Oestrogen receptor, progesterone receptor, pS2, ERD5, HSP27 and cathepsin D in invasive ductal breast carcinomas

Hormonal receptors and markers for prognostic evaluation were detected immunohistochemically in 196 infiltrating ductal breast carcinomas. Immunohistochemical detection of progesterone and oestrogen receptor is a method giving results generally concordant with those of the binding assay. However, immunohistochemical detection seems better. It allows the detection of hormonal receptors on small carcinomas, it is not modified by the endogenous hormones, and it has a slightly better correlation with prognosis and with the response to hormone therapy. Immunohistochemical detection of progesterone receptor has a prognostic value, sorting a negative subgroup with a poor prognosis from the oestrogen receptor positive tumours. These results can be obtained without quantitative immunohistological methods. ERD5, pS2, HSP27 and cathepsin D are associated with oestrogen receptor positivity. pS2 and HSP27 are interesting markers. They characterize a subgroup of oestrogen receptor negative tumours with a good prognosis. Moreover, pS2 is a marker of response to hormone therapy. ERD5 and cathepsin D do not appear to be of value as markers of prognosis.     

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.     

Expression of pS2 protein in transitional cell bladder tumours

A series of 201 bladder cancer biopsy specimens was analysed immunohistochemically for the expression of pS2 protein. Altogether, 61 per cent of the tumours were pS2-negative; in 16 per cent less than 1 per cent and in 23 per cent of cases more than 1 per cent of cells were pS2-positive. Normal transitional epithelium was negative for pS2. The fraction of positive cells was higher in poorly differentiated non-papillary tumours and in invasive tumours with pelvic lymph-node (P=0.05) and distant metastasis (P=0.10). pS2 expression was not related to sex, while patients aged 60–70 years had low fractions of pS2-positive cells (P=0.03). DNA ploidy, S-phase fraction, mitotic index, morphometric nuclear features, and expression of c-erbB-2, p53, and epidermal growth factor receptor were independent of expression of pS2. Tumours expressing pS2 in over 10 per cent of cells had a lower survival probability (P=0.0486). The results show that pS2 is expressed in 40 per cent of transitional cell bladder tumours, but that this marker has no clinical significance over established prognostic factors.     

Membranous and cytoplasmic staining of Ki67 is associated with HER2 and ER status in invasive breast carcinoma

Aims:  Membranous and cytoplasmic Ki67 immunoreactivity has recently been observed in a number of histopathological entities, but frequency of occurrence and relationship to prognosis in more common cancers have not been described. The aim was to describe the pattern and frequency of membranous/cytoplasmic Ki67 in a cohort of invasive breast carcinomas, and their associations with grade, HER2 amplification and oestrogen receptor (ER) expression.
Methods and results:  Three hundred and twenty-two cases of invasive ductal carcinoma were assessed for histological grade, Ki67 (MIB-1 clone) proliferation index and pattern of immunoreactivity, ER expression by immunohistochemistry, and HER2 amplification status by fluorescence in situ hybridization. Overall, 26/322 (8%) breast carcinomas showed membranous/cytoplasmic Ki67, and expression was significantly associated with grade 3, HER2-amplified and ER− tumours. Membranous/cytoplasmic Ki67 was not, however, an independent prognostic factor on multivariate analysis.
Conclusions:  Membranous/cytoplasmic Ki67 identifies a group of breast carcinomas that may be important to consider separately in prognostic and predictive studies. The mechanism of subcellular Ki67 relocalization remains elusive and further studies are required to establish both the cause and effect of this unusual pattern of Ki67 immunoreactivity.     

