance of TLR3 in NSCLC, also according with smoking habits. Italy 5)Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. In a cohort of 194 NSCLC stage I patients, TLR3 immunohistochemistry expression Pascale”-IRCCS, Naples, Italy 6)Università della Campania “L. Vanvitelli”, Naples, on tumor cells predicted a favorable outcome in NSCLC, whereas that on immune Italy 7)Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. cells that infiltrated the tumor stroma was associated with poor overall survival. Pascale”-IRCCS, Naples, Italy 8)Università della Campania “L. Vanvitelli”, Naples, While no association between TLR3 expression levels and smoking habit was Italy 9)IRCCS Casa Sollievo della Sofferenza, Italy 10)Università della Campania found, opposing prognostic functions of this receptor were limited in smokers. In “L. Vanvitelli”, Naples, Italy 11)Cell Biology and Biotherapy Unit, Istituto Nazionale nonsmokers or ex-smokers, no significant association between TLR3 expression Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy in the tumor infiltrate or tumor cells and prognosis was observed. We found that TLR3 expression on lung cancer cells induced apoptosis, which in turn activated Background. Metastatic colorectal cancer (mCRC) KRAS exon 2 wild-type (wt) CD103+ lung dendritic cells, as demonstrated by flow cytometry analysis after in patients enrolled in the Cetuximab After Progression in KRAS wild type colorectal vitro co-culture. We identified the TLR3-expressing immune cells as macrophages, cancer (CAPRI) received first-line FOLFIRI-cetuximab. Retrospective analysis on which TLR3 expression associates with the immunosuppressive cytokine IL-10. showed that mCRC patients with KRAS/NRAS/BRAF/PIK3CA wt (quadruple-wt) In conclusion, TLR3 has opposing prognostic significance in NCSCL, depending tumors had a better prognosis when treated with anti-EGFR agents. In order to on its distinct functions in immune and tumor cells. The effects of TLR3 agonists identify additional mechanisms that might allow to select patients who benefit of in NSCLC patients should be examined further with regard to TLR3 expression in anti-EGFR monoclonal antibodies, we analyzed quadruple-wt tumors from the cancer and immune cells, and considering smoking habits. CAPRI clinical trial with a large targeted sequencing panel and correlated results with patients’ outcome. Methods. NGS analysis was performed by the Oncomine Comprehensive Panel. Q9 The panel covers 143 cancer genes providing information on hotspot mutations of Identification of a 3-miRNAs signature in human osteosarcoma 73 oncogenes, copy number variation (CNV) of 49 genes, full-length sequence of discriminating patient prognosis 26 tumor suppressor genes, and sequence of 22 fusion driver genes. Data analysis 1)Chiadini V. 2)Romano G. 3)Veneziano D. 4)Nigita G. 5)Fadda P. 6)Croce C.M. 7) was carried out by Ion Reporter™ Software. Scotlandi K.. Results: Tumors from 21 quadruple-wt patients enrolled in the CAPRI clinical trial 1)CRS Development of Biomolecular Therapies, Experimental Oncology were sequenced. Median progression-free survival (mPFS) of this cohort of patients Laboratory, Rizzoli Orthopaedic Inst., Bologna, Italy 2)Division of Pulmonary was 10.7 months (mos) and median overall survival (mOS) of 32.6 mos, which is Disease and Critical Care Medicine, School of Medicine, Virginia Commonwealth comparable to the whole cohort of CAPRI patients. The analysis revealed in all 21 University, Richmond, Virginia, USA 3)Department of Cancer Biology and Genetics, tumors the presence of at least one mutation, and in 10/21 showed the presence Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA 4) of at least one CNV. Department of Cancer Biology and Genetics, Comprehensive Cancer Center, The Variants were detected in the following genes: TP53 (22 mutations), APC (19), Ohio State University, Columbus, Ohio, USA 5)CCC Genomics Shared Resource, FBXW7 (3), MAP2K1 (1), PTPN11 (1), PTCH1 (2), ATM (1), CTNNB1 (1), PIK3R1 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA 6) (1), PTEN (1), CDKN2A (1), KRAS (1), NF1 (2). CNVs were found in APC, TP53, Department of Cancer Biology and Genetics, Comprehensive Cancer Center, The PIK3R1, BCL2L1, GAS6, MYC, ZNF217, FLT3, ERBB2, APEX1. Ohio State University, Columbus, Ohio, USA 7)CRS Development of Biomolecular We observed that patients (N.7) with the shortest PFS (≤ 8 mos) had peculiar Therapies, Experimental Oncology Laboratory, Rizzoli Orthopaedic Inst., Bologna, genetic alterations that might be associated with resistance to anti-EGFR agents. In Italy particular, this subgroup was significantly enriched for mutations in genes involved in RAS/RAF/MEK (KRAS, MAP2K1 and NF1), Sonic Hedgehog (Shh) (PTCH1) and Osteosarcoma (OS) is the most frequent primary bone tumor and is characterized mTOR (FBXW7) pathways as compared with patients with longer PFS. by an aggressive and metastatic potential. A way to improve the prognosis of OS Conclusions. Our preliminary data show that quadruple-wt CRC carry genetic patients is the discovery of novel biomarkers able to predict patient prognosis. alterations that potentially can drive resistance to anti-EGFR monoclonal antibodies. Prognostic relevance of miRNAs has emerged in recent years, due to their tissue- While the significance of the identified variants needs to be explored in experimental and cell-specificity. The aim of this study is to identify a miRNA signature at diagnosis models, these findings do suggest that wide genetic profiling of CRC might improve of human osteosarcoma, able to predict patients with high probability to relapse. our ability to select patients who are highly sensitive to EGFR inhibition. As a pilot study, a total of 14 samples derived from OS biopsies were submitted for small RNA Illumina sequencing. Patients were affected by high grade OS, localized at the extremities and with no evidence of metastatic disease at diagnosis. The age at diagnosis was under 40 years and all patients were treated following
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neoadjuvant protocol. The median of time of recurrence for relapsed (REL) patients glioma model, to assess intratumoral HIF-1α activity as an early biomarker of GBM is of 23 months (range 14-31 months), while median of follow up for patients with no responsiveness to TMZ. evidence of disease (NED) is of 151 months (range 44-207 months). Methods. We performed in vitro experiments in a panel of glioma cell lines after The bioinformatics analyses performed compared NED vs REL groups. As a result, specific gene silencing and pharmacological treatments. In vivo experiments were seven miRNAs (hsa-miR-183-5p, hsa-miR-130b-5p, hsa-miR-99b-3p, hsa-miR- performed in orthotopic glioma model expressing concomitantly the luciferase 139-5p, hsa-miR-195-5p, hsa-miR-3687, hsa-miR-335-3p) were found significantly reporter gene under control of HRE (Hypoxia Responsive Elements) sequences and deregulated between the two groups. This signature has been evaluated by qPCR the m-Cherry fluorescent gene under control of a constitutive promoter. Fluorescent in a larger OS cohort. The following validation was conducted on 78 RNA samples injectable probes were used to simultaneously study glioma features by means of derived from OS biopsies: 35 patients belonging to the NED group, while 43 to the optical non-invasive imaging. REL group. Quantitative expression analysis was performed using 2^- (delta Ct) Results. In vitro analyses highlighted the relationship between HIF-1α and luciferase method in order to compare the average of the two groups. Three of seven miRNAs activity, validating our cellular models as an imaging tool to selectively monitor HIF- were validated and were significantly differentially expressed in the two groups: hsa- 1α activity. Moreover, the reliability of HIF-1α as early biomarker of TMZ response miR-99b-3p (Student’s t test p-value = 0.001), hsa-miR-130-3p (p-value = 0.016), in glioma models has been validated after multiple treatments, in vitro and in vivo, hsa-miR-139-5p (p-value = 0.045). by means of optical imaging techniques. HIF-1α was also identified as a key player To better understand the functions of validated miRNAs, a gene expression profiling in GBM responsiveness to TMZ, since its depletion by silencing is able to revert the experiment was performed using HTA 2.0 Affimetrix microarrays for the same resistant cells to a “sensitive-like” pattern. samples used for the small RNA-seq pilot study. Gene set enrichment analyses was Finally, we suggested the chaperone mediated-autophagy (CMA) as the main performed via Ingenuity® Pathway Software (IPA) for putative and anticorrelated mechanism by which HIF-1α is degraded after TMZ treatment in sensitive cells, target genes of miR-130b-5p and miR-139-5p. Results showed the involvement of since CMA impairing is able to revert sensitive cells to a “resistant-like” behaviour. these miRNAs in immunological processes and cellular metabolism, underlining the Conclusions. The powerful imaging strategy described herein will be suitable for in importance of these mechanisms in the pathogenesis and response to therapy in OS. vivo monitoring of key tumoral features related to responsiveness to treatment and Grants: NIH/NCI R35CA197706 to CMC and AIRC_IG18451 to KS. HIF-1α activity with the aim to validate specific new treatments with fast translatable potential. Furthermore, our findings demonstrate the central role of HIF-1α activity Q10 in maintaining the TMZ-resistance of GBM cells, and CMA activity as the main MICRORNA-567 DYSREGULATION CONTRIBUTES TO CARCINOGENESIS mechanism mediating HIF-1α modulation after TMZ treatment in sensitive cells. OF BREAST CANCER, TARGETING TUMOR CELL PROLIFERATION, AND Acknowledgement. This study was supported in part by the FP7-funded INSERT MIGRATION project (HEALTH-2012- INNOVATION-1, GA305311). 1)Bertoli G. 2)Cava C. 3)Diceglie C. 4)Martelli C. 5)Rizzo G. 6)Piccotti F. 7)Ottobrini L. 8)Castiglioni I. 1)Institute of Molecular Bioimaging and Physiology, National Research Council Q12 (IBFM-CNR), Segrate-Milan, Milan, Italy 2)Institute of Molecular Bioimaging and Impact of TRF2 on miRNAs regulation in colorectal cancer Physiology, National Research Council (IBFM-CNR), Segrate-Milan, Milan, Italy 3) Dinami R., Zizza P., Ganci F., Petti E., Sacconi A., Blandino G., Biroccio.A. Tecnomed Foundation, University of Milano-Bicocca, Monza, Italy 4)Department of Oncogenomic and Epigenetic Unit, Regina Elena National Cancer Institute, Rome, Pathophysiology and Transplantation, University of Milan, Milan, Italy 5)Department Italy of Medical Oncology, IRCCS Fondazione Maugeri, Pavia, Italy 6)Laboratory of Experimental Oncology and Pharmacogenomics, Institutional Oncologic Bio-Bank The management of metastatic colorectal cancer (mCRC) patients remains “Bruno Boerci”, IRCCS Fondazione Salvatore Maugeri, Pavia, Italy 7)Department challenging by the emergence of primary or secondary resistance to conventional of Pathophysiology and Transplantation, University of Milan, Milan, Italy 8)Institute and/or targeted therapies, thus making necessary more effective treatments. of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), The Telomeric Repeat binding Factor 2 (TRF2), one of the main regulators of Segrate-Milan, Milan, Italy telomere integrity, has been found overexpressed in various human malignancies, including CRC. Interestingly, the tumorigenic functions of TRF2 do not depend Purpose. We demonstrated that Hsa-miR-567 expression is significantly solely on telomere protection but also on extra-telomeric functions such as the downregulated in poor prognosis breast cancer, compared to better prognosis transcriptional regulation. breast cancer, having a role in the control of cell proliferation and migration by Here, we investigated the impact of TRF2 on the regulation of miRNAs, a class of regulating KPNA4 gene. small non-coding RNAs targeting specific “genes”, that play a key role as predictive Methods and results. In this study, based on our previously published in silico and prognostic biomarkers. results, we proved both in vitro (cell line studies) and ex vivo (clinical studies), that Preliminary experiment preformed on HCT116 CRC cells silenced for TRF2, Hsa-miR-567 expression is significantly downregulated in breast cancer with poor evidenced that TRF2 deregulates a panel of miRNAs. Among them, miR-193b-3p prognosis when compared to breast cancer with better prognosis. More intriguingly, shows a positive correlation with TRF2 we demonstrated that the ectopic expression of Hsa-miR analysis performed on The Cancer Genome Atlas (TCGA) CRC dataset. Moreover, 567 in poor prognosis breast cancer cell line strongly inhibits in vitro cell proliferation increased levels of miR-193b-3p were found to correlate with tumor stage, and migration. Furthermore, we showed in vivo that breast cancer cells, stably metastasis, and lymph node positivity. expressing Hsa-miR-567, xenografted in mouse, reduce tumor growth ability. According to these data, in vitro experiments suggest that miR-193b-3p can drive Consistently, we found that karyopherin 4 (KPNA4), predicted target gene of Hsa- the cells toward an aggressive phenotype, opening new strategy for patients miR-567 as identified by our in silico analysis, is upregulated in highly aggressive resistant to conventional and /or targeted therapies. MDA-MB-231 breast cancer cell line and patient tissues with poor prognosis with respect to those with good prognosis. Conclusions. Our results suggest a potential role of Hsa-miR-567 as a novel prognostic biomarker for breast cancer and as regulator of KPNA4. Q11 HIF-1a ACTIVITY AS A MOLECULAR AND OPTICAL IMAGING BIOMARKER OF GBM RESPONSIVENESS TO TMZ 1)Lo Dico A. 2)Salvatore D. 3)Martelli C. 4)Diceglie C. 5)Lucignani G. 6)Ottobrini L. 1)Molecular Bioimaging and Physiology (IBFM), CNR, Segrate-Milan, Italy 2) Molecular and Translational Medicine Doctorate School, University of Milan, Milan, Italy 3)Department of Pathophysiology and Transplantation, University of Milan, Segrate-Milan, Italy 4)Department of Pathophysiology and Transplantation, University of Milan, Segrate-Milan, Italy 5)San Paolo Hospital, Department of Diagnostic Services, Unit of Nuclear Medicine, Milan, Italy 6)Molecular Bioimaging and Physiology (IBFM), CNR, Segrate-Milan, Italy Rationale. The activity of the transcription factor, hypoxia-inducible factor (HIF)-1α, is a common driver of a number of the pathways involved in the aggressiveness of glioblastomas (GBM), and it has been suggested that the reduction in this activity, observed soon after the administration of Temozolomide (TMZ), is a biomarker of an early response in GBM models. In this research, molecular imaging has been applied to the study of preclinical
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Q13 by comparing the gene expression patterns of two patient cohorts characterized by Claudin-7 downregulation is predictive of distant metastases in extreme overall survival (OS). high-grade serous ovarian carcinoma patients Methods. We determined the gene expression profiles of 12 HGSOC long-term 1)Romani C. 2)Zizioli V. 3)Corsini M. 4)Todeschini P. 5)Zanotti L. 6)Ravaggi A. 7) (OS>7 years) and 27 short-term (OS<3 years) survivors (training set) by Affymetrix Odicino F. 8)Mitola S. 9)Bignotti E. 10)Calza S. genechip. By a generalized linear model with cross-validation, we generated a 1)Molecular and translational medicine, University of Brescia, Brescia, Italy 2) prognostic gene signature that was further evaluated on the entire “The Cancer Division of obstetrics and gynecology, Asst spedali civili, Brescia, Italy 3)Molecular Genome Atlas” (TCGA) ovarian cancer dataset. The resulting genes were then and translational medicine, University of Brescia, Brescia, Italy 4)A. Nocivelli institute verified on an independent cohort of 29 HGSOC flash frozen samples (validation set) for molecular medicine, University of Brescia, Brescia, Italy 5)A. Nocivelli institute by RT-qPCR, and in a wider panel of 38 formalin fixed paraffin embedded HGSOC for molecular medicine, University of Brescia, Brescia, Italy 6)Division of obstetrics tissues by immunohistochemistry (IHC). Correlations of biomarker expression to and gynecology, University of Brescia, Brescia, Italy 7)Division of obstetrics and clinicopathological parameters and patients’ prognosis were evaluated by Kaplan- gynecology, Asst spedali civili, Brescia, Italy 8)Molecular and translational medicine, Meier and Cox proportional-hazards analyses. University of Brescia, Brescia, Italy 9)Division of obstetrics and gynecology, Asst Results. We identified a ten-gene signature able to correctly assign 98% of patients spedali civili, Brescia, Italy 10)Molecular and translational medicine, University of within their survival class. By “in silico” validation on TCGA dataset, we confirmed Brescia, Brescia, Italy the differential expression and the prognostic power of the FXYD domain containing ion transport regulator 5 (FXYD5), one of the 10 top score genes of the signature. Background. High-grade serous carcinoma (HGSOC) is the most frequent and lethal FXYD5 mRNA was successfully validated by RT-qPCR on both the training and ovarian carcinoma histotype. The majority of patients present at an advanced stage, the validation sets. By IHC, we demonstrated, for the first time, FXYD5 protein with widespread metastasis within the peritoneal cavity. Although the abdominal overexpression in short-term survivors compared to long-term ones. FXYD5 dissemination is considered the most common, distant metastases occur in about overexpression, both at mRNA and protein level, was significantly associated with 30% of patients with newly diagnosed or recurrent HGSOC. platinum resistance and cancer progression. Noteworthy, FXYD5 overexpression Tight-junction protein claudin-3, 4 and 7 are frequently dysregulated in HGSOC and was an independent prognostic marker for poor OS and PFS in multivariate Cox functionally related to cancer progression to a metastatic disease. regression analyses. Aim. The aim of this study was to analyse expression of claudin-3, 4 and 7 in Conclusion. We demonstrated the consistent overexpression of FXYD5 in HGSOC HGSOC samples and to correlate it to the site and pattern of metastasis. short-term survivors compared to long-term ones. FXYD5 may become a useful Methods. Expression of claudin-3, 4 and 7 was evaluated in a cohort of 105 HGSOC predictive marker for a more accurate selection of HGSOC patients for adjuvant fresh-frozen biopsies, 26 fallopian tube and 9 ovarian surface epithelium (Hose), treatments, and a possible target for antibody-drug conjugated anticancer agents. using RT-qPCR and IHC. Information regarding stage, recurrence, site of metastasis and pattern of tumor spread were recorded. The relationship between claudin expression and clinicopathological parameters was evaluated using univariate and Q15 multivariate linear models and logistic models. Identification of hypoxia-regulated miRNAs associated with Results. We compared claudin mRNA expression in HGSOC, Hose and tube platinum-resistance in high-grade serous ovarian carcinoma brushings, both considered as putative site of HGSOC origin. 1)Todeschini P. 2)Salviato E. 3)Romani C. 4)Zanotti L. 5)Ravaggi A. 6)Ferrari F. 7) We found the three claudin to be correlated (Pearson’ r = 0.65-0.77, all p <0.001), Odicino F. 8)Sartori E. 9)D’incalci M. 10)Marchini S. 11)Samaja M. 12)Romualdi C. consistently highly downregulated in Hose compared to HGSOC samples (all p< 13)Bignotti E. 0.001) but markedly upregulated in normal tube compared to HGSOC (all p < 0.001). 1)Obstetrics and gynecology, Asst spedali civili of Brescia, Brescia, Italy 2)Biology, IHC performed on a subset of 39 matched FFPE tumors, 5 normal tube and 5 University of Padova, Padova, Italy 3)Molecular and translational medicine, normal ovary samples, show a positive correlation with claudin transcriptional levels University of Brescia, Brescia, Italy 4)Obstetrics and gynecology, University of as determined by RT-qPCR. Brescia, Brescia, Italy 5)Obstetrics and gynecology, University of Brescia, Brescia, We observed a tendency toward downregulation of claudin-4 and 7 in advanced Italy 6)Obstetrics and gynecology, Asst spedali civili of Brescia, Brescia, Italy 7) stage tumors (p = 0.07 and 0.09 respectively) and a downregulation of claudin-7 in Obstetrics and gynecology, University of Brescia, Brescia, Italy 8)Obstetrics and patients that relapse to distant organs compared to the abdominal cavity (p=0.016). gynecology, University of Brescia, Brescia, Italy 9)Oncology, Irccs - “Mario Negri” According to the spread pattern, decreased expression of claudin-7 is associated institute for pharmacologica, Milano, Italy 10)Oncology, Irccs - “Mario Negri” institute with a hematogenous-type diffusion modality (p=0.02). for pharmacologica, Milano, Italy 11)Health science, University of milan, Milano, Using a logistic regression we estimate a reduction in the probability of distant Italy 12)Biology, University of Padova, Padova, Italy 13)Obstetrics and gynecology, disease of 39% per unit increase in the level of claudin-7 (p = 0.03). Via bootstrap, Asst spedali civili of Brescia, Brescia, Italy we estimated the corresponding optimism-adjusted AUC to be 65.9% [CI95% 53.2% - 76.4%]. Background. High-grade serous ovarian carcinoma (HGSOC) is the most Conclusions. Our findings point towards claudin-7 downregulation being engaged in lethal gynecologic malignancy, frequently diagnosed at an advanced stage distant metastatic process possibly through hematogenous dissemination. and characterized by the early onset of chemoresistant recurrences. The rapid proliferation of HGSOC cells causes the depletion of available oxygen and the tumor molecular adaptation to hypoxia condition promotes cancer progression and Q14 chemoresistance. In this stress condition, a group of miRNAs, termed “hypoxia- FXYD5 IS A PREDICTOR OF SHORT-TERM SURVIVAL AND POOR PROGNOSIS regulated miRNAs” (HRMs) playing an important role in breast and colon cancers, IN HIGH-GRADE SEROUS OVARIAN CARCINOMA has been reported. The aim of the present study was to identify potential hypoxia- 1)Tassi RA. 2)Gambino A. 3)Ardighieri L. 4)Bugatti M. 5)Romani C. 6)Todeschini P. related miRNAs in HGSOC tissues and to evaluate their association with platinum 7)Zanotti L. 8)Gebbia F. 9)Picardo E. 10)Katsaros D. 11)Bignotti E. 12)Odicino F. resistance and survival. 13)Sartori E. 14)Romualdi C. 15)Ravaggi A. Methods. We selected a panel of 25 miRNAs, either belonging to HRMs or among 1)”A. Nocivelli” institute of molecular medicine, University of Brescia, Brescia, Italy the most reported miRNAs associated with hypoxia in ovarian cancer published 2)Division of obstetrics and gynecology, University of Brescia, Brescia, Italy 3) studies. We evaluated their expression on miRNA-Sequencing data of 178 stage III- Department of pathology, Asst spedali civili di Brescia, Brescia, Italy 4)Department IV HGSOC samples belonging to The Cancer Genome Atlas (TCGA) dataset. Then, of pathology, Asst spedali civili di Brescia, Brescia, Italy 5)Department of molecular the HGSOC-HRM signature identified was evaluated in our cohort of 74 stage III-IV and translational medicine, University of Brescia, Brescia, Italy 6)”A. Nocivelli” HGSOC tissues profiled for miRNA expression by Agilent microarray technology. institute of molecular medicine, University of Brescia, Brescia, Italy 7)”A. Nocivelli” Survival curves were plotted using Kaplan Meier and Cox proportional hazard institute of molecular medicine, University of Brescia, Brescia, Italy 8)Division of regression model was used for survival analysis. DESeq2 analysis was performed obstetrics and gynecology, University of Brescia, Brescia, Italy 9)Dep. of surgical to identify miRNAs associated with response to therapy. We intend to confirm the sciences, gynecol. oncol., University of Turin, Torino, Italy 10)Dep. of surgical HGSOC-HRM signature expression in a larger dataset of 150 HGSOC tissues by sciences, gynecol. oncol., University of Turin, Torino, Italy 11)Division of obstetrics RT-qPCR. and gynecology, Asst spedali civili di Brescia, Brescia, Italy 12)Division of obstetrics Results. On TCGA HGSOC data we identified seven hypoxia-related miRNAs and gynecology, University of Brescia, Brescia, Italy 13)Division of obstetrics and associated with platinum-response and poor survival. On our dataset of 74 gynecology, University of Brescia, Brescia, Italy 14)Department of biology, University HGSOC miRNA microarray profiles, analysis confirmed 4 out of 7 miRNAs showing of Padova, Padova, Italy 15)Division of obstetrics and gynecology, University of predictive and prognostic value. In particular, miR-181c-5p, miR-23a-3p and miR- Brescia, Brescia, Italy 30b-5p emerged overexpressed in chemoresistant samples and, together with miR- 21-5p, associated with poor overall survival in multivariate analysis (all p-values Background. High grade serous ovarian carcinoma (HGSOC) is generally associated <0.05). To date, miR-23a-3p and miR-21-5p overexpression has been confirmed with a very dismal prognosis. Nevertheless, patients with similar clinicopathological by RT-qPCR and associated with worst survival in 99 HGSOC samples (p-values characteristics can have markedly different clinical outcomes. Our aim was to <0.05). Evaluations of the four miRNAs by RT-qPCR on a larger dataset of 150 identify and independently validate the molecular determinants influencing survival HGSOC are ongoing.
