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Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using 125I-E2 and 3H-Org 2058 as tracers. The interaction of 125I-E2 with ER and plasma proteins in comparison to 3H-E2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. 125I-E2 bound the same molecular forms displayed by 3H-E2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of 125I and 3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV < 8.0 %). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less purturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.

Estrogen (ER) and progesterone (PgR) receptor content was assayed in 290 premenopausal women with primary breast cancer, in order to investigate the influence of endogenous hormones on cytoplasmic receptor concentrations throughout the menstrual cycle, subdivided into four phases of ovarian function (early and late follicular phase, early and late luteal phase). Of the total population, 231 (79.7%) patients were ER positive and 59 (20.3%) were ER negative; 220 (75.9%) were PgR positive and 70 (24.1%) were PgR negative. The percentages of positive cases were almost constant in each phase. No significant difference in mean values of ER concentration was noted throughout the cycle. Instead, the PgR concentration significantly increased from the first to the third phase (P = 0.02) and decreased from the third to the fourth phase (P = 0.01). Our results suggest that ER- and PgR- cases are homogeneously distributed and not influenced by the phase of the cycle. Moreover, they suggest that PgR measurement in the luteal phase, rather than in other phases, prevents the occurrence of false low PgR levels and, at the same time, improves its prognostic significance and response rate to endocrine therapy.