1. 만성 C 형간염 치료 심재준 경희대학교병원 소화기내과 Kyung Hee Univ. School of Medicine Department of Internal Medicine Jae-Jun Shim MD, PhD 2016.3.3. 경희의료원 내과

2. Questions  왜 C 형간염이 중요한가 ?  국내 C 형 간염의 감염 상태 ( 역학 ) 는 어떠한가 ?  C 형간염바이러스 (HCV) 의 특징은 무엇인가 ?  현재 C 형간염의 치료는 어떻게 ?  향후 C 형간염 치료의 전망은 ?  현재 국내에서 사용 가능한 치료 약제는 ?

3. 왜 C 형간염이 중요한가 ?  유병률  의료기관 내 감염 : 사회적 이슈 ( 다나의원, 원주한양정형 외과의원, 전수감시체제 전환 등 )  만성 간질환의 주요한 원인  최신 치료제 등장

4. A Nationwide Seroepidemiology of Hepatitis C Virus Infection in South Korea Using an estimated 2009 population of Korea, the age, sex and area-adjusted anti-HCV positive rate was 0.78%. Kim DY, et al. Liver Int 2013

5. HCV infection, 10% of liver-related mortality (cirrhosis or liver cancers), in a single health care center  about 1,800 deaths/year in Korea JJ Shim, KASL 2014

6. Adapted from Chen SL, Morgan TR. Int J Med Sci. 2006;3:47-52. *20%-30% of individuals are symptomatic. Acute Infection* Chronic Infection 50%-80% Clearance of HCV RNA 20%-50% Extrahepatic M anifestations Cirrhosis 20%-25% ov er 20 years Hepatocellular Carcin oma (HCC) 1%-4% per year Decompensated Cirrhosis (DCC) 5-yr survival rate 50% Natural History of HCV Infection o Oder age o Longer duration of infection o Higher inflamm. grade and fibrosis stage o Alcohol, HBV, HIV o Obesity (NASH) o Iron overload

7. C 형간염바이러스 (HCV) 의 특징은 무엇인가 ?  RNA virus  Life cycle, replication mechanism 이 잘 밝혀졌다  완치가 가능

8. Hepatitis C virus □RNA virus □Half-life: a few hours □Production and clearance of an estimated 10 12 vir ions per day in a given individual. Nature Reviews Microbiology 2007

9. Lifecycle of hepatitis C virus (HCV)

10. Takaji Wakita Dr. Volker Lohmann Science 1999 Nature Med 2005  G1b HCV RNA  Huh7 HCC cell lines  HCV RNA replication system in a cell line  G2a HCV RNA  Huh7 HCC cell lines  HCV viral replication and excretion of viral particles 대박대박 대박대박

11. 11 22 44 55 33 Asunaprevir Sofosbuvir Ledipasvir Rosen HR. N Engl J Med 2011;364:2429

12. 현재 C 형간염의 치료는 어떻게 ?

13. Standard treatment Peg-interferon (Peg-IFN) + Ribavirin (RBV) Peg-IFN-α2b (Peg-intron ®,MSD) Peg-IFN-α2a (Pegasys ®,Roche)  Inhibit viral replication  Upregulate IFN stimulated genes  Immunomodulatory effects  Dose: weight-based [15 mg/kg #2 or 1,000 mg (<75 kg), 1200 mg (≥ 75 kg)] PO G1, 4, 5, 6 G2,3: obese (BMI>25), insulin resistance, metabolic syndrome, severe fibrosis (LC)  800 mg #2 PO (fixed dose) G2 or G3  1.5 μg/kg every 1 week (Wt based)  180 μg/kg every 1 week (fixed dose) 13/35

14. Direct-acting Antiviral Agents (DAAs) NS3 serine protease inhibitors Boceprevir, telaprevir (FDA 2011) Simeprevir (FDA 2013) Paritaprevir (ABT450)/r (FDA 2014) Asunaprevir (not FDA, only approved in Japan) NS5A inhibitors Daclatasvir (2015) Ledipasvir (2014) Ombitasvir (2014) Velpatasvir (in 2016) NS5B polymerase inhibitors Nucleoside/nucleotide analogue Sofosbuvir (FDA 2013) Non-nucleoside analogue Dasabuvir (ABT-333) (FDA 2014) Rosen HR. N Engl J Med 2011;364:2429 ~previr ~asvir ~buvir