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Conclusions. We determined a signature of miRNAs related to hypoxia with potential gene expression of 16 ABCs. predictive and prognostic impact in HGSOC tissues, using independent datasets High levels of ABCA6 and ABCA7 resulted unexpectedly correlated with prolonged and different methods of miRNA quantification. EFS and OVS, further confirmed by univariate analysis in a validation set of 103 tumor samples. Moreover, multivariate analysis revealed that patients with low levels of both Q16 transporters presented a markedly worse outcome. Soluble stroma-related biomarkers of pancreatic cancer Our findings point to ABCA6 and ABCA7 as potentially indicators of prognosis in 1)Resovi A. 2)Bani M. 3)Porcu L. 4)Anastasia A. 5)Minoli L. 6)Allavena P. 7)Cappello ES, supporting a different role from the canonical function of ABC drug extruders. P. 8)Falanga A. 9)Taraboletti G. 10)Belotti D. 11)Giavazzi R. To confirm the prognostic role, we evaluated their expression in a representative 1)Department of Oncology, IRCCS - Istituto di Ricerche Farmacologiche Mario panel of PDX-derived cell lines. Expression levels of both ABCs resulted inversely Negri, Bergamo and Milan, Italy 2)Department of Oncology, IRCCS - Istituto di correlated with ES malignancy, assessed by cellular migration and anchorage- Ricerche Farmacologiche Mario Negri, Bergamo and Milan, Italy 3)Department of independent growth. In addition, they were associated with enhanced sensitivity to Oncology, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Bergamo and several DNA damaging chemotherapeutics, such as Doxorubicin. Milan, Italy 4)Department of Oncology, IRCCS - Istituto di Ricerche Farmacologiche When the analysis was extended to patient-derived cell lines with different TP53 Mario Negri, Bergamo and Milan, Italy 5)Fondazione Filarete and Department mutation status, we found that expression of ABCA7 was higher in TP53 wild- of Veterinary Pathology, University of Milan, Italy 6)Humanitas Clinical and type cells. Accordingly, a higher recruitment of p53 on ABCA7 promoter was Research Center, Rozzano, Italy 7)CeRMS, University of Turin, Italy 8)Department demonstrated after doxorubicin-induced p53 activation, suggesting the involvement of Immunohematology and Transfusion Medicine, Hospital Papa Giovanni of p53 in ABCA7 transcriptional regulation. XXIII, Bergamo, Italy 9)Department of Oncology, IRCCS - Istituto di Ricerche Even though ABCA6 and ABCA7 functions are still unclear, some evidence of a role Farmacologiche Mario Negri, Bergamo and Milan, Italy 10)Department of Oncology, in lipid homeostasis has been recently described. Therefore, we are investigating IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Bergamo and Milan, Italy the association between ABCA7 expression and increased cellular ceramide, a 11)Department of Oncology, IRCCS - Istituto di Ricerche Farmacologiche Mario sphingolipid involved in several processes such as inhibition of cell adhesion and Negri, Bergamo and Milan, Italy doxorubicin-induced apoptosis. Funded by: AIRC IG2016 18451 and Transcan EU project Provabes. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive epithelial malignancies characterized by a relevant amount of tumor stroma. It is usually diagnosed late and has limited treatment options. This study investigates the Q18 potential value of soluble stroma-related molecules as PDAC biomarkers. EXPLORING miR-9 DRIVEN RESISTANCE TO γ-RADIATION AND EGFR Thirty-eight stroma-related candidate biomarkers were selected from published INHIBITORS IN HNSCC proteomic studies on PDAC and analyzed by ELISA and Multiplex Luminex assays 1)Citron F. 2)Rampioni Vinciguerra G.L. 3)Fanetti G. 4)Belletti B. 5)Franchin G. 6) in a first cohort of 25 PDAC patients and 16 healthy subjects. Twelve molecules Baldassarre G. – extracellular matrix proteins and proteolytic fragments, matrix-degrading 1)Division of Molecular Oncology, Department of Translational Research, CRO- enzymes and their inhibitors, growth factors and adhesion molecules – were Aviano National Cancer Institute, IRCCS, Aviano, Italy 2)Division of Molecular found overexpressed in PDAC patients’ plasma compared to healthy subjects. A Oncology, Department of Translational Research, CRO-Aviano National Cancer second confirmatory analysis on an independent cohort of 131 PDAC patients, Institute, IRCCS, Aviano, Italy 3)Division of Radiotherapy, CRO-Aviano National 30 chronic pancreatitis and 131 healthy subjects confirmed the PDAC association Cancer Institute, IRCCS, Aviano, Italy 4)Division of Molecular Oncology, Department for MMP7, CCN2, IGFBP2, TSP2, sICAM1, TIMP1 and PLG. Multivariable of Translational Research, CRO-Aviano National Cancer Institute, IRCCS, Aviano, logistic regression model identified biomarker panels discriminating respectively Italy 5)Division of Radiotherapy, CRO-Aviano National Cancer Institute, IRCCS, PDAC vs healthy subjects (MMP7+CA19.9, AUC=0.99, 99% CI=0.98-1.00), Aviano, Italy 6)Division of Molecular Oncology, Department of Translational (CCN2+CA19.9, AUC=0.96, 99% CI=0.92-0.99) and PDAC vs chronic pancreatitis Research, CRO-Aviano National Cancer Institute, IRCCS, Aviano, Italy (CCN2+PLG+FN+Col4+CA19.9, AUC=0.94, 99% CI=0.88-0.99). MMP7, CCN2, IGFBP2, TSP2 and TIMP1 were found upregulated in plasma Introduction. In Head and Neck Squamous Cell Carcinoma (HNSCC) most and tumor tissues of two genetically engineered mouse models of pancreatic patients are diagnosed with a locally advanced disease. Despite aggressive cancer [Pdx-1-Cre;LSL-KrasG12D and Pdx-1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+] in schedules, the 5-year overall survival is 45%, mainly due to the appearance association with PanIN development, suggesting their potential role in the early of loco-regional recurrences. Clinical and experimental evidences support that detection of PDAC. These markers were also elevated in the plasma and in the HNSCC progression is a stepwise process, in which the regulation of cancer cell tumors of mice bearing patient derived orthotopic PDAC xenografts. plasticity through reversible Epithelial to Mesenchymal Transition (EMT) favors cell In conclusion, the identified stroma-related biomarkers represent potential tools for distant dissemination and mediates drug resistance, possibly leading to recurrence PDAC detection. formation. In our previous study, we validated a signature of 4 EMT-related microRNAs (miRs), that acts as an independent risk factor able to stratify patients at high risk to develop Q17 recurrence in HNSCC. In particular, we demonstrated that miR-9 is significantly High levels of ABCA6 and ABCA7 predict good prognosis in expressed in primary tumors from patients who experienced recurrence respect to primary Ewing sarcoma those that had not. 1)Giudice A.M. 2)Pasello M. 3)Manara M.C. 4)AParra A. 5)Serra M. 6)Picci P. 7) Materials and Methods. We used FaDu and SCC9 cells as a model to evaluate the Scotlandi K. role of miR-9 expression in response to selective pressure of radio- and/or chemo- 1)CRS Development of Biomolecular Therapies, Experimental Oncology Laboratory, therapies, testing both cisplatin and EGFR-targeting agents. Primary HNSCC Istituto Ortopedico Rizzoli, Bologna, Italy 2)CRS Development of Biomolecular samples were used to confirm in vitro evidences. Therapies, Experimental Oncology Laboratory, Istituto Ortopedico Rizzoli, Bologna, Results and Discussion. In vitro experiments show that miR-9 expression Italy 3)CRS Development of Biomolecular Therapies, Experimental Oncology modulates EMT in HNSCC, both impinging on SASH1 and KRT13 expression, and Laboratory, Istituto Ortopedico Rizzoli, Bologna, Italy 4)CRS Development of triggering an up-regulation of N-Cadherin and SP1. Thus, we investigate whether Biomolecular Therapies, Experimental Oncology Laboratory, Istituto Ortopedico miR-9 expression could be linked to survival advantage and tumorigenic potential Rizzoli, Bologna, Italy 5)Pharmacogenomics and Pharmacogenetics Research Unit, of tumor cells, eventually representing an escape mechanism leading to radio- or Experimental Oncology Laboratory, Istituto Ortopedico Rizzoli, Bologna, Italy 6) drug-resistance. We observe that miR-9 knockdown decreases clonogenic survival Pharmacogenomics and Pharmacogenetics Research Unit, Experimental Oncology capability in 2D and 3D growth, both in FaDu and SCC9 cells. Accordingly, in a in Laboratory, Istituto Ortopedico Rizzoli, Bologna, Italy 7)CRS Development of vivo model of HNSCC, miR-9 knockdown dampens tumor cells growth. Biomolecular Therapies, Experimental Oncology Laboratory, Istituto Ortopedico When challenged to growth under the selective pressure of γ-radiation or in Rizzoli, Bologna, Italy presence of EGFR inhibitors, antimiR-9 cells display a strong sensitivity to these treatments and a reduction in cell viability. In vivo validations are in progress. Ewing sarcoma (ES) is a childhood bone tumor characterized by an aggressive Conclusions. Altogether these data strongly suggest that in HNSCC, a subpopulation and metastatic behavior. Overcoming resistance to conventional drugs and of miR-9 expressing cells is intrinsically resistant to the standard radio- and targeted- identifying molecular predictors of outcome represent a primary clinical challenge. therapy, thus confirming the potential prognostic value of miR-9 expression and Canonical ATP-binding cassette (ABC) transporters (e.g., ABCB1, ABCC1) are would possibly lead to the identification of widely recognized as mediators of multidrug resistance in cancer, but they do novel clinical treatment for this group of patients who may benefit for a more not play relevant roles in ES. However, novel functions proposed for several ABC accurate personalized therapy, avoiding unfaithful toxic therapies. suggest that they might also be important for cancer initiation, progression and metastatization, extending their contribution to cancer biology beyond drug export. In a pilot study of 27 patients with primary localized ES, we analysed by qRT-PCR
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Q19 In the multivariate analysis, high IP10 was confirmed as negatively associated Comparative study of high-throughput and serum/plasma with OS (HR=3.097, P=0.014) and IL4 and TNFa remain negatively (HR=2.75, focus qPCR-based platforms for the detection of circulating P=0.002) and positively (HR=0.224, P=0.049) associated with DFS, respectively. human microRNAs Simultaneous expression of low IL4 and high TNFa identified patients with best 1)Mariancini A. 2)Gargiuli C. 3)De Cecco L. 4)Sensi M. 5)Boeri M. 6)Sozzi G. 7)Dugo M. prognosis (HR=0.313, P<0.0001). 1)Platform of Integrated Biology, Department of Applied Research and Technology Conclusions. We demonstrated that, among a series of cytokines, IL4 is the most Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 2) significant independent prognostic factor for DFS in resectable PDAC patients, and Platform of Integrated Biology, Department of Applied Research and Technology it could be useful to select patients with high risk of early recurrence who may avoid Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 3) an unnecessary resection. Platform of Integrated Biology, Department of Applied Research and Technology Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 4) Platform of Integrated Biology, Department of Applied Research and Technology Q21 Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 5) IDENTIFICATION OF CIRCULATING MICRORNAs AS EARLY BIOMARKERS OF Unit of Tumour Genomics, Department of Research, Fondazione IRCCS Istituto BREAST CANCER DEVELOPMENT IN A PRE-CLINICAL MOUSE MODEL Nazionale dei Tumori, Milano, Italy 6)Unit of Tumour Genomics, Department 1)Renzi T.A. 2)Tomirotti A.M. 3)Chiodoni C. 4)Dugo M. 5)Cancila V. 6)Perrone M. 7) of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 7) M. Milani 8)Tripodo C. 9)Colombo M.P. Platform of Integrated Biology, Department of Applied Research and Technology 1)Fondazione IRCCS Istituto Nazionale dei Tumori, Molecular Immunology Unit, Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy MILANO, Italy 2)Fondazione IRCCS Istituto Nazionale dei Tumori, Molecular Immunology Unit, MILANO, Italy 3)Fondazione IRCCS Istituto Nazionale dei Background. In the last years circulating microRNAs (cmiRNAs) are emerging as Tumori, Molecular Immunology Unit, MILANO, Italy 4)Fondazione IRCCS Istituto potential non-invasive biomarkers in cancer. Several platforms have been developed Nazionale dei Tumori, Functional Genomics and Bioinformatics Core Facility, to determine the presence of miRNAs in biological fluids, including specific focus MILANO, Italy 5)University of Palermo, Department of Human Pathology, Palermo, panels. In this pilot study, we aimed at comparing different quantitative real-time Italy 6)Fondazione IRCCS Istituto Nazionale dei Tumori, Molecular Immunology PCR (qPCR)-based platforms for detection of miRNAs in blood. Unit, MILANO, Italy 7)Fondazione IRCCS Istituto Nazionale dei Tumori, Molecular Methods. RNA samples were isolated from 8 plasma samples obtained from Immunology Unit, MILANO, Italy 8)University of Palermo, Department of Human lung cancer patients (n=4) and healthy heavy smokers (n=4). Four different Pathology, Palermo, Italy 9)Fondazione IRCCS Istituto Nazionale dei Tumori, qPCR platforms were used to detect human cmiRNAs: two high-throughput Molecular Immunology Unit, MILANO, Italy technologies, i.e. TaqMan OpenArray (OA) and OA Advanced Human miRNA Panels (ThermoFisher Scientific), both allowing to profile 754 miRNAs, and two During tumorigenesis, newly transformed cells initiate an active cross-talk with the serum/plasma focus PCR panels, i.e TaqMan Advanced miRNA Human Card (192 adjacent stroma through the secretion of a variety of factors, microRNAs (miRNA) miRNAs, ThermoFisher) and miRCURY LNA miRNA PCR Panel (179 miRNAs, included. The characterization of circulating miRNAs that are relevant for the Qiagen). cDNA synthesis, pre-amplification (for TaqMan assays) and real-time progression of nascent breast cancers could lead to the identification of prognostic/ qPCR were performed according to manufacturer’s instructions for biofluids. predictive biomarkers. Results. For comparison we restricted the analysis to the subset of 155 miRNAs To investigate circulating miRNA deregulation during different phases of mammary present in all platforms. Each platform was analyzed independently using specific cancer development, we analysed the plasma of transgenic MMTV-NeuT (NeuT) recommended thresholds. For each sample the number of miRNAs detected by mice, which spontaneously develop mammary carcinomas with a well-defined all platforms ranged from 73 to 94 and a core of 46 miRNAs was detected by all progression, mimicking the human pathology. Plasma miRNA profile was performed platforms in all samples. The highest level of correlation was observed between the with Agilent microarrays on NeuT and WT sibling controls, collected at early (6 and two TaqMan Advanced assays (average r=0.8). Focusing on the core of 46 miRNAs, 12 weeks) and late (24 weeks) time-points of cancer progression. We identified correlation values were greater than 0.9 in all samples (range: 0.92-0.96). Finally, a signature composed of 8 and 10 up-regulated miRNAs in early and late NeuT in addition to miRNAs present in the two plasma-focused platforms, the OA and OA samples, respectively, with 2 miRNAs in common. Histopathological analyses on Advanced high-throughput platforms were able to detect, on average, 127 and 86 the primary lesions of each mouse showed a considerable degree of individual supplementary miRNAs, respectively. variability in the onset and extension of nascent lesions within the mammary glands Conclusions. This pilot study is one of the first comparing high-throughput with of early time points. For this reason, we have re-classified the samples on the basis serum/plasma focus miRNA platforms. A high-throughput approach can be of the severity of the primary lesions, identifying 3 different categories (normal, preferred for discovery studies, whereas analysis of predefined known microRNAs mild/moderate dysplasia and severe dysplasia/early carcinoma). Class comparison can benefit from using specific designed panels. The chemistry and assay design analysis confirmed that specific circulating miRNAs increased along transformation of the selected panel must be considered. Further studies involving digital barcode from normal to severe dysplasia/early carcinoma. Moreover, we found a significant (i.e. NanoString Technologies) and sequencing-based platforms are ongoing. correlation among the deregulated circulating miRNAs and the tumor burden, even This work was supported by AIRC Special Program “Innovative Tools for Cancer before the tumor became palpable. Risk Assessment and early Diagnosis” 5X1000 (no. 12162). These results demonstrate that during the early phases of tumor transformation a significant modification in circulating miRNAs can be detected and is strictly correlated with the stage and severity of the disease. Circulating miRNAs could be Q20 not only a possible diagnostic tool but also a classification tool used to discriminate Cytokines profile analysis identifies circulating IL4 as between different stages of breast cancer progression. predictive factor of early relapse in resectable pancreatic ductal adenocarcinoma patients Piro G., Simionato F., Carbone C., Frizziero M., Malleo G., Santoro R., Zecchetto C., Q22 Merz V., Scarpa A., Bassi C., Tortora G., Melisi D. THE SANIST PLATFORM FOR SERUM STEROID RATIO PROFILING IN PROSTATE Digestive Molecular Clinical Oncology Research Unit, Department of Medicine, CANCER: TOWARD A NEW DIAGNOSTIC TOOL FOR A PERSONALIZED Università degli studi di Verona, Verona, Italy MEDICINE APPROACH 1)Albini A. 2)Bruno A. 3)Bassani B. 4)D’Ambrosio G. 5)Pelosi G. 6)Consonni P. 7) Background. Surgery is the only potentially curative option for patients with Castellani L. 8)Conti M. 9)Cristoni S. 10)Noonan D.M. pancreatic ductal adenocarcinoma (PDAC), but metastatic relapse remains 1)School of Medicine and Surgery, University Milano-Bicocca, Monza, Italy 2)IRCCS common. We hypothesized that the expression levels of inflammatory cytokines MultiMedica, Milan, Italy 3)IRCCS MultiMedica, Milan, Italy 4)IRCCS MultiMedica, could predict recurrence of PDAC, thus allowing to select patients who most likely Milan, Italy 5)Department of Oncology and Hemato-Oncology, University of Milan, could benefit from surgical resection. Milan, Italy 6)IRCCS MultiMedica, Milan, Italy 7)IRCCS MultiMedica, Milan, Italy Methods. We prospectively collected plasma at diagnosis from two-hundred eighty- 8)Sant’Orsola Hospital, Bologna, Italy 9)I.S.B.—Ion Source & Biotechnologies, seven patients with pancreatic resectable neoplasms. The expression levels of 23 Bresso, Italy 10)Department of Biotechnologies and Life Sciences, University of cytokines were measured in ninety patients with PDAC by using a multiplex analyte Insubria, Varese, Italy profiling assay. Results. Levels higher than cutoff identified of the TH2 cytokines interleukin (IL)4, Steroids are molecules involved in prostate cancer (PCa). Steroids are characterized IL5, IL6, of macrophage inflammatory protein (MIP)1a, granulocyte-macrophage by complex biological pathways and stoichiometry, thus it is important to efficiently colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein detect their quantitative ratio. In this context, mass spectrometry (MS) currently (MCP)1, and of IL17a, IFN-γ- induced protein (IP)10, and IL1b were significantly represents the elicited technology to analyse serum steroid presence in clinical associated with a shorter median OS. In particular, levels of IL4 and IP10 higher samples. than cutoff identified, and level of TH1 cytokines TNFa and INFg, and of IL9 and Here, we employed our recently developed and validated he Surface Activated IL1Ra lower than cutoff identified were significantly associated with a shorter DFS. Chemical Ionization-Electrospray-NIST (SANIST) platform, to obtain quantitative
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serum steroid ratio relationship profiles, associated with a machine learning Q24 Bayesian model, aimed at discriminating patients with PCa. An approach focused Can the poor chemotherapeutic response to gemcitabine/ on steroid relationship profiles and disease association was used. The study was nabpaclitaxel depend on the interaction of the pancreatic performed on serum samples from patients being subjected to a prostate biopsy tumor with the microenvironment? for PCa diagnosis at the Urology Unit of the MultiMedica Castellanza, Varese, 1)Iacobazzi R.M. 2)Porcelli L. 3)Di Fonte R. 4)Serratì S. 5)Garofoli M. 6)Mazzotta Italy. Individuals with autoimmune diseases, hypersensitivities, and other immune- A.7)Argentiero A. 8)Silvestris N. 9)Azzariti A. mediated physical states were excluded from the study. Controls included healthy 1)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, subjects undergoing PSA dosage at our Urology unit that had a PSA lower than Italy 2)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, 2.5 ng/mL or less than 4 ng/mL and free PSA greater than 15%, and patients with Bari, Italy 3)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo benign prostate hyperplasia (BPH). II”, Bari, Italy 4)Nanotecnology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, We quantitated the levels of 10 steroids by our SANIST platform: Aldosterone, Italy 5)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Corticosterone, Cortisol, 11-deoxycortisol, Androstenedione, Testosterone, Bari, Italy 6)Experimental Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo dehydroepiandrosterone, dehydroepiandrosterone sulfate (DHEAS), 17-OH- II”, Bari, Italy 7)Medical Oncology Unit, Istituto Tumori “Giovanni Paolo II”, Bari, Italy 8) Progesterone and Progesterone. We found that our machine learning approach, Medical Oncology Unit, Istituto Tumori “Giovanni Paolo II”, Bari, Italy 9)Experimental based on the steroid relationships, was much more accurate than the PSA, DHEAS, Pharmacology Laboratory, Istituto Tumori “Giovanni Paolo II”, Bari, Italy and direct absolute value match method in separating the PCa, BPH and control subjects, with specificity and sensitivity of 90% and 84%, respectively. Scientists search for an exhaustive explanation to the poor response to gemcitabine/ Finally, our technology, if applied to a larger number of samples will be able to nabpaclitaxel of some PDAC patients, by analyzing the mutational landscape of detect the individual enzymatic disequilibrium associated with the steroid ratio and PDAC for the identification of specific subtypes and by identifying the molecular correlate it with the disease providing a personalized medicine setting. events responsible for the initiation and progression of PDAC but today it is still an open question. We approach this topic shifting from studies in which the tumor is the main actor to other in which it is a co-actor together with stroma cells. PDAC Q23 has an excessive (up to 80%) amount of scar tissue (“desmoplasia”) surrounding Circulating biomarkers in Giant Cell Tumours the malignant cells consisting predominantly of cancer-associated fibroblasts 1)Conti A. 2)Luchini A. 3)Benassi M.S. 4)Magagnoli G. 5)Pierini M. 6)Piccinni- (CAFs), mast cells, inflammatory cells, small blood vessels, and extracellular Leopardi M. 7)Quattrini I. 8)Pollino S. 9)Rhoden K.J. 10)Picci P. 11) Liotta L.A. 12) matrix. We have focused on the relevance of mast cells in modulating the response Pazzaglia L. to anticancer gemcitabine and nabpaclitaxel-based regimen in pancreatic cancer 1)Department of Medical and Surgical Sciences, DIMEC, University of Bologna, models. The experimental approach included the analysis of cell proliferation, Bologna, Italy 2)Center for Applied Proteomics and Molecular Medicine, George induction of apoptosis and the determination of the role of tryptase and TGF-beta1, Mason University, Manassas, VA, USA 3)Experimental Oncology Laboratory, Rizzoli released by mast cells, in reducing cellular response to drugs administration. Orthopaedic Institute, Bologna, Italy 4)Experimental Oncology Laboratory, Rizzoli The addition of mast cells conditioned medium (CM-HCM-1) to single and drugs Orthopaedic Institute, Bologna, Italy 5)Chemotherapy Unit, Rizzoli Orthopedic combination impacted on cell response with a strong reduction of the treatments Institute, Bologna, Italy 6)Chemotherapy Unit, Rizzoli Orthopedic Institute, Bologna, effectiveness in CFPac, MiaPaCa-2 and Panc-1 cells as respect to Capan-1 and Italy 7)Experimental Oncology Laboratory, Rizzoli Orthopaedic Institute, Bologna, AsPC-1. Such effect was evidenced in MiaPaCa-2 cell as an inhibition of apoptosis Italy 8)Experimental Oncology Laboratory, Rizzoli Orthopaedic Institute, Bologna, induction. Italy 9)Department of Medical and Surgical Sciences, DIMEC, University of Bologna, The relevance of tryptase and/or TGF-beta1 as determinants responsible of the Bologna, Italy 10)Experimental Oncology Laboratory, Rizzoli Orthopaedic Institute, “protection” from drugs-induced cell death was confirmed by results showing that Bologna, Italy 11)Center for Applied Proteomics and Molecular Medicine, George the gemcitabine/nabpaclitaxel, when given in the presence of CM-HCM-1, induced Mason University, Manassas, VA, USA 12)Experimental Oncology Laboratory, a strong activation of Erk1/2 and Akt, both down streams effectors of tryptase Rizzoli Orthopaedic Institute, Bologna, Italy through Par-2, and of TGF-beta1 through Smad3. Thus, the study assesses the role of mast cells as modulator of chemotherapeutic Giant cell tumours of bone (GCT), a benign neoplasm occurring in the long response in PDAC cellular models, as well as their potential as predictive markers bone and in the axial skeleton of young adults, develop pulmonary metastases for the future development of PDAC treatment strategies. in approximately 5% of cases. Although many biomarkers have been proposed, identification of low abundance circulating molecules generating from tumour cells and tissues, may be useful to predict metastasis non-invasively, providing Q25 information on tumour progression and stratifying patients at high risk of metastasis Outcome prediction of metastatic colorectal cancer patients development. undergoing liver resection by analyzing serum metabolomics The hydrogel nanoparticle technique followed by mass spectrometry was used to 1)Costantini S. 2)Sorice A. 3)Capone F. 4)Di Gennaro E. 5)Vitagliano C. 6)De detect low molecular weight serum proteins or protein fragments in serum of 20 GCT Stefano A. 7)Delrio P. 8)Bianco F. 9)Izzo F.10)Avallone A. 11)Budillon A. patients with different clinical course and in 10 healthy sera used as controls. The 1)Experimental Pharmacology Unit, Istituto Nazionale Tumori “Fondazione G. most representative low-abundant de novo or differentially abundant proteins were Pascale” - IRCCS, Napoli, Italy 2)Experimental Pharmacology Unit, Istituto submitted to String database in order to define protein-protein interaction network. Nazionale Tumori “Fondazione G. Pascale” - IRCCS, Napoli, Italy 3)Experimental Cluster analysis was performed to identify prognostic groups of patients with similar Pharmacology Unit, Istituto Nazionale Tumori “Fondazione G. Pascale” - IRCCS, abundance of proteins that significantly discriminate between the groups. Napoli, Italy 4)Experimental Pharmacology Unit, Istituto Nazionale Tumori Proteoma cluster analysis separated metastasis-free from metastatic GCT patients “Fondazione G. Pascale” - IRCCS, Napoli, Italy 5)Experimental Pharmacology in two well-defined groups, where serum levels of signalling transduction mediators Unit, Istituto Nazionale Tumori “Fondazione G. Pascale” - IRCCS, Napoli, Italy and regulators of kinase activity presented a high discriminatory power. Increased 6)Abdominal Oncology Department, Istituto Nazionale Tumori “Fondazione G. expression of proteins STAT5B, GRB2 and OXSR1, presented a high discriminatory Pascale” IRCCS, Napoli, Italy 7)Abdominal Oncology Department, Istituto Nazionale power in identifying metastatic patients, thus improving efficacy of the treatment Tumori “Fondazione G. Pascale” IRCCS, Napoli, Italy 8)Abdominal Oncology protocols, expectancy and quality of life. Multivariate analysis demonstrated that Department, Istituto Nazionale Tumori “Fondazione G. Pascale” IRCCS, Napoli, tumour grade and STAT5B were independent prognostic factors and STAT5B role Italy 9)Abdominal Oncology Department, Istituto Nazionale Tumori “Fondazione was further validated by ELISA in an independent GCT subset. G. Pascale” IRCCS, Napoli, Italy 10)Abdominal Oncology Department, Istituto In conclusion, using a non-invasive nanoparticle technique, we identified differentially Nazionale Tumori “Fondazione G. Pascale” IRCCS, Napoli, Italy 11)Experimental abundant serum biomarkers, also providing prognostic information in patients Pharmacology Unit, Istituto Nazionale Tumori “Fondazione G. Pascale” - IRCCS, with GCT of bone. Furthermore, this technique could also monitor the biological Napoli, Italy response to a specific treatment through the determination of abundance and/or activation state of selected biomarkers. Future studies are needed to establish the In metastatic colorectal cancer (mCRC) patients (pts), treatment strategies interplay between these biomarkers in order to fully understand the mechanism integrating liver resection with more effective therapies offer better 5-year survival involved in tumour development and to focus on the planning of tailored therapies rates than palliative chemotherapy alone. However, resectability, established only that should be more effective and less toxic. on clinical-morphovolumetric criteria, is a complex and costly procedure and relapse occurs in almost 2/3 of pts after potentially curative resection. Therefore, prompt identification of pts at higher risk of recurrence is critical to avoid not-beneficial, expensive procedures. Aberrant metabolism is an emerging hallmark of cancer and recent observations suggest that specific metabolic changes can be used to stratify pts for prognosis and drug-response. We evaluated by 600MHz NMR spectroscopy the metabolomics profiling on sera from 30 mCRC pts, enrolled in the Obelics trial (NCT01718873), which investigated different schedules of bevacizumab
80 81 ABSTRACT BOOK in combination with oxaliplatin plus fluoropyrimidines (FOLFOX-6 or OXXEL) regimens, and subdivided on the basis of outcome, in good (R) vs bad responders SIGNAL TRANSDUCTION AND (NR) according to PFS: 12 months or longer (R, n = 12) and shorter than 12 months INTRACELLULAR TRAFFICKING (NR, n = 18). We compared the samples of the two mCRC pts groups, collected at response evaluation when resectability was established, and demonstrated by R1 Principal Component Analysis and sPLS-DA plots that R and NR pts grouped into AKT1 PHOSPHORYLATION OF THE ADENOSINE DEAMINASES, ADAR-1P110 two different clusters, suggesting the presence of some metabolites with statistically AND ADAR-2 ALTERS EDITASE ACTIVITY different levels between the two sub-groups. Indeed, loading plots evidenced such 1)Blalock WB. 2)Bavelloni AB. 3)Piazzi MP. 4)Focaccia EF. 5)Tomaselli ST. 6) metabolites. Thus, to identify the cutoff levels of these significant metabolites Cesarini VC. 7)Faenza IF. 8)Cocco LC. 9)Gallo AG. we performed ROC curves that evidenced AUC values ranging between 0.741 1)Institute of Molecular Genetics, National Research Council of Italy, Bologna, Italy and 0.63, and using these cutoff we identified the metabolites with a significant 2)Department of Musculoskeletal Cell Biology, Ircss, Rizzoli Orthopedic Institute, correlation with the patient survival. Kaplan-Meier curves of overall survival Bologna, Italy 3)Institute of Molecular Genetics, National Research Council of Italy, evidenced that lower levels of 3-hydroxybutyrate (p-value=0.0067) and higher levels Bologna, Italy of histidine (p-value=0.011) correlated with a good outcome. Analysis of immune 4)Institute of Molecular Genetics, National Research Council of Italy, Bologna, Italy state within resected metastases by nanostring-based gene expression profiling, 5)Oncohematology Department, Ircss, Bambino Gesù Pediatric Hospital, Rome, and of cytokines profiling on blood samples is ongoing and will be correlated with Italy 6)Oncohematology Department, Ircss, Bambino Gesù Pediatric Hospital, the metabolic alterations described above. Overall, these data suggest that NMR- Rome, Italy based metabolomics is a potent and affordable method that could play a role in the 7)Department of Biomedical and Neuromotor Sciences, University of Bologna, prediction of mCRC outcome. Bologna, Italy 8)Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 9)Oncohematology Department, Ircss, Bambino Gesù Pediatric Hospital, Rome, Italy RNA editing is a mechanism by which RNA molecules are post-transcriptionally modified through the deamination of select adenosine and cytosine residues. The adenosine deaminase acting on dsRNA (ADAR) family of enzymes account for the vast majority of these post-transcriptional nucleotide modifications. These enzymes convert adenosine residues at specific sites within RNA molecules of complex secondary structure to inosine, a non-canonical nucleoside that pairs with cytosine and is interpreted as guanine by the cell’s molecular machinery, thus accounting for an A to G transition. These modifications can alter RNA secondary structure, lead to alternative splicing of mRNAs or change the amino acid(s) present in a particular protein. Moreover, ADAR editase activity can influence miRNA expression and substrate specificity as well as gene transcription and nuclear export of hyperedited RNAs. The expression and/or activity of both ADAR1 and ADAR2 have been found to be altered in diverse cancers with ADAR1 expression elevated and ADAR2 activity suppressed. While the effects of ADAR expression and activity have been studied, little information exists regarding the regulation of these proteins by post- translational modifications. In this study, we follow-up our recent findings that the AKT family kinases (AKT1, AKT2 and AKT3) interact with and phosphorylate ADAR1p110 and ADAR2. Using site-directed mutagenesis of both suspected AKT phosphorylation sites and in vitro kinase assays, our data demonstrate that AKT1 phosphorylates ADAR1p110 and ADAR2 at multiple sites. Overexpression of the certain phosphomutants of ADAR1p110 and ADAR2 resulted in an 80% reduction in editase activity toward known substrates of these two editases in a U87MG glioblastoma model. Thus, activation of AKT can have a direct impact on RNA editing.
R2 Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling 1)Carbone C. 2)Piro G. 3)Ligorio F. 4)Santoro R. 5)Torroni L. 6)Simionato F. 7)Merz V. 8)Zecchetto C. 9)Di Nicolantonio F. 10)Scarpa A. 11)Tortora G. 12)Melisi D. 1)Medicine, University of Verona, Verona, Italy 2)Medicine, University of Verona, Verona, Italy 3)Medicine, University of Verona, Verona, Italy 4)Medicine, University of Verona, Verona, Italy 5)Medicine, University of Verona, Verona, Italy 6)Medicine, University of Verona, Verona, Italy 7)Medicine, University of Verona, Verona, Italy 8) Medicine, University of Verona, Verona, Italy 9)Oncology, Candiolo cancer institute, Candiolo, Torino, Italy 10)Pathology and diagnostics, University of Verona, Verona, Italy 11)Medicine, University of Verona, Verona, Italy 12)Medicine, University of Verona, Verona, Italy Background. Solid epidemiological evidences connect obesity with incidence, stage, and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. Methods. We studied the role of factors secreted by two adipogenic model systems from primary human Bone Marrow Stromal Cells (hBMSC) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS). Results. We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway,
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including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced SYSTEM BIOLOGY the overexpression and the nuclear translocation of the Frizzled family member AND OMICS TECHNOLOGIES Receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear S1 translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. Combined approach for the analysis of copy number alteration Conclusions. We demonstrated that adipocytes could induce EMT and heterogeneity in circulating tumor cells aggressiveness in models of pancreatic preneoplastic lesions by orchestrating 1)Silvestri M. 2)Reduzzi C. 3)Vismara M. 4)Niger M. 5)Miodini P. 6)Motta R. 7) a complex paracrine signaling of soluble modulators of the non-canonical WNT Cappelletti V. 8)Daidone M.G. signaling pathway that determine, in turn, the activation and nuclear translocation of 1)Biomarkers Unit, Department of Applied Research and Technological ROR2. This signaling pathway could represent a novel target for pancreatic cancer Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 2) chemoprevention. Most importantly, these factors could serve as novel biomarkers to Biomarkers Unit, Department of Applied Research and Technological Development, select a risk population among obese subjects for screening and, thus, early Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 3)Biomarkers Unit, diagnosis of pancreatic cancer. Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 4)Medical Oncology Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 5)Biomarkers Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 6)Biomarkers Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 7)Biomarkers Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy 8)Biomarkers Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Background. Whole genome sequencing (WGS) of circulating tumor cells (CTCs) represents a non-invasive approach to study tumor progression and treatment resistance in clinical practice. In particular, copy number alteration (CNA) analysis offers a way to investigate the presence of heterogeneity between different groups of single CTCs. Since standard analysis pipelines for such a purpose are still lacking we aimed at developing a bioinformatic approach allowing to obtaining biologically and clinically useful information. Methods. We collected 57 CTCs from 18 patients with advanced biliary tract cancer. Recovered cells were subjected to whole genome amplification (WGA) employing the Ampli1Ô WGA kit. Amplified DNA passing the quality control check were processed by low-coverage WGS (0.1X) using the IonTorrent Ion S5Ô system. WGS sequences were aligned to the human reference genome (hg19) using tmap aligner tool on Torrent_Suite 5.4.0 for copy number variations prediction by Control- FREEC 11.0. Clustering analysis was used to group CTCs as a function of CNA logRatio values considering firstly whole genome and thereafter genes with variance >0.95 percentile. Using frequency alteration analysis, regions altered in at least 70% of CTCs grouped by treatment, response and anatomical site were identified and consequently subjected to enrichment analysis. Finally each specific signature was further refined by intersecting with the list of the most variable genes extracted by clustering analysis. Results. Whole genome clustering analysis revealed a difficult-to-manage level of heterogeneity among CTCs. Better results in the separation of different CTC groups were obtained by reducing the feature dimension selecting only the most variable genes. Frequency alteration analysis increased the informative content on heterogeneity and allowed performing gene enrichment analysis. This way, hints on gene pathways distinguishing clinically defined CTCs groups were obtained. The intersection between signatures extracted from frequency and clustering analysis returned three genes lists useful for extraction of the minimum number of features distinguishing CTCs. Conclusions. CNA analysis of CTCs represents a promising way for obtaining information on the evolution of the disease under treatment pressure. In particular, the developed bioinformatic pipeline based on combination of clustering and frequency analysis identifies genes and pathways specific for distinct clinical categories.
