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Breast cyst fluid from 35 women was stratified into risk groups based on personal and family history of breast cancer. Mitogenic activity in breast cyst fluid of women at highest risk to develop breast cancer was significantly higher than the activity in the lowest-risk group. There was a direct dose-dependent relationship between mitogenic activity and increased risk of developing breast cancer. Size-exclusion chromatography showed that breast cyst fluid from women at highest risk contained two peaks of growth factor activity: less than 6 kilodaltons (kd), identified as human EGF (epidermal growth factor), and 6 to 18 kd. Moderate-risk group samples demonstrated only the single less than 6 kd peak, whereas the lowest-risk group had insignificant growth-promoting activity. Breast cancer tissue analyzed in a similar manner revealed a predominant 6- to 14-kd peak of mitogenic activity demonstrating the same acid- and heat-stability found in breast cyst fluid.

Two types of human fibronectin have been detected by the reactivity of specific monoclonal antibody FDC-6: (a) those present in normal plasma and adult tissues, which do not react with FDC-6 (normal fibronectin or norFN); and (b) those present in fetal tissues and in tumoral cell lines, which do react with FDC-6 (oncofetal fibronectin or onfFN) (H. Matsuura and S. I. Hakomori, Proc. Natl. Acad. Sci. USA, 82: 6517-6521, 1985). We compared the distribution of norFN and onfFN in normal breast tissue (15 samples), breast fibroadenoma (ten samples), and breast adenocarcinoma (80 samples), using an immunofluorescence technique with monoclonal antibody FDC-6. A polyclonal antiserum against human normal fibronectin was used as a control. While norFN was diffusely present in normal gland and in benign and malignant tumors, onfFN was absent in normal gland and in benign tumors. In carcinomas, however, its presence was frequent, as we found it in 60% of cases. Among classical prognosis factors in breast carcinomas, onfFN distribution was significantly correlated with histological grade. Indeed, the presence of FDC-6 labeling was significantly linked with intermediary and high malignancy grades, while its absence was significantly linked with low malignancy grade. onfFN could be considered a marker of the neoplastic state; its immunohistological detection may represent a new prognosis factor in breast carcinomas.

Digoxinlike materials (DLMs) are compounds found in animals that (1) cross-react with digoxin antibodies in specific radioimmunoassay procedures and (2) bind to Na+, K(+)-ATPase and inhibit its activity. DLMs may play a part in the regulation of electrolyte composition. We have determined DLM concentrations in breast cyst fluids and found that these fluids were a rich source of DLMs. Total DLMs were elevated in fluids with type 1 electrolyte composition (high K+ and low Na+ content) when compared to fluids with type 2 composition (low K+ and high Na+ content). Chromatographic studies showed that the basis for the increase was the presence of an additional DLM in the type 1 cyst fluids rather than an increased concentration of the same DLM present in both types of fluids. We propose that this additional DLM may have a role in the accumulation of K+ ions in type 1 fluids. In addition, 7 of 80 cyst fluid samples subjected to high-performance liquid chromatography had a large UV-absorbing peak at 6.4 minutes. Four of these samples were taken from women who later developed breast cancer; information on the subsequent medical history of the other 3 women was unavailable. We suggest that the presence of this material in cyst fluid may be a marker of risk for development of breast cancer.

In our study we have examined 314 samples of cyst fluid taken from women suffering from fibrocystic breast disease (gross cystic disease). We have subdivided the cyst fluid with respect to epithelial coating and we have related trophoblastic protein content of the cyst fluid with age, seriousness of illness, and cytology of epithelial lining. We have performed RIA analysis of the trophoblastic proteins betahCG, beta1-SP-1, and alphahCG and in a smaller (n=84) group of specimens we have also tested for CEA, TPA, and ferritin. Trophoblastic proteins were positive in cystic fluids but the biological meaning of this is not known and the values are not related to clinical manifestations, except in a group of patients with apocrine metaplasia in which we tried to find a relationship between fertile age and increased betahCG. This finding presumably has a prognostic meaning that can be further understood by epidemiologic studies (of dietary intake and evaluation of lipid metabolites) and by information about inflammatory state of cystic fluid.

