1. New treatment approaches in Hepatocellular Carcinoma Dr. Francesco Caponigro Istituto Tumori Napoli

2. F. Caponigro - Istituto Tumori Napoli Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and the fifth most prevalent cancer worldwide with > 80% of patients presenting with advanced disease. Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and the fifth most prevalent cancer worldwide with > 80% of patients presenting with advanced disease. Late diagnosis and the frequent coexistence of cirrhosis result in a poor prognosis for patients with HCC Late diagnosis and the frequent coexistence of cirrhosis result in a poor prognosis for patients with HCC Drug-resistance genes such as multidrug resistance-1 are common in liver cells, causing HCC tumors to be inherently chemoresistant Drug-resistance genes such as multidrug resistance-1 are common in liver cells, causing HCC tumors to be inherently chemoresistant

3. F. Caponigro - Istituto Tumori Napoli Phase II/III Studies of Systemic Treatments in Advanced HCC Treatment Phase of Study Number of Patients Overall Response Rate Systemic Chemotherapy Doxorubicin as a single agent II/III > 1000 10%-18% Doxorubicin in combination II/III14426% CisplatinII4810% EpirubicinII6211% MitoxantroneII11816% 5-FU/paclitaxel/irinotecanII/III- < 10% AntiandrogenIII376 InterferonIII60 OctreotideIII60 < 5% SeocalcitolIII746

4. F. Caponigro - Istituto Tumori Napoli Sorafenib (BAY 43-9006, Nexavar): a multikinase inhibitor with activity against Raf, VEGFR-2, VEGFR-3, PDGFR, c-Kit The turning-point in the management of advanced HCC

5. F. Caponigro - Istituto Tumori Napoli Signaling pathways in HCC Villanueva A et al, Semin Liver Dis 2007 VEGF EGFR 1. 2. 5. 3. 4.

6. F. Caponigro - Istituto Tumori Napoli SORAFENIB Farmacokinetics Low absorption T1/2: 24-36 h Steady state: after 7 days Time of assumption: 1-2 hours before or after meals Elimination: mainly as glucuronate

7. F. Caponigro - Istituto Tumori Napoli SORAFENIB Safety Data Sorafenib is well tolerated and grade 3-4 adverse events have been reported in 30% of cases in phase I studies. The main side effects are: Skin rash Hand and foot syndrome Diarrhea - nausea Fatigue Hypertension

8. F. Caponigro - Istituto Tumori Napoli SORAFENIB Clinical studies in HCC Phase II Study of Sorafenib in non resectable HCC Abou-Alfa GK, et al. J Clin Oncol 2006

9. F. Caponigro - Istituto Tumori Napoli SORAFENIB Clinical studies in HCC Phase II Study of Sorafenib in non resectable HCC 3/137 pts (2.2%) partial response 8/137 pts (5.8%) minor response 46/137 (33.6%) long-term stable disease (> 16 weeks) Time to progression (TTP) 4.2 months Overall survival 9.2 months Grade ¾ toxicity: fatigue (9.5%), diarrhea (8.0%) hand and foot syndrome (5.1%) Abou-Alfa GK, et al. J Clin Oncol 2006

10. F. Caponigro - Istituto Tumori Napoli SORAFENIB Clinical studies in HCC Pre-treatment pERK was correlated to TTP Although Sorafenib activity was really modest, both the manageable toxicity profile and the peculiarity of its mechanism of action support further trials Abou-Alfa GK, et al. J Clin Oncol 2006

11. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Study Design Primary endpoints: overall survival, time to symptomatic progression (FHSI8-TSP) Primary endpoints: overall survival, time to symptomatic progression (FHSI8-TSP) Secondary endpoint: time to progression (independent review) Secondary endpoint: time to progression (independent review) Stratify by: Macroscopic vascular invasion (portal vein) and/or extrahepatic spread ECOG PS Geographic region Placebo 2 tablets p.o. b.i.d. continuous dosing (n = 303) Sorafenib 400 mg p.o., b.i.d. continuous dosing (n = 299) RANDOMIZERANDOMIZE

12. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Baseline characteristics of patients CharacteristicsSorafenib(n=299) Placebo (n=303) Age (yr, median) Age (yr, median)6566 Male/Female (%) Male/Female (%)87/1387/13 Region (Europe/N. America/other; %) Region (Europe/N. America/other; %)88/9/387/10/3 Etiology (%) Etiology (%) Viral hepatitis (HCV/HBV) Alcohol/other29/1926/2627/1826/29 Llovet JM et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

13. Phase III SHARP Trial Baseline characteristics of patients CharacteristicsSorafenib(n=299)Placebo(n=303) Child-Pugh (A/B; %) Child-Pugh (A/B; %)95/598/2 Prior therapies: Surgical resection Prior therapies: Surgical resection Loco-regional therapies 19%39%21%41% ECOG PS (%) ECOG 0 ECOG 1 ECOG 2 ECOG PS (%) ECOG 0 ECOG 1 ECOG 2 54 38 8 54 39 7 Vascular invasion / extrahepatic spread Present Absent Vascular invasion / extrahepatic spread Present Absent 70 30 Llovet JM et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain. Llovet JM and Bruix J, BCLC Group, Semin Liver Dis. 1999.

14. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Efficacy (Intent-to-Treat Population) Sorafenib (n = 299) Placebo (n = 303) Median Overall Survival (95% CI) 10.7 (9.4-13.3)months 7.9 (6.8-9.1)months HR 0.69; 95% CI, 0.55-0.88; P =.00058 Median Time to Progression (95% CI) 5.5 (4.1-6.9)months 2.8 (2.7-3.9)months HR 0.58; 95% CI, 0.44-0.74; P =.000007 Response Complete response Complete response00 Partial response Partial response 7 (2%) 2 (< 1%) Stable Disease 211 (71%) 204 (67%) Progressive Disease 54 (18%) 73 (24%) Progression-Free Rate at 4 Months 62%42% Median Duration of Treatment 5.3 months 4.4 months

15. F. Caponigro - Istituto Tumori Napoli Survival Probability Weeks Hazard ratio (S/P): 0.69 (95% CI: 0.55, 0.87) P=0.00058* Sorafenib Median: 46.3 weeks (10.7 mo) (95% CI: 40.9, 57.9) Placebo Median: 34.4 weeks (7.9 mo) (95% CI: 29.4, 39.4) 1.00 0 0.75 0.50 0.25 08081624324048566472 02742412051611086738120 Patients at risk Sorafenib: 027622417912678472572 Placebo: 299 303 Phase III SHARP Trial Overall survival (Intention-to-treat) *O’Brien-Fleming threshold for statistical significance was P=0.0077 Llovet JM et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain

16. F. Caponigro - Istituto Tumori Napoli Placebo Median: 12.3 weeks (2.8 mo) (95% CI: 11.7, 17.1) Sorafenib Median: 24.0 weeks (5.5 mo) (95% CI: 18.0, 30.0) 01961268050281482 0192101573112821 Probability of Progression Hazard ratio (S/P): 0.58 (95% CI: 0.45, 0.74) P=0.000007 546121824303642480 Weeks 1.00 0 0.75 0.50 0.25 Patients at risk Sorafenib: Placebo: 299 303 Phase III SHARP Trial Time to progression (Independent central review) Llovet JM et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

17. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Exploratory subgroup survival analysis Sorafenib benefitPlacebo benefit Hazard Ratio (95% CI) ECOG PS 0 ECOG PS 1 & 2 Macroscopic vascular invasion No macroscopic vascular invasion No macroscopic VI/extrahepatic spread Macroscopic VI/extrahepatic spread No extrahepatic spread Extrahepatic spread 0.51.01.50.0 Llovet JM et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

18. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Adverse Events (1/2) Sorafenib ( % ) ( n = 297 ) Placebo ( % ) ( n = 302) Drug-Related Treatment-Emergent Serious Adverse Events 13%9% Drug-Related Adverse Events All Grade 3 All Diarrhea Diarrhea39%8%11%2% Pain (abdomen) Pain (abdomen)8%2%3% < 1% Weight loss Weight loss9%2% < 1% 0% Anorexia Anorexia14% < 1% 3%

19. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Adverse Events (2/2) Sorafenib ( % ) ( n = 297 ) Placebo ( % ) ( n = 302) Drug-Related Adverse Events All Grade 3 All Nausea Nausea11% < 1% 8%1% Hand-foot skin reaction Hand-foot skin reaction21%8%3% < 1% Vomiting Vomiting5%1%3% < 1% Alopecia Alopecia14%0%2%0% Liver dysfunction Liver dysfunction < 1% 0%0% Bleeding Bleeding7% < 1% 4%<1%* * Less than 1 % of patients also experienced grade 4 bleeding

