Identification of the major protein components in breast secretions from women with benign and malignant breast diseases.

The protein composition of breast secretions from 99 premenopausal women with benign or malignant breast diseases and from 70 control women without breast pathologies has been studied by using polyacrylamide gel electrophoresis. These fluids have been classified into two types according to their major polypeptide components. Type I fluids are defined by three major distinctive bands at Mr 44,000, 24,000, and 17,000, while those designated Type II present distinctive bands at Mr 80,000, 15,000, and 14,000. Amino acid sequencing and immunoblotting analysis demonstrated that proteins in Type I secretions correspond to Zn-alpha 2-glycoprotein, apolipoprotein D, and gross cystic disease fluid protein-15, while those from Type II fluids have been identified as lactoferrin, lysozyme, and alpha-lactalbumin. Most women (93%) without breast pathology and most patients (88%) with benign diseases had secretions with a Type I polypeptide pattern. By contrast, a large percentage (57%) of secretions from women with breast carcinoma presented a Type II protein pattern. Further studies with a large number of women will be useful for corroborating the potential clinical interest of breast fluid protein analysis.     

Aromatase and prostaglandin inter-relationships in breast adipose tissue: significance for breast cancer development

The role of prostaglandins in the development of breast cancer is a topic of growing interest. Stimulation of aromatase expression within the breast has been proposed as a mechanism whereby prostaglandins could influence breast cancer growth. In the present study, we show that PGE₂ is a powerful stimulator of aromatase expression in human breast adipose stromal cells. Moreover, TNF, which also stimulates aromatase expression in breast adipose stromal cells, acts to increase the secretion of PGE₂ by these cells, as well as the expression of COX 2 and PGE synthase, but not that of COX 1. On the other hand, class I cytokines had no effect, either by themselves or in the presence of estradiol. These factors had little influence on secretion of 15-deoxy-^12,14-PGJ₂, which is inhibitory of aromatase expression by breast adipose stromal cells. These results further substantiate an important role for PGE₂ to stimulate estrogen biosynthesis within the local environment of the breast.     

Molecular forms of prostate-specific antigen in the serum of women with benign and malignant breast diseases.

Using a highly sensitive immunofluorometric procedure, we measured the total prostate-specific antigen (PSA) concentration in 632 sera obtained from female blood donors and women with idiopathic hirsutism, breast cancer or benign breast diseases. A total of 50 sera with total PSA > 15 ng l(-1) were fractionated by high-performance liquid chromatography (HPLC) in order to resolve the two immunoreactive molecular forms, i.e. free PSA (approximately 30 kDa) and PSA bound to alpha1-antichymotrypsin (PSA-ACT, 100 kDa). We found that breast cancer patients have presurgical serum total PSA levels similar to those of blood donors. Total serum PSA concentration decreases with age in women with idiopathic hirsutism, in cancer patients and in patients with benign breast diseases. The major molecular form of PSA in the serum of all normal and hirsute women (n = 15) is PSA bound to the proteinase inhibitor alpha1-antichymotrypsin. The major molecular form in 44% of presurgical cancer patient sera is free PSA. A total of 58% of benign breast disease patients also have in their serum mainly free PSA. We conclude that about half the patients with breast cancer or benign breast diseases have free PSA as the major molecular form in their serum, whereas patients without breast pathologies (normal blood donors, idiopathic hirsutism) have PSA bound to alpha1-antichymotrypsin as the major molecular form. The ratio of PSA/PSA-ACT may have value as a simple biochemical test for diagnosis of breast pathologies including breast cancer.     

Gamma-glutamyl transpeptidase immunoreactivity in benign and malignant breast tissue