15. Development of Tx strategy in the era of DAAs (Directly Acting Antiviral agents) 1 1 PegIFN + RBV + DAA 2 2 3 3 IFN-free (DAAs ± RBV) One pill (FDC)

16. Peg-IFN + RBV + PI (Boceprevir or telaprevir) SPRINT-2 1 Naïve G1 BOC, boceprevir (protease inhibitor, PI); TVR, telaprevir; PEG, peginterferon; RBV, ribavirin; All, both naïve and previously treated patients; RGT: response guided therapy ADVANCE 2 Naïve G1 ILLUMINATE 3 Naïve G1 1.N Engl J Med 2011;364:1195-206. 2.N Engl J Med 2011;364:2405-16. 3.N Engl J Med 2011;365:1014-24 SVR of control grp 44 40

17. Simeprevir + PEG-IFN + RBV CONCERTO-1 1 Naïve, Japan G1b SMV, simeprevir (NS3/4A inhibitor); PEG, peginterferon; RBV, ribavirin; RGT, response- guided therapy QUEST-1 2 Naïve G1 PROMISE 3 Relapse G1 1.Hayashi N. et al. J Hepatol 2014 Epub 2.Lancet 2014;384:403-13 3.Gastroenterology 2014;146:1669-1679 4.Lancet 2014; 384: 414–26. QUEST-2 4 Relapse G1 SVR of control grp 50 * No additional benefit in G1a, Q80K mutation (SVR 52-75% vs 50-53%) 62 50

18. Development of Tx strategy in the era of DAAs (Directly Acting Antiviral agents) 1 1 PegIFN + RBV + DAA 2 2 3 3 IFN-free (DAAs ± RBV) One pill (FDC)

19. DAAs 특징 NS3/4A PINS5A InhibitorNS5B NINS5B NNI Potency +++ ++ CoverageNarrow (G1)G1~6 Narrow Genetic barrierLow HighLow Drugs Boceprevir Telaprevir Simeprevir Paritaprevir Asunaprevir Grazoprevir Daclatasvir Ledipasvir Ombitasvir Velpatasvir Elbasvir Sofosbuvir Beclabuvir Dasabuvir PI, protease inhibitor; NI, nucleos(t)ide analogue inhibitor; NNI, non-nucleoside analogue inhibitor

20. Paritaprevir (ABT-450) /r/Ombitasvir + Dasabuvir ± RBV SAPPHIRE-I 1 Naïve, LC(-) G1 ABT, ABT-450 (NS3/4A inhibitor, paritaprevir); LC, liver cirrhosis; SOF, sofosbuvir (NS5B inhibitor); PEG, peginterferon; RBV, ribavirin; R, ritonavir; OBT, ombitasvir (NS5A inhibitor); DBV, dasabuvir (NS5B inhibitor) SAPPHIRE-II 2 Prior treated G1 PEARL-III 3 Naïve, LC(-) G1b 1.N Engl J Med 2014;370:1594-603. 2.N Engl J Med 2014;370:1604-14. 3.N Engl J Med 2014;370:1983-92. 4.N Engl J Med 2014;370:1973-82. PEARL-IV 3 Naïve, LC(-) G1a TURQUOISE-II 4 LC(+) G1 $83,320

21. Sofosbuvir + (PEG + RBV) NEUTRINO 1 Naïve G1, G4, G5, G6 SOF, sofosbuvir (NS5B inhibitor); PEG, peginterferon; RBV, ribavirin wt based (1000 mg 75kg); All, both naïve and previously treated patients FISSION 1 Naïve G2 POSITRON 2 PEG ineligible G2 VALENCE 3 All G3 1.N Engl J Med 2013;368:1878-87. 2.N Engl J Med 2013;368:1867-77. 3.N Engl J Med 2014;370:1993-2001. $84,000 \3,800,000