20. F. Caponigro - Istituto Tumori Napoli Phase III SHARP Trial Conclusions Sorafenib prolonged median overall survival compared to placebo in patients with advanced HCC: Sorafenib prolonged median overall survival compared to placebo in patients with advanced HCC: Median overall survival: 10.7 months vs. 7.9 months Median overall survival: 10.7 months vs. 7.9 months Hazard ratio 0.69; P =.00058 Hazard ratio 0.69; P =.00058 44% increase in median overall survival 44% increase in median overall survival Sorafenib prolonged median time to progression compared to placebo in patients with advanced HCC: Sorafenib prolonged median time to progression compared to placebo in patients with advanced HCC: Median time to progression: 5.5 months vs. 2.8 months Median time to progression: 5.5 months vs. 2.8 months Hazard ratio 0.58; P =.000007 Hazard ratio 0.58; P =.000007 73% prolongation in median time to progression 73% prolongation in median time to progression Sorafenib was tolerable, with manageable side effects Sorafenib was tolerable, with manageable side effects Sorafenib is the first systemic therapy to prolong survival in patients with HCC Sorafenib is the first systemic therapy to prolong survival in patients with HCC

21. F. Caponigro - Istituto Tumori Napoli Sorafenib + Dox Placebo + Dox PRIMARY ENDPOINT Time to Progression (TTP) SECONDARY ENDPOINT ■ Overall Survival (OS) ■ Time To Symptom Progression (TTSP) ■ Quality of life (QoL) ~ 90 pts with advanced HCC ECOG PS: 0,1,2 Phase II randomized study of Sorafenib + Doxorubicin versus Doxorubicin + Placebo in HCC Phase II randomized study of Sorafenib + Doxorubicin versus Doxorubicin + Placebo in HCC Randomization 1:1 Abou-Alfa GK et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain

22. F. Caponigro - Istituto Tumori Napoli Demographics (n=96) DXR/sorafenib (n=47) DXR/placebo (n=49) Gender n(%) Male 31 (66) 42 (86) Female 16 (34) 7 (14) Age (years) Mean6362 ECOG n(%) n(%) 0 22 (47) 16 (33) 1 18 (38) 25 (51) 2/3 4 (8.5) 4 (8) Missing 4 (8.5) 4 (8) Child’s Pugh n(%) A 47 (100) 48 (98) B 0 (0) 1 (2) Extrahepatic disease n(%) Yes24(51) 32 (65) No 23 (49) 17 (35) Macroscopic Vascular Invasion n(%) Yes 13 (28) 16 (33) No 33 (70) 32 (65) Missing 1 (2) DXR=Doxorubicin Abou-Alfa GK et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

23. F. Caponigro - Istituto Tumori Napoli Results DXR/sorafenib(n=47) DXR/placeb o (n=49) TTP (months) 8.64.8 OS (months) 13.76.5 PFS (months) 6.92.8 Response (CR+PR) n(%) 2 (4) 1 (2) Stable Disease (SD) n (%) 36 (77) 27 (55) Definitive analysis (data from March 2007 cutoff, independent assessment, TTP: 38 events, OS: 50 events, PFS: 70 events) Abou-Alfa GK et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

24. F. Caponigro - Istituto Tumori NapoliToxicities DXR/sorafenib (n=47) DXR/placebo (n=49) n(%) All grades Grade 3-4 All grades Grade 3-4 Fatigue 35 (75) 7 (15) 31 (65) 7 (15) Abdominal pain 16 (34) 5 (10) 14 (29) 4 (8) Neutropenia 31 (66) 25 (53) 29 (60) 22 (46) Febrile Neutropenia 2 (4) 7 (15) 5 (10) Diarrhea 25 (51) 5 (11) 12 (25) 5 (10) Bilirubin 16 (34) 5 (11) 15 (31) 3 (6) Hand foot 14 (30) 4 (9) 2 (4) 0 (0) LV Dysfunction 9 (19) 1 (2) 0 (0) Hypertension 8 (17) 0 (0) NCI-CTC (version 3); NCI-CTC=National Cancer Institute–Common Toxicity Criteria Abou-Alfa GK et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

25. F. Caponigro - Istituto Tumori Napoli Conclusions This randomized phase II study of Doxorubicin plus Sorafenib and Doxorubicin plus placebo, showed encouraging TTP and OS outcome for the Doxorubicin plus Sorafenib This randomized phase II study of Doxorubicin plus Sorafenib and Doxorubicin plus placebo, showed encouraging TTP and OS outcome for the Doxorubicin plus Sorafenib This trial supports the growing body of evidence of the activity of Sorafenib in HCC This trial supports the growing body of evidence of the activity of Sorafenib in HCC Any synergistic role between Sorafenib plus doxorubicin in HCC needs to be further defined Any synergistic role between Sorafenib plus doxorubicin in HCC needs to be further defined Abou-Alfa GK et al. Presented at: ECCO; September 23-27, 2007; Barcelona, Spain.

