Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer.

Phosphorylation of oestrogen receptor alpha at serine 305 (ERalphaS305-P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERalphaS305-P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n=248) and patients with advanced disease (n=129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence-free survival (RFS) for ERalphaS305-P-negative tumours (multivariate HR=0.53, 95% CI 0.32-0.86, p=0.010), but not for ERalphaS305-P-positive tumours (multivariate HR=1.01, 95% CI 0.33-3.05, p=0.99) (interaction p=0.131). Notably, ERalphaS305-P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR=0.64, 95% CI 0.30-1.37, p=0.248), indicating that ERalphaS305-P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERalphaS305-P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERalphaS305-P-positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup.

Validation of 70-gene prognosis signature in node-negative breast cancer.

PURPOSE: The 70-gene prognosis signature (van't Veer et al., Nature 415(6871):530-536, 2002) may improve the selection of lymph node-negative breast cancer patients for adjuvant systemic therapy. Optimal validation of prognostic classifiers is of great importance and we therefore wished to evaluate the prognostic value of the 70-gene prognosis signature in a series of relatively recently diagnosed lymph node negative breast cancer patients. METHODS: We evaluated the 70-gene prognosis signature in an independent representative series of patients with invasive breast cancer (N = 123; <55 years; pT1-2N0; diagnosed between 1996 and 1999; median follow-up 5.8 years) by classifying these patients as having a good or poor prognosis signature. In addition, we updated the follow-up of the node-negative patients of the previously published validation-series (Van de Vijver et al., N Engl J Med 347(25):1999-2009, 2002; N = 151; median follow-up 10.2 years). The prognostic value of the 70-gene prognosis signature was compared with that of four commonly used clinicopathological risk indexes. The endpoints were distant metastasis (as first event) free percentage (DMFP) and overall survival (OS). RESULTS: The 5-year OS was 82 +/- 5% in poor (48%) and 97 +/- 2% in good prognosis signature (52%) patients (HR 3.4; 95% CI 1.2-9.6; P = 0.021). The 5-years DMFP was 78 +/- 6% in poor and 98 +/- 2% in good prognosis signature patients (HR 5.7; 95% CI 1.6-20; P = 0.007). In the updated series (N = 151; 60% poor vs. 40% good), the 10-year OS was 51 +/- 5% and 94 +/- 3% (HR 10.7; 95% CI 3.9-30; P < 0.01), respectively. The DMFP was 50 +/- 6% in poor and 86 +/- 5% in good prognosis signature patients (HR 5.5; 95% CI 2.5-12; P < 0.01). In multivariate analysis, the prognosis signature was a strong independent prognostic factor in both series, outperforming the clinicopathological risk indexes. CONCLUSION: The 70-gene prognosis signature is also an independent prognostic factor in node-negative breast cancer patients for women diagnosed in recent years.

The impact of preoperative MRI on breast-conserving surgery of invasive cancer: a comparative cohort study.

AIM: To assess whether preoperative contrast-enhanced magnetic resonance imaging (MRI) of the breast influences the rate of incomplete tumor excision. METHODS: In a cohort of 349 women with invasive breast cancer, patients eligible for breast-conserving therapy (BCT) on the basis of conventional imaging and palpation only (N = 176) were compared to those who had an additional preoperative MRI (N = 173). Multivariate analysis was applied to explore associations with incomplete tumor excision. RESULTS: MRI detected larger extent of breast cancer in 19 women (11.0%), leading to treatment change: mastectomy (8.7%) or wider excision (2.3%). Tumor excision was incomplete in 22/159 (13.8%) wide local excisions in the MRI group and in 35/180 (19.4%) in the non-MRI group (P = 0.17). Stratified to tumor type, incompletely excised infiltrating ductal carcinoma (IDC) was significantly associated with absence of MRI: 11/136 (8.1%) versus 2/126 (1.6%) (MRI present) (P = 0.02). No significant factors explained incomplete excision of other tumor types. CONCLUSION: Preoperative MRI did not significantly affect the overall rate of incomplete tumor excision, but it yielded significantly lower rate of incompletely excised IDC. The reduction of incomplete excisions after MRI was smaller than the rate of a prior treatment change incurred by MRI.

