1. Early stage HCC management

2. BCLC Staging and Treatment Schedule Adapted from Llovet JM et al. J Natl Cancer Inst 2008;100: 698 – 711 HCC Stage 0 PST 0, Child-Pugh A Stage A-C PST 0-2, Child-Pugh A-B Stage D PST>2, Child-Pugh C Early stage (A) Single or 3 nodules < 3 cm, PS 0 Intermediate stage (B) Multinodular, Ps 0 Advanced stage (C) Portal invasion, N1, M1, PS 1-2 Terminal stage (D) Very early stage (O) Single < 2 cm Carcinoma in situ Single 3 modules  3 cm Portal pressure/bilirubin Normal No Yes Associated diseases Increased Resection Liver Transplantation (CLT/LDLT) PEI/RF ChemoembolizationSorafenib Curative Treatments (30%) 5-yr survival: 50-70% Randomized controlled trials (50%) 3 yr survival: 20-40% Symptomatic ttc (20%) 1 yr survival: 10-20% ttc: treatment

3. Levels of evidence in the assessment of benefits in HCC treatment Llovet JM et al J Natl Cancer Inst 2008;100: 698 – 711 Treatments assessedBenefitEvidence Surgical treatments Surgical resection Adjuvant therapies Liver transplantation Neoadjuvant therapies Increased survival Uncertain Increased survival Treatment response 3iiA 1iiA 3iiA 2iiDiii Locoregional treatments Percutaneous ablation Ethanol injection Radiofrequency ablation Chemoembolization Arterial chemotherapy Internal radiation (I131, Y90) Increased survival Better local control Increased survival Treatment response 3iiA 1iiD 1iiA 3iiDiii Systemic treatments Sorafenib Tamoxifen Systemic chemotherapy Interferon Increased survival No benefit 1iA 1iiA Classification of evidence adapted from the National Cancer Institute (from Llovet JM, et al. J Natl Cancer Inst 2008;100:698-711) Level 1 = Randomized, controlled trial, meta-analysis (double-blinded, 1i; non-blinded, 1ii) Level 2 = Non-randomized controlled trial Level 3 = Case series (population-based, 3i; non-population-based, consecutive; 3ii; non-population-based, non-consecutive, 3iii) Endpoints: A = Survival, B = Cause-specific mortality, C = quality of life, D = indirect surrogates (DFS, PFS, tumor response)

4. Surgical resection

5. Surgical Resection Optimal candidates: BCLC stage 0 or A –Child-Pugh A –Performance status 0 –Single tumors (< 3 cm) –Normal portal pressure –Normal bilirubin Excellent functional reserve 5-year survival 60-70% High recurrence rate 50% at 3 years 70% at 5 years Bruix J et al. J Hepatol 2001; 35: 421-430; Llovet JM. J Gastroenterol 2005; 40: 225-235

6. Resection in Child A Patients offers good survival Poon RT et al. Ann Surg 2002; 235(3): 373-82. 70%

7. Hazard ratio 95% CI Microscopic vascular invasion 2.361.62 – 3.45 Serum AFP value ≥ 32 ng/ml 1.831.25 – 2.68 Non anatomical resection 1.651.13 – 2.40 Factors contributing to early phase (<2 years) recurrence Risk Factors Contributing to HCC Early Phase Intrahepatic Recurrence after Hepatectomy Imamura H et al. J Hepatol 2003; 38: 200-207

8. Yamamoto M et al. Ann Surg. 2004; 239(3): 395-9 The survival rate of patients with early HCC undergoing Liver Resection decreases 5 years after surgery. This phenomenon is explained by occurrence of second primary HCCs that should be prevented Early HCC Small advanced HCC Is resection only a palliation? 186 patients with HCC  2 cm treated with curative hepatectomy

9. Liver transplantation

10. Liver Transplantation Illustration Copyright © 2007 Nucleus Medical Art, All rights reserved. www.nucleusinc.com. 5-year survival 70% Recurrence rate < 15% Bruix J, Sherman M. Hepatology 2005; 42: 1208-1236; Llovet JM. J Gastroenterol 2005; 40: 225-235; Mazzaferro V et al. N Engl J Med 1996; 334: 693-699 Optimal candidates: BCLC Stage A disease No vascular invasion No metastases Fulfill the Milan criteria –Solitary tumor < 5 cm or –≤ 3 nodules < 3 cm AdvantageRemoval of the diseased liver together with the tumor DisadvantageLong waiting lists

