TCOS, Shanghai, May 20 2010

1. Cancer Biomarkers : Opportunities and Challenges TCOS, Shanghai, May 20 2010 Ann-Lii Cheng M.D., Ph.D. Department of Oncology and Department of Internal Medicine, National Taiwan University Hospital; Taipei, Taiwan.

2. Cancer Biomarkers- the opportunities • A new era in which physicians no longer make treatment choices that are based on population-based statistics but rather on the specific characteristics of individual patients and their tumors. Dalton W Science 2006

3. First-line study of gefitinib vs carboplatin / paclitaxel in patients with advanced non-small cell lung cancer (IPASS) EGFR wild-type EGFR mutation-positive Gefitinib (n=132) Carboplatin/paclitaxel (n=129) Gefitinib (n=91) Carboplatin/paclitaxel (n=85) 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 HR (95% CI) = 0.48 (0.36, 0.64)p<0.0001 HR (95% CI) = 2.85 (2.05, 3.98)p<0.0001 Probability of PFS Probability of PFS 0 4 8 12 16 20 24 0 4 8 12 16 20 24 Months Months At risk: GefitinibC/P 132 108 71 31 11 3 0 129 103 37 7 2 1 0 91 21 4 2 1 0 085 58 14 1 0 0 0 Mok et. al. ESMO 2008

4. First-line treatment of mCRC with FOLFOX  cetuximab(OPUS Study) K-Ras mutant K-Ras wild-type 1.0 0.8 0.6 0.4 0.2 0 1.0 0.8 0.6 0.4 0.2 0 Cetuximab + FOLFOX FOLFOX Cetuximab + FOLFOX FOLFOX Kaplan-Meier estimate HR=1.83; p=0.0192 FOLFOX: 8.6 m Cetuximab + FOLFOX: 5.5 m HR=0.57; p=0.016 FOLFOX: 7.2 mCetuximab + FOLFOX: 7.7 m 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Progression-free time (months) Progression-free time (months) Bokemeyer C, et al. JCO 2009

5. Cancer Biomarkers- the challenges • Clinically a must in an increasing number of cancer types. • Cost, workload, and workflow. • Technic issues – reliability of methods • Ethnic and geographic issues – how far can data be extrapolated from area to area ? Non-Small cell lung cancer Colorectal cancer Breast cancer Gastric cancer Renal cell carcinoma GI stromal tumor Leukemia Lymphoma Melanoma Basal cell carcinoma Medulloblastoma …………

6. Reliability of Biomarkers on Surgically Resected Tumor Specimens • Ischemia during operation – unknown in most specimens • Unknown processing of specimens Warm ischemia Specimen aging

7. Comparison of Biopsy vs. Hepatectomy HCC Tissues Study design: • 46 paired HCC tissues (‘03~’08) • Biopsy taken within 3 months before hepatectomy • Hepatectomy • IHC studies: • p-Akt (S473): rabbit antibody (Santa Cruz) • p-ERK1/2 (T202/Y204): rabbit antibody (Santa Cruz) ~ Shao YY et al: AACR 2010, Abstract 3759.

8. Comparison of Biopsy vs. Hepatectomy HCC Tissues • Poor correlation • Higher expression in biopsy tissues phospho-ERK phospho-Akt ~ Shao YY et al: AACR 2010, Abstract 3759.

9. p-ERK Biopsy tissue Hepatectomy tissue

10. Tissue Ischemia Affects Gene Expression within Minutes Following CRC Excision─ Microarray analysis Spruessel A, et al. BioTechniques 2004;36:1030-1037

11. Tissue Ischemia Affects Protein Expression within Minutes Following CRC Excision─ SELDI-TOF MS analysis Spruessel A, et al. BioTechniques 2004;36:1030-1037

12. Slide Aging Affects Immunohistochemistry HER2 ER E-Cadherin Mirlacher M, et al. Mod Pathol, 2004;17:1414-1420

13. Slide Aging Affects Immunohistochemistry Figure 2.Influence of slide aging on the fraction of positive cases. For each antibody, the frequency of positive cases is shown as separate bars for old (O) and fresh (F) sections. Mirlacher M, et al. Mod Pathol, 2004;17:1414-1420

14. Slide Aging Affect p53 Immunostaining of Breast Cancer Tissues Jacob TW et al J, Natl Cancer Inst 1996;88:1054-09

15. The Problems of Genomic Biomarkers- minimal reproducibility • Numerous gene signatures have been reported. • Only marginal overlap of reports • poor study design lack of a standard technology platform different ways of tumor collection different statistical methods • Only few validated into clinic practice. Dalton W. science 2006;312:1165-8

16. Toward Robust Genomic Biomarkers • Share samples • Common technology platform • Ask similar questions • Use similar criteria for patient enrollment • Partnership among academic groups, pharmaceutical and biotechnology companies, as well as government agencies. Dalton W. Science 2006;312:1165-8

17. Challenges of Cancer Biomarkers- ethnic and geographic issues - East vs West Lung cancer Breast cancer Hepatocelluar carcinoma

18. Gefitinib Placebo 1.0 0.8 0.6 Proportion surviving 0.4 0.2 0.0 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Time (months) Importance of Ethnicity for MTT─ the lessons of ISEL trial Asian (n=342) Non-Asian (n=1350)

19. EGFR mutation rates in each subgroup • Shih et al, IJC 2005 • Chou et al, CCR 2005 • Han et al, JCO 2005 • Takano et al, JCO 2005 • Mitsudomi et al, JCO 2005 • Lung et al, PAACR 2005 • Mu et al, CCR 2005 • Machetti et al, JCO 2005 *only AdenoCa

