Global and gene specific DNA methylation as a screening tool for early detection of liver cancer.

B136 Background: Global hypomethylation and CpG island promoter hypermethylation have been identified as hallmarks of human cancer. The genome of the transformed cell undergoes a global genomic hypomethylation and a dense hypermethylation of CpG islands associated with gene regulatory regions. Although the temporal association between these two events has not been elucidated, it is widely recognized that global hypomethylation is one of the earliest epigenetic changes in the transformation to a diseased cell, followed by a progressive increase in gene specific hypermethylation. There are no studies that examine the relationship between these two events in liver cancer as a potential indicator of early carcinogenesis. Previously, we reported hypermethylation of PGP9.5 and NMDAR2B in human esophageal cancer.
 >Objective: In this study we aim to validate the use of PGP9.5 and NMDAR2B together with global methylation levels as a potential biomarker panel for early detection of liver cancer.
 >Methods: Ten frozen tumor tissues from liver cancer patients and 10 frozen liver tissue control samples from non-cancerous patients were obtained from the Cooperative Human Tissue Network. DNA was extracted using standard methods. Genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global DNA hypomethylation was quantified using HPLC for fraction separation and Mass Spectrometry for quantification. We compared the promoter methylation status of PGP9.5 and NMDAR2B in pre-malignant liver with that in liver tumor tissues by using quantitative methylation-specific PCR (Q-MSP). The methylation status of p16, p15, and RASSF1A was also examined in parallel.
 >Results: A global methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p-value = 0.0001) for all tumor tissue, mean = 2.39 (95% CI, 1.99, 2.80), when compared to tissue from non-cancerous liver, mean = 3.54 (95% CI, 3.16, 3.93). In liver tumor, PGP9.5 methylation was observed with highest frequency (100 %). The methylation frequency in NMDAR2B and RASSF1A was also very high (83%), but p16 or p15 methylation was relatively low (33% in p16 and 0% in p15), Interestingly, RASSF1A methylation was detected in all non-cancerous liver tissue tested (100%). NMDAR2B and p15 methylation was observed in (57%), and (14%) non-cancerous liver. PGP9.5 and p16 methylation was not detected in non-cancerous liver tissue.
 >Conclusion: The results of this proof-of-principle study suggest that an epigenetic panel of global and gene specific methylation changes may be a useful biomarker of early detection in cancer. RASSF1A and NMDAR2B are possibly more sensitive markers than p15 or p16 in detection of liver cancer.