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In 1990, 215 patients with operable breast cancer were entered into a prospective study of the prognostic significance of five biochemical markers and 15 other factors (pathological/chronological/patient). After a median follow-up of 6.6 years, there were 77 recurrences and 77 deaths (59 breast cancer-related). By univariate analysis, patient outcome related significantly to 13 factors. By multivariate analysis, the most important of nine independent factors were: number of nodes involved, steroid receptors (for oestrogen or progestogen), age, clinical or pathological tumour size and grade. Receptors and grade exerted their influence only in the first 3 years. Progestogen receptors (immunohistochemical) and oestrogen receptors (biochemical) were of similar prognostic significance. The two receptors were correlated (r=+0.50, P=0.001) and displaced each other from the analytical model but some evidence for the additivity of their prognostic values was seen when their levels were discordant.

A method to determine the binding of epidermal growth factor (EGF) to the particulate fraction of the cell has been established and evaluated using rat liver, human placenta, and tumours of human breast and brain. Little EGF receptor (EGFR) activity was detected in normal or benign tumour tissues except for meningioma (positive in 95% samples), but EGFR were present in 43% of 131 breast tumours and 75% of 55 primary cerebral tumours. Despite the strong inverse correlation between EGFR activity and oestrogen receptors in breast tumours and a tendency for high levels of EGFR activity to be associated with glioblastoma multiforme, analysis showed that EGFR was of little prognostic significance in patients with tumours of either breast or brain.

The incidence of mammary tumours developing after administration of the carcinogen DMBA (at 50 days of age) has been determined in 2 strains of Sprague-Dawley rat. Untreated animals of each strain were exsanguinated in dioestrus at a time corresponding to the early post-carcinogen stage (at 70 days of age) and the plasma concentrations of prolactin, oestradiol-17B and progesterone were measured by radioimmunoassay. In an inbred strain of rats, tumour-induction rate was 6-4% and plasma prolactin concentration was 2-5 x lower than that found in a random-bred strain with a tumour-induction rate of 41-6%. No difference was found between the 2 strains in the level of either ovarian hormone. It is concluded that the difference between these strains in mammary gland susceptibility to DMBA may be related to plasma prolactin concentration, but it is unlikely to be determined by the ovarian hormones.

The soluble oestrogen-receptor content of common breast tumours has been measured by 5 different laboratories, each using their own assay procedure. Good agreement was achieved on whether a sample was positive or negative for oestrogen receptor. Qualitative differences between laboratories could be explained by differences in thiol-reagent content of assay medium and by the method of homogenization. Recommendations are made on some of the factors involved in the routine assay of receptors in breast tumours.

The reproducibility of measurements of oestrogen-receptor activity has been examined in multiple specimens from a rabbit uterus, a rat mammary tumour and human breast tumours. The relationship between receptor concentration and tumour histology has also been investigated in 11 large primary tumours. In the animal tissues, receptor measurements were relatively reproducible (coefficient of variance: wet wt. basis 16-17%, protein basis 16-21%) but in human breast tumours receptor activity varied considerably (c.v.: wet wt. basis, 22-125%; protein basis, 28-72%). In addition to these variations in receptor activity within tumours, there was a difference between tumours, as demonstrated by an analysis of variance (P less than 0.01). In the 11 primary breast cancers selected for study, the level of receptor activity was related to menopausal status and the tumour content of the specimen. We conclude that the receptor activity detected varies within a tumour and depends upon the tumour content of the biopsy specimen. Predictions based on precise quantitation of receptor concentrations may therefore necessitate replicate tumour sampling and correction for the fraction of non-tumour tissue in each sample.

The relationship between oestrogen-receptor protein (ER) content of the tumour and the response to endocrine therapy was determined in 119 patients, in a collaborative prospective study. Twenty-eight of the 80 patients with measurable ER responded to treatment according to UICC criteria, compared with only 3/39 without ER. It was found that site of biopsy did not influence the result, but tumour content of the tissue sample was significantly related to the presence of receptors. The organizational problems of such a study are discussed.

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) were measured by Gas Liquid Chromatography-Mass Spectrometry (GLC-MS) in extracts of 100 human mammary carcinomas. All tumours contained measurable amounts of both prostaglandins but wide variations between individual tumours were observed. Values for PGE2 ranged from 7 to 762 ng g-1 tissue with a median of 100 ng g-1 tissue. Values for PGF2 alpha ranged from 3 to 475 ng g-1 tissue (median 60 ng g-1 tissue). There was a highly significant positive correlation between amounts of the 2 prostaglandins in individual tumours. Amounts of both PGE2 and PGF2 alpha were not significantly related to the menopausal status of the patients or the presence of oestrogen and progesterone receptors.

Radioimmunoassay methods for the determination of oestradiol-17 beta and prolactin have been examined for their reliability and applied to the measurement of hormone concentrations in the plasmas from male and female rats. Prolactin was detectable in all samples (greater than 7 ng RP-1 ng/ml) but the concentration of oestradiol-17 beta was below the sensitivity of the method (greater than 0-10 ng/100 ml) in ovariectomized females. Plasma oestradiol-17 beta concentration rose gradually from metoestrus to proestrus and fell to barely detectable levels in oestrus. Plasma prolactin concentration was very variable even for rats in the same stage of the oestrous cycle but values were minimal in the afternoon of diestrus and a surge in secretion occurred in the afternoon of proestrus. In addition to the stage of the oestrous cycle, the prolactin concentration was influenced by mode of blood collection, degree of haemolysis and choice of serum or plasma. There was no correlation between the concentration of prolactin and that of oestradiol-17 beta in the same sample of plasma.

Four albumin conjugates of oestradiol. Labelled with fluorescein or peroxidase to permit visualization under light or fluorescence microscopy, were synthesized. These were used to examine the feasibility of identifying oestrogen binding in frozen section by two published histochemical techniques. In a variety of experimental tissues and human breast cancers, binding of the oestrogen conjugates was demonstrable, but it appeared nonspecific (i.e., rarely displaceable by competitor) and unrelated to oestrogen receptor (RE) status of the tissue as determined biochemically by assay with dextran-coated charcoal. Investigation of the fate of the RE through the various steps of a histochemical assay, demonstrated major losses of RE from unfixed tissue or after tissue fixation. The RE also exhibited a 10-50-fold poorer affinity for the conjugates synthesized than for oestradiol-17 beta and, at the concentrations of conjugate routinely used in histochemical assays, it seems likely that considerable nonspecific binding takes place. These factors may combine to make it (1) difficult to implement such histochemical assays and (2) unlikely that the RE is being detected.