S2 CONSERVATION OF COLORECTAL CANCER CELL-INTRISINC TRANSCRIPTIONAL TRAITS IN PAIRED IN VITRO / IN VIVO CELLULAR MODELS 1)Isella C. 2)Petti C. 3)Cancelliere C. 4)Cantarella D. 5)Porporato R. 6)Trusolino L. 7)Bertotti A. 8)Bardelli A. 9)Medico E. 1)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 2)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 3)University of Turin, Candiolo cancer institute - irccs, Turin, Italy 4)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 5)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 6)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 7) University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 8)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy 9)University of Turin, Candiolo cancer institute - IRCCS, Turin, Italy
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The best clinical and molecular criteria currently adopted in the diagnosis of colorectal cancer (CRC) often fail to predict the natural history of the disease, TUMOR HETEROGENEITY or to provide information regarding the molecular mechanisms inducing cancer T1 onset and progression. To address these issues, molecular taxonomies based ANALYSIS OF cfDNA REVEALS HIGH LEVELS OF TUMOR HETEROGENEITY on transcriptional CRC subtypes have been developed; however, since the CRC IN NSCLC transcriptome is heavily affected by mRNAs of stromal origin, these transcriptional 1)Pasquale R. 2)Bergantino F. 3)Fenizia F. 4)Forgione L. 5)Roma C. 6)De Luca A. 7) traits are not specifically associated with cancer cell-intrisic features. To overcome this limitation, we recently employed gene expression profiles of CRC tissues Azzaro R. 8)Iervolino V. 9)Rocco G. 10)MorabitoA. 11)Normanno N. specifically deprived of stromal transcripts to define “colorectal cancer cell-intrisinc 1)Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione subtypes” (CRIS). CRIS classification is not only bestowed with high prognositc and G. Pascale”-IRCCS, Naples, Italy 2)Cell Biology and Biotherapy Unit, Istituto predictive value: it also conveys biological and molecular information on the subtypes, Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy 3)Cell Biology and that may be tailored for specific therapies. To assess reliability of CRIS subtyping in Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, preclinical CRC models, we designed a “XenoLine” platform, composed of 60 CRC Italy 4)Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione cell lines representative of each CRIS class, grown in vitro and implanted in NOD- G. Pascale”-IRCCS, Naples, Italy 5)Cell Biology and Biotherapy Unit, Istituto SCID mice to obtain xenograft tissues. After subcutaneous injection of 5 milion cells, Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy 6)Cell Biology xenografts were allowed to grow until they reached 2500 mm3 and were explanted. and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Engraftments were performed in duplicate and had a success rate around 85%. Naples, Italy 7)Transfusion Service, Department of Hematology and Developmental Further engraftments are ongoing. RNA-seq analysis was employed to compare Therapeutics, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, in vivo- and in vitro-grown cells, and evaluate the stability of CRIS subtypes. The Italy 8)Transfusion Service, Department of Hematology and Developmental XenoLine RNA-seq profiles were in-silicomicrodissected, discriminating transcripts Therapeutics, Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, of epithelial cancer cell origin (human reads) from transcripts of stromal origin Italy 9)Department of Thoracic Surgery, Istituto Nazionale Tumori “Fondazione (mouse reads). Comparing paired in vitro / in vivo gene expression profiles, we G. Pascale”-IRCCS, Naples, Italy 10)Department of Thoracic Medical Oncology, observed that: 1) matched model pair correlation was systematically higher that Istituto Nazionale Tumori “Fondazione G. Pascale”-IRCCS, Naples, Italy 11)Cell random pair correlations; 2) for most genes, variation of expression across cells Biology and Biotherapy Unit, Istituto Nazionale Tumori “Fondazione G. Pascale”- was consistent between in vitro and in vivo profiles; 3) CRIS classification was IRCCS, Naples, Italy substantially maintained upon xenograft propagation. In conclusion, the molecular Background. Circulating cell free DNA (cfDNA) is an alternative to tumor tissue features distinguishing CRIS subtypes are resilient to major changes in cell growth for molecular profiling in non-small cell lung cancer (NSCLC). In addition, cfDNA conditions, such as changes in the microenvironment and acquisition of supporting analysis might better recapitulate intra-tumor heterogeneity as compared with tissue stroma. The XenoLine platform was successfully established and will be further biopsy in patients with multiple metastatic lesions. extended to better ascertain differences between in vitro- and in vivo- grown CRC Material and methods. Next generation sequencing (NGS) with targeted panels was cells of variable molecular makeup. performed on tissue or cytology specimens with the Oncomine Solid Tumour DNA kit, and on plasma-derived cfDNA with the Oncomine Lung cfDNA Assay. Droplet Digital PCR (ddPCR) was performed with the QX200 System to solve discordant cases. Results. We performed targeted-sequencing analysis of tumor samples and matched cfDNA from 97 patients with metastatic NSCLC before systemic treatment. We also analyzed plasma-derived cfDNA from 25 healthy donors, in order to define appropriate mutation detection cutoffs. We observed the presence of at least one variant in genes covered by both panels in 68% of tumor samples and in 61.9% of plasma samples from NSCLC patients. EGFR mutations were found in 18/24 plasma samples from EGFR-mutant patients (sensitivity 75%) and in 3/73 plasma samples from EGFR wild type (wt) patients (specificity 95.9%), with a concordance rate of 90.7%. In the 3 false-positive cases (EGFR mutant-plasma/wt-tissue), ddPCR confirmed the EGFR mutations in 2/2 available tissue samples thus confirming the specificity of the NGS cfDNA-panel and suggesting tumor heterogeneity. We next evaluated in the cohort of 73 EGFR wt tumors the concordance between tumor and plasma for mutations in genes other than EGFR covered by both NGS panels. The mean concordance was low (59.7%). In particular, we observed a high discordance among KRAS mutations. Of 11 cases with KRAS mutations in tissue, only 3 showed KRAS mutations in plasma. ddPCR analysis confirmed KRAS mutations in the cfDNA from 4/5 available samples that were negative by NGS, suggesting a low sensitivity of the panel for RAS mutations. Plasma NGS analysis and ddPCR also revealed KRAS mutations in 3 cases that were negative on tissue, implying tumor heterogeneity for these variants. Conclusions. These data suggest that targeted sequencing with specific panels is a suitable method for EGFR genotyping in NSCLC starting from cfDNA. The relatively low sensitivity and high tumor heterogeneity must be taken into account for KRAS variants.
T2 Heterogeneity of ascites from epithelial ovarian cancer: from cells to microenvironment 1) Napoli A. 2) Roggiani F. 3) Bagnoli M. 4) Raspagliesi F. 5) Invernizzi A.M. 6) Mezzanzanica D. 7) Tomassetti A. 1)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Unit of Gynecologic Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 5)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 7)Unit of Molecular Therapies, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and a
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very heterogeneous malignancy. More than two third of patients with advanced of drug type or resistance mechanisms. Pharmacological blockade of WNT ligands disease present secondary lesions associated to malignant ascites rich of tumor secretion resulted decreased of β-catenin dependent Tcf/LEF transcriptional activity, multicellular aggregates (MCAs) together with a mixture of cell populations which and cell growth impairment despite massive molecular drift of resistant derivatives. include fibroblasts, endothelial or mesothelial cells, adipocytes, immune cells as Concomitant blockade of WNT and MAPK signalling restrained evolution of drug- well as soluble factors. These components can promote EOC progression. In resistant clones. this context, we wanted to characterize EOC cells of MCAs. In MCAs from EOC In summary, reliance of CRC on the WNT pathway is preserved in later phases of E-cadherin was present in all samples while few samples also co-expressed colorectal carcinogenesis when tumors face genomic bottlenecks and sub-clonal N-cadh. Immunofluorescence on high grade EOC (HGEOC) MCAs showed evolution driven by administration of target therapies; thus offering the possibility of E-cadherin expression at the site of cell-cell junctions together with the adherens a common therapeutic strategy to overcome secondary resistance to treatments. junction partner β-catenin while in the solid primary tumors from the same patient co-expression of E-and N-cadherin was observed. These data, consistent with those obtained at transcript levels, indicate that EOC MCAs tend to maintain an epithelial- like phenotype. In order to identify factor/s responsible for EOC growth in the ascites and/or MCA formation, we applied different cell culture techniques for isolation and propagation of tumor cells derived from EOC patients’ ascites assessing possible biological changes during their long-term in vitro culture. Ascites-derived cells present two populations: one with an epithelial-like morphology; the other showing a mesenchymal-like morphology that was most prevalent after a number of cell doubling in vitro. After an average of 4 in vitro passages, surviving cells revealed a progressive decrease of the expression of the epithelial markers together with an increase of vimentin and calretinin indicating the selection of non-tumor cell population. With the aim to maintain tumor cell survival, we cultured ascites- derived cells with different growth-promoting factors including the ascitic fluids. We observed that some ascitic fluids might increase cell vitality and the capability to form MCAs. When tested on selected EOC cell lines, some ascitic fluids were able to promote the activation of several intracellular signaling pathways as well as to induce the formation of MCAs indicating the presence of tumor activating factors. So far, our results suggest that systematic studies of ascites and the generation of patient-derived MCAs can shed light in the understanding of ovarian cancer biology. Partially supported by AIRC, Cariplo Foundation and 5x1000 Institutional Research Funding.
T3 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies 1)Corti G. 2)Russo M. 3)Lamba S. 4)Lorenzato A. 5)Sogari A. 6)Rospo G. 7)Montone M. 8)Lazzari L. 9)Arena S. 10)Oddo D. 11)Linnebacher M. 12)Sartore-Bianchi A. 13) Pietrantonio F. 14)Siena S. 15)Di Nicolantonio F. 16)Bardelli A. 1)Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Turin, Italy 2)FIRC Institute of Molecular Oncology (IFOM), Milan, Italy 3)Candiolo Cancer Institute- FPO, IRCCS, 10060 Candiolo, Turin, Italy 4)Department of Oncology, University of Torino, Candiolo, Turin, Italy 5)Department of Oncology, University of Torino, Candiolo, Turin, Italy 6)Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Turin, Italy 7)Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Turin, Italy 8) Department of Oncology, University of Torino, Candiolo, Turin, Italy 9)Department of Oncology, University of Torino, Candiolo, Turin, Italy 10)Candiolo Cancer Institute- FPO, IRCCS, 10060 Candiolo, Turin, Italy 11)Department of General Surgery, University of Rostock, Rostock, Germany 12)Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy 13)Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumouri, Milan, Italy 14)Department of Oncology and Hemato-Oncology, Università degli Studi di Milano, Milan, Italy 15) Candiolo Cancer Institute-FPO, IRCCS, 10060 Candiolo, Turin, Italy 16)Department of Oncology, University of Torino, Candiolo, Turin, Italy
Clinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift. To test our assumption, we first generated a panel of CRC cells resistant to commonly used targeted agents, including EGFR and BRAF inhibitors. Treatment with targeted therapies, although initially effective, fuels clonal evolution and further amplifies molecular diversity. Molecular profiling, performed by whole exome sequencing, unveiled SNV and copy-number variations associated with the emergence of resistance to drug treatment. Starting from these data, we used bioinformatic algorithms to infer phylogenetic trees on resistance cell populations. The mutation’s co-occurrence/ exclusivity patterns predicted by the bioinformatics analysis was then confirmed by single cell dilution of the resistant cells. These phylogenetic results showed a complex sub-clonal architecture indicating a parallel evolution of multiple independent cellular lineages, associated with distinct genomic landscapes. Functional and pharmacological modulation of WNT signaling induces cell death in CRC preclinical models from patients that relapsed during treatment, regardless
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and metastasis often fatal. In recent years, the development of immune checkpoint TUMOR IMMUNOLOGY AND IMMUNOTHERAPY inhibitors has changed this poor prognosis, though only in a limited subset of MM U1 patients, while most exhibit innate resistance and disease progression. Therefore, Blockade of Intrinsic PD-1 Promotes Oxaliplatinum and new therapies are needed. In our studies, the feasibility and the anti-tumor potential 5-Fluoruracil Chemoresistance in human colon cancer cells of targeting the MM-associated oncoantigen chondroitin sulfate proteoglycan 1)Monaco G. 2)Ieranò C. 3)Napolitano M. 4)Portella L. 5)Rea G. 6)Maiolino P. 7) (CSPG)4 with DNA vaccines delivered by electroporation (EP) have been evaluated. Due to the many similarities with its human (Hu) counterpart, canine (Do) MM offers Scala S. an excellent opportunity for translational clinical investigations, being the CSPG4 an 1)Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Tumori, attractive immunotherapy target expressed by both human and canine MM patients. Fondazione “ G. Pascale”- IRCCS, Naples, Italy 2)Functional Genomics, Istituto We have previously demonstrated the safety and the clinical relevance of the Nazionale per lo Studio e la Cura dei Tumori, Fondazione “ G. Pascale”- IRCCS, intramuscular injection of a Hu-CSPG4 plasmid followed by EP in dogs with stage Naples, Italy 3)Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei II-III surgically resected CSPG4+ oral MM. Hu-CSPG4 EP caused no side effects Tumori, Fondazione “ G. Pascale”- IRCCS, Naples, Italy 4)Functional Genomics, and resulted in a significantly longer disease-free (DFI) and overall survival (OS) Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “ G. Pascale”- of vaccinated dogs as compared to controls. However, Hu-CSPG4 vaccine was IRCCS, Naples, Italy 5)Functional Genomics, Istituto Nazionale per lo Studio e not effective in activating human T cells from healthy donors in vitro. To increase la Cura dei Tumori, Fondazione “ G. Pascale”- IRCCS, Naples, Italy 6)Pharmacy the translational power of our approach, we employed a hybrid plasmid coding for -Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “ G. Pascale”- a chimeric HuDo-CSPG4 protein, expected to be effective in both veterinary and IRCCS, Naples, Italy 7)Functional Genomics, Istituto Nazionale per lo Studio e la human settings. Cura dei Tumori, Fondazione “ G. Pascale”- IRCCS, Naples, Italy We tested the immunogenicity and the anti-tumor potential of HuDo-CSPG4 intramuscular DNA EP in mice, in surgically resected CSPG4+ oral MM canine A considerable proportion of colorectal cancer patients develop local recurrence patients and in an in vitro human setting. Chimeric HuDo-CSPG4 EP is immunogenic and distant metastasis within 5 years after surgical treatment. Nivolumab is in mice. In dogs the procedure is safe and induces antibodies (Ab) against both indicated for treatment of patients with metastatic colorectal cancer carrying DNA Hu- and Do-CSPG4, with a higher affinity and anti-tumor potential as compared to mismatch repair–deficient (dMMR)/microsatellite instability-high (MSI-H) whose Hu-CSPG4. From the clinical point of view, the hybrid HuDo-CSPG4 is effective in disease has progressed on fluoropyrimidine, oxaliplatin, and irinotecan. Thus only a increasing both the DFI and the OS of vaccinated canine MM patients as compared minority of patients with CRC will benefit from the immunotherapy. Recent evidence demonstrate that PD-1 is expressed, not only by immune cells, but also on human to non-vaccinated controls. Interestingly, data obtained in vitro with T cells from cancer cells, such as melanoma cells, in which PD-1 transduces on mTOR pathway human healthy donors also suggest HuDo-CSPG4 is more immunogenic than Hu- inducing cell survival. Thus, it is possible to speculate that colon cancer immune CSPG4 plasmid. resistance could partially reside on intrinsic colon cancer cells PD-1 overexpression. In conclusion, the HuDo-CSPG4 EP is an effective way to break immune tolerance PD-1 expression was evaluated through RT-PCR and flow cytometry in human colon against the self antigen in dogs and humans and could move this approach to cancer cell lines (HT29, HCT116, SW620, Colo205, KM12). PD-1 was detectable by standard therapies in both veterinary and human clinical settings. flow cytometry in HT29 (74,4%), in HCT116 (57,5%) and in SW620 (62,9%) while PD-L1 was weakly expressed in these cell lines. Treatment with recombinant PD- U3 L1 fusion protein significantly decreased the proliferation of HT29 and HCT116 in Maternal immunization against ALK delays tumor progression culture, while the cells were protected by PD-L1 effect in the presence of Nivolumab. in neuroblastoma-prone offspring To investigate the effect of PD-1 on oxaliplatin or 5-fluoruracil cytotoxicity, HT29 and 1)Barutello G. 2)Riccardo F. 3)Voena C. 4)Bolli E. 5)Quaglino E. 6)Chiarle R. 7) HCT116 cells were cultured with Nivolumab (10uM) plus oxaliplatin or 5-fluoruracil. Cavallo F. Unexpectedly, Nivolumab significantly protected cancer cells from cytotoxic drugs. 1)Molecular Biotechnology Center, Department of Molecular Biotechnology and The oxaliplatin and 5-fluoruracil IC50 significantly increased in nivolumab –treated Health Sciences, University of Torino, Torino, Italy 2)Molecular Biotechnology HT29 and HCT116 cells (IC50 in HT29 oxa: 0,7 to 2,5µM; 5-fluo:1,9 to 2,8µM; IC50 in HCT116 oxa: 0,8 to 1,6µM; 5-fluo:6,4 to 12,9µM). Conversely, in Colo38 cells, Center, Department of Molecular Biotechnology and Health Sciences, University of expressing very low levels of PD-1, no survival difference were detected in the Torino, Torino, Italy 3)Department of Molecular Biotechnology and Health Sciences, presence of Nivolumab. PD-1-mediated signaling are under evaluation focusing Center for Experimental Research and Medical Studies, University of Torino, Torino, on Nivolumab effect on cell apoptosis, cell-cycle arrest and mTOR signaling. Italy 4)Molecular Biotechnology Center, Department of Molecular Biotechnology In addition, the effect of Nivolumab on HT29, human colon cancer cells, will be and Health Sciences, University of Torino, Torino, Italy 5)Molecular Biotechnology evaluated in vivo. Taken together, our preliminary results demonstrate that PD-1 Center, Department of Molecular Biotechnology and Health Sciences, University axis activation decrease cancer cell proliferation and Nivolumab protects the cells of Torino, Torino, Italy 6)Children’s Hospital Boston, Boston MA, USA 7)Molecular from the anti-tumor effects of chemotherapy in PD-1 positive colon cancer cells. Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, These results reveal a possible tumor-promoting effect from anti-PD-1 therapy on University of Torino, Torino, Italy PD1-overexpressing colon cancer. Neuroblastoma (NB) is the most common pediatric solid tumor, usually diagnosed within the first year of life. One of the most predisposing genetic alterations towards NB development is a somatic mutation in the anaplastic lymphoma kinase (ALK) U2 gene, resulting in a phenylalanine-to-leucine substitution at codon 1174 (ALKF1174L). DNA ELECTROVACCINATION WITH A CHIMERIC CSPG4 PLASMID FOR This mutation potentiates the oncogenic activity of MYCN tumor driver, as well THE TREATMENT OF MALIGNANT MELANOMA IN DOGS: A COMPARATIVE F1174L ONCOLOGY TRIAL FOR TRANSLATIONAL MEDICINE documented in the ALK /MYCN preclinical model of spontaneous NB. 1)Riccardo F. 2)Barutello G. 3)Tarone L. 4)Rolih V. 5)Bolli E. 6)Arigoni M. 7)Occhipinti Since it has been shown that DNA immunization against ALK oncoantigen is able to induce a strong specific immune response against different mutated form of S. 8)Ferrone S. 9)Buracco P. 10)Cavallo F. ALK, imparing the growth of ALK-positive tumors and enhancing survival in mouse 1)Department of Molecular Biotechnology and Health Sciences, Molecular models of non-small cell lung cancer and anaplastic large cell lymphoma, ALK Biotechnology Center, University of Torino, Torino, Italy 2)Department of Molecular targeting could be exploited against NB. Biotechnology and Health Sciences, Molecular Biotechnology Center, University of As NB development occurs very early during life or even during fetal life, we sought Torino, Torino, Italy 3)Department of Molecular Biotechnology and Health Sciences, to evaluate whether maternal immunization (MI) against ALK could be applied to Molecular Biotechnology Center, University of Torino, Torino, Italy 4)Department of cancer immune-prevention in a model of neonatal NB (ALKF1174L/MYCN). Indeed, Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, as a proof of concept of the effect of MI against an oncoantigen in cencer-prone University of Torino, Torino, Italy 5)Department of Molecular Biotechnology and offspring, we previously demonstrated the efficacy of MI against Her-2/neu in Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, hampering tumor progression in a mouse model of Her-2/neu-driven mammary Italy 6)Department of Molecular Biotechnology and Health Sciences, Molecular cancer. Biotechnology Center, University of Torino, Torino, Italy 7)Center for Experimental Pre-birth immunization against ALK, using a DNA plasmid coding for the extracellular Research and Medical Studies (CERMS), Città della Salute e della Scienza di and transmembrane domains of ALK (ALK-ECTM) followed by electroporation, Torino, Torino, Italy 8)Department of Surgery, Massachusetts General Hospital, leads to an extended tumor-free survival and a lower tumor growth kinetic in Harvard Medical School, Boston, MA, USA 9)Department of Veterinary Sciences, ALKF1174L/MYCN offspring born from and fed by ALK-ECTM-vaccinated mothers, University of Torino, Grugliasco, Italy 10)Department of Molecular Biotechnology as compared to controls born from control empty vector vaccinated mothers. and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Maternally derived anti-ALK antibodies were successfully transferred from mothers Italy to newborns, as well as immune IgG-ALK protein complexes. Moreover, MI against ALK induces a decrease in ALK expression in tumor harbored in ALKF1174L/MYCN Malignant melanoma (MM) is the sixth most frequently diagnosed cancer and after offspring born by ALK-ECTM vaccinated mothers. primary tumor resection, about 30% of patients will experience disease recurrence Overall, these results indicate MI as a possible intervention to hamper NB
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development in genetically predestined offspring and could be a valuable treatment well as in human and murine tissues, in CSCs and more differentiated prostate for this cancer type for which efficient preventive options are currently unavailable. cancer cells by immunohistochemistry, immunofluorescence, flow cytometry and ImageStream. Galectin-3 was indeed expressed in CSCs, and in human and mouse prostate intraepithelial neoplasia lesions. Interestingly, Galectin-3 was found U4 strongly expressed at the invading edge of LN metastasis. Galectin-3 impacted The ADCC-mediated activity of the de-fucosylated monoclonal CSC tumorigenic and metastatic potential in vivo as Galectin-3 silencing in CSCs antibody MEN1112/OBT357 is increased by pre-treatment with reduced both tumor growth and nodal invasion. Galectin-3 was also involved in 5-Azacytidine and Decitabine in acute myeloid leukemia cell CSC-mediated immune suppression because either Galectin-3 silencing in CSCs lines or co-culture of CSCs and T cells in the presence of the Galectin-3 inhibitor LacNAc Carrisi C., Bellarosa D., Pellacani A., Binaschi M. rescued T cell proliferation. LacNAc also rescued the proliferation of T cells from Experimental Oncology and Traslational Research Department, Menarini Ricerche, LN of mice affected by prostate intraepithelial neoplasia. All together these findings Pomezia, Roma, Italy identify Galectin-3 as a key molecule and potential therapeutic target in the early phases of prostate cancer progression and metastasis. Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. It has been demonstrated that AML cells and patient samples from U6 peripheral blood and bone marrow express the myeloid marker CD157, making INCREASED SOLUBLE AND MEMBRANE-BOUND PD-L1 EXPRESSION IN them susceptible to CD157-target therapy, thus a de-fucosylated, humanized NSCLC CELL LINES TREATED WITH PEMETREXED monoclonal antibody, MEN1112/OBT357, was generated to act through the ADCC 1)Cavazzoni A. 2)Digiacomo G. 3)Petronini P.G. 4)Fiorentino M. 5)Ardizzoni A. 6) mechanism. Alfieri R. Indeed, previously, blast depletion by MEN1112/OBT357 in AML samples was 1)Medicine and Surgery, University of Parma, Parma, Italy 2)Medicine and demonstrated, thus supporting, together with a favorable toxicity profile, the current Surgery, University of Parma, Parma, Italy 3)Medicine and Surgery, University of phase I clinical trial ARMY. Parma, Parma, Italy 4)Experimental, diagnostic and specialty medicine, Univesity Hypomethylating agents (HMA) such as 5-Azacytidine and Decitabine, which of Bologna, Bologna, Italy 5)Experimental, diagnostic and specialty medicine, are the standard of care for AML patients, can improve the activity of therapeutic University of Bologna, Bologna, Italy 6)Medicine and Surgery, University of Parma, antibodies through modulation of membrane antigens. To determine whether the Parma, Italy combination of MEN1112/OBT357 and HMA, could be beneficial, an in vitro ADCC model was used to evaluate the synergism of MEN1112/OBT357 in combination Immune check-point inhibitors, such as PD-1/PD-L1 targeting monoclonal with Decitabine (DEC) and 5-Azacytidine (AZA). The ADCC reaction was performed antibodies, represent a significant step forward in cancer immunotherapy and a using CD157 positive cell lines with different AML Fab classification as target cells major breakthrough in the treatment of several solid tumors, including Non-Small and allogenic PBMCs from healthy donors as effector cells. Both target and effector Cell Lung Cancer (NSCLC). The efficacy of these agents appears to be restricted cells were pre-treated in vitro with DEC or AZA at concentrations corresponding to to a minority of patients and, particularly, to those with significant PD-L1 tissue the plasmatic levels observed in clinical studies (2-0.5 uM). After pre-treatment with expression (score ≥50%). Therefore, there is an urgent need for developing HMA, target and effector cells were incubated together for 4 hours in the presence strategies to sensitize NSCLC to immune-check-point inhibitors by boosting tumor of MEN1112/OBT357 antibody and cell death was measured by LDH release. We immune-infiltrate and up-regulating PD-L1 tumor cell expression. observed a significant synergism at different concentrations of MEN1112/OBT357 In this study we evaluated whether chemotherapy may change PD-L1 expression in in combination both with DEC or AZA on different AML cell lines. In particular, a order to guide optimal combination/sequencing with Immune check-point inhibitors statistically significant synergism was observed in SKNO-1, HL60 and K052 cells. in the clinic. The efficacy of the combinations seems to be explained by a general up-regulation Firstly we evaluated, membrane PD-L1 (mPD-L1) level in NSCLC cell lines with both of CD157 antigen and of innate immunity-related molecules involved in NK- adenocarcinoma histotype by flow cytometry. mPD-L1 was expressed at different mediated cytotoxicity (ULBP-1 and MICA/B), observed on AML cell lines by FACS levels in all the cancer cells irrespectively of EGFR mutational status. We then tested analysis following incubation with DEC or AZA. These results seem to confirm the the effect of gemcitabine, pemetrexed, paclitaxel, vinorelbine, and cisplatin on PD-L1 immune-modulatory role of HMA and the possibility to sensitize leukemic cells expression by RT-PCR, immunoblotting and flow cytometry. Cells were treated with to MEN1112/OBT357 therapy, promoting an increase of tumor cell killing when each compound at the corresponding IC50 value and only pemetrexed up-regulated administered before immunotherapy. In conclusion, these findings provide the mPD-L1 expression (5 Fold Increase) and soluble PD-L1 (sPD-L1) levels (8 FI) in rationale to evaluate these combinations in clinical trial. all the cell lines tested. Even when cells were treated with drugs at doses higher than IC50, only pemetrexed significantly enhanced membrane PD-L1 expression suggesting that modulation of PD-L1 was not related to a general cytotoxic effect of U5 chemotherapy but to a specific signalling activated by pemetrexed. STAT3 signaling Galectin-3 contributes to prostate cancer malignancy and was found to play an important role in PD-L1 regulation by pemetrexed. We also immunosuppression in the early phases of disease development demonstrated that after pemetrexed removal, PD-L1 was maintained at high level, and metastasis indicating that PD-L1 once induced is a stable protein. 1)Caputo S. 2)Grioni M. 3)Brambillasca C.S. 4)Jachetti E. 5)Freschi M. 6)Doglioni C. Our data indicate that pemetrexed effectively induces the expression of mPD-L1 7)Galli R. 8)Bellone M. and sPD-L1 in NSCLC cell lines suggesting a possible positive interaction with 1)Cellular Immunology Unit, Division of Immunology, Transplantation and immune-check-point inhibitors. Recent results from KEYNOTE-189 trial indicates Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy 2)Cellular that the addition of pembrolizumab to pemetrexed and platinum-based drug results Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, in longer OS and PFS that chemotherapy alone even in originally PD-L1 negative IRCCS San Raffaele Scientific Institute, Milan, Italy 3)Cellular Immunology Unit, patients (Gandhi L. et al. NEJM 2018). These clinical data might be explained by an Division of Immunology, Transplantation and Infectious Diseases, IRCCS San up-regulation of PD-L1 induced by pemetrexed treatment. Raffaele Scientific Institute, Milan, Italy 4)Cellular Immunology Unit, Division This study is supported by AIRC IG 2016, A.Ardizzoni. of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy 5)Unità Operativa Anatomia Patologica, IRCCS San Raffaele Scientific Institute, Milan, Italy 6)Unità Operativa Anatomia Patologica, U7 IRCCS San Raffaele Scientific Institute, Milan, Italy 7)Neural Stem Cell Biology Newly developed CXCR4 antagonist potentiates T-cell Unit, Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS trafficking and enhances PD-1 blockade efficacy in poor/high San Raffaele Scientific Institute, Milan, Italy 8)Cellular Immunology Unit, Division immunoresponsive mouse models of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele 1)D’Alterio C. 2)Napolitano M. 3)Portella L. 4)G. Rea G. 5)Barbieri A. 6)Luciano A. Scientific Institute, Milan, Italy 7)Arra C. 8)Scognamiglio G. 9)Tatangelo F. 10)Anniciello A. 11)Monaco M. 12)Gabriele L. 13)Buoncervello M. 14)Romagnoli G. 15)Cavalcanti E. 16)Botti G. Galectin-3 is a glycan-binding protein with several pro-tumoral and 17)Scala S. immunosuppressive functions. We previously reported that Galectin-3 transcript 1)Functional Genomics Unit, Department of Pathology and Laboratory Diagnostics, was overexpressed in immunosuppressive prostate cancer stem-like cells (CSCs) Istituto NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 2)Functional precociously colonizing prostate-draining lymph nodes (LN) in mice affected by Genomics Unit, Department of Pathology and Laboratory Diagnostics, Istituto autochthonous prostate intraepithelial neoplasia (Jachetti, Caputo et al. Cancer Res NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 3)Functional 2015). Here we asked if, already at the stage of prostate intraepithelial neoplasia, Genomics Unit, Department of Pathology and Laboratory Diagnostics, Istituto Galectin-3 promotes cancer progression and evasion from immune surveillance NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 4)Functional locally and at sites of dissemination. Galectin-3 expression and functions were Genomics Unit, Department of Pathology and Laboratory Diagnostics, Istituto investigated in immunocompetent and immunodeficient murine models, as NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 5)Animal Facility,
