Wang Ying, Yamaguchi Yasushi, Watanabe Hiroyuki, Ohtsubo Koushiro, Wakabayashi Tokio, Sawabu Norio
Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan.
J Gastroenterol. 2002;37(10):831-9. doi: 10.1007/s005350200137.
The sensitivity of bile cytology for the diagnosis of biliary tract carcinoma (BTCa) is still low. In addition, the incidence of detection of genetic mutations in the bile of BTCa is not satisfactory yet. To improve the molecular diagnosis of BTCa, we analyzed p53 and K- ras mutations in DNA extracted from not only the sediment but the supernatant of bile samples.
Polymerase chain reaction-single-strand conformation polymorphism and direct sequencing were used for analyses of p53 mutations in exons 5 through 8. K- ras mutations at codon 12 were examined by mutant allele-specific amplification.
In bile supernatant from patients with BTCa, p53 and K- ras mutations were detected in 50.0% (15/30) and 56.7% (17/30) of cases, respectively. The incidence of p53and K- ras mutations in the sediment was 33.3% and 43.3%, respectively. When a combination assay with both genes was used, molecular abnormalities were detected in 80.0% of cases, including 3 in which p53 alone was positive. In addition, either p53 or K- rasmutations were detected in 12 of 15 (80.0%) cases of BTCa in which the cytologic diagnoses were negative. p53 mutations were detected in neither supernatant nor sediment in 20 patients with cholelithiasis, although the incidence of K- ras mutations in the sediment was 20%.
The incidence of p53 and K- ras mutations is higher in the supernatant than in the sediment, and simultaneous analyses of p53 and K- ras in the two bile fractions could enhance the genetic diagnosis of BTCa. Notably, the specificity of p53 mutations for cancer was very high in bile samples, and the sensitivity was also relatively high.
胆汁细胞学检查对胆道癌（BTCa）诊断的敏感性仍然较低。此外，BTCa患者胆汁中基因突变的检出率仍不尽人意。为改善BTCa的分子诊断，我们不仅分析了胆汁样本沉淀物中的DNA，还分析了上清液中的p53和K-ras基因突变情况。
采用聚合酶链反应-单链构象多态性分析及直接测序法分析外显子5至8的p53基因突变情况。通过突变等位基因特异性扩增检测第12密码子的K-ras基因突变。
在BTCa患者的胆汁上清液中，p53和K-ras基因突变的检出率分别为50.0%（15/30）和56.7%（17/30）。沉淀物中p53和K-ras基因突变的检出率分别为33.3%和43.3%。当同时检测这两个基因时，80.0%的病例检测到分子异常，其中3例仅p53基因呈阳性。此外，在15例细胞学诊断为阴性的BTCa病例中，有12例（80.0%）检测到p53或K-ras基因突变。20例胆石症患者的上清液和沉淀物中均未检测到p53基因突变，不过沉淀物中K-ras基因突变的检出率为20%。
上清液中p53和K-ras基因突变的检出率高于沉淀物，同时分析胆汁两个部分中的p53和K-ras基因可提高BTCa的基因诊断水平。值得注意的是，在胆汁样本中p53基因突变对癌症的特异性非常高，敏感性也相对较高。