26. F. Caponigro - Istituto Tumori Napoli A Phase II Study of Sorafenib Plus Tegafur/Uracil for the Treatment of Advanced or Metastatic Hepatocellular Carcinoma NCT00464919 Ongoing trial Unresectable and/or metastatic HCC PS 0-2 ECOG No metastatic brain tumors Prior systemic treatment for HCC  Inclusion criteria  Primary end-point PFS  Secondary end-points Toxicity, ORR, SDR, OS Tegafur/uracil, which has potential anti-HCC activity (ORR 0-18% ~, in phase II studies) and interesting anti-angiogenesis activity (in several preclinical models), is an ideal candidate drug to improve the efficacy of sorafenib in HCC  Rationale Enrollment50 Start Date April 2007 Expected Completion Date December 2008

27. F. Caponigro - Istituto Tumori Napoli Beyond Sorafenib NEW POTENTIAL TARGETED THERAPIES IN HCC

28. F. Caponigro - Istituto Tumori Napoli Signaling pathways in HCC Villanueva A et al, Semin Liver Dis 2007 VEGF EGFR 1. 2. 5. 3. 4.

29. F. Caponigro - Istituto Tumori Napoli 1. EGFR pathway EGFR is overexpressed in HCC and is associated with increased cell proliferation and reduced apoptosis Schmidt CM et al, Biochem Biophys Res Commun 1997 Ito Y et al, Br J Cancer 2001

30. F. Caponigro - Istituto Tumori Napoli In HepG2 cells, Gefitinib causes: Inhibition of EGFR by gefitinib induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines Hopfner M et al, J Hepatol 2004 A)time- and dose-dependent growth inhibition B)dose-dependent accumulation in the G0/G1-phase of the cell cycle. C)dose-dependently induced caspase-3 activation A B C

31. F. Caponigro - Istituto Tumori Napoli Phase II Study of Erlotinib in Patients With Advanced Hepatocellular Cancer (N = 38) Philip et al, JCO 2005 ■ Patients with unresectable or metastatic HCC ■ 47% of patients received prior chemotherapy ■ Erlotinib given continuously at of 150 mg/day orally ■ Primary end-point: proportion of patients progression-free at 6 months PFS at 6 months: 32% (CI 95%, 18 to 49) Median OS = 13 months

32. F. Caponigro - Istituto Tumori Napoli Erlotinib in Combination With Docetaxel in Advanced Hepatocellular and Biliary Tract Carcinomas NCT00532441 Phase II ongoing study  Inclusion criteria ■ HCC or biliary tract carcinomas, not amenable to curative resection or transplantation ■ ≤ 2 prior chemotherapy regimens ■ PS  2 ECOG ■ Child-Pugh A cirrhosis  Primary end-point  Treatment protocol ■ Erlotinib: 150 mg p.o., days 2-7, 9-14, 16-28 q28 ■ Docetaxel: 30 mg/m2 iv d 1, 8, 15 q28 ■ PFS rate at 16 weeks  Secondary end-points ■ ORR; d RR; disease control; OS ■ Safety and toxicity ■ Correlation of response with biomarkers

33. Key regulator of cell proliferation 2. The mTOR pathway Rapamycin inhibits HCC growth in vivo tumor- bearing control animal −Bjornsti MA et al, Nat Rev Cancer 2004 −Sahin F et al, Clin Cancer Res 2004 −Semela D et al, J Hepatol 2007

34. F. Caponigro - Istituto Tumori Napoli A Phase I/II Study of RAD001 in Advanced Hepatocellular Carcinoma NCT00516165 Ongoing study  Primary end-points ■ To determine the maximum tolerated dose of RAD001 ■ To determine PFS rate at 24 weeks  Inclusion criteria ■ Unresectable of metastatic HCC ■ 0-2 prior systemic therapy for HCC ■ PS  2 ECOG RAD001 (Everolimus) is an oral kinase inhibitor that blocks the mTOR, a key component of PI3K/AKT pathway Enrollment: Enrollment: 40 Start Date: Start Date: August 2007

35. F. Caponigro - Istituto Tumori Napoli Nuclear  -catenin accumulation, a hallmark of the activeted Wnt signaling, has been observed in 33-67% of HCC. 3. The Wnt pathway In the absence of ligand,  -catenin is degraded through ubiquitin-proteasome systems after phosphorylation by the APC/AXIN/GSK3b complex. Upon Wnt ligand stimulation,  -catenin accumulates in the cytosol and translocates into the nucleus. Intranuclear b-catenin leads to modifications in expression of numerous genes related to cell proliferation, cell cycling, apoptosis, and differentiation (Survivin, c-myc, Cyclin D1, etc). Moreover, mutations of components of the Wnt pathway (  -catenin, AXIN 1, AXIN 2) have been described in HCC Breuhahn K et al, Oncogene 2006 Miyoshi Y et al, cancer Res 1998 Lee HC et al, Front Biosci 2006

