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.     

Demonstration of oestrogen receptor in symptomatic breast carcinoma, using fine needle aspiration cytology

Oestrogen receptor immunocytochemical assay (ER-ICA) was used to determine oestrogen receptor (ER) content of cells in fine needle aspirate (FNA) specimens from 88 breast carcinomas. In 49 of these the radioligand binding assay for oestradiol was available for comparison. The predictive value of ER-ICA staining for a positive radioligand binding assay (greater than 10 fmol/mg protein) was 95%. Although the predictive value of negative staining was only 66%, 34 out of 37 ER-ICA negative tumours had radioligand binding assays below 60 fmol/mg protein. ER-ICA staining showed a strong positive correlation with age of the patient, positivity being rare before the menopause. There was a weak inverse correlation with tumour grade but none with tumour size or lymph node status. The assessment of ER by immunocytochemistry using FNA cytology is a rapid technique, which may easily be repeated and provides a pre-operative assessment of ER status. It allows confirmation that tumour cells are present in the sample and an assessment of tumour heterogeneity.     

Use of the dinitrophenyl hapten sandwich staining procedure (DHSS) to localize estrogen receptors in paraffin-embedded tissues.

To date, reliable and sensitive methods to localize the estrogen receptor (ER) in rat tissues and human breast cancers have required the use of frozen sections. This not only incurs poor tissue structure but also precludes the study of small breast lesions that are usually paraffin embedded for histological evaluation. We have developed and optimized a dinitrophenyl hapten sandwich staining (DHSS) immunocytochemical procedure to demonstrate ER in paraffin-embedded, hormone-sensitive tissues of the rat and in human breast cancers. The method was applicable to formalin- and Bouins-fixed material, with trypsinization of sections being essential. The immunocytochemical system utilized a dinitrophenyl (DNP) hapten-labeled monoclonal antibody to the receptor. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase complex, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This highly sensitive method localized the ER within paraffin-embedded rat uterus, fallopian tube, vagina, and normal and cancerous mammary gland. Furthermore, excellent staining was generated in human breast cancers in accordance with their ER-ICA status. Control sections involving simultaneous incubation with DNP-labeled and unlabeled H222 were background free, while uteri from castrated rats demonstrated reduced receptor immunostaining. Staining was also absent in ER-negative human breast tumors.     

Detection of the level of estrogen receptor and functional variants in human breast cancers by novel assays

The level of estrogen receptor (ER) is a key determinant for the management of ER-positive [ER(+)] breast cancer patients. Growth of many human breast cancers is regulated by estrogen (E2) and progesterone (Pr). Generally, the ER in ER(+) breast cancer patients is targeted for therapy with antihormones. However 40% of ER(+) patients do not respond to antihormone therapy. Thus, the identification of antihormone resistant ER(+) breast cancers is essential for therapeutic predictions. Although 3H-E2 binding and immunodetection can identify ER, these procedures do not assess the functional state of the receptor molecule. In this study we describe a novel and rapid assay for the detection of ER and its functional state on the basis of the downstream interaction with its response element (ERE) based on the preferential binding of DNA-protein complex (ERE-ER) to a nitrocellulose membrane (NMBA). This method permits measurement of both the total and the functional fraction of ER. The ER status was examined in breast cancer cell lines and in breast cancer biopsy specimens by (i) 3H-E2 binding assay, (ii) immunodetection assays and (iii) by its interaction with 32P-ERE. The sensitive NMBA assay was validated with well-characterized ER(+) breast cancer cell lines and also identified functional variants of ER among breast tumor biopsy specimens.     

Cytologic diagnosis of estrogen and progesterone receptors in breast imprints

OBJECTIVE: To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. STUDY DESIGN: Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. RESULTS: Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. CONCLUSION: ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.     

Variation of ER status between primary and metastatic breast cancer and relationship to p53 expression*.

OBJECTIVE: Estrogen-dependent growth of breast cancer can be blocked by anti-estrogens. Estrogen receptor (ER) presence in breast cancer implies responsiveness to endocrine therapy. However, for those patients who ultimately develop resistance to endocrine therapy, the mechanisms remain unclear. The present study attempted to compare the expression status of ER mRNA in a series of primary breast tumors with matched metastases and explored the relation between ER and mutant p53 expression. METHODS: In situ hybridization using a digoxigenin-labeled estrogen receptor cDNA probe was employed to determine the expression of ER mRNA in 52 cases of primary tumors and their matched axillary lymph node metastases. Immunohistochemical staining using a monoclonal antibody against ER was also performed. RESULTS: ER expression was observed in 53.8% (28/52) of primary tumors and 48% (25/52) of metastases, while 57.7% (30/52) of primary tumors and 53.8% (28/52) of metastases showed ER mRNA positivity. There were variations in ER status between in situ hybridization and immunohistochemistry measurements and between primary tumors and metastases. Mutant p53 expression was inversely associated with ER-negative, high-grade tumors. CONCLUSIONS: In situ hybridization may be a more specific and sensitive method for determination of ER status than immunohistochemistry. It is possible that the biologic properties of ER change, and these changes may influence tumor response to endocrine therapy. In view of the ER variation, it was suggested that the ER status of metastatic tumors in addition to primary tumors should be taken into consideration in order to better determine the benefit of clinical endocrine therapy.     

