Identification of oestrogen receptors in cells of paraffin-processed breast cancers by IGSS

Oestrogen receptor (ER) analysis of breast cancers by the standard dextran coated charcoal (DCC) method and the oestrogen receptor immunocytochemical assay (ERICA), shows that ERICA is more sensitive. We find that the immunogold-silver staining technique (IGSS), which is used on paraffin sections, is applicable to the ERICA antibody and that the DCC and IGSS methods have comparable sensitivity. Reasons for wishing to develop an improved method for oestrogen receptor localisation in paraffin sections and its advantages are given.     

An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody.

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.     

Identification of oestrogen receptors in cells of paraffin-processed breast cancers by IGSS

Summary Oestrogen receptor (ER) analysis of breast cancers by the standard dextran coated charcoal (DCC) method and the oestrogen receptor immunocytochemical assay (ERICA), shows that ERICA is more sensitive. We find that the immunogold-silver staining technique (IGSS), which is used on paraffin sections, is applicable to the ERICA antibody and that the DCC and IGSS methods have comparable sensitivity. Reasons for wishing to develop an improved method for oestrogen receptor localisation in paraffin sections and its advantages are given.     

Structure and dynamics of the estrogen receptor

To evaluate the structure and function of estrogen receptor (ER) in various mammalian systems, the cytosolic forms of receptor from calf uterus and from MCF-7 human breast cancer cells have been purified to virtual homogeneity by sequential selective adsorption to estradiol-Sepharose and heparin-Sepharose. In both cases, the purified steroid-receptor complex appears to exist as an activated 5S homo- or heterodimer of mol. wt 65,000 (4S) steroid-binding subunits. Purified ER has high affinity for DNA and serves as a substrate for phosphorylation by a purified rat brain kinase. Several monoclonal antibodies prepared against affinity-purified MCF-7 cytosol ER have been used to localize receptor by an indirect immunoperoxidase technique in fixed, frozen sections of human breast tumors, human uterus, rabbit uterus and in other mammalian reproductive tissues and cancers, as well as in fixed MCF-7 cell cultures and in paraffin-embedded sections of breast tumors and human endometrium. In all cases, we have observed only nuclear localization of immunoreactive receptor in tissues and whole cells, even under conditions in which virtually all of the receptor is found in a low-salt extract (cytosol) of the target cells. Treatment of cells or tissues in vivo or in vitro with estradiol alters the intensity but not the distribution of specific staining for ER. By immunoelectron microscopy, receptor was localized in the euchromatin, but not in the marginated heterochromatin or nucleoli of MCF-7 nuclei and epithelial and stromal nuclei of postmenopausal human endometrium. These observations suggest that the majority of the unoccupied receptor may actually reside in the nucleus, rather than in the cytoplasm as previously thought. Thus, hormone action may involve binding of the steroid directly to receptor loosely associated with nuclear components, followed by conversion of the steroid-receptor complex to an activated form which becomes more tightly associated with chromatin.     

Immunocytochemical detection of estrogen receptors by staining with monoclonal antibodies on cytologic specimens of human breast cancer

A study was undertaken to test the possibility of determining the estrogen receptor (ER) content in human breast cancers by staining with commercial specific monoclonal antibodies (MAbs) on cytologic specimens (touch imprints and fine needle aspirates). The aspirates were suspended in a cell culture medium and cytocentrifuged onto slides to preserve their morphologic characteristics and to allow a proper immunocytochemical staining for ERs. MAb staining for ER was also performed on the respective surgical samples. The staining of cytologic samples for ER showed 100% specificity and 95% sensitivity in comparison to the staining of the histologic samples. Moreover, comparison of the percentage of stained cells in the cytologic specimens to the ER content in the respective surgical specimens, as assayed by the dextran-coated charcoal method, showed the MAb staining of cytologic samples to have 94% specificity and 100% sensitivity. These results support the reliability of MAb staining for ERs in cytologic samples and suggest that it could be the assay of choice in particular clinical settings in the evaluation of primary and recurrent breast cancers.     

