Expression of the pS2 peptide in primary breast carcinomas: Comparison of membrane and cytoplasmic staining patterns

The pS2 protein is oestrogen-regulated in breast cancer cell lines. Previous studies have shown a relationship to oestrogen receptor in primary breast carcinomas. This study examined 178 breast carcinomas for pS2 using immunohistochemistry. A high frequency (77 per cent) of positive tumours was found, using a 10 per cent cut-off point to define a positive tumour. There was no relationship with menopausal status or node status, a significant association with differentiation, a weak association with oestrogen receptor, and no association with progesterone receptor or overall survival. Two patterns of cellular localization were observed: cytoplasmic and membrane. The former showed a stronger relationship with oestrogen receptor status, although there were oestrogen receptornegative tumours with marked pS2 staining. Membrane staining showed a stronger relationship with differentiation, with a staining pattern similar to that observed for milk fat globule membrane. The staining patterns observed may support a role for pS2 in a secretory mechanism. However, the expression and function of pS2 in breast carcinomas emain complex, and are not simply related to oestrogen regulation.     

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.     

Methodology of immunohistological detection of oestrogen receptor in human breast carcinoma in formalin-fixed, paraffin-embedded tissue: a comparison with frozen section methodology

We describe a method of immunohistochemically assessing oestrogen receptor status on routinely processed formalinfixed tissue, using a commercially available monoclonal antibody (Abbott H222). with pronase predigestion of tissue sections and overnight antibody incubation. The staining was assessed using the H score system. A series of 94 cases of breast cancer were analysed and the results were compared with assessment by oestrogen receptor immunocytochemical assay performed on frozen section. Direct comparison of the paired sets of H scores obtained with frozen tissue and formalin-fixed tissue showed a highly significant correlation of 0.8 ( P < 0.001) between the two methods of oestrogen receptor assessment. Chi-squared analysis using H score cut off points of 50 and 100 also showed a similar significant association ( P < 0.001). We conclude that this oestrogen receptor method, applicable to formalin-fixed, paraffin-embedded tissue, gives accurate results on routinely fixed tissue and could be used as an alternative to other methods.     

Oestrogen receptor in breast cancer: prognostic studies using a new immunohistochemical assay

A recently developed and validated histochemical method--immunogold-streptavidin enhancement--was used to determine oestrogen receptor content in paraffin sections of breast cancers. The selection of the best cut-off point to define oestrogen receptor status as rich or poor was made on the basis of survival data, using the following indices: survival to term; disease-free interval; survival at 5 years; and disease-free interval at 5 years. Oestrogen receptor status was examined in relation to histological grade, lymph node status, menopausal status and tumour size and these four indices were considered as independent prognostic factors. Semi-quantitative assay of receptor content showed that increasing content was related to better prognosis. Adjuvant therapy alone had no effect on patient outcome. Independently, histological grade and lymph node status, but not menopausal status or tumour size, were prognostic indicators. Oestrogen receptor rich status, as measured by immunogold-streptavidin, in conjunction with certain of these factors indicated a better prognosis. This was comparable with results in reports using other methods of receptor assay. We found the oestrogen receptor status and menopausal status more significant at 5 years than at term. The advantage of immunogold-streptavidin enhancement, which we found as reliable as other methods for oestrogen receptor assay, is that it can be used on archival, routinely paraffin-processed material.     

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.     

Oestrogen receptor and its potential role in breast cancer development

The first studies of oestrogen receptor (ER) focused on measurements of levels in breast cancers and relationship to hormonal response, but the introduction of antibodies which can be used on fixed tissue has meant that ER can be studied in detail in normal and precancerous tissue. Cloning and sequencing of ER has resulted in more detailed understanding of structure and function, with the identification of variant forms in both tumours and normal, followed by the discovery that ER has two forms, the classical ER-α and ER-β. Analyses of both forms in normal and preneoplastic breast will be important for our understanding of the role of ER in the development of breast cancer and its possible prevention. Copyright © 1999 John Wiley & Sons, Ltd.     

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 and epidermal growth factor receptor expression in male breast carcinoma.

Male breast carcinomas are probably hormone-dependent, but receptor studies are few because this is a relatively rare tumour. We have studied 21 cases of male breast carcinoma immunohistochemically for oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) expression employing the antibodies ER-ICA and 12E on formalin-fixed, paraffin-embedded material. In our series, 86 per cent of male breast cancers were ER-positive and 76 per cent were EGFR-positive. Male breast carcinomas do not exhibit the inverse correlation between ER and EGFR expression that characterizes female breast carcinomas. Owing to the limitations of a small series, we were unable to comment on the relationship between ER and EGFR expression and patient survival. However, the relatively high incidence of ER expression may provide a growth advantage for this tumour in a male environment characterized by low levels of oestrogen. In addition, high EGFR expression may also contribute to a poor prognosis independent of ER status.     

