Identification and Validation of Potentially Clinically Relevant CpG Regions within the Class 2 Tumor Suppressor Gene SFRP1 in Pancreatic Cancer

SIMPLE SUMMARY: Pancreatic cancer is one of the deadliest cancer entities with five-year survival rates of less than 11%. Besides standardly used surgical therapy, available chemotherapies are increasingly used to prolong overall survival. Promoter hypermethylation of the tumor suppressor gene Secreted frizzled-related protein 1 (SFRP1) seems to be correlated with poor response to gemcitabine treatment in stage IV pancreatic cancer. The aim of this study was to find and characterize key CpG sites in the promoter region of the SFRP1 gene. We identified a core CpG island whose DNA methylation may have a decisive influence on SFRP1 expression loss and unfavorable overall survival. Its specific analysis may predict response of stage IV tumors to chemotherapy in the future. ABSTRACT: 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 SFRP1 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 SFRP1 promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the SFRP1 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 SFRP1 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 SFRP1 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 SFRP1 methylation group compared to the high SFRP1 methylation group (702 versus 517 days, p = 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 SFRP1 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.