Refinement of breast cancer classification by molecular characterization of histological special types.

Most invasive breast cancers are classified as invasive ductal carcinoma not otherwise specified (IDC NOS), whereas about 25% are defined as histological 'special types'. These special-type breast cancers are categorized into at least 17 discrete pathological entities; however, whether these also constitute discrete molecular entities remains to be determined. Current therapy decision-making is increasingly governed by the molecular classification of breast cancer (luminal, basal-like, HER2+). The molecular classification is derived from mainly IDC NOS and it is unknown whether this classification applies to all histological subtypes. We aimed to refine the breast cancer classification systems by analysing a series of 11 histological special types [invasive lobular carcinoma (ILC), tubular, mucinous A, mucinous B, neuroendocrine, apocrine, IDC with osteoclastic giant cells, micropapillary, adenoid cystic, metaplastic, and medullary carcinoma] using immunohistochemistry and genome-wide gene expression profiling. Hierarchical clustering analysis confirmed that some histological special types constitute discrete entities, such as micropapillary carcinoma, but also revealed that others, including tubular and lobular carcinoma, are very similar at the transcriptome level. When classified by expression profiling, IDC NOS and ILC contain all molecular breast cancer types (ie luminal, basal-like, HER2+), whereas histological special-type cancers, apart from apocrine carcinoma, are homogeneous and only belong to one molecular subtype. Our analysis also revealed that some special types associated with a good prognosis, such as medullary and adenoid cystic carcinomas, display a poor prognosis basal-like transcriptome, providing strong circumstantial evidence that basal-like cancers constitute a heterogeneous group. Taken together, our results imply that the correct classification of breast cancers of special histological type will allow a more accurate prognostication of breast cancer patients and facilitate the identification of optimal therapeutic strategies.

Hormone replacement therapy, mammography screening and changing age-specific incidence rates of breast cancer: an ecological study comparing two European populations.

BACKGROUND: In 2003, for the first time, US breast cancer incidence rates have fallen. Experts argue whether this is due to the reduced uptake of screening mammography or to lower use of Hormone Replacement Therapy (HRT). This study aims to disentangle the respective impact of screening and HRT on age-incidence rates and histology of breast cancer, by comparing two populations with comparably high levels of screening mammography, but with different prevalence of HRT. METHODS: We included all invasive breast cancers recorded at the Geneva cancer registry (n = 4,909) and the Netherlands Cancer Registry (n = 152,428) between 1989-2003. We compared age-specific incidence rates and trends in histological subtyping between the two populations. RESULTS: Between 1989-1991, incidence rates increased with age in both populations. In 2001-2003, women aged 60-64 years showed highest incidence rates in Geneva, while in the Netherlands incidence rates continued to increase with age. The annual increase in ductal cancer incidence was similar in the Netherlands (2.3%) and Geneva (2.5%), but the annual increase in lobular cancer was sharper in Geneva (10%) than in the Netherlands (5%). CONCLUSION: The sharp differences in age distribution and histological subtyping of breast cancer between two European populations are not attributable to screening, since both populations have a high uptake of mammography screening. Since the prevalence of HRT use is very high in Geneva and rather low in the Netherlands, HRT may explain these discrepancies. However, other etiological factors and differences in histological assessment may also have played a role.

Immunohistochemical categorisation of ductal carcinoma in situ of the breast.

The aim of this study is to analyse whether immunohistochemistry (IHC) applying a broad set of markers could be used to categorise ductal carcinoma in situ (DCIS) of the breast in distinct subgroups corresponding to the recently defined molecular categories of invasive carcinoma. Immunohistochemistry of pure DCIS cases constructed in tissue arrays was performed with 16 markers (oestrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), Bcl-2, p53, Her2, insulin-like growth factor receptor, E-cadherin, epithelial membrane antigen (EMA), CA125, keratins 5/6, 14, 19, epidermal growth factor receptor, S100, and CD31). Results in 163 cases were analysed by unsupervised hierarchical clustering. Histological classification was performed by review of whole tissue sections and identified 36 well-, 55 intermediately, and 72 poorly differentiated DCISs. Unsupervised hierarchical cluster analysis categorised DCIS into two major groups that could be further subdivided into subgroups based on the expression of six markers (ER, PR, AR, Bcl-2, p53, and Her2). In the major predominantly ER/Bcl-2-positive (luminal) group, three subgroups (AR-positive (n=33), AR-negative (n=40), and mixed (n=34)) could be identified and included 34 well-differentiated DCISs. Within the major predominantly ER/Bcl-2-negative (nonluminal) group, a Her2-positive subgroup (n=34) was characterised by 31 poorly differentiated lesions. Eight triple-negative lesions, including one positive for keratin 5/6 and two positive for p53, were encountered. Intermediately differentiated DCIS shared a comparable IHC staining pattern with well-differentiated DCIS that was distinct from poorly differentiated DCIS (P<0.001). Ductal carcinoma in situ could be categorised by IHC into two major groups and five subgroups using six markers. Morphologically, intermediately differentiated DCIS seems to have more biological similarities with well-differentiated lesions as compared to poorly differentiated lesions.

