Real-time quantification of CK-19 mRNA-positive cells in peripheral blood of breast cancer patients using the lightcycler system

Clin Cancer Res. 2003 Nov 1;9(14):5145-51.


Purpose: The purpose of this research was to develop a quantitative real-time reverse transcription-PCR (RT-PCR) for CK19-mRNA and evaluate its clinical potential for the molecular detection of occult carcinoma cells in the peripheral blood of breast cancer patients.

Experimental design: The method is based on real-time monitoring during PCR of fluorescently labeled specific hybridization probes for CK19-mRNA. The breast cancer cell line MCF-7 was used for the development and analytical evaluation of the assay. We analyzed blood samples from 89 healthy blood donors, 77 patients with early breast cancer (stage I-II) postoperatively, and 47 patients with previously untreated metastatic disease (stage IV) before and after chemotherapy. All of the samples were also analyzed by nested RT-PCR.

Results: The method is highly sensitive and specific, because only 2 of 89 (2.2%) of the healthy control subjects had detectable CK19-mRNA+ cells. In 77 patients with early breast cancer, CK19-mRNA+ cells were detected in 24 (31.2%) before and 5 (6.5%) after adjuvant chemotherapy, and their levels differed significantly (P < 0.001, Wilcoxon test). In 47 patients with verified metastases 19 (40.4%) and 20 (42.6%) were found positive before and after chemotherapy, and no significant difference in CK19-mRNA+ cell levels was observed (P = 0.96, Wilcoxon test). Results obtained by the proposed real-time RT-PCR method correlated well with those obtained for the same samples by nested RT-PCR [concordance in 312 of 337 (92.6%); P = 0.69, McNemar test].

Conclusions: The developed method is highly sensitive and specific, and can be used for high-throughput continuous monitoring and quantification of circulating epithelial cells in the peripheral blood of breast cancer patients.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Antineoplastic Agents / therapeutic use
  • Breast Neoplasms / blood*
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / genetics
  • DNA Primers
  • Female
  • Humans
  • Keratins / blood*
  • Keratins / genetics
  • Middle Aged
  • Neoplasm Staging
  • Neoplastic Cells, Circulating*
  • RNA, Messenger / analysis*
  • RNA, Neoplasm
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Tumor Cells, Cultured


  • Antineoplastic Agents
  • DNA Primers
  • RNA, Messenger
  • RNA, Neoplasm
  • Keratins