22. Sofosbuvir + Ledipasvir ION-1 1 Naïve G1 SOF, sofosbuvir (NS5B inhibitor); LDV, ledipasvir (NS5A inhibitor) ION-2 1 Previously Tx G1 ION-3 2 No cirrhosis G1 1.N Engl J Med 2014;370:1889-98. 2.N Engl J Med 2014;370:1483-93. 3.N Engl J Med 2014;370:1879-88. 4.N Engl J Med 2015;373:705-13 ION-4 4 HIV coinfected G1, G4 $94,000 \4,600,000

23. Simeprevir + Sofosbuvir COSMOS 1 Naïve or NR G1 SMV, simeprevir (NS3/4A inhibitor); SOF, sofosbuvir (NS5B inhibitor); RBV, ribavirin; NR, previously not responded COSMOS 1 Naïve, fibrosis(+) G1 1.Lancet 2014; 384: 1756–65 2.AASLD 2014 Abstract #46 TRIO-N 2 Naïve G1 Real life Exp 1 Any G1

24. Daclatasvir + asunaprevir ± beclabuvir HALLMARK DUAL 1 Naïve Non-response Ineligible G1b* DCV, daclatasvir (NS5A inhibitor); ASV, asunaprevir (NS3/4A inhibitor); BEC, beclabuvir (NS5B inhibitor); RBV, ribavirin 1.Lancet 2014;384:1597-1605 2.JAMA. 2015;313:1736-1744 * SVR in NS5A-Y93H (8%), L31M,V (5%) = 38%, 41%, respectively UNITY-2 2 Naïve Tx-experienced G1a or G1b Fixed-dose combination twice daily (DCV-TRIO)

25. Daclatasvir + sofosbuvir DCV, daclatasvir (NS5A inhibitor); SOF, sofosbuvir (NS5B inhibitor); RBV, ribavirin 1. N Engl J Med 2014;370:211-21. 2. N Engl J Med 2015;373:714-25 3. HEPATOLOGY 2015;61:1127-1135 AI444040 1 Naïve G1 AI444040 1 Naïve G2, G3 * Adding RBV or 24 weeks of therapy did not improve SVR rate. ALLY-2 2 HIV-1 and Naïve or previously treated G1,G2,G3,G4 ALLY-3 3 Naïve or previously treated G3, LC(-)** ** G3 with LC (+): very low SVR12 (63%)

26. Grazoprevir + Elbasvir GZVR, grazoprevir (NS3/4A protease inhibitor); EBVR, elbasvir (NS5A inhibitor) 1.Ann Intern Med. 2015;163:1-13 2.Lancet 2015; 386: 1537–45 C-EDGE 1 Naïve G1,G4,G6 Fixed-dose combination once daily C-SURFER 2 CKD (stage 4-5) G1

27. C 형간염 경구 칵테일 요법의 현재  인터페론 없이 90% 이상의 높은 완치율 (SVR) 을 보임.  두통, 피로감 외에 약물 부작용 거의 없음.  치료 전후 필요한 검사 유전자형 (G1a vs. G1b vs. G2 vs. G3 vs. others) 검사가 필수. RAV ( 돌연변이종 바이러스 ) 검사가 사전에 필요 (ASV+DCV 요법 ). 정기적인 간기능 검사 필요 (ASV+DCV 요법 ). 간경변 여부도 확인 필요.

28. 향후 C 형간염 치료의 전망은 ?

29. Development of Tx strategy in the era of DAAs (Directly Acting Antiviral agents) 1 1 PegIFN + RBV + DAA 2 2 3 3 IFN-free (DAAs ± RBV) One pill (FDC)

30. Test and Cure No genotyping, No imaging nor biopsy 1b, 2a 1b, 3a,2a,2b,2c 1a, 1b, 2a,2b,3a 3a, 1b 1b, 6 HEPATOLOGY 2015;61:77-87 Global prevalence of each HCV genotypes