The expression pattern of MUC1 (EMA) is related to tumour characteristics and clinical outcome of invasive ductal breast carcinoma.

AIMS: To clarify MUC1 patterns in invasive ductal breast carcinoma and to relate them to clinicopathological parameters, coexpression of other biological markers and prognosis. METHODS AND RESULTS: Samples from 243 consecutive patients with primary ductal carcinoma were incorporated into tissue microarrays (TMAs). Slides were stained for MUC1, oestrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53 and cyclin D1. Apical membrane MUC1 expression was associated with smaller tumours (P = 0.001), lower tumour grades (P < 0.001), PR positivity (P = 0.003) and increased overall survival (OS; P = 0.030). Diffuse cytoplasmic MUC1 expression was associated with cyclin D1 positivity (P = 0.009) and increased relapse-free survival (RFS; P = 0.034). Negativity for MUC1 was associated with ER negativity (P = 0.004), PR negativity (P = 0.001) and cyclin D1 negativity (P = 0.009). In stepwise multivariate analysis MUC1 negativity was an independent predictor of both RFS [hazard ratio (HR) 3.5, 95% confidence interval (CI) 1.5, 8.5; P = 0.005] and OS (HR 14.7, 95% CI 4.9, 44.1; P < 0.001). CONCLUSIONS: The expression pattern of MUC1 in invasive ductal breast carcinoma is related to tumour characteristics and clinical outcome. In addition, negative MUC1 expression is an independent risk factor for poor RFS and OS, besides 'classical' prognostic indicators.

The expression pattern of MUC1 (EMA) is related to tumour characteristics and clinical outcome in 'pure' ductal carcinoma in situ of the breast.

AIMS: To classify MUC1 according to five predefined expression patterns in ductal carcinoma in situ (DCIS) and related clinicopathological parameters, coexpression of other biological markers and prognosis. METHODS AND RESULTS: With a manual tissue arrayer, 92% (n = 80) of the 87 DCIS samples were successfully targeted. Immunohistochemistry was carried out for MUC1, oestrogen receptor (ER), progesterone receptor (PR), Her2/Neu, p53 and cyclin D1. Entire membrane expression was related to Her2/neu negativity (P =0.042). Apical membrane expression was associated with low grade (P = 0.027), Her2/neu negativity (P = 0.014) and PR positivity (P = 0.005). Focal cytoplasmic expression was related to high grade (P = 0.006). Diffuse cytoplasmic expression was associated with high grade (P = 0.004), large tumour size (P = 0.046), Her2/neu positivity (P =0.042) and cyclin D1 positivity (P = 0.002). On the basis of these analyses the four patterns were reclassified as membranous or cytoplasmic expression. On multivariate analysis, cytoplasmic MUC1 expression (hazard ratio 8.5, 95% confidence interval 1.0, 73.0; P = 0.04) was the only independent predictor of local recurrence. CONCLUSIONS: Four patterns of MUC1 expression are recognized in DCIS that suggest a relationship to functional differentiation and can be simplified into two types that are clinically relevant and could therefore be helpful in the distinction between different subgroups of DCIS.

Risk of invasion and axillary lymph node metastasis in ductal carcinoma in situ diagnosed by core-needle biopsy.

