TUMORI RARI LESEMPIO DEL COLANGIOCARCINOMA PROBLEMATICHE MEDICHE E

TUMORI RARI L’ESEMPIO DEL COLANGIOCARCINOMA PROBLEMATICHE MEDICHE E SOCIALI ROMA, 28 NOVEMBRE 2018 Dai fattori di rischio (incluso amianto) ai cambiamenti del genoma Prof GIOVANNI BRANDI, MD, Ph. D UNIVERSITY OF BOLOGNA 1

RISK FACTORS IN CHOLANGIOCARCINOMA (CC) § ICC incidence in progressive increase in almost all the world § Risk factors induce carcinogenesis through the pathway of chronic inflammation and cholestasis § Over 50% of patients affected in the West have no known risk factor Known Unknown A Modificato da: Farioli & Brandi, Risk Factors for 2 Cholangiocarcinoma. In “Cholangiocarcinoma!” NOVA Publ ; NY, 2015

Intrahepatic Cholangiocarcinoma (ICC) and NAFLD/NASH Intrahepatic cholangiocarcinoma Author Year Type Reddy et al 2013 Kinoshita et al 2016 ICC group Control group OR (C. I. 95%) p value Total NASH (case-control) Cohort 181 31 (17, 1%) / / Case. Control 88 15 (17%) 83* 13 (15, 6%) 3, 36 (1, 15 -10, 2) 0, 0269 * Patients with metastatic liver tumor

i r ie ti t n an e p gn ieri r ca inse ent m e c vetrai i c i l u a idr meccanici conciatori edili elicotteristi tipografi marinai militari chimici palom ferro bari can vieri tier c ald isti o d m on ais et to ti all te ur cn gic ici i ge c olo a r gi r o z z a i A MIANTO i l i s tes fi ora & COLANGIOCARCINOMA

CARCINOGENIC MECHANISMS OF ASBESTOS FIBERS chrysotile actinolite amosite tremolite anthophyllite crocidolite CRONIC INFLAMMATION Osmond-Mc. Leod et al. Parr and Fibre Toxicol 2011 REACTIVE OXYGEN SPECIES (ROS) PRODUCTION ASBESTOS FIBERS CHROMOSOMIC ABERRATIONS Nagai et al. Cancer Sci 2011 Cortez BA et al. Oncotarget 2016 ACTIVATION OF TK RECEPTORS BINDING AFFINITY FOR DNA, RNA AND NUCLEAR PROTEINS Mossman BT et al. Am J Pathol 2013 Kubo et al. J Clin Biochem Nutr 2012 ABSORPTION OF CARCINOGENS IMMUNE RESPONSE REDUCTION AND IONIZING RADIATIONS Matsuzaki et al. E. H and Prev Med 2017 Nakamura et al. Proc. Japan Acad. Ser. 2009 INITIATION CARCINOGENESIS PROMOTION 5

Asbestos and primary liver cancer COHORT STUDIES 1 Autore (anno) Tipo Studio Period o Industria Tipo asbesto Oss/Att SMR o SIR* (95% CI) Correlazione Note Selikoff I. et al (1991) Coorte 1967 -87 17800 (M) Coibentatori - 269/191 1. 4. (p<0, 001) + GI+fegato+ vie biliari Battista G. (1999) Coorte 1970 -97 Decoibentatori ferrovie (G. O) Crisotilo, Crocidolite 9/3. 74 2. 41 (1. 26 -4. 20) + Fegato Berry G. et al (2000) Coorte 1933 -80 5000 (M/F) Operai fibrocemento Amosite, crisotilo 10/3. 76 2, 66 (1. 28 -4. 89) + Fegato, + colecisti+ vie biliari Wingren G. (2004) Coorte 1964 -97 1229 (M/F) [9151] Vetrai Amianto (ND) 3/1. 50 (M) 3/0. 69 (F) 2. 00 (0. 41 -5. 84) (M) 4. 35 (0, 75 -10. 59) (F) - Fegato+ vie biliari Hein MJ et al (2007) Coorte 1940 -01 3072 (M/F) Tessili Crisotilo, Crocidolite 10/9. 5 1. 05 (0. 51 -1. 94) - Fegato + vie biliari Pira E. et al (2007) Coorte 1946 -84 1966 (M/F) [62025] Tessili Crocidolite 11/4. 6(M/F) 2. 37 (1. 18 -4. 25) + Fegato Clin B. et al (2009) Operai fibrocemento Crisotilo, Anfibolo 13/8 1. 62 (0. 86 -2. 75) - Fegato Coorte 2024 (M/F) 1978 -04 [24513] 3/1, 57 1. 92 (0. 38 -5. 6) - Vie biliari Wang X. et al (2013). Coorte Tessili Crisotilo 15/11 (M) 0/1. 02 (F) 1. 34 (0. 81 -2. 21) - - Fegato + vie biliari *Fegato= HCC+ICC 1972 -58 Coorte [P/Y] [contd…] 734 (M) 586 (M) 272 (F)