The present report deals with the concentrations of C-21, C-19 and C-18 steroids as well as steroid glucuronides, namely androstane-3 alpha,17 beta-diol glucuronide, androsterone glucuronide, estradiol glucuronide, and estrone glucuronide (E1-G) in breast cyst fluid (BCF). The concentration of the gross breast cystic disease protein 15 (GCDFP-15) was also measured and its value was correlated with concentrations of unconjugated steroids as well as steroid conjugates. The present data indicate that for a large number of unconjugated steroids (namely pregnenolone, progesterone, dehydroepiandrosterone (DHEA), androsterone, androstane-3 alpha,17 beta-diol, estrone and estradiol) there is an important accumulation in BCF. Our data permit us to demonstrate a statistical relationship between the concentrations of DHEA and its metabolites (namely, androstenedione [4-ene-Dione]), thus suggesting an activity of the enzyme 3 beta-hydroxysteroid dehydrogenase-4-ene-5-ene-isomerase in the breast tissue. Further examination of the relationship between the concentrations of 4-ene-Dione and its metabolites, namely testosterone and 5 alpha-reduced steroid metabolites, as well as their glucuronide derivatives, strongly suggests that the metabolism of androgens in the breast tissue occurs mainly via 5 alpha-reductase and glucuronyl transferase activities. The concentration of GCDFP-15 in BCF found in the present investigation was 2745 +/- 234 micrograms/ml and we have shown that a negative relationship exists in the BCF between E1-G and GCDFP-15 levels.

Two hundred women with breast cysts proven by aspiration were entered into a randomized double-blind trial of Efamol (evening primrose oil) at a dose of 6 capsules daily or equivalent placebo dose for a year. Cysts were categorized by initial electrolyte composition, and follow-up continued for 1 year posttherapy. Recurrent cyst formation in the first year was slightly (but not significantly) lower in the Efamol group compared with the placebo-treated group. The Efamol treatment was well tolerated as the dropout rate was only 7% and equal in both the active and placebo groups. The initial electrolyte composition did not predict for cyst recurrence.

We briefly review some biochemical aspects of benign breast disease (BBD), mainly focusing on free and conjugate estrogen content of breast cyst fluid (BCF), also in relation to cyst type. Evidence is reported that high K(+)-type I-cysts clearly associate with low Cl- levels and accumulate significantly higher quantities of dehydroepiandrosterone sulfate (DHAS) and estrone-3-sulfate (E1S). In spite of the limited number of cases, both increasing DHAS and E1S levels correlate with the increment of K+ to Na+ ratio. A positive correlation was also found between DHAS and E1S. Using electrochemical detection (ECD) on-line to high performance liquid chromatography (HPLC) in the reverse phase mode, we also studied the free estrogen profile. We observed that in type I BCF there are significantly increased amounts of free estrone (E1). The E1S to E1 ratio was significantly different in the two cyst subpopulations; again, a positive correlation was found between free and sulfated E1 (r = 0.820, p less than 10(-6). This last, together with other experimental observations, allows us to hypothesize that in BCF a main pathway of steroids should be E1S----E1. Besides, high specific activity of sulfatase, as well as beta-glucuronidase enzymes, has been demonstrated for BBD. Preliminary information is also reported concerning the BCF pattern of free estrogens, including the highly polar ones, i.e., catecholestrogens (CCE) and the parent methoxy (MeO) conjugates, which represent, in BCF, a predominant portion of all free estrogens. Both CCE levels and ratios appear unevenly distributed in the two different cyst types. In addition, some BCFs show very high concentrations of 16 alpha-OH-E1. Further studies are needed to answer the main question: whether estrogen patterns could represent additive parameters to further categorize breast cystic disease (BCD) or whether they are of minor interest to determine patients' risk of developing breast cancer.

A simple, one-step fractionation technique suitable for the finest quantitation of type V collagen from acidic mixtures of interstitial collagens is reported. The technique is based on the higher solubility of this collagen type in alkaline KCl-phosphate solution, and the purity of the sample has been monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analyses. The latter reveal that the majority of the molecules are endowed with a thread portion and a terminal knob, and show a low tendency to originate ordered and reproducible polymers at low temperature.

The cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins of dysplastic (control) and neoplastic human breast tissue cytosols were investigated after photoaffinity labeling with 8-azido-cyclic adenosine 3':5'-[32P]monophosphate (8-N3-cAMP) by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Four main binding proteins, all specific for cAMP, were identified, with molecular weights of 52,000, 49,000, 39,000, and 37,000. According to their molecular weights, elution on diethylaminoethyl cellulose, and in vitro phosphorylation, the Mr 49,000 and 52,000 species correspond to the regulatory subunits (R-I, R-II) of cAMP-dependent protein kinases types I and II. The smaller cAMP receptors (Mr 39,000 and 37,000) are proteolytic fragments of the intact R-proteins. Dissociation constants (Kd) with 8-N3-cAMP of 0.8 nM for R-I, and 0.12 microM for R-II were obtained; the proteolytic fragments exhibited Kd's similar to that of R-I. No difference in the 8-N3-cAMP affinities and labeling efficiencies was found between control and neoplastic tissues. Although the average incorporation of 8-N3-cAMP was 0.29 +/- 0.02 (S.E.) pmol/mg protein for control and 0.45 +/- 0.06 pmol/mg protein for neoplastic breast tissue cytosol, this difference does not reflect different cellular concentrations of cAMP receptors since the content of blood protein components is lower in tumor tissue. However, tumor cytosols exhibited an increased content of proteolytic R-fragments, and the ratio of intact cAMP receptors versus proteolyzed R-proteins was significantly (p less than 0.01) higher in control (8.3 +/- 0.9) than in tumor (3.0 +/- 0.5) tissue. The average R-I/R-II ratio was greater than 1 in each case, but no significant difference was observed between control and neoplastic tissue. Inverse relationships were obtained, especially between proteolyzed R-fragments and estrogen receptors, when the contents and ratios of cAMP-binding proteins were correlated with the contents of estrogen and progesterone receptors in tumor tissue by a Spearman rank correlation coefficient r = -0.55 (significance of difference from zero being p less than 0.01).

Concentration of dehydroepiandrosterone sulphate (DHAS), cortisol (F), aldosterone (A) and prolactin (PRL) were measured in 70 samples of breast cyst fluid (BCF) obtained by needle aspiration from patients having gross cystic disease of the breast. DHAS concentrations in our series of BCF were scattered over a broad range, up to values 200-fold higher than in plasma, with ample interindividual variability; cortisol was present in BCF in very small amounts. Aldosterone and PRL values were in the same order of plasma levels; moreover, a significant correlation was found between DHAS and PRL concentrations in BCF.

Amniotic fluid was analyzed for the presence of the 15,000 monomer molecular size glycoprotein found in human breast gross cystic disease fluid (GCDFP-15). From 24 weeks' gestation a log-linear increase in levels of GCDFP-15 was noted. The levels of GCDFP-15 doubled every 16 to 28 days, and the highest value recorded was 7,200 ng/ml. At delivery, levels of GCDFP-15 in cord blood plasma were in a background range (mean, 8 ng/ml). Maternal plasma levels of GCDFP-15 were one to tenfold higher in the third trimester of pregnancy when compared to those in nonpregnant women. Since saliva is known to contain high concentrations of GCDFP-15 (10 to 70 micrograms/ml), it is proposed that the levels in amniotic fluid originate from saliva and tracheobronchial secretions.

The levels of estradiol-17 beta, progesterone and glucocorticoids were assayed in tumor tissues from 41 patients with benign breast tumors. Histologically, 21 tumors were identified as fibroadenomas of various types, 14--as fibrous-cystic non-proliferative mastopathy and 6--proliferative mastopathy. Tumor tissue contained estradiol receptors in 51% of cases, all three types of receptors--in 7% and none of the receptors was found in 29% of tumors. The levels of estrogen and glucocorticoids in cases of benign tumors were lower than those in cancer patients. However, in proliferative fibrous-cystic mastopathy group, estrogen receptor level was higher than in the other two groups.

Cystic fluid from 30 Greek women suffering from macrocystic disease was tested for mutagenicity in the Salmonella typhimurium mutagenicity assay using three bacterial strains in the presence or absence of liver homogenate. None of the samples tested showed mutagenic potential in this test supporting the absence of potential carcinogens in the cyst fluids.

