© The Author 2006. Published by Oxford University Press.
ARTICLE |
Re-evaluating Adjuvant Breast Cancer Trials: Assessing Hormone Receptor Status by Immunohistochemical Versus Extraction Assays
For the International Breast Cancer Study Group (IBCSG)
Affiliations of authors: IBCSG Statistical Center, Dana-Farber Cancer Institute, Boston, MA (MMR, RDG, KNP); Harvard School of Public Health, Boston, MA (MMR, RDG); Department of Pathology, European Institute of Oncology, University of Milan, Milan, Italy (GV, MGM); Department of Pathology, University of Bari, Bari, Italy (EM); The Institute of Oncology, Ljubljana, Slovenia (RG); Division of Pathology, Centro di Riferimento Oncologico, Aviano, Italy (AC); Department of Pathology, Istituto Nazionale Tumori, Milan, Italy (AC); Melbourne Pathology, Collingwood, Victoria, Australia (BB); Department of Pathology, Göteborg/Sahlgrenska University Hospital, Göteborg, Sweden (MS); Department of Anatomical Pathology, University of Cape Town, and Groote Schuur Hospital, Cape Town, South Africa (GL); Bern Institut für Pathologie der Universität Bern, Swiss Group for Clinical Cancer Research, Bern, Switzerland (LM); Australian New Zealand Breast Cancer Trials Group, University of Newcastle, Newcastle, and Anatomical Pathology, Hunter Area Pathology Service, John Hunter Hospital, New Lambton Heights, New South Wales, Australia (SB); Oncologia Medica-Spedali Civili, Brescia, Italy (PG); Frontier Science and Technology Research Foundation, Boston, MA (RDG, KNP); IBCSG Coordinating Center, Bern, Switzerland (MCG); The Cancer Council Australia, Australian New Zealand Breast Cancer Trials Group and School of Public Health, University of Sydney, Sydney, Australia (ASC); Oncology Institute of Southern Switzerland, Bellinzona, Lugano, Mendrisio, Switzerland (AG); European Institute of Oncology, Milan, Italy (AG); Division of Cancer Sciences and Molecular Pathology, Western Infirmary, University of Glasgow, UK (BG).
Correspondence to: Meredith M. Regan, ScD, IBCSG Statistical Center, Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115 (e-mail: mregan{at}jimmy.harvard.edu).
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ABSTRACT |
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 TopNotes Abstract IntroductionPatients and methodsResultsDiscussionAppendixReferences |
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Background: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%–30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node–negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)–negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. Methods: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. Results: Concordance of hormone receptor status determined by both assays ranged from 74% (
= 0.48) for PgR among postmenopausal patients to 88% ( = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. Conclusions: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed—immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.
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INTRODUCTION |
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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The levels of estrogen receptor (ER) and progesterone receptor (PgR) in the primary tumor of a patient with early-stage invasive breast cancer are powerful predictors of that patient's response to adjuvant endocrine therapies and chemosensitivity of the primary tumor (1–3). The 2005 International Expert Consensus on the Primary Therapy of Early Breast Cancer recognized that endocrine responsiveness of the primary tumor should be the first consideration for selecting adjuvant systemic therapies (4).
Early studies establishing the predictive and prognostic value of steroid hormone receptors measured levels of ER and PgR in tumor cytosols with extraction assays (5). Over the past 30 years, when the importance of evaluating steroid hormone receptor levels was being established and when the need to assess the value of adjuvant therapies according to receptor level in clinical trials was being recognized, the standard method of determining hormone receptors changed from extraction assays to immunohistochemical assays. This change was brought about by the development of immunohistochemical assays in general, followed by the wide commercial availability of ER antibodies and PgR antibodies in the 1990s. Immunohistochemical assays are less labor intensive and less expensive than extraction assays. They are also suitable for small tumors, allow morphologic correlation, and can be used on retrospectively collected specimens.
When hormone receptor status of tumor tissue determined by immunohistochemical assay has been compared with that determined by extraction assays (6–10), discordance of 10%–30% has been found for both ER and PgR status. There has been remarkable consistency in this discordance across the literature, especially when one considers that previous studies were predominantly cohort studies, in which patient cohorts varied widely across and within studies (e.g., by disease stage and characteristics, treatments received, age, and menopausal status), and that assay methods varied widely (e.g., type of tissue and tissue preparation, type of extraction assays, antibodies used in the immunohistochemical assay, and scoring and cut points used to classify tumors as negative or positive) (6–14).
Hormone receptor status determined by immunohistochemical assay has also been found consistently to have similar or superior predictive and prognostic value than that determined by extraction assays (6,7,14–16). It is important to note, however, that many of these studies were limited by short follow-up and that the predictive and prognostic value was confounded by the inclusion in the cohort of women treated with a range of therapies (11).
We therefore decided to use specimens from well-defined randomized clinical trial cohorts to investigate the extent to which the method of assessing hormone receptor status may affect trial conclusions. The clinical trials that we selected were two International Breast Cancer Study Group (IBCSG) randomized clinical trials of adjuvant therapy in pre- and perimenopausal patients (Trial VIII) and in postmenopausal patients (Trial IX) with lymph node–negative breast cancer. In such patients, the receipt of chemotherapy in addition to endocrine therapy is highly dependent on hormone receptor status. IBCSG Trial VIII concluded that premenopausal women with ER-negative (i.e., endocrine nonresponsive), lymph node–negative breast cancer should receive adjuvant chemotherapy, whereas for patients with ER-positive (i.e., endocrine responsive) disease, the combination of chemotherapy with ovarian function suppression or other endocrine agents and the use of endocrine therapy alone should be studied further (17). IBCSG Trial IX concluded that postmenopausal patients with lymph node–negative breast cancer benefited substantially from adjuvant chemotherapy if their cancer was ER negative (i.e., endocrine nonresponsive); in contrast, if their cancer was ER positive (i.e., endocrine responsive), they obtained no benefit from the combination treatment compared with endocrine therapy alone (18).
