1. Outline Biomarkers for early detection of HCC - Currently so called HCC biomarker - AFP - Our proposed HCC biomarkers - PIVKAII - GGTII - Detection of double mutation of HBV based on SNP-ELISA assay

2. Why do we need biomarker for HCC? For early diagnostic before severity of disease occurs. Non invading technique To measure the progression of disease.

3. Biomarker for HCC - fetoprotein (AFP) and Ultrasonography ( US) has been used since 1980s. AFP is not enough sensitivity and low specificity for HCC tumors less than 3 cm but is still widely use because it is cheap and easy to measure. US is too expensive for most people in Asia and Africa to afford.

4. Biomarkers for HCC There are several molecules that are considered to be tumor markers for diagnostic of small HCC tumor. Protein induced by vitamin K absence (PIVKAII) or des-gamma carboxy prothombin (DCP) Gamma-glutamyl transferase (GGTII)

5. PIVKAII and GGTII Serum PIVKAII has high specificity and lack of correlation with serum AFP level. Using enzyme immunoassay (EIA) to determined serum PIVKAII Using polyacrylamide stage gel plate to determined serum GGTII ( dot blot ELISA)

6. Correlation between tumor size and PIVKAII level R Cui et al.,Bjcancer(2003) There are a correlation between tumor size and PIVKAII

7. Correlation between PIVKAII and AFP level R Cui et al.,Bjcancer(2003) There is no correlation between AFP and PIVKAII

8. Sensitivity and specificity of serum PIVKAII, GGTII and AFP R Cui et al.,Bjcancer(2003)

9. PIVKAII and GGTII compare with AFP Protein induced by vitamin K absence ( PIVKAII) and gamma-glutamyl transferase (GGTII) are useful for early diagnostic for HCC when combined information with AFP. For HCC patients with tumors less than 3 cm in dimension, combining the information of PIVKAII, GGTII and AFP is significantly increase the sensitivity of diagnostic above AFP alone. R Cui et al.,BJcancer(2003)

10. Double Mutation of HBV Associated with accelerated carcinogenesis among HBV carriers Increased inflammatory response lead to liver damage, progresses from chronic hepatitis to cirrhosis Double mutation of HBV usually occurs at the position 1762T( adenine to thymine)/1764A (guanine to adenine) Detect in plasma of patients up to 8 years before the diagnostic of HCC Cancer Epidemiol Biomarkers Prev 2005;14(2). February 2005

11. How to detect double mutation? Use SNP- based approach with ELISA technique. Holliday junction will be occurred and stable if the sample contains DNA mutation by PCR reaction. Yellow color will be occurred after apply the stop solution in 96 well plates. Detect at OD 450 nm by a plate reader.

12. SNP-ELISA assay www.panomics.com

13. Components in test Primers for target ( for amplifying target DNA sequence) Primers for internal control ( for positive control) Primers for reference (for correcting background) Ruv A protein 96-well plate with pre-coated RUVA antibody Use PCR-grade water as a negative control instead of Viral DNA Streptavidin-HRP (horseradish peroxidase) conjugate TMB ( 3,35,5-tetramethylbenzidine) substrate solution Stop solution

14. Summary Combining PIVKAII and GGTII as biomarkers for HCC can significantly increase sensitivity of early detection and diagnostic of HCC. Double mutation of HBV can be detected in plasma routinely by SNP- ELIZA assay up to 8 years before diagnostic of HCC. These biomarkers and double mutation detection can be useful for early detection of HCC in normal, cirrhosis, and hepatitis patients.