20. EGFR Mutations in NSCLC Patients Born or Domiciled in the USA • Other ethnicities 14/159 (9%) • East Asians 12/22 (55%) Adi Gazdar, 2005

21. Ethnic Difference in Mutational Pattern of Lung Adenocarcinoma East Asians Other Countries EGFR mutation EGFR mutation KRAS mutation KRAS mutation

22. Trends in Breast Cancer Incidence • Incidence of breast cancer has been increasing around the world including Latina America, Middle East and Asia • Incidence now stabilizing in some Western populations • Incidence continues to increase in many Asian populations Country Incidence Incidence% 1973–77 1993–97Increase Japan (Miyagima) 17.5 34.497 Japan (Osaka) 12.7 28.7126 Singapore (Chinese) 21.9 45.9 110 China (Shanghai) 19.6 27.239 Cancer Incidence in Five Continents (Vol. IV, VIII) Katanoda et al, Jpn J Clin Oncol. 2007 Aug;37(8):638-9

23. Age-specific Breast Cancer Incidence in Taiwan

24. Comparison of Molecular Subtypes between American and Taiwanese

25. High frequency of ER expression in Taiwanese YFBC: validated by Taiwan Cancer Database Taiwan Cancer DataBase 2005

26. Time Trend of Increase Frequency of ER expressions in Young Breast Cancer at NTUH (2004-2006) (1992-2000) 74 75 61 62 58 54 Preliminary data

27. ER expression : a poor prognostic factor for DFS among very young patients receiving adjuvant chemotherapy alone in Western Countries IBCSG data Lancet 2000

28. ER expression : a favorable prognostic factor for OS among very young (<35y) patients in Taiwan 178 very young (<35 years) breast cancer patients diagnosed at NTUH in 1997-2006 Proceeding ASCO 2009

29. Hepatocellular Carcinoma— Distinct Geographic Distribution Europe and United States (%) Japan (%) Asia and Africa (%) Estimate Range 22 4~-58 60 12~72 45 8~57 12 0~14 --- 10~50 Limited exposure < 5 --- Estimate Range 20 18~-44 63 48~94 20 15~33 40 9~51 --- --- Limited exposure --- --- Estimate Range 60 40~90 20 9~56 --- 11~41 22 --- 8 --- Important exposure < 5 --- Risk Factors Hepatitis B virus Hepatitis C virus Alcohol Tobacco Oral contraceptives Aflatoxin Other and emerging risk factors/cofactors Bosch FX et al. Gastroenterology 2004;127:S5-16.

30. HCV core HCV core By Hsu C, Shen YC, Cheng AL Viral Proteins And Signal Transduction Pathways

31. Transcriptome classification of HCC ~ Boyault S et al: Hepatol 2007;45:42. based on120 surgically resected HCC, including transcriptome analysis on 57 HCCs and 3 adenomas, and qRT-PCR validation in additional 63 HCCs 31

32. HBV-related HCC Is HBV-related HCC a more aggressive tumor ? Is HBV-related HCC associated with molecular changes which affect molecular therapy ?

33. HCC ─ East vs West Long-term results after surgical treatment were similar in West and East when clinicopatnologic factors were accounted for. Pawlik TM et al Liver Transplantation 2004;10(suppl 1) 74-80 Taeck D et al Liver Transplantation 2004;10(suppl 1) 58-63

34. HBV vs. HCV HCCItalian Liver Cancer group Survival in patients with advanced HCC. HBV-HCC patients had a lower survival than HCV-HCC patients (p=0.025) • Patients with HBV-HCC tended to have poor prognosis; and the difference became statistically significant among patients with advanced HCC ~ Cantarini MC et al: Am J Gastroenterol 2006;101:91-8.

35. HBV vs. HCV HCC in NTUHSurvival for patients with advanced ds. 927 patients receiving supportive care or chemotherapy. Etiology Median survival (M) 1 year (%) 3 year (%) 5 year (%) 10 year (%) HBV 2.5 12.4 3.4 0.8 0.5 HCV 3.4 21.7 8.5 2.2 1.1 B+C 3.4 10.8 3.1 1.5 0 NBNC 2.6 11.2 3.1 1.0 1.0 HCV+ HBV+ HCV-HCC patients had better survival than HBV-HCC patients Chen CH et al.Eur J Cancer. 2006 Oct;42(15):2524-9. Epub 2006 Aug 22

36. Sorafenib phase II HCC studyHCV- vs. HBV-related HCC ~ Huitzil-Melendez FD et al: ASCO-2007 GI Symposium Abstract# 173. A retrospective analysis

37. Thalidomide phase II HCC studyHCV- vs. HBV-related HCC HBV+ 61 53.6 13.1 8.3 W 21.4 W P 61 < .001 .09 .03 .08 P’t No. Median age Objective response+ AFP response (%) TTP (median) OS (median) HCV+ 33 67.5 27.3 14.1 W 32.6 W ~ Hsu C et al: Proc. ASCO 2004: Abs#4198.

38. Conclusions─ Cancer biomarkers • Provide excellent opportunity for personalized care. • Cost, workload, and workflow remain a problem. • Technic issues should not be overlooked. • Significant ethnic and geographic difference exist.

39. Cancer Biomarkers : Opportunities and Challenges TCOS, Shanghai, May 20 2010