Cancer care Ontario guideline recommendations for hormone receptor testing in breast cancer.
Topic: Hormone Therapy

Hormone receptor testing (oestrogen and progesterone) in breast cancer at the time of primary diagnosis is used to guide treatment decisions. Accurate and standardised testing methods are critical to ensure the proper classification of the patient's hormone receptor status. Recommendations were developed to improve the quality and accuracy of hormone receptor testing based on a systematic review conducted jointly by the American Society of Clinical Oncology/College of American Pathologists and Cancer Care Ontario's Program in Evidence-Based Care. Evidence-based recommendations were formulated to set standards for optimising immunohistochemistry in assessing hormone receptor status, as well as assuring quality and proficiency between and within laboratories. A formal external review was conducted to validate the relevance of these recommendations. It is anticipated that widespread adoption of these guidelines will further improve the accuracy of hormone receptor testing in Canada.

PICO
Population: Patients with breast cancer undergoing hormone receptor testing at the time of primary diagnosis
Intervention: Implementation of Cancer Care Ontario guideline recommendations for hormone receptor testing
Comparison: Standard hormone receptor testing methods without following the guidelines
Outcomes: Improved accuracy and quality of hormone receptor testing in breast cancer patients

Included studies:
title: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer.
abstract: The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor-positive tumors is poorly defined by clinical and histopathological measures.
title: Immunohistochemical quantitation of oestrogen receptors and proliferative activity in oestrogen receptor positive breast cancer.
abstract: To evaluate the effect of the duration of formalin fixation and of tumour heterogeneity on quantitative estimates of oestrogen receptor content (oestrogen receptor index) and proliferative activity (MIB-1 index) in breast cancer.
title: Correlation of immunocytochemical and immunohistochemical determination of estrogen and progesterone receptors in breast cancer.
abstract: To evaluate prospectively the degree of correlation between immunocytochemical (ICC) and immunohistochemical (IHC) determination of estrogen (ER) and progesterone receptors (PR) in breast cancer.
title: Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue.
abstract: Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy.
title: Association between standard clinical and pathologic characteristics and the 21-gene recurrence score in breast cancer patients: a population-based study.
abstract: The 21-gene recurrence score (RS) assay has been reported to accurately predict the risk of disease recurrence and chemotherapy benefit in women with estrogen receptor (ER)-positive, lymph node (LN)-negative breast cancer who are treated with tamoxifen. To the authors' knowledge, the association between the RS and clinicopathologic characteristics has been studied in randomized and case-control trials, but not in the general population.
title: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.
abstract: To develop a guideline to improve the accuracy of immunohistochemical (IHC) estrogen receptor (ER) and progesterone receptor (PgR) testing in breast cancer and the utility of these receptors as predictive markers.
title: Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study.
abstract: Progesterone receptor is a surrogate marker of estrogen receptor activity in breast cancer and its utility in helping predict clinical outcome has been established using biochemical assays. However, most laboratories worldwide have switched to immunohistochemistry to assess progesterone receptor, but unfortunately no validated immunohistochemical assay exists for progesterone receptor. The purpose of this study was to develop and validate an immunohistochemical assay for progesterone receptor in breast cancer. The assay was based on monoclonal antibody 1294 (DakoCytomation) and slides were scored microscopically using the 'Allred score' on a scale of 0-8. The assay was compared to ligand-binding assay in 1235 breast cancers, and a subset (n=362) that received only hormonal therapy was used to define a cutoff for progesterone receptor-positive. Clinical utility was validated in an independent set of samples (n=423) from a clinical trial randomizing premenopausal breast cancer patients to tamoxifen+oophorectomy vs observation following surgery. A cutoff of >2 (corresponding to >1% positive cells) dichotomized patients with significantly better or worse clinical outcome (P=0.0014). Progesterone receptor by immunohistochemistry provided significantly better results than progesterone receptor by ligand-binding assay in predicting clinical outcome. In the clinical trial, a positive result in univariate analyses was associated with significantly improved disease-free and overall survival both in untreated (hazard ratios/P=0.656/0.060 and 0.479/0.005, respectively) and hormonally treated patients (hazard ratios/P=0.529/0.017 and 0.451/0.007, respectively). Positive progesterone receptor remained significant for improved disease-free and overall survival (hazard ratios/P=0.666/0.038 and 0.549/0.007, respectively) in multivariate analyses including the standard variables of tumor size, nodal status, treatment, histological grade, and HER-2/neu status. Estrogen and progesterone receptors are codependent variables and progesterone receptor was a weaker predictor of response to endocrine therapy than estrogen receptor when both were included in multivariate analysis. This is the first comprehensive study assessing the clinical usefulness of progesterone receptor by immunohistochemistry in archival tissue in breast cancer. Progesterone receptor assessed by immunohistochemistry provides useful information about clinical outcome and it is better than progesterone receptor measured by ligand-binding assay.
