Abstract 3366: Loci-specific differences in blood DNA methylation for early detection of hepatocellular carcinoma
Authors: Lubecka Katarzyna , Beetch Megan , Qiu Jay , Kurzava Lucinda , Flower Kirsty , Gawrieh Samer , Liangpunsakul Suthat , Chalasani Naga , Flanagan James M. , Stefanska Barbara 
Published: July 2017

Abstract
Late onset of clinical symptoms in hepatocellular carcinoma (HCC) results in late diagnosis and poor disease outcome. It is estimated that early detection of HCC would increase the cure rate from 5% to 80%. Thus, identifying new effective tools with reliable and quantifiable biomarkers is of high importance. Such tools are currently missing. 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 diagnostic markers would have a high application in clinics only if detectable by minimally invasive tests like a blood test. In the present study, we performed a comprehensive evaluation of DNA methylation profiles in blood DNA collected from 24 HCC patients (cases) who provided samples after diagnosis and from 24 controls, enrolled by the Indiana Biobank. Cases were matched with controls on gender, age, ethnicity, hepatitis C infection, and diabetes. We used Human Methylation 450K BeadChip array for genome-wide DNA methylation analysis and pyrosequencing for validation of DNA methylation differences. We identified 7,047 CpG sites differentially methylated between cases and controls with p&lt;0.05 and intraclass correlation coefficient (ICC) &gt;0.5. The change in DNA methylation within the top 14 CpG sites corresponding to 12 genes (probes) was technically validated using pyrosequencing. Five probes were hypomethylated and 7 probes were hypermethylated in cases vs. controls. Those probes constitute candidate biomarkers that could be used in early detection of HCC only if detectable at pre-diagnostic stages. We therefore selected 4 out of 12 probes that presented the highest differences in post-diagnostic cases. We then verified DNA methylation changes at those 4 probes in blood samples collected from 21 patients at the time when they were clinically considered cancer free (pre-diagnostic) and developed HCC within 4 years of follow-up. We confirmed that methylation at all 4 selected probes discriminates pre-diagnostic cases from matched healthy controls. One of the strongest differences was detected within an enhancer of PCGF3 (difference=-0.12, ICC=0.84). PCGF3 is a component of a Polycomb group multiprotein repressive PRC1-like complex and was found to be over-expressed in HCC tumors and in many other types of cancer according to Oncomine database. Our present study establishes for the first time differences in DNA methylation at specific CpG sites that are detectable in blood of individuals both before and after conventional diagnosis with HCC as compared with healthy individuals. The identified probes have high potential to be developed into early detection HCC biomarkers once validated in a larger cohort of individuals at risk. This research was supported by the ACS Institutional Research Grant and Showalter Trust Award granted to BS.
Citation Format: Katarzyna Lubecka, Megan Beetch, Jay Qiu, Lucinda Kurzava, Kirsty Flower, Samer Gawrieh, Suthat Liangpunsakul, Naga Chalasani, James M. Flanagan, Barbara Stefanska. Loci-specific differences in blood DNA methylation for early detection of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3366. doi:10.1158/1538-7445.AM2017-3366