Identification and Validation of Potentially Clinically Relevant CpG Regions Within the Class 2 Tumor Suppressor Gene in Pancreatic Cancer

In pancreatic cancer treatment, tumor stage-dependent chemotherapies are used to prolong overall survival. By measuring DNA promoter hypermethylation in the plasma of patients with stage IV pancreatic cancer, it was recently shown that promoter DNA methylation of the tumor suppressor gene has a high value for predicting failure of drug treatment with gemcitabine. In this study, we therefore aimed to identify as precisely as possible the region in the promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the transcription start site that were significantly more methylated in pancreatic cancer compared to normal pancreatic acinar tissue. A core CpG island was identified that exhibited abundant tumor DNA methylation and anti-correlation of mRNA expression. To validate our in silico results, we performed bisulfide conversion followed by DNA pyrosequencing of 28 matched formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer cases and six pancreatic cancer cell lines. A defined block of seven CpG sites within the core CpG island was identified, which confirmed our in silico results by showing significantly higher methylation in pancreatic cancer specimens than in normal pancreatic tissue. By selecting this core CpG island, we were able to determine a median overall survival benefit for the low methylation group compared to the high methylation group (702 versus 517 days, = 0.01) in the TCGA pancreatic cancer cohort. We propose a compact pyrosequencing assay that can be used in the future to further investigate the prognostic value of promoter hypermethylation in predicting pancreatic cancer chemoresistance. Therefore, instead of DNA analysis from blood (liquid biopsy), DNA easily extractable from cancer tissue blocks (FFPE material) could be used.