Biochemical study of cyst fluid in human breast cystic disease: a review

Summary Gross cystic disease of the breast may sometimes indicate an increased risk of breast cancer. Biochemical analysis of the cyst fluid could suggest which cysts are associated with breast cancer risk, as well as providing insights into the pathophysiology of this condition. The Na⁺/K⁺ ratio appears to be associated with the histological classification of the cyst. Sulfoconjugated estrogens and androgens, especially DHEA-S, are often found at high levels. A number of gross cystic disease fluid proteins (GCDFPs) have been described, and several polypeptide growth factors including EGF and IGF-I are frequently found. It is hoped that biochemical analysis of these components of breast cyst fluids will shed further light on the role of gross cysts in relation to breast cancer.     

Conjugated bile acids in breast cyst fluids: relationship to cation-related cyst subpopulations

Gross cystic breast disease is a benign lesion occurring in 7% of adult women. Apocrine changes of epithelium lining the breast cysts cause a higher risk of developing breast cancer. According to the possible role of bile acids in the pathogenesis of cancer, we analysed breast cyst fluids aspirated from 96 women for distribution of conjugated bile acid concentrations in the two subsets of breast cysts. Bile acid levels were correlated to K+ concentrations (P < 0.0001) and mean value was higher in Na/K < 3 metabolically active apocrine cyst as compared with Na/K > 3 flattened cyst (P < 0.001). Because bile acids could play an important role in the pathogenesis and growth of breast cancer, the significantly higher intracystic concentrations of these carcinogen compounds in apocrine Type I cysts might provide a further biological explanation as to why women with apocrine changes may be at higher breast cancer risk and could be useful for the biochemical knowledge occurring in the different functional stages of the gross breast cysts.     

Lipids status in human breast cyst fluids

Benign mammary gross cystic disease is the most common breast lesion; women with apocrine changes of epithelium lining the cysts are at higher risk for developing breast cancer than the normal population. Total cholesterol, high- and low-density lipoproteins fractions, triglycerides and phospholipids, lipase activity and total lipid concentrations were measured in cyst fluids and sera from 89 women affected by gross cystic breast disease. Total cholesterol and high-density lipoprotein content were significantly (P<0.001) greater in pooled cyst fluids than normal sera. Moreover, data analyses show a significant increase in the mean values of total lipids and lipase activity in metabolically active apocrine cysts, when compared to the flattened cysts (P<0.001). The lipids feature of apocrine cysts could represent an altered expression of biosynthetic activity of the surrounding apocrine cell surface glycolipid and steroidogenic metabolism and may provide further knowledge about the functional stage changes of gross breast cysts.     

Presence of immunoassayable transforming growth factor-β1 (tgf-β1) in breast cyst fluid (BCF): Relationship with the intracystic electrolyte and epidermal-growth-factor (EGF) content

We evaluated the presence and distribution of transforming growth factor-β1 (TGF-β1) in breast cyst fluid (BCF), and its relationship with intracystic epidermal growth factor (EGF). EGF and TGF-β1 were determined by radioimmunoassay on 47 BCFs (27 of the Na⁺/K⁺ <3 type and 20 of the Na⁺/K⁺ >3 type). As expected, EGF levels were inversely correlated with the Na⁺/K⁺ ratio, and were consequently higher in Na⁺/K⁺ <3 cysts as compared with Na⁺/K⁺ >3 cysts, (p < 0.005). By contrast, TGF-β1 levels were directly correlated with the Na⁺/K⁺ ratio (p < 0.01), being higher in Na⁺/K⁺ >3 cysts, though not significantly (p = 0.057). A significant negative relationship was found between EGF and TGF-β1 concentration. When the analysis was performed separately in the 2 cyst sub-populations, EGF and TGF-β1 were found to be negatively and significantly correlated in Na⁺/K⁺ <3 cysts only (p < 0.01). Our results demonstrate that Na⁺/K⁺ <3 cysts contain high levels of EGF, a growth-stimulating factor, and very low levels of TGF-β1, a growth-inhibiting factor. This may provide an explanation for the higher risk of breast cancer observed in women with Na⁺/K⁺ <3 cysts. Our results also suggest that EGF accumulation in this type of cysts might be regulated by TGF-β1.     

