Prostate-specific antigen and insulin-like growth factor binding protein-3 in nipple aspirate fluid are associated with breast cancer

Insulin-like growth factor-1 (IGF-1) is an important growth factor for breast cancer cells and insulin-like growth factor binding protein-3 (IGFBP-3) its most prevalent binding protein. Prostate-specific antigen (PSA) enzymatically cleaves IGFBP-3 into fragments (BP3-FR). Our purpose was to determine the association of these markers in nipple aspirate fluid (NAF) and serum with the presence of breast cancer. NAF from 175 and serum from 215 subjects were collected from women with or without breast cancer. In unadjusted analysis low NAFPSA (P < 0.001) and high NAFIGFBP-3 (P = 0.023) were associated with breast cancer. Low serum PSA was associated with postmenopausal breast cancer (P = 0.034). In separate multivariate analyses, controlling for age, menopausal status, and age at menarche, NAF PSA and IGFBP-3 were each associated with breast cancer. The association was significant for NAF IGFBP-3 in all women (P = 0.031), but for NAF PSA only in premenopausal women (P < 0.001). When considered jointly, only NAF PSA was significant. Therefore, NAF PSA, and to a lesser extent NAF IGFBP-3 and serum PSA, seem to be important predictors of breast cancer.     

PTHrP and the PTH/PTHrP receptor are co-expressed in human breast and colon tumours.

Using RNA extracted from human tumour samples removed during surgery, we have analysed expression of mRNA for parathyroid hormone-related protein (PTHrP) and for the PTH/PTHrP receptor by RT-PCR in a panel of human breast and colon tumours. All but 1 of 18 breast tumour samples expressed PTHrP, whereas receptor expression was detected in 11 of these. Expression of the PTH/PTHrP receptor was found in three out of four metastatic lesions, including one sample in which no receptor was detected in the primary tumour. PTHrP expression was also detected in five colon tumours, and receptor expression detected in two of these. These results demonstrate that PTHrP and the PTHrP receptor are also co-expressed in breast tumours in vivo and provide further evidence that PTHrP may be an important autocrine/paracrine growth factor in breast cancer.     

Prostate-specific antigen in breast cyst fluid: Possible role of prostate-specific antigen in hormone-dependent breast cancer

Prostate-specific antigen (PSA) is a 33 kDa serine protease which is produced by many different tissues in the body and has been shown to be present in low concentrations in breast milk and in about 30% of breast cancers. The presence of PSA in breast cancers is associated with the presence of steroid-hormone receptors and may be a favourable prognostic indicator. In this study, PSA immunoreactivity was measured in breast cyst fluid obtained from women with palpable breast cysts which is the most common benign breast disease. PSA was found to be present in very low concentrations in breast cyst fluid. In an attempt to understand the possible role of PSA in the breast, the effect of PSA on growth of the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 human breast cancer cell lines was studied. In addition, the effect of PSA on oestrone sulphatase activity and oestrogen 17-oxidoreductase activity in these cell lines was investigated. PSA, in low concentrations, was found to inhibit MCF-7 cell growth and to stimulate the conversion of oestradiol to the less potent oestrogen oestrone in this cell line. PSA had no effect on the MDA-MB-231 cell line. Our findings suggest that PSA may act as a negative growth regulator in hormone-dependent breast cancers. © 1996 Wiley-Liss, Inc.     

Presence of alpha-lactalbumin, epidermal growth factor, epithelial membrane antigen, and gross cystic disease fluid protein (15,000 daltons) in breast cyst fluid

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.     

Human gross cyst breast disease and cystic fluid: bio-molecular, morphological, and clinical studies

Summary For more than one and a half century the cystic disease of the breast has been recognized as the most frequent female benign breast lesion. Although some conundrums and controversies exist about the relation between gross cysts and breast cancer, recent evidence suggests that the multidisciplinary study of gross cystic breast disease (GCBD) may be a powerful tool for predicting the natural history of the multifaceted gross cyst pathology. A lot of papers have been published on breast cyst fluids (BCF) concerning biochemical, hormonal and morphological aspects, demonstrating that the intracystic fluid contains a wide variety of components (such as ions, lipids, proteins, enzymes, growth factors and antigens) and suggesting that their profile provides additional knowledge on both physiopathology and etiologic pathways of human gross cystic breast disease. The aim of this overview is the critical evaluation of all data accumulated in the last thirty years, in order to highlight the utility of biochemical and epidemiological studies to identify gross cysts, if any, at higher breast cancer risk.“Qui addit scientiam, addit et laborem” “He that increaseth knowledge increaseth also sorrow” (Ecclesiastes 1:18, Bible)     

