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

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

Evaluation of estrogen receptor immunocytochemical assay (ER-ICA) and estrogen receptor enzyme immunoassay (ER-EIA) for the determination of estrogen receptor status in breast carcinoma. A comparative study on frozen sections, cytological smears and tissue homogenates.

One hundred thirty three cases of primary breast cancer were analyzed for estrogen receptors (ERs) by two different techniques: monoclonal ER immunocytochemical assay (ER-ICA) and ER enzyme immunoassay (ER-EIA). The overall concordance between ER-ICA and ER-EIA was 89.5% despite the semiquantitative nature of the immunocytochemical evaluations. There was a significant correlation between these two methods in linear regression analysis. The correlations were evident both for ER detection on frozen sections as well, as on fine needle aspirates. Correlations between histoscores calculated independently by three pathologists might overcome the objections of subjectivity of ER-ICA evaluations. The results suggest that ER-ICA can be useful in assessing ERs in breast cancer, especially in small primary tumours, primary inoperable breast cancer, recurrences and metastases.     

Whither cytosolic estrogen receptor assays? A comparison of commercially available kits for estrogen receptor assay

Frozen tissue sections and cytosols from 89 specimens of breast and ovarian tumours have been assayed for the presence of estrogen receptor (ER) or related protein using four commercially available monoclonal antibody methods. These were estrogen receptor enzyme immunoassay (ER-EIA), estrogen receptor enzyme immunocytochemical assay (ER-ICA), ER D5 antigen immunoradiometric assay and ER D5 antigen immunocytochemical assay. The results have been compared with those obtained using a standard dextran coated charcoal steroid binding assay (ER-DCC). The correlation coefficient (r) between ER-DCC and ER-EIA results was 0.72 while that of both monoclonal antibody cytosol methods and their respective immunocytochemical assays was 0.66. ER-ICA gave additional valuable information concerning receptor heterogeneity in breast cancer sections. However, the correlation between ER D5 antigen assays and both ER-DCC and ER-EIA was weak (r less than 0.4). We conclude that there are a number of methodological advantages in using the kit systems including their ability to detect receptor presence in small tumour specimens (e.g., "Tru-cut" biopsies) but that their usefulness is limited by the current lack of widely available monoclonal based methods for the concurrent determination of progestogen receptor. We believe that, once these are available, immunocytochemical technology could offer an alternative method of determining the steroid receptor concentration in both ovarian and breast tumours, thus obviating the need for costly and time-consuming cytosolic methods, with their inherent difficulties of quality control.     

Immunocytochemical evaluation of estrogen receptor on archival Papanicolaou-stained fine-needle aspirate smears

The availability of limited fine-needle aspirate smears necessitates the selection of immunocytochemical (IC) methods that allow reuse of Pap-stained smears to assess the estrogen receptor (ER) status of breast carcinoma. The objective of the current study was to compare IC evaluation of ER status on FNA smears by three methods: 1) ER-ICA using H222 monoclonal antibody performed on slides fixed in formaldehyde-methanol-acetone; 2) destained Pap slides using 1D5 antibody; and 3) Pap-stained slides without destaining using the same 1D5 antibody. Two representative Pap smears of breast carcinoma were selected from 48 cases of breast carcinoma in which ER was previously evaluated by ER-ICA. One of these Pap smears was used as such and the other was destained prior to immunostaining by a modified ABC method using 1D5 monoclonal antibody. The number of cells with positive nuclear staining was expressed as a percentage and the intensity of staining was semiquantitatively scored on a scale of 1+ to 3+. The degree of agreement between the three methods was evaluated statistically by weighted kappa statistics. Thirty cases (63%) showed varying degrees of positive staining while 18 cases (38%) were entirely negative by all three methods. Significant discrepancies in the number of cells with positive staining and in the intensity of staining between the three methods occurred in 40% and 23% of the cases and was mainly due to a reduction in the number of cells with positive staining and the intensity of staining using Pap slides in comparison to ER-ICA. Weighted kappa agreement of the percentage of cells with positive staining using Pap-stained slides and destained Pap-slides in comparison to ER-ICA was 0.75 and 0.64, respectively, and that for the intensity of staining was 0.75 and 0.66, respectively. Therefore, IC evaluation of ER using Pap-stained smears as such or destained Pap smears compared favorably with ER-ICA. However, Pap-stained smears used as such for ER immunostaining showed a slightly better agreement with ER-ICA than destained Pap smears. Because significant differences in ER-IC staining can occur with any of the immunocytochemical methods, a negative result is less reliable as an indicator of true ER status than a positive result.     