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.     

Immunocytochemistry In the Assessment of Ps2 Protein Expression In Fine Needle Aspiration Cytology From Breast Carcinoma

pS2 protein is a cysteine-rich polypeptide, of unknown function, the expression of which is induced in the human cancer cell line MCF-7 by oestrogen. the availability of a murine monoclonal antibody to human pS2 protein has prompted us to evaluate its expression in 47 cases of primary breast carcinoma. Using a double indirect immunoperoxidase technique, we compared the expression of pS2 protein in fine needle aspiration (FNA) cytology smears with that in formalin-fixed, paraffin-embedded sections from subsequently excised tumours from the same patients. We also compared the expression of pS2 protein and oestrogen receptor (ER) status using immunocytochemical assay (ER-ICA) in formalin-fixed, paraffin-embedded sections from 22 primary breast carcinomas. We found the application of immunocytochemistry in the assessment of pS2 protein expression in FNA cytology to be a reliable and cost-effective technique, having a sensitivity of 84% and a specificity of 100%. There was also a good correlation between the expression of pS2 protein and ER status.     

A system for accurate immunolocalization of Tamm-Horsfall protein in renal biopsies

Summary The distribution of Tamm-Horsfall protein (THP) was studied in the human kidney using formalin-fixed, paraffin-embedded sections with a monoclonal antibody specific for human THP applied in conjunction with a modified dinitrophenyl hapten sandwich staining (DHSS) procedure.The method was found to be highly sensitive producing very strong specific staining at antibody dilations up to 1 in 64 000. Counterstaining with Haematoxylin and Eosin was possible without significant masking of the specific staining. This provided excellent structural definition of the background tissue, which proved especially important in the study of THP localization in randomly oriented biopsy material. THP was found in all segments of the thick ascending limbs of loops of Henle, most segments of distal convoluted tubules and occasionally in distended collecting ducts and in the glomerular capsular space. Maculae densae did not contain THP.The combination of the modified DHSS procedure and the human THP specific antibody represents a highly sensitive and reliable method for specific staining of the THP in kidney sections.     

Value of ER-D5 immunocytochemical determination in routine tissue sections of human breast cancer

ER-D5 is a recently identified protein related to estrogen receptors (ER). Generally ER measurement requires fresh frozen tissue and for ER-D5 assay ethanol (E) fixation of the specimen is recommended. We evaluated the possibility of immunocytochemical detection of ER-D5 in routine formalin-fixed (F) sections in 51 breast cancers comparing the results with those obtained in the same specimens using E as fixative. The results of ER-D5 assay were expressed by the staining index (SI) taking values greater than or equal to 5 as positive. In all tumors ER was also assayed by a biochemical method (DCCA). The sensitivity of ER-D5 detection in F was only 33.3%, while the specificity was 94.4%. A lower cut-off value of SI for F sections (greater than or equal to 2) increased the sensitivity to 66.6%, leaving the specificity unchanged. A strong correlation was found between the SI of ER-D5 in E and F (p less than 0.001). The SI of ER-D5 in F sections was also well correlated with ER concentrations (p less than 0.001). These results suggest that immunocytochemical determination of ER-D5 in routine sections may be useful in retrospective studies of hormone dependence in breast cancer.     

Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue

Summary In a preliminary paper (Teasdale et al. 1987) comparing the oestrogen receptor (ER) content of breast cancers by the biochemical dextran coated charcoal (DCC) method and by two histochemical methods, peroxidase immunocytochemistry (ERICA) and immunogold-silver staining (IGSS), it was indicated that ERICA is more sensitive than DCC and that IGSS is as specific as ERICA but less sensitive. This paper describes the comparison of the above three assay methods with two other biochemical methods, iso-electric focusing (IEF) and an enzyme immuno-assay (EIA) on a larger number of cancers. All methods gave statistically comparable results except that IGSS remained less sensitive than the rest. Various modifications to IGSS showed that an immunogold streptavidin enhancement method (IG-SAM) produced sensitivity and specificity equal to that of ERICA. Since IGSS and its modifications are the only methods which can be used on archival paraffin-embedded cancers and IG-SAM gives results highly comparable to ERICA, retrospective studies can be performed on patients whose outcome and response to various treatments are known. Most recent studies have shown that ER positive results can be obtained from 10-year-old paraffin blocks.     