title: High progesterone receptor expression correlates to the effect of adjuvant tamoxifen in premenopausal breast cancer patients.
abstract: Tamoxifen has long been the drug of choice in adjuvant endocrine therapy of steroid hormone receptor-positive breast cancer, and it still remains important due to its well-documented beneficial effect. Hormone receptor status is often reported as "positive" or "negative" using 10% positive nuclei as a cutoff. In this study, we aimed to assess whether a further subclassification of hormone receptor status could enhance the treatment predictive value.
title: Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: a pilot study.
abstract: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment.
title: Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients.
abstract: Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.
title: Estrogen and progesterone hormone receptor status in breast carcinoma: comparison of immunocytochemistry and immunohistochemistry.
abstract: Estrogen receptors (ER) and progesterone receptors (PR) play a significant role in the prognosis of breast cancer. For preoperative chemotherapy in locally advanced lesions, trucut biopsy is used to localize the ER and PR receptors by immunohistochemistry. Immunocytochemistry can be a better alternative to immunohistochemistry as it better fixes cells.
title: Wet autoclave pretreatment for immunohistochemical demonstration of oestrogen receptors in routinely processed breast carcinoma tissue.
abstract: The immunohistochemical demonstration of oestrogen receptor (OR) was performed on 32 randomly selected and routinely processed breast carcinomas after wet autoclave pretreatment of sections. The autoclave method was compared to the OR status found on frozen sections as well as to alternative pretreatment methods such as enzymatic predigestion and microwave irradiation. Using four different monoclonal antibody clones (H222, LH1, CC4-5, 1D5.26), the OR status was evaluated for each of the various pretreatment methods applied. All cases with a high OR content on frozen sections (n = 11) also showed a high OR status on wet autoclave-pretreated paraffin tissues using antibody clones 1D5.26 and CC4-5; in cases with low OR content on frozen sections, no false-negative cases were recorded using only the antibody 1D5.26 neither after wet autoclave nor microwave pretreatment. In addition, with this antibody, OR was detectable after autoclave pretreatment in two cases which were considered to be OR-negative even on frozen sections. When the primary antibody was omitted, no false-positive cases were observed after wet autoclave pretreatment. Thus, in our hands, wet autoclave pretreatment, in combination with the antibody 1D5.26, offers a highly sensitive method for the immunohistochemical demonstration of OR in routinely formalin-fixed, paraffin-embedded sections of breast carcinomas.
title: Preoperative profiling of symptomatic breast cancer by diagnostic core biopsy.
abstract: Precise preoperative profiling of breast tumors could facilitate fuller consideration of (neo)adjuvant therapies.
title: Semi-quantitative scoring of potentially predictive markers for endocrine treatment of breast cancer: a comparison between whole sections and tissue microarrays.
abstract: To assess whether immunohistochemically stained tissue microarrays (TMA) of 2 mm cores from paraffin embedded tumour tissue may replace whole sections in semi-quantitative evaluation of selected potential markers for endocrine treatment.
title: Objective measurement of breast cancer oestrogen receptor status through digital image analysis.
abstract: The authors have previously described quantitative, computer-assisted analysis of oestrogen receptor status in immuno-histochemically stained sections in patients with primary breast cancer. The aim of this study was to validate the aforementioned system against the commonly used methods of assessing oestrogen receptor status.
title: Quantitative immunohistochemical assay for hormonal receptors: technical aspects and biological significance.
