Clinical Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Infection and Cancer: Biology, Therapeutics, and Prevention
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Clinical Cancer Research 13, 1987-1994, April 1, 2007. doi: 10.1158/1078-0432.CCR-06-1823
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data, Gruverberger-Saal, et al.
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gruvberger-Saal, S. K.
Right arrow Articles by Fernö, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gruvberger-Saal, S. K.
Right arrow Articles by Fernö, M.

Human Cancer Biology

Estrogen Receptor ß Expression Is Associated with Tamoxifen Response in ER{alpha}-Negative Breast Carcinoma

Sofia K. Gruvberger-Saal1, Pär-Ola Bendahl1, Lao H. Saal1,3, Mervi Laakso4,5, Cecilia Hegardt1, Patrik Edén2, Carsten Peterson2, Per Malmström1, Jorma Isola4, Åke Borg1 and Mårten Fernö1

Authors' Affiliations: Departments of 1 Oncology and 2 Theoretical Physics, Lund University, Lund, Sweden; 3 Institute for Cancer Genetics, Columbia University, New York, New York; 4 Institute of Medical Technology, University of Tampere, Tampere, Finland; and 5 Department of Pathology, Seinäjoki Central Hospital, Seinäjoki, Finland

Requests for reprints: Sofia K. Gruvberger-Saal, Institute for Cancer Genetics, Columbia University, 1130 Saint Nicholas Avenue, Irving Cancer Research Center, Suite 406, New York, NY 10032. Phone: 212-851-5263; Fax: 212-851-5267; E-mail: sg2414{at}columbia.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Endocrine therapies, such as tamoxifen, are commonly given to most patients with estrogen receptor (ER{alpha})–positive breast carcinoma but are not indicated for persons with ER{alpha}-negative cancer. The factors responsible for response to tamoxifen in 5% to 10% of patients with ER{alpha}-negative tumors are not clear. The aim of the present study was to elucidate the biology and prognostic role of the second ER, ERß, in patients treated with adjuvant tamoxifen.

Experimental Design: We investigated ERß by immunohistochemistry in 353 stage II primary breast tumors from patients treated with 2 years adjuvant tamoxifen, and generated gene expression profiles for a representative subset of 88 tumors.

Results: ERß was associated with increased survival (distant disease-free survival, P = 0.01; overall survival, P = 0.22), and in particular within ER{alpha}-negative patients (P = 0.003; P = 0.04), but not in the ER{alpha}-positive subgroup (P = 0.49; P = 0.88). Lack of ERß conferred early relapse (hazard ratio, 14; 95% confidence interval, 1.8-106; P = 0.01) within the ER{alpha}-negative subgroup even after adjustment for other markers. ER{alpha} was an independent marker only within the ERß-negative tumors (hazard ratio, 0.44; 95% confidence interval, 0.21-0.89; P = 0.02). An ERß gene expression profile was identified and was markedly different from the ER{alpha} signature.

Conclusion: Expression of ERß is an independent marker for favorable prognosis after adjuvant tamoxifen treatment in ER{alpha}-negative breast cancer patients and involves a gene expression program distinct from ER{alpha}. These results may be highly clinically significant, because in the United States alone, ~10,000 women are diagnosed annually with ER{alpha}-negative/ERß-positive breast carcinoma and may benefit from adjuvant tamoxifen.

Estrogens play an important role for the development and progression of breast carcinoma. Their effects on growth and proliferation are mediated through the two estrogen receptors (ER) {alpha} and ß (1–3), which function as transcription factors and modulate the expression of target genes in response to estrogens. The biology of ER{alpha} has been studied for decades and evaluation of tumor ER{alpha} content is a mainstay of clinical practice as a marker associated with prognosis and response to endocrine therapies such as tamoxifen (4).