31. Sofosbuvir + Velpatasvir ASTRAL-1 1 Any G1,2,4,5,6 SOF, sofosbuvir (NS5B inhibitor); VEL, velpatasvir (NS5A inhibitor) ASTRAL-2 2 Any G2 ASTRAL-3 2 Any G3 1.N Engl J Med. 2015;373:2599-607 2.N Engl J Med. 2015;373:2608-17 3.N Engl J Med. 2015;373:2618-28 ASTRAL-4 3 Decompensated LC G1,2,3,4,6 ** No RBV: SVR 83%, esp. in G3 with LC (+): 50%

32. Development of Tx strategy in the era of DAAs (Directly Acting Antiviral agents) 1 1 PegIFN + RBV + DAA 2 2 3 3 IFN-free (DAAs ± RBV) One pill (FDC)

33. 현재 국내에서 사용 가능한 치료 약제는 ?

34. Distribution of genotypes in Korea Genotype (N = 421) Genotype (n = 491) Others During 2007 ~ 2011, HCV infection (n=930) Current status of Rep. of Korea Seong et al. J Med Virol 2013;85:1724 G1b 86% (n = 422) G2a/c 63.2% (n = 266)

35. Daclatasvir NS5A G1b Daclatasvir NS5A G1b DAAs approved in Korea (2015~) Asunaprevir NS3A PI G1b Asunaprevir NS3A PI G1b Sofosbuvir NS5B G1,2,3,4 Sofosbuvir NS5B G1,2,3,4 Ledipasvir NS5A G1 Ledipasvir NS5A G1 Sofosbuvir NS5B G1,2,3,4 Sofosbuvir NS5B G1,2,3,4 (Product information_ http://drug.mfds.go.kr, S.Korea) http://drug.mfds.go.kr Harvoni® Sovaldi® Daklinza® Sunvepra®

36. a HCV RNA < LLOQ (25 IU/mL) TD or TND; responder/treated; b Patients with baseline sequence data a nd excluding non-virologic failures. 13% (16/125) and 0% (0/125) of patients had key NS5A and NS3 RAVs at baseline, respectively Higher SVR rates seen in patients without key baseline NS5A RAVs Y93 or L31 (92%) Korean and Taiwanese patients combined (N = 138) < 65≥ 65MaleFemale<800K≥800K AgeGenderHCV RNA 87 82 AbsentPresent Cirrhosis 77 89 40 49 83 100 34 38 53 61 64 77 25 26 92 112 96 WithWithout Key NS5A RAV 1,b (Y93H or L31F/I/M/V) 38 92 6 16 99 108 SVR12, % a Asunaprevir (ASV) + daclatasvir (DCV) for 24 weeks Virologic Response by Baseline Factors – RAV (Resistance-Associated Variants) Kao JH, et al. APASL 2015. Oral 1424.

37. Safety Profile by Ethnicity Risk of acute hepatic injury Parameter Korean N = 65 Taiwanese N = 71 Total Global N = 645 Deaths000 SAEs a 5 (7.7)6 (8.5)39 (6.0) AEs leading to discontinuation b 0 (0)2 (2.8)10 (1.6) Common AEs (≥10% overall) Headache Fatigue Diarrhea Nausea 7 (10.8) 6 (9.2) 5 (7.7) 2 (3.1) 12 (16.9) 16 (22.5) 20 (28.2) 1 (1.4) 159 (24.7) 140 (21.7) 103 (16.0) 75 (11.6) Grade 3/4 laboratory abnormalities Lymphocytes (< 0.5 × 10 ⁹ cells/L) Platelet count (< 50 × 10 ⁹ cells/L) ALT (≥ 5xULN) AST (≥ 5xULN) Total bilirubin (≥ 2.5xULN) Lipase (≥ 3.1xULN) 3 (4.6) 2 (3.1) 0 1 (1.4) 2 (2.8) 0 1 (1.4) 8 (1.2) 11 (1.7) 15 (2.3) 12 (1.9) 3 (0.5) 18 (2.8) a All SAEs were reported in 1 patient each with the exception of hepatocellular carcinoma which occurred in 2 patients; b Discontinuation due to adverse events occurred in 2 patients, both Taiwanese (ALT increase and constipation/bronchiectasis; AEs resolved following discontinuation and both patients achieved SVR12). Kao JH, et al. APASL 2015. Oral 1424.

