Quantitation of estradiol receptors alpha and beta and progesterone receptors in human breast tumors by real-time reverse transcription-polymerase chain reaction. Correlation with protein assays.

Hormone receptor content is the most useful parameter for predicting hormone response therapy in breast cancer. The high frequency of primary and secondary resistance to treatment makes it necessary to find out other parameters in order to improve the prediction of response to treatment. The newly described estrogen receptor beta (ERbeta) is a potential candidate. Using real-time quantitative RT-PCR, we evaluated estrogen receptor alpha (ERalpha), ERbeta, and progesterone receptors (PR) in comparison with ERalpha and PR protein content measured with the enzyme immunoassay (EIA), in a retrospective series of 98 breast tumors. We obtained a highly significant correlation between mRNA and EIA assays for ERalpha and PR (r=0.79 and r=0.71, respectively; P<0.001). We confirmed the low level of ERbeta mRNA transcripts in comparison to ERalpha in breast tumors. We did not find any statistically significant correlation between the absolute ERbeta mRNA level and ERalpha or PR mRNA level, and ERalpha or PR-EIA. We found a significant correlation between ERalpha mRNA and PR mRNA expressions. We did not find any correlation between ERbeta mRNA and clinical features of the patients (age, menopausal status, tumor size, and nodal status), nor with the histological type of the tumor. In conclusion, the accuracy of the present quantitative RT-PCR assay makes it suitable for a routine clinical use. In addition, the present results suggest that, ERbeta mRNA expression is independent of the classical parameters.

Real-time quantitative PCR determination of urokinase-type plasminogen activator receptor (uPAR) expression of isolated micrometastatic cells from bone marrow of breast cancer patients.

Disseminated tumor cells (DTC) in bone marrow are independently related to poor outcome in patients with breast cancer. Phenotypic characterization of DTC may be useful to improve evaluation of the metastasizing potential of DTC and also to more accurately target aggressive tumor cells. DTC were screened in bone marrow aspirates from breast cancer patients by immunocytochemistry with an anticytokeratin (anti-CK) antibody (A45B/B3). Because the cell permeabilization and fixation required for intracellular CK staining is deleterious for mRNA, we used microaspiration to isolate single tumor cells stained with a monoclonal antibody directed against a membrane epitope, epithelial cell adhesion molecule (EpCAM), in CK-positive cases. Urokinase-type plasminogen activator receptor (uPAR) was quantified by real-time quantitative RT-PCR. The SKBR3 human breast cancer cell line was used to calibrate RT-PCR. A linear relationship was observed between the cycle threshold (Ct) of uPAR and 18S gene expression and SKBR3 cells spiked (1, 3, 7, 10 and 20) in control patient bone marrow. EpCAM-positive cells were aspirated in 21 out of 25 bone marrow specimens from breast cancer patients with CK-positive cells and uPAR mRNA expression was determined in 16 cases. A high level of uPAR mRNA in DTC was detected in 8 out of 16 patients (50%) and was associated with a more aggressive primary tumor phenotype (estrogen receptor [ER]-negative, progesterone receptor [PR]-negative or HER2-positive) (p = 0.01). We demonstrated that real-time quantitative RT-PCR was reliably adapted to phenotype analysis of isolated micrometastatic cells. A larger study would be useful to confirm the importance of uPAR to define higher risk subgroups of breast cancer patients with micrometastatic disease.