Tamoxifen is a selective ER modulator and is the most frequently prescribed drug for treatment of breast cancer. Tamoxifen is known to inhibit estrogen-stimulated growth of breast cancer cells by competitively binding to and blocking ER{alpha} (4). Patients with tumors lacking ER{alpha} in general do not benefit from tamoxifen therapy, although a fraction of ER{alpha}-negative tumors do seem to be sensitive to tamoxifen (5–7). The factors responsible for these responders are debated and no means of identifying this group is currently known. Therefore, tamoxifen is not indicated for patients with ER{alpha}-negative tumors in the adjuvant or metastatic setting.

Since the discovery of ERß (3), which has a similar binding affinity as ER{alpha} for estrogens (8), several studies have focused on its biological function in relation to ER{alpha}. The two ER proteins share a high degree of homology in the DNA-binding regions but differ considerably in the NH2-terminal activation function 1 region, where interactions with other proteins in the transcriptional machinery takes place, and to a certain degree in the ligand-binding region (9), indicating that these receptors may share some similar functions but may not be entirely redundant. Indeed, they have been shown to respond differently in ligand-induced activation at activator protein 1 sites (10). In addition, ER{alpha} and ERß can exist as a heterodimer (11–13), suggesting a possible role for ERß as a modulator of ER{alpha} activity.

Recently, several studies have measured ERß in breast cancer specimens and sought to clarify the relationship between ERß and other clinicopathologic features and its role in response to endocrine treatment; however, some of the results have been conflicting and most focused on ERß as a resistance marker in ER{alpha}-positive tumors (refs. 14–16, and those reviewed in ref. 17). ERß has been shown to bind tamoxifen (18), and it has been suggested that low levels of ERß associates with tamoxifen resistance (14). Conversely, Hopp et al. (15) showed that expression of ERß had a beneficial effect on disease-free and overall survival in a group of 186 tamoxifen-treated tumors; however, they found no such association in their set of 119 untreated patients, suggesting a role for ERß as a predictive marker for tamoxifen sensitivity but not as a prognostic marker. Importantly, the patients studied by Hopp et al. (15) were predominantly ER{alpha}-positive and the numbers were limited; thus, they were unable to perform an analysis stratified by ER{alpha} status. To our knowledge, no study has investigated the role of ERß as a predictive marker for tamoxifen response for patients with ER{alpha}-negative tumors.

In the present report, we investigated ERß protein levels as predictor of therapy response in a large patient set, including both ER{alpha}-positive and ER{alpha}-negative tumors all treated uniformly with 2 years of adjuvant tamoxifen. Furthermore, we sought to identify a gene expression signature for ERß status and compare it with the ER{alpha}-associated expression signature.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. We studied a cohort of 425 women with stage II breast cancer collected by the participating departments of the South Swedish Health Care Region after approval of the Lund University Hospital Ethics Committee. These women had been part of two randomized trials of adjuvant tamoxifen monotherapy (19, 20), and were selected for this study with the following criteria: 2-year tamoxifen treatment arms (n = 995), complete follow-up data (n = 992), receipt of fresh-frozen sample from primary tumor (n = 783), and uniform method for hormone receptor content determination (n = 537). From these, all the premenopausal women (n = 79) and a random selection of the postmenopausal women (n = 346) were included in the study. Losses due to nonevaluable ERß immunostaining reduced the final cohort to 353 cases (Table 1 ). Patients were operated with either modified radical mastectomy or breast conservation surgery in combination with axillary lymph node dissection. Radiotherapy was offered to all patients treated with breast conservation surgery and to patients with lymph node metastases treated with modified radical mastectomy. The median follow-up for patients free from distant recurrence was 5.7 years and for patients alive at the end of the study was 14.5 years.