Estrogen receptor immunocytochemistry for preoperative determination of estrogen receptor status on fine-needle aspirates of breast cancer

Fine-needle aspirates (FNAs) of 84 primary breast carcinomas were analyzed immunocytochemically for estrogen receptor (ER) using (ER-ICA) monoclonal antireceptor antibodies. ER-ICA in FNAs was concordant to ER-ICA in histologic biopsies in 87% (P less than 0.0001). In most of the carcinomas, biochemically determined ER status also correlated to ER-ICA. There was no false positive ER-ICA in FNAs compared with ER-ICA in histologic biopsies. In 11 FNAs, ER-ICA was negative, whereas it showed positivity in histologic specimens. The most frequent contributing factors to false negative ER-ICAs of FNAs were ER-ICA-low results in histologic biopsies, a prominent stroma component in these tumors, and low cellularity of FNAs. The biochemical ER values in these cases never exceeded 90 fmol/mg protein. In a minority of cases, false negative results were inexplicable.     

A simplified histoscore for the estrogen receptor assay in breast cancer.

Different histoscores combining the number of positive cells and the intensity of staining have been used to evaluate the estrogen receptor immunocytochemical assay (ER-ICA). Our aim was to investigate if the simple estimation of the amount of positive cells could be sufficient for the semiquantitative analysis of ER-ICA. Tissue from 51 women with ductal breast carcinoma was used. Half of each sample was processed with the quantitative assay (ER-EIA) and the other half with ER-ICA. Microscopical analysis was performed by two independent observers and classified on a simple scale from 0 to 4+. With EIA 31 cases (60.78%) were positive and 20 (39.21%) negative. With ER-ICA 29 (56.86%) had immunostaining, whereas 22 (43.13%) did not. 95.83% of the ER-ICA positive cases and 77.7% of ER-ICA negative had a good correlation with EIA values. Statistical analysis showed a high degree of correlation (r = 0.88 p 0.001). Hence, simple semiquantitative estimation in ER-ICA is sufficient to provide useful information for clinical use about ER content in tissue sections.     

Comparative evaluation of ER-ICA and enzyme immunoassay for the quantitation of estrogen receptors in breast cancer

Evaluation of estrogen receptors (ERs) is essential for the treatment and prognosis of human breast cancer. The authors have undertaken a study of 225 primary breast cancers to compare the receptor values using immunohistochemistry and enzyme immunoassay (EIA) techniques. The results are compared with the biochemical assay with the use of the dextran-coated charcoal (DCC) method done on the same specimens. The overall concordance between ER-ICA and DCC was 77%. Ninety-seven percent concordance was observed for specimens with a positive ER-ICA score (greater than 70 and the DCC more than 10 fmol/mg protein). Ninety-two percent of tumors had negative results by ER-ICA when the DCC was less than 10 fmol/mg protein. When the ER-ICA score was plotted against the DCC or EIA value, a significant correlation was obtained with a P value of less than 0.001. The correlation between the ER-ICA score and the total ER content (cytosol and nuclear) assayed by EIA was similar to comparison with cytosol alone. The authors conclude that the ER-ICA can be useful in assessing ERs in breast especially in hypocellular or small tumors and in assessing the degree of heterogeneity in a given tumor. The immunohistochemical and biochemical assay methods are complementary and provide greatly needed information for the management of breast cancer.     