11. Mazzaferro V et al. N Engl J Med. 1996; 334(11): 693-9 MILAN Criteria Unresectable HCC single nodule <5cm or <3nodules <3cm No vascular invasion or node mets

12. Overall Survival % Mazzaferro V et al. N Engl J Med. 1996; 334(11): 693-9 Survival of patients with single HCC < 5 cm or 3 < 3 cm (n= 48) 0 20 40 60 80 100 012243648 months 75% Liver Transplantation

13. …The 5-year survival of liver transplantation for HCC has improved with time (1987-2001). It is possible that the published criteria for patient selection may have contributed to the better outcome. Yoo HY et al. J Clin Oncol. 2003; 21(23): 4329-35 Liver Transplantation

14. Non surgical treatments

15. Non Surgical Treatments: Percutaneous Ablation Radiofrequency ablation (RFA) Percutaneous ethanol injection (PEI) Optimal candidates: Child-Pugh A Single tumors < 3 cm in diameter Llovet JM. J Gastroenterol. 2005; 40(3): 225-35; Bruix J et al. Hepatology 2005; 42(5): 1208-36; Bruix J et al. J Hepatol. 2001; 35(3): 421-30 Illustration Copyright © 2007 Nucleus Medical Art, All rights reserved. www.nucleusinc.com.

16. Non Surgical Treatments: Percutaneous Ablation Radiofrequency ablation (RFA) Percutaneous ethanol injection (PEI) Optimal candidates: Child-Pugh A Single tumors < 3 cm in diameter PEI 5-year survival 40-50% High recurrence rate 50% at 3 years 70% at 5 years Llovet JM. J Gastroenterol. 2005; 40(3): 225-35; Bruix J et al. Hepatology 2005; 42(5): 1208-36; Bruix J et al. J Hepatol. 2001; 35(3): 421-30

17. Recurrence rates

18. Recurrence of HCC after curative treatment PEI/RFA Liver transplantation Resection Very early stage (0) Single <2 cm carcinoma in situ Early stage (A) 1–3 nodules <3 cm, PS 0 Single 3 nodules ≤3 cm Portal pressure/bilirubin Increased Associated diseases Normal NoYes Potentially curative treatment 5-year recurrence Possible causes contributing to recurrence HCC stage >70% <15% Proliferation of residual microscopic disease Neovascularization Mazzaferro V et al. N Engl J Med 1996;334:693–9; Zavaglia C et al. Am J Gastroenterol 2005;100:2708–16; Cherqui D et al. Ann Surg 2009;250:738–46; Imamura H et al. J Hepatol 2003;38:200–07; Forner A & Bruix J. Hepatology 2008;44:5–7; Qin LX & Tang ZY. Curr Cancer Ther Rev 2005;1:71–80; Poon R et al. J Clin Oncol 2002;20:1775–85

19. Poon et al, 2002 Llovet et al, 1999 After resection After ablation Shiina et al, 2005 100 60 40 20 0 80 0122432604872 Probability (%) Months Magnitude of the problem: the unmet need of prevention of recurrence Llovet J et al. Hepatology 1999;29:62–7; Poon R et al. Ann Surg 2002;235:373–82; Lencioni R et al. Radiology 2005;234:961–7; Shiina S et al. Gastroenterology 2005; 129:122–30 Lencioni et al, 2005 Months

20. Predictors of EARLY recurrencePredictors of LATE recurrence Microscopic vascular invasion Serum AFP value ≥32 ng/mL Non-anatomical resection Grade of hepatitis activity Aetiology of hepatitis Age Patterns of HCC recurrence Imamura H et al. J Hepatol 2003;38: 200–07; Mazzaferro V et al. Hepatology 2006;44:1543–54; Cucchetti A et al. Ann Surg Oncol 2009;16:413–22

21. Patients who developed recurrencePatients who did not develop recurrence  AFP-expressing tumour cells are disseminated mostly post-operatively  This may potentially be the source of recurrence or metastasis Haematogenous dissemination of tumour cells after resection of HCC Wong GL et al. Clin Cancer Res 1999;5:4021–7