We investigated the concordance of hormone receptor status as determined by extraction assays originally in local pathology offices when patients were randomly assigned to treatment and as determined, more recently, by immunohistochemical assay in the IBCSG Central Pathology Laboratory. We examined the extent to which the assessment of treatment responsiveness in the two randomized trials differed when ER status was determined by immunohistochemical assay and by extraction assays. We further examined the assessment of treatment responsiveness according to PgR status, which was not considered in the primary trial analyses, because, in particular, we wanted to clarify whether PgR status as determined by either assay could predict treatment responsiveness. PgR requires a more technically difficult extraction assay than that for ER, and the PgR level in the primary tumor has been discounted by some as not useful in clinical practice (19), although it has been endorsed by others as supplementing information on ER level (20–22).
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PATIENTS AND METHODS |
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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Trial Cohorts
The designs of IBCSG Trials VIII (17) and IX (18) have been described. Briefly, Trial VIII enrolled pre- and perimenopausal patients with lymph node–negative breast cancer. The trial evaluated whether sequential treatment with six 28-day courses of classical chemotherapy with cyclophosphamide, methotrexate, and fluorouracil (CMF) followed by 18 monthly subcutaneous implants of the gonadotropin-releasing hormone analog goserelin was statistically significantly associated with longer disease-free survival than six 28-day courses of classical CMF chemotherapy alone or 24 monthly implants of goserelin alone. From March 1, 1990, through October 1, 1999, a total of 1063 assessable patients were randomly assigned to treatment. Trial IX enrolled postmenopausal patients with lymph node–negative breast cancer and evaluated whether sequential treatment with three 28-day courses of classical CMF chemotherapy followed by tamoxifen for 57 months was associated with statistically significantly longer disease-free survival than tamoxifen alone for 60 months. From October 1, 1988, through August 1, 1999, a total of 1669 eligible and assessable patients were randomly assigned to treatment. Previously reported trial results were presented according to CONSORT guidelines and included participant flow diagrams (17,18). Institutional review boards reviewed and approved the protocols, and informed consent was required according to the criteria established within the individual countries.
In both trials, patients with ER-positive, ER-negative, or ER-unknown tumors (ER-unknown status was allowed only if ER determination was not possible because of the lack of tumor material) were eligible until August 1, 1998. At that time, protocol amendments restricted enrollment to patients with ER-positive tumors because other trials had found that, for patients with ER-negative tumors, tamoxifen was not effective and ovarian ablation also might not be effective (23,24). More than 94% of patients in these two trials were randomly assigned to treatment before release of the amendments. Randomization was stratified according to ER status and Trial IX was statistically powered for subset analyses according to ER status (the entire trial cohort had 80% power to detect a relative reduction in relapse risk of 33% for the ER-positive stratum and 50% for the ER-negative stratum).
Pathology Methods
Before randomization in both trials, concentrations of steroid hormone receptor in the primary tumors were determined by local pathologists with the use of standard assay methods (25,26). At the trials' outset, ER status was determined by extraction assays, including a sucrose-gradient, dextran-coated charcoal assay or an enzyme-linked immunosorbent assay (ELISA); determination of ER status by immunohistochemical assay was allowed later in the study. A case report form collected data about all available quantitative and qualitative results from steroid hormone receptor assays. In Trials VIII and IX, 65% and 71% of patients, respectively, had their hormone receptor levels determined by an extraction assay. The assay method used was dextran-coated charcoal assay or ELISA in approximately 55% and 45% of these patients, respectively.
More than 80% of patients randomly assigned to treatment in Trials VIII and IX had archival tumor material available for immunohistochemical hormone receptor evaluation. Retrospective tissue collection was carried out in accordance with institutional guidelines and national laws. Immunohistochemical assays of the expression of ER and PgR in the primary tumors were carried out at the IBCSG Central Pathology Laboratory in Milan, Italy, in a blinded manner, as previously described (27). Tumor specimens of 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and PgR levels before patients were randomly assigned to treatment were re-evaluated with an immunohistochemical assay in the IBCSG Central Pathology Laboratory.
To assess hormone receptor levels in archival formalin-fixed and paraffin-embedded tumor tissue specimens, sections were dewaxed, pretreated with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase activity, and then treated with 0.001 M EDTA (pH 8.0) at 99 °C for 20 minutes to retrieve antigenicity. Sections were then incubated with specific primary mouse monoclonal antibodies against human ER (clone 1D5, 1 : 100 dilution) or human PgR (clone 1A6, 1 : 800 dilution) (both obtained from Dako, Glostrup, Denmark) for 30 minutes at room temperature by use of an automatic immunostainer (Autostainer, Dako). Antibody–antigen complexes were subsequently visualized with a high-sensitivity detection kit (EnVision+ System-HRP; Dako) according to the manufacturer's instructions. Peroxidase activity was visualized as a brown–black product by use of 3,3'-diaminobenzidine and copper sulfate (Sigma Chemical Co, St Louis, MO) as the chromogen.
