Abstract 2959: Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk
Authors: Lubecka-Pietruszewska Katarzyna , Kurzava Lucinda , Buvala Hannah , Flower Kirsty , Gawrieh Samer , Mansfield Jennifer , Chalasani Naga , Flanagan James M. , Stefanska Barbara 
Published: August 2015

Abstract
Hepatocellular carcinoma (HCC), one of the most prevalent types of primary liver cancer, is the sixth most common cancer worldwide and the third leading cause of cancer death with rising mortality and morbidity rates. As late onset of HCC accounts for late diagnosis and poor prognosis and early detection increases cure rate from 5% to 80%, identifying reliable and quantifiable biomarkers of risk prediction is of high interest. Aberrations in the DNA methylation patterns, an important early event in carcinogenesis, have been shown to differentiate HCC tumors from normal tissues. However, these changes as predictive markers would have a high application in clinics only if detectable by minimally invasive tests like blood test.
In the present study, we performed a comprehensive evaluation of DNA methylation profiles in blood DNA collected before diagnosis with HCC. Aberrant methylation was investigated in DNA isolated from blood of 21 HCC patients (cases) who provided samples between 1-4 years prior to diagnosis and 21 controls enrolled by the Indiana Biobank of Indiana CTSI. Cases were matched with controls for gender, age, ethnicity, hepatitis C infection, and diabetes. We used Infinium Human Methylation 450K BeadChip array for genome-wide DNA methylation analysis and pyrosequencing for validation of DNA methylation differences.
We found 966 probes differentially methylated between cases and controls with p&lt;0.05 and intraclass correlation coefficient &gt;0.5. Among these significant changes, 732 CpG sites are hypomethylated in HCC cases compared to matched controls and include 130 CpGs corresponding to 75 genes with delta beta value (differential methylation) ≥0.1. Forty six CpG sites out of 234 significantly hypermethylated probes show delta beta value ≥0.1 and correspond to 46 genes. Functional analyses using GO, KEGG and DAVID knowledgebase indicates that hypomethylated genes are associated with Wnt signaling, cell adhesion, blood coagulation, and regulation of transcription, whereas hypermethylated genes are enriched with cytoskeleton organization and small GTPase mediated signal transduction. One of the genes hypomethylated in blood DNA of HCC cases prior to diagnosis is TET1 that was found to be 8-fold overexpressed in HCC tumors in our previous studies and implicated in gene-specific hypomethylation. Interestingly, 75% of differentially methylated sites in blood DNA of HCC cases are hypomethylated prior to diagnosis. Validation by pyrosequencing of four hypomethylated probes including CpGs in the CpG island shore of BRUNOL5 that was linked to fatty liver disease in earlier reports and found demethylated and overexpressed in HCC tumors, suggests their predictive potential.
Our results establish the possible predictive value of aberrant methylation, in particular DNA hypomethylation, in blood DNA for risk of HCC. This study was supported by Showalter Trust and Purdue Center for Cancer Research Awards granted to BS.
Citation Format: Katarzyna Lubecka-Pietruszewska, Lucinda Kurzava, Hannah Buvala, Kirsty Flower, Samer Gawrieh, Jennifer Mansfield, Naga Chalasani, James M. Flanagan, Barbara Stefanska. Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2959. doi:10.1158/1538-7445.AM2015-2959