Dingemans A M, Brakenhoff R H, Postmus P E, Giaccone G
Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Lab Invest. 1997 Sep;77(3):213-20.
Conflicting results have been reported on the use of cytokeratin-19 (CK-19) in the detection of tumor cells in the peripheral blood of patients with solid tumors. We investigated the expression of CK-19 in lung cancer cell lines and in human lung tumor samples using a nested reverse transcriptase (RT)-PCR to determine the sensitivity and specificity of this method. In addition, blood samples of lung cancer patients and healthy controls were analyzed for the presence of CK-19 transcripts. Amplification products were visualized by ethidium bromide staining and radioactive hybridization with a CK-19-specific probe. Application of a previously described nested RT-PCR for the detection of CK-19 resulted in amplification of the processed pseudogene. Therefore, a more stringent RT-PCR was developed by increasing the annealing temperature. RT-PCR amplification products for CK-19 were detected in 38 of 41 lung cancer cell lines. The three negative cell lines were all variant small-cell lung cancer cell lines. Concordant results were observed between CK-19 detection by immunohistochemistry and by RT-PCR. In serial RNA dilution experiments, CK-19 transcripts could be detected in 18 to 80 pg of total cellular RNA in three cell lines and in 60 ng total RNA in one cell line. The nested RT-PCR had the sensitivity of detecting 50 tumor cells in 10(6) peripheral blood mononuclear cells (PBMNC), and CK-19 transcripts were randomly detected in normal PBMNC. This study shows the necessity in processing parallel samples without reverse transcriptase enzyme to avoid amplification of pseudogenes. A serious problem in the detection of tissue-specific transcripts in PBMNC is the detection of illegitimate transcription levels. In conclusion, although CK-19 may be a useful marker for the detection of lung cancer cells, its application for the detection of circulating tumor cells is not recommended.
关于细胞角蛋白-19（CK-19）在实体瘤患者外周血中肿瘤细胞检测方面的应用，已有相互矛盾的结果报道。我们使用巢式逆转录酶（RT）-PCR研究了CK-19在肺癌细胞系和人肺肿瘤样本中的表达，以确定该方法的敏感性和特异性。此外，还分析了肺癌患者和健康对照者的血样中是否存在CK-19转录本。扩增产物通过溴化乙锭染色和与CK-19特异性探针的放射性杂交进行可视化。应用先前描述的用于检测CK-19的巢式RT-PCR导致加工后的假基因扩增。因此，通过提高退火温度开发了一种更严格的RT-PCR。在41个肺癌细胞系中的38个中检测到了CK-19的RT-PCR扩增产物。三个阴性细胞系均为变异型小细胞肺癌细胞系。免疫组织化学和RT-PCR检测CK-19的结果一致。在连续RNA稀释实验中，在三个细胞系中18至80 pg的总细胞RNA以及在一个细胞系中60 ng总RNA中均可检测到CK-19转录本。巢式RT-PCR检测10(6)个外周血单个核细胞（PBMNC）中50个肿瘤细胞的敏感性，并且在正常PBMNC中随机检测到CK-19转录本。本研究表明，处理无逆转录酶的平行样本以避免假基因扩增的必要性。在PBMNC中检测组织特异性转录本的一个严重问题是非法转录水平的检测。总之，尽管CK-19可能是检测肺癌细胞的有用标志物，但不建议将其用于检测循环肿瘤细胞。