According to the recommendations for all IBCSG trials, we evaluated immunohistochemical results by the following protocol. 1) Tissue was checked for expected immunostaining of non-neoplastic breast tissue (intense nuclear staining of at least a minor percentage of luminal epithelial cells) and lack of immunoreactivity of myoepithelial, stromal, and inflammatory (if any) cells. 2) The staining pattern of the neoplastic component was evaluated at low or intermediate magnification (x100 to x250) that took into account any biologically significant heterogeneity of staining in different parts of the invasive tumor. Staining of the intraductal (or in situ) component was not considered. 3) At higher magnification (x400), any definite nuclear localization of the immunostaining was assessed, without considering as specific any membrane or cytoplasmic staining. This nonspecific staining may appear on occasion (especially when apocrine or squamous metaplasia is present), and it does not interfere with the specific nuclear staining of the hormone-responsive neoplastic cells. 4) For tumors with homogeneous staining throughout, at least 10 high-power fields (containing a minimum of 2000 invasive tumor cells) were randomly selected, and the number of cells showing nuclear immunostaining (irrespective of the staining intensity) was counted and compared with the total number of neoplastic cells. The results were recorded as an overall percentage. For tumors with biologically significant staining heterogeneity, 10 high-power fields were selected to mirror the degree of staining heterogeneity. For example, if only 20% of the invasive tumor area showed diffuse nuclear staining and the remaining tumor had only occasional (or no) immunoreactive cells, then only two high-power fields were counted in the former area and eight high-power fields were counted in the latter area. In tissues with about 10% immunoreactive cells, additional fields were counted and/or another pathologist checked the results.
Statistical Considerations
Extraction assay values (expressed as femtomoles per milligram of cytosol protein) and immunohistochemistry values (expressed as the percentage of immunoreactive cells) were initially categorized as follows: 0 = none; 1–9 = low; 10 or more = high, with units corresponding to the assay used. The choice of cut points for the extraction assays reflected the standard practice: a value of 10 fmol/mg of cytosol protein or more was used to indicate a hormone receptor–positive status and a value of less than 10 fmol/mg of cytosol protein was used to indicate a hormone receptor–negative status. The choice of cut point for the immunohistochemical assay also reflected the frequent practice: 10% immunoreactive cells or more were used to indicate a hormone receptor–positive status and less than 10% immunoreactive cells were used to indicate a hormone receptor–negative status, as well as evidence that the presence of any immunoreactive cells appeared to indicate the endocrine responsiveness of the tumor (11). Thus for extraction assays, our dichotomization was negative (<10 fmol/mg of cytosol protein) and positive (
10 fmol/mg of cytosol protein). For immunohistochemical assays, our dichotomization was absent (0% stained cells) and present (>0% stained cells) or was negative (<10% stained cells) and positive (10% stained cells).
Concordance denoted the number or percentage of tumors with the same classification by the two assay methods. Agreement denoted the chance-corrected concordance as measured by a statistic, which was weighted (w) for ordinal hormone receptor status variables and unweighted for dichotomous variables; 95% confidence intervals (CIs) were also calculated.
The primary outcome in both trials was disease-free survival, defined as the length of time from the date of randomization to any recurrent disease (including ipsilateral breast recurrence), the appearance of a second primary cancer (including contralateral breast cancer), or death, whichever occurred first. Hazard ratios (HRs) of recurrence or death (with corresponding 95% confidence intervals) and log-rank tests were reported for comparisons of disease-free survival between treatment arms according to hormone receptor status. Cox proportional hazards regression models were used to estimate hazard ratios and 95% confidence intervals. To check assumptions of proportionality, curves of the log of the cumulative hazard versus time were plotted and assessed visually to determine if the vertical shift between the curves was constant over time and by testing for an interaction term of treatment arm with time in the models.
The discriminatory ability of hormone receptor status to predict disease-free survival was evaluated separately according to treatment by use of the c index (28,29). In this study, the c index was interpreted as an estimate of the probability that, for two randomly chosen patients on the same treatment, the patient with a receptor-positive (or a receptor present) tumor will have longer disease-free survival than the patient with a receptor-negative (or a receptor absent) tumor. The c indices were compared between assay methods by use of a U statistic (28,29). Disease-free survival among patients whose tumors were concordant or discordant by the two assay methods according to treatment were summarized by use of Kaplan–Meier plots. All statistical tests were two-sided, and P values of less than .05 were considered to be statistically significant.
All analyses were undertaken separately for the two trials. The data analysis used SAS version 9.1 (SAS Institute Inc, Cary, NC) and S-PLUS version 6.1 (Insightful Corp, Seattle, WA).
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RESULTS |
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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Among the pre- and perimenopausal patients randomly assigned to treatment in Trial VIII and the postmenopausal patients randomly assigned to treatment in Trial IX, 571 (54%) of 1063 patients and 976 (58%) of 1669 patients, respectively, had hormone receptor levels measured both locally by an extraction assay and centrally by an immunohistochemical assay and were included in these analyses. The clinical characteristics of patients in this study and those of the trial cohorts were comparable, although, in this analysis cohort, there were slightly fewer patients (123 [8%] of 1547 total patients) with tumors that were 0–1 cm in diameter than there were in the overall trial cohorts. Median duration of follow-up was 8.2 and 9.4 years in the Trials VIII and IX analysis cohorts, respectively; follow-up was more than 1.5 years longer in these analysis cohorts than among patients who were not included in our study because patients in the analysis cohorts likely entered the trials earlier when only extraction assays were used to establish ER status at randomization.
