A novel flow cytometric steroid hormone receptor assay for paraffin-embedded breast carcinomas: An objective quantification of the steroid hormone receptors and direct correlation to ploidy status and proliferative capacity in a single-tube assay

Semiquantitative estimation of steroid hormone receptors by immunohistochem istry applied to paraffin sections is common practice in surgical pathology Flow cytometric (FCM) analysis of estrogen receptor (ER) and progesterone receptor (PR) levels provides a faster and more objective quantitative assa y. However, a major problem in such FCM analyses of solid tumor samples is the admixture of tumor cells with normal epithelial, stromal, and inflammat ory cells. The aim of the underlying study was to investigate the applicabi lity of a recently developed multiparameter flow cytometric methodology for the accurate estimation of the fraction of steroid hormone receptor-positi ve tumor cells and to explore whether this multiparameter approach allows t he detection of specific, clinically relevant subsets of tumors, based on a combination of ploidy level, steroid hormone receptor status, and cell cyc le characteristics. For this purpose, samples of 42 breast cancer patients, from which routine immunohis tochemistry for ER and PR also was available, were analyzed. From each case, a cell suspension was prepared from the par affin block by applying a heating and short pepsin digestion step to 50-mu m-thick sections. These cell suspensions were double-immunostained for cyto keratin to identify the epithelial cells, and ER or PR, whereas DNA was qua ntitatively stained with propidium iodide using an optimized protocol. In t he entire group of breast tumors, the percentages of ER- and PR-positive ce lls were registrated in the epithelial subfraction, in combination with DNA ploidy and S phase fraction (SPF). A significant correlation was found bet ween the fraction of hormone receptor-positive cells as found by the immuno histochemical and FCM procedures. For ER, a correlation coefficient of r = .87 was found, and for PR r = .62, both P < .0001. It became dear that all the diploid breast tumors had more than 30% tumor cells positive for ER wit h a SPF lower than 10%, whereas aneuploid tumors contained on average a sma ller percentage of steroid hormone receptor-positive cells, and simultaneou sly an SPF greater than 10%. Our results show that this multiparameter FCM analysis allows an objective and reproducible quantification of the fractio n of steroid hormone receptor-positive cells in the relevant epithelial cel l compartment in relation to DNA ploidy status and proliferative capacity i n a single-tube assay. Copyright (C) 2000 by W.B. Saunders Company.