Quantification of prostate-specific antigen immunoreactivity in human breast cyst fluids

Summary The frequency of gross cystic breast disease in premenopausal women and its possible association with in-creased breast cancer risk emphasises the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured the presence of prostate-specific antigen immuno-reactivity in sixty-four human breast cyst fluids. Data analyses show that 35% of samples presented a level of this antigen < 0.05 µg/L, whereas 42 out of 64 cysts show a significant increase in the mean value of metabolically active apocrine cysts when compared to flattened cysts (p < 0.01). We report the first evidence that breast epithelium of gross cysts produces, secretes, and accumulates large amounts of prostate-specific antigen, a glycoprotein produced by prostatic tissue but recently detected in breast tumours, normal tissues, and during pregnancy. The production and intracystic accumulation of this serine protease in biosynthetically active apocrine type cyst can play a feasible role in the natural history of gross cystic breast disease as well as in the mechanism of cyst formation, enlargement, and transformation.     

Prostate‐specific antigen found in Type I breast cyst fluids is a secretory product of the apocrine cells lining breast gross cysts

Prostatespecific antigen (PSA), a serine protease thought to be exclusively produced by the prostate epithelial cells, has been recently found in human breast tissues and fluids. PSA in breast cancer is associated with the presence of steroidhormones and receptors, and its presence seems to be a favourable prognostic indicator. In order to clarify whether the cells lining breast cysts may represent the source of PSA found in human breast cyst fluid, we performed an ultrastructural immunolocalization of PSA in the cells surrounding Type I breast cysts, obtained from breast cyst fluids of women affected by breast gross cystic disease, the most commonly occurring benign breast lesions associated with increased cancer risk. These apocrine cells show morphological features typical of actively synthesizing and secreting cells, and a PSA labelling distributed on free ribosomes, RER cisternae, and secretory granules, indicating that the metabolically active apocrine cells lining the Type I cysts are responsible for the production and secretion of PSA in Type I breast cyst fluids. The synthesis and intracystic accumulation of this serine protease in biosynthetically active apocrine Type I cysts can play an important role in the natural history of breast gross cystic disease as well as in the mechanism of cyst evolution.     

Relationship between epidermal growth factor and dehydroepiandrosterone and its sulphate in breast cyst fluid

Gross cystic breast disease is a common condition. Women with apocrine breast cysts may be at higher risk of breast cancer than women with cysts which are lined by flattened epithelium. Apocrine cysts have been shown to be associated with higher intracystic levels of dehydroepiandrosterone sulphate and intracystic sodium to potassium ratios of less than 3. In this study we measured the concentrations of epidermal growth factor, dehydroepiandrosterone and its sulphate in breast cyst fluid. The concentrations of all three analytes were significantly higher in cysts with intracystic Na+/K+ ratios of less than 3 than in cysts with electrolyte ratios of greater than or equal to 3 (P less than 0.001). The higher levels of EGF in cysts with low intracystic electrolyte ratios may provide an explanation of why women with apocrine cysts may be at greater risk of breast cancer. Positive correlations were obtained between concentrations of EGF and DHAS and between EGF and DHA, compatible with the view that intracystic EGF levels may be androgen-modulated. A positive correlation was also obtained between DHA and DHAS concentrations which supports the view that DHA in cyst fluid may be derived from the metabolism of DHAS in the breast cyst wall.     

Presence of epidermal growth factor receptor in breast smears of cyst fluids: relationship to electrolyte ratios and pH concentration.