Immunohistochemistry of pS2 in normal human breast and in various histological forms of breast tumours

Expression of pS2 was studied by immunocytochemistry in normal breast tissue ( n = 20), benign tumours ( n = 9) and 145 breast cancers representative of the different histological types. pS2 immunostaining was scored as negative (D1 = 0-5% stained cells), positive (D2 = 5-75% stained cells) or highly positive (D3 > 75% stained cells). pS2 protein was evident in all normal breast samples examined. Six of nine benign lesions showed pS2 staining. In both cases, immunostaining was weaker than in breast cancers. Of breast cancers, 77/145 (53.1%) were pS2 positive, including 33.1% with intense staining. The presence of pS2 was not correlated with the age of patients, the size of the primary tumour, or lymph node status, but was correlated with histological grading and nuclear grading. pS2 expression was also correlated with menopausal status and oestrogen receptor status (59% of receptor-positive tumours were pS2 positive), but not to progesterone receptor status. pS2 expression in breast carcinomas is not a characteristic of specific histological types. Although this protein is predominantly expressed in oestrogen receptor-positive and differentiated tumours, it shows oestrogen-independent expression in about 30% of cases.     

The relationship between growth fractions and oestrogen receptors in human breast carcinoma, as determined by immunohistochemical staining

Measurements of the growth fraction (GF) and oestrogen receptor (OR) status were performed by immunohistochemical staining, using the monoclonal antibodies Ki-67 and anti-OR, in consecutive cryostat sections of biopsies from 74 women with primary breast cancer, and 12 women with benign breast disease. A significant inverse correlation was identified between the GF and OR status (r = -0.452, P less than 0.0001), but a group of OR-positive tumours with high GFs was also observed. There was a strong positive correlation between immunocytochemically determined OR percentages and values obtained with a cytosolic OR biochemical assay for the 70 cases in which both techniques were performed (r = 0.801, P less than 0.0001). A significant relationship was also revealed between the histological grade of infiltrating ductal carcinomas and their associated GFs (r = 0.622, P less than 0.0001). No correlation was found between the GF and the number of positive axillary nodes. Our findings suggest that GF and OR status are useful independent prognostic parameters in breast cancer, but the former probably has an overriding influence. Both parameters may be useful guides for selection of appropriate endocrine and/or adjuvant chemotherapy.     

Immunohistochemical demonstration of glutathione S-transferases in primary human breast carcinomas.

The expression of cytosolic glutathione S-transferase (GST) isoenzymes has been assessed in a series of 74 primary human breast carcinomas using an immunohistochemical method. GST pi was detected in sections from all 74 tumours; it was expressed by non-epithelial (stromal and inflammatory) cells in 62 tumours (84 per cent), but by tumour epithelium in only 35 (47 per cent). Non-neoplastic mammary epithelium was uniformly positive for GST pi. Expression of GST alpha and mu was observed in 19 and 42 per cent of the tumours, respectively, and was largely confined to the neoplastic component. Lack of staining of tumour epithelium for GST pi was significantly associated with poorer tumour differentiation (higher grade). There was no association between expression of any of the three isoenzymes and either menopausal status or expression of c-erbB-2 oncogene protein product. Immunohistochemistry is a useful method for the investigation of expression and cellular localization of GSTs within tumours; such data are needed to improve our understanding of the role of these enzymes in neoplasia and in resistance to cytotoxic drug therapy.     

pS2 protein in gastric carcinoma and normal gastric mucosa: Association with clinicopathological parameters and patient survival

The presence of pS2 protein (pS2) was studied in a total of 120 consecutive patients with gastric carcinomas. This immunohistochemical study found pS2 expression in 48 per cent (n=58) of carcinomas. pS2 expression was also detected in normal gastric mucosa in 95 per cent (n=102) of specimens in upper antral mucopeptic glands and deep foveolar cells of the gastric pits but not in intestinal metaplasia. There was a significant statistical correlation between pS2 expression and extent of tumour growth (pT state) and expression of pepsinogen II by the tumours. There was no statistical correlation with clinical features such as patient age or sex or other pathological parameters (tumour stage, size, grade, and localization or growth pattern according to histological classification). There were no statistically significant differences in survival times between patients with pS2-positive and pS2-negative tumours. In contrast to findings concerning breast cancer, pS2 expression in gastric carcinomas has no influence on the patient's prognosis. On the other hand, strong expression of pS2 by surface epithelium of normal gastric mucosa may indicate that pS2 might play a role in physiological cell renewal of normal gastric mucosa.     