GGT 129, a polyclonal antibody directed against gamma-glutamyltranspeptidase (GGT), was used to study GGT expression in formalin-fixedparaffin-embedded tissues from normal breast, 24 benign lesions, 27 in situcarcinomas or atypical hyperplasias, and 79 infiltrating mammary carcinomas.Epithelium of the ducts and ductules in normal breast tissue showedimmunoreactivity along the apical surface. There was a strong correlation (P< 0.01) between the histologic classification of the tissue and GGTexpression. All of the benign breast lesions stained positive for GGT. Amongin situ carcinomas and atypical hyperplasias, 5/27 (19%) werenegative for GGT while 22/27 were immunopositive. Infiltrating carcinomasshowed the greatest deviation from normal tissue with 23/79 (29%)negative for GGT. GGT expression in benign and malignant breast tissue wasnot correlated with the age of the patient, suggesting that menopausalstatus does not influence expression of GGT. Correlation of GGTimmunoreactivity with tubule formation, nuclear pleomorphism, mitoses,grade, size of tumor, lymph node status, and ER/PR status was performed for69 cases of infiltrating ductal adenocarcinoma. There were no statisticallysignificant relationships between the level of GGT immunoreactivity and anyof the parameters. The loss of GGT in some of the cases is evidence thatthis enzyme is not required for mammary tumor development or maintenance.However, as GGT is a component of the pathways that metabolize glutathioneand glutathione-conjugates, the difference in levels of the enzyme ininvasive breast cancers may be one explanation for the variation inchemotherapy response that has been observed in patients treated foradvanced mammary cancer.     

Serum oestradiol-17 beta in women with benign and malignant breast disease

Serum concentrations of oestradiol-17β were measured daily throughout one menstrual cycle in 32 normal women, 31 women with benign disease of the breast and 10 with cancer of the breast. The concentrations were found to differ significantly from normal in women with cysts and to a lesser extent in those with cancer of the breast. In 25 normal post-menopausal and in 27 post-menopausal women with cancer repeated assays disclosed consistently low levels of oestradiol-17β.     

Trace elements and superoxide dismutase in benign and malignant breast diseases

This study was planned to determine the probable changes in trace elementlevels and superoxide dismutase (SOD) activity in women with neoplasticbreast diseases. Measurements were performed in three different groups. Thefirst group consisted of 20 healthy women, control group, the second groupcontained 16 patients with benign breast disease and the third groupcontained 39 patients with malignant breast disease. The trace elementconcentrations were determined by using atomic absorption spectrophotometryand SOD activity by using spectrophotometry. When compared with the controlvalues, the plasma copper levels were slightly increased in the second groupand significantly in the third group (p < 0.001). The difference betweenthe benign and malignant groups was also significant (p < 0.001). The redcell copper values showed a marked decrease in both groups (p < 0.001).Although there were increases in the plasma zinc levels of both patientgroups, the differences were not significant statistically. But, the redcell zinc values showed an significant increase in benign and malignantpatients compared to the control group (p < 0.001) (p < 0.001). Theplasma magnesium and red cell magnesium values did not show significantdifferences. The red cell SOD activity showed an significant increase in thebenign and malignant patient groups (p < 0.001). The results of thisstudy suggested that reactive oxygen metabolites may play a pathogeneticrole in both benign and malignant tumor development, which is reflected bythe change in SOD activity, and in trace element concentrations.     

Endogenous sugar-binding proteins in human breast tissue and benign and malignant breast lesions

Binding of carbohydrate moieties was detected in tissue sections of human breast by employing two types of labeled ligands: neoglycoproteins (chemically glycosylated, histochemically inert carrier protein) and desialylated naturally occurring glycoproteins. Paraffin-embedded, formalin-fixed sections from 40 benign and malignant breast lesions were examined for the presence and distribution of endogenous sugar receptors, employing a panel of 13 biotinylated neoglycoproteins, representing carbohydrates commonly found in naturally occurring glycoconjugates, and four biotinylated glycoproteins. Benign and malignant breast lesions revealed staining with mannosylated carrier neoglycoprotein in comparison to normal breast. A mixed pattern of staining localization and intensity was seen for different types of malignancy with this neoglycoprotein. Similarly, receptors for lactose and N-acetylglucosamine could only be detected within the cytoplasm for certain types of malignancy. Their nuclear localization, however, could also be seen in normal breast specimens. The extent of staining with different glycoproteins, containing different types of galactoside-terminal sugar chains, also appeared to differ between various types of breast cancer. The detection of endogenous sugar receptors by neoglycoproteins is proposed to contribute to an understanding of malignancy-associated alterations in the structure of their potential physiological ligands, the glycoconjugates. Changes in the structure and abundance of such glycoconjugates have commonly been detected with the use of plant lectins in histopathologic studies.     