BACKGROUND: The aim of the study was to assess the risk of invasion and axillary lymph node metastasis in patients with ductal carcinoma in situ (DCIS) diagnosed by preoperative core-needle biopsy. The data were used to select criteria for patients in whom sentinel node (SN) biopsy might be indicated. METHODS: One hundred and seventy-one women with 172 DCIS lesions diagnosed by core-needle biopsy were analysed. Axillary staging was performed by SN biopsy, axillary node sampling, or level 1-2 axillary lymph node dissection. RESULTS: Invasive breast cancer was found in the surgical specimens from 45 tumours (26.2 per cent). Risk factors for invasion were a palpable lesion (odds ratio (OR) 2.95 (95 per cent confidence interval 1.20 to 7.26); P = 0.019), presence of a mass on mammography (OR 3.06 (1.43 to 6.56); P = 0.004), and intermediate (OR 5.81 (1.18 to 28.57); P = 0.030) or poorly differentiated (OR 5.46 (1.17 to 25.64); P = 0.031) tumour grade. Lymph node metastases were found in ten women with DCIS and invasion on final pathology. Factors associated with metastases were age 55 years or less (P = 0.030), invasion of 1.0 cm or more (P < 0.001) and the presence of vascular invasion (P = 0.001). CONCLUSION: SN biopsy should be considered in women with an initial diagnosis of DCIS on core-needle biopsy who are at risk for invasion; this includes women with a palpable lump, a mass on mammography, and intermediate or poor tumour grade.

The correlation of mammographic-and histologic patterns of breast cancers in BRCA1 gene mutation carriers, compared to age-matched sporadic controls.

Breast cancers in BRCA1 gene mutation carriers often have specific histologic features: grade III tumors with pushing margins. Our purpose was to compare the mammographic and histologic features of breast cancers in carriers with those in age-matched sporadic controls. The features of breast cancers in 27 BRCA1 carriers found during annual surveillance were compared to those in 107 age-matched sporadic controls. The carriers had no (classic) spiculated mammographic lesions, a high percentage of well-defined masses and hardly any masses with microcalcifications, whereas the controls had significantly fewer well-defined ones and only in 27% spiculated lesions on the mammogram. The well-defined mammographic tumors correlated in 83% of the carriers and in 70% of the controls with histologic circumscribed tumor margins. Spiculated mammographic lesions in the controls were in 90% grade I or II tumors. DCIS with or without infiltration was seen in 22% of the carriers and in 45% of the controls. In conclusion, breast cancers diagnosed in BRCA1 carriers do not have classic malignant mammographic features. A minority of the young sporadic controls show the classic malignant lesion on the mammogram. Both carriers and controls generally show a good correlation between their mammographic- and histologic tumor pattern.

The incidence and significance of micrometastases in lymph nodes of patients with ductal carcinoma in situ and T1a carcinoma of the breast.

AIM: To report the incidence and predictive value of positive axillary nodes in ductal carcinoma in situ (DCIS) and T1a carcinoma of the breast. METHODS: Cases from The Netherlands Cancer Institute were used to determine the incidence of lymph-node metastases. All consecutive patients with primary breast cancer that were treated between 1989 and 1998 and who had undergone axillary dissection were selected. Patients were identified with pure DCIS (n = 71), DCIS with small invasion (n = 12), invasive ductal/lobular carcinoma (IDC/ILC) < or =5 mm (n = 18) or tubular carcinoma < or =10 mm (n = 17). All archived lymph nodes of these patients were re-evaluated using immunohistochemistry (IHC). RESULTS: In DCIS the incidence increased from 1.4% with routine staining to 11% with IHC. For DCIS with small invasion it was 0 vs 27%, respectively. In IDC/ILC sized 2-5 mm the incidence rose from 6 to 12% and in tubular carcinoma < or =10 mm from 0 to 12%. All but one of the immunohistochemically detected metastases were isolated tumour cells (n = 9) or small (micro)metastases (n = 4). Maximally two nodes per patient were affected. None of the patients with positive lymph nodes died during follow-up (mean 102 months). CONCLUSIONS: Survival of our patients appeared not to be influenced by the finding of micrometastases in the lymph nodes by IHC. Immunohistochemistry of the sentinel node seems not contributive to further treatment in these patients.

Efficacy of high-dose alkylating chemotherapy in HER2/neu-negative breast cancer.