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Department of Pathology and Laboratory Diagnostics, Istituto NazionaleTumori Hepatology, Antwerp Univ. Hospital, Edegem, Belgium 14)EA3826 - Thérapeutiques “Fondazione G. Pascale”, IRCCS, Naples, Italy 6)Animal Facility, Department of cliniques et expérimentales des infections, Univ of Nantes Nantes, France 15) Pathology and Laboratory Diagnostics, Istituto NazionaleTumori “Fondazione Immatics Biotechnologies GmbH, Tübingen, Germany 16)Immatics Biotechnologies G. Pascale”, IRCCS, Naples, Italy 7)Animal Facility, Department of Pathology GmbH, Tübingen, Germany 17)CUREVAC AG, Tübingen, Germany 18)Immatics and Laboratory Diagnostics, Istituto NazionaleTumori “Fondazione G. Pascale”, Biotechnologies GmbH, Tübingen, Germany 19)Cancer Immunoregulation Unit, Ist. IRCCS, Naples, Italy 8)Pathology, Department of Pathology and Laboratory Naz. Tumori “Pascale”, Napoli, Italy Diagnostics, Istituto NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 9)Pathology, Department of Pathology and Laboratory Diagnostics, Istituto Background. HCC is the third leading cause of death from cancer globally with an NazionaleTumori “Fondazione G. Pascale”, IRCCS, Naples, Italy 10)Pathology, extremely variable 5-year survival rate. Immunotherapy strategies for HCC may Department of Pathology and Laboratory Diagnostics, Istituto NazionaleTumori represent a key therapeutic tool to improve clinical outcome in HCC patients. The “Fondazione G. Pascale”, IRCCS, Naples, Italy 11)Functional Genomics Unit, HepaVac-101 phase I/II, first-in-man, single-arm clinical trial is performed as part Department of Pathology and Laboratory Diagnostics, Istituto NazionaleTumori of the HepaVac project, funded by the European Commission’s 7th Framework “Fondazione G. Pascale”, IRCCS, Naples, Italy 12)Department of Haematology, Program under the Grant Agreement Nr. 602893 (www.hepavac.eu). The Oncology and Molecular Biology Istituto Superiore di Sanità, Rome Italy 13) HepaVac-101 trial identification numbers are NCT03203005 (Clinical trials.gov) and Department of Haematology, Oncology and Molecular Biology Istituto Superiore 2015-003389-10 (EudraCT). di Sanità, Rome Italy 14)Department of Haematology, Oncology and Molecular Method. The therapeutic cancer vaccine IMA970A is a multi-peptide-based HCC Biology Istituto Superiore di Sanità, Rome Italy 15)Division of Laboratory Medicine, vaccine composed of 16 newly discovered and overexpressed tumor-associated Department of Pathology and Laboratory Diagnostics, Istituto NazionaleTumori peptides (TUMAPs) directly identified from resected HCC tissues. Of these TUMAPs, “Fondazione G. Pascale”, IRCCS, Naples, Italy 16)Pathology, Department of 7 are restricted to HLA-A*02, 5 to HLA-A*24 and 4 to HLA class II. CV8102 is a Pathology and Laboratory Diagnostics, Istituto NazionaleTumori “Fondazione novel ribonucleic acid (RNA) based immunostimulatory agent inducing a balanced G. Pascale”, IRCCS, Naples, Italy 17)Functional Genomics Unit, Department of Th1/Th2 immune response. Patients with very early, early and intermediate stage Pathology and Laboratory Diagnostics, Istituto NazionaleTumori “Fondazione G. of HCC are enrolled to be treated with a single pre-vaccination infusion of low- Pascale”, IRCCS, Naples, Italy dose cyclophosphamide, followed by 9 intradermal vaccinations consisting of IMA970A plus CV8102. The study drugs are applied without concomitant anti- Inefficient Tcell access to the tumor microenvironment (TME) is a mechanism of tumor therapy, in order to reduce risk of tumor recurrence/progression in patients immuneresistance and the chemokine CXCL12was reported to regulate T cell access having received all indicated standard treatments and without evidence of active and immunosuppressive population Tregs and MDSC recruit. CXCR4 antagonists disease. The primary endpoints of the HepaVac-101 clinical trial are safety, were developed (PCT/IB2011/000120/ EP2528936B1/US2013/0079292A1). To tolerability, and immunogenicity. Secondary/exploratory endpoints are additional evaluate the role of CXCR4 inhibition through Peptide R in modulating T cell access immunological parameters in circulation (e.g. regulatory T-cells, myeloid-derived to TME, improving anti PD1 efficacy; anti PD-1 treatment was associated with suppressor cells, impact of the standard therapy on the natural immune response), PeptideR in two syngeneic murine models, the poor-immunoresponsive B16BL6/ infiltrating T-lymphocytes in tumor tissue, biomarkers in blood and tissue, disease- F10 murine melanoma and the immunoresponsive MC38, murine colorectal cancer. free survival/progression-free survival and overall survival. Overall, it is planned to C57Bl/6 were subcutaneously inoculated with MC38 1*10^6 or with B16-human- enroll 40 patients. Suitable patients enrolled at Tuebingen are invited to participate CXCR4 5*10^5. PeptideR (2mg/kg ip, 5 day/week), Anti–PD-1 (RMP1-14, BioXCell, in a trial extension investigating an actively personalized vaccine (APVAC). The 5mg/kg ip, twice weekly) treatment was initiated at mean tumor volume~250 HepaVac-101 trial is conducted in 6 centers located in 5 European countries. mm^3 (well established tumor), or at palpable mass (rapidly growing model). In Results of the trial will be presented. poor immunogenic B16-hCXCR4 mice, PepR potentiates efficacy of anti PD-1 in reducing tumor growth. Tumor volume(mm^3 mean±SEM) for PepR+antiPD-1 group was mean±SEM 373±451 n=10,vs vehicle 845±417 n=12, and anti–PD-1 U9 605±397, n=10. In B16hCXCR4 tumors PepR+anti-PD-1 group, higher number The diverse functions of PD-1 depend on CD28 molecule of Granzyme B+ cells [mean cells/mm^2±SEM (16.3±3.6 vs antiPD-1 7.02±1.2 expression in Ag-specific CD8+ T cells vs CTRL 3.2±1.2, or PepR 7.44±1.2)]were detected; and lower Tregs as FoxP3+ 1)Palermo B.2)Franzese O. 3)Panetta M. 4)Ferraresi V.5)Alessandrini G. 6)Facciolo cells were detected (4.08±1.7vs8.7±2.2 vehicle). Similar results were detected F. 7)Ciliberto G. 8)Nisticò P. in immunogenic MC38 tumors. To investigate on intrinsic efficacy of PepR+PD-1 1)Unit of Tumor Immunology and Immunotherapy, Department of Research, AthymicNude-Foxn1nu mice were subcutaneously inoculated with 1*10^6 human Advanced Diagnostics and Technological Innovation, IRCCS Regina Elena melanoma cells PES43. The last generated CXCR4 antagonist with high affinity National Cancer Institute, Rome, Italy 2)Department of Systems Medicine, School for human, Peptide R54, was evaluated (2mg/kg ip, 5 day/week) w/wo Nivolumab of Medicine, University of Tor Vergata, Rome, Italy 3)Unit of Tumor Immunology (5mg/kg ip, twice weekly) initiated at tumors volume (~500 mm^3). After 3 weeks and Immunotherapy, Department of Research, Advanced Diagnostics and of treatments combined treatment reduced tumor volume (207.39±54.44, n=8) as Technological Innovation, IRCCS Regina Elena National Cancer Institute, Rome, compared anti–PD-1 monotherapy (297±58.57, n=7) and vehicle (418.32±104.95, Italy 4)Department of Medical Oncology 1, IRCCS Regina Elena National Cancer n=9). Decrease in lung/liver metastasis (%MSCP) was observed in PepR54group Institute, Rome, Italy 5)Thoracic Surgery Unit, IRCCS Regina Elena National Cancer vs CTRL: lungs 0.06±0,0002vs0.1±0,03 and liver 0.172±0,065 vs 0.299±0,106 Institute, Rome, Italy 6)Thoracic Surgery Unit, IRCCS Regina Elena National Cancer respectively). In conclusions CXCR4 inhibition (PepR) improves the anti PD-1 Institute, Rome, Italy 7)Scientific Direction, IRCCS Regina Elena National Cancer efficacy reducing tumor growth and potentiating immuneresponse in both Institute, Rome, Italy 8)Unit of Tumor Immunology and Immunotherapy, Department immunosensitive and immunoresistant syngeneic models. In the PES43 human of Research, Advanced Diagnostics and Technological Innovation, IRCCS Regina melanoma xenograft PepR54+Nivolumab decreased tumor growth suggesting a Elena National Cancer Institute, Rome, Italy direct effect on neoplastic cells and on TME not Tcell dependent. We have recently demonstrated that CD8+ T-cell anti-tumor functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen U8 nature, T-cell immune-checkpoint phenotype, TCR repertoire diversity and anti- HepaVac-101 first-in-man therapeutic cancer vaccine Phase I/II tumor T-cell quality. We observed that the co-expression of PD-1 with CD28 confers clinical trial for hepatocellular carcinoma patients an exhausted phenotype and defective anti-tumor functionality to the Ag-specific 1)Mayer-Mokler A. 2)Accolla R. 3)Ma Y. T. 4)Heidenreich R. 5)Ascierto P. 6) cells, which may be reverted by the PD-1 blockade. In contrast, the expression Koenigsrainer A. 7)Loeffler M. 8)Gouttefangeas C. 9)Flohr C. 10)Ludwig J. 11) of PD-1 in the absence of CD28 does not impair anti-tumor T-cell functionality. Rammensee H.-G. 12)Sangro B. 13)Francque S. 14)Chaumette T. 15)Weinschenk In particular, we showed that CD8+ CD28-PD-1+ T cells represent the T-cell T. 16)Reinhardt C. 17)Gnad-Vogt U. 18)Singh-Jasuja H. 19)Buonaguro L. subpopulation with the highest proliferative capability and an AKT-dependent anti- 1)Immatics Biotechnologies GmbH, Tübingen, Germany 2)Dept. Surgical and tumor polyfunctionality sustained by ICOS. Morphological Sciences, Univ. dell’Insubria, Varese, Italy 3)NIHR Biomedical Considering that CD8+ T-cell PD-1 expression does not only reflect an impaired Research Unit in Liver Disease, School of Immunity and Infection, Univ. of anti-tumor functionality, but also a specific differentiation and/or activation status, Birmingham, UK 4)CUREVAC AG, Tübingen, Germany 5)Melanoma, Cancer herein we elucidated the modulatory PD-1 role in relationship to the differentiation Immunotherapy and Development Therapeutics Unit, Ist. Naz. Tumori “Pascale”, phenotype, focusing on CD28 expression. We performed our study both in a panel Napoli, Italy 6)Dept. of General, Visceral and Transplant Surgery Univ. Hospital of antigen-specific (Melan-A and gp100) T-cell clones isolated from vaccinated Tübingen, Tübingen, Germany 7)Dept. of General, Visceral and Transplant Surgery melanoma patients and in “ex vivo” PBMC and TILs isolated Univ. Hospital Tübingen, Tübingen, Germany 8)Dept of Immunology, Eberhard Karls from patients with different types of cancer, by flow cytometry, biochemical and Univ., Tübingen, Germany 9)Immatics Biotechnologies GmbH, Tübingen, Germany functional analyses. 10)Immatics Biotechnologies GmbH, Tübingen, Germany 11)Dept of Immunology, Molecular analysis of the different CD8+T-cell subsets defined in terms of PD-1/ Eberhard Karls Univ., Tübingen, Germany 12)Liver Unit, Clinica Universidad CD28 co-expression have revealed that the AKT pathway was not activated de Navarra, and CIBEREHD, Pamplona, Spain 13)Div. of Gastroenterology and when PD-1 and CD28 were co-expressed, and confirmed our previous results
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demonstrating that this kinase was activated when PD-1 was expressed in the U11 absence of CD28. A comparative functional analysis, irrespective of Ag-specificity, Predictive value of circulating tumor cells and circulating revealed that clones expressing PD-1 in the presence of CD28 were not able to lyse free DNA in NSCLC patients treated with Nivolumab tumor cells, whereas clones expressing high levels of PD-1 in the absence of CD28 Dal Bello M.G., Coco S., Alama A., Vanni I., Boccardo S., Rijavec E., Barletta G., were tumor-reactive. We are currently evaluating the role of these PD1/CD28 T-cell Genova C., Biello F., Rossi G., Tagliamento M., Francesco Grossi F. subsets in peripheral blood and TIL of cancer patients. Lung Cancer Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy Our results highlight the complex role of PD-1 in regulating T-cell functionality and the need to determine the activation/inhibition pathways of this molecule to tailor Background. Immunotherapy represents a dramatic change in the treatment of non inhibitory blockade therapies in cancer patients. small cell lung cancer (NSCLC), but nowadays prognostic and predictive factors of response to immune checkpoint inhibitors (ICIs) are still under debate. It has been observed that circulating biomarkers might have a prognostic role in lung cancer. U10 The aim of this study was to determine whether circulating tumor cells (CTCs) as Antibody-Fc/FcR Interaction on Macrophages as a Mechanism well as circulating free DNA (cfDNA) might predict the outcome of patients with for Hyperprogressive Disease in Non-Small Cell Lung Cancer advanced NSCLC treated with Nivolumab. Subsequent to PD-1/PD-L1 Blockade Methods. From May 2015 to April 2017 89 NSCLC patients were treated with 1)Moro M. 2)Lo Russo G. 3)Sommariva M. 4)Cancila V. 5)Boeri M. 6)Centonze G. Nivolumab as a second or further line of therapy. All patients underwent blood 7)Ganzinelli M. 8)Milione M. 9)Pruneri G. 10)Proto C. 11)Sangaletti S. 12)Storti C. sample collection before the start of treatment (baseline) and after 4 and 7 cycles of 13)Torri V. 14)Anichini A. 15)Rivoltini L. 16)Tripodo C. 17)Colombo M.P. 18)Balsari Nivolumab to evaluate both biomarkers. CTCs were isolated from 3mL of blood by A. 19)Sozzi G. 20)Garassino M.C. the filtration-based device ScreenCell Cyto (ScreenCell) according to manufacturer’s 1)Department of research, Fondazione IRCCS istituto nazionale dei tumori, Milan, protocol. cfDNA was extracted from plasma using the QIAamp DNA Blood Mini Kit Italy 2)Medical oncology department, Fondazione IRCCS istituto nazionale dei (Qiagen) and quantified (ng/mL) by qPCR method, using hTERT single copy gene. tumori, Milan, Italy 3)Department of biomedical sciences for health, University The median baseline CTC number and cfDNA content were used as cut-off values of Milan, Milan, Italy 4)Department of health sciences, University of Palermo, to discriminate patients with different outcomes. An univariate analysis was done to Palermo, Italy 5)Department of research, Fondazione IRCCS istituto nazionale evaluate the overall survival (OS) in the study population based on CTC and cfDNA dei tumori, Milan, Italy 6)Department of research, Fondazione IRCCS istituto using the Kaplan Meyer method. nazionale dei tumori, Milan, Italy 7)Medical oncology department, Fondazione Results. The median CTC number and cfDNA at baseline were 2/3mL and 836.5 ng/ IRCCS istituto nazionale dei tumori, Milan, Italy 8)Department of pathology and mL, respectively. Median OS was 8.8 and 6.2 months for patients with baseline CTC laboratory medicine, Fondazione IRCCS istituto nazionale dei tumori, Milan, Italy ≤2 and >2, respectively (HR 1.53, 95% CI 0.96-2.42; p=0.072). Similarly, patients 9)Department of pathology and laboratory medicine, Fondazione IRCCS istituto with high level of cfDNA > 836.5 ng/ml showed a worse OS as compared with those nazionale dei tumori, Milan, Italy 10)Medical oncology department, Fondazione having lower cfDNA: 5.1 vs 9.4 months (HR 1.63, 95% CI 1.02-2.59; p=0.040). IRCCS istituto nazionale dei tumori, Milan, Italy 11)Department of research, Patients with both CTC and cfDNA over the median had worse OS (HR 2.23, 95% Fondazione IRCCS istituto nazionale dei tumori, Milan, Italy 12)Department CI 1.21-4.11; p=0.037) than patients with both markers under the median. of research, Fondazione IRCCS istituto nazionale dei tumori, Milan, Italy 13) Conclusions. A statistically significant advantage in terms of OS was observed in Department of oncology, Istituto di ricerche farmacologiche Mario Negri, Milan, Italy the group of patients with baseline cfDNA below the median value. A longer survival 14)Department of research, Fondazione IRCCS istituto nazionale dei tumori, Milan, of borderline significance was observed in the group of patients with baseline CTC Italy 15)Department of research, Fondazione IRCCS istituto nazionale dei tumori, ≤2. Furthermore, the combined presence of high CTC and cfDNA was associated Milan, Italy 16)Department of health sciences, University of Palermo, Palermo, Italy with worse OS. 17)Department of research, Fondazione IRCCS istituto nazionale dei tumori, Milan, Italy 18)Department of biomedical sciences for health, University of Milan, Milan, Italy 19)Department of research, Fondazione IRCCS istituto nazionale dei tumori, U12 Milan, Italy 20)Medical oncology department, Fondazione IRCCS istituto nazionale Retargeting T cells against leukemia by lipid-specific TCR dei tumori, Milan, Italy transfer 1)Consonni M. 2)Garavaglia C. 3)de Lalla C. 4)Bigi A. 5)Casucci M. 6)Bondanza A. Background. The advent of immune checkpoint inhibitors (ICI) has radically changed 7)Lepore M. 8)Mori L. 9)De Libero G. 10)Ciceri F. 11)Dellabona P. 12)Casorati G. the paradigm of care for patients with non-small cell lung cancer (NSCLC). However, 1)Division of Immunology, Transplantation, and Infectious Diseases, Experimental a paradoxical boost in tumor growth was described in a subset of patients treated Immunology Unit, San Raffaele Scientific Institute, Milan, Italy 2)Division of with immune checkpoint inhibitors (ICI), a phenomenon known as hyperprogression Immunology, Transplantation, and Infectious Diseases, Experimental Immunology (HP). Data already published in the field mainly focused on adaptive immunity Unit, San Raffaele Scientific Institute, Milan, Italy 3)Division of Immunology, without finding any characteristics to predict a priori HP. The aim of this study was to Transplantation, and Infectious Diseases, Experimental Immunology Unit, San investigate, at the clinical and pathological level, the phenomenon of HP in NSCLC Raffaele Scientific Institute, Milan, Italy 4)Division of Immunology, Transplantation, patients and to evaluate the role of innate immunity during ICI treatment and Infectious Diseases, Experimental Immunology Unit, San Raffaele Scientific Methods. Among 187 patients with non-small cell lung cancer (NSCLC) treated with Institute, Milan, Italy 5)Division of Immunology, Transplantation, and Infectious ICI at our Institute, cases with HP were identified according to clinical and radiological Diseases, Innovative Immunotherapies Unit, San Raffaele Scientific Institute, Milan, criteria. Baseline histological samples from patients treated with ICI were evaluated Italy 6)Division of Immunology, Transplantation, and Infectious Diseases, Innovative by immunohistochemistry (IHC) for myeloid and lymphoid markers. T-cell deficient Immunotherapies Unit, San Raffaele Scientific Institute, Milan, Italy 7)Experimental mice, injected with human lung cancer cells and patient-derived xenografts (PDXs) Immunology, Department of Biomedicine, University Hospital, Basel, Switzerland 8) belonging to specific mutational subsets, were assessed for tumor growth after Experimental Immunology, Department of Biomedicine, University Hospital, Basel, treatment with antibodies against mouse and human programmed death receptor-1 Switzerland 9)Experimental Immunology, Department of Biomedicine, University (PD-1). The immune microenvironment was evaluated by flow cytometry and IHC. Hospital, Basel, Switzerland 10)Hematology and Bone Marrow transplantation Results. Among 187 patients, 152 were evaluable for clinical response. We Unit, San Raffaele Scientific Institute, Milan, Italy 11)Division of Immunology, identified 4 categories: 32 cases were defined as Responders (21%), 42 patients Transplantation, and Infectious Diseases, Experimental Immunology Unit, San with Stable Disease (27.7%), 39 cases defined as Progressors (25.7%) and 39 Raffaele Scientific Institute, Milan, Italy 12)Division of Immunology, Transplantation, patients with HP (25.7%). Pre-treatment tissue samples from all patients with HP and Infectious Diseases, Experimental Immunology Unit, San Raffaele Scientific showed tumor-infiltration by M2-like CD163+CD33+PD-L1+ clustered epithelioid Institute, Milan, Italy macrophages. Enrichment by tumor-associated macrophages (TAM) was observed even in tumor nodules from immunodeficient mice injected with human lung cancer CD1-restricted T cells specific for self-lipids could play a role in cancer immune cells and with PDXs. Moreover, anti-PD-1 treated PDXs showed HP-like growth surveillance, where self-antigens are the targets of immune responses. We showed with dissemination to lung and regional (iliac) lymph node metastases, as well as an that primary acute myelogenous (AML) and B-lymphoblastic (B-ALL) leukemia increase in F4/80+/CD206+ TAMs aggregating in fibrotic-like areas. Interestingly, in blasts express CD1c and are recognized by a group of CD1c self-reactive T these in vivo models, HP-like growth was triggered by treatment with anti-PD-1, but cells specific for methyl-lysophosphatidic acids (mLPAs), a novel class of self- not by anti-PD-1 F(ab)2-fragments. lipid antigens that accumulate in malignant cells, which control leukemia growth Conclusions. These results suggest a crucial role of TAM reprogramming, upon in vitro and in vivo. These findings point to CD1c and self-lipids as new potential FcR engagement by ICI, eventually inducing HP and provide clues on a distinctive targets for leukemia immunotherapy. The little polymorphism of CD1 molecules immunophenotype potentially able to predict HP. and their expression on mature leukocytes are indeed highly attractive for adoptive cell therapy (ACT) with such T cells in the context of stem cell transplantation for hematological malignancies. To assess the feasibility of ACT for acute leukemia with mLPA-specific T cells, we generated a library of lentiviral vectors encoding a panel of human mLPA-specific TCRs. Upon TCR transduction, either Jurkat T
88 89 ABSTRACT BOOK cells or human primary T cells were specifically retargeted against CD1c-expressing U14 malignant targets in vitro, highlighting a lead mLPA-specific TCR suitable for Immune Profile of peripheral blood T lymphocytes in Melanoma adoptive immunotherapy. Primary T cells transduced with this TCR killed CD1c- and NSCLC patients treated with anti PD-1 antibodies expressing malignant targets in vitro and significantly delayed leukemia progression 1)Grazia G. 2)Tassi E. 3)Vegetti C. 4)Molla A. 5)Perotti V. 6)Sgambelluri F. 7) in NSG mice. To gain further insight into the efficacy and safety of mLPA-specific Cimminiello C. 8)Di Guardo L. 9)Indini A. 10)Signorelli D. 11)Proto C. 12)Lo Russo ACT, we generated transgenic mice expressing CD1c with a pattern similar to the G. 13)Del Vecchio M. 14)Garassino M.C. 15)Mortarini R.16)Anichini A. human one, which harbored functional APCs recognized by mLPA-specific T cells 1)Human Tumors Immunobiology Unit, Research Department, Fondazione and selected a CD1c self-reactive peripheral T cell repertoire. IRCCS Istituto Nazionale dei Tumori, Milan, Italy 2)Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital, Milan, Italy 3)Human Tumors Immunobiology Unit, Research U13 Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 4)Human FUNCTIONAL CHARACTERIZATION OF PERIPHERAL BLOOD T CELLS Tumors Immunobiology Unit, Research Department, Fondazione IRCCS Istituto FROM MELANOMA AND NON SMALL CELL LUNG CANCER PATIENTS IN THE Nazionale dei Tumori, Milan, Italy 5)Human Tumors Immunobiology Unit, Research CONTEXT OF IMMUNE CHECKPOINT BLOCKADE Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Human 1)Sgambelluri F. 2)Vegetti C. 3)Grazia G. 4)Perotti V. 5)Molla A. 6)Cimminiello C. 7) Tumors Immunobiology Unit, Research Department, Fondazione IRCCS Istituto Di Guardo L. 8)Indini A. 9)Signorelli D. 10)Proto C. 11)Lo Russo G. 12)Del Vecchio Nazionale dei Tumori, Milan, Italy 7)Department of Medical Oncology, Fondazione M. 13)Garassino MC. 14)Mortarini R. 15)Anichini A. IRCCS Istituto Nazionale dei Tumori, Milan, Italy 8)Department of Medical Oncology, 1)Department of research, Fondazione IRCCS istituto nazionale dei tumori, Milano, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 9)Department of Italy 2)Department of research, Fondazione IRCCS istituto nazionale dei tumori, Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Milano, Italy 3)Department of research, Fondazione IRCCS istituto nazionale dei 10)Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei tumori, Milano, Italy 4)Department of research, Fondazione IRCCS istituto nazionale Tumori, Milan, Italy 11)Department of Medical Oncology, Fondazione IRCCS dei tumori, Milano, Italy 5)Department of research, Fondazione IRCCS istituto Istituto Nazionale dei Tumori, Milan, Italy 12)Department of Medical Oncology, nazionale dei tumori, Milano, Italy 6)Department of medical oncology, Fondazione Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 13)Department of IRCCS stituto nazionale dei tumori, Milano, Italy 7)Department of medical oncology, Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Fondazione IRCCS istituto nazionale dei tumori, Milano, Italy 8)Department of 14)Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei medical oncology, Fondazione IRCCS istituto nazionale dei tumori, Milano, Italy Tumori, Milan, Italy 15)Human Tumors Immunobiology Unit, Research Department, 9)Department of medical oncology, Fondazione IRCCS istituto nazionale dei Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 16)Human Tumors tumori, Milano, Italy 10)Department of medical oncology, Fondazione IRCCS Immunobiology Unit, Research Department, Fondazione IRCCS Istituto Nazionale istituto nazionale dei tumori, Milano, Italy 11)Department of medical oncology, dei Tumori, Milan, Italy Fondazione IRCCS istituto nazionale dei tumori, Milano, Italy 12)Department of medical oncology, Fondazione IRCCS istituto nazionale dei tumori, Milano, Italy 13) Despite remarkable improvements in clinical management of advanced disease in Department of medical oncology, Fondazione IRCCS istituto nazionale dei tumori, both melanoma and non-small cell lung cancer (NSCLC), following the introduition Milano, Italy 14)Department of research, Fondazione IRCCS istituto nazionale immunotherapy with antibodies blocking the programmed death (PD)-1 pathway, dei tumori, Milano, Italy 15)Department of research, Fondazione IRCCS istituto still clinical benefit is limited to a fraction of patients. Primary and acquired resistance nazionale dei tumori, Milano, Italy to immunological treatment is common in these diseases and it is thought to be in part related to functional impairment of anti-tumor T cells and/or to presence Therapeutic blockade of PD-1 with monoclonal antibodies leads to durable tumor of immunosuppressive cells. A possible approach to verify this hypothesis is to regressions in patients with several types of cancer. This type of treatment is compare the functional profile of peripheral blood lymphocytes (PBL) isolated from based on the functional re-activation of inhibitory receptor+, exhausted T cells that patients (before treatment with immunotherapy) to PBL from healthy donors. To test accumulate at tumor site and that can be identified even in periphery. Since only a this hypothesis, we used 10 color flow cytometry to profile pre-therapy peripheral fraction of patients respond to this type of immunological treatment, one hypothesis blood T lymphocytes from NSCLC (n=48) and melanoma (n=23) patients treated is that cancer patients may have defective T cells that cannot be rescued to anti- with anti-PD-1 antibody, and of 10 healthy donors as controls. Our experiments tumor function by the therapeutic antibodies. Defective function in T cells can be evaluated the presence and frequency of CD8+ and CD4+ T lymphocytes as well tested by measuring the modulation of surface activation molecules as well as the as of CD4+ T regulatory cells and their expression of markers of activation (ie: production of relevant immune mediators (IFN-g and TNF-a), upon induction by HLA-DR, CD137), proliferation (ie Ki67), differentiation (ie: KLRG1, CD127) and polyclonal stimuli (PMA-ionomycin). To this end we used 10-color flow cytometry exhaustion (ie: inhibitory receptors, CD39). Compared to donors, in the CD4+ T to assess the modulation of several activation/functional differentiation molecules cell compartment of both melanoma and NSCLC patients, we found an enrichment (CD137, CD39, PD-1, CD28) and the intra-cellular expression of IFN-g and TNF-a in Tregs (CD4+FOXP3+CD39+) and in CD4+ conventional T cells an increased in T cells from pre-therapy peripheral blood samples from 22 melanoma patients, 45 expression of Inhibitory receptors (TIGIT, TIM3) was observed. In the CD8+ fraction non-small cell lung cancer (NSCLC) patients, compared to samples from 10 healthy of patients in both diseases, a higher frequency of T cells expressing IRs as PD-1, donors. In agreement with the hypothesis several differences were observed in TIGIT and TIM3, as well as the exhaustion-related marker CD3 was found. CD8+ the response to PMA-Ionomycin in both melanoma and NSCLC blood samples T cells from patients were also enriched in activated (HLA-DR+) and proliferating compared to healthy donors. Specifically, CD8+ T cells as well as CD8+ PD-1+ (Ki67+) lymphocytes, and also showed a significant increase of CD8+PD-1+HLA- lymphocytes from cancer patients were selectively defective in the modulation of DR+ and of CD8+PD-1+Ki67+ cells. These results suggest that cancer patients CD28 and of PD-1, compared to donors. In contrast, interestingly, CD8+ T cells have a peripheral immune profile skewed towards enhanced representation of from melanoma and NSCLC patients showed enhanced modulation of CD137, a immunosupressive T cells (Tregs), and of IR+ CD4+ and CD8+ T cells that may marker associated with presence of anti-tumor T cells when evaluated at tumor be functionally impaired. However, within the CD8+ subset, cancer patients also site, compared to donors. Enhanced modulation of CD137 in cancer patients’ have a limited set of recently activated, proliferating CD8+ T cells that may signal lymphocytes was observed even among Tregs (CD8-CD39+). Furthermore, for an ongoing functional immune reponse, as we recently described at tumor disease-specific effects were also observed: TNF-a and IFN-g were often induced site in NSCLC. The association of these immune profiles with clinical benefit after at higher levels in CD8+ CD28+ T cells (i.e. the subset functionally competent to immunotherapy is being currently investigated. This work was supported by AIRC respond to anti-PD-1) from NSCLC patients compared to donors. Taken together grant 17431 to AA and FIRC fellowship 8122 to GG. these data suggest that testing the response to polyclonal stimuli, of peripheral blood lymphocytes from cancer patients, is a valuable approach to detect altered T cell function in the context of immunotherapy. The association of these functional U15 profiles with clinical benefit from immunotherapy is currently being investigated. The mutational landscape of nivolumab response in non-small Acknowledgments. This work was supported by AIRC grant 17431 to AA. cell lung cancer 1)Laprovitera N. 2)Cinausero M. 3)Riefolo M. 4)De Maglio G. 5)Fiorentino M. 6) Ferracin M. 7)Ardizzoni A. 1)Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 2)Department of Pathology, University Hospital of Udine, Udine, Italy 3)Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 4)Department of Pathology, University Hospital of Udine, Udine, Italy 5)Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 6)Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 7)Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy
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tumor biopsies at resistance. N=20 patients are expected to bear after TMZ priming Programmed cell death -1 (PD-1) inhibitors represent a therapeutic option for >20 mutations/megabase/tumor and will proceed to trial-phase for pembrolizumab advanced non-small cell lung cancer (NSCLC) patients, providing an overall survival treatment. Overall response rate (primary outcome), Progression Free, and benefit independently from programmed cell death 1 ligand (PD-L1) expression. Overall Survival, and treatment related toxicities (secondary outcomes) in MMRp However, about 50% patients fail to respond to immune checkpoint inhibitors and pembrolizumab-treated patients will be calculated. Treatment efficacy and toxicity rapidly progress. within pembrolizumab-treated MMRd cohort will serve as comparison. Pre- and A high (>10/Mb) tumor non-synonymous mutation burden (TBM) has been recently post-TMZ biopsies and longitudinal blood and stool collection during priming and proposed as biomarker for response to anti-PD-1/PD-L1 therapy. However, there is trial phases will allow for discovery of predictive molecular markers and for the the possibility that other genetic alterations could identify patients who can obtain a assessment of integrated tumor and (immune)environment evolution in response clinical benefit from immunotherapy. to therapy. To verify this hypothesis, we collected DNA from NSCLC neoplastic lesions obtained from advanced, pre-treated NSCLC patients enrolled in a clinical trial with anti PD-1 monoclonal antibody nivolumab at Bologna and Udine hospitals. U17 We analyzed the mutational status of 22 genes (92 amplicons and more than 500 TN EXPRESSION IN SERUM EXOSOMES FROM BREAST CANCER PATIENTS variants) using the CE-IVD Oncomine Solid Tumour DNA kit (Thermo Fisher). This Rabassa ME., Isla Larrain MT., Insaurralde F., Croce MV. panel includes selected receptor tyrosine kinase genes (EGFR, ERBB2, ERBB4, CINIBA, Faculty of Medical Sciences, UNLP, La Plata, Buenos Aires, Argentina MET, FGFR1, FGFR2, FGFR3, DDR2 and ALK), receptor tyrosine kinase pathway genes (KRAS, NRAS, PIK3CA, BRAF, PTEN MAP2K1 and AKT1), and other Tumor cells secrete exosomes, small vesicles involved in tumor dissemination and cancer-related genes (TP53, STK11, CTNNB1, SMAD4, FBXW7, NOTCH). immune evasion. It has been found that glycoproteins may be O-glycan carriers at We used our results to determine the landscape of genetic alterations in nivolumab- the exosomal membrane. One of them is the Tn antigen, a well-known tumor antigen treated NSCLC, providing a correlation between gene mutations and anti PD-1 constituted by the attachment of GalNAc to Ser/Thr. Also, Tn is recognized by the resistance. major galactose binding lectin (MGL). With the aim to study Tn antigen expression in In our cohort, the most frequently mutated genes were TP53, KRAS, ERBB2, circulating exosomes, we isolated exosomes from 27 plasma samples from breast SMAD4, DDR2, NOTCH1, STK11, which were mutated in more than 10% of untreated cancer patients. We employed size exclusion chromatography in a 20 mL patients; while EGFR, BRAF, ERBB4, NRAS, PIK3CA, MET, ALK, FGFR2, FGFR3, CL4B agarose column; the void volume was collected in 0,5 mL fractions. Protein AKT1, FBXW7 and PTEN mutations were shared between two patients or patient- content was analyzed employing a Qubit assay and 330 and 260/280 absorbance specific. ratio was calculated for fraction selection; high 330 and 260/280 fractions were We believe that patient stratification according to their specific mutational pattern evaluated by electron microscopy. Presence of exosomal markers CD9 and CD6, as can become a determinant of therapeutic choice also in immunotherapy settings. well as Tn expression was assessed by dotblot. Tn positive samples were selected for Western blot. From 27 samples 11 (41%) were positive for CD63, 13/27 (48%) for CD9 and 7/27 (26%) were Tn+. All Tn+ samples were also CD63+ and CD9+ U16 and showed exosome like microvesicles by electron miscroscopy. CD63+ samples Pharmacological Inactivation of DNA repair to improve had more protein content than CD63- (5,7 vs 3,5 ug/mL). Western blot analysis RESPONSE TO IMMUNOTHERAPY: the arethusa trial in metastatic showed several molecular weight bands from 110 to 60 kD, suggesting that Tn may colorectal CANCER (mCRC) patients be attached to different glycoproteins in breast cancer exosomes. In conclusion, we 1)Siena S. 2)Sartore-Bianchi A. 3)Amatu A. 4)Ghezzi S. 5)Bencardino K. 6)Zagonel could detect Pan-cancer Tn antigen in breast cancer plasma circulating exosome V. 7)Lonardi S. 8)Loupkis F. 9)De braud F. 10)Pietrantonio F. 11)Germano G. 12) containing fractions. Crisafulli G. 13)Rospo G. 14)Barault L. 15)Di Nicolantonio F. 16)Amirouchene- Angelozzi N. 17)Luraghi P. 18)Luca L. 19)Marsoni S. 20)Bardelli A. 1)Medical oncology, Grande Ospedale Metropolitano Niguarda, Milano, Italy 2) Medical oncology, Grande Ospedale Metropolitano Niguarda, Milano, Italy 3) Medical oncology, Grande Ospedale Metropolitano Niguarda, Milano, Italy 4) Medical oncology, Grande Ospedale Metropolitano Niguarda, Milano, Italy 5) Medical oncology, Grande Ospedale Metropolitano Niguarda, Milano, Italy 6)Medical oncology, Iov- istituto oncologico veneto, Padova, Italy 7)Medical oncology, Iov- istituto oncologico veneto, Padova, Italy 8)Medical oncology, Iov- istituto oncologico veneto, Padova, Italy 9)Medical oncology, Istituto nazionale tumori, Milano, Italy 10)Medical oncology, Istituto nazionale tumori, Milano, Italy 11)Molecular oncology, Candiolo cancer center, Candiolo, Italy 12)Molecular oncology, Candiolo cancer center, Candiolo, Italy 13)Molecular oncology, Candiolo cancer center, Candiolo, Italy 14)Cancer epigenetics, Candiolo cancer center, Candiolo, Italy 15)Cancer epigenetics, Candiolo cancer center, Candiolo, Italy 16)Precision oncology, Ifom (istituto firc di oncologia molecolare), Milano, Italy 17)Precision oncology, Ifom (istituto firc di oncologia molecolare), Milano, Italy 18)Precision oncology, Ifom (istituto firc di oncologia molecolare), Milano, Italy 19)Precision oncology, Ifom (istituto firc di oncologia molecolare), Milano, Italy 20)Molecular oncology, Candiolo cancer center, Candiolo, Italy Background. mCRC remains mostly not curable despite improvement in survival in genetically selected patients. Recently remarkable responses to immunotherapy had been witnessed in mismatch repair deficient (MMRd) tumors that represent less than 5% of mCRCs. Nonetheless we showed in preclinical models that pharmacological inactivation of MMR with the drug temozolomide (TMZ) foster an increase in immunogenic neoantigens and prime MMR proficient (MMRp) tumors for response to checkpoint inhibitors. Accordingly, mCRC patients recruited in clinical trials with TMZ acquired alterations of MMR genes upon treatment and showed remarkable increase in tumor mutational burden (TMB) at disease progression (PD). We thus designed ARETHUSA (NCT03519412) to test whether a priming course with TMZ in patients can sensitize mCRC to anti-PD1 inhibitor pembrolizumab. Methods. Arethusa is a 2-cohorts, phase II trial consisting of three different phases. During screening-phase, 344 mCRC patients with RAS-extended mutations who failed standard therapies will be tested for MMR status. MMRd CRC patients will proceed directly to trial-phase for immediate pembrolizumab treatment (expected N=14). MMR-proficient (MMRp) patients will be further tested for TMZ sensitivity via assessment of expression of O6-methylguanine-DNA methyltransferase (MGMT) by immunohistochemistry and by promoter methylation analysis. Expected 67 IHC-negative, promoter methylation-positive MMRp patients will thus be eligible for priming-phase and will receive TMZ until PD; TMB will then be assessed on
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to endosomes/lysosomes, where they physically interact with TORC1-associated TUMOR MICROENVIRONMENT: RagA and LAMTOR p18, and with the autophagy-related adapter p62. Accordingly, INFLAMMATION, HYPOXIA OCTN1 overexpression led to constitutive, aminoacid-independent phosphorylation of downstream effector S6K and S6, while dumping physiological response to AND ANGIOGENESIS aminoacids. Of note, these interactions were overall enhanced in the 503F variant. Furthermore, OCTN1, and to an even higher extent the 503F variant, promoted IL1b V1 secretion in an aminoacid- and mTOR-dependent fashion in engineered 293T, and p140Cap affects breast cancer features through the mTOR signaling was downregulated in OCTN1-silenced Caco-2 colon carcinoma regulation of the tu mor microen vironmen t comp osition cells and in the intestine of OCTN1 KO mice. 1)Salemme V. 2)Chapelle J. 3)Vedelago M. 4)Conti L. 5)Cavallo F. 6)Angelini C. 7) Taken together these finding highlight OCTN1 and its cancer associated variant Lamolinara A. 8)Moiso E. 9)Iezzi M. 10)Turco E. 11)Defilippi P. as novel players in mTORC1-dependent metabolic and proinflammatory signaling 1)Dept. of Molecular Biotechnology and Health Sciences, University of Torino, Torino, within the intestinal tumor microenvironment, and by extension as potential Italy 2)Dept. of Molecular Biotechnology and Health Sciences, University of Torino, candidates for the rational design of OCTN1-targeted personalized therapies. Torino, Italy 3)Dept. of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 4)Dept. of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 5)Dept. of Molecular Biotechnology and Health V3 Sciences, University of Torino, Torino, Italy 6)Dept. of Molecular Biotechnology Dissecting and targeting Toll-like receptors signaling in and Health Sciences, University of Torino, Torino, Italy 7)Dept. of Medicine and chronic lymphocytic leukemia Aging Sciences, G. D’Annunzio University of Chieti-Pescara, Chieti, Italy 8)Dept. of Delvecchio S., Sana I., Vilia M.G., Mantione M.E., Ranghetti P., Rovida A., Angelillo Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 9) P., Scarfò L., Ghia P., Muzio M. Dept. of Medicine and Aging Sciences, G. D’Annunzio University of Chieti-Pescara, Division of Experimental Oncology, IRCCS San Raffaele Hospital, Milan, Italy Chieti, Italy 10)Dept. of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 11)Dept. of Molecular Biotechnology and Health Sciences, Toll like receptors (TLR) are master regulators of innate immunity while at the same University of Torino, Torino, Italy time they co-stimulate immune signaling in B-lymphocytes. TLR are also expressed in B-cell lymphoma cells, including chronic lymphocytic leukemia (CLL). CLL cells Increasing data indicate the key role of the tumor microenvironment in cancer depend on external stimuli for survival and proliferation, and both antigen receptor progression, metastasis formation and treatment sensitivity, where the tumor stroma and TLR are involved. In particular, the TLR9 ligand CpG can modulate cell viability, and the immune infiltrate strongly influence tumor features. proliferation, apoptosis and chemoresistance in vitro. TLR9 stimulation can also Our recent studies indicate that the adaptor protein p140Cap is highly expressed in induce NFKBIZ expression and IgM secretion thus regulating a central signaling 50% of ERBB2-amplified breast cancer patients. High levels of p140Cap correlate route in leukemic cells pathobiology. We herein aimed at dissecting and targeting with increased patient survival and decreased probability to develop metastasis. TLR-mediated signaling pathways in CLL. To inhibit TLR activation, we targeted In the NeuT preclinical mouse model,p140Cap negatively regulates breast tumor IRAK4, the most upstream mediator of TLR signaling complex; specifically, we used growth, Epithelial to Mesenchymal transition (EMT) and spontaneous metastasis small molecule drugs in different preclinical models of CLL including cell lines and formation. primary leukemic cells. Leukemic cell lines and primary malignant B-cells isolated Here we exploit Mock and p140Cap-expressing TuBo cells, as syngeneic ERBB2+ from the peripheral blood of CLL patients (n=44 cases) were cultured in vitro with or breast cancer models. Weshow that p140Capimpacts on the G-CSF levels (mRNA without IRAK4 inhibitor in the presence or absence of the TLR9 ligand CpG. IRAK4 and protein) and affects theTuBotumorimmune infiltrate composition. Indeed, we inhibitor induced cell death in a dose dependent manner in both primary leukemic observed a reducedmobilization of G-CSF-dependent myeloid-derived suppressor cells and cell lines; in detail, at a concentration of 10 microM, leukemic cells viability cells (MDSCs-CD11b+Ly6G+Ly6Clo) from bone marrow and a decreased level of decreased to a mean of 40% as compared to untreated cells. The monocytic cell line gMDSCs in tumor, blood and spleen of mice orthotopically injected with p140Cap Thp-1 used as internal control was not significantly affected by the treatment (cell TuBo cells, compared to controls.This “less” immune suppressive microenvironment viability >90%), while both Mec-1 and Mec-2 CLL cell lines showed an increased associated to p140Cap-expression, was also confirmed by an increase in CD8+T cell death after IRAK4 inhibition (cell viability <50%). Administration of CpG caused lymphocytes, M1 macrophages and Natural Killer lymphocytes,with a concomitant heterogeneous responses as previously observed by different groups including decrease in M2 macrophages and FoxP3 immunosuppressive regulatory T cells. ours; regardless, IRAK4 inhibitor blocked the protective effect of CpG on cell Interestingly, p140Cap significantly reduces the fraction ofcancer stem cells (CSC) viability. Co-administration of increasing doses of IRAK4 inhibitor and fludarabine and their sphere and tumor initiationfrequency. Since that the CSC population exerted a synergic effect in both Mec-1 and primary leukemic cells, as measured by exhibits an enhanced G-CSF production, these data indicate that p140Cap, by the Combination Index (CI<1). To note, TLR-induced NFKBIZ expression decreased regulating the CSC compartment, may limit tumor aggressiveness by creating a when cells were treated with IRAK4 inhibitor in both cell lines and primary CLL cells, “less” immunosuppressive tumor microenvironment,adverse to tumor progression. thus representing a specific biomarker of drug response. These data open novel potential therapeutic perspectives for the use of TLR signaling inhibitors in this type of leukemia, either alone or in combination with currently used drugs. V2 OCTN1 and its disease-associated variant L503F as novel modulators of mTOR/TORC1 signaling at the crossroad of V4 energy metabolism, inflammation and colorectal cancer Tumor microenvironment stroma cells increase aggressiveness Fidaleo M., Cavallucci V., Samengo D., Pani G. of pheochromocytoma spheroids Institute of general Pathology, Laboratory of Cell Signaling, Università Cattolica 1)Martinelli S. 2)Riverso M. 3)Richter S. 4)Canu L. 5)Pacak K. 6)Eisenhofer G. 7) School of Medicine, Rome, Italy Mannelli M. 8)Rapizzi E. 1) Mello T. 1)Department of Experimental and Clinical Biomedical Sciences, University of Genetic variants in signaling pathways relevant to innate immunity, inflammation and Florence, Florence, Italy 2)Department of Experimental and Clinical Biomedical cell metabolism may shape the individual risk of cancer development , progression, Sciences, University of Florence, Florence, Italy 3)Institute of Clinical Chemistry and and response to therapy. Identification of these variants and clarification of their Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität molecular networks and environmental co-factors represent therefore emerging Dresden, Dresden, Germany 4)Department of Experimental and Clinical Biomedical priorities for current cancer research in a personalized medicine perspective. Sciences, University of Florence, Florence, Italy 5)Eunice Kennedy Shriver National The relatively common variant L503F (c1672t, rs1050152) of the Organic Cation/ Institute of Child Health and Human Development, National Institutes of Health, Ergothioneine Transporter Slc22a4/OCTN1 is enriched among individuals affected Bethesda, USA 6)Institute of Clinical Chemistry and Laboratory Medicine, University by Inflammatory Bowel Disease (IBD) and Rheumatoid Arthritis, and our previous Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany work has linked this allele to increased risk of both IBD-associated and sporadic 7)Department of Experimental and Clinical Biomedical Sciences, University of colorectal cancer (CRC) especially in young (< 50 year old) individuals. However Florence, Florence, Italy 8)Department of Experimental and Clinical Medicine, the molecular underpinnings of these important associations remain ill defined, nor University of Florence, Florence, Italy is known how and through which molecular partners OCTN1 variants, expressed in both intestinal epithelial cells and monocytes, impact on inflammatory and growth- Paragangliomas (PGL) are rare neuroendocrine tumors derived from neural crest related circuitries conducive to colorectal cancer. cells: if localized in the adrenal medulla they are called Pheocromocytomas (Pheo). Both inflammation and cancer are mechanistically linked to aberrant nutrient The 30-40% of Pheo/PGL is mutated in one of the susceptibility genes among which signaling and cell energy metabolism. Intriguingly, another carrier structurally related there are genes encoding for the four subunits (A, B, C and D) of the succinate to OCTN1, SLC38A9, has been recently identified as an essential component of the dehydrogenase (SDH). Normally these tumors are benign, but mutations of SDHB aminoacid sensing mTOR/TORC1 complex at the lysosomal surface. We found that are metastatic in about 80% of the cases. Surgery is the current therapy, but in a significant fraction of OCTN1 molecules overexpressed in 293T cells localizes presence of metastasis there is no effective treatment.
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In this study, we first evaluated the effects on cell metabolism and function ofSDHB V6 and SDHD silencing in a three dimension (3D) culture using tumor spheroids. We Breast cancer-adipocytes crosstalk as promoter of cancer used a mouse pheochromocytoma cell line (MTT) model silenced or not (wild type progression = Wt) for the catalytic SDHB or the anchoring SDHD subunits. Rybinska I., Regondi V., Ghirelli C., Tagliabue E., Triulzi T. Moreover, we evaluated the role of the microenvironment in modulating spheroid Molecular Targeting Unit, Department of Research, Fondazione IRCCS Istituto migration/invasion processes, by co-culturing SDHB or SDHD silenced spheroids Nazionale dei Tumori, Milan, Italy with primary cancer-activated fibroblasts (CAFs), and comparing them with Wt ones. To address this issue, we measured matrigel invasion of spheroid cells by Adipocytes are the major breast component and are emerging as critical players the computation of the migratory areas. We found a significant increase in migratory in the breast cancer (BC) microenvironment. Crosstalk between BC cells and capability of SDHB silenced spheroids compared with Wt ones and we observed that adipocytes has been recently described. However, key molecular mechanisms SDHD silenced spheroids had an intermediate migration pattern compared to SDHB of this interaction are still to be defined. To date, murine preadipocytes 3T3-L1 and Wt spheroids. To study migration in details, we examined the type of migration mimicking adipogenesis has provided proper tool to study the interaction between by confocal microscopy. Interestingly, we found that SDHB silenced cells invaded the adipocytes and tumor cells in vitro. Our data showed that conditioned medium surrounding space moving collectively, unlike the Wt and SDHD silenced spheroids, (CM) obtained from triple negative (TN) 4T1 cells resulted in dedifferentiation where cells tended to move individually. In particular, SDHB silenced spheroids of 3T3-L1, observed as a reduced expression of specific differentiation markers developed long filamentous formations along which cells migrated far away from the (FABP4, APN, PPARγ, CEBPα) by qPCR, and a reduced triglyceride content, as spheroid in clusters, whereas Wt and SDHD silenced spheroids develop filaments as assessed by oil red O staining, compared to mature adipocytes. Exosome depletion well, but cells move along them in a single cell manner. in CM did not impact the dedifferentiation process indicating that the crosstalk is In this work, we demonstrated that despite in single culture SDHB and SDHD silenced exosome-unrelated. Similar results were obtained in human adipose tissue-derived spheroids show some analogies, they respond very differently in the presence of mesenchymal stem cells-ASC52 cultured with TN MDAMB231 CM. Gene expression tumor microenvironment stroma cells. These preliminary results suggest that tumor analysis with Clariom D arrays were used to identify differences in dedifferentiated microenvironment plays a pivotal role in tuning the aggressiveness and the metastasizing ASC52 (DED), compared to mature adipocytes (MA) and not adipogenic-stimulated potential of SDHB silenced tumor cells, opening new approaches for therapy. cells (NT). While pathways related to lipid metabolism and proliferation were modulated during adipogenesis (NT vs MA), in DED cells these pathways were comparable to NT cells. Solely DED were featured by upmodulation of protumoral V5 genes mainly related to inflammatory response. Accordingly, we observed a CROSS-TALK OF MACROPHAGES AND THYROID CELLS IN EARLY AND two-fold higher migration of PBMCs towards DED vs MA in a Boyden migration LATE THYROID TUMOR STAGES assay. We then analyzed the BC cell ability to dedifferentiate ASC52 across BC 1)Mauro G. 2)Mazzoni M. 3)Erreni M. 4)Romeo P. 5)Avigni R. 6)Rizzetti M.G. 7) subtypes using luminal A (MCF7, T47D), Her2+ (SkBr3, HCC1954, MDAMB453), Borrello M.G. 8)Allavena P. 9)Greco A. luminal B (MDAMB361, ZR75.30, BT474) and TN (MDAMB231, BT549, HCC1937, 1)Molecular Mechanisms Unit, Research Department, Fondazione IRCCS Istituto MDAMB468, MDAMB157) cell lines. CM from all but not luminal B cells induced Nazionale Tumori, Milan, Italy 2)Molecular Mechanisms Unit, Research Department, adipocyte dedifferentiation, characterized by delipidation and decreased expression Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy 3)Department of of differentiation markers. Transwell culture with adipocytes stimulated the faculty of Immunology, IRCCS Clinical and Research Institute Humanitas, Rozzano, Milan, Italy luminal B cells to dedifferentiate adipocytes. MCF7 cells after co-culture with ASC52 4)Molecular Mechanisms Unit, Research Department, Fondazione IRCCS Istituto expressed higher levels of EMT markers and reduced proliferation rate than MCF7 Nazionale Tumori, Milan, Italy 5)Department of Immunology, IRCCS Clinical and grown alone. These findings highlight that adipocyte dedifferentiation caused by BC Research Institute Humanitas, Rozzano, Milan, Italy 6)Molecular Mechanisms Unit, cells represent common feature of all kind of BC cells and results in provision of Research Department, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy protumorigenic stimuli thereby supporting cancer progression. 7)Molecular Mechanisms Unit, Research Department, Fondazione IRCCS Istituto Supported by AIRC. Nazionale Tumori, Milan, Italy 8)Department of Immunology, IRCCS Clinical and Research Institute Humanitas, Rozzano, Milan, Italy 9)Molecular Mechanisms Unit, Research Department, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy V7 NATURAL KILLER CELLS FROM MALIGNANT PLEURAL EFFUSION ARE Inflammation plays a critical role in many tumor types, including thyroid cancer. SWITCHED TOWARD AN NK PROANGIOGENIC POLARIZATION Conditions predisposing to cancer, as well as causative genetic events, contribute 1)Mortara L. 2)Bosi A. 3)Zanellato S. 4)Gallazzi M. 5)De Leo M. 6)Imperatori A. 7) to the construction of an inflammatory microenvironment that facilitates thyroid Dominioni L. 8)Albini A. 9)Noonan D.M. 10)Bruno A. tumor progression by limiting immune surveillance and therapy response. 1)Department of Biotechnology and Life Sciences, University of Insubria, Varese, Thyroid carcinoma, the most frequent endocrine neoplasia, includes several Italy 2)Department of Biotechnology and Life Sciences, University of Insubria, variants characterized by different biological and clinical features: from the early Varese, Italy 3)Department of Medicine and Surgery, University of Insubria, Varese, indolent stage papillary thyroid microcarcinoma to undifferentiated and aggressive Italy 4)Department of Biotechnology and Life Sciences, University of Insubria, anaplastic carcinoma. Varese, Italy 5)Department of Biotechnology and Life Sciences, University of Recent studies have documented the occurrence in thyroid cancer of oncogene Insubria, Varese, Italy 6)Department of Medicine and Surgery, University of Insubria, induced senescence, a mechanism acting as a barrier in cancer progression, tightly Varese, Italy 7)Department of Medicine and Surgery, University of Insubria, Varese, connected with inflammation, as it triggers a complex pro-inflammatory programme Italy 8)School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy known as the “senescence associated secretory phenotype”. Recently, we have 9)Scientific and Technology Pole, IRCCS MultiMedica, Milano, Italy 10)Scientific proposed senescent thyrocytes as an in vitro model of early stage of thyroid cancer. and Technology Pole, IRCCS MultiMedica, Milano, Italy Here we characterized the in vitro interplay between macrophages and cells representative of early and late thyroid tumor stages, modeled by senescent Natural killer (NK) cells are effector lymphocytes crucial in tumor recognition and thyrocytes and tumor cell lines, respectively. Purified peripheral blood-derived eradication. However, NK cell activity is often impaired in cancer patients and in human monocytes were exposed to thyroid cells conditioned medium (CM), and their NSCLC and colorectal cancer patients, we recently demonstrated that they acquire phenotype was assessed by different approaches. By flow cytometry, we found that, proangiogenic phenotype and functions. upon exposure to thyroid cell CM, human monocytes undergo M2-like polarization, We characterized NK cells from peripheral blood (PB) and pleural effusions (PE) as shown by high level of CD206 and low level of MHC II surface markers; by of patients with either inflammatory diseases (iPE, n = 18), primary tumor (ptPE, ELISA assay we detected the upregulation of CCL17 secretion. By gene expression n = 18), and metastatic tumor (mtPE, n = 27) by multicolor flow cytometry, for analysis, we also observed upregulation of several genes, including: M-CSF surface antigens expression and cytokine release, along with functional assay for (CSF1), genes coding for chemokines (CCL20, CCL3, CCL4, CXCL1, CXCL8) angiogenesis.We also investigated whether NK cells from different PEs can acquire and cytokines (IL-6, IL-23, IL-24), Osteopontin, VEGF and the prostaglandin- decidual-like NK phenotype and whether exposure to IL-2 can restore their killing endoperoxide synthase enzyme (PTGS2), known also as cyclooxygenase-2. We ability in the presence of pleural fluids. focused on the role of PTGS2, that is involved in the convertion of arachidonic We found that CD56brightCD16- were predominant in mtPE (60%), while representing acid to prostaglandins (e.g. PGE2), and we investigated the effect of the inhibition the 40% and 35% of total NKs from ptPE and iPE, respectively. NK cells from both of PGE2 production on the M2-polarizing ability. By exposing monocytes to CM of ptPE and mtPE showed increased expression for the CD49a and CD69 decidual- thyroid cells treated with Celecoxib, a specific PTGS2 inhibitor, we observed a clear like NK markers and decreased levels of the CD57 maturation marker. NK from all reduction of CD206 expression and of CCL17 secretion. Overall our results suggest the PE analyzed showed impaired degranulation capability and reduced perforin that senescence thyrocytes and thyroid tumor cell lines trigger M2-like macrophage release. PE-NK cells efficiently responded to IL-2 stimulation in vitro, but addition polarization through PGE2 production. of TGFβ or cell-free pleural fluid blocked IL-2-mediated degranulation and IFNγ release. We observed the same effects when using healthy donor-derived NK cells (n = 14), exposed to PEs. We found that mtPE-NK cells produce VEGF and support the formation of capillary-like structures in human umbilical vein endothelial cells.