Expression of oestrogen receptor-beta in apocrine carcinomas of the breast

AIMS: Apocrine carcinoma of the breast seldom expresses oestrogen receptors (ER) or progesterone receptors (PR), but frequently expresses androgen receptors (AR). Because of this unusual hormone receptor status, it has been suggested that oestrogens have a less important role in the pathogenesis of apocrine carcinoma. The ER status of apocrine carcinoma has been studied for one kind of ER, the classic receptor now named ER-alpha; however, the status of ER-beta, a secondary oestrogen receptor, has not been examined systematically in apocrine carcinoma. The aim was to study ER-beta status in apocrine carcinoma. METHODS AND RESULTS: The expression of ER-beta was examined immunohistochemically in 48 apocrine carcinomas and compared with clinicopathological factors and ER-alpha, PR and AR status. ER-beta positivity was observed in 35 cases (73%), regardless of any clinicopathological factors or the status of other receptors. The results of ER-beta mRNA analysis supported the immunohistochemical results. CONCLUSIONS: The significance of oestrogens in apocrine carcinoma should not be dismissed at present when the role of ER-beta remains to be determined. Studying the action of oestrogen or antioestrogen in apocrine carcinoma may reveal a role for ER-beta independent of ER-alpha and raise the potential of hormonal therapy for these tumours.     

Expression of p53 protein in infiltrating and in-situ breast carcinomas.

Five antibodies directed against the whole or part of p53 protein have been used to detect the protein immunohistochemically in 70 infiltrating breast carcinomas and 10 ductal carcinomas in situ. Mutations are known to occur in different conserved domains, and the antibodies employed spanned the expected sites. p53 protein was identified in 53 per cent of infiltrating carcinomas using the antibodies PAb 240, PAb 1801, C19, and JG8. The antibody PAb 421 detected the protein in 31.5 per cent; all positive with the other antibodies. Well-differentiated oestrogen receptor-positive tumours had a low incidence of p53 detection. Variation in the percentage of reactivity was seen between carcinomas and in some cases between different antibodies in the same cancer. Those carcinomas with a high percentage of positive cells with all antibodies were more likely to have metastasized to nodes, be at an advanced stage, and be oestrogen receptor-negative/epidermal growth factor receptor-positive. There was no significant correlation with c-erbB-2 protein expression or retinoblastoma protein loss. p53 protein was detected in a high proportion of cells in three of the six comedo ductal carcinomas in situ studied but either not at all or at a lower level in tumours of the cribriform type. p53 mutations are common in breast carcinomas, but heterogeneity within individual tumours is frequent. Marked expression of p53 appears to relate to tumour progression.     

Evaluation of ER, PR, MIB-1, pS2, and nuclear grade in FNA specimens of cT1 breast carcinomas: clinicopathological correlation

Objectives were to determine oestrogen (ER), progesterone receptors (PR), and antigen related to ER (pS2) and to characterize their relationship with the cellular proliferation marker MIB-1 and the nuclear grade (NG) of the cancer cells, using fine-needle aspirates (FNA), as well as the evaluation of their clinical usefulness. The expression of ER, PR, pS2, and MIB-1 was preoperatively detected by immunocytochemistry in FNAs of 70 patients with breast adenocarcinoma and clinical tumor size up to 2 cm. The NG of the tumor cells was also assessed in these samples. We analyzed whether there was any correlation between these biocytologic markers and the invasion of ipsillateral axillary lymph nodes (LN), which were histologically identified after standard surgical treatment in each case. Of the 70 patients 50, 42.85, 50, and 41.42% were positive for ER, PR, pS2, and MIB-1, respectively. Only NG alone was strongly related to the invasion of the LN (P < 0.001). All the patients with NG1 (100%) tumors presented free LN, whereas the majority of those with NG3 (72.72%) had invaded LN (P < 0.001). Patients (14.28%) with NG1 expressed MIB-1, 85.71% ER or PR, and 71.42% pS2. Among the MIB-1-positive tumors a high proportion of NG3 (65.51%) was observed. This finding underlined a relationship between MIB-1 and NG (P < 0.05), identifying an aggressive cancer type. Remarkably 93.33% of the patients with positive MIB-1 and invaded axilla had NG3, whereas 66.66% of them expressed ER or PR and 40% pS2. The findings of the present prospective, multivariate study indicate that NG of the tumor cells, obtained from the preoperative FNAs of breast cancer patients, is a strong predictive marker for the axillary status and in parallel with MIB-1 expression can with sufficient accuracy be of clinical utility. ER, PR, or pS2 on the other hand did not show any relation to the LN status and were not dependent to NG or MIB-1.     

