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The levels of alpha 1-antitrypsin, transferrin, alkaline phosphatase, phosphohexoseisomerase and gamma-glutamyltransferase were measured in 32 samples of breast cyst fluid, and a wide range of values was obtained. The levels observed in some samples for these parameters, being similar to those of normal serum, might suggest a mechanism of plasma exudation for the formation of breast cyst fluid. Nevertheless, a comparison with the maximum normal serum reference value revealed very high levels of gamma-glutamyltransferase in all of the samples; about 50% also exhibited high levels of phosphohexoseisomerase. These results indicate that the formation of breast cyst fluid takes place with a specific local production, in addition to the mechanism of selective plasma exudation. Moreover, elevated transferrin levels in some cyst fluids are probably the expression of a high biosynthetic activity and could be diagnostically useful in the course of breast gross cystic disease. The importance of these observations from the point of view of diagnostic and prognostic trends are discussed.

The frequency of gross cystic breast disease in premenopausal women and its possible association with increased breast cancer risk emphasize the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured four proteins the presence of which in human milk was well documented. Breast cyst fluid specimens from 266 breast cyst disease patients were assayed and compared as to concentrations of alpha-lactalbumin, gross cystic disease fluid protein (GCDFP-15), epidermal growth factor (EGF), and epithelial membrane antigen (EMA). All the analyzed cyst fluids contained GCDFP-15, EMA, and EGF whereas alpha-lactalbumin was detected in only 14.2% of fluids assayed. Positive correlations were observed between GCDFP-15 and EMA concentrations (P less than 0.005), as well as GCDFP-15 and EGF concentrations (P less than 0.0005). The cyst fluid GCDFP-15 and EGF levels were higher when alpha-lactalbumin concentrations were below detection limits. This association was statistically significant for GCDFP-15 (P less than 0.03) and for EGF (P less than 0.001). These results suggest that GCDFP-15 and EMA could be the biochemical expression of apocrine metaplasia and epithelial hyperplasia, respectively, two histopathological features which characterize breast cystic disease. On the other hand, the occasional presence of alpha-lactalbumin in the cyst fluid would reflect the persistence of differentiated cells in the epithelium surrounding the cyst and would be inversely proportional to the degree of cellular proliferation. The omnipresence of EGF in the cyst fluid argues for the hypothesis of its production by the mammary gland. The highly significant relationship between GCDFP-15 and EGF levels in the medium remains to be elucidated but may be related to an androgen sensitivity in the breast epithelium surrounding the cyst.

Tissue specimens from 27 patients with mammary fibrocystic disease were assayed for estrogen (ER) and progestin (PR) receptors. Seventeen patients (63%) had ER-positive lesions (greater than 5 fmol/mg protein). A progression in ER presence was noted from fibrocystic disease (35%) and fibrosing adenosis (50%) to lobular and ductal hyperplasia (85%). Similarly, ER content increased from 14.5 +/- 13 fmol/mg cytosol protein in fibrocystic disease and fibrosing adenosis to 31.5 +/- 23 fmol/mg protein in epithelial hyperplastic lesions. Furthermore, the ER-PR couple was only encountered in lobular and ductal hyperplasia. The authors' data indicate that the incidence of steroid receptor parallels the intensity of hyperplasia in benign breast disease.

Estrogen receptor content of breast lesions was estimated using a fluorescent cytochemical technique and a competitive protein binding assay. Of 48 cancers examined, an equal proportion contained significant quantities of receptor by either method (62.5%). The concordance between methods for individual patients was also 62.5%. A greater proportion (26%) of patients younger than 45 years of age had receptor-positive cancers using the cytochemical method than were found by the biochemical method (10%). Benign breast disease was also studied using the fluorescent cytochemical method. A greater proportion of lesions containing estrogen receptors was found compared with that cited in the literature for the competitive protein-binding assay. Because of the methodologic simplicity of the fluorescent cytochemical method, further study for routine use is indicated.