Asbestos and primary liver cancer COHORT STUDIES 2 Tipo Studio Autore (anno) Periodo Coorte [P/Y] Industria Tipo asbesto SMR o SIR* (95% CI) Correlazione Note 18/7. 3 2, 48 (1. 47 -3. 91) + Fegato 2/1. 3 1. 6 (0. 19 -5. 78) - Vie biliari extraep. 18/9. 75 1. 85 (1. 09 -2. 92) + Fegato 4/1. 41 2. 84 (0. 76 -7. 26) - Vie biliari Hogstedt et al (2013) Coorte Boulanger M. et al. (2015) Coorte Wu W. et al (2015) Coorte 1975 -89 4427(M/F) Navalmeccanici [109932] Amianto (ND) - 1. 6 (1. 08 -2. 36) [↑TEP] + Fegato Pira E. et al. (2016) Coorte 1946 -13 1977 (M/F) [74126] Tessili Amianto (ND) 12/- 1. 06 (0. 55 -1. 86) - Fegato Lin S. et al (2017) Coorte 1981 -06 1539 (M/F) [34736] Minatori Crisotilo 18/15. 69 1. 15 (0, 73 -1. 81) - Fegato Pira E. et al (2017) [update 2009] Coorte 1946 -14 1056 (M) Minatori Crisotilo - Fegato *Fegato= HCC+ICC 1981 -06 6320 (M/F) 1978 -09 2024 (M/F) Spazzacamini Tessili Amianto (ND) Oss/Att Amianto (ND) 5/7. 7 0, 65 (0. 21 -1. 51)

Brandi G, Tavolari S, Biasco G, Mattioli S, Violante F; Farioli A “Putative role of asbestos exposure in the development of ICC” In “Asbestos” , Book ( Nova Pub , NY, 2016)

ASBESTOS AND ICC: data from case-control studies NOCCA STUDY: ICC Brandi et al. Cancer Causes Control 2013 Farioli A, Straif K, Brandi G, et al. Occup Envir Med 2017) CARA STUDY: ICC Brandi G, et al. Submitted 163 CC : 112 ICC; 51 ECC 185 controls year AAdjusted for sex and age; for sex, age and factors extra-professional (previous viral hepatitis, steatosis, ) B Adjusted 9

ASBESTOS FIBERS IN BILE DUCTS AND GALLBLADDER Szendröi M. et al. ♀ 55 Yrs. (BUDAPEST) Asbestos bodies in a bile duct cancer after occupational exposure. Environ Res. 1983; 30: 270 -280. Ca. colecisti + asbestosi ♀ 80 Yrs. (MPM) Grosso F et al. ♀ 73 Yrs. (MPM) Asbestos fibres detected by scanning electron microscopy in the gallbladder of patients with malignant pleural mesothelioma (MPM). Journal of Microscopy. 2017 Apr 1; 266(1): 48– 54. ♀ 30 -75 Yrs. (CASALE MONFERRATO) ♀ 15 Yrs. Grosso F et al. Asbestos fibers in the gallbladder of patients affected by benign biliary tract diseases. Eur J Gastroenterol Hepatol. 2015 Jul; 27(7): 860– 4. NO fibers NO fiibers 10

PUTATIVE WAYS THE FIBERS CAN REACH THE LIVER Brandi G. and Tavolari S. in “Cholangiocarcinoma” NOVA Publ ; NY, 2015 11

PERIBILIARY GLANDS EXTRAHEPATIC ILAR CANAL OF HERING INTRAHEPATIC DIFFERENT CARCINOGENETIC PATWHAYS FOR ICC(+HCC) AND ECC Adapted from 1) Bragazzi MC et al. Ann Gastroenterol 2018 2) Tavolari S. , Venturi M, Brandi G. , Carcinogenesis of cholangiocarcinoma, in Cholangiocarcinoma (2015) 12

Genetic variability in CC could be the result of: a. the existence of genetically distinct stem cell niches along the biliary tree b. tumor clonal heterogeneity, either in the primary tumor (intratumoral heterogeneity), or in the same metastasis (intrametastatic heterogeneity) Genetic variability can also occur among metastases derived from the same primary tumor c. stochastic mutations and genomic instability d. tumor microenvironment and cancer treatment

NEXT GENERATION SEQUENCING PER STUDIO DEL GENOMA Shyr et al. Biol Proced 2013 WHOLE EXOME SEQUENCING • Sequenziamento dell’esoma, cioe’ di quella parte del genoma che codifica proteine (circa 1% dell’intero genoma di un individuo) • Si stima che l’esoma contenga circa l’ 85% delle mutazioni associate a patologia.