The distributions of ER and PgR values are summarized in Table 1. Spearman's rank correlation coefficients for the quantitative values obtained by the two assay methods were .46 for ER and .61 for PgR among premenopausal patients and were .53 for ER and .61 for PgR among postmenopausal patients, indicating moderately strong positive associations between the quantitative assay values.
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Concordance and Agreement
The classification of tumors by hormone receptor status is presented in Table 2. The concordance and agreement of hormone receptor status as determined by extraction and immunohistochemical assays are presented in Table 3. For the different hormone receptor status variables in both pre- and postmenopausal patients, few tumors were classified into the low category by immunohistochemical assay (1%–9% of cells stained), and few tumors were classified as none (0 fmol/mg of cytosol protein) by extraction assays. Thus, the concordance of the three categories was in the range of 55%–74% (w, indicating agreement, ranged from 0.33 to 0.47).
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In contrast, concordance and agreement were better for extraction assay results dichotomized as negative or positive compared with immunohistochemical assay results dichotomized as absent or present or as negative or positive; concordance ranged from 74% to 88% (
ranged from 0.48 to 0.66). For ER status, concordance and agreement were observed to be higher among postmenopausal patients (88%; = 0.66) than among premenopausal patients (81%; = 0.53). However, concordance and agreement for PgR status were lower among postmenopausal patients (76%; = 0.49) than among premenopausal patients (80%; = 0.51), and concordance for ER status and PgR status was similar among premenopausal patients (Fig. 1).
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ER status from extraction assays indicated that 179 (32%) of 562 tumors in premenopausal patients were ER negative and 231 (24%) of 959 tumors in postmenopausal patients were ER negative. When the ER status of these ER-negative tumors was determined by immunohistochemical assay, 76 (42%) of the 179 ER-negative tumors in premenopausal patients were ER present and 72 (31%) of the 231 ER-negative tumors in postmenopausal patients were ER present. Of 383 and 728 tumors in premenopausal and postmenopausal patients, respectively, as determined by extraction assays to be ER positive, 30 (8%) and 44 (6%) were ER absent by immunohistochemical assay. Thus, approximately one-third of the 25%–30% of patients who would not have been eligible for the trials after ER-negative tumors were deemed ineligible would have been considered as appropriate candidates for the trials according to immunohistochemical assay results, and approximately 10% of patients who were eligible according to extraction assay results would not have been considered as appropriate trial candidates using immunohistochemical assays.
PgR results from extraction assays indicated that 162 (29%) of the 557 tumors in premenopausal patients were PgR negative and that 337 (37%) of the 919 tumors in postmenopausal patients were PgR negative. Among the 162 PgR-negative tumors in premenopausal patients, 58 (36%) were PgR present by immunohistochemical assay, and among the 337 PgR-negative tumors in postmenopausal patients, 113 (34%) were PgR present by immunohistochemical assay. Among the 395 and 582 PgR-positive tumors by extraction assays in premenopausal and postmenopausal patients, respectively, 53 (13%) and 105 (18%) were PgR absent by immunohistochemical assay.
Among the few tumors staining ER low by immunohistochemical assay, six (40%) of the 15 such tumors in premenopausal patients and nine (50%) of the 18 such tumors in postmenopausal patients were ER positive by the extraction assay. Among tumors staining PgR low by immunohistochemical assay, 20 (59%) of the 34 such tumors in premenopausal patients and 53 (60%) of the 89 such tumors in postmenopausal patients were PgR positive by the extraction assay. This finding suggests that it was reasonable to consider tumors classified as hormone receptor low by immunohistochemical assay as also endocrine responsive. Thus, in subsequent analyses, we focused on immunohistochemical assay values that were dichotomized as absent versus present and extraction assay values that were dichotomized standardly as negative versus positive at a cut point of 10 fmol/mg of cytosol protein.
Trial Results
The forest plot in Fig. 2 summarizes evaluations of outcomes for Trials VIII and IX that are based on the patients included in this analysis. For each trial, disease-free survival was compared between treatment arms according to hormone receptor status, which was determined by extraction assays (negative or positive) and by an immunohistochemical assay (absent or present). Hazard ratios of recurrence or death and corresponding 95% confidence intervals are presented.
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In Trial VIII, we observed remarkably similar treatment comparison results between the two assay methods among subgroups of premenopausal patients defined by ER status, i.e., the estimated hazard ratios and 95% confidence intervals comparing treatments among the ER-negative subgroup defined by extraction assays are virtually identical to the those among ER-absent subgroup defined by immunohistochemical assay (e.g., for the comparison CMF versus goserelin: ER-negative HR = 0.60, 95% CI = 0.30 to 1.17; ER-absent HR = 0.61, 95% CI = 0.30 to 1.26). In contrast, we observed different results among subgroups defined by PgR status that were determined by the two assay methods, especially in comparisons that included goserelin. In the PgR-absent subgroup as defined by immunohistochemical assay, the decreased hazard of recurrence or death in the CMF-alone arm or the CMF–goserelin arm, as compared with that in the goserelin-alone arm, was more striking (for the comparison CMF versus goserelin, HR = 0.62, 95% CI = 0.32 to 1.19; for the comparison CMF–goserelin versus goserelin, HR = 0.46, 95% CI = 0.23 to 0.90); in the PgR-present subgroup defined by immunohistochemical assay, the ratios of hazards of recurrence or death were not statistically different from unity (for the comparison of CMF versus goserelin, HR = 0.92, 95% CI = 0.55 to 1.54; for the comparison CMF–goserelin versus goserelin, HR = 0.99, 95% CI = 0.60 to 1.65).