A mouse monoclonal antibody (MAb) recognizing alpha-smooth-muscle actin has been used to study smooth-muscle differentiation features in the stromal cells of desmoplastic reactions accompanying mammary tumors. We have studied, by the same immunohistochemical technique, a series of malignant and non-malignant human breast tissues. Cells composing the desmoplastic reaction were found to express alpha-smooth-muscle actin in all the 11 breast carcinomas examined, whereas no immunostain was demonstrated in the stromal cells of 7 breast tissue samples histologically defined as normal. Three of 9 cases of fibrocystic disease showed a minority of positively stained stromal cells, generally in association with epithelial hyperplasia. All the 7 cases of sclerosing adenosis, 3 of 4 cases of diffuse papillomatosis and all 3 intraductal papillomas exhibited a majority of immunoreactive stromal cells. Numerous stromal cells in 3 of 11 circumscribed fibroadenomas analyzed expressed low amounts of alpha-smooth-muscle actin. The factor(s) responsible for smooth-muscle differentiation in stromal cells are presently unknown, but the detection of this previously unsuspected stromal cell phenotype in nonmalignant mammary tissues might help in characterizing the variant morphological aspects designated under the label "fibrocystic disease" and in understanding the biology of premalignant or early malignant lesions of the breast.

Although cystic lesions of the breast are not precancerous per se, statistical studies have indicated that this condition predisposes a 2- to 4-fold greater risk for breast cancer. Seeking a hormonal etiology to this correlation, investigators have analyzed breast cyst fluid ( BCF ) for steroids and have compared the levels to those in the blood. The 17-ketosteroids-androsterone, dehydroisoandrosterone and their sulfates are elevated in BCF . The same is true for estrone sulfate and estradiol-3-sulfate. We have found the most dramatic differences with estriol-3-sulfate (E3-3S), the concentration of which ranged from 187-6134 pg/ml in over 40 specimens analyzed, whereas in 12 serum specimens from normal women, E3-3S was barely detectable. The origin of E3-3S is not known. [3H]E3-3S is not concentrated in BCF following its injection into an arm vein. The blood half-life of [3H]E3-3S is much lower than that of estrone sulfate. Samples of breast nipple aspirates from normal women were also analyzed for E3-3S. None could be detected. The best explanation of the data accumulated thus far is that E3-3S is synthesized at the epithelial lining of the cyst and released into the BCF , from which its efflux is inefficient.

All breast cysts aspirated from a series of 100 patients followed for a minimum period of 2 years were classified on the basis of electrolyte composition as apocrine or flattened, this being the nature of the epithelium lining the two populations of breast cysts. Patients with a single cyst were more than 3 times as likely to have a flattened rather than an apocrine cyst. Multiple cysts, whether simultaneous or sequential in any individual patient, were usually all of the same type, and were more commonly apocrine than flattened. A comparison of the frequency of subsequent cysts in patients whose initial cysts were of either apocrine or flattened type showed further cysts were over 5 times more common in patients who presented with apocrine cysts. These observations suggest that the natural history of cystic disease is closely related to cyst type.

Because of a lack of suitable archival material, it is rarely possible to make retrospective studies of the correlation between the prognosis for a patient with mammary carcinoma and the distribution of nuclear DNA in the cells of the neoplasm. An investigation of the possibility of using sections cut from paraffin-embedded specimens showed that such sections are not suitable for use in retrospective studies of breast carcinoma. Because of such factors as the heterogeneity in size and shape of the nuclei from neoplastic cells and their tendency to mold around each other, determinations of DNA content of cells in sections were extremely difficult; in this particular carcinoma it was found that the distribution of nuclear DNA as obtained from a Feulgen-stained histologic section was not the same as that obtained from a Feulgen-stained imprint smear, and some polyploid tumors were erroneously classified as aneuploid.