MUTAZIONI DRIVER NEI TUMORI DELLE VIE BILIARI Valle et al. Cancer Discov. 2017

DISTRIBUTION OF MUTATIONS IN BTC IDENTIFIED BY NGS (1) Not stratified by site or risk factors 107 by WES, 39 by WGS, 266 by targeted sequencing N= 412 (194 Italian and 218 Japanese) Deleterious germline mutations of cancer-predisposing genes detected in 11% of BTC patients 17% 14% Wardell et al. Journal of Hepatology 2018

DISTRIBUTION OF MUTATIONS IN BTC IDENTIFIED BY NGS (2) ( BY ION TORRENT) 19% 14% Simbolo et al. Oncotarget 2014

DISTRIBUTION OF MUTATIONS IN BTC IDENTIFIED BY NGS (3) FFPE bloks from 84 pts: NGS+q. PCR N=55 >30% Pd 1/Pdl 1 Papadopoulou et al. Am J Cancer Res 2018 N=15 N=14

NGS in ICC N=38 BTC ( 32 ICC) WES + Rna seq 3 p loss in all cases of bap 1 mutation Farshidfar et al. Cell 2017 N=496 ICC 165 home (150 TES+15 WES) vs 262 WES public database Analysis 122/150 TES for FGFR-2 (Sanger) FGFR-2 + =15% Nepal and Andersen. Hepatology 201819

Nepal and Andersen. Hepatology 2018 Poor prognosis 20

O. Viverrini Chan-On et al. Nature Genetics 2013 Over 60% of cases from Romania and Greek

CHOLANGIOCARCINOMA - CLUSTER SUBTYPES (Potential Role of Etiology) Whole-exom sequencing + RNA -seq -> 239 BTC (Japanese patients) Whole-genome sequencing -> 71 CCA tumors (several countries) EXTRA & INTRAHEPATIC MOSTLY INTRAHEPATIC Different mutation clusters within same histological subtypes Nakamura et al. Nature Genetics 2015 Jusakul et al. Cancer Discov 2017 22

ETHERBIL STUDY (NCT 02184871) ASBESTOS AND INTRAHEPATIC CHOLANGIOCARCINOMA: SEARCHING FOR MOLECULAR BIOMARKERS BY NEXT GENERATION SEQUENCING APPROACHES 45 patients planned / enrolled excluded from analysis 1 ADENOSQUAMOUS CA. 3 HCC/CC 2 HCC DISCOVERY PHASE 39 ICC (37 cases succesfully undergone WES analysis) (Sanger sequencing) Basing on CARA study, we expect that about 50% of ICC enrolled pts will be exposed to asbestos. Assuming that mutations of promising genes (such as BAP 1) would be mostly related to asbestos, we calculated that we will need to enroll in the discovery phase of the study a total of 92 ICC patients to demonstrate a statistically significant difference assuming an alpha error of 0. 05 (twosided test) and a beta error of VALIDATION PHASE 0. 20 (power 80%). 23

IDH 1 MUTATION AND ICC CARCINOGENESIS α-chetaglutarate 2 -hydroxyglutarate IDH-1 R 132 C IDH-1 R 132 H Saha et al. Cell cycle 2014 Valle et al. Cancer Discovery 2017 2 -HG: oncometabolite measurable (ng/ml) in the serum of patients Possible effect at distant organs (liver and others) 24

Conclusions v Distinct putative or established risk factors are linked to BTCs. v These risk factors are commonly different between ICC and ECC. v In Western populations more than 50% of BTCs are diagnosed without any known risk factor; v Epidemiological evidences strongly suggest a role of asbestos in ICC pathogenesis; v In our series over 50% of patients were exposed to asbestos according to the Re. Na. M; v In asbestos exposed ICC patients, the most recurrent mutated genes (by (WES analysis) were BAP-1 and IDH 1. . already considered driver genes; v BAP-1 and IDH 1 mutations were mutually exclusive and found exclusively in asbestos exposed patients, thus representing putative candidate biomarkers of asbestos exposure and deserves further investigation in a larger study population; v Being a BAP-1/IDH 1 impairing mitochondrial function it can be hypothesized that the co-existence of damaging mutations in both genes may strongly impair energy production thus leading to cell death and failing to become neoplastic. 25

26