A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood
A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood
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DOI：
10.1023/a:1008317512253
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发表时间：
2001-01-01
影响因子：
50.5
通讯作者：
Vandekerckhove, F
中科院分区：
文献类型：
--
作者：
Aerts, J;Wynendaele, W;Vandekerckhove, F
背景资料：乳腺癌患者隐匿性癌细胞的检测已被证明可以预测疾病的复发和转移.Materials and Methods：To improve on molecular detection of breast carcinoma cells in blood，we have developed a sensitive and quantitative assay using real-time quantitative RT-PCR identifying transcripts of the cytokeratin-19（CK 19）gene.Results：这种实时定量RT-PCR灵敏、准确，在宽的动态范围内具有高重现性，允许对不同输入浓度的样品进行同时定量分析。此外，该方法提供了优于经典定量PCR方法（竞争性RT-PCR、北方印迹）的若干技术优势，例如由于不存在PCR后操作而降低污染的可能性，由于不存在PCR后处理时间（无琼脂糖凝胶电泳）而提高样品通量。在这项初步研究中，我们在< 10%的志愿者中检测到CK 19转录水平显著升高。结论：采用这种真实的时间定量RT-PCR分析CK 19 mRNA可能被证明在评估乳腺癌患者外周血中循环肿瘤细胞、骨髓或淋巴结中的微转移中具有临床意义。该技术在临床人群中的应用可能会改善转移性乳腺癌的诊断和监测，其验证目前正在进行中。
Background: The detection of occult carcinoma cells in patients with breast cancer has been shown to predict disease recurrence and metastasis.Materials and methods: To improve on molecular detection of breast carcinoma cells in blood, we have developed a sensitive and quantitative assay using real-time quantitative RT-PCR identifying transcripts of the cytokeratin-19 (CK19) gene.Results: This real-time quantitative RT-PCR is sensitive, accurate and has a high reproducibility within a wide dynamic range, which permits simultaneous quantitative analysis of samples with varying input concentrations. Furthermore, the procedure offers several technical advantages over classic quantitative PCR methods (competitive RT-PCR, Northern blotting) such as decreased likelihood of contamination due to absence of post-PCR manipulations, high sample throughput because of absence of post-PCR processing time (no agarose gel electrophoresis). In this pilot study, we detected significantly elevated CK19 transcript levels in < 10% of the volunteers, in . 30% of stage I-IIIa patients preoperatively and in > 70% of the and stage IV breast cancer patients.Conclusions: Analyses using this real time quantitative RT-PCR for CK19 mRNA may prove to have clinical implications in the assessment of circulating tumour cells in peripheral blood, micrometastases in bone marrow or lymph nodes in breast cancer patients. Application of this technique in a clinical population may improve diagnosis and monitoring of metastatic breast cancer and its validation is currently ongoing.