Progesterone receptor detection and quantification in breast tumors by bivariate immunofluorescence/DNA flow cytometry

A method was developed for the detection of progesterone receptors (PgR) by flow cytometry (FCM) in cell suspensions obtained from mechanically dispersed fragments of operated breast cancers. Two monoclonal antibodies were tested for sensitivity and specificity on four breast cancer cell lines of known PgR expression and a calibration curve thus established. A simple procedure was used to calculate the level of PgR expression, taking into account the relative displacement of total cellular fluorescence compared to nonspecific fluorescence for each sample and the average DNA content of the cells derived from the corresponding histograms. The PgR-specific immunofluorescence of the tumor specimens measured in arbitrary units (channels) was then transformed to fmoles/mg DNA by comparison with the calibration curve. The FCM-derived results were compared with those of a conventional immunoenzymatic PgR assay on 30 surgical samples. PgR content ranged from 10 to 22,000 fmoles/mg DNA and linear regression analysis yielded a good correlation (r = 0.86). With a threshold of positivity of 300 fmoles/mg DNA, the two methods concurred for 28 of 30 tumors (93%). Nine specimens were analyzed repeatedly, showing good reproducibility. This method could prove to be more useful than the biochemical assays on homogenates, since it allows the simultaneous analysis of receptor expression in individual cells and of DNA index (ploidy).