Development of genetic diagnosis system for pancreatic cancer using modified telomeric repeat amplification protocols and electrochemical array chip

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To establish highly sensitive assays for the diagnosis of pancreatic cancer, we developed HPA/TRAP assay, real time TRAP assay, methods for quantitative measurements of mRNA in pancreatic juice, and electrochemical analysis of k-ras mutations.Telomeric repeat amplification protocol combined with hybridization protection assay (HPA/TRAP) was highly sensitive and rapid to detect telomerase activity in pancreatic juice. HPA/TRAP assay was useful to differentiate pancreatic cancer from IPMN or pancreatitis. TRAP with real-time PCR (real time TRAP) was also sensitive and more rapid to detect the telomerase activity. Futhermore, we quantitatively measured mRNA of hTERT, subunits of telomerase, in pancreatic juice. We found significant difference in hTERT expression among pancreatic cancer, IPMN and chronic pancreatitis.Several mRNAs of pancreatic cancer related genes were also quantitatively measured in pancreatic juice. MUC1 and MUC5AC were highly expressed in pancreatic cancer and quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer. S100A6,a member of S100 family, was significantly higher in microdissected pancreatic cancer cells. Quantification of S100A6 mRNA in pancreatic juice is also a promising tool for diagnosis of pancreatic cancer.To investigate the validity of the electrochemical array (ECA) chip, k-ras muations were detected with ECA chip in pancreatic cancer tissues. K-ras mutations were identified in 85% of pancreatic cancer, which was identical to the results obtained by PCR-dependent preferential homoduplex formation assay. We found that ECA chip is a sensitive, rapid, and reliable for screening point mutations in clinical samples.