Analysis of Tumor-Related Genes In Pancreatic Juice Aiming to be Useful for Early Diagnosis of Pancreatic Cancer

我们分析了DNA提取物中p53的突变，这些DNA提取物不仅来自沉积物（Sed），也来自ERCP抽取的胰液（PJ）上清（sup）和PTC或ERBD获得的胆汁。在胰腺癌（PCa）患者PJ的Sup和Sed中p53突变的发生率分别为43%（9 / 21）和29%(6 / 21)，而在Sup和Sed联合检测中p53突变的发生率为52%（11 / 21）。此外，在细胞逻辑诊断为阴性的PCa病例中，有47%（15例中的7例）检测到p53突变。在25例慢性胰腺炎（CP）患者中，没有p53突变体。在胆道癌患者的胆汁中，p53突变的阳性率在Sup组为50%(15 / 30)，在Sed组为33%（10 / 30）。另一方面，没有胆石症患者携带p53突变体。这些结果表明，p53突变在Sup中的发生率高于Sed， p53突变在Sup中的特异性高于Sed， p53突变在Sed中的特异性很高，p53突变对癌症的特异性很高。21例前列腺癌患者中有11例（52%）在PJ中表达间皮素mRNA， 11例导管内乳头状粘液瘤（IPMN）患者中有5例（45%）在PJ中表达间皮素mRNA， 22例CP患者中有3例（14%）在PJ中表达间皮素mRNA。我们采用实时RT-PCR技术对h-TERT mRNA进行了高灵敏度的定量分析，并测量了血清和PJ中h-TERT mRNA的含量。h-TERT mRNA水平在几乎所有PCa病例中升高，但在相当多的良性病变病例中也升高，表明h-TERT mRNA对癌症的特异性较低。良性病变体液中h-TERT mRNA来源于淋巴细胞。接下来，我们利用甲基化特异性PCR分析了PJ样本中ppENK、SARP2和NPTX2三个CpG岛的甲基化状态。异常甲基化的ppENK PJ中检测出14(50%)的患者28 PCa, 4(27%)的15 IPMN和1(15%)的20与CP和NPTX2中检测出24(77%)的患者35 PCa, 10(63%)的16 IPMN 6(43%)的13和CP。异常甲基化的发生率在PCa ppENK或NPTX2明显高于在CP。此外,我们发现异常甲基化PJ的SARP2 24 PCa 35(74%)的患者,16例IPMN患者中有15例（94%），19例CP患者中有2例（11%）。PCa或IPMN患者SARP2异常甲基化的发生率明显高于CP患者。尽管PCa患者PJ中肿瘤相关基因异常甲基化的发生率非常高，但其对癌症的特异性还不够。良性和恶性IPMN组间无显著差异，提示这些基因的异常甲基化是PCa发展的早期事件，定量检测PJ异常甲基化可能对PCa有潜在的诊断意义。少

We analyzed mutations of p53 in DNA extract not only from the sediment(Sed) but also from the supernatant(sup) of aspirated pancreatic juice(PJ) by ERCP and bile obtained by PTC or ERBD. The incidence of p53 mutations was 43%(9 of 21) and 29%(6 of 21) in the Sup and the Sed of PJ from patients with pancreatic cancer(PCa), respectively, and nurse to 52%(11 of 21) in the combination assay with Sup and Sed. Moreover, p53 mutations were detected in 47%(7 of 15) of PCa cases in which the cyto logic diagnosis was negative. Among 25 patients with chronic pancreatitis(CP), none harbored mutant p53. In bile abtained from patients with biliary tract cancer, the positive rate of p53 mutations was 50%(15 of 30) in the Sup and 33%(10 of 30) in the Sed. On the other hand, there were no pantients with cholelithiasis harboring mutant p53. These results could indicate that the incidence of p53 mutation was higher in the Sup than in the Sed, and the specificity of p53 mutation was higher in the Sup than … More in the Sed, and the specificity of p53 mutation for cancer was very high. Mesothlin mRNA expression in the PJ was discovered in 11(52%) of 21 patients with PCa,5(45%) of 11 with intraductal papillary mucinous neoplasm(IPMN) and 3(14%) of 22 with CP. There was significant difference between PCa and CP for mesothelin mRNA. We developed an highly sensitive quantitative analysis for h-TERT mRNA using real-time RT-PCR and measured the amount of h-TERT mRNA in serum and PJ. The levels of h-TERT mRNA were elevated in almost all cases with PCa but also in considerable cases with benign lesions, suggesting low specificity of h-TERT mRNA for cancer. h-TERT mRNA in body fluids from benign lesions originates from lymphocyte.Using methylation-specific PCR, we nextly analyzed the methylation status of 3 CpG islands of ppENK, SARP2 and NPTX2 in PJ samples. Aberrant methylation of ppENK in the PJ was detected in 14(50%) of 28 patients with PCa, 4(27%) of 15 with IPMN and 1(15%) of 20 with CP, and that of NPTX2 was detected in 24(77%) of 35 patients with PCa, 10(63%) of 16 with IPMN and 6(43%) of 13 with CP. The incidence of aberrant methylation of ppENK or NPTX2 was significantly higher in PCa than in CP. Furthermore, we found aberrant methylation of SARP2 in PJ in 24(74%) of 35 patients with PCa, 15(94%) of 16 with IPMN and 2(11%) of 19 with CP. The incidence of aberrant methylation of SARP2 was significantly higher in PCa or IPMN than in CP. Although the prevalence of aberrant methylation of the tumor-related genes in PJ from PCa was strikingly high, its specificity for cancer was not sufficient. There was no significant difference between benign and malignant groups of IPMN, suggesting that aberrant menthylation of these genes is a early event in PCa development, and quantitative detection of aberrant methylation in PJ may have potential diagnostic implication for PCa. Less