These observations were confirmed in an assessment of how well ER status and PgR status predicted disease-free survival within a given treatment arm (Table 4) and in a comparison of hormone receptor status as determined by extraction assays and by immunohistochemical assay. Among patients treated with goserelin alone, ER status as determined by extraction assays and that determined by immunohistochemical assay had similar discriminatory ability (c index = 0.54 and 0.56, respectively; P = .87). However, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better than that determined by extraction assays (c index = 0.60 and 0.51, respectively; P = .003), with PgR status determined by extraction assays having little ability to predict disease-free survival among goserelin-treated patients. To illustrate these results, disease-free survival among patients randomly assigned to the goserelin-alone arm was summarized (Fig. 3) for ER status and PgR status as classified by the two assay methods. Among the 15 patients whose PgR status was PgR present by the immunohistochemical assay and PgR negative by the extraction assay (Fig. 3, B), only one patient experienced a recurrence or death; whereas, among the 15 patients whose PgR status was PgR absent by the immunohistochemical assay and PgR positive by the extraction assay, eight patients experienced recurrence or death (Fig. 3, B).
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Among postmenopausal patients in Trial IX, similar estimates of hazard ratios were obtained when the two assay methods were used to determine hormone receptor status, and the same conclusions would be reached—i.e., adjuvant CMF in addition to tamoxifen increased disease-free survival compared with tamoxifen alone among patients with ER-negative tumors but not among patients with ER-positive tumors (Fig. 2). A similar pattern was found for PgR status, although results that were based on PgR status appeared more discrepant. Among patients on the tamoxifen-alone arm, ER status and PgR status as determined by extraction assay or by an immunohistochemical assay could predict response to tamoxifen, with c index values ranging from 0.55 to 0.58, but values were not statistically significantly different between assay methods (for ER status, P = .37; for PgR status, P = .23; Table 4). As an illustration, disease-free survival among patients in the tamoxifen-alone arm was summarized according to ER status and PgR status as classified by both assay methods (Fig. 3). Disease-free survival appeared slightly, although not statistically significantly, better for patients whose ER status was ER present by an immunohistochemical assay and ER negative by the extraction assay than for patients whose ER status was ER absent by an immunohistochemical assay and ER positive by the extraction assay (Fig. 3, C).
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DISCUSSION |
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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In this re-evaluation of data from two IBCSG randomized clinical trials of adjuvant therapy in premenopausal (Trial VIII) and postmenopausal (Trial IX) patients with lymph node–negative breast cancer, conclusions that were based on ER status determined by immunohistochemical assay performed at a central laboratory confirmed those that were based on ER status determined by extraction assays performed at local pathology laboratories (i.e., premenopausal and postmenopausal women with ER-negative, lymph node–negative breast cancer benefited [in terms of better disease-free survival] from the addition of adjuvant chemotherapy to endocrine therapy, whereas if their cancer was ER positive, postmenopausal women obtained no benefit from the combination treatment compared with endocrine therapy alone and for premenopausal women the use of combination treatment or endocrine therapy alone required further study). When disease-free survival was evaluated according to PgR status of the primary tumor, which was not examined in the primary trial analyses, PgR status that was determined by an immunohistochemical assay could predict response to endocrine therapy better than that determined by extraction assays, particularly among pre- and perimenopausal patients. These results provide confidence in the use of steroid hormone receptor status determined by immunohistochemical assay to select adjuvant treatment, even though the clinical trials on which these evidence-based recommendations were established had determined hormone receptor status by locally performed extraction assays, which are expected to have a discordance of 10%–30% with respect to immunohistochemical assay.
We investigated data from two randomized clinical trial populations of patients with lymph node–negative, early-stage invasive breast cancer. The trial populations were pre- and perimenopausal patients and postmenopausal patients who underwent similar well-defined treatment strategies to evaluate the combination of chemoendocrine therapy compared with endocrine therapy alone. Only two other studies (14,30) used randomized clinical trial populations who received adjuvant therapy, but the cohorts of patients with lymph node–positive disease were smaller in both studies than those in Trials VIII and IX. In the IBCSG trial populations of patients with lymph node–negative breast cancer, immunohistochemical assay–determined hormone receptor status and extraction assay–determined status had rates of concordance and agreement that were consistent with those previously reported (6–14), and we confirmed that immunohistochemical assay–determined hormone receptor status was a valid discriminator of endocrine responsiveness and chemosensitivity.
In particular, among pre- and perimenopausal patients in Trial VIII, the benefit of increased disease-free survival associated with CMF followed by goserelin, compared with goserelin alone, was most clearly evident among PgR-absent tumors, as determined by immunohistochemical assay. In contrast, among postmenopausal patients in Trial IX, ER status demonstrated the benefit of increased disease-free survival associated with adjuvant CMF among patients with ER-negative (or ER-absent) tumors, regardless of assay method. Colleoni et al. (31) also found that PgR status was more predictive of response to perioperative chemotherapy than ER status among premenopausal patients with lymph node–negative disease, whereas ER status was more predictive of response than PgR status among postmenopausal patients.