Identification of immunoassayable estrogen receptor lacking hormone binding ability in tamoxifen-treated rat uterus

Using two different monoclonal antibodies to human estrogen receptor (ER), the enzymeimmunoassay was performed. The values of ER contents in human breast cancer and untreated rat uteri obtained by this procedure were correlated well with those by [3H] estradiol binding assay. When estradiol was injected to immature rats, the enzymeimmunoassay showed the uterine receptor dynamic pattern similar to those analyzed by exchange assays. In contrast, tamoxifen administration induced the immunoassayable but nonsteroid binding form of ER. This ER-like antigen was the heat-labile molecule with the sedimentation constant of 7 S while ER in untreated rat uterine cytosol sedimented at 9 S. These results suggest the presence of unique molecular state of ER induced by tamoxifen.     

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.     

Quantitative image analysis of estrogen and progesterone receptors as a prognostic tool for selecting breast cancer patients for therapy

OBJECTIVE: To assess estrogen and progesterone receptor presence in human breast tumors using immunocytochemical analysis. STUDY DESIGN: For both estrogen (ER) and progesterone (PR) receptor assay, percent of stained cells and intensity of staining were estimated on a series of 251 consecutive breast cancer cases from the M. Ascoli Cancer Hospital Center in Palermo using the CAS 200 image analysis system. RESULTS: Cytochemical assay revealed a differential distribution of both ER and PR, by menopausal status of the patients; premenopause (PreM) was mostly ER negative (63%), and postmenopause (PostM) > 10 years was mostly ER and PR positive (64%). The percent of cells stained for ER was significantly different between PreM and PostM patients when they were considered as a whole. By contrast, no difference emerged for PR staining among menopausal groups. Overall, patients whose tumors were PR positive showed a significantly (P < .03) longer interval free of relapse. CONCLUSION: The present results suggest that PRs behave as better indicators than ERs of early relapse in breast cancer patients. Further studies, with longer follow-up, are needed, however, to validate this concept.     

Comparison of two methods of storing breast fine needle aspirates (FNAs) using oestrogen receptor immunocytochemical assay as a method of evaluating the storage methods

Two methods of storing fine needle aspirates were compared in 14 patients with breast cancer. the methods of storage were: (1) as a Cytospin slide prepared immediately from the aspirated material and stored at −80°C; (2) as a suspension of cells in tissue culture medium, stored at −80°C. the effect of storage on the cells was assessed by means of an oestrogen receptor immunocytochemical assay (ER-ICA). an ER positivity of 100% was obtained by ER-ICA staining of cells after storage method 1, whilst all of the specimens stored by method 2 were ER-negative. the data demonstrate that cells stored in tissue culture medium at −80°C are not suitable for ER measurement. the storage method of choice for specimens intended for ERICA is as a Cytospin slide. the ER status of cells deposited on Cytospin slides prepared immediately and stored at −80°C for 2 years could be demonstrated despite the delay in processing the specimen.     

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.     

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.     

Estrogen and progestin receptors in human uterus: reference ranges of clinical conditions

Estrogen receptor (ER) and progestin receptor (PR) levels and their respective dissociation constants (Kd) were determined by titration assay and Scatchard analyses in 319 human uteri. Levels of receptors were neither age nor uterine weight dependent. ER was higher in postmenopausal patients while PR levels were lower in women under 25 years of age. ER ranged from undetectable to 560 fmol/mg cytosol protein (mcp) while PR levels were generally 10-fold greater with a mean of 791 fmol/mcp. The mean of the Kd of ER was 4.0 X 10(-10) M while that for the PR was 9.2 X 10(-10) M. Receptor levels were not correlated with their respective Kd values nor with the Kd of the other receptor; therefore ligand affinities were not receptor concentration dependent. A population of patients was identified (12.5% of the total) in which the ER levels were undetectable while their corresponding PR ranged from 38 to 2,100 fmol/mcp. This suggests the existence of a type of PR which may not require ER for its expression and is independent of the phase of the cycle. Both ER and PR content were significantly elevated in the proliferative phase of the cycle. Evaluation of results as a function of histopathological features showed no relationship between the ER and PR of patients with anovulatory bleeding versus pathology. Uteri with leiomyomas contained ER and PR at levels comparable to those of histologically normal uteri. Adenomyosis patients tended to have lower ER and higher PR levels than the normal uteri. Reference ranges have been established for these receptors in uteri as a corollary to studies of these proteins in endometrial cancer.     