Immunohistochemical staining patterns of tenascin in invasive breast carcinomas

Summary Eighty-two cases of primary invasive breast carcinoma and adjacent normal mammary glands were examined immunohistochemically for tenascin expression and distribution. Formalin-fixed tissues pretreated with actinase were processed by the avidin-biotin complex method using anti-human tenascin monoclonal antibody (RBC1). In normal mammary glands, tenascin was distributed around the ducts and ductules but not around the acini. In carcinomas, a high incidence of tenascin-positive cases (>67%) was seen with various histological appearances, with the exception of lobular carcinoma where a low incidence was found (25%). Although intense staining was seen around cancerous foci when compared with normal mammary glands, tenascin was often expressed at cancer-mesenchymal junctions with dense fibrotic stroma, but not at junctions with active inflammatory change and a loose fibrotic stroma. Tenascin expression is not an all-or-none marker for mammary malignancy and the staining pattern suggests either a role in stimulating cancer cells or a host defence mechanism accompanied by a desmoplastic response to them.     

Comparison of molecular markers expression in vacuum-assisted biopsies and surgical specimens of human breast carcinomas

In this study, we examined whether vacuum-assisted breast biopsy (VABB) specimens provide an accurate immunohistochemical assessment of estrogen receptors (ER), progesterone receptors (PR), c-erbB-2, and p53 proteins.     

Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry.

Five peptides, corresponding to regions of the predicted protein sequence of the oestrogen-regulated pNR-2 protein which is expressed in oestrogen-responsive human breast cancer cells, were synthesized. Two peptides were immunogenic in rabbits and antisera against one peptide reacted with the pNR-2 protein in sections of formalin-fixed, paraffin-embedded breast tumour. There was a significant correlation between the extent of pNR-2 protein expression detected by immunohistochemistry and pNR-2 mRNA levels determined by hybridization with a cDNA probe in a series of primary breast tumours. pNR-2 expression was assessed immunohistochemically in a panel of normal tissues. Expression was detected in normal breast, small intestine, and stomach (body and antrum).     

In vivo patterns of Bcl-2 family protein expression in breast carcinomas in relation to apoptosis

The expression of the pro-apoptotic proteins (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bcl-X, Mcl-1) was studied by immunohistochemistry in 110 invasive ductal breast carcinomas. The results were correlated with tumour grade, expression of oestrogen receptor (ER) and p53 protein, and the apoptotic index by combined morphology, immunohistochemistry, and a terminal UTP nick end labelling (TUNEL) procedure. Overall, Bcl-2, Bcl-X, Mcl-1, Bax, Bak, ER, and p53 were detected in 62, 75, 68, 75, 60, 68 and 26 per cent of the cases respectively, but at different levels in each case. A high apoptotic index was correlated with high tumour grade (p < 0·001), overexpression of p53 (p < 0·001), Bak expression (p < 0·001), and low expression of Bcl-2 (p < 0·001) and ER (p < 0·001). No correlation was found between the apoptotic index and Bax, Bcl-X, and Mcl-1 immunostaining results. The expression of Bcl-2 and Bcl-X was correlated to that of ER. Overall, the results of this study strongly suggest that Bcl-2 and Bak expression is critical in regulating apoptosis in breast carcinomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Comparison of the expression of prognostic biomarkers between primary tumor and axillary lymph node metastases in breast cancer

