Steroid patterns of benign breast disease.

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.

DNA in situ sensitivity to denaturation as a marker of human breast tumors.

DNA content and in situ sensitivity to denaturation were analyzed by flow cytometry of individual cell nuclei isolated from 40 breast carcinomas, nine fibroadenomas, and 14 samples of normal breast tissue. The extent of DNA denaturation induced by acid was expressed as alpha t, which represents the fraction of DNA staining metachromatically red with the fluorochrome acridine orange. In all cases of normal breast tissue DNA was very sensitive to denaturation and the frequency distribution of alpha t values was unimodal with over 90% of cells having alpha t above 0.6. All fibroadenomas were diploid; four had unimodal alpha t as in normal tissue and five had a bimodal distribution with an additional peak below 0.6. Twenty-seven adenocarcinomas (67%) had a DNA index above 1.0; of these 24 had bimodal alpha t distributions. Among 13 diploid carcinomas 10 had bimodal alpha t distributions. Statistically significant differences were observed in alpha t distributions of normal versus tumor breast tissue (P less than 0.005). In normal tissue and in all tumors a predominant proportion of cells with S and G2 + M DNA content were characterized by DNA resistant to denaturation (alpha t below 0.6). Of interest, the diploid cells from aneuploid tumors which may represent reactive host cells often displayed bimodal distributions of alpha t. These results may be interpreted in light of earlier studies demonstrating increased resistance of DNA to denaturation in diffuse chromatin of proliferating and/or transcriptionally active cells, and greater sensitivity to denaturation of DNA in condensed chromatin of quiescent cells. Thus, the presence of the second peaks representing cells with low alpha t values in breast tumors may indicate a high proportion of proliferating cells, whereas high alpha t populations may represent quiescent and differentiating (condensed chromatin) or dying (pycnotic nuclei) cells. It is likely that the low alpha t diploid cells detected in aneuploid tumors may represent the reactive (transcriptionally active and/or proliferating) infiltrating host cells (i.e., lymphocytes, monocytes) whose presence may also be of prognostic value. The data suggest that a DNA denaturability assay may be useful to characterize tumor and infiltrating host cell populations.

Estrogen receptor immunocytochemical assay on cytologic material from primary and metastatic breast cancer.

The determination of estrogen receptor (ER) status in primary and metastatic breast tumours has been facilitated by the recent advent of monoclonal antibodies to ER. The aim of this study was to determine the feasibility of estrogen receptor immunocytochemical assay (ER-ICA) applied to cytologic specimens from primary and metastatic breast tumours. One hundred and sixty specimens from 133 patients were evaluated by cytologic ER-ICA. Comparison with histologic ER-ICA was available for 28 of the specimens and with cytosol assay for 27 specimens. Some 101 of the 160 samples were breast lesions of which 87 had a definitive diagnosis of breast carcinoma. Of these, 68% were considered positive for ER. Metastatic breast cancers comprised 59 of the 160 specimens of which 37% were found to be positive for ER. The predominant staining intensity (SI) of the nuclei of the tumour cells added to the percentage of cells (PC) stained gave an estrogen receptor score (ERS) in both cytologic and histologic specimens. A positive threshold was determined for an ERS greater than 2, equivalent to ER levels greater than 10 fmol/mg of protein. We observed very good correlation between cytologic ERS and the corresponding cytosol assay values (r = 0.74; p less than 0.001; n = 27). The sensitivity was 95% and the specificity 88%. Correlation with histologic ER-ICA was also very high (r = 0.83; p less than 0.001; n = 28). We assessed the role of video image analysis (VIA) and did not find any additional advantages in evaluating cytologic ER-ICA.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of enhancement of the ras oncogene protein product (p21) in a spectrum of human tumors.

