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

为了建立高灵敏度的胰腺癌诊断方法，我们建立了HPA/TRAP法、实时TRAP法、胰液中mRNA定量检测方法和k-ras突变的电化学分析方法。端粒重复序列扩增结合杂交保护试验(HPA/TRAP)是检测胰液中端粒酶活性的高灵敏度和快速方法。HPA/TRAP检测有助于胰腺癌与IPMN或胰腺炎的鉴别。TRAP结合实时定量聚合酶链式反应(Real-Time TRAP，Real-time TRAP)检测端粒酶活性也是一种灵敏、快速的方法。此外，我们还定量检测了胰液中端粒酶亚基hTERT的mRNA。我们发现hTERT在胰腺癌、IPMN和慢性胰腺炎中的表达有显著差异，并对胰液中几个胰腺癌相关基因的mRNA进行了定量检测。MUC1和MUC5AC在胰腺癌组织中高表达，定量检测胰液中MUC1mRNA和MUC5AC基因的表达对胰腺癌的术前诊断有较高的价值。S100家族成员S100A6在显微切割的胰腺癌细胞中表达显著升高。为探讨电化学芯片检测胰腺癌组织中k-ras基因突变的有效性，采用电化学芯片检测胰腺癌组织中的k-ras基因突变。在85%的胰腺癌组织中检测到K-ras基因突变，这与依赖于聚合酶链式反应的优先同源双链形成试验的结果一致。我们发现ECA芯片是一种敏感、快速、可靠的筛查临床样本中点突变的方法。

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.