When we compared the results of locally assessed extraction assays with those of centrally assessed immunohistochemical assay in these trial populations of patients with lymph node–negative disease, the immunohistochemical assay appeared to increase the fraction of ER-present and PgR-present tumors in both pre- and perimenopausal patients and in postmenopausal patients, most likely because of the overall higher sensitivity of the immunohistochemical assay. The fraction of tumors that scored positive by use of extraction assays and absent by use of an immunohistochemical assay was much higher for PgR levels than for ER levels in both trials, suggesting that extraction assays were more likely to yield false-positive results for PgR status than for ER status. This result appeared to be especially true among pre- and perimenopausal patients because, when extraction assays were used, the fraction of patients with PgR-positive tumors was greater than the fraction of patients with ER-positive tumors, a result that is at variance with the current knowledge on the coordinate expression of the two receptors (32). The false-positive results for PgR status as determined by extraction assays may explain its inability to predict disease-free survival among pre- and perimenopausal patients treated with endocrine therapy alone and its inability to demonstrate disease-free survival benefit of chemoendocrine therapy compared with endocrine therapy alone in PgR-negative tumors. These abilities emerged clearly when we reassessed PgR status among pre- and perimenopausal patients by immunohistochemical assay.
The main advantages of immunohistochemical assays compared with extraction assays are that immunohistochemical assays are easier and quicker to perform and may be performed on very small tumors or tumor specimens, such as those obtained by fine-needle aspiration biopsy or core biopsy examinations. Immunohistochemical assays allow precise determination of hormone receptor status at the individual cell level, by taking into account only the neoplastic cells of the invasive component of the tumor, and thereby accommodate the heterogeneity of hormone receptor expression in tumors, which cannot be evaluated by extraction assays. Extraction assays, in contrast, generally provide an overall score for the entire cell population of the tumor tissue, including non-neoplastic cells and in situ components of the tumor, and thus may lead to false-positive or false-negative findings, depending on the relative proportions of truly invasive tumor cells and other cell types in the tumor tissue. Extraction assays also quantitatively evaluate the hormone receptor content of the sample, which may be more difficult to achieve by immunohistochemical assay. In fact, the quantitative or semiquantitative assessment of the percentage of immunoreactive cells may not be truly representative of the quantitative differences in hormone receptor content of different tumors. Both intensity of staining and number of immunostained cells in different tumor samples may be affected by preanalytical (e.g., type and length of fixation) and analytical (e.g., type and concentration of the immunohistochemical reagents or the immunostaining protocol) variables and by interpretative bias. As a consequence, intra- and interlaboratory reproducibility of the results of immunohistochemical assays for hormone receptor levels may reduce the predictive power of hormone receptor status and thus affect the choice of the most appropriate therapeutic intervention. It is essential that the reproducibility of immunohistochemical assay results be substantially improved by active participation of all involved laboratories in quality assurance and quality control programs and that standardized procedures, such as those used by IBCSG and also used in this study, be adopted to satisfy the continual need for accurate evaluation of receptor status. The standardized approach to evaluation and immunostaining used by IBCSG minimized interlaboratory technical variations and underscores the value of tissue collection and central review of hormone receptors as part of randomized clinical trials evaluating adjuvant systemic therapies for breast cancer.
When interpreting the results of this study, certain limitations should be taken into account. First, we were unable to determine whether a cut point of 1% or more or of 10% or more stained cells in immunohistochemical assays was more concordant with tumors classified as ER positive (10 fmol/mg of cytosol protein) by extraction assays because too few tumors were classified into the ER low category by immunohistochemical assay (1%–9% of cells stained). Second, the small numbers of tumors with discordant classification prevented direct comparisons of disease-free survival between patients whose tumors were hormone receptor absent by immunohistochemical assay and positive by extraction assay with patients whose tumors were hormone receptor present by immunohistochemical assay and negative by extraction assay. Finally, about 20% of patients randomly assigned to treatment in Trials VIII and IX did not have archival tumor material available for hormone receptor evaluation, though the clinical characteristics of patients included in this study and those of the overall trial cohorts were comparable, with the exception that there were slightly fewer patients with tumors that were 1 cm or less in diameter in this study.
Despite the 10%–30% discordance in steroid hormone receptor status determined by immunohistochemical assay versus extraction assays, on average in this population of lymph node–negative invasive breast cancers, the presence or absence of ER determined by an immunohistochemical assay confirmed randomized clinical trial results that were based on ER status determined by extraction assays. PgR status determined by immunohistochemical assay improved the ability of PgR to predict response to endocrine therapy, particularly among pre- and perimenopausal patients.
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APPENDIX |
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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International Breast Cancer Study Group—Participants and Authors
Scientific Committee: A. Goldhirsch, A. S. Coates (Co-Chairs).
Foundation Council: B. Thürlimann (President), M. Castiglione, A. S. Coates, J. P. Collins, H. Cortés-Funes, R. D. Gelber, A. Goldhirsch, M. Green, A. Hiltbrunner, S. B. Holmberg, D. K. Hossfeld, I. Láng, J. Lindtner, F. Paganetti, C.-M. Rudenstam, R. Stahel, H.-J. Senn, A. Veronesi.
Coordinating Center, Bern, Switzerland: M. Castiglione-Gertsch (CEO and Study Chair), A. Hiltbrunner (Director); G. Egli, M. Rabaglio, R. Maibach; R. Studer, C. Perron; Pathology Office: R. Kammler (Project Manager), H.-R. Pauli, A. Aeschbacher, S. Oelhafen.
Statistical Center, Harvard School of Public Health, and Dana-Farber Cancer Institute, Boston, MA: R. D. Gelber (Group Statistician), K. Price (Director of Scientific Administration), M. Regan, D. Zahrieh, S. Gelber, A. Keshaviah, Z. Sun, B. Cole, L. Nickerson.
Data Management Center, Frontier Science and Technology Research Foundation, Amherst, NY: L. Blacher (Director), R. Hinkle (Trial Data Manager), S. Lippert, J. Celano.