View this table:
[in this window]
[in a new window]

  		Table 1. Patient and tumor characteristics for the 353 patients

 
ERß immunohistochemical analysis. Formalin-fixed, paraffin-embedded tumor blocks from the 425 cases were used to generate tissue microarrays, with three 0.6-mm-diameter cores taken per tumor. Antigens were retrieved by heat pretreatment [102°C for 30 min with 1 mmol/L Tris EDTA buffer (pH 9)] in the PT Module device (LabVision, Fremont, CA). Immunostainings were carried out with an Autostainer (LabVision) using a cocktail of two monoclonal anti-ERß antibodies (clone 14C8 from GeneTex, San Antonio, TX, which is pan-specific for ERß isoforms, and PPG5/10 from Serotec, Oxford, United Kingdom, which is specific for ERß1), both diluted 1:2,000 from the manufacturers' stock. PowerVision+ (Immunovision Technologies, Daly City, CA) was used for immunodetection according to the manufacturer's instructions. The diaminobenzidine reaction product was enhanced with 0.5% copper sulfate for 5 min at room temperature, and the tissue was counterstained with hematoxylin. Losses due to lack of sufficient invasive tumor cells in the cores or detachment of tissue cores left 353 cases that could be evaluated. The scoring was done by one person (M.L.), blinded to all patient data, with the score ERß-negative (ERß–) defined as no to weak staining reaction (over background) in less than 20% of carcinoma cells, ERß moderately positive (ERß+) as weak staining intensity in 20% to 100%, and ERß strongly positive (ERß++) as intense staining intensity in 20% to 100%, using high-resolution digitized images and a virtual microscopy system (21).

Determination of other tumor markers. Steroid receptor protein [ER{alpha} and progesterone receptor (PgR)] determinations using enzyme immunoassay (22), and flow cytometric analysis of S-phase fraction and DNA ploidy analysis (23), were done as part of the routine tumor evaluation. ER{alpha} and PgR status, S-phase fraction status, and DNA ploidy status were classified as previously described (22, 24). ERBB2 amplification was measured using chromogenic in situ hybridization analysis (25).

Statistical analysis. The {chi}2 test for association and {chi}2 test for trend, Mann-Whitney U test, and Kruskal-Wallis test were used to assess associations between tumor ERß or ER{alpha} content and other variables. All factors were used as categorized variables in the statistical analysis except for age, which was also analyzed as a continuous variable. The Kaplan-Meier method was used to estimate distant disease-free survival and overall survival and the log-rank test was used to compare survival between two strata. The log-rank test for trend was used to compare survival in more than two strata. To test whether the effects on distant disease-free survival of ER{alpha} and ERß changed significantly with time, Schoenfeld's test for time dependence was applied. The association of the level of ERß with patient outcome, adjusted for other prognostic factors and for interaction between ERß and ER{alpha}, was assessed in a multivariate analysis using Cox proportional hazards model. All tests were two-sided and P values <0.05 were considered significant. Statistical analyses were carried out using Stata 8.0 (Stata Corporation, College Station, TX).

Microarrays. cDNA microarrays with 27,648 spots were produced in the SWEGENE Microarray Facility, Department of Oncology, Lund University. The gene set consisted of 24,301 sequence-verified IMAGE clones (Research Genetics, Huntsville, AL) and 1,296 internally generated clones, together representing {approx}15,000 UniGene clusters (build 180) and {approx}1,200 unclustered expressed sequence tags, and were PCR amplified using vector-specific primers essentially as previously described (26) with some modifications. Tissue processing for the 88 breast tumor samples, RNA labeling, and microarray hybridization protocols are described in detail in Supplementary Materials and Methods.

Microarray data analysis. Microarray data are available through National Center for Biotechnology Information Gene Expression Omnibus,6 accession no. GSE6577. The ERß+ and ERß++ tumors were grouped together and analyzed as a single ERß-positive (ERß+/++) entity. Thus, the distribution of the 88 tumors between the four ER{alpha}/ERß groups was as follows: 10 ER{alpha}–/ERß–, 36 ER{alpha}–/ERß+/++, 8 ER{alpha}+/ERß– and 34 ER{alpha}+/ERß+/++.

