1. Epigenetic approaches in liver cancer epidemiology: Global and gene specific quantification of DNA methylation and histone modifications Rafael Guerrero-Preston Dr PH, MPH APHA 135th Annual Meeting & Exposition November 6, 2007 Washington, DC

2. Hepatocellular carcinoma (HCC) Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, with 564,000 new cases worldwide per year Its age-adjusted incidence rates in the United States (US) have doubled over the last 20 years. Hepatitis C, hepatitis B and alcohol-related cirrhosis are the major known risk factors for HCC Prognosis is poor Non-alcoholic fatty liver disease is becoming widely recognized as another potential risk factor for liver cancer

3. El-Serag, H. B. et al. Arch Intern Med 2007;167:1983-1989 Estimated Annual Percentage Change HCC Incidence by Race/Ethnicity in US 1992-2002 The largest increase has been observed among Hispanic women, followed by Black males

4. Hispanics show fastest rise in HCC incidence in US The fastest rise in the incidence of HCC in the US has been observed among Hispanic men and women, followed by non-Hispanic whites and blacks. Asians/Pacific Islanders experienced the least increase in the incidence of HCC The highest rates, as well as most of the increase in the incidence of HCC, were seen in native rather than immigrant Hispanics El-Serag, H. B. et al. Arch Intern Med 2007;167:1983-1989

5. Screening for HCC in US Approximately only 28% of patients are screened for HCC within the 3-years before HCC diagnosis Davila JA, Weston A, Smalley W, El-Serag HB, J Clin Gastroenterol. 2007 Sep;41(8):777-82

6. DNA hypomethylation in hepatocarcinogenesis Global DNA hypomethylation is a tissue specific early epigenetic event, both in human and experimental hepatocarcinogenesis. Genetic and epigenetic alterations following global DNA hypomethylation in non-coding regions have been shown to play a significant role in animal models of hepatocarcinogenesis. Global DNA methylation levels could be used as a screening biomaker for early detection in liver cancer.

7. Epigenomic Era Baylin, S and Schuebel KE, Nature 448, 548-549 (2007 ) Best understood epigenetic marks DNA methylation Histone modifications micro RNA silencing

8. Epigenetics: The science of change Epigenetics refers to the inheritance of information based on gene expression levels without changes in DNA sequence, as opposed to genetics which refers to information transmitted on the basis of gene sequence DNA methylation is tissue specific and can be quantified globally, at gene-promoter regions and at “non-coding” regions of the DNA

9. Determinants of global DNA hypomethylation and its consequences in a transformed cell Guerrero-Preston, et al, (in press), Epigenetics

10. Global DNA methylation as a screening tool for liver cancer We used HPLC/MS/MS for quantification of a global methylation index to distinguish cases from controls A global methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p-value = 0.0001) for all cases, mean = 2.39 (95% CI, 1.99, 2.80), when compared to controls, mean = 3.54 (95% CI, 3.16, 3.93)

11. DNA Chromatograms Figure 2. LC-ESI/MS chromatograms for a control sample. Panel A: HPLC separation of deoxycytidine (dC), 5-methyl 2´deoxycytidine (mdC), deoxyadenosine (dA), deoxyguanosine (dG) and thymidine (dT). Full-scan spectra of dC (Panel B) and mdC (Panel C).

12. Discussion We have been able to distinguish between liver cancer cases from controls using a global DNA methylation assay Global DNA hypomethylation may be a useful biomarker for cancer screening and cancer epidemiology research

13. Future Directions Global Quantification of Histones by HPLC/MS/MS Controls Cases Histones begin to elute at minute 35 (red arrow)

14. Copyright ©2007 American Association for Cancer Research Rivenbark, A. G. et al. Clin Cancer Res 2007;13:2309-2312 Future Directions Gene specific methylation

15. We have analyzed some of the reported hypermethylated genes in HCC using a candidate gene approach. Our preliminary results indicate that RASSF1A and NMDAR2B are possibly more sensitive markers than p15 or p16 in detection of liver cancer We will continue to test more candidate genes and analyze a larger number of cases and controls Future Directions Gene specific methylation

16. Acknowledgements Maria Berdasco, Esteban Ballestar, Mario Fraga, and Manel Esteller at the Cancer Epigenetics Laboratory of the Spanish National Cancer Institute - (CNIO) Regina Santella, Paul Brandt-Rauf, Frederica Perera and Andrew Gelman – Columbia University Augusto Elias and Adriana Baez – UPR Cancer Epigenetics Laboratory at CNIO David Sidransky, Myoung Sook Kim and Cynthia Lebrón at the Head and Neck Cancer Research Laboratory of Johns Hopkins School of Medicine