Pathology Office, European Institute of Oncology, Milan, Italy: G. Viale, E. Maiorano, M. Mastropasqua, S. Andrighetto, G. Peruzzotti, R. Ghisini, E. Scarano, P. Dell'Orto, B. Del Curto.
Pathology Office, University of Glasgow, Scotland, UK: B. Gusterson, E. Mallon.
The Ontario Cancer Treatment and Research Foundation, Toronto Sunnybrook Regional Cancer Centre, Toronto, Canada: K. Pritchard, D. Sutherland, C. Sawka, G. Taylor, R. Choo, C. Catzavelos, K. Roche, H. Wedad.
National Institute of Oncology, Budapest, Hungary: I. Láng, E. Hitre, E. Juhos, I. Szamel, J. Toth, Z. Orosz, I. Peter.
Centro di Riferimento Oncologico, Aviano, Italy: D. Crivellari, M. D. Magri, A. Veronesi, A. Buonadonna. S. Massarut, E. Candiani, A. Carbone, T. Perin, M. Roncadin, M. Arcicasa, F. Coran, S. Morassut, V. Canzonieri.
Spedali Civili & Fondazione Beretta, Brescia, Italy: E. Simoncini, G. Marini, P. Marpicati, M. Braga, P. Grigolato, L. Lucini.
General Hospital, Gorizia, Italy: S. Foladore, L. Foghin, G. Pamich, C. Bianchi, B. Marino, A. Murgia, V. Milan.
European Institute of Oncology, Milan, Italy: A. Goldhirsch, M. Colleoni, G. Martinelli, L. Orlando, F. Nolé, A. Luini, R. Orecchia, G. Viale, G. Renne, G. Mazzarol, F. Peccatori, F. de Braud, A. Costa, S. Zurrida, P. Veronesi, V. Sacchini, V. Galimberti, M. Intra, S. Cinieri, G. Peruzzotti, U. Veronesi.
Ospedale Infermi, Rimini, Italy: A. Ravaioli, D. Tassinari, G. Oliverio, F. Barbanti, P. Rinaldi, L. Gianni, G. Drudi.
Ospedale S. Eugenio, Rome, Italy: M. Antimi, M. Minelli, V. Bellini, R. Porzio, E. Pernazza, G. Santeusanio, L.G. Spagnoli.
Ospedale S. Bortolo, Vicenza, Italy: M. Magazu, V. Fosser, P. Morandi, G. Scalco, M. Balli, E. S. G. d'Amore, S. Meli, G. Torsello.
The Institute of Oncology, Ljubljana, Slovenia: J. Lindtner, D. Erzen, E. Majdic, B. Stabuc, A. Plesnicar, R. Golouh, J. Lamovec, J. Jancar, I. Vrhovec, M. Kramberger.
Groote Schuur Hospital and University of Cape Town, Cape Town, Republic of South Africa: D. M. Dent, A. Gudgeon, E. Murray, G. Langman, I. D. Werner, P. Steynor, J. Toop, E. McEvoy.
Sandton Oncology Center, Johannesburg, Republic of South Africa: D. Vorobiof, M. Chasen, C. Mohammed, A. Rosengarten, C. Thatcher.
Madrid Breast Cancer Group, Madrid, Spain: H. Cortés-Funes, C. Mendiola, J. Hornedo, R. Colomer, F. Cruz Vigo, P. Miranda, A. Sierra, F. Martinez-Tello, A. Garzon, S. Alonso, A. Ferrero.
West Swedish Breast Cancer Study Group, Göteborg, Sweden: C.-M. Rudenstam, M. Suurküla, Ö. Sjukhuset, G. Havel, S. Persson, J. H. Svensson, G. Östberg, S. B. Holmberg, A. Wallgren, S. Ottosson-Lönn, R. Hultborn, G. Colldahl-Jäderström, E. Cahlin, J. Mattsson, L. Ivarsson, O. Ruusvik, L. G. Niklasson, S. Dahlin, G. Karlsson, B. Lindberg, A. Sundbäck, S. Bergegårdh, H. Salander, C. Andersson, M. Heideman, Y. Hessman, O. Nelzén, G. Claes, T. Ramhult, A. Kovacs, P. Liedberg.
Swiss Group for Clinical Cancer Research member institutions—Inselspital, Bern, Switzerland: M. F. Fey, M. Castiglione-Gertsch, E. Dreher, H. Schneider, S. Aebi, J. Ludin, G. Beck, A. Haenel, J. M. Lüthi, L. Mazzucchelli, J. P. Musy, H. J. Altermatt, M. Nandedkar, K. Buser.
Kantonsspital, St Gallen, Switzerland: H.-J. Senn, B. Thürlimann, Ch. Oehlschlegel, G. Ries, M. Töpfer, U. Lorenz, O. Schiltknecht, B. Späti, A. Ehrsam, M. Bamert, W. F. Jungi.
Istituto Oncologico della Svizzera Italiana, Bellinzona, Switzerland: F. Cavalli, O. Pagani, H. Neuenschwander, L. Bronz, C. Sessa, M. Ghielmini, T. Rusca, P. Rey, J. Bernier, E. Pedrinis, T. Gyr, L. Leidi, G. Pastorelli, G. Caccia, A. Goldhirsch.
Kantonsspital, Basel, Switzerland: R. Herrmann, C. F. Rochlitz, J. F. Harder, S. Bartens, U. Eppenberger, J. Torhorst, H. Moch.
Hôpital des Cadolles, Neuchâtel, Switzerland: D. Piguet, P. Siegenthaler, V. Barrelet, R. P. Baumann, B. Christen.