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations

The assessment of multiple variables on breast carcinoma fine needle aspiration (FNA) cytology specimens: method, preliminary results and prognostic associations
We have assessed multiple biological variables on breast carcinoma FNA specimens using a Cytoblock technique. The growth fraction (MIBI), oestrogen receptor (ER), progesterone receptor (PR), p53 mutant protein, c-erbB-2, epidermal growth factor receptor (EGFR), NCRCl Vepithelial membrane antigen (EMA) and DNA plopidy were examined. Objective quantification using image analysis (CAS 200) was applied as appropriate. Fifty cases were examined in this preliminary study. Excellent correlation between the Cytoblock preparations and parallel tissue sections was seen. Of the cancers, 81% were aneuploid with only 19% diploid in character, but 67% of the carcinomas were of histological grade 3. The mean nuclear area staining with MIBl was 31.3% and with ER was 26.7%. Twenty-four percent (24.1%) of the nuclear area showed immunoreactivity with PR. Significant immunostaining was seen in 38%, 46%, 38% and 95% of carcinomas with c-erbB-2, p53, EGFR and EMA, respectively. A significant association between histological grade of the resected tumours and both MIBl (P=0.04) and EGFR (P=0.02) expression in the Cytoblock samples was seen. p53 (P = 0.03) and EGFR (P=0.01) immunoreactivity showed an association with tumour size. EGFR (P=0.04) immunostaining also showed a relationship with the lymph node status of the patient. The technique is, we believe, a useful one for the assessment of multiple variables on breast cytology specimens; these preliminary data suggest that some of these may be useful in predicting prognosis in breast cancer patients.     

An estrogen receptor (ER)alpha deoxyribonucleic acid-binding domain knock-in mutation provides evidence for nonclassical ER pathway signaling in vivo

We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis. The uteri are enlarged with evidence of cystic endometrial hyperplasia, and the mammary glands are hypoplastic. These phenotypic features indicate differential ER effects on growth and development in various estrogen-responsive tissues. These findings suggest that nonclassical ER signaling pathways play an important physiological role in the development and function of the reproductive system.     

The expression of PCNA, c-erbB-2, p53, ER and PR as well as atypical hyperplasia in tissues nearby the breast cancer

To examine the proper margin for breast conservative surgery in Chinese women population. 40 breast cancer specimens were collected and each sample was dissected into several groups: primary tumor group, 1 cm paracarcinoma, 2 cm paracarcinoma, 3 cm paracarcinoma and excessive 3 cm paracarcinoma groups. The immunohistochemistry staining was performed to measure the expression levels of proliferating cell nuclear antigen (PCNA), c-erbB-2, p53, estrogen receptor (ER) and progesterone receptor (PR). The gene expressions of PCNA, c-erbB-2, and p53 gradually decreased with the increased distance from primary tumor (P < 0.05). The 1 and 2 cm paracarcinoma group (no differences between the two, P > 0.05) showed higher risk factors (c-erbB-2, p53) than the 3 cm and excessive 3 cm paracarcinoma groups (P < 0.05). The expression of PCNA, ER, and PR showed no correlation with cancer progression (P > 0.05). Beyond the paracarcinoma 2 cm distance, the tissues showed significant decreases in tumor gene expression, which could represent the appropriate region for breast conservative surgery.     

Comparison of estrogen receptor immunocytochemical assay in frozen and paraffin sections.

Frozen and paraffin sections of 11 breast carcinomas were stained for estrogen receptors (ER) using the same rat monoclonal primary antibody, D75P3, as the marker and alkaline phosphatase as the chromogen-linking enzyme. The results of this staining process were assessed visually and with the microTICAS image analysis system to determine the degree of correlation between frozen and paraffin-embedded tissue. In all specimens, some fraction of the nuclei stained positively. This included two specimens selected for their biochemically negative assay; one of them stained strongly positively with D75P3. The results of quantitative analysis support the visually apparent correlation between the two types of samples in terms of both overall staining pattern and intensity of nuclear staining. Although the conclusions of this pilot study are limited because of the small number of cases, this method of staining establishes the feasibility of representative ER determination in archival paraffin-processed material. The additional information provided by this method is potentially useful in stratifying patients in prospective studies on the basis of the efficacy of hormonal therapy in biochemically ER positive breast tumors.     

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.     

Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry

Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.