The purpose of this study was to evaluate and correlate different high risk factors in breast cysts to produce a more accurate prognosis. We considered the cytology of the lining epithelium (apocrine or flattened), the Na+/K+ ratio (less than 3; greater than or equal to 3), the pH (lower, equal, or higher than 7.30), and the EGFr (present or not) in an attempt to reach better prognoses and diagnoses by evaluating more than one risk factor. Our material of 40 macrocysts consisted of 23 simple cysts with flattened epithelium and 17 complex cysts with apocrine or hyperplastic epithelium. In the simple cysts, the Na+/K+ ratio was greater than or equal to 3, while in the complex cysts the Na+/K+ ratio was, in all cases but one, less than 3. The pH was not significantly lower than neutral in the complex cysts. The EGFr was detected in 5 of 23 simple cysts and in 12 of 17 complex cysts. In conclusion, reaction of EGFr in smears of cyst fluids with the low intracystic electrolyte ratios may provide an explanation of why women with apocrine metaplasia or epithelial hyperplasia may be at higher risk of breast cancer than women with cysts which are lined by flattened epithelium.     

Epidermal growth factor levels in human breast cyst fluid

Epidermal growth factor (EGF) seems to modulate the in vitro and in vivo growth of normal and neoplastic breast cells. We determined, by a radio-receptor assay, EGF levels in cyst fluid and in plasma of patients with gross cystic disease of the breast. The mean levels of EGF were lower in plasma than in breast cyst fluid (BCF) (p less than 0.001). In BCF of apocrine cysts we found higher EGF levels than in flattened cysts (p less than 0.001). The EGF content of apocrine BCF seems to be under sex steroid hormone control, being higher in reproductive age than in post menopause (p less than 0.05). Since it has been reported that patients with apocrine cysts are at a greater risk of developing breast cancer, we hypothesize that the high EGF concentration in apocrine BCF may play a role in the autocrine breast cyst epithelium growth control and neoplastic transformation.     

Sodium/potasium ATPase (Na+, K+-ATPase) and ouabain/related cardiac glycosides: a new paradigm for development of anti- breast cancer drugs?

Summary Prolonged exposure to 17β-estradiol (E₂) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERα and ERβ. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na⁺, K⁺-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na⁺, K⁺-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na⁺, K⁺-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na⁺, K⁺-ATPase inhibitors and ER antagonists.     

Enterolactone in breast cyst fluid: correlation with EGF and breast cancer risk

The purpose of our study was to investigate whether enterolactone does accumulate into breast cyst fluid and whether it correlates with breast cancer risk. We included 258 women who had at least one cyst aspiration and known intracystic cation and epidermal growth factor (EGF) concentration values. For 191 of such women serum aliquots were also available. The median value of serum enterolactone was 17 nM/l (range 1–140 nM/l). The median intracystic level of enterolactone was much higher (63 nM/l, range 0–872 nM/l) and was significantly higher in type I cysts (p = 0.000). This cyst type contained also significantly higher levels of EGF (p = 0.000). A direct relationship was found between serum and cyst fluid enterolactone levels (p = 0.000) and between cyst enterolactone and EGF levels (p = 0.03), the latter correlation being evident especially in type II cysts. Twelve patients in the cohort of women were found to have developed a breast cancer. After univariate analysis breast cancer risk was associated with cyst type and especially with EGF concentration. No association was evident for enterolactone concentration. However, enterolactone concentration appeared to significantly decrease the risk of patients with high EGF concentrations. Our results show that enterolactone does accumulate in breast cysts, and that it modulates the risk related to the intracystic level of EGF, which is confirmed to be a strong predictor of breast cancer risk.     

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.     