Prostate-specific antigen expression in nipple aspirate fluid is associated with advanced breast cancer

We previously demonstrated that prostate-specific antigen (PSA) is present in breast nipple aspirate fluid (NAF) and its expression is inversely associated with the presence of breast cancer. The purpose of this study was to determine if PSA levels in NAF decrease with disease progression from DCIS to metastatic breast cancer. One hundred and forty-nine women underwent nipple aspiration before or in conjunction with surgery to treat their breast cancer. PSA levels decreased with more advanced disease stage (P = 0.016), larger tumor size (P = 0.031), and nodal involvement (P = 0.041). PSA levels were lower in women with than without distant disease spread (P = 0.049). We also evaluated the association of PSA with these clinical parameters based on menopausal status. In general, PSA predicted disease involvement better in pre- than in post-menopausal women. There was no association between PSA and race. Spearman's rank analysis demonstrated that PSA was inversely related to tumor size (P = 0.009), nodal status (P = 0.005), disease stage (P = 0.004), and distant metastases (P = 0.04). NAF PSA provides useful prognostic information which may assist with breast cancer treatment.     

Prostate-specific antigen in the cerebrospinal fluid: A marker of local disease

Leptomeningeal involvement from prostate cancer is a rare complication with dismal prognosis. Prostate-specific antigen in the cerebrospinal fluid may be found elevated and can be used as a marker for local disease. We present the case of a patient with prostate cancer and leptomeningeal metastases who had high levels of prostate-specific antigen in the cerebrospinal fluid.     

The epidemiology of gross cystic disease of the breast confirmed by biopsy or by aspiration of cyst fluid.

This study concerns 3443 patients treated by 1 physician for benign breast conditions, with follow-up on 94% of the patients averaging 19 years. Over three quarters of these patients had gross cystic disease (GCD) of the breast confirmed by aspiration of cyst fluid or by biopsy. Diagnosis of GCD by microscopic pathology review alone is shown to have high error rates when compared with information in the operative report and the gross pathology report. The risk of subsequent breast cancer for GCD patients was more than double the rates in the general population, and about the same as that of patients with no GCD who had biopsy findings of benign proliferative disease. Among patients with aspirations for GCD and no biopsy, there was a trend of increasing risk of subsequent breast cancer with increasing numbers of aspirations, with a sixfold relative risk for patients with ten or more aspirations.     

Prostate-specific antigen in serum of women with breast cancer.

Prostate-specific antigen (PSA) was recently found in 30% of female breast tumours. In this study we have examined if PSA circulates in the blood of breast cancer patients and if serum PSA has any clinical application. We have compared serum PSA levels between women with and without breast cancer, between women with PSA-positive and PSA-negative breast cancer and between women with breast cancer before and after surgical removal of the tumour. We found that for women > or = 50 years, there is no difference in serum PSA between normal or breast cancer patients. We also could not find any difference in presurgical or post-surgical serum PSA between women who have PSA-positive or PSA-negative breast cancer. We found no correlation between PSA concentrations in matched presurgical and post-surgical sera, between presurgical sera and tumour cytosols and between post-surgical sera and tumour cytosols. High-performance liquid chromatography has shown that PSA in normal male serum consists mostly of PSA bound to alpha 1-antichymotrypsin (molecular weight approximately 100,000), and PSA in breast tumours and presurgical and post-surgical serum consists mostly of free PSA (molecular weight approximately 33,000). These data suggest that female serum PSA is not associated with tumour PSA levels. We speculate that most of the circulating PSA in women originates from the normal breast. It appears that serum PSA in women does not have potential for breast cancer diagnosis or monitoring, but our previous data are consistent with the view that tumour PSA concentration is a favourable prognostic indicator in women with breast cancer.     