38. Sofosbuvir + RBV in Asian G2 Overall 1 Any G2 SOF, sofosbuvir (NS5B inhibitor); RBV, ribavirin, wt based Naïve 1 Any G2 Tx-Exp 1 Any G2 1.EASL 2015 April 22-26 Vienna Austria Asian Patients with Genotype 2 HCV Infection Receiving 12 W eeks of Sofosbuvir Plus Ribavirin: Results From an Internation al, Multicenter Phase 3 Study

39. 유전자 2 형의 치료 G2: SOF+RBV, 12w  초치료, 간경변 여부 상관 없이  과거 치료 실패 여부 상관 없이 12 주 Sofosbuvir 1T (400 mg) qd With or without food Ribavirin <75kg=1000 mg #2 ≥75kg=1200 mg #2 With food 98%

40. 유전자 1b 형의 치료 G1b: ASV+DCV for 24w; SOF/LDV for 12w Asunaprevir NS3A PI G1b Asunaprevir NS3A PI G1b Daklinza®Sunvepra® Harvoni® Daclatasvir NS5A G1b Sofosbuvir / Ledipasvir NS5B/NS5A inhibitor G1,2,3,4/G1 ASV 1 Cap (100 mg) every 12 hr DSV 1T (60 mg) qd With or without food Resistant-Associated Variant assay RAV (Y93, L31) Acute hepatic injury (ASV) LFT F/U every 2w CIx: decompensated LC 24 주 90% SOF/LDV 1T (400/90mg) qd With or without food Naïve with or without cirrhosis (LC) = 12w Naïve, HCV<6,000,000 IU/mL, no LC = 8w Tx experienced without LC = 12w Tx experienced with LC = 24w 12 주 96%

41. In the future One pill one day –Irrespective of genotype (no need of genotyping) –Shorter treatment course (2~3 months) –Fewer adverse events (no need of specialists) Jayasekera CR N Engl J Med 2014 Apr

42. 만성 C 형간염의 치료

43. Application of Gene Expression Data Analysis in Liver Diseases Jae-Jun Shim, MD, PhD Kyung Hee University Hospital Department of Internal Medicine 2016.3.3.

57. Cancer is a genetic disease □ Oncogenes RAS, WNT, MYC, ERK BCR-ABL DNAJB1-PRKACA: fibrolamella HCC IDH1: hydroxyglutarate  DNA hypermet hylation □Tumor suppressor genes P53, Rb, APC, BRCA1,-2, PTEN

58. Gene expression analysis Southern blotting Quantitative RT-PCR method Microarray RNA Seq (NGS)

59. Microarray

60. Fudan data (HCC, n = 228)

62. 수 십 ~ 수 백 개의 샘플 2 ~ 3 만개의 Gene expressions

63. Pattern recognition

64. Clustering analysis Expression data  clustering visualization (Heat map)

65. Unsupervised analysis 91 HCC tumors (NCI) HEPATOLOGY 2004;40:667– 676

66. HCC survival gene set (n=406)

67. Nature Med 2006

69. Gene signatures in HCC Signature (uniq genes) CategoryMethodsRef NCI-P (406) Prediction of general prognos is (OS or PFS) Unsupervised clustering of NCI-HCC cohort Original cohort, selected genes associated with survival Lee, et al. 2004, Hepatology HS (907) Stem cell signature Comparing Rat hepatoblasts, Transgenic mouse HCCs, and Human HCCs Lee, et al. 2006, Nature Me d SNU-R (628) Cancer recurrence Original cohort, selected genes associated with early recurr ence(<1year) Woo, et al. 2008, Clin Cancer Res BC Beta-catennin activation SOH (610) Silence of Hippo path. Transgenic mouse models (Mst1/2-/- and Sav1-/-)Sohn, Shim et al. 2015, Clin Cancer Res HIR (233) Hepatic injury and regenerati on signature Serial biopsy before and after hepatectomy, LTKim, Sohn et al. 2014, Plos Med IR Immune response VA_fib Fibrosis Liver biopsy in patients with chronic hepatitis C CC-like Cholangiocarcinoma-like TCGA data