Mitogenic peptides in breast cyst fluid: relationship with intracystic electrolyte ratios

Women with palpable breast cysts which are lined with apocrine epithelium may be at higher risk of developing breast cancer than women with breast cysts which are lined with flattened epithelium, the former group being characterized by intracystic sodium to potassium ratios below 3, while the latter group has intracystic sodium to potassium ratios above 3. In this study the distribution of intracystic concentrations of the mitogenic peptides, epidermal growth factor, endothelin and gastrin-releasing peptide in the 2 groups of breast cysts were compared to see whether differences in concentrations between the 2 cyst groups might provide an explanation for the higher risk of breast cancer observed in women with "apocrine" breast cysts. The concentrations of epidermal growth factor and gastrin-releasing peptide were significantly higher in the low electrolyte ratio group (p less than 0.001). There was no difference in endothelin concentrations between the 2 groups. Negative correlations were found between epidermal growth factor concentrations and Na+/K+ and between gastrin-releasing peptide concentrations and Na+/K+ (p less than 0.001). A positive correlation was found between gastrin-releasing peptide and epidermal growth factor concentrations in breast cyst fluid (p less than 0.001). The significantly higher intracystic concentrations of both epidermal growth factor and gastrin-releasing peptide in the low-electrolyte-ratio group may provide an explanation for the higher risk of breast cancer which has been observed in women with "apocrine" breast cysts.     

Identification of breast cyst subpopulations: biochemical and morphological features

The concentration of insulin-like growth factor-I (IGF-I) and its relationship to other biochemical parameters of cyst fluids was investigated in 94 cyst fluids of 86 women with gross cystic breast disease. The relationship between the biochemical parameters and the cytological features of breast fluids (presence or absence of apocrine cells) was also studied. IGF-I was detected in all tested fluids, with a concentration 50 to 100 times lower than that found in plasma. IGF-I concentration was higher in cysts with a Na+/K+ ratio greater than 3 (greater than 3) and was inversely related to both dehydroepiandrosterone sulfate (DHEA-S) and epidermal growth factor (EGF) concentration (p less than 0.001). It is suggested that Na+/K+ greater than 3 cysts have a higher permeability to plasma and extracellular fluids compared to Na+/K+ less than 3 cysts. Apocrine cells were found in 78% of Na+/K+ less than 3 fluids as well as in 53% of Na+/K+ greater than 3 fluids. A well-defined relationship was found between the biochemical parameters of breast fluids, but the presence of either IGF-I or EGF was not related to the morphology of breast cysts as assessed by cytological examination.     

Zinc alpha-2 glycoprotein levels in serum and breast fluids: a potential marker of apocrine activity

Zinc alpha-2 glycoprotein (ZnGP) was measured in human breast microcysts, breast secretions, breast cyst fluid and serum. Detectable amounts of ZnGP were found in all fluids but the highest levels were found in microcysts. Apocrine macrocysts had a higher ZnGP level than flattened macrocysts. In both cysts and secretions levels of ZnGP correlated with those of dehydroepiandrosterone sulphate. Levels were significantly higher in cyst fluids from women who developed further cysts during follow-up compared with those in fluid from women who did not. Concentrations of ZnGP in serum from breast cancer patients were significantly higher than controls but not women with breast cysts. Women with node positive breast cancer had higher serum levels compared with those in node negative patients. Women with more advanced breast cancer had higher serum ZnGP levels than those with earlier disease. ZnGP is a serum and breast marker of apocrine activity and may prove to be a useful prognostic marker in breast cancer.     

Using biological measurements, can patients with benign breast disease who are at high risk for breast cancer be identified?

In order to address the question of whether biological measurements might identify women with benign breast disease (BBD) at particular risk for breast cancer, analyses were performed on cyst fluids aspirated from patients presenting with palpable breast cysts. Electrolyte profiles showed that cyst fluids may be divided into major subpopulations which differ in terms of histological appearance of cyst lining epithelium, pattern of cyst presentation, and levels of other fluid constituents such as androgen conjugates and epidermal growth factor. Analysis of cyst fluids from 18 patients who subsequently developed breast cancer 1 to 8 years later showed that 12 individuals had group A cysts, three had group B cysts, and three presented with a mixture of the two types. Therefore, although this represents an increased proportion of group A cysts as compared with the total population of cyst fluids studied over the same time period, individuals subsequently developing breast cancer were not confined to one subgroup of cysts. Androgen conjugate and growth factor content also did not predict for subsequent cancer. At the present time, it is therefore concluded that biochemical measurements in cyst fluids cannot accurately identify women likely to develop breast cancer. However, the routine aspiration of cysts does provide the opportunity to monitor the local microenvironment of the breast.     