Ultrastructural characterization and biochemical profile of human gross cystic breast disease

Human gross cystic breast disease is a benign condition affecting about 7–10% of adult women occurring with the highest incidence in the premenopausal decade. Although breast cysts do not represent a preneoplastic condition per se, several studies indicate an increased breast cancer risk in women affected by this pathology. In this report we study 115 breast cystic fluid samples obtained by needle-aspiration from women with gross cystic breast disease. The samples were analysed biochemically and the cells contained therein were observed at the electron microscope. According to their biochemical profiles, the cysts were subdivided into three types: Type I, showing a Na/K ratio < 0.5 and a typical protein content; Type II, showing a Na/K ratio >10 and a protein content quite similar to plasma; Type III, showing a Na/K ratio between 1 and 7 and an intermediate protein content. The electron microscopic examination demonstrated that Type I cystic fluid cells exhibit morphological features typical of actively synthesising and secreting cells, while the characteristics of Type II cells indicate a low metabolic activity. Type III cells have characteristics typical of both Type I and Type II cells, thereby confirming the intermediate nature of this cyst type. We hypothesise that these cyst types could represent different developmental stages of a structural evolution pathway, during which the biosynthetically active 'apocrine stage' would be the key step to cell neoplastic transformation.     

Circulating levels and breast cyst fluid concentrations of human chorionic gonadotropin, progesterone and testosterone in women with gross cystic breast disease

Circulating levels and cyst fluid concentrations of human chorionic gonadotropin, progesterone and testosterone were measured in 30 premenopausal women with gross cystic breast disease. Specific radioimmunoassay procedures were used. Amounts of human chorionic gonadotropin exceeding 5.0 mIU/ml were found in cyst fluid of many patients, but not in serum. Progesterone and testosterone concentrations in breast cyst fluid were significantly higher than in serum (p less than 0.01 and p less than 0.001, respectively).     

Proteases in cyst fluid from human gross cyst breast disease

Cyst fluid from women with gross cystic breast disease was found to contain protease activity when assayed against [14C]albumin. At least six different proteases were detected when the fluid was fractionated by a combination of S-300 Sephacel, hydroxylapatite, and DEAE-Sephacel chromatographic techniques. The distribution of the proteases appeared to be related to the ionic composition of the fluids. A major protease component, found in both high Na and high K fluids, was isolated. It showed chymotryptic cleavage characteristics against the beta-chain of insulin. It was partially inhibited by alpha 2-macroglobulin, N-tosyl-L-phenylalanine chloromethyl ketone, and benzamidine but not by leupeptin, pepstatin, N-tosyl-L-lysine chloromethyl ketone, or alpha 1-protease inhibitor. The protease has an apparent molecular weight of 110,000 with Mr 24,000 subunits. This protease may be identical or closely associated with Haagensen's GCDFP-24 progesterone binding protein which was isolated in a similar manner. An imbalance between protease and protease inhibitors in cyst fluid may account for gross cyst formation and may be involved in the tumorigenic process. The accumulation of poorly diffusible peptide fragments, as a result of protease activity, would increase the oncotic pressure leading to enlargement of the cyst cavity as water enters to reestablish osmotic equilibrium.     

Immunologic and steroid binding properties of the GCDFP-24 protein isolated from human breast gross cystic disease fluid

Summary A major protein of human breast cyst fluid, termed GCDFP-24, has the property of specifically binding progestins. The purified glycoprotein, of 24,000 apparent molecular weight, bound pregnenolone and progesterone with highest affinity. The association constant for binding of progesterone was 1 × 10⁶ L/mol by Scatchard analysis, and there was one binding site per molecule. Changes to the progesterone structure at C-17, C-20, or C-21 interfered with binding. The pH optimum for binding was 4–4.5. The purified protein was highly stable and was not irreversibly denatured by 50% methanol or 3M guanidine. However, dithiothreitol reversibly interfered with progesterone binding. Rabbit antiserum produced against the glycoprotein recognized an immunologically identical component in normal human sera, and partially cross-reacting components in normal monkey and baboon sera. The component in human sera was present in Cohn fractions IV and VI.     