Comparative histological,histochemical, immunohistochemical and biochemical studies on oestrogen receptors,lectin receptors,and Barr bodies in human breast cancer

Summary The present study performed on a total of 567 cases of human female breast cancer compares the results of the biochemical assay (dextran-coated charcoal assay=DCC) for oestrogen receptor (ER) with those of several morphological methods developed for the detection of the ER or for the prediction of prognosis by use of other systems (FSA= fluorescent ligand binding assay, ER-ICA=monoclonal antibody assay for ER, LRA=lectin receptor assay using peanut agglutinin, and Barr body estimation). Whereas no correlation at all was observed among the results of the DCC and those of the FSA and Barr body estimation, the ER-ICA and the LRA showed an unanimous tendency towards higher values of ER with increasing intensity of the staining product. The results of the ER-ICA may be expressed by an immuno-reactive score (IRS) calculated from the staining intensity (SI) and the percentage of positive cells (PP). The morphological methods are evaluated with special regard to their correlation with the DCC, their theoretical basis, and their practical application. In summary, the ER-ICA appears to be the sole method directly visualizing the ER protein and - in contrast to the DCC - is therefore completely independent of the content of endogenous or exogenous oestrogens in the tumor tissue. The LRA provides valuable additional information concerning tumour differentiationDedicated to Professor Karl Lennert, Kiel, on the occasion of his 65th birthdaySupported in part by the German Cancer Fund (Deutsche Krebshilfe)     

Estrogen receptor analysis on biopsies and fine-needle aspirates from human breast carcinoma. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.

Monoclonal antibodies against estrogen receptor (ER) were used for determination of ER status immunocytochemically in histologic specimens from 192 primary breast carcinomas. All tumors were also assayed biochemically for ER with the dextran-coated charcoal method (DCC). The comparison of biochemically and immunocytochemically determined ER status showed concordant results in 80% (P less than 0.0001). In only 2 cases (1%) with low ER levels (less than 20 fmol/mg protein) immunocytochemistry failed to detect ER. ER positivity determined with a semiquantified approach based on intensity and heterogeneity of immunocytochemical staining correlated significantly with biochemically determined ER levels (P = 0.0001). In a series of fine-needle aspirates of 34 breast carcinomas sufficient cell material was available for ER immunocytochemistry (ER-ICA). Overall, the results of ER-ICA in fine-needle aspirates were concordant with ER-ICA in histologic specimens in 88% of the samples. In a few cases with weak positivity of ER-ICA in histologic specimens, ER-ICA was negative in fine-needle aspirates. In no case was there a false-positive immunocytochemical ER determination in a tumor aspirate. Thus, ER-ICA seems to be a reliable assay which can be performed in histologic and cytologic specimens.     

Oestrogen and progesterone receptors in screen-detected breast carcinoma: an immunohistological study using paraffin sections

The presence of oestrogen and progesterone receptors was studied in paraffin sections of 81 screen-detected breast carcinomas using the monoclonal antibodies ER-ICA and PgR-ICA (Abbott) and the immunoperoxidase technique. The immunohistological results were compared with the results of the standard dextran-coated charcoal biochemical assay in 28 tumours which were big enough to provide tumour tissue for this assay. Sixty-three cases (78%) were oestrogen receptor positive and 62 (77%) were progesterone receptor positive. There was no statistical difference between receptor positivity in palpable or impalpable, in situ or invasive tumours. In the 28 cases where the biochemical assay was carried out, the two methods gave similar results in 23 (82%) and 21 (75%) tumours for oestrogen and progesterone receptors respectively. The majority of the remaining tumours, with one exception, were positive with immunohistology and negative with biochemistry. A good correlation was also present between the mean numerical biochemical values and the semiquantitative histological scores for both receptors. It is concluded that assessment of receptor status of small screen-detected carcinomas is feasible using routinely processed paraffin sections. There is reasonably good correlation with the results obtained by the standard dextran-coated charcoal biochemical assay, but more genuine receptor positive cases are detected by immunohistology.     