22.  Micrometastases could spread via invasion of portal vein branches at an early stage even when the tumour is solitary and small Micrometastases/microsatellites could spread via invasion of portal vein branches at an early stage Sasaki A et al. Cancer 2005;103:299–306; Shi M et al. World J Surg 2004;28:376–81 Size of main tumour (mm) Distance of microsatellite (mm) 50 01020304060 40 30 20 10 –10 N=100 y = –0.344 + 0.24x r 2 = 0.084 P<0.001 Distance of spread (cm) Number of micrometastases (n) 80 0.00 Micrometastases in proximal area 70 60 40 20 0 50 30 10 0.250.50 0.751.001.25 1.501.752.002.252.50 2.75 3.00 3.25 3.50 Micrometastases in distal area 0 50

23. LOW RISK <2 risk factors HIGH RISK ≥2 risk factors  Risk factors for and incidence of late recurrence after surgery overlap with those associated with HCC first occurrence in cirrhosis (Resected patients) (HCC occurrence) Late recurrence of HCC after surgery Cucchetti A et al. Ann Surg Oncol 2009;16:413–22

24. Strong predictors of HCC recurrence after curative resection: Microvascular invasion Grade of differentiation Microsatellites Microvascular invasion Grade of differentation Microsatellites Prognostic factors associated with risk of recurrence Lauwers GY et al (The International Cooperative Study Group on Hepatocellular Carcinoma). Am J Surg Pathol 2002;26:25–34; Bruix J & Sherman M. Hepatology 2005;42:1208–36 Nuclear grade 1 Nuclear grade 2 Nuclear grade 3

25. Incidence of mVI and G3 tumours are parallel and increase significantly with size-and-number features of HCC 1083 pts Mazzaferro V et al. Lancet Oncol 2009;10:35–43 Morphology: pathology correlation The metroticket experience: 1556 HCCs studied with explant pathology 17%

26. Microvascular invasion and outcome Roayaie S et al. Gastroenterology 2009;137:850–5 The degree of mVI predicts outcome after resection and could be useful to select patients for salvage transplant or to enrol patients in trials evaluating new molecular targeted therapies Immunoreactivity for anti-smooth muscle actin antibody to asses presence of muscle in the wall

27. Can mVI invasion be predicted by imaging? Kim H et al. Eur Radiol 2009;19:1744–51 NMR findings of circumferential peritumoural enhancement showed statistical correlation with microscopic vascular invasion Wedge-shaped peritumoural enhancement is triangular enhancement with the base headed way from the tumour Irregular circumferential peritumoural enhancement: (polygonal shape parallel to the tumour border)

28. S1 tumours exhibited more vascular invasion and satellite lesion These results may suggest that the S1 subclass is associated with a more invasive/disseminative phenotype Integrative transcriptome analysis reveals common molecular subclasses of human HCC Hoshida Y et al. Cancer Res 2009;69:7385–92

29. A reproducible gene signature correlated with survival in liver tissue adjacent to the tumour Molecular markers of late recurrence Hoshida Y et al. N Engl J Med 2008 359:1995–2004 A Expression pattern of 186 gene-survival- signature B OS according to the level of expression of the 186 genes among 225 tissue validation samples C OS according to the level of expression of the 186 genes among 168 pts with longer duration of follow-up D Probability of late-recurrence according to the expression of the late- recurrence gene signature

30. Taub R et al. Nat Rev Mol Cell Biol 2004;5:836–47 Liver regeneration pathways after resection are partially shared by HCC cell proliferation A Growth factor-dependent B Cytokine-dependent Hepatocyte TGF  uPA/plasminogen HGF Met Stellate cell Pro-HGF P13K AKT S6 kinase TGF  AP1 JNK pERK C/EBP  IGFBP1 PAI SCF STAT3 SOCS3 Endothelial cell VEGF TOR? Hepatocyte Cyclin E P27 Cyclin D IL-6 TNF  Kupffer cell LPS C3a C5a ICAM A: Growth factor-dependent HGF activate hepatocyte regeneration through downstream pathways (PI3K, pERK, AKT) VEGF activate proliferation of endothelial cells B: Cytokine-dependent IL-6 and TNFα are crucial priming stimuli activating STAT 3, MAPK and pERK

