实时荧光定量PCR检测大肠癌腹腔微转移及其临床意义

Quantitative Detection of Disseminated Cancer Cells in Peritoneal Washes with Real-Time RT-PCR for Patients with Colorectal Carcinoma and Its Clinical Significance

[目的]应用实时荧光定量PCR技术检测大肠癌腹腔内游离癌细胞并探讨其临床意义。[方法]2003年5月至2004年1月,收集在日本爱知癌症中心手术81例大肠癌病例的腹腔冲洗液,每个样本的cDNA均应用两种引物合成(随机引物和寡核苷酸引物),并在罗氏实时荧光定量PCR(LightCycler)上进行定量分析。每个样本同时进行常规细胞学检查。所有病例术后经过平均为1年的随访。[结果]CEA mRNA和CK-20 mRNA的阳性率和阳性值均与肿瘤的浸润深度(T)、分期以及淋巴结转移相关。在一个合理界定值上,CEA和CK-20检测的敏感度与特异度均为100%,而常规的细胞学检查则为33%和100%。[结论]CEA和CK-20 mRNA定量分析是检测大肠癌腹腔微转移的敏感和特异的方法,CEA和CK-20 mRNA水平的异常与术后的无瘤生存率显著相关。

[Purpose ] To detect the free cancer cells in peritoneal cavity in patients with colorectal cancer by quantitative real-time RT-PCR method and investigate its clinical significance. [Methods ] From May 2003 to Jan. 2004, peritoneal wash was obtained from 81 cases with colorectal cancer in Aichi cancer center, cDNA was synthesized from mRNA extracted from peritoneal washes by 2 kinds of primers （random primer and oligo primer）. Quantification of CEA and CK-20 mRNA was performed on a LightCycler instrument with hybridization probe. A part of each peritoneal wash was also examined by conventional cytology. All patients were followed up with an average period of 12 months. [Results] Both CEA mRNA and CK-20 mRNA positive rates and values were significantly correlated with depth of tumor invasion, staging, lymph node metastasis. Sensitivity and specificity of CEA and CK-20 realtime RT-PCR with an appropriate cutoff value were all 100%, whereas those of conventional cytology were 33%, and 100%, respectively. [Conclusion] Both CEA and CK-20 mRNA quantifications are sensitive and specific methods for detecting micrometastasis in the peritoneal washes with colorectal cancer. Abnormality of CEA and CK-20 mRNA in peritoneal washes is correlated with disease free survival for colorectal cancer.