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

采用真实的实时定量逆转录聚合酶链反应（RT-PCR）检测骨髓和外周血中播散性癌细胞。首先，我们比较了4种胃癌细胞系和3种正常组织细胞系的CEA、CK 18和CK 20的表达，以选择最适合检测骨髓中播散的胃癌细胞的标志物。CK 20被证明是最有希望的标记物，因为正常细胞系的表达水平极低，并且在胃癌细胞系和正常组织细胞系之间发现约50- 100倍的差异。我们还使用该系统筛选了47例胃癌患者的骨髓RNA。在所检测的三种肿瘤标志物中，仅在CK 20左右检测到27例阳性。接下来，我们采用定量RT-PCR方法检测了胃癌患者外周血中循环肿瘤细胞。用于检测胃癌中少量癌细胞的RT-PCR的常见mRNA靶标是CK 18、CK 19、CK 20和CEA。与健康志愿者相比，非根治性手术或胃癌复发患者的CK 19和CK 20 mRNA拷贝数显著增加（CK 19;p=0.0087，CK 20;p=0.0022）。术前诊断CK 19阳性与阴性患者的肿瘤分期差异有统计学意义。CK 19阳性或阴性患者的5年生存率分别为50.0%和79.0%（p=0.0347）。结论：应用实时荧光定量RT-PCR技术可以检测外周血中胃癌的微转移。CK 19是一种较CK 18、CK 20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方案。

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.