BACKGROUND: High-dose chemotherapy in the adjuvant treatment of breast cancer has been abandoned by many. PATIENTS AND METHODS: 885 patients with stage III primary breast cancer and four or more axillary lymph node metastases were randomised to receive either five courses of FEC (fluorouracil, epirubicin and cyclophosphamide) followed by radiation therapy and tamoxifen, or the same treatment but with high-dose alkylating chemotherapy (cyclophosphamide, thiotepa and carboplatin) replacing the fifth course of FEC. Of these patients, 621 had HER2/neu-negative disease, as determined by immunohistochemistry and chromogenic in situ hybridisation. RESULTS: At a median follow-up of 84 months, a trend for a better relapse-free survival was observed in the high-dose arm: (hazard ratio (HR) 0.84, P = 0.076, two-sided). The 621 patients with HER2/neu-negative disease benefited from high-dose therapy, while patients with HER2/neu-positive disease did not (test for interaction, P = 0.006). There was a marked relapse-free survival benefit for patients with HER2/neu-negative disease (71.5% versus 59.1%, 5 years after randomisation; HR 0.68, P = 0.002) and also a survival benefit (78.2% versus 71.0% at 5 years; HR 0.72, P = 0.02). CONCLUSIONS: The findings from this subgroup analysis provide additional evidence that HER2/neu-positive breast cancer is relatively resistant to alkylating agents. For HER2/neu-negative tumours, however, high-dose chemotherapy should remain the subject of clinical studies.

No common denominator for breast cancer lymph node metastasis.

The axillary lymph node status is the most powerful prognostic factor for breast cancer patients to date. The molecular mechanisms that control lymph node metastasis, however, remain poorly understood. To define patterns of genes or gene regulatory pathways that drive breast cancer lymph node metastasis, we compared the gene expression profiles of 15 primary breast carcinomas and their matching lymph node metastases using microarrays. In general, primary breast carcinomas and lymph node metastases do not differ at the transcriptional level by a common subset of genes. No classifier or single gene discriminating the group of primary tumours from those of the lymph node metastases could be identified. Also, in a series of 295 breast tumours, no classifier predicting lymph node metastasis could be developed. However, subtle differences in the expression of genes involved in extracellular-matrix organisation and growth factor signalling are detected in individual pairs of matching primary and metastatic tumours. Surprisingly, however, different sets of these genes are either up- or downregulated in lymph node metastases. Our data suggest that breast carcinomas do not use a shared gene set to accomplish lymph node metastasis.

Long-term prognosis of patients with local recurrence after conservative surgery and radiotherapy for early breast cancer.

We have studied the long-term prognosis of 266 patients considered to have isolated local recurrence in the breast following conservative surgery and radiotherapy for early breast cancer. The median follow-up of the patients still alive after diagnosis of local relapse was 11.2 years. At 10 years from the date of salvage treatment, the overall survival rate for the 226 patients with invasive local recurrence was 39% (95% CI, 32-46), the distant recurrence-free survival rate was 36% (95% CI, 29-42), and the local control rate (i.e., survival without subsequent local recurrence or local progression) was 68% (95% CI, 62-75). Among patients with a local recurrence at or near the original tumour site a better distant disease-free survival was observed for patients with recurrences measuring 1cm or less, compared to those with larger recurrences. This suggests, though does not prove, that early detection of local recurrence can improve the treatment outcome but might as well point towards a different biologic behaviour, facilitating early detection.

Changing patterns in diagnosis and treatment of ductal carcinoma in situ of the breast.

BACKGROUND: The increased incidence of ductal carcinoma in situ (DCIS) of the breast and the emergence of new diagnostic and therapeutic tools like mammographic screening, stereotactic core biopsy and reconstructive surgery prompted us to investigate how these developments influenced diagnosis and treatment. METHODS: Clinical and pathological characteristics of 403 patients with DCIS consecutively treated at The Netherlands Cancer Institute between 1986 and 2002 were evaluated and the effect of introduction of mammographic screening, stereotactic core biopsy and reconstruction on diagnosis and treatment was studied. RESULTS: Following the nationwide introduction of mammographic screening the number of non-symptomatic DCIS increased from 47 to 77%. Introduction of stereotactic core biopsy resulted in a rise of one-step procedures from 26 to 52%. Mastectomy rate did not change over time: 59% overall. However, reconstruction rate increased from 17 to 39%. CONCLUSION: This study shows a steep rise in diagnosis of non-symptomatic DCIS after introduction of screening. Further, the introduction of pre-operative diagnosis by stereotactic core biopsy resulted in a decrease of multiple surgical procedures. Mastectomy, with increasing application of breast reconstructions, remains an important treatment modality in the management of DCIS despite advancements in detection and diagnosis.