Secretion of breast gross cystic disease fluid proteins by T47D breast cancer cells in culture — modulation by steroid hormones

Summary The effect of steroid hormones on modulating the secretion rates of three human breast gross cystic disease fluid proteins (GCDFP-15, GCDFP-24, and GCDFP-44) by T47D breast carcinoma cells in tissue culture was evaluated. Androgens (dihydrotestosterone or fluoxymesterone) were capable of stimulating the secretion rates for all three GCDFP's while showing a minimal trend toward slowing the growth rate of T47D cells. This is the first study which shows that androgens can specifically stimulate all three of the major breast GCDFP's concomitantly. Progesterone, and three synthetic progestins, all showed inhibition of the growth rate of T47D cells while causing enhancement of the secretion of GCDFP-15 and GCDFP-44, and only minimal effect on the secretion rate of GCDFP-24. Estradiol was essentially neutral to the growth rate of the T47D cells in our test system. Estradiol did cause a mild enhancement of GCDFP-44 secretion rate, with no appreciable effect on GCDFP-15 or GCDFP-24 secretion rates. These findings suggest that an androgenic stimulus may be involved in the secretion of GCDFP's associated with breast gross cystic disease.     

Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein.

Human breast gross cystic disease (GCD) fluid was analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, and four major proteins (GCDFP-70), GCDFP-44, GCDFP-24, and GCDFP-15) were identified. By fractionation techniques, these proteins were separated from one another. The GCDFP-70 was immunologically identical to human albumin and was present in GCD fluid at approximately a 100-fold lower concentration than in plasma. The GCDFP-44 was immunologically identical to human plasma Zn-alpha2-glycoprotein; however, it was present in GCD fluid at an approximately 50-fold higher concentration than in plasma. The GCDFP-24 was the major component protein of GCD fluid. It had progesterone binding activity, and immunologically it was identical to a component of human plasma; however, antisera that identified 30 separate components of plasma failed to identify the GCDFP-24 as one of these plasma proteins. The GCDFP-24 concentration in GCD fluid was approximately 100-fold higher than the plasma analog. The GCDFP-15 component was immunologically distinct from any plasma components, as judged by Ouchterlony analysis. It was, however, immunologically identical with a component of both human milk and saliva. As revealed by radioimmunoassay, plasma levels in normal subjects were 7-85 ng/ml. In patients with metastatic breast carcinoma, markedly plasma levels (150-30,000 ng/ml) of this protein were detected. Short-term tissue cultures of breast carcinoma explants released this protein into the culture medium.     

Mitogenic effect of the 15-kDa gross cystic disease fluid protein (GCDFP-15) on breast-cancer cell lines and on immortal mammary cells

The biological significance of a major protein component in the fluid of gross cystic breast disease and a recognized marker of apocrine metaplasia, i.e. the 15-kDa glycoprotein (GCDFP-15), is presently unknown. We have added GCDFP-15 to cell culture medium and tested its effect on proliferation of 4 human breast-cancer cell lines (MCF7, BT474, MDA-MB231 and T47D) and a “normal” human immortal breast-cell line (MCF10A). These breast-cell lines showed a mitogenic response to GCDFP-15 (10 μ/ml). GCDFP-15 enhanced cell growth of the MCF10A, MCF7, BT474 and MDA-MB231 cell lines at both 48 and 96 hr of exposure. The glycoprotein exerted a mitogenic effect on the T47D cell line at 48 hr but not at 96 hr. This may be due to an auto-regulatory effect of endogenous GCDFP-15 synthesized by the T47D cells. GCDFP-15 was ineffective on 2 colon-cancer cell lines (HT29 and NIC-H7I6), on the IMR32 neuroblastoma cell line and on the NIC-H209 small-cell lung carcinoma cells. A separate major breast cystic disease fluid protein of 24 kDa (GCDFP-24) was tested, following the same experimental design, on the 5 breast-cell lines, and showed no mitogenic activity. The mitogenic effect of GCDFP-15 observed in this study in both “normal” and malignant breast epithelial cells suggests a possible relationship between apocrine metaplasia in breast cystic disease and the development of breast epithelial hyperplasia. In addition, a possible role of GCDFP-15 in breast-cancer progression should be considered. © 1995 Wiley-Liss, Inc.     

Prostate-specific antigen in nipple aspiration fluid: menstrual cycle variability and correlation with serum prostate-specific antigen.

