Helfrich W, ten Poele R, Meersma G J, Mulder N H, de Vries E G, de Leij L, Smit E F
Department of Clinical Immunology, University Hospital Groningen, The Netherlands.
Br J Cancer. 1997;76(1):29-35. doi: 10.1038/bjc.1997.331.
The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples.
循环系统中肿瘤细胞的存在可能预示疾病复发和转移。为改进现有的细胞学或免疫细胞学检测方法，我们开发了一种灵敏且定量的技术，用于检测血液中的癌细胞，即利用逆转录聚合酶链反应（RT-PCR）来鉴定泛癌相关肿瘤标志物EGP-2（KSA或17-1A抗原）的转录本。使用已知浓度的内部重组竞争RNA标准品对EGP2 mRNA的量进行定量，该标准品在同一反应中既被逆转录又被共同扩增，从而能够可靠地评估样品中EGP2 mRNA的初始量。用MCF-7乳腺癌细胞接种血液进行的校准研究表明，该检测方法能够在1.0×10⁶个白细胞中检测出10个肿瘤细胞。PCR检测显示，正常骨髓表达低水平的EGP2 mRNA，尽管用抗EGP2单克隆抗体MOC31进行免疫细胞化学检测未发现任何阳性染色细胞。使用这种RT-PCR检测方法进行分析可能在临床样本中循环肿瘤细胞的评估方面具有应用价值。