Comparison of age at first full-term pregnancy between women with breast cancer and women with benign breast diseases

Benign breast diseases have a broadly similar risk profile to that of breast cancer, possibly reflecting a similar underlying endocrine milieu. We have hypothesized that a crucial distinction between breast cancer and benign breast diseases is that mammary gland terminal differentiation has not been successfully accomplished among women who tend to develop breast cancer. From October 2001 to December 2002, information concerning breast cancer risk factors and sociodemographic characteristics was collected from 174 women with breast cancer and 116 women with benign breast diseases, all 30 years old or older, who were histologically diagnosed at a major prevention center in Athens, Greece. Among the examined breast cancer risk factors, only age at first full-term pregnancy was significantly associated with the odds of having breast cancer rather than benign breast disease, and the association was evident among premenopausal [odds ratio (OR) per 5 years = 1.76, 95% confidence interval (CI) 1.10-2.93] and postmenopausal (OR = 2.10, 95% CI 1.16-3.71) women, as well as among all women (OR = 1.93, 95% CI 1.34-2.70). There was no evidence that any of the remaining breast cancer risk factors could discriminate between breast cancer and benign breast diseases. We conclude that early age at first pregnancy may convey substantial protection against breast cancer risk among women with benign breast diseases, probably operating through induction of terminal differentiation of mammary gland cells. The finding is accentuated by the fact that women with benign breast diseases are already at a relatively high risk for breast cancer.     

还氧合酶-2在乳腺良恶性病变中的表达及意义

目的 探讨环氧合酶2(COX-2)在乳腺良恶性病变中的表达和分布情况及COX-2表达与乳腺癌临床病理因素、激素受体等的关系.方法 分别对8例副乳腺、31例乳腺良性增生性疾病(BPBD,包括腺病15例,纤维腺瘤16例)、70例浸润性导管癌(IDC),其中35例伴有导管原位癌(DCIS)标本进行COX-2免疫组化标记,观察COX-2在这些组织中的表达情况和分布特征并与C-erbB-2、雌激素受体(ER)、孕激素受体(PR)、临床病理因素等进行对比.结果 ①COX-2表达8例副乳腺全部阴性;BPBD阳性率为96.5%(30/31),其中腺病93.3%(14/15),纤维腺瘤100%(16/16);DCIS阳性率为85.7%(30/35),表达强度≥相应癌组织;IDC阳性率为84.3%(59/70),癌周非癌腺体有不同程度的COX-2表达.②COX-2在BPBD的阳性率明显高于IDC,两者间差异有统计学意义(χ2=9.39,P＜0.025);③COX-2阳性表达在DCIS中与组织分化低(χ2=10.98,P＜0.005)、PR阴性(P=0.019)及C-erbB-2阳性(P=0.0008)有关.在IDC中主要与淋巴结转移(χ2=4.09,P＜0.05)、组织分化低(P=0.004)、PR阴性(χ2=6.91,P＜0.01)及C-erbB-2阳性(χ2=5.94,P＜0.025)等有关.结论 COX-2在正常乳腺组织不表达,在BPBD、DCIS和IDC中均有较高表达,提示COX-2表达上调既参与乳腺致癌,又促进其发展和转移,并可能参与了PR阴性乳腺癌对辅助性内分泌治疗的抵抗性而使治疗反应性下降.     

Aromatase activity and its gene expression in tumor tissue of smokers and non-smokers with breast cancer

Tissue was sampled from 121 tumors of the breast. The activity of aromatase (estrogen synthetase) was assessed by radiobiochemical means in 61 cases; gene expression was evaluated with the aid of polymerase chain reaction in 14 and the same--by the dot-blot procedure in 46 patients. Inveterate smokers (15 years and more) made up 16.5%. The smokers revealed a distinct tendency towards aromatase activity decreasing in tumor tissue (chiefly in menopausal patients) as well as lower intensity of aromatase gene expression assessed in terms of polymerase chain reaction. Alongside with other evidence, our finding point to long-term-smoking-related sensitivity of intratissular estrogen synthesis being higher than in general circulation. It also demonstrated local aromatization inhibition in tumor tissue, the latter being a possible mechanism which causes tumor tissue hormone sensitivity to change in smokers and affects course of disease.     

