DNA Methylation Biomarker Panels for the Differentiation of Hepatocellular Carcinoma and Cholangiocarcinoma from Liver Metastases from Colorectal Carcinoma and Pancreatic Adenocarcinoma

Abstract

Background DNA methylation biomarkers are one of the most promising tools for the diagnosis and differentiation of adenocarcinomas of the liver, which are among the most common malignancies worldwide. Their differentiation is important because of the different prognosis and treatment options. This study validates novel diagnostic DNA methylation panels that focus on DNA hypermethylation in cancer and successfully differentiate between the two most common primary liver cancers (hepatocellular carcinoma and cholangiocarcinoma), two common metastatic liver cancers (from colorectal and pancreatic ductal adenocarcinomas) and healthy liver tissue. Moreover, this study investigates whether hypermethylation of selected DNA methylation biomarkers of primary colorectal carcinoma and pancreatic ductal adenocarcinoma are preserved in their liver metastases. Methods Our study included a cohort of 149 formalin-fixed, paraffin-embedded tissue samples. The methylation status of the samples was experimentally determined by methylation-sensitive high-resolution melting and methylation-specific digital PCR. The digital PCR results were additionally validated by bioinformatic analysis using an independent dataset of 487 samples from the TCGA and GEO databases. The sensitivities, specificities and diagnostic accuracies of the panels for individual cancer types were calculated. Results The methylation-sensitive high-resolution melting analysis led to the selection of the best biomarker candidates and enabled the development of panels that exhibit a sensitivity of 60–93% and a specificity of 85–98% for all included primary tumors and paired normal tissues. The panels tested with digital PCR show a sensitivity of 66.7–100%, a specificity of 94.9–100% and a diagnostic accuracy of 93–100% for hepatocellular carcinoma, cholangiocarcinoma, healthy liver tissue, colorectal liver metastases and liver metastases from pancreatic ductal adenocarcinoma. The bioinformatic analysis revealed similar sensitivities (64-97.4%), specificities (85–98%) and diagnostic accuracies (86–98%). Furthermore, the results show that DNA hypermethylation of the investigated promoter regions is preserved from primary colorectal carcinoma and pancreatic ductal adenocarcinoma to their liver metastases. Conclusions The new methylation biomarker panels exhibit high sensitivity, specificity and diagnostic accuracy and enable successful differentiation between primary and metastatic adenocarcinomas of the liver using methylation-specific digital PCR. A high concordance between methylation-sensitive high-resolution melting analysis, digital PCR and bioinformatic results from publicly available databases was achieved.