Data analysis was done using BioArray Software Environment (27). Data preprocessing and filtering procedures, described in the Supplementary Materials and Methods, left 10,493 informative genes. The genes were ranked based on the signal-to-noise statistic (28), which calculates a correlation score between gene expression and the tumor annotation of interest. To evaluate the significance of the expression signatures between two annotation classes (e.g., ERß status), 1,000 permutations were run whereby the samples were randomly given an annotation label and the P value for a score was calculated as the average number of reporters exceeding the score in the permutation test, divided by the total number of reporters in the gene list. The false discovery rate (FDR) was calculated by random permutations, controlled according to Benjamini et al. (29), and used as an indicator of the robustness of the gene expression profile. An FDR of 0% indicates no false positives, whereas an FDR of 100% indicates complete random signal. To test the dependence of FDR on number of samples for the ERß analysis in the two ER{alpha} status subgroups, two ERß-negative and two ERß-positive samples were randomly removed from the ER{alpha}-negative cohort, making both these two groups equal in size to the corresponding groups in the ER{alpha}-positive cohort. For the reduced ER{alpha}-negative data set, genes were ranked and FDRs based on 1,000 permutations were calculated; the procedure was repeated 200 times with different random selections of removed samples. The Significance Analysis of Microarrays algorithm (30) implemented in TIGR Multiexperiment Viewer 3.1 (31) with 1,000 permutations and default settings was also used to generate comparative FDR plots. Hierarchical clustering and data visualization are described in the Supplementary Materials and Methods.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Association between ERß and clinicopathologic variables. We evaluated total ERß protein levels by performing immunohistochemistry using a cocktail of two well-characterized monoclonal anti-ERß antibodies, clones 14C8 and PPG5/10, previously shown to be the best-performing antibodies for this application (32). Seventy-four percent of the tumors stained positive for ERß (ERß+, 54%; ERß++, 20%) and 26% were ERß– (Table 1; Supplementary Fig. S1). Seventy percent of the tumors were ER{alpha} positive, and the association between the expression of ERß and ER{alpha} was nonsignificant (P = 0.18). Although ER{alpha} positivity was associated with several clinical variables such as increasing age (P = 0.003), postmenopausal status (P = 0.007), a greater number of lymph nodes with metastases (P = 0.006), PgR positivity (P < 0.001), a low S-phase fraction (P < 0.001), and nonamplified ERBB2 (P < 0.001), ERß expression did not correlate significantly with any of the clinical variables tested (Table 1). In a subgroup analysis, increasing ERß level was associated with high S-phase fraction within the ER{alpha}-negative group (P = 0.03) but not with any other markers (data not shown).

Analysis of distant disease-free survival. Among all cases, ERß expression was significantly associated with an increased distant disease-free survival (P = 0.01; Fig. 1A ). When stratified by ER{alpha} status, ERß level was significantly associated with better distant disease-free survival (P = 0.003) in the ER{alpha}-negative group (Fig. 1B). A multivariate Cox regression analysis of distant disease-free survival, including lymph node status, menopausal status, tumor size, ERBB2 amplification, ER{alpha} status, two dummy variables for ERß– (versus ++ and + versus ++), and two interaction terms for ERß with ER{alpha} status (Table 2 ) showed a significantly worse distant disease-free survival for the ERß– group compared with ERß++ in the ER{alpha}-negative subgroup, with a hazard ratio (HR) of 14 [95% confidence interval (95% CI), 1.8-106; P = 0.01]. Between the ERß+ and ERß++ groups in the ER{alpha}-negative subgroup, there was a similar but nonsignificant trend (HR, 6.1; 95% CI, 0.79-46; P = 0.08). In contrast, as extrapolated from the model presented in Table 2, there was no effect of ERß on distant disease-free survival in the ER{alpha}-positive group (Fig. 1C), neither between the ERß and ERß++ groups (HR, 1.2; 95% CI, 0.57-2.5; P = 0.70) nor between the ERß+ and ERß++ groups (HR, 1.0; 95% CI, 0.05-2.0; P = 1.00). The Cox regression model also showed that the effect of ERß++ status (compared with ERß–) was significantly different in the two ER{alpha} subgroups (P = 0.02; Table 2).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Kaplan-Meier estimates of distant disease-free survival (DDFS; A-G) and overall survival (OS; H-J) for ERß and ER{alpha} status in the whole patient group (All) and in the two ER{alpha} and three ERß subgroups. Distant disease-free survival according to ERß status for the whole patient group (A), for the ER{alpha}-negative group (B), and for the ER{alpha}-positive group (C). ER{alpha} effects on distant disease-free survival in all tumors (D), in the ERß– group (E), in the ERß+ group (F), and in the ERß++ group (G). Estimates of overall survival for ERß status in the whole patient group (H), in the ER{alpha}-negative subgroup (I), and in the ER{alpha}-positive group (J). P values were calculated using log-rank test for trend (A-C and H-J) or the log-rank test (D-G). Numbers below each graph, number of patients remaining at risk in each group at each time point.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Cox multivariate analysis of distant disease-free survival in 347 tamoxifen-treated patients with stage II breast cancer

