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A highly specific real‐time RT‐PCR method for the quantitative determination of CK‐19 mRNA positive cells in peripheral blood of patients with operable breast cancer
Author(s) -
Stathopoulou Aliki,
Ntoulia Maria,
Perraki Maria,
Apostolaki Stella,
Mavroudis Dimitris,
Malamos Nikos,
Georgoulias Vassilis,
Lianidou Evi S.
Publication year - 2006
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.22017
Subject(s) - breast cancer , primer (cosmetics) , false positive paradox , cancer , taqman , amplicon , concordance , mcnemar's test , medicine , biology , microbiology and biotechnology , real time polymerase chain reaction , polymerase chain reaction , pathology , oncology , gene , genetics , chemistry , statistics , mathematics , organic chemistry , machine learning , computer science
Abstract 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. © 2006 Wiley‐Liss, Inc.