Abstract 4086: Location matters: The impact of the location of CpG sites on the specificity of methylation biomarkers for HCC

Abstract

Abstract Hepatocellular carcinoma (HCC) is the second fastest growing cancer and one of the top 10 causes of cancer deaths in the United States. The 5-year survival rate is 14%, ranging from 26% among early stage tumors to only 2% in late stage HCC. The only available biomarker for screening, serum alpha-fetoprotein (AFP), has a low sensitivity of detection (40%-60%), so the need for a better detection method is urgent. Given the heterogeneous nature of HCC, a panel of genetic and epigenetic biomarkers would be needed to obtain high sensitivity for the early diagnosis of HCC. In the course of assembling a panel of epigenetic markers for HCC, we noticed variable HCC specificity of three extensively studied potential methylation biomarkers, APC, GSTP1, and RASSF1A. We hypothesized that the specificity of promoter methylation of the tumor suppressor genes APC, GSTP1, and RASSF1A as a biomarker for HCC could depend upon the location of the CpG sites analyzed. To test this hypothesis, we used bisulfite-PCR sequencing to compare the methylation status of the promoter region of these three genes in DNA isolated from HCC, matched adjacent non-HCC, cirrhosis, hepatitis, and normal liver. We identified HCC-specific CpG methylation sites and unique liver-specific methylation patterns in the promoter region of the APC (Jain et. al, PloSOne 2011) and GSTP1 genes and in a more HCC-specific promoter region (p < 0.05 by Fisher's exact test) of the RASSF1A gene. Together, these results proved our hypothesis and provided an interpretation for the variability in the literature. Using PCR-based technology, we have developed assays for a panel of genetic and epigenetic markers for liver cancer screening. These assays were first tested in tissue to determine their ability to distinguish HCC (n=120) from cirrhosis (n=35) and hepatitis (n=35). The AUROC is 0.809 for mAPC, 0.756 for mGSTP1, 0.887 for mRASSF1, and 0.733 for TP53. When combined, this panel of DNA markers had an AUROC of 0.931 and identified 90% of AFP-negative tumors. This study clearly demonstrates (1) the importance of location of CpG site methylation for HCC specificity and the way in which liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC and (2) the potential of a sensitive DNA test for HCC screening. An analysis of the performance of this panel of markers in peripherals (blood and urine) for HCC screening is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4086. doi:1538-7445.AM2012-4086