Using a direct binding liquid competition radioimmunoassay, the amount of the ras oncogene protein product, p21, was quantitated in a variety of human tumors and adjacent apparently normal tissues. In 48 of 50 matched tumor and normal tissue biopsy specimens from 50 patients, more ras p21 was detected in the tumor than in its normal counterpart. Twenty-five of 28 breast tumors demonstrated more ras p21 than the average of the values obtained for fibroadenomas. Furthermore, in 17 of the 19 cases studied, over 20% more ras p21 was observed in breast carcinomas compared with their respective normal counterparts. More ras p21 was also demonstrated in the majority of tumors of the stomach, lung, colon and bladder compared with their respective adjacent normal tissues. Our data therefore indicate that ras p21 expression is quantitatively enhanced in many human tumors originating from several different tissue types.

The cytokeratin profiles of ovarian common "epithelial" tumors.

An improved immunohistochemical determination of the cytokeratin profiles of epithelia and their neoplasms is possible using monoclonal antibodies that will either identify all 19 cytokeratins (AE1/3) or delineate specific subsets (35 beta H11, 34 beta E12, 34 beta B4 and Cam 5.2). Ovarian common "epithelial" tumors (CET) contain cytokeratin filaments. To determine the nature and differences in the cytokeratin profiles of ovarian CET, eight benign Brenner tumors, four serous cystadenofibromas, 28 mucinous tumors, 27 serous tumors and six endometrioid, five clear cell and five undifferentiated carcinomas, as well as nine normal ovaries were immunostained with the above five antibodies. AE1/3 staining was predominant, while Cam 5.2 and 35 beta H11 displayed the most frequent staining thereafter. Statistically significant staining differences were found between a number of tumor groups using the antibodies 35 beta H11, 34 beta E12 and Cam 5.2. In this study, all ovarian CET, except the benign Brenner tumors, displayed a predominantly low molecular weight cytokeratin profile. The same profile in the normal surface epithelium lends credence to the belief that these tumors are derived from this epithelium. A significant staining difference between some of the tumor types using some of the antibodies suggests a possible ancillary, diagnostic role of cytokeratin profiling in situations where exact tumor typing is difficult.

[Enzymo-immunoassay of progesterone receptors in human breast lesions and tumors. Comparison with a biochemical method]. / Enzymo-immunodosage des récepteurs à la progestérone dans les lésions et tumeurs mammaires humaines comparison à la méthode biochimique.

The determination of progesterone receptors (RP) was performed on 80 benign and malignant human breast tumors with a single saturating dose method (10 nM) using dextran-coated charcoal (PR-Bio) and an enzymo-immunoassay (PgR-EIA). There was a significant correlation between the 2 methods qualitatively (P less than 0.001) and quantitatively (r = 0.79). However the results were significantly higher using the PgR-EIA method than the PR-Bio method (P = 0.04) with a regression line Y = 0.81 x +0.58.

Immunohistochemical studies on oncogene products (c-erbB-2, EGFR, c-myc) and estrogen receptor in benign and malignant breast lesions. With special reference to their prognostic significance in carcinoma.

It is a matter of debate whether the amplification of c-erbB-2 oncogene or production of the oncoprotein in breast cancers correlate with the presence of lymph node metastasis and with a poor prognosis. This study was aimed at elucidating the immunohistochemical localization of oncogene products which are related to cell growth, c-erbB-2 product, epidermal growth factor receptor (EGFR), c-myc protein and estrogen receptor (ER), in benign and malignant lesions of the breast. Fresh frozen sections of 25 breast cancers and 11 fibroadenomas from Japanese women were studied by indirect immunoperoxidase method with proper fixation. C-erbB-2 product and EGFR were localized on the cell membrane whereas c-myc protein and ER were observed in the nuclei. Immunohistochemical expression of oncogene products and ER were not only observed in the mammary carcinomas but also in the fibroadenomas. However immunoreactivities of EGFR and ER were more frequently seen in the fibroadenomas (p less than 0.05). In breast cancers, the incidence of immunoreactivity for c-erbB-2 was higher in the cases with lymph node metastasis than cases without nodal metastasis (p less than 0.05) and there was reciprocal correlation between the expressions of EGFR and ER (p less than 0.05). Regarding the size of the primary tumour, there was no statistically significant correlation with the expressions of c-erbB-2, EGFR, c-myc or ER. Histological grade correlated only with the expression of ER (p less than 0.05).

