Relationship between flow cytometric parameters, steroid receptors, and menopausal status in breast cancers.

Of 163 human breast cancers examined, 68% contained detectable aneuploid populations, whilst 32% had apparently normal DNA distributions. A slightly higher incidence of aneuploidy was observed in pre-menopausal patients (83%) than in post-menopausal patients (66%). Also, pre-menopausal patients had slightly higher proportions of S-phase or cycling (S + G2 + M) cells. Estradiol receptor negative (ER-) tumours from post-menopausal patients were found to have the lowest incidence of aneuploidy (59%) and ER- tumours from pre-menopausal patients the highest (91%). There were no significant differences in the proportions of cycling nuclei when receptor status and menopausal status were considered. A weak relationship is shown to exist between flow cytometric data and two common prognostic indicators of breast cancer, namely receptor status and menopausal status.

Value of hormone receptors in the management of breast cancer--I. Advanced breast cancer.

A review of 152 women with locally advanced or metastatic breast cancer is presented. Only 32% of unselected women responded to appropriate endocrine therapy; 64% responded if ER was present and 71% if both ER and PR were present. Survival for the endocrine responders was prolonged and of good quality. Chemotherapy, used subsequently as initial treatment in ER negative women, significantly improved response rates but without a survival advantage. However, there was a strong clinical impression that good performance status was maintained until shortly before death. This data suggests therapy given according to hormone receptor status is of value and confirms the validity of our hormone receptor assays as applied to clinical practice.

The effect of tamoxifen on the growth of human malignant melanoma in vitro.

The effect of tamoxifen on the growth of malignant melanoma was investigated using human cell lines and single-cell suspensions prepared from patients' tumours cultured in soft agar. Tamoxifen stimulated both [3H]-thymidine incorporation and cell numbers in all of the cell lines tested. Cytoplasmic oestrogen receptor (ER) was detected in one of the responding lines and progesterone receptor (PR) in another. Tumour colony formation in soft agar culture was satisfactorily established from tumour cell suspensions from 13 of 21 patients, only one of which had detectable cytoplasmic ER. Greater than 50% reduction in colony formation with 5 X 10(-7) M tamoxifen occurred in two tumours, neither of which contained ER. These results indicate that tamoxifen has the potential to either retard or accelerate the growth of human malignant melanoma.

Properties and interpretation of the Woolf and Scatchard plots in analysing data from steroid receptor assays.

The Woolf plot has been shown to have superior statistical properties compared with the Scatchard plot in analysing data from steroid receptor assays. This paper examines the properties of the Woolf plot, with a view to aiding its interpretation and improving familiarity with it so that its use may become more widespread. The Woolf plot contains the same information as the Scatchard plot, and deviations from linearity of the plot due to positive cooperativity, second binding sites, or non-specific binding are readily identifiable, and are described. It is emphasised, however, that the range of incubating concentrations should be chosen to give an even spread of data points on the plot. There is shown to be a relatively stable error distribution along the Woolf plot, and the implications of this for analysis of the data points are discussed. This error distribution is associated with consistently better fitting of a straight line to the data points when they are presented on the Woolf, as opposed to the Scatchard scale. This has further implications in the definition of receptor positivity, and in determining the reliability of a receptor assay result. Interpretation of the Woolf plot is easy, with the receptor site concentration being obtained from the slope of the plot, and the dissociation constant from the intersection of the extrapolated line with the abscissa. Familiarity with the plot should lead to its wider use.

Analysing data from hormone-receptor assays.

Assay results for hormone receptors in human breast cancer are generally plotted in the form of a Scatchard plot. Usually a line is fitted to the data points by least squares regression and thence the concentration of binding sites present in the cancer is calculated. The subsequent treatment of the patient with hormones depends on this value, so a good estimate of it is necessary. In this study, three methods of representing the data, namely the Scatchard plot, the reciprocal plot and the Woolf plot, were investigated, and three ways of fitting lines to the data points of each graph, namely least squares regression, an unweighted robust procedure and a weighted robust procedure, were examined. When the data were well-behaved all plots gave similar answers for the concentration of binding sites, irrespective of the regression technique used. However, when there were up to three outlying points, the Scatchard plot, particularly with least squares regression analysis, performed poorly. The robust regression analyses yielded more consistent results on all plots; in particular the hitherto mistrusted reciprocal scale seemed to perform well under robust regression, but other evidence indicated instability in the plot. It is concluded that the most reliable way of representing binding data in these experiments is by a Woolf plot, and that the subsequent line fitting is made resistant by an unweighted robust regression analysis.

Inter-site variation of oestrogen receptors in human breast cancers.

When large human breast cancers were assayed for oestrogen receptors at multiple sites, 5-fold differences were found in the numbers of oestrogen receptors, between the site within a tumour. This may result from variations in the cell:stroma ratio from site to site. Such differences could be significant when receptor levels in the tumour are low (less than 50 fmol oestradiol bound mg cytosol protein) since the classification distinction between hormone-sensitive and hormone-insensitive breast cancers is based upon numbers of oestrogen receptors detected by the assay. This problem might be remedied by assessment of the cell:stroma ratio in all assayed tumours, and by the combination of the cytoplasmic oestrogen-receptor assay with other hormone-receptor assays.

PIP: Both human mammary gland tumors and human myometrium were used to examine the inherent variability of the estrogen-receptor assay of cytosol fractions derived from the tumors and to report results of assays on multiple sites within large breast cancers. Cytosol and tissue assays of human myometrium showed only small variability in binding site numbers between assays of the same fractions. However, estrogen-receptor assays performed on multiple sites in large receptor-positive mammary gland tumors showed marked intersite variation within each tumor, with up to a 4-fold difference (e.g., a range of 175-523 sites in 1 tumor). This difference may result from variation in the cell:stroma ratio from site to site. In the majority of tumors (5), this variability did not affect classification of receptor status, but in 2 tumors, this variation was significant where receptor levels in the tumors were low ( 50 fmol estradiol bound). Resolution of this assay problem may be achieved by combining: 1) assessment of cell:stroma ratio of the assayed site; 2) estimation of nuclear receptors for estrogen; and 3) estimation of progesterone receptors.

Effect of snap freezing, dithiothreitol and storage on estimations of estrogen receptor sites.

Estrogen receptor assays were performed on human myometrium in order to determine optimal conditions for certain steps of the assay. The findings indicate that: (1) tissue should be snap-frozen in liquid nitrogen prior to assay, (2) dithiothreitol does not increase measurable binding site numbers, (3) estrogen receptors in the cytosol fraction are unstable and should be stored in excess of 1 day; (4) if storage prior to receptor assay is unavoidable, tissue should be stored at -20 degrees C or -70 degrees C for no longer than 1 week.

Hormone receptors and breast cancer.

The hormones of reproduction have been implicated in breast cancers of women. A short reproductive life, and a pregnancy prior to the age of 30 years, both reduce the risk of breast cancer (Doll, 1975). In addition, removal of endogenous sources of oestrogens, and administration of anti-oestrogens, cause some breast cancers to regress (Stoll, 1969). Such observations have led to an investigation of the role of the hormones of reproduction, and of oestrogen in particular, in the maintenance of breast cancers.