Expression of oestrogen receptor and loss of E-cadherin are diagnostic for gastric metastasis of breast carcinoma.

AIMS: To investigate whether immunohistochemical staining for oestrogen receptor (ER)alpha, progesterone receptor (PgR) and E-cadherin might be useful to differentiate between metastatic breast carcinoma and primary gastric carcinoma. METHODS: Gastric biopsies of 75 patients containing adenocarcinoma were stained for ERalpha, PgR and E-cadherin. Included were: Group A, 28 patients with primary gastric cancer; Group B, 28 patients with an adenocarcinoma containing gastric biopsy and a clinical diagnosis of metastatic breast carcinoma; Group C, all consecutive patients with a positive gastric biopsy in 2001 (n = 19) without clinical history of breast carcinoma and not followed by gastric resection (control group). RESULTS: All ERalpha+ or PgR+ carcinomas (n = 20) were of patients with a previous or concurrent history of breast carcinoma: 19 in group B, one in group C. In addition, absence of E-cadherin staining was seen significantly more often in patients with metastatic breast carcinoma than in patients with primary gastric cancer (P < 0.001). CONCLUSION: Positive immunohistochemical staining for ERalpha or PgR of an adenocarcinoma in a gastric biopsy is diagnostic for metastatic breast carcinoma. Moreover, when carcinoma in a gastric biopsy is negative for E-cadherin staining, metastatic breast carcinoma should be considered.

Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology.

AIMS: To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision. METHODS: A questionnaire was circulated by mail or electronically by the authors in their respective countries. Replies from pathology units dealing with SLN specimens were evaluated further. RESULTS: Of the 382 respondents, 240 European pathology units were dealing with SLN specimens. Sixty per cent of these units carried out intraoperative assessment, most commonly consisting of frozen sections. Most units slice larger SLNs into pieces and only 12% assess these slices on a single haematoxylin and eosin (HE) stained slide. Seventy one per cent of the units routinely use immunohistochemistry in all cases negative by HE. The terms micrometastasis, submicrometastasis, and isolated tumour cells (ITCs) are used in 93%, 22%, and 71% of units, respectively, but have a rather heterogeneous interpretation. Molecular SLN staging was reported by only 10 units (4%). Most institutions have their own guidelines for SLN processing, but some countries also have well recognised national guidelines. CONCLUSIONS: Pathological examination of SLNs throughout Europe varies considerably and is not standardised. The European guidelines should focus on standardising examination. They should recommend techniques that identify metastases > 2 mm as a minimum standard. Uniform reporting of additional findings may also be important, because micrometastases and ITCs may in the future be shown to have clinical relevance.

Consistency of staining and reporting of oestrogen receptor immunocytochemistry within the European Union--an inter-laboratory study.

To assess the variability of oestrogen receptor (ER) testing using immunocytochemistry, centrally stained and unstained slides from breast cancers were circulated to the members of the European Working Group for Breast Screening Pathology, who were asked to report on both slides. The results showed that there was almost complete concordance among readers (kappa=0.95) in ER-negative tumours on the stained slide and excellent concordance among readers (kappa=0.82) on the slides stained in each individual laboratory. Tumours showing strong positivity were reasonably well assessed (kappa=0.57 and 0.4, respectively), but there was less concordance in tumours with moderate and low levels of ER, especially when these were heterogeneous in their staining. Because of the variation, the Working Group recommends that laboratories performing these stains should take part in a external quality assurance scheme for immunocytochemistry, should include a tumour with low ER levels as a weak positive control and should audit the percentage positive tumours in their laboratory against the accepted norms annually. The Quick score method of receptor assessment may also have too many categories for good concordance, and grouping of these into fewer categories may remove some of the variation among laboratories.