OBJECTIVES: To determine the variability of prostate-specific antigen (PSA) in the breast ductal fluid (nipple aspiration fluid, NAF) during the menstrual cycles of healthy premenopausal women. METHODS: Fifteen female volunteers underwent weekly nipple aspiration of ductal fluid from both breasts for the duration of two menstrual cycles. A highly sensitive and specific Third Generation PSA Assay (IMMULITE); Diagnostic Products Corporation, DPC) was used to detect NAF and serum PSA. Associations between NAF PSA and paired serum hormone levels of the pituitary-ovarian axis were tested using the Spearman rank correlation and the Wilcoxon signed rank test. Analysis of variance on log transformed NAF PSA was used to determine the intra- and intervolunteer variability. RESULTS: NAF PSA ranged from <0.003 to 133,330 ng PSA/g total protein (median 2,030 ng/g). No repeatable pattern of change was observed for individual volunteers and no significant association between NAF PSA and any pituitary-ovarian axis serum hormone was detected. There was no correlation between serum and NAF PSA. CONCLUSIONS: Weekly NAF sampling in healthy premenopausal women to provide adequate volumes for PSA analysis was successfully achieved. Considerable variation was observed for NAF PSA, which may limit the future potential for this tumor marker in NAF. This variability was not associated with hormones of the pituitary-ovarian axis and did not show repeated cyclical variability during the menstrual cycle. Serum PSA does not appear to be an acceptable indicator of NAF PSA levels.     

p53 and P-glycoprotein are often co-expressed and are associated with poor prognosis in breast cancer.

Expression of both P-glycoprotein (P-gp) and mutant p53 have recently been reported to be associated with poor prognosis of breast cancer. The expression of P-gp is associated in vitro and in vivo with cross-resistance to several anti-cancer drugs. p53 plays a regulatory role in apoptosis, and mutant p53 has been suggested to be involved in drug resistance. Interestingly, in vitro experiments have shown that mutant p53 can activate the promoter of the MDR1 gene, which encodes P-gp. We investigated whether p53 and P-gp are simultaneously expressed in primary breast cancer cells and analysed the impact of the co-expression on patients prognosis. Immunohistochemistry was used to investigate P-gp expression (JSB-1, C219) and nuclear p53 accumulation (DO-7) in 20 operable chemotherapy untreated and 30 locally advanced breast cancers undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. Double immunostaining showed that P-gp expression and nuclear p53 accumulation often occur concomitantly in the same tumour cells. A correlation between p53 and P-gp expression was found in all 50 breast cancers (P = 0.003; Fisher''s exact test). P-gp expression, nuclear p53 accumulation, and co-expression of p53 and P-gp were more frequently observed in locally advanced breast cancers than in operable breast cancers (P = 0.0004, P = 0.048; P = 0.002 respectively. Fisher''s exact test). Co-expression of p53 and P-gp was the strongest prognostic factor for shorter survival by multivariate analysis (P = 0.004) in the group of locally advanced breast cancers (univariate analysis: P = 0.0007). Only 3 out of 13 samples sequentially taken before and after chemotherapy displayed a change in P-gp or p53 staining. In conclusion, nuclear p53 accumulation is often associated with P-gp expression in primary breast cancer, and simultaneous expression of p53 and P-gp is associated with shorter survival in locally advanced breast cancer patients. Co-expression of P-gp and mutant p53 belong to a series of molecular events resulting in a more aggressive phenotype, drug resistance and poor prognosis.     

Association of cyst type with risk factors for breast cancer and relapse rate in women with gross cystic disease of the breast.

The concentration of potassium (K+) and sodium (Na+) was measured in breast cyst fluid (BCF) from 611 cysts greater than 3 ml aspirated in 520 women with gross cystic disease of the breast. These women were enrolled, from 1983 on, in a cohort study aimed at assessing the relationship between cyst type, as defined by the K+/Na+ ratio in BCF, and the risk of breast cancer. The inverse relationship between K+ and Na+ and the bimodal distribution of the K+/Na+ ratio in BCF were confirmed. Type I cysts were defined as cysts with a K+/Na+ greater than 1.5 in BCF. Among women with type I cysts, a higher proportion of women with one or no births, of women with a history of apocrine cysts, of current smokers, and of women who do not drink coffee was found, as compared to women with other types of cysts. The risk of cyst relapse was significantly higher among women with type I cysts than among women with other types of cysts and among women with multiple cysts at presentation. These findings indicate that type I BCF is a marker of "active" gross cystic disease of the breast and suggest that it may be associated with increased breast cancer risk.