The prognosis and prediction of axillary lymph node (ALN) metastases in breast cancer is traditionally based upon the biomarkers status of the primary tumor. Some retrospective studies showed significant discordance in receptor expression between primary and metastatic tumors. We aim to prospectively assess the incidence of discordant biomarkers status in primary tumor and ALN metastases and to evaluate the role of ALN biopsies for the reassessment of receptor status. Tissue arrays were constructed from 54 breast cancer patients with ALN metastases diagnosed. Arrays were immuno-stained to compare protein expression of four biomarkers including estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 by immunohistochemistry. The kappa value of consistency in the primary tumor and the metastatic lymph nodes were 0.465 for ER, 0.445 for PR, and 0.706 for HER2. Good consistency was shown for Ki67 expression in primary and metastases regions with T test. No significant difference is existed between primary tumor and ALN metastases. It is concluded that the good consistency is present for ER, PR, HER2 and Ki67 between the primary tumor and the metastatic lymph nodes, suggesting that ER, PR, HER2, or Ki67 status in primary tumors could reflect their status in ALN metastases.     

Expression of MHC class I and class II antigens in primary breast carcinomas and synchronous nodal metastases

Expression of the major histocompatibility (MHC) class I and class II antigens was studied by immunohistochemistry in a series of 70 primary breast carcinomas and in nodal metastases. In particular, the expression of class I (HLA A-B-C) and class II (DP, DQ and DR) molecules was compared in: a) primary breast cancers devoid of nodal metastases (n = 36) and tumors exhibiting metastatic deposits (n = 34) at the time of surgery, and b) primary breast carcinomas and their corresponding synchronous axillary nodal metastases. Reduced or absent HLA A-B-C antigen expression was seen in approximately 54.3% of primary breast carcinomas, whereas a partial or complete induction of class II products was observed in 18.5% (DQ), 30% (DP) or 48.5% (DR) of the same cases. An almost complete overlap of antigen expression was observed in breast tumors in which no metastases were found by histological examination of axillary nodes and in neoplasms showing histologically-diagnosed synchronous metastases. The reactivity for class I and class II antigens in nodal metastases roughly paralleled that exhibited by corresponding primary tumors. A discordant expression was seen in 11 cases (32%) stained for HLA A-B-C and in 8 (24%), 7 (21%) and 6 (18%) cases assayed for DP, DQ and DR products, respectively. When a discordant expression was detected, either decreased or increased staining patterns were observed in metastases. The finding of overlapping MHC antigenic profiles in the majority of primary breast tumors and nodal metastases casts doubts on the hypothesis that loss of MHC antigens can play an important role in the seeding and growth of metastatic breast carcinoma cells.     

Demonstration of oestrogen and progesterone receptors in freeze-dried, paraffin-embedded sections of breast cancer

Immunohistochemical evaluation of oestrogen and progesterone receptors is of importance in evaluating human breast tumours. Staining techniques can be performed on snap-frozen, cryostat-cut tissues or, as recently reported, on formalin-fixed, paraffin-embedded tissues. These methods are, however, limited by several drawbacks, including difficulties in retrospective studies and in storage of the material, and the relatively high frequency of false negative results for chemically fixed specimens. We therefore investigated the application of freeze-drying technology to assess the feasibility and reliability of this technique as an alternative method for diagnostic breast pathology. Morphological and immunohistochemical studies were performed on snap-frozen, freeze-dried and paraffin-embedded tissue obtained from 16 cases of benign and malignant breast neoplasms. Our results showed good preservation of tissue morphology, similar to standard formalin fixation, and excellent preservation of antigenic reactivity of nuclear receptors, comparable to that obtained with cryostat sections. We therefore suggest that freeze drying and paraffin embedding of frozen tissue blocks is equivalent or even preferable to formalin fixation for the demonstration of oestrogen and progesterone receptors, at least in the case of small tumours.