The distribution of lectin receptor sites in human breast lesions.

Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.

Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues.

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.

Glycosaminoglycans in human breast cancer.

Abnormal concentrations of glycosaminoglycans (GAG) have been reported for various types of tumors, suggesting that they may play a role in neoplasia. The correlation between the content of individual GAGs was studied in breast tumor tissues. The total content of GAG was estimated by uronic acid analysis. The relative distributions of dermatan sulphate, heparan sulphate, hyaluronic acid and chondroitin sulphate were measured after cellulose acetate electrophoresis. Mammary tissue samples were obtained at the time of surgery from 11 women, 6 with fibroadenomata and 5 with carcinoma. From each patient, biopsies were obtained centrally in the tumor and perilesional areas adjacent to the tumor, and also from clinically uninvolved tissue in the same region. In the central areas, it was found that carcinoma had a significant increase in chondroitin sulphate and uronic acid content, and a significant decrease in dermatan sulphate content, as compared with fibroadenoma. The chondroitin sulphate content in perilesional carcinomatous tissue was significantly greater than in clinically uninvolved tissue.

Immunohistochemical localization of myoepithelial cells and basement membrane in normal, benign and malignant human breast lesions.

Distributions of actin and type IV collagen were investigated immunohistochemically as markers for myoepithelial cells and basement membranes. Carnoy's and Methacarn-fixed, paraffin-embedded tissues from 103 human breast lesions from 103 patients were examined; 65 with carcinomas, 27 with mastopathies, 9 with fibroadenomas and 2 with phyllodes tumours. Fifty-five samples of the normal mammary gland tissue adjacent to tumours were also included for comparison. In normal breast and benign breast diseases, type IV collagen was identified around the mammary glandular cells and actin-positive cells were demonstrated to attach to basement membranes. In noninvasive carcinomas, type IV collagen was found as a continuous lining around a cell nest, while actin-positive cells were usually absent in ductal but quite numerous in lobular carcinomas. In invasive carcinomas, type IV collagen was fragmented or absent and actin-positive cells were very uncommon around the fragmentary basement membranes. These results suggest that the different distributions of myoepithelial cells and basement membrane material is useful in the differential diagnosis of surgical pathology of the breast.

Proton NMR of human breast tumors: correlation with clinical prognostic parameters.

Proton NMR spectroscopy and imaging of human breast tissue have provided new methods in studying breast carcinomas. Continuous wave proton NMR spectroscopy in this study is able to discriminate breast carcinomas from normal breast tissue on the basis of the integrated area under the water and lipid peaks, width at half height of the water peak, and chemical shift of the lipid peak. In addition, the NMR parameters were correlated with the following clinical and pathologic prognostic indices: TNM tumor stage, nuclear grade, and estrogen receptor status (ER). Width at half height of the lipid peak (1/2 delta lipid) correlated with tumor content and ER. Studies using higher resolution proton or phosphorus NMR spectra may separate signals that can correlate with biological information on breast neoplasms useful to the clinician. Chemical shift of the lipid peak may be used to sharpen contrast on MRI of breast tumors.

Tenascin is a stromal marker for epithelial malignancy in the mammary gland.

Tenascin is an extracellular matrix glycoprotein that is not present in the normal mature rat mammary gland. The distribution of tenascin was examined by immunohistochemistry in mammary tumors from carcinogen-treated and untreated rats, in virus-induced mammary tumors from mice, and in a variety of mammary gland lesions from humans. Tenascin was detectable in the stroma of the malignant but not of the benign tumors from all species. An inhibition ELISA, testing homogenates of rat tumors, confirmed that tenascin was present in malignant but not in benign tumors. Thus, tenascin was consistently found to be a stromal marker for epithelial malignancy in the mammary gland. It is concluded that tenascin may be involved in the interactions between the epithelial and mesenchyme-derived (stromal) components of the mammary gland, which are known to influence epithelial carcinogenesis in this organ.