Immunohistochemical demonstration of cell proliferation and estrogen receptor status in human breast cancer. Analysis of 45 cases

Cell proliferation and estrogen receptor (ER) status was investigated in 45 invasive ductal carcinomas of the breast by immunohistochemical methods using monoclonal antibodies Ki-67 (anti-human proliferating cell antibody) and ER-ICA. The results were assessed on the basis of nuclear staining intensity and the percentage of positively stained tumor cell nuclei (index score). There was a significant inverse correlation between the Ki-67 and ER-ICA index scores, although 4 cases showed high index scores for both markers. We conclude that ER-positive cells do not always have low proliferation activity, which may be one of the reasons why endocrine therapy is not effective against all ER-positive breast cancers.     

Densitometric and morphometric study of immunocytochemical estrogen receptors detection in breast carcinomas.

Immunohistochemical quantitative evaluation of estrogen receptors (ER) detected in tissue sections from 30 breast tumors by monoclonal antibody was performed using a densitometric method. In particular, ER concentration was calculated by nuclear mean optical density (nMOD), while heterogeneity in ER content was calculated by the coefficient of variation (CV) of the nuclear optical density histogram. Tumors which showed more than 60% of positive cells had a mean value of ER-nMOD of 0.116 +/- 0.002 a.u. and of ER-CV of 33.74 +/- 0.68. Tumors which showed 30% to 60% of positive cells had a mean value of ER-nMOD of 0.082 +/- 0.006 a.u. (arbitrary units) and of ER-CV of 36.25 +/- 3.44. Tumors showing less than 30% of positive cells had ER-nMOD of 0.052 +/- 0.009 a.u. and ER-CV of 48.49 +/- 5.61. These results indicate that the greater the concentration the lower the ER heterogeneity within the tumor sample. No significant differences between ER-ICA results, nuclear size and form factors were found.     

Discrepancies of the biochemical and immunohistochemical estrogen receptor assays in breast cancer

We examined the estrogen receptor (ER) content of 124 primary breast cancers by hormone binding and immunohistochemical (ER-ICA) assays. Both assays were in agreement in 110 tumors (89%; P less than .0001); 68 tumors were positive and 42 were negative. In 14 cases (11%), the assays yielded discordant results. Three tumors showed hormone binding in the absence of immunohistochemically detectable ER; the false positive hormone binding resulted from the presence of normal epithelium adjacent to ER-ICA negative malignant cells. Eleven tumors failed to show hormone binding but were ER-ICA positive. Four of these were from premenopausal patients whose circulating endogenous estrogen may occupy the receptor, giving rise to false negative hormone binding assays. In four cases, the discrepancy of negative hormone binding assay and positive ER-ICA assay was attributed to scant tumor cells in the tissue sample. The remaining three discrepancies could not be resolved with certainty, but possibly resulted from alteration of the hormone binding site with preservation of the immunoreactive epitope on the ER molecule. These results indicate that the ER-ICA assay is more accurate than the hormone binding assay in identifying the presence of ER in cancer cells. The heterogeneous immunostaining of ER in tumor sections, which may reflect mosaicism of tumor cells, rate of cell proliferation, or phase of cell cycle, remains unexplained.     

Quantification of estrogen receptors on paraffin-embedded tumors by image analysis

Emphasis on early detection of breast carcinomas has increased the number of instances in which an insufficient amount of tissue is available for biochemical estrogen receptor (ER) assay. Image analysis, used together with immunohistochemistry, introduces the possibility of quantifying molecules, such as ER, in routinely processed tissues. To explore that possibility, sections from 40 formalin-fixed, paraffin-embedded breast carcinomas with biochemically determined ER values were reacted with H222 anti-ER antibody (ER-ICA) and quantified on a CAS 200 image analysis system. A minimum of ten fields comprising at least 15,000 microns 2 of nuclear area were analyzed in each case. If the antigen distribution were not homogeneous, proportional sampling of the different tumor areas was carried out. Of the 31 ER-positive (10 to 344 fmol/mg) tumors, 27 (87%) were immunoreactive by the ER-ICA assay (greater than 5% positive nuclear area), which represents an improvement over simple microscopic evaluation (68%). Bivariate analyses showed statistically significant, albeit only modest, correlation between ER values and the percentages of positive area (r = 0.556) and positive stain (r = 0.518). The following obstacles were found to interfere with a proper correlation: (a) antigen loss during fixation and processing; (b) intratumoral antigenic heterogeneity; and directly associated with it, (c) interobserver variability. Uniform tissue handling or, alternatively, the use of internal controls to compensate for fixation-induced differences, together with thorough assessment of the tissue, should reduce these obstacles and facilitate accurate and reproducible quantification.     

Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin-embedded sections of breast carcinoma.

Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5-microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of proliferative rate of breast cancer by Ki-67 monoclonal antibody

The proliferative activity of 163 primary breast cancers was assessed by immunocytochemistry with the mouse monoclonal antibody Ki-67, which recognizes a nuclear antigen expressed in all phases of the cell cycle except for Go. The overall frequency distribution of Ki-67 staining was of exponential type, with percentage of positive staining cells ranging from 0.3 to 88.3%, with a median value of 10.1%. No relationship was observed between Ki-67 values and menopausal status of patients. A significant positive correlation was found between Ki-67 values and tumor grade, especially mitotic grade. Estrogen Receptors (ER) were assayed by immunocytochemistry with ER-ICA method and by dextran-coated charcoal method (DCC) in 129 and 141 tumors, respectively. A negative correlation was found between the ER content by both methods and Ki-67 score. Eighty-nine percent of cases with Ki-67 value less than 10.1% contained more than 10% ER-ICA-positive cells. Progesterone receptors (PgR) were assayed by immunocytochemistry with PgR-ICA method and by DCC in 62 and 141 tumors, respectively. A negative correlation was observed between the PgR content by both methods and Ki-67 score. No correlation was found between Ki-67 score and lymph node involvement by tumor. These findings suggests that Ki-67 score could be used as an independent prognostic marker, useful to distinguish different risk for recurrence within the two clinically heterogeneous groups of N- and N+ patients.     

Estrogen receptors in skin appendage tumors and extramammary Paget's disease.

Determination of estrogen receptors (ER) in breast carcinoma is valuable in the management of patients. However, little is known about the presence of these receptors in other tumors. Normal skin appendages and their neoplasms, including extramammary Paget's disease (EPD), might be expected to express ER since the breast is histogenetically related to sweat glands. In this study, 41 cases of skin appendage tumors (SAT) and 11 cases of EPD were stained using the ER-ICA monoclonal kit (Abbott, Chicago, IL) with a modified technique for paraffin-embedded sections. Controls included 10 biopsies of primary breast carcinoma and 4 cases of metastatic breast carcinoma to skin, all positive for ER. None of the samples of SAT or EPD showed staining for ER. Normal skin appendages were also negative. Normal vaginal epithelium in one case of EPD showed positive nuclear staining for ER. ER determination using immunohistochemical technique in paraffin-embedded sections may be useful in the differential diagnosis between malignant SAT and metastatic breast carcinoma in the skin. The absence of ER in normal skin appendages suggests that its apparition is a feature of specialized differentiation of breast epithelium.     

Oestrogen receptor content and cancer cell/stroma ratio in mammary carcinoma

In a study of 86 human breast cancers the volume fraction of the neoplastic epithelium was estimated semiquantitatively and a significant association was found between the oestrogen receptor (ER) content and volume fraction of cancer cells of tumours (p less than 0.00001). Tumours with very low cellularity could thus be falsely ER negative. When quantitative ER concentration is used to predict the response of tumours to hormonal therapy the cellularity of tumours should be taken in consideration. The cellularity was not related to ER positivity. Although a correlation was seen between the contents of oestrogen and progesterone (PR) receptors, no correlation between PR content and volume fraction of the neoplastic epithelium was observed.     