Tissue carcinoembryonic antigen levels in benign and malignant diseases of the breast

The peroxidase-antiperoxidase histochemical method of staining for tissue carcinoembryonic antigen (CEA) was performed on 20 samples of malignant breast tissue, 20 samples of fibroadenomatous breast tissue, and 15 samples of breast tissue that variably contained minimal fibrosis (N = 7), ductal ectasia (N = 5), and sclerosing adenosis (N = 3; the fibrocystic changes of the breast). The intensity of staining was described to be either negative, weak, intermediate, or strong and was assigned a point value of 1, 2, 3, or 4, respectively. The following weighted average values of tissue CEA were obtained: carcinoma, 3.35 +/- 0.88; fibroadenomas, 2.85 +/- 0.67; fibrocystic changes, 2.13 +/- 0.52. Carcinomatous tissue is likely (55%) to display intense tissue staining, whereas fibrocystic disease (0%) or fibroadenoma (15%) are unlikely to exhibit such a reaction. The tissue CEA content between carcinoma and fibrocystic changes (P less than 0.01), carcinoma and fibroadenoma (P less than 0.01), and fibroadenoma and fibrocystic changes (P less than 0.05) are statistically significant.     

Differential expression of cellular oncogenes in benign and malignant human breast tissue

We have examined 62 specimens of benign fibrocystic breast tissue, fibroadenomas, carcinomas and surrounding non-malignant tissue excised from 50 patients to determine the level of expression of 4 cellular oncogenes, c-myc, c-H-ras, c-K-ras, and c-N-ras. Our results demonstrate that in breast carcinoma the frequency and relative level of expression of these oncogenes are significantly greater than those found for benign breast tissue. However, some fibrocystic specimens having prominent hyperplastic features also exhibited enhanced oncogene expression. In view of the association between the increased frequency of carcinoma of the breast in women with a previous history of benign breast disease, it will be interesting to follow up donors of benign specimens to see if there is any relationship between the expression of oncogenes in such lesions and the development of carcinomas.     

An immunohistological study of leukocyte localization in benign and malignant breast tissue

Using morphometric and immunohistological techniques, we investigated the distribution of T lymphocytes and subsets, B lymphocytes, macrophages and plasma cells in sections of human breast tissue. In normal lobules and ducts, leukocytes were found in greater density in the epithelium than in the stroma. The intra-epithelial cells consisted largely of suppressor/cytotoxic T lymphocytes with a smaller number of macrophages; inducer T cells were seen in only 1 out of 10 subjects and were present in very low numbers. In the stroma, there were roughly equal numbers of suppressor/cytotoxic cells, macrophages and plasma cells containing IgA or IgM. Small numbers of stromal inducer lymphocytes were seen in only 2 cases. Although the density of suppressor/cytotoxic cells in the epithelium was considerably greater than in the stroma, macrophages were present in roughly similar density in both locations. In carcinoma, the ratio of epithelial to stromal leukocyte density was reversed, due to a marked reduction in intra-epithelial cells and to an increase in those in the stroma. Between 10% and 30% of lymphocytes expressed activation markers in contrast to the normal breast in which virtually all were negative. Inducer lymphocytes were identified in 9/10 carcinomas. The possible relationship of inducer cells to breast carcinoma and pre-neoplasia is discussed and warrants further investigation. No alteration in stromal plasma cell numbers was observed.     

Immunohistochemical demonstration of metallothionein in normal human breast tissue and benign and malignant breast lesions

Metallothioneins (MTs) are a set of low molecular weight proteins with a high binding affinity to metal ions. MT over-expression has been recently demonstrated in invasive ductal carcinoma of the breast with poor clinical prognosis. In the present study, MTs have been immunohistochemically investigated in normal human breast tissue and a variety of benign, pre-invasive, and malignant breast lesions. In normal breast tissue, MTs were present in myoepithelial cells whereas the vast majority of luminal cells were MT negative. In lesions without increased cancer risk (adenosis and scleradenosis), MT was only immunolocalized in myoepithelial cells. In papillomas, MT was also found exclusively in myoepithelial cells. In most cases of epitheliosis, both the luminal and myoepithelial cells expressed MT. Atypicallobular hyperplasia, lobular carcinomain situ, and 13/15 invasive lobular carcinomas showed no MT over-expression. The two invasive lobular carcinomas with MT over-expression were classified as pleomorphic lobular carcinomas with apocrine differentiation. In contrast to lobular cancerization, 12/24 ductalin situ carcinomas and 9/20 invasive ductal carcinomas showed MT over-expression.In situ components found within invasive ductal carcinomas usually reflected the MT status of their invasive counterpart. It is concluded from our immunohistochemical results that breast carcinoma cases with MT overexpression arise from lesions which also show MT overexpression. Thus MT expression in carcinomas may be regarded as a genuine feature of the tumour cells and seems not to be related to endogenous or exogenous factors known to induce MT synthesis.     