Estrogen receptor in dissociated and cultured human breast fibroadenoma epithelial cells.

Estrogen binding was measured by a whole cell receptor assay in epithelial cells isolated from 20 premenopausal patients with breast fibroadenomas. A high affinity specific binding for estrogens was detected in the epithelial cells isolated from all 20 fibroadenomas. A relationship between estrogen binding and the phase in the menstrual cycle of the patient has been observed. Cell culture experiments using serum-free medium have also shown that estrogen binding can be augmented by cortisol.

Eccrine syringofibroadenoma. Immunohistological study of a new case.

Eccrine syringofibroadenoma is a rare tumor considered to originate from the excretory portion of the eccrine sweat gland. A new case of this lesion, whose acrosyringeal differentiation was underlined by an immunohistological study using antibodies to keratin and involucrin, is reported herein.

Estrogen staining in breast carcinoma by PAP methods compared to CEA and ferritin staining.

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

Stromal proliferations of the breast: an ultrastructural and immunohistochemical evaluation of cystosarcoma phyllodes, juvenile fibroadenoma, and fibroadenoma.

The stromal cells of three cystosarcoma phyllodes, five typical fibroadenomas, and one juvenile fibroadenoma were studied by light and electron microscopy. Immunohistochemical staining for the S-100 protein also was performed on tissues from each of the three categories. On ultrastructural examination, cells comprising the three varieties of lesions were similar. Cells with fibroblastic features predominated in all cases. Myoid differentiation was present in two cases, one of cystosarcoma and one of fibroadenoma. Junctional complexes were present in the cystosarcomas but not in the fibroadenomas. Basal lamina was focally present around stromal cells in the cystosarcoma phyllodes but was not evident around cells of the typical fibroadenomas or the juvenile fibroadenoma. Stromal cells of the fibroadenomas and the cystosarcoma phyllodes did not stain for S-100 protein. The results support the hypothesis that the proliferating cells in all three tumor categories are similar and have features of fibroblasts. The lack of staining for S-100 protein would suggest an origin different from the myoepithelia. The latter conclusion, however, must be interpreted with a degree of reservation as we have shown that not all myoepithelial cells stain with certain monoclonal antibodies directed against the alpha and beta chain of S-100 protein.

[Cyclic nucleotides during radiotherapy of breast cancer]. / Tsiklicheskie nukleotidy pri luchevoi terapii raka molochnoi zhelezy.

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.

Cytophotometric measurements of nuclear DNA content values in human breast lesions.

Quantitative measurements of nuclear DNA content values have been evaluated in cytological smears from 33 women with normal breast, fibrocystic disease, fibroadenoma and different types of breast carcinomas. The DNA amount for normal breast was found to be in the diploid region. For fibrocystic disease and fibroadenoma the cytochemical data were established to range from diploid to tetraploid regions, with peaks at 2 C and 3 C, respectively. For the different types of malignant breast lesions the quantitative DNA amounts were presented in the various regions of the histograms: from diploid to tetraploid for papillar carcinomas and metaplastic carcinomas--fusiformcellular and epidermoid types, with peaks at 2 C; from diploid to octaploid for intracanalicular carcinomas with peaks at 2 C, 4 C and 6 C. Dynamic changes in the pathway from the normal to cancer have been determined after quantitative cytochemical measurements of various benign and malignant breast lesions.

Immunocytochemical demonstration and significance of p21 ras family oncogene product in benign and malignant breast disease.

It has been suggested that the immunocytochemical demonstration of the p21 ras oncogene product is a useful marker of malignancy in breast disease. We have studied the reactivity of a series of specimens of benign and malignant breast disease with the anti ras p21 monoclonal antibody Y13-259, and shown widespread positive staining in both benign and malignant (including metastatic) disease as well as in adjacent 'normal' epithelium. In addition some staining of stromal cells as well as nerve fibres was observed. Our results suggest that the presence of ras p21 protein as demonstrated by this antibody is not a useful marker of malignancy or of proliferating epithelium but is rather a normal feature of certain cell types.