Epidermal growth factor content of breast cyst fluids from women with breast cancer or proliferative disease of the breast

Summary The intracystic electrolyte content is generally used to identify different breast cyst subpopulations: cysts containing high K+ levels have been associated with an increased risk of subsequent breast cancer. In order to define whether other biochemical features of breast cyst fluid (BCF) might further explain such an increased risk, we determined the content of epidermal growth factor (EGF), a known mitogenic factor for normal and transformed breast epithelium, in cysts of women with breast cancer or proliferative lesions of the breast (atypical ductal or lobular hyperplasia and proliferative disease without atypia).Median intracystic EGF levels were significantly higher in patients with breast cancer or atypical hyperplasia than in cysts of women without any clinical or instrumental evidence of proliferative disease chosen as controls (p < 0.05 and p < 0.01, respectively). In patients affected by proliferative disease without atypia, intracystic EGF levels were not different either from controls or from the other study groups. No significant difference among groups was observed in the prevalence of Na+/K+< 3 cysts, this being the most frequently observed type of cysts in all groups except in that with proliferative disease without atypia. No significant difference in EGF levels between cysts ipsilateral or contralateral to the biopsy was observed within each histological group.Our results indicate that EGF levels are higher in cysts aspirated from breasts with an associated proliferative pathology, either benign or neoplastic. The determination of intracystic EGF, combined with that of electrolyte content, might help to identify a subset of patients with gross cystic disease of the breast at potentially higher risk of developing breast cancer.     

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)     

Contribution of Na+,HCO3−‐cotransport to cellular pH control in human breast cancer: A role for the breast cancer susceptibility locus NBCn1 (SLC4A7)

Genome‐wide association studies recently linked the locus for Na⁺,HCO₃^?‐cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO₃^? into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na⁺,HCO₃^?‐cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20–30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na⁺/H⁺‐exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH‐sensitive fluorophores, we showed that Na⁺,HCO₃^?‐cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid in human primary breast carcinomas. At intracellular pH (pH_i) levels >6.6, CO₂/HCO₃^?‐dependent mechanisms accounted for >90% of total net acid extrusion. Na⁺/H⁺‐exchange activity was prominent only at lower pH_i‐values. Furthermore, steady‐state pH_i was 0.35 ± 0.06 units lower in the absence than in the presence of CO₂/HCO₃^?. In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na⁺,HCO₃^?‐cotransport is a major determinant of pH_i in breast cancer and the modest DIDS‐sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.     

The biochemistry of breast cyst fluids and duct secretions

Summary The ratio of potassium to sodium concentrations in breast fluids has led other investigators to the subclassification of cysts into two types: 1) apocrine (secretory) cysts with high potassium and low sodium, and 2) attenuated (flattened) cell cysts with low potassium and high sodium content. Apocrine cells are thought by some to actively secrete potassium. Cell typing is considered important as apocrine cysts are more likely to be bilateral, multiple, recurrent, and serve as markers for epithelial cell atypia.A retrospective study of the biochemical analyses of 58 cyst fluids and 28 duct secretions obtained by nipple aspiration was conducted. Potassium and sodium concentrations obtained from 12 cyst fluids were statistically correlated with creatinine concentrations. Evidence is presented indicating that micro cysts are initially apocrine in cell type and are more likely in continuity with the terminal ductal-lobular unit. It is postulated that apocrine cysts undergo cellular desquamation and lysis, becoming attenuated cysts. The ratio of potassium to sodium is altered by cell degradation rather than active secretory processes. Biochemical contents of cysts and nipple aspiration fluids are compared.We regret to report that Dr. Sartorius died on Dec. 16.1994.