70. Inactivation of Hippo pathway is significantly associated with poor prognosis in hepatocellular carcinoma Bo Hwa Sohn 1,2+, Shim Jae-Jun 1,2, Sang-Bae Kim 1,2+, Kyu Yun Jang 3, Soo Mi Kim 4, Ji Hoon Kim 5,, Hwang Jun Eul 1,2, Hee-Jin Jang 1,2, Hyun-Sung Lee 1,2, Sang-Cheol Kim 6, Woojin Jeong 7, Sung Soo Kim 8, Eun Sung Park 9, Jeonghoon Heo 10, Yoon Jun Kim 11, Dae-Ghon Kim 12, Sun- Hee Leem 13, Ahmed Kaseb 14, In-Sun Chu 15, Randy L. Johnson 16, Yun-Yong Park 17 *, and Ju- Seog Lee 1,2,8 * 1 Department of Systems Biology, 2 Kleberg Center for Molecular Markers, 14 Department of Gastrointestinal Medical Oncology, and 16 Department of Biochemistry and Molecular Biology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 3 Department of Pathology, 4 Department of Physiology, and 12 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chonbuk National University Medical School and Hospital, Jeonju, Korea. 5 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Korea University College of Medicine, Seoul, Korea. 6 Samsung Genome Institute, Samsung Medical Center, Gangnam-Gu, Seoul, Korea. 7 Department of Life Sciences, Division of Life and Pharmaceutical Sciences, Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, Korea. 8 Department of Biochemistry and Molecular Biology, Medical Research Center and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Korea. 9 Institute for Medical Convergence, Yonsei University College of Medicine, Seoul, South Korea. 10 Departments of Molecular Biology and Immunology, Kosin University College of Medicine, Busan, Korea. 11 Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea. 13 Department of Biological Science, Dong-A University, Busan, Korea. 15 Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. 17 ASAN Institute for Life Sciences, ASAN Medical Center, Department of Medicine, University of Ulsan College of Medicine, Seoul, Korea

71. The Hippo pathway is a tumor suppressor in the liver, as demonstrated by the development of hepatocellular carcinoma (HCC) after the pathway is inactivated in the livers of mice. However, the clinical significance of Hippo pathway inactivation in HCC is currently unknown. In current study, we aim to assess the clinical significance of inactivation of Hippo pathway in HCC. Background & Aims

72. The Hippo Pathway Genes & Dev. 2010. 24: 862-874

73. We analyzed gene expression data from Mst1/2-/- and Sav1-/- mice and identified a 610-gene expression signature that reflected Hippo pathway inactivation in the liver (silence of Hippo [SOH] signature). By integrating gene expression data from mouse models with those from human HCC tissues, we developed a prediction model that could identify HCC patients with an inactivated Hippo pathway and used it to test its significance in four independent cohorts of HCC patients (n = 546), via univariate and multi-variate Cox analyses. (  Fig. 1) Methods

74. Figure 1. Gene expression patterns that were significantly associated with the silence of Hippo pathway in the mouse liver. (A)Venn diagram of genes selected using a two-sample t-test with a multivariate permutation test (10,000 random permutations). The blue circle (gene list X) represents genes that were differentially expressed between normal livers from wild-type (WT) mice and non-tumor tissues from mutant (Sav1 -/- and Mst1/2 -/- ) mice (ST). The red circle (gene list Y) represents genes that were differentially expressed between all non- tumor liver tissues and all hepatocellular carcinoma tissues from mutant (Sav1 -/- and Mst1/2 -/- ) mice. A stringent cut-off (P < 0.001 and 2-fold difference) was used to retain only the genes whose expression was significantly different between the two groups of tissues examined. The 737 probes (610 unique genes) in the X not Y category displayed unique expression patterns that are significantly associated with the silence of Hippo pathway. (B)Expression patterns of selected genes in the Venn diagram. Colored bars at the top of the heat map represent the tissues indicated.

