Molecular characterization of NASH-HCC

1. Molecular characterization of NASH-HCC Josep M Llovet, MD, PhD Professor of Medicine. Director, Liver Cancer Program. ISM at Mount Sinai, NYC Professor of Research-ICREA. Head, Translational research in hepatic oncology. Liver Unit. Hospital Clínic Barcelona.

2. Molecular characterization of NASH-HCC • Epidemiology • Molecular pathogenesis: from NAFLD to HCC 3. Molecular characterization of NASH-HCC a) Molecular subclasses and pathways b) Are there unique drivers? c) Specific genotoxicity or cancer field effect?

3. Incidence and mortality of HCC • Epidemiology of liver Cancer • Liver cancer is the sixth most common cancer globally and the fourth leading cause of cancer-related mortality. • Over 850,000 new cases of liver cancer (2018), around 1M (2025) • Eastern Asia: 570,000 • Europe: 68,000 • United States: 32,000 • Liver cancer is the leading cause of death in cirrhotic patients • The incidence of HCC is increasing globally IARC, https://gco.iarc.fr/today/home WHO, http://www.who.int/healthinfo/global_burden_disease/projections/en/ Villanueva A, NEJM 2019

4. Incidence of HCC Incidence rates of HCC according to geographical area. Llovet JM et al. Nat Rev Dis Primers 2016

5. Incidence and riskfactors-HCC Incidence rates of HCC according to geographical area. 2016: 850K cases 2025: >1M cases HBV Alcohol AB1 NASH HCV Llovet JM et al. Nat Rev Dis Primers 2016

6. Global NAFLD prevalence Younossi Z et al, NatRevGastroenterolHepatol, 2018 • Summary: • Global prevalence of NAFLD: 25% of global population (highestMiddle East) • Estimated NAFLD population in the US by 2030: 100 million • PNPLA3 SNPsis more prevalent in Hispanics

7. Risk of HCC in NAFLD Kanwal F et al, Gastroenterology 2018 • Summary: • Risk of HCC: HR: 7.6 NAFLD vs controls (Highestrisk: NASH-Cirrhosis in hispanics) • 20% of NAFLD-HCC are non cirrhotics

8. Risk of HCC in NAFLD NASH-HCC withcirrhosis: 70-80% NASH-HCC without cirrhosis: 20-30% D’Avola D et al, ClinLivDis 2016

9. Molecular characterization of NASH-HCC • Epidemiology • Molecular pathogenesis: from NAFLD to HCC 3. Molecular characterization of NASH-HCC a) Molecular subclasses and pathways b) Are there unique drivers? c) Specific genotoxicity or cancer field effect?

10. Molecular pathogenesis From NAFLD to HCC Oxidative stress Hepatic substrate overload Chronic liver damage Chronic inflammation Anstee Q, Nat Rev Gastro & Hepatol 2019

11. Molecular pathogenesis Key molecular factors 3) Inflammatory processes 1) Metabolic processes 4) Activation of HSC, autophagy, cell death 2) Oxidative stress Anstee Q, Nat Rev Gastro & Hepatol 2019

12. Molecular pathogenesis Role of PNPLA3 Prevalence of PNPLA3 SNP* General population NAFLD NASH NASH-HCC 20-30% 30-50% 50% 60% NASH-HCC Meta-analysis : 2,937 patients PNPLA3: associatedwithrisk of HCC in NASH/ alcohol cirrosis: HR 1.67 (95% CI 1.27-2.21) Singal A, Am J Gastroenterol 2014 Mann J, Nat Rev Gastro & Hepatol 2017; Sookolian et al., Hepatology, 2011; Rotman et al., Hepatology, 2010 • * MOA PNPLA3: enzyme metabolites liver tryglicerides. • PNPLA3- SNP variant rs738409:accumulation of lipid droplets in the hepatocytes due to lack of ubiquitylation and of proteasomal degradation.

13. Targets and therapies Targets for therapies and ongoing clinical trials for NASH • Ongoing phase III trials • Targets for molecular therapies • Management recommendations: • Control MetabolicSd • Phase III improvement of fibrosis/NASH- positive: Pioglitazone , Obeticholicacid (FXR agonist), • Phase II ongoing: Cenicriviroc (CCR2/5 inh), elafibranor (PARP inh) Friedman SL, Nature Medicine 2018

14. Molecular characterization of NASH-HCC • Epidemiology • Molecular pathogenesis: from NAFLD to HCC 3. Molecular characterization of NASH-HCC a) Molecular subclasses and pathways b) Are there unique drivers? c) Specific genotoxicity or cancer field effect?