 
Having identified an ER{alpha}-dependent effect for ERß, we tested for the inverse dependency: The correlation of ER{alpha} status with distant disease-free survival in the whole patient group and in the different ERß subgroups (Fig. 1D-G). Interestingly, using the Kaplan-Meier method and log-rank test, ER{alpha} was significantly associated with a better prognosis exclusively in the ERß– subgroup (P = 0.05; Fig. 1E) but not in the whole cohort or any of the other ERß subgroups (Figs. 1D and F-G, respectively). From the Cox regression model with interaction between ER{alpha} and ERß (Table 2), the predictive value of ER{alpha} for distant disease-free survival in the different ERß subgroups could be interpreted. In agreement with the results from the Kaplan-Meier analysis, we found that ER{alpha} positivity was a significant independent predictive marker for improved distant disease-free survival only within the ERß– group (HR, 0.44; 95% CI, 0.21-0.89; P = 0.02), whereas no significant effect was seen in the other two subgroups (ERß+: HR, 0.86; 95% CI, 0.45-1.7; P = 0.70; ERß++: HR, 5.2; 95% CI, 0.67-40; P = 0.12).

The effect of ERß on distant disease-free survival in the ER{alpha}-negative group did not change significantly with time (P = 0.21; Schoenfeld's test), whereas the effect of ER{alpha} expression on distant disease-free survival in the ERß– group proved to be time dependent (P = 0.005) and diminished over a long follow-up time. Furthermore, as the distributions of ER{alpha} and PgR concentrations between the ERß subgroups within the ER{alpha}-negative tumors were similar (P = 0.68 for ER{alpha} and P = 0.41 for PgR, Kruskal-Wallis test), we concluded that residual low levels of ER{alpha} or PgR protein in the three ERß subgroups did not contribute to the prognostic effect of ERß within the ER{alpha}-negative tumors.

In addition to ERß++ status compared with ERß– in the ER{alpha}-negative subgroup, four or more lymph nodes (compared with 0) and menopausal status had independent prognostic value after tamoxifen treatment in the multivariate analysis (Table 2). No other effects were significant; however, ERBB2 amplification and ERß+ status (compared with ERß++) in the ER{alpha}-negative subgroup showed a tendency (P < 0.10) to carry prognostic information (Table 2). S-phase fraction, age as a continuous variable, PgR, and DNA ploidy status were not included in the multivariate analysis as they showed no significant association with distant disease-free survival in univariate analysis.

Analysis of overall survival. Using the Kaplan-Meier method and log-rank test, a beneficial effect of ERß positivity on overall survival was seen among ER{alpha}-negative tumors (P = 0.04; Fig. 1I) but not in the whole tumor group (P = 0.22; Fig. 1H) or among ER{alpha} positives (P = 0.88; Fig. 1J).

