Real time RT-PCR for absolute quantification of hormone receptors in breast cancer

C Caraion MD^a,b, N Vincent PhD^a, C Lambert MD^a, S Ursoniu MD PhD^b, C Genin MD PhD^a

^aImmunology Laboratory, Universitary Hospital of St. Etienne, France, ^bUniversity of Medicine and Pharmacy of Timisoara, Romania

Introduction: In breast cancer, the adjuvant treatment of choice is the endocrine therapy. This decision is made after the hormone receptor content determination that has a predictive value for the response to the hormonal therapy. The breast tumors expressing Estrogen (ER)a and Progesterone Receptor (PR) are the most probably "good responders". Nevertheless, approximately a third of this group is primarily resistant to the endocrine agents and another part becomes secondly resistant. The hormone receptor status (ER and PR) is usually determined by immunohistochemistry, a semi-quantitative method. Aim: The aim of this study was to evaluate an absolute quantification of ERa, b, PR and Androgen (AR) receptor expression in breast cancer samples by real-time RT-PCR. We have addressed the question if the precise determination of the hormonal receptors’ expression at molecular level can improve the selection of good candidates for endocrine therapy? Methods: breast tumor samples (10) and breast cancer cell lines (MCF-7 and MDA-MB 231) stored at -80°C were used for RNA extraction. After reverse transcription, the cDNA was used in a real-time PCR with SybrGreen detection system on a Light Cycler (RocheDiagnostics, USA) with specific primers corresponding to the steroid binding domain of the receptors. For absolute quantification we used as standards of concentration newly constructed recombinant plasmids with ERa, b, PR and AR gene inserts. The ARN quality and inter-samples normalization were performed using the housekeeping RPLPO gene as an internal control. Results: Preliminary results show that this method allows us to calculate the expression level of ERa, b, PR and AR genes as gene copies number/microg ARN extract in the breast cancer cell lines and 10 breast tumor samples. Conclusion: real-time RT-PCR quantification assay appears to be an accurate and easily standardisable quantitative method and complementary to IHC for routine clinical analysis. Determination of an extended panel of hormonal receptor expression allows a better characterization of tumors’ molecular profile; the clinical and therapeutical relevance of the absolute quantitative determination of hormonal receptors is in study.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 893 (Molecular pathology).