abstract: Over 250 breast carcinomas were studied in order to establish whether or not quantitative immunohistochemical assays (ICA) for estrogen and progesterone receptors (ER and PR) with computer-assisted image analysis could favorably compare with standard cytosolic assays. Initially, variable antigenic preservation secondary to improper tissue fixation and processing led to irregular receptor preservation and unevenly stained areas indistinguishable from true intratumor antigenic heterogeneity. As a direct consequence of the field selections chosen for analysis, assay reproducibility was less than optimal. Proper tissue fixation and handling eliminated most of the irregular staining; selection of fields to analyze became less cumbersome and more reproducible. Differences in staining intensity due to minimal variations in the ICA also resulted in difficult reproducibility. Standardizing the technique and using an automatic stainer notably eliminated that problem. The second and equally important question was to establish if quantitative ER-ICA had relevance as a predictor for prognosis. The Kaplan-Meier product limit estimator for quantitated ER values and Cox regression for risk of mortality and disease progression were performed. The results obtained discriminated high- and low-risk groups for overall survival (p = 0.016) better than the dextran-coated charcoal assay. Elimination of two major obstacles and proof of the predictive value of quantitative ICA has transformed the assay into a valid alternative to cytosolic methods; however, before that takes place it is critical to establish standard procedures for both ICA and quantitation so interlaboratory variability is reduced to a minimum.
title: Immunohistochemical assessment of oestrogen and progesterone receptors: correlations with the DCC method and clinical outcome in primary breast cancer patients.
abstract: Two different methods to determine steroid receptors were analysed with respect to their ability to estimate prognosis in primary breast cancer patients. The immunohistochemical assay (IHA) was compared with the dextran-coated charcoal (DCC) method of receptor determination. A random sample of 281 patients with invasive ductal carcinoma was drawn from 841 consecutive patients with primary breast carcinoma treated at Odense University Hospital between 1 January 1980 and 31 December 1990. Receptor determination by the DCC method had been carried out previously in 164 patients for the oestrogen receptor and in 132 patients for the progesterone receptor. The former group was reassessed by IHA with the antibody ER1D5, and the latter with the antibody PgR-ICA. The median follow-up time was 8.3 years (range 2.9-12.9 years). A cutoff of zero was used for the DCC method. Immunohistochemical results were quantified by counting in systematically random sampled fields of vision and values above zero were considered to be positive. Overall agreement of positive and negative cases was 86% for the oestrogen receptor and 83% for the progesterone receptor. Although the study included a limited number of patients, receptor positive cases fared better than negative cases in all situations. Investigation of the prognostic power revealed that classification based on IHA allowed better discrimination of patients than classification based on the DCC method. The reason for this difference might be because distinction between benign and malignant tissue is possible using the IHAmethod. Thus, IHAresults appear to be more clinically relevant.
title: Prognostic and predictive value of centrally reviewed expression of estrogen and progesterone receptors in a randomized trial comparing letrozole and tamoxifen adjuvant therapy for postmenopausal early breast cancer: BIG 1-98.
abstract: To evaluate locally versus centrally assessed estrogen (ER) and progesterone (PgR) receptor status and the impact of PgR on letrozole adjuvant therapy compared with tamoxifen in postmenopausal women with early breast cancer.
title: A comparison of prognostic tumor markers obtained on image-guided breast biopsies and final surgical specimens.
abstract: This study was initiated to determine whether tumor markers obtained on image-guided breast biopsy specimens provide accurate prognostic information for women with invasive breast cancer.
title: A simple index using video image analysis to predict disease outcome in primary breast cancer.
abstract: Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour-suppressor protein and tumour cell proliferation (MIB-1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow-up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Cox's univariate analysis indicated that tumour size, number of cancer-involved nodes, MIB-1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse-free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB-1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut-point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut-point of 30% positive nuclear area and MIB-1 cut-point of 10% positive nuclear area. Kaplan-Meier analysis of this image-based prognostic index identified 4 risk groups with predicted 5-year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient.
title: Oestrogen receptor analysis of paraffin sections and cytosol samples of primary breast cancer in relation to outcome after adjuvant tamoxifen treatment. The South Sweden Breast Cancer Group.