Prognostic markers in histologic and cytologic specimens of breast cancer

OBJECTIVE: To correlate histologic and cytologic specimens of breast cancer by the expression of prognostic factors, such as estrogen receptor (ER), progesterone receptor (PR) and c-erbB-2, with immunochemical staining. STUDY DESIGN: Cytologic and histologic specimens from 83 patients were analyzed for expression of ER and PR, and 30 cases were analyzed for overexpression of c-erbB-2 using a standard immunochemical method. The material used for immunocytochemical staining was taken from the needle and syringe after each aspiration and smear preparation. The material was washed into a small container with preservative solution. Immunohistochemical staining was performed on paraffin-embedded specimens. RESULTS: A significant association was found between the histologic specimens and cytologic specimens by means of the expression of immunochemical markers. The best correlation between cytologic and histologic specimens was found when using c-erbB-2. CONCLUSION: A reliable and rapid evaluation of markers for breast cancer can be achieved by immunocytochemical staining on cytologic material. A good association was found between histologic and cytologic specimens using immunostaining.     

Development and validation of a sensitive immunohistochemical oestrogen receptor assay for use on archival breast cancer tissue

In a preliminary paper (Teasdale et al. 1987) comparing the oestrogen receptor (ER) content of breast cancers by the biochemical dextran coated charcoal (DCC) method and by two histochemical methods, peroxidase immunocytochemistry (ERICA) and immunogold-silver staining (IGSS), it was indicated that ERICA is more sensitive than DCC and that IGSS is as specific as ERICA but less sensitive. This paper describes the comparison of the above three assay methods with two other biochemical methods, iso-electric focusing (IEF) and an enzyme immuno-assay (EIA) on a larger number of cancers. All methods gave statistically comparable results except that IGSS remained less sensitive than the rest. Various modifications to IGSS showed that an immunogold streptavidin enhancement method (IG-SAM) produced sensitivity and specificity equal to that of ERICA. Since IGSS and its modifications are the only methods which can be used on archival paraffin-embedded cancers and IG-SAM gives results highly comparable to ERICA, retrospective studies can be performed on patients whose outcome and response to various treatments are known. Most recent studies have shown that ER positive results can be obtained from 10-year-old paraffin blocks.     

Comparison of Four Immunochemical Methods For the Measurement of Oestrogen Receptor Levels In Breast Cancer

Four methods of assessing oestrogen receptor (ER) status were compared in 33 patients with operable primary breast cancer. The methods used to assess the ER status were immunocytochemical assay (ER-ICA) of frozen sections, fine needle aspirates and imprint material and enzyme immunoassay (ER-EIA) of tumour tissue. A mean overall ER positivity of 45% (15 out of 33), 41% (13 out of 32) and 21% (six out of 29) was obtained by immunocytochemical (ER-ICA) staining of frozen sections, fine needle aspirates and tumour imprints, respectively, and a mean overall ER positivity of 42% (14 out of 33) was obtained by ER-EIA. The concordance of ER positivity in pairs of data obtained from different method combinations was found to range between 72 and 91%. However, statistically there was no significant difference between the four methods on the basis of the data obtained. Good comparability has been shown between the three tissue analyses and therefore the method of choice is technically not immediately apparent.     