C-erbB-2 oncoprotein amplification in infiltrating ductal carcinoma of breast relates to high histological grade and loss of oestrogen receptor protein

Eighty-six infiltrating ductal carcinoma of breast were studied by the standard avidin-biotin complex immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections, for oestrogen receptor (ER) protein and c-erbB-2 oncoprotein expression. They were categorized according to the modified Bloom and Richardson criteria into three histological grades. 21% tumours were ER positive while 44% were c-erbB-2 positive. Of ER positive tumours, 33.3% were c-erbB-2 positive whereas the c-erbB-2 positivity rate was much higher (47.1%) in ER negative tumours. Only 16% of c-erbB-2 positive tumours were ER positive while 25% of c-erbB-2 negative tumours were ER positive. This negative relationship between ER and c-erbB-2 expression was statistically significant (Mc Nemar's test, p < 0.005). The ER positivity rate did not vary significantly with histological grade. However, c-erbB-2 overexpression was significantly more prevalent in grade III tumours compared with grade I and II tumours (Chi-square test, p < 0.005). Since the c-erbB-2 oncogene has extensive structural homology to the epidermal growth factor receptor (EGFR) gene, we expect that c-erbB-2 oncoprotein would share functional similarities with EGFR leading to both loss of oestrogen receptor and poor prognosis in breast cancer. Its overexpression can be expected to relate to more aggressive tumour proliferation and may explain its correlation with high histological grade, a known indicator of aggressive cancer behaviour. As there is no indication that ER protein activity contributes to advancement in histological grade, it would appear that cellular dedifferentiation precedes ER loss during malignant transformation. It has been mooted that ER positive breast cancers which also show c-erbB-2 oncoprotein overexpression have a poorer response to hormonal therapy. The use of this parameter in the routine assessment of breast cancer patients may identify subsets of patients for more aggressive therapy.     

Immunohistochemical demonstration of oestrogen and progesterone receptors: correlation of standards achieved on in house tumours with that achieved on external quality assessment material in over 150 laboratories from 26 countries

AIMS: To investigate the sensitivity of immunohistochemical (IHC) assays for oestrogen receptors (ER) and progesterone receptors (PR) achieved by laboratories on breast tumours fixed and processed in their own department, and to compare this with the degree of sensitivity they achieve on tumours circulated as part of an external quality assessment (EQA) programme. METHODS: On 10 occasions between April 1994 and June 1998, histological sections from breast cancers showing various degrees of expression of ER and PR were circulated for IHC staining to laboratories participating in the UK national external quality assessment scheme for immunocytochemistry (UK NEQAS-ICC). The staining of these tumours, in addition to that of tumours fixed and processed in the participants own laboratories (in house tumours), was assessed by a panel of four assessors, using the established UK NEQAS-ICC scoring system. For a selected assessment run, the degree of expression of participants in house tumours was evaluated by means of the semiquantitative quick score method. RESULTS: Although the scores awarded for the staining of in house tumours were generally higher than those awarded for the staining of UK NEQAS tumours, there was also a significant positive correlation between the two sets of scores. Using the quick score method of evaluation for one of the assessment runs, 47% of in house tumours were classified as having a high degree of ER expression. Of the remaining cases, a significant proportion initially classified as having only low or medium expression of ER were found to have higher expression when stained by the organising laboratory. The UK NEQAS-ICC centre's routine assay for hormonal receptors was found to be 90-100% efficient in achieving optimal demonstration of breast tumours from over 150 different laboratories. CONCLUSIONS: The significant positive correlation between the results obtained on the UK NEQAS tumours and the in house tumours provides evidence for the view that results achieved on EQA material are accurate indicators of in house laboratory performance. Although most laboratories adequately detected tumours with high receptor expression, a large proportion of in house tumours classified initially by participants' staining as being of low or medium ER expression had a higher degree of expression when stained by the UK NEQAS-ICC centre. The efficiency of the organising centre's routine IHC method for ER and PR in optimally demonstrating participants in house breast tumours shows that variations in fixation and tissue preparation are not limiting factors preventing a different laboratory achieving optimal demonstration.