Prof GIOVANNI BRANDI, MD, PhD UNIVERSITY OF BOLOGNA

1. 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, PhD UNIVERSITY OF BOLOGNA

2. 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 Cholangiocarcinoma. In “Cholangiocarcinoma!” NOVA Publ ; NY, 2015

3. IntrahepaticCholangiocarcinoma (ICC) and NAFLD/NASH * Patients with metastatic liver tumor Intrahepatic cholangiocarcinoma

4. elicotteristi edili tipografi meccanici militari chimici conciatori marinai palombari ferrovieri idraulici vetrai cementieri cantieristi insegnanti caldaisti odontotecnici carpentieri metallurgici AMIANTO & carrozzai tessili geologi orafi COLANGIOCARCINOMA

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

6. Asbestos and primary liver cancer COHORT STUDIES 1 [contd…] *Fegato= HCC+ICC

7. Asbestos and primary liver cancer COHORT STUDIES 2 *Fegato= HCC+ICC

8. 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)

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

10. ASBESTOS FIBERS IN BILE DUCTS AND GALLBLADDER ♀ 55 Yrs. (BUDAPEST) Szendröi M. et al. 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. 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. ♀ 73 Yrs.(MPM) ♀ 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 fiibers NO fibers

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

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

13. 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

14. 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.

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

16. 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

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

18. DISTRIBUTION OF MUTATIONS IN BTC IDENTIFIED BY NGS (3) FFPE bloks from 84 pts: NGS+qPCR Papadopoulou et al. AmJCancer Res 2018 N=55 N=15 N=14 >30% Pd1/Pdl1

19. NGS in ICC N=496 ICC 165 home (150 TES+15 WES) vs 262 WES public database Analysis 122/150 TES for FGFR-2 (Sanger) N=38 BTC ( 32 ICC) WES + Rna seq FGFR-2 + =15% 3p loss in all cases of bap1 mutation Nepal and Andersen. Hepatology 2018 Farshidfar et al. Cell 2017

20. Nepal and Andersen. Hepatology 2018 Poor prognosis

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

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

23. ETHERBIL STUDY (NCT02184871) 45 patients planned / enrolled 39 ICC (37 cases succesfully undergone WES analysis) ASBESTOS AND INTRAHEPATIC CHOLANGIOCARCINOMA: SEARCHING FOR MOLECULAR BIOMARKERS BY NEXT GENERATION SEQUENCING APPROACHES excluded from analysis 1 ADENOSQUAMOUS CA. 3 HCC/CC 2 HCC 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 (two-sided test) and a beta error of 0.20 (power 80%). DISCOVERY PHASE (Sanger sequencing) VALIDATION PHASE

24. IDH1 MUTATION AND ICC CARCINOGENESIS 2-hydroxyglutarate α-chetaglutarate IDH-1 R132C IDH-1 R132H 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)

25. Conclusions • Distinct putative or established risk factors are linked to BTCs. • These risk factors are commonly different between ICC and ECC. • In Western populations more than 50% of BTCs are diagnosed without any known risk factor; • Epidemiological evidences strongly suggest a role of asbestos in ICC pathogenesis; • In our series over 50% of patients were exposed to asbestos according to the ReNaM; • In asbestos exposed ICC patients, the most recurrent mutated genes (by (WES analysis) were BAP-1 and IDH1.. already considered driver genes; • BAP-1 and IDH1 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; • Being a BAP-1/IDH1 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.