Detection and clinical application of circulating cancer cells, using highy pricise cancer cell determination system

用实时定量逆转录聚合酶链式反应(RT-PCR)检测骨髓和外周血中的播散性癌细胞。首先，我们比较了4个胃癌细胞系和3个正常组织细胞系CEA、CK18和CK20的表达，以选择最适合检测骨髓播散性胃癌细胞的标志物。CK20是最有前景的标记物，因为正常细胞系的表达水平极低，胃癌细胞系与正常组织细胞系之间的差异约为50-100倍。我们还利用该系统对47例胃癌患者的骨髓RNA进行了筛选。在我们检测的三种标志物中，只有大约CK20才能发现47例患者中有27例阳性。接下来，我们研究了用定量RT-PCR检测胃癌患者外周血中的肿瘤细胞。用于检测少量胃癌细胞的RT-PCR常见的信使核糖核酸靶点是CK18、CK19、CK20和CEA。CK19和CK20mRNA拷贝数在未手术或复发的胃癌患者中显著高于正常对照组(P=0.0087，P=0.0022)。CK19阳性和阴性的根治性疾病患者术前诊断的肿瘤分期有显著差异。CK19阳性和阴性患者的5年生存率分别为50.0%和79.0%(P=0.0347)。综上所述，实时定量RT-PCR可以检测到外周血中胃癌的微转移。CK19是一种比CK18、CK20和CEA更好的外周血标志物，可用于临床判断预后或制定术后辅助治疗策略。

Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect disseminated cancer cells in bone marrow and peripheral blood. First of all, we compared CEA, CK18 and CK20 expression using 4 gastric cancer cell lines and 3 normal tissue cell lines in order to select the most appropriate marker for detection of disseminated gastric cancer cell in bone marrow. CK20 proved to be the most promising marker since the expression level of normal cell lines was extremely low and about 50- to 100-fold differences were found between gastric carcinoma cell lines and normal tissue cell lines. We also screened bone-marrow RNA of 47 patients with gastric cancers, using this system. Among the three markers we tested, with only about CK20 could we find that 27 of 47 patients were positive.Next, we studied the detection of circulating tumor cells in peripheral blood in patients with gastric cancer using Quantitative RT-PCR. Common mRNA targets for RT-PCR for detection of small numbers of cancer cells in gastric cancer are CK18,CK19,CK20, and CEA. The number of CK19 and CK20 mRNA copies was significantly increased in patients with a non-curative operation or recurrence of gastric cancer (CK19;p=0.0087, CK20;p=0.0022) compared with healthy volunteers. There was a significant difference in tumor stages between CK19 positive and negative patients with curative disease on preoperative diagnosis. Five-year survival rates of patients with CK19 positive or negative cases were 50.0% or 79.0%, respectively (p=0.0347). In conclusion, micrometastases of gastric cancer can be detected in circulating peripheral blood using quantitative real-time RT-PCR. CK19 is a better marker than CK18,CK20 and CEA for peripheral blood, and could be clinically useful to estimate prognosis or to make a postoperative strategy of adjuvant treatment.