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

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-

SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer

“…mRNA was isolated from the total RNA by use of the Dynabeads mRNA purification kit (Invitrogen) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems) and was used for keratin 19 (KRT19, also known as CK-19) expression studies in CTCs as previously described (21 ).…”

“…We evaluated KRT19 mRNA expression in all matched EpCAM-positive immune-magnetically isolated CTC fractions, because this epithelial marker has been extensively used to verify the presence of CTCs (1,4,5,20,21 ). In early breast cancer we found 20 (36.4%) of 55 samples to be positive for KRT19 expression, whereas in the group of patients with verified metastasis 24 (40.7%) of 59 were positive.…”

Circulating Tumor Cells in Breast Cancer: Detection Systems, Molecular Characterization, and Future Challenges

“…Our group developed an RT-qPCR assay for KRT19 mRNA (36,37 ) and evaluated both its analytical and diagnostic sensitivity and its specificity and clinical potential for the molecular detection of occult carcinoma cells in peripheral blood of breast cancer patients (10 -13 ).…”

DNA Methylation of Tumor Suppressor and Metastasis Suppressor Genes in Circulating Tumor Cells

“…Peripheral blood (20 mL in EDTA) was obtained at the middle of vein puncture after the first 5 mL of blood was discarded. All blood samples were processed within 4 h of collection as described (31,32 ). All individuals signed an informed consent to participate in the study, which was approved by the ethics and scientific committees of our institutions.…”