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Our results suggest that the PE tumor microenvironment can shape NK cell research center, Milan, Italy 9)Immunohematology and transfusional medicine polarization towards a low cytotoxic, decidual-like, highly proangiogenic phenotype unit, Irccs Regina Elena national cancer institute, Rome, Italy 10)Department of and that IL-2 treatment is not sufficient to limit this process. pharmaceutical sciences, Università del Piemonte orientale Amedeo Avogadro, Novara, Italy 11)Preclinical models and new therapeutic agents unit, Irccs Regina Elena national cancer institute, Rome, Italy V8 SEMAPHORINE 5A DRIVES MELANOMA PROGRESSION: ROLE OF MIR-204 Introduction. Cutaneous melanoma is an highly aggressive cancer with metastatic AND C-MYB behavior. Despite advances in the therapy, patients diagnosed at advanced stages 1)Valentini E. 2)D’ Aguanno S. 3)Tupone MG. 4)Desideri M. 5)Di Martile M. 6) display low survival rates. Therefore, the identification of signaling molecules Spagnuolo M. 7)Rizzo MG. 8)De Dominici M. 9)Calabretta B. 10)Falcone I. 11) involved in the acquisition of a metastatic phenotype by melanoma cells is of primary Milella M. 12)Trisciuoglio D. 13)Del Bufalo D. importance. In this context, we previously demonstrated that the overexpression of bcl-2, one of the most crucial regulators of cell apoptosis, increases melanoma 1)Preclinical models and new therapeutic agents uni, Irccs Regina Elena national progression and metastatization. Being a relevant constituent of the tumor cancer institut, Rome, Italy 2)Preclinical models and new therapeutic agents uni, microenvironment, tumor-associated macrophage (TAM) may regulate melanoma Irccs Regina Elena national cancer institut, Rome, Italy 3)Preclinical models and progression, through distinct proinflammatory (M1) vs protumor (M2) polarized new therapeutic agents uni, Irccs Regina Elena national cancer institut, Rome, programs. To date, there is no information about the role of bcl-2-expressing tumor Italy 4)Preclinical models and new therapeutic agents uni, Irccs Regina Elena cells on TAM. The aim of our study was to test whether bcl-2 overexpression in national cancer institut, Rome, Italy 5)Preclinical models and new therapeutic melanoma cells might affect TAM functions. agents uni, Irccs Regina Elena national cancer institut, Rome, Italy 6)Oncogenomic Results. Conditioned Media (CM) from bcl-2 overexpressing melanoma cells and epigenetic unit, Irccs Regina Elena national cancer institut, Rome, Italy 7) promoted M2 polarization of Monocyte-Derived Macrophages (M-DM) in vitro. Oncogenomic and epigenetic unit, Irccs Regina Elena national cancer institut, In particular, compared to control CM, macrophages exposed to CM from bcl-2 Rome, Italy 8)Department of cancer biology, Sidney kimmel cancer center, Thomas overexpressing melanoma cells expressed high levels of M2 markers, as opposed Jefferson univ, Philadelphia, Usa 9)Department of cancer biology, Sidney kimmel to low levels of M1 markers. To identify melanoma-derived factors with M2-polarizing cancer center, Thomas Jefferson univ, Philadelphia, Usa 10)Medical oncology 1, activity, mRNA expression and protein secretion of several cytokines were evaluated Irccs Regina Elena national cancer institut, Rome, Italy 11)Medical oncology 1, Irccs in melanoma cells. Bcl-2 overexpressing melanoma cells showed an increased Regina Elena national cancer institut, Rome, Italy 12)Institute of molecular biology expression of interleukin-1β (IL-1β), interleukin-8 (IL-8) and interleukin-17 (IL-17), and pathology, National research council, Rome, Italy 13)Preclinical models and at mRNA as well as at protein levels. Noteworthy, after forced expression of bcl-2, new therapeutic agents uni, Irccs Regina Elena national cancer institut, Rome, Italy melanoma cells displayed also enhanced expression of CCL2, a molecule involved in macrophages recruitment. In agreement, chromatin immunoprecipitation assay Introduction. Melanoma, the most aggressive form of skin cancer, is characterized demonstrated an increased NF-κB recruitment at the IL-1β, IL-8, COX2 and CCL2 by high rates of metastasis, drug resistance, and mortality. Semaphorins (SEMAs) promoters in bcl-2 transfectants, and the use of a mutated super-repressor form of are a large family of phylogenetically conserved proteins, which are involved in IkBα corroborated the involvement of NF-κB on the activation of these molecules different physiological and developmental functions including regulation of nervous by bcl-2. Of note, high levels of bcl-2 protein also correlated with increased and immune systems, and angiogenesis. In tumor, SEMAs and their receptors expression of COX2 and secretion of prostaglandin E2. In support of bcl-2 as a control several hallmarks of cancer, including tumor growth, angiogenesis, and cancer-associated orchestrator of TAM recruitment and functions, a massive in vivo metastasis. Here we focused our attention on Semaphorin 5A (Sema5A), for which recruitment of M2-polarized macrophages was confirmed in bcl-2-overexpressing aberrant expression has been reported in different tumors including glioma, lung, melanoma xenografts. prostate, pancreas, and gastric cancers, to elucidate the involvement of SEMA5A in Conclusions. Overexpression of bcl-2 in melanoma cells recruits monocytes and melanoma progression and factors involved in its regulation. promotes their polarization into M2-like TAM. Results. A correlation of Sema5A mRNA expression and melanoma progression was observed by performing microarray analysis using public GEO profile dataset. Moreover, endogenous Sema5A protein expression was found in a panel V10 of commercially available and patient-derived melanoma cell lines, previously thrombospondin-1 DELAYS tumor growth and improveS tumor established in our Institute from surgical specimens obtained from primary or response to chemotherapy BY REMODELING THE TUMOR VASCULATURE metastatic tumors. By either overexpressing the Sema5A transmembrane domain 1)Pinessi D. 2)Resovi A. 3)Morosi L. 4)Borsotti P. 5)Berndt A. 6)Giavazzi R. 7) or reducing Sema5A expression by siRNA, we demonstrated that Sema5A has Zucchetti M. 8)Taraboletti G. an essential role in promoting migration and invasion in vitro. We also found an 1)Tumor Angiogenesis Unit, Laboratory of Biology and Therapy of Metastasis, increased level of Sema5A at both mRNA and protein level after forced expression IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy 2)Tumor of Bcl-2, an antiapoptotic protein associated with resistance to apoptosis, poor Angiogenesis Unit, Laboratory of Biology and Therapy of Metastasis, IRCCS- prognosis and melanoma progression. The use of transcriptional and proteasome Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy 3)Clinical Cancer inhibitors demonstrated that Bcl-2 increases the stabilization of Sema5A mRNA and Pharmacology Unit, Laboratory of Cancer Pharmacology, IRCCS-Istituto di protein. Moreover, by chromatin immunoprecipitation experiments we demonstrated Ricerche Farmacologiche Mario Negri, Milano, Italy 4)Tumor Angiogenesis Unit, that Sema5A expression is under the control of the transcription factor c-Myb, and Laboratory of Biology and Therapy of Metastasis, IRCCS-Istituto di Ricerche that its recruitment on Sema5A promoter is increased in Bcl-2 overexpressing cells. Farmacologiche Mario Negri, Bergamo, Italy 5)Institute of Forensic Medicine, Finally, a concomitant reduction of both Sema5A and c-Myb proteins was observed University Hospital Jena, Jena, Germany 6)Tumor Angiogenesis Unit, Laboratory in melanoma cells after overexpression of the synthetic miR-204, a microRNA of Biology and Therapy of Metastasis, IRCCS-Istituto di Ricerche Farmacologiche recently reported to regulate c-Myb in prostate cancer. Conclusions. Overall, our Mario Negri, Bergamo, Italy 7)Clinical Cancer Pharmacology Unit, Laboratory of data provide for the first time, evidence of both the role of Sema5A in melanoma Cancer Pharmacology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, progression and the molecular mechanism regulating its expression involving Bcl-2, Milano, Italy 8)Tumor Angiogenesis Unit, Laboratory of Biology and Therapy of miR-204 and c-Myb. Metastasis, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy Antiangiogenic agents can induce a transient normalization of tumor vessels, an V9 important strategy to improve the delivery and efficacy of cytotoxic therapy. Our BCL-2 OVEREXPRESSION IN MELANOMA CELLS PROMOTES RECRUITMENT previous studies identified an FGF-2 binding site in the type III repeats (T3R) domain AND DIFFERENTIATION OF MACROPHAGES TOWARDS A M2-LIKE of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Aim PHENOTYPE of this study was to investigate the role of the T3R domain in tumor growth and response to chemotherapy, with particular interest for its activity on tumor vessels. 1)Di Martile M. 2)Farini V. 3)Trisciuoglio D. 4)Desideri M. 5)Valentini E. 6)Buglioni S. The human ovarian carcinoma cell line (A2780-1A9) was transfected to express 7)Ercolani C. 8)Consonni FM. 9)Foddai ML. 10)Sica A. 11)Del Bufalo D. and secrete T3R (1A9-T3R) and implanted in immunodeficient mice. Expression of 1)Preclinical models and new therapeutic agents unit, Irccs Regina Elena national T3R delayed tumor growth and improved tumor response to paclitaxel (PTX 10 mg/ cancer institute, Rome, Italy 2)Preclinical models and new therapeutic agents unit, kg Q4x3, i.v.) and cisplatin (DDP 4 mg/kg Q4x3, i.v.) compared to control. By HPLC Irccs Regina Elena national cancer institute, Rome, Italy 3)Institute of molecular mass spectrometry and MALDI imaging mass spectrometry analysis, a higher biology and pathology, National research council, Rome, Italy 4)Preclinical models concentration and a more homogeneous drug distribution was observed in tumors and new therapeutic agents unit, Irccs Regina Elena national cancer institute, expressing T3R, 4h after a single administration of PTX (60 mg/kg, i.v.). Evidence of Rome, Italy 5)Preclinical models and new therapeutic agents unit, Irccs Regina vascular reorganization was confirmed by histological analysis in tumors expressing Elena national cancer institute, Rome, Italy 6)Pathology unit, Irccs Regina Elena T3R that showed a decreased vessel area and vessel diameter and increased national cancer institute, Rome, Italy 7)Pathology unit, Irccs Regina Elena national number of CD31-positive vessels. These findings indicate the ability of T3R to cancer institute, Rome, Italy 8)Molecular immunology lab, Humanitas clinical and reduce tumor growth and improve tumor response to chemotherapy by remodeling
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the tumor vasculature. This opens new perspectives for the development of T3R- properties, and their ability to target the surrounding microenvironment. Through based antiangiogenic compounds to be used in combination therapies. metabolomics approaches we had previously found that in serum samples from Supported by AIRC. DP is recipient of the AIRC fellowship ‘’Lina e Giovanni Giasini’’. prostate cancer (PCa) patients, three carnitine family members were significantly decreased, suggesting a potential protective role of carnitine against PCa. Acetyl- L-carnitine (ALCAR) is an acetic acid ester of carnitine with high bioavailability and V11 is involved in the transport of fatty acids across the inner mitochondrial membrane. TUMOR INFILTRATING (TINKs) AND TUMOR ASSOCIATED (TANKs) FROM Here we investigated the ability of ALCAR to limit key functional steps of inflammatory COLORECTAL CANCER PATIENTS ARE PROANGIOGENIC AND EXPRESS angiogenesis and identified the molecular mechanisms involved. ANGIOGENIN AND THE MMP9-TIMP2, SIMILAR TO DECIDUAL NK CELLS The effects of ALCAR on cytokine-activated human umbilical vein endothelial 1)Bruno A. 2)Bassani B. 3)Gallazzi M. 4)Pelosi G. 5)Boni L. 6)Dominioni L. 7) cells (HUVEC) were investigated in vitro using functional angiogenesis assays Mortara L. 8)Noonan D. 9)Albini A. (morphogenesis on matrigel, migration and invasion assays), molecular and 1)Vascular Biology and Angiogenesis Laboratory, Science and Technology Pole biochemical approaches (RT-PCR, FACS, Immunofluorescence, western blot). (PST) IRCCS MultiMedica, Milano, Italy 2)Vascular Biology and Angiogenesis Data from in vitro studies were confirmed in vivo using the angiogenesis matrigel Laboratory, Science and Technology Pole (PST) IRCCS MultiMedica, Milano, Italy 3) sponge assay. Vascular Biology and Angiogenesis Laboratory, Science and Technology Pole (PST) We found that ALCAR significantly inhibited angiogenesis in vitro, by targeting IRCCS MultiMedica, Milano, Italy 4)Department of Oncology and Hemato-Oncology, VEGF/VEGFR2 axis and their key down-stream protein kinases, such as University of Milan, Milan, Italy 5)IRCCS - Ca’ Granda - Polyclinic Hospital Milan, pTyr397-FAK, pTyr416-Src, p-38 MAPK, and p-Ser1248-PLCγ1. Crucial steps of Italy 6)Department of Surgical and Morphological Sciences, University of Insubria, angiogenesis such as migration and invasion, and key regulators of pro-angiogenic Varese, Italy 7)Department of Biotechnology and Life Sciences, University of migratory phenotype, such as CXCR4 and its ligand, CXCL12, were also inhibited Insubria, Varese, Italy 8)Department of Biotechnology and Life Sciences, University by ALCAR. Furthermore, in TNFα -activated HUVECs, ALCAR blocked NF-κB of Insubria, Varese, Italy 9)School of Medicine and Surgery, University of Milano- activation and nuclear translocation, and lowered ICAM-1 expression. These results Bicocca, Monza, Italy were consistent with in vivo data where we found that ALCAR significantly lowered endothelial cell and macrophage recruitment into the matrigel plugs. Extensive evidence has demonstrated that chronic inflammation represents a Our results highlight the anti-angiogenic and anti-inflammatory properties of ALCAR hallmark of cancer and a key risk factor for CRC development. The immune and allow the identification of multiple and overlapping mechanisms of action contexture (including type, density, and location of immune cells within the tumor through which ALCAR inhibits inflammatory angiogenesis. Our findings provide the samples) within tumors has been shown to significantly influence CRC progression, rational for the employment of ALCAR, as a possible supplement for approaches of determining the fate and survival of the patient and the response to therapy. interception in subjects at high risk to develop cancer. Natural Killer (NK) cells are effector lymphocytes involved in tumor immunesurveillance. In solid malignancies, tumor associated (TANK; peripheral blood) and tumor infiltrating (TINK) NK cells acquire altered phenotype and V13 functions. hMENA is a key regulator of tumor cell-cancer associated fibroblast dialogue We previously reported that NKs from Non-Small Cell Lung Cancer (NSCLC) can via Gas6/Axl paracrine axis acquire a decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNglow phenotype and 1)Melchionna R. 2)Spada S. 3)Di Modugno F. 4)Panetta M. 5)Di Carlo A. 6)Mileo
that TGFb1 is a relevant orchestrator in the NK angiogenic switch. A.M. 7)Sperduti I. 8)Antoniani B. 9)Lawlor R.T. 10)Piemonti L. 11)Diodoro M.G. 12) Here, we functionally and molecularly characterize TINK and TANKs from blood and D’Andrea D. 13)Visca P. 14)Milella M. 15)Grazi G.L. 16)Facciolo F. 17)Chen E. 18) tissue samples of colorectal cancer (CRC) patients, compared to NK isolated from Scarpa A. 19)Nisticò P. control and non-oncologic inflammatory bowel disease patients. 1)Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National NK subset distribution and cytokine profiling were detected by multicolor flow Cancer Institute, Rome, Italy 2)Tumor Immunology and Immunotherapy Unit, cytometry, using peripheral blood and tissue samples from CRC patients, for IRCCS Regina Elena National Cancer Institute, Rome, Italy 3)Tumor Immunology surface antigen and cytokine profiling characterization. Conditioned media (CM) and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, from FACS-sorted NKs were used either for secretome profiling, using antibody Italy 4)Tumor Immunology and Immunotherapy Unit, IRCCS Regina Elena National membrane arrays or in functional in vitro angiogenesis assays. Cancer Institute, Rome, Italy 5)Tumor Immunology and Immunotherapy Unit, IRCCS CRC TINKs/TANKs showed decreased levels of the activation marker NKG2D, Regina Elena National Cancer Institute, Rome, Italy 6)Tumor Immunology and impaired degranulation activity, a decidual-like NK polarization toward the Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy 7) CD56brightCD16dim/- CD9+CD49+ subset. TINKs and TANKs supernatants induced Biostatistics and Scientific Direction, IRCCS Regina Elena National Cancer Institute, endothelial cell proliferation, migration, adhesion and formation of capillary-like Rome, Italy 8)Pathology Unit, IRCCS Regina Elena National Cancer Institute, structures in vitro. It has been reported that dNK release proangiogenic factors and Rome, Italy 9)ARC-NET Research Centre, Dept of Pathology and Diagnostics, metastasis-associated (MMP-9, TIMP1-2) proteins. Univ. of Verona, Verona, Italy 10)Diabetes Research Institute, IRCCS San Raffaele We describe for the first time the CRC derived NK cells express angiogenin and Scientific Institute, Milan, Italy 11)Pathology Unit, IRCCS Regina Elena National MMP2, MMP9, TIMP1 and TIMP2. This could be a phenotype relevant to their Cancer Institute, Rome, Italy 12)Dept. of Medicine, Centre for Cell Signaling and invasive capabilities and pro-angiogenic function. STAT-3/STAT-5 activation Inflammation, Imperial College London, UK 13)Pathology Unit, IRCCS Regina was observed in TANKs, and inhibition of the STAT5 pathway by pimozide, an Elena National Cancer Institute, Rome, Italy 14)Dept. of Medical Oncology 1, antipsychotic drug, reduced proangiogenic factor VEGF and angiogenin production IRCCS Regina Elena National Cancer Institute, Rome, Italy 15)Hepato-Pancreato- and capillary-like structure formation, but did not affect the levels of TIMP-1, Biliary Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy TIMP-2 and MMP-9, suggesting the utility of a combination of MMP-inhibitors and 16)Thoracic-Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, immunotherapy. Italy 17)Dept. of Pharmacology Herbert Irving Comprehensive Cancer Center, Columbia University medical Centre NY, USA 18)ARC-NET Research Centre, Dept of Pathology and Diagnostics, Univ. of Verona, Verona, Italy 19)Tumor Immunology V12 and Immunotherapy Unit, IRCCS Regina Elena National Cancer Institute, Rome, VEGFR2 AND CXCR4 PATHWAYS ARE MODULATED BY ACETYL-L- Italy CARNITINE THAT ACTS AS AN ANTI-ANGIOGENIC AGENT 1)Baci D. 2)Bruno A. 3)Bassani B. 4)Tramacere M. 5)Mortara L. 6)Albini A. 7) The dynamic interplay between the neoplastic epithelial cells and stromal cells, Noonan D.M. including cancer-associated fibroblasts (CAF) may either favor or hinder tumor 1)Vascular Biology and Angiogenesis Laboratory, Science and Technology Pole initiation, progression and drug resistance. Tumor cells trigger stromal cell (PST), IRCCS MultiMedica, Milano, Italy 2)Vascular Biology and Angiogenesis activation, which reciprocally regulate tumor cell signaling, establishing paracrine Laboratory, Science and Technology Pole (PST), IRCCS MultiMedica, Milano, Italy 3) pathways that are crucial in cancer. The identification of phenotypical and functional Vascular Biology and Angiogenesis Laboratory, Science and Technology Pole (PST), subtypes is of great relevance for the development of microenvironment-related IRCCS MultiMedica, Milano, Italy 4)Vascular Biology and Angiogenesis Laboratory, anti-tumor treatment. Science and Technology Pole (PST), IRCCS MultiMedica, Milano, Italy 5)Department We have previously demonstrated that hMENA, a member of the actin-regulatory of Biotechnology and Life Sciences, University of Insubria, Varese, Italy 6)School of protein ENA/VASP family, and its tissues specific isoforms influence a number Medicine and Surgery, University of Milano-Bicocca, Monza, Italy 7)Department of of intracellular signaling pathways involved in cancer cell proliferation, survival, Biotechnology and Life Sciences, University of Insubria, Varese, Italy invasion and epithelial mesenchymal transition (EMT).Of clinical relevance, we have suggested that the pattern of hMENA isoform expression in tumor cells may Angiogenesis, a process characterized by the formation of new blood vessels have prognostic value in early stage NSCLC and PDAC. from pre-existing ones, is a crucial step in tumor growth and dissemination. Many Here, we define a novel function of hMENA in stromal fibroblast activation and in the efforts have been addressed on the identification of novel active compounds from regulation of CAF-tumor cell cross-talk, via the modulation of the paracrine Gas6- natural sources, endowed with anti-proliferative, anti-oxidant and anti-angiogenic Axl signaling.
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We demonstrated that hMENA/hMENADv6 is overexpressed in CAFs with respect V15 to normal fibroblasts, identifying a subset of CAFs with pro-tumoral functions. CAFs ROLE OF JAG1 AND JAG2 LINGANDS IN PROMOTING THE ANGIOGENIC over-expressing hMENADv6 secret the Axl ligand Gas6, favoring the invasiveness SWITCH IN MULTIPLE MYELOMA of Axl-expressing NSCLC and PDAC cells. 1)Palano M.T. 2)Colombo M. 3)Neri A. 4)Chiaramonte R. Reciprocally, tumor cells overexpressing hMENA/hMENADv6 secrete factors which 1)Department of Health Sciences, University of Milano, Milano, Italy 2)Department up-regulate hMENA/hMENADv6 in CAFs, that in turn increase Gas6 secretion. Of of Health Sciences, University of Milano, Milano, Italy 3)Department of Oncology relevance we demonstrated that hMENA/hMENADv6 regulate Axl expression in and Hemato-Oncology, University of Milano; Hematology, Fondazione Cà Granda tumor cells, sustaining Gas6-Axl, a paracrine axis described as crucial in EMT, drug IRCCS Policlinico, Milano, Italy 4)Department of Health Sciences, University of resistance and immune evasion. Milano, Milano, Italy From a clinical point of view, we found that a high hMENA/Gas6/Axl gene expression signature is associated with a poor prognosis in PDAC patients. Multiple myeloma(MM) is an incurable hematological malignancy that develops in Our findings indicate that the pattern of hMENA isoform expression in both tumor the bone marrow (BM) and displays “angiogenic switch” during its progression. It cells and CAFs may reveal tumor mesenchymal traits and that hMENA is crucial has been demonstrated that an increase in BM angiogenesis correlates and with in the reciprocal signaling between tumor cells and CAF. Thus, the network-based adverse prognosis and with metastatization. Another feature of MM cells is the on hMENA/Gas6/Axl expression may represent novel prognostic and therapeutic aberrant activation of Notch pathway triggered by the overexpressed JAG1 and targets for tailored therapies. JAG2 (J1/2) ligands. This overexpression promotes reciprocal support between MM cells and BM microenvironment. Thus, Jag-induced Notch activation may occur in nearby healthy cells including BM stromal cells (BMSCs) and osteoclasts and leads V14 to the instauration of protective mechanisms inducing MM cell survival, proliferation INHIBITION OF EXPORTIN-1 (XPO1) ACTIVITY REVERSES PRO-TUMORIGENIC and drug resistance. While the outcome on tumor vessels formation still need to be SECRETORY PHENOTYPE OF LUNG CANCER FIBROBLASTS clarified. 1)Bruccoleri A. 2)Andriani F. 3)Facchinetti F. 4)Rondinone O. 5)Pastorino U. 6)Sozzi This work aims to study the role of MM-derived Jag1/2 in stimulating endothelium G. 7)Roz L. and in promoting a pro-angiogenic behavior in BMSC. 1)Tumor Genomics Unit, Department of Research, Fondazione IRCCS Istituto We used RPMI8226 and U266 cell lines as models for MM; primary human Nazionale dei Tumori, Milan, Italy 2)Tumor Genomics Unit, Department of Research, pulmonary artery endothelial cells (HPAECs) as ECs model; HS5 cell line stably Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 3)Tumor Genomics expressing GFP as BMSCs. JAG1/2 were silenced in MM cells (MMJAG1/2KD) using Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori, short interfering RNAs. We studied the outcome of the stimulation of MM-derived Milan, Italy 4)Tumor Genomics Unit, Department of Research, Fondazione IRCCS JAG1/2 in modulating EC adhesion on fibronectin, motility and tube organization Istituto Nazionale dei Tumori, Milan, Italy 5)Thoracic Surgery Unit, Department of on Matrigel by analyzing the effect of a direct cell-cell contact and the contribution Surgery, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy 6)Tumor of the conditioned media (CM). Secondly, we focused our attention on MM-BMSC Genomics Unit, Department of Research, Fondazione IRCCS Istituto Nazionale dei co-culture system to study if Jag1/2 can induce a pro-angiogenic behavior in BMSC Tumori, Milan, Italy 7)Tumor Genomics Unit, Department of Research, Fondazione stimulating the release of VEGF. IRCCS Istituto Nazionale dei Tumori, Milan, Italy Direct contact and CM from MM cells improves ECs adhesion, motility and organization into a grid of tube-like structures. These effects are significantly reduced Cancer-associated fibroblasts (CAFs) actively participate in tumor development, when using MMJAG1/2KD cells indicating that MM-derived JAG1/2 play a key role in the progression and metastasis through several interactions with cancer cells driven modulation of ECs behavior and the promotion of malignant phenotype. MM can by remodeling of the extracellular matrix and changes in their secretory phenotype upregulate the amount of BMSC-derived VEGF and promote BMSC pro-angiogenic affecting multiple soluble factors. Specific molecular determinants of their tumor- potential, but MMJAG1/2KD cells lost this ability. Our results indicate a novel contribution promoting function in lung cancer are however not well characterized. Using co- of MM-derived JAG1/2 in promoting the formation of new vessels stimulating the injection experiments with lung cancer cells in immunocompromised mice and activation of the Notch signaling pathway in the same tumor cells and in the tumoral gene expression profiling, we previously identified increased levels of the nuclear microenvironment. The involvement of Notch-Jag axis in the angiogenic switch of transporter Exportin-1 (XPO1) in patient-derived fibroblasts with an in vivo pro- MM suggests that a novel therapy targeted to interrupt this molecular interaction tumorigenic phenotype. XPO1 mediates nuclear export of numerous regulatory could contrast the support of neo vessel formation to MM progression. proteins and oncosuppressors and may represent a master regulator of tumor stroma metabolism. To investigate the role of XPO1-related signaling in secretome changes of stromal cells, we tested the effects of a selective inhibitor of XPO1 (KPT- V16 276) on lung fibroblasts. Notch signaling promotes bone marrow-induced drug Pharmacological inhibition of XPO1 resulted in general nuclear accumulation of its resistance in multiple myeloma through the regulation of the cargo proteins (such as p53 and p62) associated with reduced long-term proliferation CXCR4/CXCL12 system (G2/M block) and increased senescence (2-4 fold increase in β-galactosidase 1)Colombo M. 2)Mazzola M. 3)Barbieri M. 4)Platonova N. 5)Palano M.T. 6)Garavelli positive cells) selectively in activated fibroblasts compared to control fibroblasts. S. 7)Giannandrea D. 8)Lazzari E. 9)Basile A. 10)Pistocchi A. 11)Neri A. 12) Interestingly, this senescent phenotype was not associated with induction of pro- Chiaramonte R. tumorigenic senescence-associated secretory phenotype (SASP) but rather with a 1)Department of Health Sciences, Università degli Studi di Milano, Milan, Italy 2) downregulation of the signature SASP factors, like IL-8, CXCL1 and CCL2. These Department of Medical Biotechnology and Translational Medicine, Università degli effects followed nuclear p53 accumulation and increased CDKN1a transcription. Studi di Milano, Milan, Italy 3)Department of Oncology and Hemato-Oncology, XPO1 inhibition also reduced extracellular acidification and lactate production in Università degli Studi di Milano, Milan, Italy 4)Department of Health Sciences, concordance with cellular relocalization of p62/SQSTM1, a master regulator of Università degli Studi di Milano, Milan, Italy 5)Department of Health Sciences, energy metabolism. In addition, upon KPT-276 treatment, we observed a reduction Università degli Studi di Milano, Milan, Italy 6)Department of Health Sciences, of SDF-1 expression, a chemokine acting as a crucial regulator of cancer cell Università degli Studi di Milano, Milan, Italy 7)Department of Health Sciences, migration and invasion. Consistently, in heterotypic co-cultures experiments, we Università degli Studi di Milano, Milan, Italy 8)Department of Health Sciences, found that XPO1 inhibition prevented the ability of activated fibroblasts to stimulate Università degli Studi di Milano, Milan, Italy 9)Department of Health Sciences, migration of lung cancer cell lines and to increase the frequency of CD133+ cancer Università degli Studi di Milano, Milan, Italy 10)Department of Medical Biotechnology stem cells, thereby also reducing their in vivo pro-tumorigenic activity. and Translational Medicine, Università degli Studi di Milano, Milan, Italy 11) Overall these evidences suggest that XPO1 inhibition could represent a promising Hematology, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, therapeutic strategy to reduce tumor stroma cross-talk by re-educating fibroblast Italy 12)Department of Health Sciences, Università degli Studi di Milano, Milan, Italy secretome towards a non pro-tumorigenic phenotype. Multiple myeloma (MM) is a diffuse hematologic tumor that is still incurable, despite the development of innovative therapies. One of the most relevant obstacles to currently available treatments is the protection from drug-induced apoptosis caused by the crosstalk of MM cells and bone marrow stromal cells (BMSCs), which provides a source of pro-survival and growth factors that promotes drug resistance, that causes patients’ relapse and contribute to MM fatal outcome. The overexpression of the two Notch ligands Jag1 and Jag2 by tumor cells causes the hyperactivation of the Notch signaling both in the neighboring MM cells and in the cells of the tumor microenvironment. To test if Jag1/2 double-silencing (Jag1/2KD) in MM cells prevented BMSC- mediated drug resistance, we established a co-culture system composed of human myeloma cell lines (HMCLs) and the human BMSC line HS5, and compared the
96 97 ABSTRACT BOOK
efficacy of commonly-used drugs, i.e. bortezomib, melphalan and lenalidomide between control and Jag1/2KD co-cultures. While HS5 are able to protect MM cells, decreasing the apoptotic rate in response to the therapy compared to single culture, Jag1/2KD is able to revert this effect, abolishing the ability of BMSCs to rescue HMCL from drug-induced death. At molecular level, we detected an increase in Notch activation in co-cultured BMSCs, which was significantly impaired when using J1/2KD MM cells. Such Notch activation correlated with a boost in BMSCs ability to produce one of the key chemokine in MM, CXCL12, which was hampered when Jag1/2 have been silenced in co-cultured HMCLs. This findings are particularly relevant since our results indicate that CXCL12 promotes the expression of the anti-apoptotic genes Bcl2, Survivin, and ABCC1 by MM cells, finally improves their resistance to therapies. Remarkably, Jag1/2KD also causes a decrease in the expression of CXCL12 receptor, CXCR4, potentiating the anti-tumor effect. The improvement in drug-response promoted by Jag1/2KD can be observed also in ex-vivo co-cultures of cells from MM patients, that further validate the key role of Jag1/2 in this process. Finally, a in vivo zebrafish xenograft model of MM allowed us to confirm that Jag1/2KD causes increase in MM cells response to bortezomib in vivo, decreasing tumor burden. These results provide the proof-of-principle for Jag1/2 represents promising therapeutic targets in MM, that may allow to restore response to chemotherapy and finally improve patient’s overall survival.
V17 GADD45ß LOSS ABLATES INNATE IMMUNOSUPPRESSION IN CANCER 1)Verzella D. 2)Bennett J. 3)Fischietti M. 4)Thotakura A.K. 5)Recordati C. 6) Pasqualini F. 7)Capece D. 8)Vecchiotti D. 9)D’Andrea D. 10)Di Francesco B. 11)De Maglie M. 12)Begalli F. 13)Tornatore L. 14)Papa S. 15)Lawrence T. 16)Forbes S.J. 17)Sica A. 18)Alesse E. 19)Franzoso G. 20)Zazzeroni F. 1)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 2)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 3)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 4)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 5)Mouse & Animal Pathology Laboratory, Fondazione Filarete, Milan, Italy 6)Department of Inflammation and Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy 7)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 8)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 9)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 10) Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 11)Mouse & Animal Pathology Laboratory, Fondazione Filarete, Milan, Italy 12)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 13)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 14)Leeds Institute of Cancer and Pathology (LICAP), University of Leeds, Leeds, UK 15)Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, Inserm, CNRS, Marseille, France 16)Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK 17)Department of Pharmaceutical Sciences, Università del Piemonte Orientale “Amedeo Avogadro”, Novara, Italy 18)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy 19)Centre for Cell Signalling and Inflammation, Department of Medicine, Imperial College London, London, UK 20)Department of Biotechnological and Applied Clinical Sciences (DISCAB), University of L’Aquila, L’Aquila, Italy To arise and evolve, cancer must evade immune attack. T-cell exclusion from the tumor microenvironment (TME) is a major barrier to overcoming immune escape. Here, we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45β (1,2) that restricts tumor-associated inflammation and T-cell trafficking into tumors. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of proinflammatory tumor-associated macrophages (TAM) and intratumoral immune infiltration, thereby diminishing oncogenesis. Since we previously showed that GADD45β additionally mediates the NF-κB antiapoptotic function in cancer cells (3), our current findings identify GADD45β as a pivotal downstream signalling hub integrating the NF-κB oncogenic functions linking cancer and inflammation. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Together, these results reveal a pathogenically critical, innate immunity “checkpoint” governed by GADD45β that is amenable to therapeutic intervention to “re-educate” TAMs and thereby overcome TME-dependent immunosuppression, with profound implications for anticancer therapy.