75. Table 1. Baseline clinical and pathological features of HCC patients Variable NCI cohort Korean cohort FULCI cohort MSH cohort Number of Patients 113100 24291 male81 (72%)83 (83%) 211 (87%)27 (30%) female32 (28%)17 (17%) 31 (13%)54 (59%) NA 10 (11%) + AgeMedian5655 5068 range26-8525-70 21-7742-80 AFP* (>300 ng/ml)+51 (45%)32 (32%) 110 (45%)15 (17%) -53 (47%)68 (68%) 128 (53%)54 (59%) NA9 (8%)0 4 (2%)22 (24%) HBV # + (%)54 (48%)58 (58%) 218 (90%) - (%)59 (52 %)17 (17 %) 6 (2.5%) NA 25 (25%) 18 (7.5%)91 (100%) AJCC StageI 35 (35%) 96 (40%) II 17 (17%) 78 (32%) III 48 (48%) 51 (21%) IV 0 (0%) NA113 (100%) 17 (7%)91 (100%) *Alpha-fetoprotein. # Hepatitis B virus + Patient Sex information is not available for patient who received liver transplantation NA, not applicable Variable NCI cohort Korean cohort FULCI cohort MSH cohort BCLC Stage0 0 (0%) 20 (8%) A 53 (53%) 152 (63%) B 37 (37%) 24 (10%) C 6 (6%) 29 (12%) D 4 (4%) 0 (0%) NA113 (100%) 17 (7%)91 (100%) Death 7143 96 Median Follow-up (Month) 23.548.5 51.6

76. Figure 2. Significant association between the silence of Hippo (SOH) signature and prognosis in patients with hepatocellular carcinoma. (A)Schematic overview of the strategy used to predict the probability that SOH was present in human HCC on the basis of gene expression signatures. KO, knockout; WT, wild type; BCCP, Bayesian compound covariate predictor. (B)Receiver operating characteristic curve analysis of SOH probability over 3-year recurrence of HCC in the National Cancer Institute cohort. (C) (D) Kaplan-Meier plots of recurrence-free survival and overall survival of SOH and AH subtype patients in the National Cancer Institute cohort. P values were obtained from the log-rank test. The + symbols in the panel indicate censored data.

77. Figure 3. Validation of robustness of silence of Hippo signature in independent cohorts. (A, B) Kaplan-Meier plots of recurrence-free survival and overall survival of patients with the silence of Hippo and active Hippo subtypes in the Korean cohort. (C, D) Kaplan-Meier plots of recurrence-free survival and overall survival of the two subtypes of patients in the Fundan University Liver Cancer Institute cohort. P values were obtained from the log-rank test. The + symbols in the panel indicate censored data.

78. Table 2. Univariate and Multivariate Cox Regression Analyses of Recurrence Free Survival. UnivariateMultivariate Hazard Ratio (95% CI)P-valueHazard Ratio (95% CI)P-value Patient Sex (M or F) 1.4 (0.89 – 2.18)0.141.22 (0.78 – 1.93)0.37 Age (>60 years) 0.88 (0.62 - 1.25)0.490.88 (0.61 – 1.29)0.52 AFP (>300 ng/ml) 1.25 (0.94 – 1.67)0.121.01 (0.75 – 1.38)0.9 Cirrhosis (Yes or No) 1.08 (0.72 – 1.64)0.681.1 (0.71 – 1.68)0.67 Tumor size (>5 cm) 1.34 (1.0 – 1.79)0.041.11 (0.81 – 1.54)0.5 BCLC stage (0,A,B,C,D) 1.37 (1.16 – 1.7)1.3 x 10 -4 1.28 (1.08 – 1.53)0.003 SOH signature (SOH or AH) 1.83 (1.31 –2.5)3.2 x 10 -4 1.6 (1.12– 2.28)0.008 CI, confidence interval AFP, alpha-fetoprotein BCLC, Barcelona Clinic Liver Cancer stage SOH, silence of Hippo pathway AH, active Hippo pathway

79. Inactivation of the Hippo pathway in HCC is significantly associated with poor prognosis. Conclusion