15. Molecular events -hepatocarcinogenesis Llovet, J. M. et al, Nat. Rev. Dis. Primers 2016

16. Molecular targets for HCC Signaling pathways: molecular targets for new therapies. • Signalingpathways (mut): • Telomerasemaintenance: 60% • Cellcycle gene: 49% • Wnt-B-Catenin: 54% • Epigeneticmodifier: 32% • Akt/mTOR: 51% • MAPK: 43% • Signalingpathways (other): • NOTCH: 30% • TGF-Beta: 17% • MET: 50% • IGF Signaling : 15% (IGF2 epi-driver) Schulze K, Nat Genetics 2015 Villanueva, Gastrotenterology 2012; Coulouarn et al, Hepatology 2008

17. Molecular classification of HCC Llovet JM et al, NatRev Clin Oncol 2018

18. Signaling-Pathways in NASH-HCC tumors NASH-HCC patients Expressionarray Tumors (n=53) • Summary: • 50% of NASH-HCC belong to the proliferative subclass whereas 50% belong to the non-proliferative subclass • Molecular classes are similar than in Hepatitis –HCC, but increase of S1-TGFB Torrecilla S et al, ILCA-Abstract #9

19. Molecular characterization of NASH-HCC • Epidemiology • Molecular pathogenesis: from NAFLD to HCC 3. Molecular characterization of NASH-HCC a) Molecular subclasses and pathways b) Are there unique drivers? c) Specific genotoxicity or cancer field effect?

20. Landscape of mutations in HCC • Genomesequencing in HCC (n=250) 35-40 Undruggablemutations Schulze K et al, Nat Genetics 2015 Vogelstein et al, Science 2013

21. Lanscape of mutations and actionable drivers in HCC Meta-analysis of 928 cases: 25% potential actionable targets!

22. Landscape of mutations in NASH-HCC Driver mutated genes in NASH-HCC (n=50) • 4 driver genes were altered in ≥10% of NASH-HCC cases • ACVR2A was mutated in 10% of NASH-HCC • Median number of 3 mutated driver genes per sample (ranging from 0-10) Torrecilla S et al, ILCA-Abstract #9

23. Landscape of mutations in HCC Torrecilla S et al, ILCA-Abstract #9

24. Molecular characterization of NASH-HCC • Epidemiology • Molecular pathogenesis: from NAFLD to HCC 3. Molecular characterization of NASH-HCC a) Molecular subclasses and pathways b) Are there unique drivers? c) Specific genotoxicity or cancer field effect?

25. Mutational signatures in HCC Dhanasekaran R et al, Gastroenterology 2019

26. Mutational signatures Prevalence of mutational signatures in NASH-HCC Torrecilla S et al, ILCA-Abstract #9

27. Mutational signatures Signature 3- significantly enriched in NASH-HCC * In-house cohort and Public Dataset (ICGC) Torrecilla S et al, ILCA-Abstract #9

28. Mutational signatures Signature 3- associated with alterations in DNA repair Ma J, et al. Nat Communications 2018

29. Cancer field effect Cancer field in HCC - predicts survival after resection Training set (n=82) p<0.01 Gene signature-poor prognosis: # 186 genes Gene Set Enrichment : 1. Inflammation 2. NF-KB signaling 3. interferon-related immune response 4. Oxidative stress and 5. Proliferative signals(IL6, EGF) Hoshida et al, NEJM 2008

30. Cancer field effect Immune mediated- cancer field predicts 1st HCC occurrence Immune-mediatedcancerfield- poor prognosis: # 172 genes Moeini A et al , Gastroenterology 2019 (in press)

31. Cancer field effect Immune mediated- cancer field NASH vs NASH-HCC Torrecilla S et al, ILCA-Abstract #9

32. Cancer field effect Immune mediated- cancer field in NASH vs HBV/HCV * In-house cohorts Torrecilla S et al, ILCA-Abstract #9

33. Conclusions • The global epidemic of obesity is associated with an increase in the incidence of NASH-HCC • Molecular traits of NASH-HCC, as opposed to hepatitis-related HCC, have been seldomly reported • NASH tumors –compared to non-NASH HCC tumors- present: • Similar molecular classes • Distinct mutational profile • a) Higher prevalence of ACVR2Amutations (10% vs 2%) • b) Lower of TP53mutations (18% vs 32%) • c) Three driver genes exclusively mutated in NASH-HCC (AMER1, E2F3, MOB3B) • d) Higher frequency of Mutational Signature 3 as their main genotoxic-related signature • Distinct cancer field effect traits : enrichment of Immunossuppressed Cancer-Field • More studies are needed to confirm these results & decipher the unique traits involved in the development of NASH-HCC