96 97 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Abbona A. ����������������������������������L1 Balsamo A. ���������������������������������C20 Bigi A. �����������������������������������������U12 Accolla R. �����������������������������������U8 Balsari A. �����������������������������������J2, Q8, U10 Bignotti E. ����������������������������������C4, C5, C8, Q13, Acito M. ��������������������������������������B4 Bani M. ��������������������������������������Q16 Q14, Q15 Acquati F. �����������������������������������F13 Bani MR. ������������������������������������A4, G8 Binaschi M. ��������������������������������U4 Addi L. ����������������������������������������I4 Baraldi C. �����������������������������������K6 Biroccio A. ����������������������������������C9, F10, Q12 Affatato R. ����������������������������������H3, L20 Baratti D. ������������������������������������E10 Bitonto V. ������������������������������������L23 Agnelli L. ������������������������������������K3 Barault L. �����������������������������������U16 Blalock W. ����������������������������������F2, R1 Agostinis C. ��������������������������������F6 Barbetta M. ��������������������������������P3 Blandino G. ��������������������������������C9, F9, L10, Q12 Alama A �������������������������������������C12, U11 Barbi S. ��������������������������������������K1 Blum G. ��������������������������������������N7 Alampi D. �����������������������������������I8 Barbieri A. ����������������������������������D3, U7 Boccardo S. �������������������������������C12, U11 Alberti G. ������������������������������������Q3, Q6 Barbieri M. ���������������������������������V16 Boeri M. �������������������������������������N1, U10, Q19 Alberti L. ������������������������������������K2 Bardelli A. �����������������������������������C20, O2, S2, T3, Bolli E. ����������������������������������������U2, U3 Alberti P. �������������������������������������G9, O3 U16 Bon G. ����������������������������������������L4 Albini A. ��������������������������������������Q22, V7, V11, V12 Bardelli D. ����������������������������������G5 Bonaldi E. ����������������������������������M2 Alcaro S. ������������������������������������L24 Barisella M. ��������������������������������C2, C14 Bonaldi T. �����������������������������������C1 Alessandrini G. ��������������������������U9 Barisone E. ��������������������������������N8, N9 Bonanno L. ��������������������������������Q3, Q6 Alesse E. ������������������������������������V17 Barletta G. ����������������������������������U11 Bonazzina E. ������������������������������C20 Alexiadis S. ��������������������������������Q8 Bartolazzi A. �������������������������������H1 Bondanza A. ������������������������������U12 Alfieri R. �������������������������������������U6 Bartolini A. ���������������������������������C20 Bonelli M. �����������������������������������L21 Alghezi DH. ��������������������������������Q2 Baruchello R. �����������������������������L3 Bonetti PB. ���������������������������������E11 Allavena P. ���������������������������������Q16, V5 Barutello G. ��������������������������������U2, U3 Bonfiglio S. ���������������������������������C12 Allegretti M. ��������������������������������K2, O1, P2 Basile A. �������������������������������������V16 Boni L. ����������������������������������������V11 Alzona M. �����������������������������������N6 Bassani B. ����������������������������������Q22, V7, V11, V12 Bonomo S. ���������������������������������L16 Amadori A. ���������������������������������Q3, Q6 Bassi C. �������������������������������������L28, Q20 Bononi I. �������������������������������������B3 Amaro A. ������������������������������������K4 Bavelloni A. ��������������������������������F2, R1 Bordo D. �������������������������������������K4 Amato R. ������������������������������������C11, L24 Begalli F. ������������������������������������V17 Borella F. �����������������������������������C21 Amatu A. ������������������������������������C20, U16 Beghini A. �����������������������������������O6 Bornaghi V. ��������������������������������L2, N1 Ambrosetti DC. ���������������������������I7 Belardinilli F. �������������������������������C6, C10, H1 Borrello MG. �������������������������������M2, V5 Amirouchene-Angelozzi N. �������U16 Belfiore A. ����������������������������������E10, K5 Borsotti P. �����������������������������������F12, V10 Amodio N. ����������������������������������K3 Bellarosa D. �������������������������������U4 Bortoluzzi S. �������������������������������A3 Amoreo C.A. ������������������������������L4, L12 Belletti B. ������������������������������������Q18 Borzi C. ��������������������������������������C7, L2 Anania MC. ��������������������������������L22 Bello AM. ������������������������������������L17 Boscolo A. ����������������������������������Q6 Anastasia A. �������������������������������A4, Q16 Bello E. ��������������������������������������C2, C14, D5, I1 Bosi A. ����������������������������������������V7 Andreoli R. ���������������������������������G5 Belloli S. �������������������������������������D2 Bosisio G. ����������������������������������A3 Andriani F. ����������������������������������V14 Bellone M. ����������������������������������J1, U5 Bossi F. ��������������������������������������F6 Anelli V. ��������������������������������������K2 Belmonte B. �������������������������������L13 Bossi G. �������������������������������������L12 Angelillo P. ���������������������������������V3 Belotti D. �������������������������������������A4, Q16 Bossi P. ��������������������������������������C3 Angelini C. ��������������������������������V1 Beltrame L. ��������������������������������C4 Botti G. ���������������������������������������U7 Anichini A. ����������������������������������F14, N2, U10, Benassi M.S. ������������������������������Q23, L5 Bottoni G. �����������������������������������P4 U13, U14 Bencardino K. ����������������������������U16 Bowen R. �����������������������������������Q2 Annaratone L. ����������������������������F1 Benevolo M. �������������������������������C9 Brambillasca C. ��������������������������U5 Anniciello A. �������������������������������U7 Benini S. ������������������������������������K2 Brancaccio M. ����������������������������F1 Annovazzi A. ������������������������������K2 Bennett J. �����������������������������������V17 Brevi A. ��������������������������������������J1 Annunziata C. ����������������������������K7 Benvenuto G. �����������������������������C5 Brich S. ��������������������������������������C2, I1 Antoniani B. �������������������������������V13 Beresford M. ������������������������������Q2 Brivio E. �������������������������������������N9 Apicella M. ���������������������������������I12 Bereshchenko O. �����������������������A1 Brizio R. �������������������������������������Q1 Appierto V. ���������������������������������M1, Q4 Beretta G. �����������������������������������L7 Broggini M. ���������������������������������H4, I6 Apuzzo T. �����������������������������������E6, E7 Bergamaschi L. ��������������������������N1 Broseghini E. �����������������������������K6 Arcangeli A. ��������������������������������L18 Bergantino F. ������������������������������T1 Bruccoleri A. ������������������������������E10, V14 Arcella P. ������������������������������������C20 Bergsagel PL. ����������������������������J1 Brundu S. �����������������������������������F15 Ardighieri L. ��������������������������������Q14 Bernardi F. ���������������������������������L6, N3 Brunelli L. �����������������������������������G8, I6 Ardizzoni A. ��������������������������������U6, U15 Bernardi T. ����������������������������������L3 Brunelli M. ����������������������������������K1 Arena S. �������������������������������������T3 Berndt A. �����������������������������������V10 Bruno A. �������������������������������������Q22, V1, V11, Argentiero A. ������������������������������Q24 Berrino E. �����������������������������������C21 V12 Arigoni M. ����������������������������������C21, E5, U2 Berrino F. �����������������������������������B4 Bruzzese F. ��������������������������������I4, L8 Arioli S. ��������������������������������������J2 Bersani I. ������������������������������������F14 Bucciotti F. ����������������������������������A3 Arra C. ���������������������������������������D3, O4, U7 Bertaina V. ���������������������������������N5 Buda A. ��������������������������������������L16 Ascierto P. ����������������������������������U8 Berto GE. �����������������������������������L23 Budillon A. ����������������������������������E3, I4, L8, L15, Attili I. �����������������������������������������Q3, Q6 Bertolaso A. �������������������������������K1 Q25 Augello G. ����������������������������������L13, L14 Bertoli G. ������������������������������������D4, Q10 Bufalieri F. ����������������������������������L6, N3 Aurilio M. ������������������������������������D3 Bertolini G. ���������������������������������E13 Bugatti M. �����������������������������������L31, Q14 Avallone A. ���������������������������������Q25 Bertotti A. �����������������������������������C20, S2 Buglioni S. ����������������������������������O1 Avigni R. ������������������������������������V5 Bettini L. �������������������������������������N9 Buglioni S. ����������������������������������P2 Ayrault O. �����������������������������������L6 Biagini R. �����������������������������������K2, L4 Buglioni S. ����������������������������������V9 Azzariti A. �����������������������������������L29, O5, Q24 Bianca P. ������������������������������������E6 Bulla R. ��������������������������������������F6 Azzaro R. �����������������������������������T1 Bianchi F. �����������������������������������C18, E2, J2, L23, Buonaguro FM. ��������������������������K7, O4 Azzolina A. ���������������������������������L13, L14 Q8 Buonaguro L. �����������������������������K7, O4, U8 Bacci M. �������������������������������������G6 Bianchi G. ����������������������������������Q4 Buoncervello M. �������������������������U7 Baci D. ���������������������������������������V12 Bianchini G. �������������������������������L32 Buonfanti G. �������������������������������L15 Bagnato A. ���������������������������������D1, F3, F9, L10 Bianco F. ������������������������������������D4, Q25 Buracco P. ����������������������������������U2 Bagnoli M. ����������������������������������G9, O3, T2 Bianco R. �����������������������������������L25 Busico A. ������������������������������������K5 Baldassari P. ������������������������������F14 Biancon G. ���������������������������������L19 Bussolati O. �������������������������������G5 Baldassarre G. ���������������������������I4, Q18 Biassoni V. ���������������������������������N2 Caderni G. ���������������������������������I5 Balestra T. ����������������������������������N4 Biello F. ��������������������������������������U11 Caforio M. ����������������������������������N5 Ballabio S. ����������������������������������C2, C14, I1 Bientinesi E. �������������������������������L5 Caimano M. �������������������������������L6 Ballestrero A. �����������������������������C12 Bifulco M. �����������������������������������L9 Caiola E. ������������������������������������H3, H4, I6, K5
98 99 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Cairoli R. ������������������������������������O6 Cavallo F. �����������������������������������E5, F1, U2, U3, Consonni M. �������������������������������U12 Calabrese F. �������������������������������Q3, Q6 V1 Consonni P. ��������������������������������Q22 Calabretta B. ������������������������������V8 Cavallucci V. �������������������������������V2 Conte P. �������������������������������������Q3, Q6 Calapà F. ������������������������������������E9 Cavazzoni A. ������������������������������U6 Conter V. ������������������������������������N8, N9 Calcinotto A. �������������������������������J1 Cavicchioli F. ������������������������������I10 Conti A. ��������������������������������������Q23 Califano D. ���������������������������������L17, O3 Cecchin E. ���������������������������������O3 Conti L. ��������������������������������������E5, F1, K2, P2, Calin GA. �����������������������������������K1 Ceni E. ���������������������������������������A1 V1 Calistri D. �����������������������������������B5 Centonze G. �������������������������������E13, K5, L2, Q8, Conti M. �������������������������������������Q22 Callegari E. ��������������������������������L28 U10 Coppa A. ������������������������������������C6, C10, H1 Calogero R. ��������������������������������C21, E5 Ceppi L. �������������������������������������C4, C8 Coppola S. ���������������������������������L18 Calura E. ������������������������������������C4, C5, C8 Cerchietti L. ��������������������������������E12 Corbetta A. ���������������������������������F11 Calza S. �������������������������������������Q13 Ceruti T. ��������������������������������������N8, N9 Corbetta E. ���������������������������������M4 Camisaschi C. ����������������������������J2, Q8 Cervello M. ���������������������������������L13, L14 Cordoba-Romero S. �������������������E12 Cammarota E. ����������������������������F11 Cesarini V. ����������������������������������R1 Corna E. ������������������������������������I8 Campese A. �������������������������������F4 Chalmers A. �������������������������������Q2 Corno C. ������������������������������������I8 Campomenosi P. ������������������������M4, P3 Chapelle J. ���������������������������������N6, V1 Corradini P. ��������������������������������L19 Cancelliere C. ����������������������������S2 Chaumette T. ������������������������������U8 Corsini M. �����������������������������������M3, Q13 Cancila V. �����������������������������������E1, E13, P1, Cheema U. ���������������������������������D1 Corso S. �������������������������������������I12 Q21, U10 Chellini L. �����������������������������������F3 Cortesi E. �����������������������������������C6 Canducci F. ��������������������������������J1 Cheloni G. ����������������������������������E4 Cortesi L. �����������������������������������B5 Canevari S. ��������������������������������C3, C16 Chen E. ��������������������������������������V13 Corti G. ��������������������������������������C20, O2, T3 Cangelosi D. ������������������������������N6 Cheshyk A. ���������������������������������B1, B2 Cosentino G. ������������������������������H5 Cantarella D. ������������������������������I10, S2 Chesi M. �������������������������������������J1 Cossa M. ������������������������������������I11 Cantoni C. ����������������������������������E8 Chiadini V. ����������������������������������Q9 Costa A. �������������������������������������I4 Canu L. ��������������������������������������V4 Chiaramonte R. ��������������������������K3, L27, V15, V16 Costa V. ��������������������������������������O4 Capalbo C. ���������������������������������C6, C10, H1 Chiaravalli AM. ���������������������������M4 Costantini C. ������������������������������L3 Capece D. ���������������������������������V17 Chiarle R. �����������������������������������U3 Costantini S. ������������������������������L8, L15, Q25 Capellero S. �������������������������������C21 Chiarugi P. ����������������������������������G6, G7, I12 Costi MP. ������������������������������������L3 Capitanio N. �������������������������������G4 Chilà R. ��������������������������������������H2 Cotroneo E. ��������������������������������C9, P2 Capone E. ���������������������������������L26, Q25 Chinnici A. ����������������������������������E6, E7 Cottone G. ����������������������������������C9, P2 Caporale A. ��������������������������������O4 Chiodoni C. ��������������������������������E1, F8, P1, Q21 Covello R. ����������������������������������C9, K2, L4 Cappelletti V. ������������������������������F7, Q5, S1 Chiorino G. ��������������������������������A4, P4 Craparotta I. �������������������������������C2, C14, I1 Cappello G. ��������������������������������C20 Chiotto AM. ��������������������������������L23 Crestani M. ��������������������������������G2 Cappello P. ���������������������������������Q16 Chiu M. ��������������������������������������G5 Cretella D. ����������������������������������L21 Caprara V. ����������������������������������F3 Cialdella A. ���������������������������������L16 Crisafulli G. ��������������������������������C20, U16 Capuano A. ��������������������������������A3 Cianfrocca R. �����������������������������F9, L10 Cristoni S. ����������������������������������Q22 Caputo S. �����������������������������������U5 Ciani Y. ���������������������������������������F6 Croce CM. ����������������������������������K1 Carbone C. ���������������������������������I2, Q20, R2 Ciardiello C. �������������������������������L8 Croce CM. ����������������������������������Q9 Carboni F. �����������������������������������P2 Ciardiello F. ��������������������������������Q7 Croce MV. ����������������������������������U17 Cardinali B. ��������������������������������H1 Ciarrocchi A. ������������������������������C23, I7 Crook T. ��������������������������������������Q1 Cardone C. ���������������������������������Q7 Ciceri F. ��������������������������������������U12 Crucitti A. �����������������������������������E9 Carenini N. ���������������������������������I8 Cicero L. ������������������������������������L13 Cusimano A. ������������������������������L13, L14 Carminati L. �������������������������������F12 Ciciola P. ������������������������������������L25 Cutrin JC. �����������������������������������L23 Carniti C. ������������������������������������L19 Cigliana G. ���������������������������������P2 Cuttano R. ����������������������������������C18 Carrassa L. ��������������������������������I1, L20 Ciliberto G. ���������������������������������O1, U9 D’ Abundo L. �������������������������������L28 Carrisi C. ������������������������������������U4 Cimminiello C. ����������������������������U13, U14 D’ Agostino D. �����������������������������L26 Carubbi C. ����������������������������������G5 Cinausero M. �����������������������������U15 D’ Aguanno S. ����������������������������V8 Casadio C. ���������������������������������C23 Cirri P. ����������������������������������������I5 D’ Alterio C. ��������������������������������D3, E13, U7 Casali P. �������������������������������������C2, I1, C14 Citron F. ��������������������������������������Q18 D’ Amato V. ���������������������������������L25 Casalini P. ����������������������������������Q8 Cittaro D. ������������������������������������C12 D’ Ambrosio C. ���������������������������C21 Casanova M. ������������������������������N1 Ciusani E. ����������������������������������I8 D’ Ambrosio G. ���������������������������Q22 Cascone F. ���������������������������������Q4, Q5 Climent MC. �������������������������������E11 D’ Amico G. ��������������������������������G5 Caselli A. ������������������������������������I5 Cocco L. �������������������������������������F2, R1 D’ Amore A. ��������������������������������F4, F5 Casini B. ������������������������������������K2 Cocco P. �������������������������������������C22 D’ Andrea D. �������������������������������V13, V17 Casini B. ������������������������������������O1 Cocco S. ������������������������������������L15, C12, U11 D’ Antona L. ��������������������������������C11, L24 Casini B. ������������������������������������P2 Cognetti F. ����������������������������������O1 D’ Antona P. ��������������������������������M4, P3 Casini T. �������������������������������������N8 Colangelo T. �������������������������������C18 D’ Incalci M. ��������������������������������C2, C4, C5, C8, Casorati G. ���������������������������������U12 Colecchia M. ������������������������������C19 C14, D5, F12, I1, Cassaro A. ���������������������������������O6 Colella F. ������������������������������������E9 L4, N8, Q15 Cassingena A. ����������������������������C20 Colella M. �����������������������������������C6 D’ Inzeo S. ���������������������������������C10 Castagnola M. ����������������������������E9 Colicchia V. ��������������������������������C6, C10, H1 Daidone MG. ������������������������������F7, M1, Q4, S1, Castagnoli L. ������������������������������E1, E12 Collini P. �������������������������������������N1 C19, Q5 Castellani G. ������������������������������I7 Colombatti A. ������������������������������A3 Dal Bello M. �������������������������������C12, U11 Castellani L. �������������������������������Q22 Colombini A. �������������������������������N8, N9 Dalprà L. ������������������������������������L16 Castellano I. �������������������������������F1 Colombo G. ��������������������������������L11 Dama E. �������������������������������������C18 Castelli C. �����������������������������������J2 Colombo MP. ������������������������������E2, F8, L27, P1, Damia G. ������������������������������������H2, H3, L16, L20 Castelli R. ����������������������������������L30 Q21, U10, V15, Dander E. �����������������������������������G5 Castiglioni I. �������������������������������D4, Q10 V16 Daniele T. �����������������������������������L32 Casucci M. ���������������������������������U12 Columbano A. ����������������������������A2, L11 Dattilo V. �������������������������������������C11, L24 Catalogna G. ������������������������������C11, L24 Comes Franchini M. �������������������H1 De Angelis C. �����������������������������L1 Cattoni M. �����������������������������������P3 Comito G. �����������������������������������G7 De Braud F. ��������������������������������U16 Cava C. ��������������������������������������D4, Q10 Comoglio P. ��������������������������������I12 De Cecco L. �������������������������������C3, C17, C19, F8, Cavadini E. ���������������������������������Q4 Conca E. ������������������������������������K5 N1, N2, O3, Q19 Cavalcanti E. ������������������������������U7 Conconi D. ���������������������������������L16 De Cesaris P. �����������������������������F4 Cavallaro U. �������������������������������E2 Condelli V. ����������������������������������G4 De Curtis I. ���������������������������������F11 Consonni FM. ����������������������������V9 De Dominici M. ��������������������������V8
98 99 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
De Lalla C. ���������������������������������U12 Dozza B. ������������������������������������L5 Forgione L. ���������������������������������T1 De Laurentiis M. �������������������������L15 Dugo M. �������������������������������������C16, C17, C19, Formisano L. ������������������������������L25 De Laurenzi V. ����������������������������L26 I11, N1, P1, Q19, Fortunato O. �������������������������������L2, N1, C7 De Leo M. ����������������������������������V7 Q21 Foschi V. ������������������������������������B3 De Libero G. ������������������������������U12 Duranti C. �����������������������������������L18 Franchin G. ��������������������������������Q18 De Luca A. ���������������������������������Q7, T1 Eberini I. ������������������������������������L27 Francque S. �������������������������������U8 De Maglie M. ������������������������������V17 Eisenhofer G. �����������������������������V4 Franzese O. �������������������������������U9 De Maglio G. ������������������������������U15 Emma MR. ���������������������������������L13, L14 Franzoso G. �������������������������������V17 De Marco C. �������������������������������Q4 Ennas MG. ��������������������������������C22 Frapolli R. �����������������������������������C2, C14 De Marco F. ��������������������������������C24 Ercolani C. ���������������������������������V9 Fratini B. �������������������������������������C10 De Maria R. �������������������������������E9 Erreni M. �����������������������������������V5 Frega S. �������������������������������������Q3, Q6 De Nicola F. �������������������������������N5 Erriquez J. ����������������������������������C21 Freschi M. ���������������������������������U5 De Placido S. �����������������������������L25 Esposito F. ���������������������������������G4, L3 Frizziero M. ��������������������������������Q20 De Rienzo S. ������������������������������E3, L8 Esposito G. ��������������������������������G3 Fronza G. �����������������������������������I3 De Salvo G. ��������������������������������Q3, Q6 Esposito I. ����������������������������������O6 Fruci D. ��������������������������������������L6 De Santis F. ��������������������������������E12 Eva A. ����������������������������������������N6 Fruscio R. ����������������������������������C4, C8, L16 De Santis MC. ����������������������������G1 Fabbri A. ������������������������������������K5 Fucà G. ��������������������������������������E12 De Stefano A. �����������������������������Q25, K8 Fabi A. ����������������������������������������O1 Fumarola C. ������������������������������L21 Decio A. �������������������������������������G8, L16 Fabretti F. �����������������������������������H1 Fusco BM. ����������������������������������L9 Defilippi P. ����������������������������������N6, V1 Facchinetti F. ������������������������������V14 Fusella F. ������������������������������������F1 Degano M. ���������������������������������F11 Facciolo F. ����������������������������������U9, V13 Fuso Nerini I. �����������������������������D5 Del Bianco P. ������������������������������Q3, Q6 Fadda A. ������������������������������������C22 Gaballo P. �����������������������������������D3 Del Bufalo D. ������������������������������L4, V8, V9 Fadda P. �������������������������������������Q9 Gaboriaud P. ������������������������������B3 Del Sal G. ����������������������������������Q1 Faedda R. ����������������������������������N3 Gabriele L. ���������������������������������U7 Del Vecchio M. ���������������������������U13, U14 Faenza I. ������������������������������������F2, R1 Gabriele P. ���������������������������������F15 Delia D. ��������������������������������������F8 Fairclough S. ������������������������������C20 Gafà R. ��������������������������������������B3 Dellabona P. �������������������������������U12 Falanga A. ����������������������������������Q16 Gaffo E. ��������������������������������������A3 Dello Sbarba P. ��������������������������E4 Falcetta F. �����������������������������������D5, N8, N9 Gaggianesi M. ����������������������������E6, E7 Delrio P. ��������������������������������������Q25 Falcioni R. ����������������������������������L4 Gai M. ����������������������������������������L23 Delvecchio S. �����������������������������V3 Falcone I. �����������������������������������V8 Galdiero F. ����������������������������������I9 Denaro N. ����������������������������������L1 Falcone M. ���������������������������������L23 Galimberti M. �����������������������������G8 Deng X. ��������������������������������������E4 Fanciulli M. ���������������������������������N5 Gallazzi M. ���������������������������������V7, V11 Deraco M. ����������������������������������E10 Fanetti G. �����������������������������������Q18 Galli A. ���������������������������������������A1 Derle A. ��������������������������������������G1 Fanti PA. �������������������������������������K6 Galli R. ���������������������������������������U5 Desideri M. ��������������������������������V8, V9 Faraci S. �������������������������������������E1, E12 Gallino F. ������������������������������������I11 Devecchi A. ��������������������������������C17 Farini V. ��������������������������������������V9 Gallo A. ��������������������������������������R1 Di Carlo A. ����������������������������������V13 Farris F. ��������������������������������������E12 Gallo Cantafio ME. ���������������������K3 Di Castro V. ��������������������������������F9, L10 Fasano E. ����������������������������������E1 Gallo E. ��������������������������������������O1 Di Cosimo S. ������������������������������F7, M1, Q4 Fassan M. ����������������������������������Q3, Q6 Gambino A. ��������������������������������Q14 Di Cunto F. ���������������������������������L23, N6 Favasuli V. ����������������������������������K3 Ganci F. ��������������������������������������C9, Q12 Di Fonte R. ���������������������������������L29, O5, Q24 Favia A. ��������������������������������������C15 Gandola L. ���������������������������������N2, L26 Di Francesco B. �������������������������V17 Fenizia F. ������������������������������������Q7, T1 Ganzinelli M. ������������������������������H3, K5, U10 Di Franco S. �������������������������������E6 Fernández-Serra A. �������������������C13 Garassino MC. ���������������������������U10, U13, U14 Di Gennaro E. ����������������������������E3, Q25 Ferracin M. ���������������������������������G6, K6, U15 Garavaglia C. �����������������������������U12 Di Guardo L. ������������������������������I11, U13, U14 Ferrandina G. �����������������������������L10 Garavelli S. ��������������������������������V16 Di Marco T. ���������������������������������L22 Ferrarese R. �������������������������������J1 Garbarino O. ������������������������������E8, I3 Di Marcotullio L. �������������������������H1, L6, N3 Ferraresi V. ���������������������������������K2, L4, U9 Garcia Perez L. ��������������������������J1 Di Maro S. ����������������������������������D3 Ferrari A. ������������������������������������N1 Garcia-Casado Z. ����������������������C13 Di Martile M. ������������������������������V8, V9 Ferrari F. �������������������������������������Q15 Garello F. ������������������������������������L23 Di Martino D. ������������������������������D3 Ferrarini F. ����������������������������������I11 Gargano G. ��������������������������������B4 Di Martino MT. ���������������������������I9 Ferraro M. ����������������������������������L11 Gargiuli C. ����������������������������������C19, I11, Q19 Di Mauro C. ��������������������������������L25 Ferrero F. ������������������������������������I11 Garibaldi E. ��������������������������������F15 Di Modugno F. ����������������������������V13 Ferretti G. �����������������������������������O1 Gariboldi M. �������������������������������E10 Di Nicola M. �������������������������������E1, E12 Ferrone L. ����������������������������������G7 Garofalo A. ���������������������������������P2 Di Nicolantonio F. �����������������������C20, R2, T3, U16 Ferrone S. ����������������������������������U2 Garofalo C. ���������������������������������N4 Di Paolo D. ���������������������������������L2 Fidaleo M. ����������������������������������V2 Garofoli M. ���������������������������������L29, O5, Q24 Di Renzo MF. �����������������������������C21 Figini M. �������������������������������������G9 Garrone O. ���������������������������������L1, Q1 Di Somma M. �����������������������������M3 Filippi M. ������������������������������������L23 Gasparini M. ������������������������������P4 Di Stefano C. ������������������������������F4 Filippini A. ����������������������������������F4, F5 Gasparini P. ��������������������������������N1, U10 Di Stefano T. �������������������������������C23 Finardi E. �����������������������������������P3 Gasparro S. �������������������������������O1 Diaz-Rodriguez E. ����������������������L26 Fiore D. ��������������������������������������L9 Gatti L. ���������������������������������������I8 Diceglie C. ���������������������������������D4, Q10 Fiore S. ��������������������������������������I12 Gazzerro P. ��������������������������������L9 Digiacomo G. �����������������������������L21, U6 Fiorentino F. �������������������������������C9 Gebbia F. �����������������������������������Q14 Dika E. ���������������������������������������K6 Fiorentino M. ������������������������������U6, U15 Genova C. ����������������������������������C12, U11 Dinami R. �����������������������������������F10, Q12 Fiori ME. ������������������������������������E9 Gentile R. �����������������������������������L26 Diodoro MG. ������������������������������L12, V13 Fischietti M. ��������������������������������V17 Gentilini D. ���������������������������������C22 Dituri F. ���������������������������������������L26 Flavell RA. ���������������������������������J1 Germano G. ������������������������������U16 Do Valle IF. ���������������������������������I7 Flohr C. �������������������������������������U8 Ghedini GC. �������������������������������L31 Doglioni C. ���������������������������������U5 Focaccia E. ��������������������������������F2, R1 Ghezzi S. �����������������������������������C20, U16 Dogrusöz M. ������������������������������K4 Foddai ML. ���������������������������������V9 Ghia P. ��������������������������������������V3 Doliana R. ����������������������������������A3 Foggetti G. ���������������������������������I3 Ghilardi C. ����������������������������������A4 Domenicotti C. ���������������������������E8, I3 Foglio E. �������������������������������������L30 Ghilardi C. ����������������������������������G8 Dominioni L. �������������������������������P3, V7, V11 Folgiero V. ����������������������������������N5 Ghirelli C. �����������������������������������V6 Donati B. ������������������������������������C23, I7 Fontana F. ����������������������������������G2, L7 Giacomini A. �������������������������������L30 Donati F. �������������������������������������C4 Fontanella R. �����������������������������F4 Giacomini P. �������������������������������K2, O1, P2 Donzelli S. ����������������������������������L10 Forbes SJ. ����������������������������������V17 Giana D. �������������������������������������L27