University Hospital, Zürich, Switzerland: B. Pestalozzi, C. Sauter, D. Fink, M. Fehr, U. Haller, U. Metzger, P. Huguenin, R. Caduff.
Centre Hospitalier Universitaire Vandois, Lausanne, Switzerland: L. Perey, S. Leyvraz, P. Anani, F. Gomez, D. Wellman, G. Chapuis, P. De Grandi, P. Reymond, M. Gillet, J. F. Delaloye, C. Genton, M. Fiche.
Hôpital Cantonal, Geneva, Switzerland: P. Alberto, H. Bonnefoi, P. Schäfer, F. Krauer, M. Forni, S. Diebold, M. Genta, M. Pelte.
Kantonsspital Graubünden, Chur, Switzerland: F. Egli, P. Forrer, A. Willi, R. Steiner, J. Allemann, T. Rüedi, A. Leutenegger, U. Dalla Torre, H. Frick.
Australian New Zealand Breast Cancer Trials Group member institutions—Operations Office, University of Newcastle, New South Wales, Australia: J. F. Forbes, D. Lindsay.
The Cancer Council Victoria (previously Anti-Cancer Council of Victoria), Clinical Trials Office, Melbourne, Australia: J. Collins, R. Snyder, B. Brown, E. Abdi, H. Armstrong, A. Barling, R. Basser, P. Bhathal, W. I. Burns, M. Chipman, J. Chirgwin, I. Davis, R. Drummond, D. Finkelde, P. Francis, D. Gee, G. Goss, M. Green, P. Gregory, J. Griffiths, S. Hart, D. Hastrich, M. Henderson, R. Holmes, P. Jeal, D. Joseph, P. Kitchen, P. Kostos, G. Lindeman, B. Mann, R. McLennan, L. Mileshkin, P. Mitchell, C. Murphy, S. Neil, I. Olver, M. Pitcher, A. Read, D. Reading, R. Reed, G. Richardson, A. Rodger, I. Russell, M. Schwarz, S. Slade, R. Stanley, M. Steele, J. Stewart, C. Underhill, J. Zalcberg, A. Zimet, C. Dow, R. Valentine.
Flinders Medical Centre, Bedford Park, South Australia, Australia: T. Malden.
Mount Hospital, Perth, Western Australia, Australia: G. Van Hazel.
Newcastle Mater Misericordiae Hospital Waratah, Newcastle, Australia: J. F. Forbes, S. Braye, J. Stewart, D. Jackson, R. Gourlay, J. Bishop, S. Cox, S. Ackland, A. Bonaventura, C. Hamilton, J. Denham, P. O'Brien, M. Back, S. Brae, R. Muragasu.
Prince of Wales, Randwick, New South Wales, Australia: M. Friedlander, B. Brigham, C. Lewis.
Royal Adelaide Hospital, Adelaide, Australia: I. N. Olver, D. Keefe, M. Brown, P. G. Gill, A. Taylor, E. Yeoh, E. Abdi, J. Cleary, F. Parnis.
Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia: M. Byrne, G. Van Hazel, J. Dewar, M. Buck, G. Sterrett, D. Ingram, D. Hastrich, D. Joseph, F. Cameron, K. B. Shilkin, P. Michell, J. Sharpio, G. Harloe, J. Lewis, B. Snowball, P. Garcia Webb, J. Harvey, W. D. De Boer, P. Robbins, N. Buxton, M. N. I. Walters.
University of Sydney, Dubbo Base Hospital and Royal Prince Alfred Hospital, Sydney, Australia: J. Beith, M. H. N. Tattersall, A. S. Coates, F. Niesche, R. West, S. Renwick, J. Donovan, P. Duval, R. J. Simes, A. Ng, D. Glenn, R. A. North, R. G. O'Connor, M. Rice, G. Stevens, J. Grassby, S. Pendlebury, C. McLeod, M. Boyer, A. Sullivan, J. Hobbs, D. Lind, J. Grace, P. McKenzie.
W.P. Holman Clinic, Launceston, Tasmania, Australia: D. Boadle, T. Brain, I. Byard, D. Byram.
Auckland Breast Cancer Study Group, Auckland, New Zealand: V. J. Harvey, R. G. Kay, P. Thompson, D. Porter, C. S. Benjamin, A. Bierre, M. Miller, B. Hochstein, A. Lethaby, J. Webber, J. P. Allen, M. Allon, J. F. Arthur, M. Gurley, P. Symmans, M. Christie, A. R. King.
Waikato Hospital, Hamilton, New Zealand: I. Kennedy, G. Round, J. Long.
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TopNotesAbstractIntroductionPatients and methodsResultsDiscussionAppendixReferences |
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This research was funded by the International Breast Cancer Study Group, which is supported by: Swiss Group for Clinical Cancer Research, Frontier Science and Technology Research Foundation, The Cancer Council Australia, Australian New Zealand Breast Cancer Trials Group (National Health Medical Research Council grants 920876, 950328, 980379, and 100925), National Cancer Institute (CA-75362), Swedish Cancer Society, Cancer Association of South Africa (for South African participation), and Oncosuisse/Cancer Research Switzerland (for collection of tumor blocks within Switzerland). The authors had full responsibility for the design of the study, collection of data, the analysis and interpretation of the data, the decision to submit the manuscript for publication, and the writing of the manuscript.
We thank the many pathologists who submitted tumor blocks and slides, Rosita Kammler and the pathology team in Bern for coordination of the pathology material transmission, and Stefania Andrighetto for data management at the pathology office in Milan. We thank the patients, physicians, nurses, and data managers who participated in the International Breast Cancer Study Group trials.
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