胰腺癌患者胰液中K-ras基因突变的检测及其意义

K ras mutation in pancreatic juice from patients with pancreatic carcinoma

目的 通过对经逆行胰胆管造影（ＥＲＣＰ） 所获胰液的Ｋｒａｓ 基因突变检测，以探索胰腺癌诊断的新方法。方法 应用聚合酶链反应（ＰＣＲ）限制性片段长度多态性的方法检测胰腺良、恶性疾病组织物、胰液上清和细胞的Ｋｒａｓ 突变。结果 胰腺癌和胰腺良性疾病标本分别有８０％ 和３３ ％的Ｋｒａｓ 突变率。胰液上清检测时，ＰＣＲ有１８％ 未获得成功，胰腺癌胰液Ｋｒａｓ 突变率为７１％ （１５／２１），而胰腺良性疾病无一例有Ｋｒａｓ 的突变。用胰液细胞有４ ％ ＰＣＲ 未能获得成功，胰腺癌胰液细胞Ｋｒａｓ的突变率为６５％ （１１／１７） ，良性疾病中有１ 例（１／７） 检测到Ｋｒａｓ 突变。综合上清与细胞的结果，在７８ ％（２１／２７） 的胰腺癌患者的胰液中检测到Ｋｒａｓ 突变，明显高于胰腺良性疾病患者的８％ （１／１３）（Ｐ＜０－００１）。Ｋｒａｓ 的突变率与肿瘤的部位及大小无统计学差异。结论 应用胰液检测Ｋｒａｓ 突变有助于胰腺癌的诊断。

Objective Pancreatic neoplasm, which is considered as one of the most malignant tumors in humans, is now increasing steadily in frequency. The 5 year survival rate is less than 5%. In order to detect it in early stage, we tried to analyze K ras mutation in pancreatic juice collected during ERCP after injection of secretin. Mutation of coden 12 in exon 1 of K ras gene was reported as high as 90% in pancreatic cancer, but much less in benign pancreatic disease. Methods 47 patients at Peking Union Medical College Hospital between 1994 and 1998 were enrolled. Pancreatic juice collected during ERCP after injection of 100u secretin , was briefly centrifuged and the sediment was resuspended with PBS. Pancreatic juice supernatant and pancreatic cells were stored at -80℃ separately. DNA of the cells and supernatant of the juice were extracted. K ras point mutation was investigated in the DNA of these supernatant and cells by means of two stage polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Results When the supernatant of the pancreatic juice was used, the PCR successful rate was 82% , K ras mutation rate of the pancreatic carcinomas was 71% (15/21) compared with 0% (0/7) in benign pancreatic disease (3 chronic pancreatitis, 3 insulinoma and 1 pancreatic cyst). When the centrifuged pancreatic cells were used, the successful rate increased to 96%, sixty five percent (11/17) of pancreatic carcinomas had K ras mutation; mutation was also detected in 1/7 of the cases with benign pancreatic diseases (an insulinoma). In total, 78% (21/27) of the cases with pancreatic carcinomas has mutant alleles compared with 8% (1/13) of the benign pancreatic diseases( P <0.001) when pancreatic juice was used to detect K ras gene point mutation. In terms of the location of the pancreatic cancer, K ras mutation rate was not statistically different between the head (80%) and the body + tail(75%). The mutation rate did not vary with the size of the tumor. Conclusion Detection of coden 12 of K ras mutation in pancreatic juice can be used in differentiating pancreatic carcinomas from benign pancreatic diseases. It has high sensitivity and specificity.