Immunocytochemical versus biochemical receptor determination in normal and tumorous tissues of the female reproductive tract and the breast

The development of highly specific and sensitive monoclonal antibodies directed against human estrogen (ER) and progesterone receptors (PR) provides a new approach in precise histochemical receptor location independent of hormone binding. Over the years receptor determination was the domain of the radioligand-binding assay, in which receptors are measured by tritiated ligand and unbound ligand is removed by the dextran-coated charcoal (DCC) procedure. Presented here are the results and experiences obtained by the classic DCC and the immunocytochemical method in the different normal and tumorous tissues of the female reproductive tract and the breast. The results of both methods were compared, and overall concordance of the results was found to vary considerably among the different types of tissue analyzed. Best agreement (86%) was found for PR determination in breast cancer, and the lowest rate of concordance for ER determination in fibrocystic disease of the breast. Special attention was directed toward the heterogeneity of receptor distribution in the specimens examined. In all tissues investigated, ER and PR were located in the nuclei of cells in both paraffin and frozen sections. Staining intensity varied among different cell types and from cell to cell for a single cell type, as well as in tumorous and normal tissues. In breast cancer, randomly scattered single cell receptor positivity was distinguished from focal/clonal positivity. Paraffin-embedded lymph node metastases showed significantly weaker staining as compared with their respective primary tumors. In the normal ovary, the corpus luteum and the stromal layer of the outer cortex were revealed as highly receptive elements for progestins, whereas ER was barely demonstrable in the normal ovary. Benign serous and mucinous ovarian tumors showed opposite ER and PR distribution among the stromal and epithelial components. Of special interest were the highly significant changes in ER and PR content in the stromal and glandular cells of the different layers of the normal endometrium throughout the menstrual cycle.     

Usefulness of estrogen receptor detection using archival Papanicolaou-stained smears.

OBJECTIVE: To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). STUDY DESIGN: Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. RESULTS: In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. CONCLUSION: Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.     

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.     

Hormone receptor status in breast cancer – a comparison between surgical specimens and fine needle aspiration biopsies

The present study was performed to evaluate the immunocytochemical analysis (ICA) of oestrogen (ER) and progesterone receptor (PR) in fine needle aspiration (FNA) biopsies from primary breast cancers as compared with the established enzyme immunoassays (ER-EIA and PR-EIA) based on cytosol homogenates from the corresponding resected tumour specimens. A total of 967 primary breast cancers were assessed for ER and PR content by both methods. Correlations between EIA and ICA expressed as percentage of tumour cells with a positive staining were highly significant (P < 0.001) for ER and PR. Staining intensity yielded only limited additional information. The concordance between the two techniques was about 80%. Evaluation of biological parameters by FNA may be useful to decide the optimal treatment for breast cancer patients.     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

A combined histological, immunocytochemical and biochemical approach in the evaluation of estrogen receptors in breast carcinomas

Correlation of structural and functional data might lead to better identification of hormone-dependent tumors. Sixty breast cancer specimens, sent to the biochemistry laboratory for estrogen receptor (ER) analysis, were studied here by a combined morpho-functional approach. Histological examination of needle biopsies on frozen tissue blocks showed that 12 cases (10%) were free of tumor cells; these cases mostly proved ER negative. On the other 48 cases, an immunocytochemical reaction was performed on the biopsy sections with a monoclonal antibody directed against p 29, an estrogen receptor related antigen. The staining values for p 29 and the biochemical ER findings were significantly correlated. A combined histological, immunocytochemical study seems to offer advantages in the selection of patients for hormonal therapy.     

Comparison of visual and CAS-200 quantitation of immunocytochemical staining in breast carcinoma samples.

Using the CAS-200 image analysis system, we compared the relationship between semiautomated computer measurements (QIC score) and visual scoring (H score) on 30 breast cancer fine needle aspirates (FNAs) and corresponding tissue specimens stained by the estrogen receptor immunocytochemical assay. These results were compared to the corresponding biochemistry assays for ER. We also investigated whether the computerized system could decrease false-negative staining for ER in 32 cryostat sections. QIC scores were generated using fixed nuclear and antibody thresholds after standardizing the illumination. Computer quantitation was essentially as precise as visual semiquantitation for FNAs: a small but significant correlation was found between tumor ER content and QIC score (r = .504, P less than .02) as compared with H score and ER content (rs = .55, P less than .005). The computerized system did not decrease the false-negative rate in cryostat sections. In all, computerized quantitation was no better than visual analysis of ER staining in these breast carcinomas.