Prognostic factors in breast cancer: immunohistochemical staining for SP1 and NCRC 11 related to survival, tumour epidermal growth factor receptor and oestrogen receptor status

It has been proposed that intense immunohistochemical staining using the monoclonal antibody NCRC 11 is indicative of a better prognosis in breast cancer, and that expression of pregnancy specific glycoprotein (SP1) and epidermal growth factor receptors (EGFR) are poor prognosis indicators. In order to evaluate the significance of staining for NCRC 11 to prognosis and to these other variables, we studied two series of breast cancer patients. In one series (n = 93), staining with NCRC 11 and for SP1 was correlated with prognosis: in neither case was there any overall relationship between staining and survival. However, when the patients were stratified by tumour histological (Bloom's) grade, SP1 expression was associated with a poorer prognosis in grade II tumours (p less than 0.025). In a further series (n = 94), NCRC 11 staining was carried out and fresh tumour samples were assayed biochemically for oestrogen receptor (OER) and EGFR. NCRC 11 showed a negative correlation with EGFR status (p = less than 0.05) but no significant association with OER status. Thus despite our demonstration of a negative correlation with an indicator of poorer prognosis, we were unable to demonstrate any direct relationship between staining with NCRC 11 and survival.     

Critical assessment of four monoclonal antibodies reactive with B-cells in formalin-fixed paraffin-embedded tissues

Four commercially available monoclonal antibodies, MB1, MB2, LN1 and LN2, were studied to determine their sensitivity and specificity for the diagnosis of B-cell lymphomas when used on formalin-fixed paraffin-embedded tissues. In addition to 125 cases of immunologically characterized non-Hodgkin's lymphoma, a range of normal tissues, reactive lymphoid proliferations, Hodgkin's disease and granulocytic sarcomas were also studied. MB1 was found to give positive results in 53.6% of B-cell lymphomas, but the staining was sometimes weak and patchy; there was also cross-reaction with 1.8% of T-cell lymphomas. MB2 reacted with 88.4% of B-cell lymphomas and the reaction was often strong and diffuse, but it showed cross-reaction with 18.2% of T-cell lymphomas. LN1 and LN2 gave positive staining of 44.9 and 46.4% of B-cell lymphomas respectively, and the results appeared to be inferior to that obtained in B5-fixed tissues; staining was sometimes weak and focal, and they also gave false-positive results in a few cases of T-cell lymphoma. This study shows that MB1, LN1 and LN2 are fairly but not entirely specific for B-cells in the non-Hodgkin's lymphomas, but are not very sensitive when applied to formalin-fixed tissues. MB2 shows a high sensitivity but only moderate specificity. Therefore, when these antibodies are used to determine the immunophenotype of malignant lymphomas, the B-cell nature can be predicted with great confidence only when two, preferably three or more, of the antibodies give positive results. The potential applications of these antibodies are discussed.     

Oestrogen receptors in benign epithelial lesions and intraduct carcinomas of the breast: an immunohistological study

We examined 198 breast lesions, representing commonly encountered benign epithelial proliferative disorders, lobular carcinoma in situ and intraduct carcinoma, immunohistologically for oestrogen receptors (ER). A mixture of three ER monoclonal antibodies--H222, D75 and D547--was used on sections of routinely processed and paraffin-embedded tissue blocks. Over 65% of the benign and malignant lesions showed some evidence of ER expression and significant staining was recorded by two observers in 28-31% of fibroadenomas, 18-28% of ductal epithelial hyperplasias, 30-40% of sclerosing adenosis cases, 38-45% of papillomas, 60% of in situ lobular carcinomas and 42-45% of intraduct carcinomas. Apocrine metaplastic cells and myoepithelial cells showed absent or only weak staining. Amongst intraduct carcinomas, less than 20% of comedo carcinomas and over 50% of cribriform, papillary and solid variants showed significant ER staining.     

