Hampton Regina, Walker Mignon, Marshall John, Juhl Hartmut
Department of Oncology, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA.
Oncogene. 2002 Nov 7;21(51):7817-23. doi: 10.1038/sj.onc.1205906.
Quantification of circulating cancer cells in whole blood samples by real time quantitative RT-PCR might be of clinical value for monitoring therapeutic effectiveness. In colon cancer patients, carcinoembrynic antigen (CEA) and cytokeratin 20 (CK20) have been frequently used for RT-PCR based tumor cell detection, but the specificity in particular for CEA has been questioned. In this study, we compared real-time RT-PCR for CEA and CK20 and analysed patients with metastatic disease (n=32) and healthy volunteers (n=17). CK20 mean values were elevated in cancer patients (P<0.001) and defined a subgroup (38%) who showed CK20 levels at least 100-fold above the highest value of the healthy control group. In contrast, only two cancer patients (6%) showed elevated CEA levels. Samples of the healthy control group showed exclusively a CEA-PCR product of 79 degrees C melting temperature. Thirty per cent of the colon cancer patients showed an additional product of 82 degrees C melting temperature. The 82 degrees C product was identical with the amplification product of CEA-cDNA and cDNA from different colon cancer cell lines. Colon cancer cells were spiked into normal blood in 10-fold dilutions that resulted in a dose dependent shift of the melt curve from 79 degrees C to the 82 degrees C. Sequencing of the PCR products showed that white blood cells express a splice variant of CEA, which hinders detection of tumor cell cDNA in whole blood samples. Our findings have implications for the use of CEA as a diagnostic molecule (e.g. by RT-PCR). The discovery of a physiologically expressed CEA splice variant might lead to a better understanding of the biological function of CEA and its family members.
通过实时定量逆转录聚合酶链反应（RT-PCR）对全血样本中的循环癌细胞进行定量分析，可能对监测治疗效果具有临床价值。在结肠癌患者中，癌胚抗原（CEA）和细胞角蛋白20（CK20）经常被用于基于RT-PCR的肿瘤细胞检测，但特别是CEA的特异性受到了质疑。在本研究中，我们比较了CEA和CK20的实时RT-PCR，并分析了转移性疾病患者（n = 32）和健康志愿者（n = 17）。癌症患者的CK20平均值升高（P<0.001），并定义了一个亚组（38%），其CK20水平至少比健康对照组的最高值高100倍。相比之下，只有两名癌症患者（6%）的CEA水平升高。健康对照组的样本仅显示出熔解温度为79℃的CEA-PCR产物。30%的结肠癌患者显示出另一种熔解温度为82℃的产物。82℃的产物与CEA-cDNA和来自不同结肠癌细胞系的cDNA的扩增产物相同。将结肠癌细胞以10倍稀释度加入正常血液中，导致熔解曲线从79℃向82℃呈剂量依赖性偏移。PCR产物测序表明，白细胞表达CEA的一种剪接变体，这阻碍了在全血样本中检测肿瘤细胞cDNA。我们的发现对将CEA用作诊断分子（例如通过RT-PCR）具有启示意义。生理表达的CEA剪接变体的发现可能有助于更好地理解CEA及其家族成员的生物学功能。