100 101 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Gianfredi V. ��������������������������������B4 Iorio J. ����������������������������������������L18 López-Guerrero JA. �������������������C13 Giannandrea D. ��������������������������L27, V16 Iorio M.V. ������������������������������������E1, H5 López-Rreig R. ���������������������������C13 Giannatempo P. �������������������������C19 Ippoliti R. ������������������������������������L26 Lorenzato A. �������������������������������C20, T3 Giannelli G. ��������������������������������L26 Ippolito L. �����������������������������������G6, G7 Lori G. ����������������������������������������I5 Gianni L. ������������������������������������L32 Isella C. ��������������������������������������I10, I12, O2, S2 Loria R. ��������������������������������������L4 Giannini G. ���������������������������������B5, C6, C10, H1 Isla Larrain MT. ��������������������������U17 Lorito N. �������������������������������������G6 Giannoni E. ��������������������������������G7, I12 Iuliano R. ������������������������������������C11, L24 Losito NS. ����������������������������������K7 Giansanti F. ��������������������������������L26 Izzo F. �����������������������������������������Q25 Lospinoso Severini L. ����������������L6, N3 Giavazzi R. ��������������������������������A4, F12, G8, L16, Izzotti A. �������������������������������������I3 Lottini T. ��������������������������������������L18 Q16, V10 Jachetti E. ����������������������������������U5 Loupkis F. �����������������������������������U16 Giordani E. ���������������������������������O1, P2 Jacobs AH. ���������������������������������D2 Luca L. ���������������������������������������U16 Giordano S. ��������������������������������I12 Jager MJ. �����������������������������������K4 Lucarelli E. ���������������������������������L5 Giovannoni R. ����������������������������L16 Jin J. ������������������������������������������N7 Lucchetti D. ��������������������������������E9 Giraudo E. ����������������������������������F15 Kang D. ��������������������������������������D5 Luchini A. �����������������������������������Q23 Giudice AM. �������������������������������Q17 Katsaros D. ��������������������������������C21, Q14 Luciani R. �����������������������������������L3 Giustetto P. ���������������������������������L18 Klein CA. ������������������������������������F7 Luciano A. ����������������������������������D3, U7 Gnad-Vogt U. �����������������������������U8 Koenigsrainer A. ������������������������U8 Lucignani G. �������������������������������D4, Q11 Gnoni R. �������������������������������������C23 Kool M. ���������������������������������������N3 Ludwig J. ������������������������������������U8 Gobbi G. ������������������������������������I7 Krampera M. ������������������������������K1 Luo M. ����������������������������������������N7 Goeman F. ��������������������������������N5 Kumar S. ������������������������������������J1 Lupi M. ���������������������������������������L20 Gontero P. ����������������������������������P4 Kusamura S. ������������������������������E10 Lupia M. �������������������������������������E2 Gouttefangeas C. �����������������������U8 Lamba S. �����������������������������������T3 Luraghi P. �����������������������������������U16 Govoni M. ����������������������������������B3 Lambertini M. �����������������������������K6 Luti S. �����������������������������������������G6 Gozzelino L. �������������������������������G1 Lambiase M. ������������������������������Q7 Ma A. �����������������������������������������N7 Gozzini A. �����������������������������������E4 Lamolinara A. �����������������������������L26, N6, V1 Ma YT. ����������������������������������������U8 Gramantieri L. ����������������������������L28 Landriscina M. ���������������������������G4, L3 Maccarinelli F. ����������������������������L30, L31 Gramignoli R. �����������������������������L13 Lanman R.B. ������������������������������C20 Maddalena F. �����������������������������G4, L3 Granchi C. ����������������������������������I12 Lanza F. �������������������������������������K4 Maffey A. ������������������������������������D4 Grassilli E. ����������������������������������L16 Lanza G. ������������������������������������B3 Magagnoli G. �����������������������������Q23 Grasso S. �����������������������������������N6 Lanzardo S. �������������������������������E5 Magnani C. ��������������������������������C22 Gray NS. ������������������������������������E4 Laprovitera N. ����������������������������U15 Magni M. ������������������������������������L19 Grazi GL. �����������������������������������V13 Laquitana V. �������������������������������L4 Magri V. ��������������������������������������C6 Grazia G. �����������������������������������U13, U14 Lastoria S. ����������������������������������D3 Maiello E. ����������������������������������Q7 Greco A. �������������������������������������L22, M2, V5 Lastraioli E. ��������������������������������L18 Maiolino P. ����������������������������������U1 Greco S. �������������������������������������F2, K1 Lavitrano M. �������������������������������L16 Maione F. �����������������������������������F15 Greenblatt J. �������������������������������C15 Lawlor RT. ����������������������������������V13 Majorini MT. �������������������������������F8 Gregnanin I. �������������������������������P4 Lawrence T. ��������������������������������V17 Malaguti P. ���������������������������������O1 Grillo E. ��������������������������������������M3 Lazarevic D. �������������������������������C12 Malapelle U. �������������������������������C6 Grioni M. ������������������������������������U5 Lazzari E. �����������������������������������V16 Malleo G. �����������������������������������Q20 Grossi F. �������������������������������������C12, U11 Lazzari L. �����������������������������������C20, T3 Malpeli G. �����������������������������������K1 Grossi S. ������������������������������������M4 Lazzaroni F. ��������������������������������O6 Manara MC. �������������������������������N4, Q17 Guaglio M. ���������������������������������E10 Le Noci V. �����������������������������������J2, Q8 Manciocco V. ������������������������������C9 Guardavaccaro D. ����������������������N3 Lebiu M. �������������������������������������N9 Mandoj C. ����������������������������������K2 Guerriero P. ��������������������������������L28 Lecis D. ��������������������������������������F8 Manenti G. ���������������������������������F8 Guffanti F. �����������������������������������H2, H3 Leone A. ������������������������������������E3, L15 Manfioletti G. ������������������������������F6 Guglielmetti S. ����������������������������J2 Leone BE. ����������������������������������L16 Mangiapane LR. ������������������������E6 Guida G. ������������������������������������O5 Leone F. �������������������������������������C20 Mannarino L. ������������������������������C2, C14, I1 Guida M. ������������������������������������L29, O5 Leonetti C. ����������������������������������F10 Mannelli M. ��������������������������������V4 Gulino A. ������������������������������������L6, L13, N3 Lepore M. �����������������������������������U12 Mano M. �������������������������������������F8 Guttieri L. �����������������������������������E9 Lepore S. �����������������������������������G4, L3 Mantione ME. �����������������������������V3 Hanbashi A. �������������������������������F5 Lettini G. ������������������������������������G4, L3 Mantovani F. �������������������������������Q1 Heidenreich R. ��������������������������U8 Li Bergolis V. ������������������������������G4 Manzoni M. ��������������������������������K3 Henderson KJ. ���������������������������J1 Li S. ��������������������������������������������E4 Marabese M. ������������������������������H4, I6 Hidalgo A. ����������������������������������H5 Licitra L. �������������������������������������C3 Marampon F. ������������������������������F4 Hirsch E. ������������������������������������G1 Ligorio F. ������������������������������������R2 Marchini S. ���������������������������������C2, C4, C5, C8, Huber S. �������������������������������������J1 Liguori P. ������������������������������������K4 I1, Q15 Iacobazzi RM. ����������������������������L29, O5, Q24 Limonta P. ����������������������������������G2, L7 Marciano R. �������������������������������L25 Iacobelli S. ���������������������������������L26 Linnebacher M. ��������������������������T3 Marengo B. ��������������������������������E8, I3 Iannaccone A. ����������������������������Q7 Liotta L.A. �����������������������������������Q23 Mariancini A. ������������������������������Q19 Iannelli F. ������������������������������������L8 Liu Z. ������������������������������������������N7 Mariniello RM. ����������������������������K8 Iannò MF. �����������������������������������N2 Lo Dico A. ����������������������������������D2, D4, Q11 Markowetz F. ������������������������������C2, C14 Ieranò C. ������������������������������������I9, L17, U1 Lo Nigro C. ���������������������������������L1, Q1 Marra G. �������������������������������������P4 Iervolino PL. �������������������������������K8 Lo Nigro L. ���������������������������������N8, N9 Marsoni S. ����������������������������������C20, U16 Iervolino V. ���������������������������������T1 Lo Russo G. �������������������������������U10, U13, U14 Martelli C. �����������������������������������D2, D4, Q10, Iezzi A. ���������������������������������������I6 Locatelli A. ���������������������������������L32 Q11 Iezzi M. ��������������������������������������L26, N6, V1 Locatelli E. ���������������������������������H1 Martinelli E. ��������������������������������Q7 Illi B. �������������������������������������������C15 Locatelli F. ����������������������������������L6, N5 Martinelli S. ��������������������������������V4 Illueca C. ������������������������������������C13 Locati LD. �����������������������������������C3 Martinetti A. ��������������������������������F7, Q5 Imperatori A. ������������������������������V7 Lococo F. �����������������������������������C23 Martínez Carrera DC. �����������������H5 Indini A. ��������������������������������������U13, U14 Lodi F. ����������������������������������������C20 Martini F. ������������������������������������B3 Indraccolo S. ������������������������������G3, Q3, Q6 Loeffler M. ����������������������������������U8 Martini M. �����������������������������������G1 Infante P. ������������������������������������H1, L6, N3 Loi E. ������������������������������������������C22 Martino C. ����������������������������������C20 Insaurralde F. ����������������������������U17 Loizidou M. ��������������������������������D1 Marullo R. ����������������������������������E12 Invernizzi A.M. ���������������������������T2 Lombardi R. �������������������������������I4, L8 Marzagalli M. �����������������������������G2, L7 Invrea F. �������������������������������������I10 Lonardi S. ����������������������������������C20, U16 Marzano V. ���������������������������������E9 Iorio E. ���������������������������������������G9 Longo R. ������������������������������������G2 Marzocchini R. ���������������������������I5
100 101 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Masgras I. ����������������������������������L11 Moroni E. �����������������������������������L11 Pandiella A. ��������������������������������L26 Massimino M. �����������������������������N1, N2 Morosi L. ������������������������������������D5, V10 Panetta M. ����������������������������������U9, V13 Matarazzo S. ������������������������������L30, L31 Morotti A. ������������������������������������F1 Pani G. ���������������������������������������V2 Matassa DS. ������������������������������L3 Mortara L. ����������������������������������V7, V11, V12 Panini N. ������������������������������������H2 Materozzi M. ������������������������������A1 Mortarini R. ��������������������������������F14, U13, U14 Paoli A. ���������������������������������������C19 Matteo C. �����������������������������������N8, N9 Mosci C. �������������������������������������K4 Paoli P. ���������������������������������������I5 Maurichi A. ���������������������������������F14 Motta R. �������������������������������������F7, Q5, S1 Paolino C. ����������������������������������K5 Mauriello A. ��������������������������������O4 Munciguerra F. ���������������������������K8 Papa S. ��������������������������������������V17 Mauro G. ������������������������������������V5 Munegato S. �������������������������������P4 Papacci F. ����������������������������������F5 Mayer-Mokler A. ������������������������U8 Mura R. ��������������������������������������N9 Pape J. ���������������������������������������D1 Mazza D. ������������������������������������F11 Muraca F. �����������������������������������L24 Paracchini L. ������������������������������C4, C5, C8 Mazzola M. ��������������������������������V16 Mussolin B. ��������������������������������C20, O2, T3 Parasole R. ��������������������������������N8 Mazzoni M. ��������������������������������M2, V5 Muzio M. ������������������������������������V3 Pardella E. ���������������������������������I5 Mazzotta A. ��������������������������������L29, O5, Q24 Naccarati A. �������������������������������P4 Pardini B. �����������������������������������P4 Medico E. �����������������������������������I10, I12, O2, S2 Nagy R.J. �����������������������������������C20 Parnigoni A. �������������������������������M4 Melchionna R. ����������������������������V13 Napoli A. ������������������������������������T2 Parra A. ��������������������������������������Q17 Melisi D. �������������������������������������I2, Q20, R2 Napolitano F. ������������������������������L25 Parravicini C. ������������������������������L27 Mello T. ���������������������������������������A1 Napolitano M. �����������������������������I9, U1, U7 Parrington J. �������������������������������F5 Mello-Grand M. �������������������������P4 Nardo G. ������������������������������������G3, Q3, Q6 Pasello G. ����������������������������������Q3, Q6 Melocchi L. ���������������������������������L31 Nasi S. ���������������������������������������C15 Pasello M. ����������������������������������Q17 Melocchi V. ���������������������������������C18 Necchi AN. ���������������������������������C19 Pasquale R. �������������������������������T1 Menichini P. ��������������������������������I3 Negrini M. ����������������������������������L28 Pasqualini F. �������������������������������V17 Merlano MC. ������������������������������L1, Q1 Neri A. ����������������������������������������K3, L22, L27, V16 Pasqualini L. ������������������������������Q3, Q6 Merz V. ���������������������������������������Q20, R2 Neri L. ����������������������������������������L28 Pastorelli R. ��������������������������������G8, I6 Mezi S. ���������������������������������������C6 Neri N. ���������������������������������������V15 Pastorino U. �������������������������������K5, L2, Q8, V14 Mezzanzanica D. �����������������������F13, G9, O3, T2 Nia SA. ���������������������������������������C22 Pavan A. �������������������������������������Q3, Q6 Micalizzi C. ���������������������������������N8, N9 Nicassio FN. ������������������������������E11 Pazzaglia L. �������������������������������L5, Q23 Michelotti A. �������������������������������L1 Nicoletti R. ���������������������������������O3 Pazzaglia S. �������������������������������L6 Micotti E. ������������������������������������A4 Nicolini G. ����������������������������������F14 Pedron S. �����������������������������������K1 Miele M. �������������������������������������I3 Nicolussi A. ��������������������������������C6, C10 Pegoraro S. ��������������������������������F6 Milanetti E. ���������������������������������C6 Nicotra A. �����������������������������������E6 Pelizzoni F. ���������������������������������K3 Milani M. ������������������������������������E1 Niger M. �������������������������������������S1 Pellacani A. ��������������������������������U4 Milani M. ������������������������������������P1 Nigita G. �������������������������������������Q9 Pelosi G. ������������������������������������Q22, V11 Milani M. ������������������������������������Q21 Nisticò C. �����������������������������������O1 Peña-Luna M. ����������������������������H5 Milano G.M. �������������������������������K2 Nistico’ P. ������������������������������������U9, V13 Perego P. ������������������������������������I8 Milella M. ������������������������������������L4, V8, V13 Noberini R. ���������������������������������C1 Perelli L. �������������������������������������E9 Mileo AM. �����������������������������������V13 Noonan DM. �������������������������������P3, Q2, V7, V11, Perotti V. �������������������������������������F14, U13, U14 Miligi L. ���������������������������������������C22 V12 Perra A. ��������������������������������������A2 Milione M. ����������������������������������E10, L2, Q8, U10 Normanno N. �����������������������������Q7, T1 Perri P. ���������������������������������������L2 Milone MR. ���������������������������������I4, L8 Novellis P. �����������������������������������C23 Perrone F. �����������������������������������K5 Minici C. �������������������������������������F11 Novello M. ����������������������������������K2, LO4 Perrone M. ���������������������������������P1, Q21 Minna E. �������������������������������������M2 Nucci D. �������������������������������������B4 Perrotti G. �����������������������������������L24 Minoli L. �������������������������������������A3, A4, F12, Q16 Nuccitelli A. ��������������������������������C9 Perrotti N. �����������������������������������C11, L24 Minutolo F. ����������������������������������I12 Occhipinti S. �������������������������������U2 Pescarmona E. ��������������������������K2, O1, P2 Miodini P. ������������������������������������Q5, S1 Oddo D. �������������������������������������T3 Peschiaroli A. �����������������������������L6 Mirandola P. �������������������������������G5 Oderda M. ����������������������������������P4 Peterlongo P. ������������������������������B5 Miranti A. ������������������������������������F15 Odicino F. �����������������������������������C5, C8, Q13, Petricci E. �����������������������������������H1 Mitchard J. ���������������������������������Q2 Q14, Q15 Petrizzo A. ����������������������������������O4 Mitola S. �������������������������������������M3, Q13 Oliva P. ���������������������������������������L3 Petroni G. �����������������������������������L18 Mittica G. ������������������������������������C21 Olivero M. ����������������������������������C21 Petroni M. �����������������������������������C6, H1 Miwa Y. ���������������������������������������J1 Orlandella FM. ���������������������������K8 Petronini PG. ������������������������������L21, U6 Moccia T. ������������������������������������E3, L8 Orrù C. ���������������������������������������A2 Petruzziello F. �����������������������������N9 Moi L. �����������������������������������������C22 Orsini A. �������������������������������������F2 Petti C. ���������������������������������������I10, O2, S2 Moiso E. �������������������������������������F1, V1 Orsini RC. ����������������������������������L25 Petti E. ���������������������������������������F10, Q12 Molla A. ��������������������������������������F14, U13, U14 Ortolan E. ����������������������������������Q4 Pezzolo A. ����������������������������������N6 Monaco G. ���������������������������������U1 Ortuso F. ������������������������������������L24 Pezzuto F. ����������������������������������K7 Monaco M. ��������������������������������U7 Ostano P. �����������������������������������A4, P4 Pfeffer U. ������������������������������������K4 Mongiovì S. ��������������������������������L13 Ottaggio L. ���������������������������������I3 Pfister SM. ���������������������������������N3 Montagna M. ������������������������������B5 Ottini L. ��������������������������������������B5, C10 Piaggio F. �����������������������������������K4 Montagnani Marelli M. ���������������G2, L7 Ottobrini L. ���������������������������������D2, D4, Q10, Piana S. �������������������������������������C23 Montalto G. ��������������������������������L13, L14 Q11 Piazza S. ������������������������������������F6 Monteleone L. ����������������������������E8 Pacak K. ������������������������������������V4 Piazzi M. ������������������������������������F2, R1 Monterisi C. �������������������������������D2 Pacelli C. ������������������������������������G4 Picardo E. ����������������������������������Q14 Monti P. ��������������������������������������I3 Padoan M. ���������������������������������C22 Picci P. ���������������������������������������Q17 Montone M. ��������������������������������C20, O2, T3 Padula F. ������������������������������������F4 Picci P. ���������������������������������������Q23 Mor M. ���������������������������������������L30 Pagano M. ����������������������������������C23 Piccinni-Leopardi M. ������������������Q23 Mora M. ��������������������������������������C12 Pagliardini S. ������������������������������L22 Piccotti F. ������������������������������������Q10 Morabito A. ���������������������������������T1 Pala V. ����������������������������������������M1 Piemonti L. ���������������������������������V13 Morandi A. ����������������������������������G6, G7 Palano MT. ���������������������������������V15, V16 Pierini M. ������������������������������������Q23 Morandi E. ���������������������������������A4 Palazzolo L. �������������������������������L27 Pierotti MA. ��������������������������������E10 Moresco RM. �����������������������������D2 Palermo B. ���������������������������������U9 Pietrafesa M. ������������������������������G4, L3 Moretti F. ������������������������������������P3 Pallavicini G. ������������������������������L23 Pietrantonio F. ����������������������������I12, T3, U16 Moretti M. �����������������������������������B4, G2 Palli D. ����������������������������������������B5 Pignata S. ����������������������������������O3 Moretti R.M. �������������������������������L7 Pallocca M. ��������������������������������N5, C9 Pilotti S. ��������������������������������������C2, C14, C17, I1 Mori L. ����������������������������������������U12 Palmerini E. �������������������������������L5 Pinazza M. ���������������������������������G3 Moro M. ��������������������������������������K5, L2, U10 Palombi F. ����������������������������������F5 Pinciroli P. ����������������������������������F8
102 103 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Pinessi D. �����������������������������������F12, V10 Regge D. ������������������������������������C20 Salvatore D. ��������������������������������Q11 Pinto C. ��������������������������������������C23 Reggiani F. ��������������������������������I7 Salvatore G. �������������������������������K8 Piro G. ����������������������������������������I2, R2, Q20 Regondi V. ���������������������������������V6 Salvatori L. ���������������������������������C15 Pisapia P. �����������������������������������C6 Reinhardt C. �������������������������������U8 Salviato E. ����������������������������������Q15 Piscopo C. ���������������������������������L9 Rena O. ��������������������������������������C23 Samaja M. ����������������������������������Q15 Pistocchi A. ��������������������������������V16 Renzi T. ��������������������������������������Q21 Samengo D. �������������������������������V2 Pitisci A. �������������������������������������N5 Renzi TA. ������������������������������������P1 Sana I. ��������������������������������������V3 Pivetta E. ������������������������������������A3 Resovi A. ������������������������������������A4, Q16, V10 Sanchez-Martin C. ���������������������L11 Platonova N. ������������������������������K3, L27, V16 Restelli V. �����������������������������������L20 Sancisi V. �����������������������������������I7 Politi LS. �������������������������������������D2 Rhoden K.J. �������������������������������Q23 Sanfilippo R. ������������������������������C2 Pollino S. ������������������������������������L5, Q23 Riccardo F. ���������������������������������E5, U2, U3 Sangaletti S. �������������������������������E1, U10 Polvani S. �����������������������������������A1 Ricci F. ���������������������������������������H3 Sangro B. �����������������������������������U8 Ponterini G. ��������������������������������L3 Ricciardi Tenore C. ���������������������E9 Santaniello A. �����������������������������L25 Ponziani S. ���������������������������������L26 Riccioli A. �����������������������������������F4 Santi S. ��������������������������������������N4 Ponzo M. ������������������������������������C2, D5, I1 Richter S. �����������������������������������V4 Santinami M. ������������������������������F14 Ponzoni M. ���������������������������������L2 Riefolo M. �����������������������������������K6, U15 Santoro R. ����������������������������������I2, Q20, R2 Porcelli L. �����������������������������������L29, O5, Q24 Riganti C. �����������������������������������N6 Sanvito F. �����������������������������������M4 Porcellini E. ��������������������������������K6 Rijavec E. �����������������������������������C12, U11 Saporiti S. ����������������������������������L27 Porcu L. ��������������������������������������Q16 Riva C. ���������������������������������������F13 Sardina F. �����������������������������������H1 Porporato PE. ����������������������������G1 Riverso M. ����������������������������������V4 Sartore-Bianchi A. ����������������������C20, T3, U16 Porporato R. �������������������������������S2 Rivoltini L. ����������������������������������I11, U10 Sartori E. ������������������������������������C8, Q14, Q15 Porru M. �������������������������������������F10 Rizzari C. �����������������������������������G5, N8, N9 Sartori P. ������������������������������������G2, L7 Portella L. �����������������������������������I9, U1, U7 Rizzetti MG. �������������������������������M2, V5 Satta A. ��������������������������������������G9 Poveda A. �����������������������������������C13 Rizzo A. ��������������������������������������G9 Satta G. ��������������������������������������C22 Pranzini E. ����������������������������������I5 Rizzo G. �������������������������������������Q10 Scagnoli F. ���������������������������������C15 Presta M. ������������������������������������L30, L31, M3 Rizzo MG. ����������������������������������V8 Scala S. �������������������������������������D3, E13, I9, L17, Prete A. ��������������������������������������C6 Rizzolo P. �����������������������������������B5 U1, U7 Privitera S. ���������������������������������C21 Roca MS. �����������������������������������E3, L8 Scanziani E. �������������������������������A3 Procacci P. ���������������������������������G2, L7 Rocca S. ������������������������������������F1 Scarfò L. ������������������������������������V3 Pronzato MA. �����������������������������E8 Rocco G. ������������������������������������T1 Scarpa A. �����������������������������������K1, Q20, R2, V13 Proto C. ��������������������������������������U10, U13, U14 Rodolfo M. ����������������������������������I11 Scavo E. ������������������������������������E6 Proto MC. �����������������������������������L9 Roggiani F. ���������������������������������F13, T2 Schamberger T. ��������������������������F7 Provenzi M. ��������������������������������N8 Rolih V. ���������������������������������������U2 Schiavello E. ������������������������������N2 Provero P. �����������������������������������F1 Rollo F. ��������������������������������������C9 Scionti F. ������������������������������������I9 Pruneri G. ����������������������������������K5, Q4, U10 Roma C. �������������������������������������Q7, T1 Scognamiglio G. ������������������������U7 Pucci B. ��������������������������������������I4, L8 Romagnoli G. �����������������������������U7 Scotlandi K. ��������������������������������N4, Q9, Q17 Puleio R. ������������������������������������L13 Romani C. ����������������������������������C8, Q13, Q14, Scuoppo C. ��������������������������������C15 Pulliero A. �����������������������������������I3 Q15 Scupoli MT. ��������������������������������K1 Puozzo A. �����������������������������������B3 Romania P. ���������������������������������L6, O1 Seclì L. ���������������������������������������F1 Pupa S.M. ����������������������������������E1, E12 Romano G. �������������������������������Q9 Selvatici R. ���������������������������������B3 Puricelli C. ����������������������������������E12 Romeo P. �����������������������������������V5 Sensi M. �������������������������������������C17, I11, Q19, Puzone R. ����������������������������������K4 Romero Cordoba SL. �����������������H5, E1 C16, C19, N1 Qiu Y. ������������������������������������������F15 Romero I. �����������������������������������C13 Serafini MS. �������������������������������C3 Quaglino E. ��������������������������������E5, U3 Romualdi C. �������������������������������C4, C5, C8, Q14, Serra M. �������������������������������������L11, Q17 Quattrini I. ����������������������������������Q23 Q15 Serratì S. �����������������������������������Q24 Queirolo P. ���������������������������������K4 Ronca R. ������������������������������������L30, L31 Servetto A. ���������������������������������L25 Rabassa ME. �����������������������������U17 Ronchetti D. �������������������������������K3 Sessa F. �������������������������������������M4 Rabusin M. ���������������������������������N9 Rondanin R. �������������������������������L3 Sestito G. �����������������������������������I6 Raccagni I. ���������������������������������D2 Rondinone O. �����������������������������E12, V14 Sestito R. �����������������������������������F9 Rachiglio AM. �����������������������������Q7 Ros G. ����������������������������������������F6 Sfondrini L. ���������������������������������J2, Q8 Radice P. ������������������������������������B5 Rosa R. �������������������������������������L25 Sgambato A. ������������������������������E9 Raggi D. �������������������������������������C19 Rosanò L. ����������������������������������D1, F3, F9, L10 Sgambelluri F. ����������������������������U13, U14 Raimondi M. �������������������������������G2, L7 Rospo G. ������������������������������������C20, T3, U16 Sgarra R. �����������������������������������F6 Raimondo D. ������������������������������C6 Rossella V. ���������������������������������C12 Sgrò F. ����������������������������������������L23 Rajkumar VS. �����������������������������J1 Rossi G. �������������������������������������U11 Sica A. ����������������������������������������V9, V17 Ramazzotti M. ����������������������������G6 Rotondo JC. �������������������������������B3 Sica L. ����������������������������������������L32 Rammensee HG. ����������������������U8 Rovida A. �����������������������������������V3 Siena S. �������������������������������������C20, T3, U16 Rampioni Vinciguerra GL. ����������Q18 Rovida E. �����������������������������������E4 Signorelli D. ��������������������������������U13, U14 Ranghetti P. ��������������������������������V3 Roz L. ����������������������������������������E10, E13, V14 Silvestri D. ����������������������������������N8, N9 Rapizzi E. �����������������������������������V4 Rugge M. �����������������������������������Q3, Q6 Silvestri M. ���������������������������������F7, Q5, S1 Rasola A. �����������������������������������L11 Ruiu R. ���������������������������������������E5 Silvestri V. ����������������������������������B5 Raspagliesi F. �����������������������������G9, O3, T2 Russillo M. ���������������������������������O1 Silvestris N. ��������������������������������Q24 Ratto E. ��������������������������������������G1 Russo A. ������������������������������������B5 Simeone I. ����������������������������������A1 Raugei G. �����������������������������������I5 Russo D. ������������������������������������L17 Simionato F. �������������������������������Q20, R2 Ravaggi A. ���������������������������������C5, C8, Q13, Russo M. ������������������������������������C20, T3, U16 Simioni C. ����������������������������������L28 Q14, Q15 Russo R. ������������������������������������I10 Simoni D. �����������������������������������L3 Ravelli A. ������������������������������������L21 Rybinska I. ��������������������������������V6 Singh-Jasuja H. �������������������������U8 Ravelli C. ������������������������������������M3 Sabbioni S. ��������������������������������L28 Siravegna G. ������������������������������C20 Ravera S. �����������������������������������E8 Sacchetto L. �������������������������������P4 Sisalli MJ. �����������������������������������L15 Razhko A. ����������������������������������B1 Sacconi A. ����������������������������������C9, F9, F10, L10, Sisinni L. ������������������������������������G4, L3 Rea G. ���������������������������������������D3, U1, U7 Q12 Soddu S. ������������������������������������H1 Recordati C. �������������������������������V17 Sachsenmeier K. �����������������������E2 Sogari A. ������������������������������������T3 Reda G. �������������������������������������O6 Sahun Roncero M. ���������������������H1 Sommariva M. ����������������������������J2, U10 Reduzzi C. ���������������������������������F7 Sala K. ���������������������������������������F11 Sonego M. ���������������������������������I4 Reduzzi C. ���������������������������������Q5 Salemme V. ��������������������������������N6, V1 Sorice A. ������������������������������������L8, Q25 Reduzzi C. ���������������������������������S1 Salvati E. ������������������������������������F10 Sorino C. ������������������������������������N5
102 103 AUTHORS INDEX
AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT AUTHOR �����������������������������������ABSTRACT
Sottile A. �������������������������������������I12 Trapella C. ����������������������������������L3 Zaitsau A. �����������������������������������B1 Sottotetti E. ���������������������������������Q5 Traverso N. ���������������������������������E8, I3 Zambelli S. ���������������������������������L32 Sozzi G. �������������������������������������C7, E10, E13, K5, Tripodo C. ����������������������������������E1, E13, P1, Zambetti M. ��������������������������������L32 L2, N1, Q8, Q19, Q21, U10 Zammataro L. ����������������������������M3 U10, V14 Trisciuoglio D. �����������������������������L4, V8, V9 Zamò A. �������������������������������������K1 Spada S. �����������������������������������V13 Triulzi T. ��������������������������������������J2, V6 Zanellato S. ��������������������������������V7 Spadaro F. ����������������������������������F3 Troncone G. �������������������������������C6 Zanieri F. ������������������������������������A1 Spadoni G. ���������������������������������L30 Trotta AM. �����������������������������������D3 Zanin R. �������������������������������������F6 Spagnuolo M. �����������������������������V8 Truini A ���������������������������������������C12 Zannini L. �����������������������������������F7 Speciale A. ���������������������������������E8, I3 Truini M. �������������������������������������C20, Q8 Zanotti L. ������������������������������������Q13, Q14, Q15 Sperduti I. ����������������������������������V13 Trusolino L. ��������������������������������C20, S2 Zanotto M. ����������������������������������I2 Spessotto P. �������������������������������A3 Tumiatti F. �����������������������������������H4 Zanta D. �������������������������������������N8 Spina A. �������������������������������������L17 Tupone MG. �������������������������������V8 Zavattari P. ���������������������������������A2, C22 Spinella F. ����������������������������������C9, P2 Turco E. ��������������������������������������F1, L23, N6, V1 Zazzeroni F. �������������������������������V17 Spinelli A. �����������������������������������B5 Turdo A. ��������������������������������������E6, E7 Zecca M. ������������������������������������N8 Spriano G. ���������������������������������C9 Tusa I. ����������������������������������������E4 Zecchetto C. �������������������������������Q20, R2 Stassi G. ������������������������������������E6, E7 Uggeri M. �����������������������������������N9 Zelli V. ����������������������������������������B5 Stefanini M. ��������������������������������L18 Ulisse A. �������������������������������������L32 Zennaro C. ���������������������������������F6 Storti C. ��������������������������������������J2, Q8 Vaira V. ���������������������������������������D2 Zinnhardt B. �������������������������������D2 Storto G. ������������������������������������G4 Valabrega G. ������������������������������C21 Ziparo E. ������������������������������������F4 Stramucci L. �������������������������������L12 Valentini E. ���������������������������������V8, V9 Zitella A. �������������������������������������P4 Strippoli S. ���������������������������������L29 Valentini V. ���������������������������������B5, C6, C10, I11 Zizioli V. ��������������������������������������Q13 Strocchio L. ��������������������������������N5 Valle M. ��������������������������������������P2 Zizza P. ��������������������������������������F10, Q12 Strom S. �������������������������������������L13 Vallelonga V. ������������������������������L27 Zoccali C. �����������������������������������L4 Szydlowska M. ���������������������������A2 Valsecchi MG. ����������������������������N8, N9 Zucca M. ������������������������������������C22 Tagliabue E. �������������������������������E1, F8, H5, J2, Valtorta E. ����������������������������������C20 Zucchetti M. �������������������������������D5, N8, N9, V10 Q8, V6 Valtorta S. ����������������������������������D2 Zucchinelli P. ������������������������������L32 Tagliamento M. ��������������������������U11, O4 Van der Velden P. �����������������������K4 Zulato E. ������������������������������������G3, Q3, Q6 Taguchi K. ����������������������������������A2 Vandone AM. �����������������������������L1 Zulato E. ������������������������������������Q6 Taiana E. ������������������������������������K3 Vanella P. �����������������������������������L1 Zupo S. ��������������������������������������K1 Takam Kamga P. ������������������������K1 Vanni I. ���������������������������������������C12, U11 Talarico G. ����������������������������������E1 Vanzati A. �����������������������������������C20 Tamborini E. �������������������������������K5 Vanzulli A. ����������������������������������C20 Taraboletti G. ������������������������������F12, Q16, V10 Varesco L. ����������������������������������B5 Taramelli R. ��������������������������������F13 Varesio L. �����������������������������������N6 Taranto S. �����������������������������������L30 Varinelli L. ����������������������������������E10 Tarocchi M. ��������������������������������A1 Vecchiotti D. �������������������������������V17 Tarone L. ������������������������������������U2 Vedelago M. �������������������������������V1 Tassi E. ��������������������������������������U14 Vegetti C. �����������������������������������F14, U13, U14 Tassi RA. ������������������������������������Q14 Veneroni S. ��������������������������������Q4 Tassone PF. �������������������������������K3 Venesio T. ����������������������������������C21 Tatangelo F. ��������������������������������U7 Veneziano D. ������������������������������Q9 Taurino G. ����������������������������������G5 Verde A. �������������������������������������K8 Terreno E. ����������������������������������L23 Vergani E. ����������������������������������I11 Thiele C. ������������������������������������N7 Vermi W. �������������������������������������L31 Thotakura AK. ����������������������������V17 Veronesi G. ��������������������������������C23 Tibiletti MG. ��������������������������������B5 Verza M. �������������������������������������G3, Q3, Q6 Tocci P. ���������������������������������������F9, L10 Verzella D. ����������������������������������V17 Todaro M. �����������������������������������E6, E7 Veschi V. ������������������������������������E6, N7 Todde S. �������������������������������������D2 Veyalkin I. �����������������������������������B1, B2 Todeschini P. ������������������������������C4, C5, C8, Q13, Viaggi S. ������������������������������������K4 Q14, Q15 Viel A. �����������������������������������������B5 Todoerti K. ���������������������������������K3, L22 Viganò L. ������������������������������������L32 Tognon G. ����������������������������������C8 Vignudelli T. ��������������������������������L3 Tognon M. ����������������������������������B3 Vilia MG. ������������������������������������V3 Tomanelli M. �������������������������������H4 Villarini A. �����������������������������������B4 Tomaselli S. �������������������������������R1 Villarini M. ����������������������������������B4 Tomassetti A. �����������������������������F13, T2 Vincenzoni F. ������������������������������E9 Tomirotti A. ���������������������������������Q21, P1 Vinci C. ��������������������������������������K3 Tomura M. ����������������������������������J1 Vinti L. ����������������������������������������N8, N9 Tonissi F. ������������������������������������L1 Visca P. ��������������������������������������V13 Tonoli D. �������������������������������������F11 Vismara M. ���������������������������������Q4, Q5, S1 Tonon F. �������������������������������������F6 Vitagliano C. ������������������������������Q25 Tonon G. ������������������������������������J1, L10 Vivenza D. ����������������������������������Q1 Torchiaro E. ��������������������������������O2 Voena C. ������������������������������������U3 Tornatore L. �������������������������������V17 Volante M. ����������������������������������I12 Tornesello ML. ���������������������������K7, O4 Volpari T. ������������������������������������E1, E12 Torri V. ����������������������������������������U10 Vuttariello E. �������������������������������I9 Torricelli F. ����������������������������������C23, I7 Weinschenk T. ����������������������������U8 Torroni L. ������������������������������������R2 Whitley P. �����������������������������������Q2 Tortora G. �����������������������������������I2, Q20, R2 Yamamoto M. �����������������������������A2 Tortoreto M. ��������������������������������E13 Yan C. ����������������������������������������N7 Tosadori G. ��������������������������������K1 Yanling Zhao D. ��������������������������C15 Touzé FA. �����������������������������������B3 Zaffaroni N. ��������������������������������I8, M1 Tramacere M. �����������������������������V12 Zago G. ��������������������������������������Q3, Q6 Trapasso F. ���������������������������������L24 Zagonel V. ����������������������������������C20, U16
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