36. F. Caponigro - Istituto Tumori Napoli 4. VEGF Pathway (angiogenesis) Microvessels stained by anti-CD34 in a tumor section of HCC, with almost no staining in the adjacent non-tumorous liver DFS after hepatic resection in pts with HCC ≤5 cm stratified into low and high MVD by CD34 immunostaining (P=0.002) HCCs are highly vascularized tumors and increased levels of VEGF and microvessel density (MVD) have been observed. High VEGF expression has been associated with inferior survival Poon RT et al, J Clin Oncol 2002 Yamaguchi R et al, Hepatology 1998 Chao Y et al, Ann Surg Oncol 2003

37. F. Caponigro - Istituto Tumori Napoli Phase II Trial of Bevacizumab in HCC Number of Patients ( n = 33) Patient Characteristics Age Age 31-81 years Gender ( M/F ) Gender ( M/F )28/5 Child-Pugh ( A/B ) Child-Pugh ( A/B )26/7 Tumor stage ( uninodular/multinodular ) Tumor stage ( uninodular/multinodular )5/28 Segmental vein invasion Segmental vein invasion5 Adverse Events Transient ischemic accident Transient ischemic accident1 Arterial hypertension Arterial hypertension1 Hepatic arterial thrombosis Hepatic arterial thrombosis1 Gastrointestinal bleeding Gastrointestinal bleeding2* Clinical Outcome Response Response 1 CR, 2 PR Stable disease ( 4-24 months ) Stable disease ( 4-24 months )21 Median TTP Median TTP 6.5 months * Including 1 death ASCO Chicago 2007

38. F. Caponigro - Istituto Tumori Napoli Phase II Study of Gemcitabine and Oxaliplatin in Combination With Bevacizumab in Patients With Advanced Hepatocellular Carcinoma Zhu AX et al, J Clin Oncol 2006 ■ 33 pts, PS 0-1, with unresectable or metastatic HCC were enrolled ■ Treatment Protocol: Bevacizumab 10 mg/kg d1 q14 Gemcitabine 100 mg/mq (10 mg/mq/min) d2, d16 q28 Oxaliplatin 85 mg/mq d2, d16 q28 Median OS = 9.6 months (95% CI, 8.0 to NA) Median PFS = 5.3 months (95% CI, 3.7 to 8.7) The most common treatment-related G3-G4 toxicities included leukopenia/neutropenia, transient elevation of aminotransferases, hypertension, and fatigue. ORR = 20% SD = 27% of pts GEMOX-B could be safely administered and had moderate antitumor activity for patients with advanced HCC

39. F. Caponigro - Istituto Tumori Napoli Bevacizumab and Erlotinib in Inoperable and Metastatic Hepatocellular Carcinoma NCT00287222 Phase II ongoing study  Primary endpoint proportion of subjects that remain free of progression at the 27th week following the onset of treatment Enrollment: Enrollment: 21 Start Date: Start Date: February 2006

40. F. Caponigro - Istituto Tumori Napoli General Conclusions (1/2) 1)A majority of HCC patients (>80%) presents with advanced disease and is inelegible for surgical or locoregional therapies 2)At this moment, there is not effective systemic chemotherapy for HCC 3)Understanding the molecular pathway of HCC is crucial to the development of novel therapies 4)The relevant molecular signaling in HCC are: EGFR, VEGFR, AKT/mTOR, Raf/MEK/ERK pathways

41. F. Caponigro - Istituto Tumori Napoli 5)Sorafenib, inhibiting both cell growth and angiogenesis, is the first agent to improve survival of patients with advanced HCC. This effect establishes Sorafenib as first- line treatment for these pts 6)The next steps include testing Sorafenib earlier in the disease and eventually in combination with other targeted therapies (Bevacizumab, Erlotinib, Sunitinib have all been tested for liver cancer in phase II studies) 7)Until now, experimental therapies for HCC have been compared with placebo. Many patients are reluctant to join clinical trials if they have a 50% chance of receiving a sugar pill. Participation in trials may now increase, with Sorafenib replacing placebo as the standard control General Conclusions (2/2)

42. F. Caponigro - Istituto Tumori Napoli Thanks for your attention Any Questions?