Oestrogen receptor/progesterone receptor and human epidermal growth factor receptor 2 status in breast cancer: a 9-year study at Princess Noorah Oncology Center, Saudi Arabia

Satti M B
(2011) Histopathology 59 , 537–542 Oestrogen receptor/progesterone receptor and human epidermal growth factor receptor 2 status in breast cancer: a 9‐year study at Princess Noorah Oncology Center, Saudi Arabia Objective: To determine the oestrogen receptor/progesterone receptor (ER/PR) and human epidermal growth factor receptor 2 (HER2) status in Saudi Arabian patients presenting with breast cancer to Princess Noorah Oncology Center (PNOC) and to explore the correlation of these markers to each other, to tumour type and to grade. Methods and results: Pathology material and records of symptomatic patients presenting to the centre during 2001–2009 were reviewed for patients’ age, tumour size, type and grade and ER/PR and HER2 immunohistochemistry (IHC) status using the Dako HercepTest Kit as well as fluorescence in situ hybridization (FISH) for HER2 IHC 2+ score cases, as per the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. There were 852 cases, with a mean age of 49 years and a mean tumour size of 3.0 cm with 75% node positivity. Of all cases, 772 (90.6%) were ductal carcinoma; 64% were ER/PR⁺ and 23% were HER2⁺; triple‐negative cases accounted for 24%. Conclusions: ER/PR and HER2 status did not differ from that reported previously, showing a direct correlation to tumour type and grade of ductal carcinoma. However, a difference exists in the relatively lower ER positivity in patients aged >50 years and the higher percentage of triple‐negative cases. This study would serve as a baseline for other future national studies and for planning strategies to targeted therapy.     

Survival in breast cancer related to tumour oestrogen receptor status and immunohistochemical staining for NCRC 11

NCRC 11 is a monoclonal antibody raised against dissociated breast cancer cells. Using this antibody, it has been shown in an earlier study that staining of a high proportion of tumour cells is strongly associated with superior survival. Many investigators have found that positive tumour oestrogen receptor (OER) status is associated with better survival in breast cancer. In the present study of 136 breast cancer patients followed up for a minimum of 30 months, tumour oestrogen receptor assays were carried out, and using the indirect immunoperoxidase technique, histological sections of paraffin embedded material were stained for NCRC 11; for the purpose of this study, tumours showing 25 per cent or less cells staining were regarded as 'negative'. Patients whose tumours were OER positive had a significantly better prognosis (logrank test P less than 0.05). Patients whose tumours were graded as negative for NCRC 11 had a poorer prognosis compared with the positive group but this did not attain significance. Our failure to demonstrate convincingly a better prognosis for the NCRC 11 positive patients may relate to the shorter duration of our study, or to different tissue fixation techniques. In this study staining for NCRC 11 was positively correlated with oestrogen receptor status (P less than 0.02).     

NB84: a new monoclonal antibody for the recognition of neuroblastoma in routinely processed material

A new monoclonal antibody NB84 recognizing an uncharacterized molecule of 57 kD was produced using human neuroblastoma tumour tissue as a source of antigen. The antibody was selected for its ability to give reliable staining on conventional formalin-fixed, paraffin-embedded material. In the present study, NB84 was assessed for its usefulness in the differential diagnosis of paediatric small cell tumours. It gave reliable staining of 17/19 (90 per cent) neuroblastomas and was negative on all other tumours apart from Ewing's sarcomas, of which 9/14 (64 per cent) showed mainly focal positivity. It is concluded that in association with a panel of markers against leucocyte, muscle, epithelial, and neural markers NB84 provides considerable assistance in the recognition of neuroblastoma and thus is important in enabling the most appropriate therapy to be given.     

Correlation between immunohistochemically determined oestrogen receptor content, using monoclonal antibodies, and qualitative and quantitative tissue features in ductal breast cancer

Previous studies have shown that oestrogen receptor content in breast cancer was correlated with qualitative and also, more strongly, with quantitative nuclear features in tissue sections. However, even with the better reproducible quantitative microscopical assessments, the variance in the correlation with oestrogen receptor was considerable. This might be due to the implicit problems of oestrogen receptor determination with the biochemical assay. Therefore, receptor content was studied using monoclonal antibodies in 50 consecutive invasive ductal breast cancers. Oestrogen receptor status was compared with qualitative features and with the mean and standard deviation of the nuclear area, morphometrically evaluated on immunostained and adjacent haematoxylin and eosin stained sections. In agreement with earlier observations, nearly all tumours with prominent elastosis were oestrogen receptor positive; but a minority of negative cases also showed elastosis. The correlation between the other qualitative features and receptor status was weak. A significant inverse correlation (P less than 0.001) existed between the receptor status and the mean and standard deviation of the nuclear area. Even with the highly reproducible morphometrical analysis, correlation between nuclear oestrogen receptor content and quantitative nuclear features was relatively weak. This might indicate that receptor status and nuclear morphometric features reflect different biological characteristics of breast cancers.     

Stability of oestrogen and progesterone receptor antigenicity in formalin‐fixed paraffin‐embedded breast cancer tissue over time

Use of archived formalin‐fixed paraffin‐embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978–2000) using a large breast cancer tissue microarray (N = 573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow‐up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time‐dependent difference in ER_IHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER‐positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PR_IHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40 years.     

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.