Serial analysis of fibronectin concentration in plasma of patients with benign and malignant breast diseases

The serial levels of fibronectin (FNp) in plasma from 65 patients with benign and malignant breast disease and from 74 healthy control women were assayed by the use of the rocket immunoelectrophoresis procedure. Mean FNp levels in patients with breast cancer were age-matched with control subjects, but no clear correlation was found between FNp levels and the presence of primary tumor. Mean FNp values for fibroadenoma patients did not differ either from controls or from patients with malignant disease. Patients were also categorized according to TNM classification, to the number of positive axillary nodes, to the histologic grade of malignancy, and to the presence of estrogen receptors. Although differences were not significant, a higher number of patients with levels greater than the normal 95% percentile were found only in the group with four or more positive axillary nodes and in the group with a greater number of histologic malignancies. Marked fluctuations in FNp concentrations were found in individual patients during the follow-up period, independently of the treatment received. FNp seems unsuitable as a tumor marker because, besides its apparent lack of specificity for cancer, it reflected neither the host-tumor burden nor the response to treatment.     

COX-2和VEGF-C基因在乳腺良恶性病变中的表达研究

目的 研究COX-2和VEGF-C mRNA在乳腺良恶性病变中的表达情况,探讨两者在乳腺肿瘤发生过程中的相互关系.方法 运用Real-time PCR方法对90例乳腺良恶性病变组织中COX-2和VEGF-C mRNA的表达进行检测.结果 8例副乳腺组织中,COX-2和VEGF-C mRNA不表达或低表达,15例腺病和16例纤维腺瘤中两者表达明显增加(P＜0.05),11例DCIS中,两者均高表达,高于腺病和纤维腺瘤(P＜0.05),40例IDC中,两者同样高表达,并且肿瘤级别越高,表达水平越高.在DCIS和IDC中,两者表达存在正相关关系(分别r=0.82,P=0.002;r=0.89,P=0.000).结论 COX-2和VEGF-C基因在乳腺良恶性病变中表达明显增高,而且在恶性肿瘤中的表达高于良性肿瘤,两者在乳腺癌组织中的表达呈正相关,表明两者参与了乳腺疾病,尤其是乳腺癌发生、发展的过程.     

Sex steroid hormone levels in breast adipose tissue and serum in postmenopausal women

Elevated levels of circulating estrogens and androgens are linked to higher breast cancer risk among postmenopausal women; however, little is known about hormone levels within the breast. Hormone concentrations within the breast may not be reflected in the blood and are likely important contributors to breast carcinogenesis. We used a previously validated method to measure levels of estrone, estradiol, androstenedione, and testosterone in adipose tissue removed as part of breast excisions performed for cancer in 100 postmenopausal women (69 ER/PR +/+ and 31 ER/PR −/−) participating in a breast cancer case–control study. We also measured the same steroid hormones, as well as estrone sulfate, and sex hormone-binding globulin (SHBG) in serum from these patients and 100 controls matched on ages at blood collection and on menopause. Overall, concentrations of serum hormones did not vary significantly between controls and cases. However, women with ER−/PR− breast cancers had lower circulating levels of all measured sex steroid hormones and higher SHBG levels than women with ER+/PR+ breast cancers and controls. Similarly, hormone concentrations in breast adipose tissue were higher among women with ER+/PR+ compared to ER−/PR− breast cancer, although differences were only significant for testosterone. These data demonstrate that high sex steroid concentrations in both serum and adipose tissues are more strongly related to ER+/PR+ than ER−/PR− breast cancers. Measurement of sex hormones in serum and in the microenvironment may help in understanding the hormonal etiology of breast cancer, suggest methods for prevention, and have value in gauging treatment response and prognosis.