Stathopoulou Aliki, Ntoulia Maria, Perraki Maria, Apostolaki Stella, Mavroudis Dimitris, Malamos Nikos, Georgoulias Vassilis, Lianidou Evi S
Laboratory of Analytical Chemistry, University of Athens, Athens, Greece.
Int J Cancer. 2006 Oct 1;119(7):1654-9. doi: 10.1002/ijc.22017.
The aim of the present study was to decrease the incidence of false positives and to better characterize marginally cytokeratin-19 (CK-19) mRNA positive peripheral blood samples from patients with early stage breast cancer. A new set of highly specific primers for CK-19, which avoids amplification of contaminating genomic DNA, was designed and evaluated to improve the specificity and sensitivity of the previously described methodology. The primers were specifically designed to avoid amplification of contaminating genomic DNA and CK-19 pseudogenes. The breast cancer cell line MCF-7 was used as positive control for the development and analytical evaluation of the assay, while peripheral blood samples from 62 healthy female individuals and 160 patients with early breast cancer were used for the evaluation of the sensitivity and specificity of the new primer pair. The novel designed primer pair was highly sensitive, as it detects up to 1 MCF-7 cell, and specific as none of the healthy individuals had detectable CK-19 mRNA positive cells in their peripheral blood. CK-19 mRNA positive cells were detected in 33 out of 160 (20.6%) patients with early breast cancer. Results obtained by the proposed optimized real-time RT-PCR protocol correlated well with those obtained in the same samples by our previously reported quantitative real-time RT-PCR [concordance in 198/222 (89.2%), p = 0.0022, McNemar test]. The improved method eliminates the incidence of false positives and is highly sensitive and specific. The method could be used in a clinical setting in the near future for continuous monitoring and quantification of circulating epithelial cells in the peripheral blood of patients with operable breast cancer, provided that a quite larger number of clinical samples with a known follow-up will be analyzed.
本研究的目的是降低假阳性率，并更好地对早期乳腺癌患者边缘性细胞角蛋白19（CK-19）mRNA阳性的外周血样本进行特征分析。设计并评估了一套新的高度特异性CK-19引物，该引物可避免污染基因组DNA的扩增，以提高先前所述方法的特异性和敏感性。这些引物经过专门设计，以避免污染基因组DNA和CK-19假基因的扩增。乳腺癌细胞系MCF-7用作该检测方法开发和分析评估的阳性对照，而来自62名健康女性个体和160名早期乳腺癌患者的外周血样本用于评估新引物对的敏感性和特异性。新设计的引物对具有高度敏感性，因为它能检测到多达1个MCF-7细胞，并且具有特异性，因为健康个体的外周血中均未检测到CK-19 mRNA阳性细胞。在160例（20.6%）早期乳腺癌患者中，有33例检测到CK-19 mRNA阳性细胞。所提出的优化实时逆转录聚合酶链反应（RT-PCR）方案所获得的结果与我们先前报道的定量实时RT-PCR在相同样本中获得的结果高度相关[一致性为198/222（89.2%），p = 0.0022，McNemar检验]。改进后的方法消除了假阳性率，具有高度敏感性和特异性。如果分析大量具有已知随访情况的临床样本，该方法在不久的将来可用于临床环境中，对可手术乳腺癌患者外周血中的循环上皮细胞进行连续监测和定量分析。
