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


METHODS
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.
Stathopoulou
Aliki
A
Laboratory of Analytical Chemistry, University of Athens, Athens, Greece.
Gizi
Anna
A
Perraki
Maria
M
Apostolaki
Stella
S
Malamos
Nikos
N
Mavroudis
Dimitris
D
Georgoulias
Vassilis
V
Lianidou
Evi S
ES
D000293:Adolescent; D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000097:blood* / Q000188:drug therapy / Q000235:genetics; D017931:DNA Primers; D005260:Female; D006801:Humans; D007633:Keratins / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D007633:Keratins