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Percutaneous fine-needle aspiration cytology or biopsy has been used for pathological confirmation in pancreatic cancer. Sometimes, it is difficult to approach the mass because of surrounding major vessels, and there is a risk of seeding. Although endoscopic retrograde cholangiopancreatography (ERCP)-guided pancreatic duct brush cytology is less invasive, its reliability is very low. Recently, aberrantly methylated genes were reported in pancreatic cancer tissue. This study was to develop a novel molecular diagnostic approach based on epigenetic characteristics.We enrolled pathologically proven 33 pancreatic cancer patients and 22 benign pancreaticobiliary disease patients. The ERCP-guided pancreatic duct brush cytology samples were obtained. Genomic DNA was extracted, and NPTX2 CpG island hypermethylation was examined quantitatively by real-time polymerase chain reaction amplification after chemical modification.Pancreatic cancer cytology samples had statistically significant higher levels of NPTX2 methylation compared with benign diseases, and the optimal cutoff value of NPTX2 methylation was 1.2%. The sensitivity was 87%, and specificity was 80%, whereas pathological examination by ERCP-guided pancreatic duct brush cytology had a sensitivity of 38%.The quantitative analysis of NPTX2 hypermethylation may play a role in making highly sensitive and less invasive diagnosis of pancreatic cancer. Therefore, NPTX2 hypermethylation could be a promising molecular diagnostic marker.