Cations (K+ and Na+) content was evaluated in 444 breast cyst fluid (BCF) specimens, aspirated from 391 patients with gross cystic disease of the breast (GCD), a benign form admittedly at major risk of cancer. In 306/444 BCF, dehydroepiandrosterone-sulfate (DHA-S) content was also evaluated. A positive correlation (P less than 0.001) was observed between log K+ vs. log DHA-S whereas a negative correlation was found between log Na+ and log DHA-S (P less than 0.001). Cysts were subdivided in three types according to their cationic concentration: most were of type I (K+/Na+ greater than 1.5) and type II (K+/Na+ less than 0.66) whereas only 10% was of the type III (intermediate). No statistical difference in subtype distribution was apparent when considering patients aspirated in the follicular vs. luteal phase of the menstrual cycle; on the contrary, a significant difference (P less than 0.001) was found between menstruating vs. menopausal patients (type I = 54.8% vs. 32.2%; type II = 34.5% vs. 58.1%, respectively). Ninety-four BCF samples were aspirated simultaneously in 41 patients bearing multiple cysts: the same cationic subtype was present in 29/41 patients. Our data confirm and extend previous observations, and provide conclusive evidence that breast macrocysts can be divided on the basis of their electrolyte composition into different types. Accordingly, the composition of BCF should always be assessed for prospective studies on GCD and breast cancer risk.

HMB-45 (melanocytic cell-specific monoclonal antibody) immunoreactivity has been detected by the immunoperoxidase technique in the cytoplasm of 2 of 100 breast carcinomas. In addition, normal breast lobules and ducts were occasionally found to have positive results in 6 of 100 cases. These findings indicate the need to exercise caution in the interpretation of HMB-45 immunoreactivity for immunohistochemical characterization of neoplasms.

A specific protein from the liquid of a mammary cyst with a molecular weight of 15,000 (GCDFP 15) was studied in normal and pathological mammary tissue using an immunohistochemical method (peroxidase-anti-peroxidase complex). An immunoreactivity of the GCDFP type was found in normal idrosadenoid glands having an apocrine secretion. Histologically normal mammary tissue was not immunoreactive. In benign breast tissue the GCDFP was found particularly in epithelium undergoing apocrine metaplasia (55/55) and in atypical lobular epithelial hyperplasia (8/10). Of the adenocarcinomas of the breast 136/161 (84%) were immunoreactive, especially lobular carcinoma (13/13). The proportion of tumors with a high percentage of immunoreactive cells (76-100%) was greater for Bloom's grade I (1/29: 34%) than for grade III (10/66: 15%). A significant correlation was found between the percentage of immunoreactive cells and the cytosolic concentration of progesterone receptors. The morphological intracellular identification of GCDFP (due to its greater sensitivity) and its correlation with progesterone receptors allowed a more precise evaluation of the functional state and the hormonal dependency of the breast cells by underlining the heterogeneity of the tumoral cell population.

One hundred human breast cyst fluids have been analysed for sodium (Na+), potassium (K+) and dehydroepiandrosterone (DHA) sulphate. Concentrations varied greatly between individual cyst fluids, Na+ from 20 to 185 mmol/l, K+ from 5 to 160 mmol/l and DHA-sulphate from 1.5 to 87 mumol/l. Analysis of the inter-relationships between Na+, K+ and DHA sulphate revealed two major sub-populations of cyst fluids--one group in which Na+ levels were markedly in excess of K+ and DHA sulphate concentrations were low and the other in which K+ was the predominant cation and DHA sulphate concentrations were high.

Prolactin receptors have been determined in 64 benign breast tumours. A specific binding of 0.5% or more (with a range of 0.5-3.3%) was found in 34.4% of the cases and was considered prolactin-receptor-positive. The binding was found to be specific only for lactogenic hormones. By Scatchard analysis the dissociation constant was 2.55 X 10(-10) mol/l and the binding capacity was 4.6 fmol/mg protein.

Levels of cAMP and cGMP were higher in breast cancer tissue as compared with benign tumors. Large-fraction irradiation was followed by a decrease in cGMP concentration matched by a rise in cAMP/cGMP ratio in cases of radiosensitive tumors. Small-fraction treatment did not affect cyclic nucleotide levels. Radiotherapy was followed by a decrease in initially raised blood plasma cyclic nucleotide concentration.

