HPLC micromethod for simultaneous measurement of estradiol, progesterone, androgen and glucocorticoid receptor levels. Application to breast cancer biopsies

Estradiol (ER), progesterone (PR), androgen (AR) and glucocorticoid receptor (GR) levels were assayed in 25 breast cancer tumors. The tissue was pulverized and homogenized in buffer, then divided into two parts: one was assayed by the standard dextran-coated charcoal method (DCC), with Scatchard plot analysis, the other was assayed by a micromethod developed in our laboratory, as described below: --incubation of the cytosol with several ligands (labelled and unlabelled) selected to avoid unwanted cross-reactions --DCC separation, followed by extraction of all receptor-bound steroids by precipitation of proteins with methanol/TCA --separation of these steroids on a high pressure liquid chromatography (HPLC) column using a methanol/water solvent --collection of the fractions of the column outlet and counting. Use of three labelled ligands and appropriate unlabelled ligands allowed assays of the four receptors. This micromethod was highly correlated with the standard method: ER = 0.985 (P less than 0.001); PR = 0.999 (P greater than 0.001); AR = 0.989 (P less than 0.001); GR = 0.867 (P less than 0.001). Thresholds of positivity were not modified. This micromethod allowed simultaneous measurement of several receptors in 40 mg biopsy specimens and can be applied to other hormone-dependent tissues.