Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients

摘要

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive cancer. There are various sub-cellular events (both genetic and epigenetic) that get dysregulated leading to tumorigenesis. Methylation in promoters of tumor suppressor genes is one of these epigenetic phenomena contributing to the pathogenesis of cancer. Genes analyzed for promoter methylation status in this study namely SPARC (Secreted Protein Acidic and Rich in Cysteine, UCHL1 (ubiquitin carboxy-terminal hydrolase L1), NPTX2 (neuronal pentraxin 2), PENK (proenkephalin) had been studied in pancreatic cancer, but there is a need to check methylation in these genes as circulatory non-invasive markers. This study analyzed the absolute quantification of methylation levels of SPARC, UCHL1, PENK, and NPTX2 genes promoters in PDAC patients as well as in chronic pancreatitis (CP) patients and healthy subjects (HC) and evaluated its clinical significance in PDAC. Materials and methods The study included 65 PDAC patients, 25 CP patients, and 25 healthy controls. DNA was extracted from their plasma samples and subsequently given bisulfite treatment. Absolute quantization of methylated and unmethylated copies of gene promoters of all the four genes was performed using real-time PCR (SYBR green) by the standard curve method. Methylation levels were expressed as methylation index (MI) for each gene in each patient. MI was calculated from absolute copy numbers as follows: MI-methylated copy number/methylated copy number + unmethylated copy number). These indices were used to compare gene methylation levels within different groups and to correlate with clinicopathological features and survival of pancreatic cancer patients. An appropriate statistical analysis was applied. Results Methylation indices for all the four genes in PDAC cases were found to be significantly higher as compared to that in healthy individuals. SPARC MI values were found to differentiate early-stage PDAC patients from CP patients. PDAC patients with the metastasized disease and stage IV disease were found to have high MI for the SPARC gene as well as for the NPTX2 gene, while a higher UCHL1 methylation index was found to correlate with an advanced stage of the disease. Higher MI values for SPARC and NPTX2 genes were found to associate with poor survival in patients with PDAC. Conclusion Methylation load in the form of MI for each of the four genes assessed in plasma may emerge as a non-invasive biomarker to differentiate pancreatic cancer from healthy individuals. But only SPARC and NPTX2 hypermethylation were able to distinguish pancreatic cancer from chronic pancreatitis. Association of aberrant methylation in SPARC and NPTX2 gene with metastasis and poor survival of patients suggest the role of methylation in these genes as prognostic markers.