abstract: The aim of the present study was to compare oestrogen receptor (ER) analysis results obtained in cytosols of frozen breast cancer tissue (using biochemical assay) with those obtained in paraffin-embedded tissue (using immunoperoxidase staining with monoclonal antibodies (DAKO-ER, 1D5), and an ER positivity cut-off level of >10% stained nuclei). In 86% (84/98) of the samples the same ER status (28 negative and 56 positive) was obtained with both procedures. In eight cases, the paraffin section was ER positive but the corresponding cytosol sample ER negative, whereas six cases showed the opposite pattern. The ER positive subgroup manifested better outcome after adjuvant treatment than the ER negative subgroup (p = 0.003 (cytosol), and p = 0.004 (paraffin)). As compared with the percentage of stained nuclei, staining intensity yielded no additional information. Although the results of ER analysis of paraffin-embedded material seem promising, it is too early to prefer it to frozen tissue, though this would be useful when no frozen tissue is available.
title: Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer.
abstract: The 21-gene recurrence score (RS) assay quantifies the likelihood of distant recurrence in women with estrogen receptor-positive, lymph node-negative breast cancer treated with adjuvant tamoxifen. The relationship between the RS and chemotherapy benefit is not known.
title: Cytological evaluation of biological prognostic markers from primary breast carcinomas.
abstract: This study was undertaken to evaluate our ability to detect multiple molecular markers of prognosis and response to treatment in fine needle aspirates (FNA) from patients with primary breast carcinomas. 147 patients with operable primary breast carcinomas who had been recruited to a randomized trial of primary medical therapy (PMT) versus adjuvant chemoendocrine therapy were analysed. FNAs were taken prior to therapy and from this multiple slides were produced using cytospin cytocentrifugation and stored at -80 degrees C for subsequent immunocytochemical analysis (ICA). ICA was performed for oestrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, and Bcl-2. Part of the aspirate was snap frozen and used for flow cytometric analysis of ploidy and S-phase fraction (SPF). In a subgroup of 50 patients who had surgery prior to systemic therapy, as well as FNAs, sections were also taken from paraffin-embedded blocks and stained by ICA for ER, PgR and p53 for validation. In these patients ER was additionally measured by enzyme immunoassay (EIA) from frozen tissue taken at surgery. ER, PgR, p53, Bcl-2, and Ki67 were successfully detected by ICA while ploidy and SPF were successfully measured by flow cytometry from FNA material. The percentage positive values obtained were reasonable and as follows: 74% for ER, 70% for PgR, 36% for p53, 80% for Bcl-2,68% of tumours were aneuploid and 32% diploid. Significant relationships between these measurements were observed in accordance with expectations. The concordance for ER, PgR, and p53 from FNA when compared to ICA of matching histological sections was 91.5%, 75.5%, and 75% respectively. For ER the concordance between measurement by ICA of cytological and histological samples and by EIA of frozen tissue was 82.5% and 84% respectively. These results indicate that multiple molecular markers can be adequately tested on cytological preparations from primary breast tumours. These markers can be used to determine prognosis and predict response to PMT.
title: Estrogen receptor in primary breast cancer estimated in paraffin-embedded tissue. A study of its usefulness compared to dextran-coated charcoal assay.
abstract: Estrogen receptor (ER) was estimated immunohistochemically in formalin-fixed and paraffin-embedded tissue from the primary breast cancer in 349 postmenopausal patients with a high risk of recurrence and compared with the results of dextran-coated charcoal assay. There was a highly significant correlation between the ER classification obtained by the two methods (p less than 10(-6)). Patients ER positive according to immunohistochemical estimate had a significantly longer disease-free survival (p less than 0.001) and survival (p less than 0.001) than ER negative patients. The DCC assay showed an advantage of ER positive patients of the same magnitude. The patients, who were followed for a median of 86 months, were a subset of 1,700 patients participating in the Danish Breast Cancer Cooperative Group's randomized trial of adjuvant tamoxifen (TAM) treatment. In the presently analyzed subset of patients there were no statistically significant difference in disease-free survival (p = 0.52) or survival (p = 0.54) between patients who received adjuvant TAM and the controls. The same was true for receptor-defined subgroups regardless if the ER receptor was estimated in paraffin-embedded tissue or by the dextran-coated charcoal method. The analyzed subset might have been too small for demonstrating a positive effect of adjuvant TAM treatment.