Serum and breast fluid obtained by nipple aspiration were collected from 46 healthy premenopausal women, and 36 patients with cystic breast disease and cyclical mastalgia of similar age. Serum prolactin and growth hormone levels determined by radioimmunoassay were similar in the two groups, but the patients showed a significant elevation in bioactive lactogenic hormones (prolactin plus growth hormone) as determined by the Nb2 rat lymphoma cell bioassay (P less than 0.001). The radioimmunoassayable prolactin levels in breast fluid were extremely variable; there was a trend towards higher concentrations in the cystic breast disease patients, but this just failed to achieve statistical significance (P = 0.06). Immunoreactive growth hormone was rarely detected in nipple aspirates obtained from either the patient or control groups. The mean breast fluid bioactive lactogenic hormone level was significantly higher in the cystic breast disease patients than in the normal controls (P less than 0.001); also ten of 46 (22%) controls, but only two of 36 (6%) patients had no detectable bioactivity. It is concluded that lactogenic hormone bioactivity, when assessed by the Nb2 cell assay, is indicative of an endocrine abnormality which is associated with benign breast disease, and is reflected in breast duct fluid, but is not identified by the conventional radioimmunoassays.

Implanted silicone breast prostheses leak silicone gel through the intact membrane. In order to determine the exact location and to achieve a semiquantitative estimation of the amount of silicone in the surrounding tissue, a method involving scanning electron microscopy (SEM) and energy dispersive analysis of X-rays (EDAX) was used. By this method we were able to demonstrate the presence of the element silicon (Si) (main component of silicone, a polymer with a general formula e.g. (CH3[Si(CH3)2O]n Si(CH3)3 along the inner border of vacuoles corresponding to the light microscopic localization of droplets and in macrophages. The tissue adjacent to the prostheses had a higher silicon content than the control tissue.

We assayed Type 1 (high K+) and Type 2 (high Na+) human breast cyst fluids for DHEA-S. When an antibody specific for the 3-sulfoconjugate end of DHEA-S was used, Type 1 cyst fluids (n = 18) showed a content of 114 +/- 68 micrograms/mL (mean +/- sigma) and Type 2 cyst fluids (n = 14) of 35 +/- 17 micrograms/mL (P less than 0.01). Using an antibody specific for the D-ring, the results were 151 +/- 91 micrograms/mL and 51 +/- 32 micrograms/mL, respectively (P less than 0.01). The apparent concentrations of DHEA-S were statistically different, even though both assays gave equal results in serum from normal adults. The presence of other compounds in individual cyst fluid samples was examined by extraction and chromatography. DHEA-S immunoreactivity was found in both early and late eluting fractions in Type 1 cyst fluids and in late eluting fractions from Type 2 cyst fluids. Only the late eluting fraction from Type 2 fluids had approximately equal immunoreactivity with both antibodies. In addition to authentic DHEA-S, breast cyst fluids contain other materials that react with DHEA-S antibodies. Radioimmunoassays for DHEA-S in cyst fluid must be specifically validated because of the presence of these compounds.

The sodium (Na) and potassium (K) concentrations were measured in the fluid of 169 breast cysts aspirated from 101 consecutive patients presenting to a single clinic. The Na/K ratio was used to define cysts as either apocrine (Na/K less than 3) or flattened (Na/K less than 3) in type. There was no consistency in the type of cyst for individual patients. Cyst type was not predictive of further cyst development, neither was a particular cyst type associated with the development of, or a history of, breast cancer.

The presence of CEA was studied in paraffin sections of 100 benign breast lesions using an immunoperoxidase technique. In fibroadenomas, CEA was found in 25% of cases as opposed to 64% with cystic disease with or without epitheliosis. Our results demonstrate a tendency for an increased frequency of CEA tissue positivity in association with a more prominent form of epithelial hyperplasia with cystic disease. The observed higher positivity of tissue CEA may reflect a higher 'cellular activity' in the latter lesions compared with fibroadenomas.

A previous study reported pH levels in human breast cyst fluid ranging from 7.00 to 7.80 with the cut off between Type I (high K+, low Cl-) and Type II (high Na+, high Cl-) cysts at pH 7.40. Our data indicate a wider range of pH levels in human breast cyst fluids with Type I cysts consistently having acidic levels below pH 7.0 while Type II cysts had pH levels greater than 7.0. Type I acidic cysts characteristically also contain greater amounts of organic acids than Type II cysts. Thus, the pH of breast cyst fluid appears to be related to the electrolyte profile.