31. Adjuvant systemic strategies Chemotherapy Adjuvant immunotherapy Vitamin chemoprevention Novel agents - HCFU - UFT - Epirubicin + cisplatin - Capecitabine ….. - Adoptive immunotherapy - Tumour vaccines - Interferon - Vitamin A - Vitamin K ….. Recurrent HCC after curative treatment: adjuvant strategies

32. Chemotherapy No evidence of benefit from adjuvant chemotherapy compared to surgery alone in improving survival rates after curative tumour resection The potential benefits of CT on tumour recurrence should be weighed against the risk of adverse reactions in patients with an underlying liver dysfunction Samuel M et al. Cochrane Database Syst Rev 2009;CD001199

33. Chemotherapy: RCT using UFT (uracil + tegafur) No evidence to support potential benefit of adjuvant UFT, an oral agent which combines uracil and 5FU prodrug Such treatment may even worsen OS Hasegawa K et al. Hepatology 2006;44:891–5 Control Years Patients (%) 100 0 80 60 40 20 0 12345678 7953383220189UFT Patients at risk 8058432919134 A. Recurrence-free survival UFT Control Years Patients (%) 100 0 80 60 40 20 0 12345678 79 78725435197UFT Patients at risk 80 79755640218 Control B. Overall survival UFT Control

34. Chemotherapy: capecitabine after curative resection Xia Y et al. Ann Surg Oncol 2010;17:3137–44 Probability of recurrenceOverall survival Multivariate analysis for DFS Post-operative adjuvant capecitabine is well tolerated and may reduce risk of recurrence with no effect on survival

35. Autologous lymphocytes activated with recombinant interleukin-2 and antibody to CD3 Adjuvant immunotherapy: adoptive immunotherapy Safe, feasible and lowers tumour recurrence No significant difference in OS Takayama T et al. Lancet 2000;356:802–7 Time after hepatectomy (years) Recurrence-free (%) 100 80 60 40 20 0 P=0.008 01234567 Immunotherapy Control

36. Cytotoxic T lymphocytes, generated by incubation of pheripheral blood lymphocytes with anti-CD3 monoclonal antibody, IL-2, IL-1 and IFN-γ Adjuvant immunotherapy: adoptive immunotherapy Hui D et al. Dig Liv Dis 2009;41:36–41  Post-operative immunotherapy with cytokine-induced killer cells may prevent recurrence/metastasis after resection of HCC  However, it cannot improve overall survival

37. Adjuvant immunotherapy: role of IFN Clavien PA. Ann Surg 2007;245:843–5

38. IFN may prevent late recurrence after HCC resection in specific subgroups of HCV cirrhosis Mazzaferro V et al. Hepatology 2006;44:1543–54 HCV pure patients

39. Oral polyprenoic acid prevents late recurrence after surgical resection or PEI Muto Y et al. N Engl J Med 1996;334:1561–7; Takai K et al. Intervirology 2005;48:39–45 Vitamin chemoprevention of recurrence: retinoids

40.  Combination treatment of vitamin K with angiotensin converting enzyme inhibitor may suppress cumulative recurrence of HCC after curative treatments Vitamin chemoprevention of recurrence: vitamin K and ACE-I combination Yoshiji H et al. J Hepatol 2009;51:315–21 Time after treatment (months) Cumulative recurrence rate (%) 100 80 60 40 20 0 P<0.01 06121824303648 Vitamin K + ACE-I (n=25) Controls (n=25) Time after treatment (months) VEGF (ng/mL) 160 120 80 40 0 012 140 100 60 20 180 42 VEGF serum levels

41.  Level II clinical evidence does not support the use of systemic adjuvant therapy, tested for resectable HCC  Based on the current evidence, there is no role for the aforementioned adjuvant strategies therapy in the management of HCC Adjuvant systemic strategies Chemotherapy Adjuvant immunotherapy Vitamin chemoprevention - HCFU - UFT - Epirubicin + cisplatin -Capecitabine ….. - Adoptive immunotherapy - Tumour vaccines - Interferon - Vitamin A - Vitamin K ….. Recurrent HCC after curative treatment: adjuvant strategies