The aims of this paper are to demonstrate the stainability of estrogen, CEA, and ferritin in breast carcinomas, fibroadenomas, and fibrocystic diseases; to examine whether the findings of endogenous estrogen using the immunohistochemical detection method are related to estrogen receptor (ER) assays; and to determine whether the stainability of estrogen, CEA, and ferritin were related to the prognosis of breast carcinomas. In breast cancer, the stainability of estrogen using the peroxidase-antiperoxidase (PAP) method was positively correlated with the dextran-coated charcoal (DCC) assay for ER. In breast cancers, the percentage of positive staining was 46% for estrogen, 48% for CEA, and 47% for ferritin. With all three stains, significant differences were observed between cancer and benign diseases. Cases that were both positive for estrogen staining and negative for CEA showed a good prognosis after the recurrence of disease. Our data suggest that the immunohistochemical staining of estrogen, CEA, and ferritin might predict the biological behavior of breast carcinomas and be a prognostically useful indicator of breast cancer patients.

Epidermal growth factor (EGF), dehydroepiandrosterone (DHA)-sulphate and [Na+] and [K+] were assayed in 78 cyst fluids from patients with a palpable breast cyst. Epidermal growth factor was detected in all but 2 cysts, the mean value +/- SEM being 506.2 +/- 39.3 ng/ml, with a range of 0-1,599 ng/ml. When the cyst fluids were sub-divided according to their [Na+]:[K+] ratio, group A cyst fluids ( [Na+]:[K+] less than 3) had a significantly higher (p less than 0.001) level of EGF than group B cyst fluids ([Na+]:[K+] greater than 3). Furthermore, the relationship between EGF and [Na+] and [K+] and between EGF and DHA-sulphate seemed to differ between the 2 cyst types and each cyst type was therefore analyzed separately.

The pattern of estrogen (ER) and progesterone receptors (PR) and their relationship to histo- and cyto-pathological parameters has been studied in 97 cases of benign breast disease and benign phyllode tumors (95 women, of whom 76 were premenopausal, and 2 men). Total (cytosolic + nuclear) ER and PR were assayed by a single-saturating dose method using a tris-KCl buffer. The cut-off between positive and negative ER and PR assay was 100 femtomoles/g tissue. All specimens were processed for histological examination: epithelial and fibroblastic proliferation, epithelial/stromal ratio and presence of focal or diffuse hyalinosis. In 33% of the 46 cases of fibrocystic disease one receptor at least was present (13% ER+, 31% PR+). All the 8 cases in which infiltrating epitheliosis was present were PR+ and 4 of them were also ER+. In 72% of the 31 fibroadenomas one receptor at least was present (19% ER+, 71% PR+). In all these cases levels of receptors were lower than in malignant tumors. An inverse correlation between PR + prevalence and fibrohyalinosis was observed; on the other hand a positive relationship between PR + and fibroblastic (p less than 0.001) or epithelial (p less than 0.01) proliferation was found. In all 5 benign phyllode tumors examined PR + were present at a very high level, almost as high as in malignant tumors. Of the 15 other benign breast lesions, all but one (1 hamartoma) were ER- and PR-.

A simple and reliable radioimmunoassay for the determination of 5-androstene-3 beta,17 beta-diol (5-Adiol) in peripheral plasma and in breast cyst fluid, after chromatography on Celite microcolum has been described and evaluated. The antiserum used was raised in the rabbit injected with dehydroepiandrosterone-15 alpha-carboxymethyl-bovine serum albumin. In men below 40 years of age the levels ranged from 0.85 to 2.80 ng/ml (mean +/- SEM: 1.52 +/- 0.11; n = 24) and from 0.50 to 2.20 ng/ml (mean: 0.93 +/- 0.09; n = 20) in men aged between 41 and 62 years. The mean level was significantly different (p less than 0.001) between the two groups. A significant correlation (r = -0.56 p less than 0.01) was demonstrated between age and all male levels. In women the mean plasma level was in the follicular phase: 0.81 +/- 0.07 ng/ml (range: 0.40 - 1.50; n = 17; age: 19-41 years) and in the luteal phase: 0.83 +/- 0.05 ng/ml (range: 0.40 - 1.30; n = 29; age: 18-43 years). No cyclical change and no correlation with age could be evidenced. A significant difference (p less than 0.001) was shown between females and the young male group. In breast cyst fluid the levels of unconjugated 5-Adiol ranged from 0.05 to 13.70 ng/ml (mean: 2.21 +/- 0.73; n = 23) whereas the sulfate concentrations ranged from 75 to 7,500 ng/ml (mean: 1,973 +/- 543; n = 18), thus demonstrating very wide inter-individual variations.