Gene expression analysis. The ERß gene expression signature in the 88 patients, representative of the original cohort (data not shown), had an FDR of 49% per top 100 discriminator genes ranked by the signal-to-noise ratio analysis score (28), and 50% per top 500 genes, indicating that ERß was associated with a unique, albeit weak, expression profile. The ER{alpha} status–associated gene expression signature from this data set had no false-positive genes in the top 1,000 genes. When stratified by ER{alpha}, ERß was associated with a detectable expression profile within the ER{alpha}-negative group (FDR of 40% per top 100 discriminator genes, and 43% per top 500; Fig. 2 ); however, there was no signal in the ER{alpha}-positive group (FDR reached >90% by the top 10 genes; Fig. 2). A similar difference in gene expression signature strength was seen when using the Significance Analysis of Microarrays algorithm (30) to generate FDR curves (data not shown).


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 2. FDRs as a function of gene rank from the signal-to-noise ratio analysis of ERß status (– versus +/++) from gene expression data from ER{alpha}-negative tumors (dashed line) and ER{alpha}-positive breast tumors (solid line). One thousand random permutations were run to estimate the FDRs, which were controlled according to Benjamini et al. (29). X axis, gene ranks.

 
To investigate whether the absence of an ERß-associated expression profile within the ER{alpha}-positive tumors was due to the ERß signal being masked by the much stronger ER{alpha}-associated profile, we used the top 50 and top 100 ERß genes generated from the ER{alpha}-negative subgroup and tested whether they could separate the tumors according to ERß status within the ER{alpha}-positive subgroup using hierarchical clustering analysis. The resulting tumor dendrogram showed a scattered distribution not correlating to ERß levels (data not shown), whereas using these genes within the ER{alpha}-negative group displayed ERß-associated tumor clusters (result for the top 50 ERß genes are shown in Fig. 3 ). The successful ERß discrimination in the ER{alpha} negatives was not due to a difference in distribution of residual ER{alpha} concentration in the ERß groups, because the distribution of ER{alpha} values in the three ERß subgroups within the ER{alpha}-negative tumors were similar (P = 0.75) as were the distribution of PgR concentrations (P = 0.58). Moreover, the ERß signal in ER{alpha}-negative tumors was still present even when top 400 ER{alpha} signature genes were removed before identifying the ERß signature (FDR of 42% per top 100 genes).


Figure 3
View larger version (67K):
[in this window]
[in a new window]

  		Fig. 3. Hierarchical clustering analysis of ER{alpha}-negative tumors using the top 50 signal-to-noise ratio–ranked genes in the ERß expression profile within ER{alpha}-negative tumors. Blue, ERß+/++ tumors; yellow, ERß– tumors. Hierarchical clustering presents the clustered samples in columns and the clustered genes in rows. Pseudocolored representation of gene expression ratios: red, high expression; green, low expression, relative to the median expression for each gene. Gray, missing data. Colorbar scale is given in relative log2 (ratio).

 
Furthermore, to test whether the lack of signal for ERß in the ER{alpha}-positive group was due to the lower number of samples in this group compared with the ER{alpha}-negative group (42 ER{alpha} positives versus 46 ER{alpha} negatives), samples were randomly removed from the ER{alpha}-negative cohort, to make the two ERß groups equal in size to the corresponding groups in the ER{alpha}-positive cohort. In none out of the 200 randomly reduced ER{alpha}-negative cohorts was the FDR, for top 10 up to top 1,000 genes or more, equal to or larger than that of the ER{alpha}-positive cohort, indicating that the difference in ERß signal strength in the two ER{alpha} subgroups is robust.

We compared the extent of overlap of genes comprising the ERß and ER{alpha} expression signatures. The two expression profiles generated from all 88 cases were substantially different: No genes were overlapping among the respective top 100 ranked genes; moreover, among the top 1,000 ranked genes, the overlap was only 6%. The ERß signature generated from within the ER{alpha}-negative tumors, which could be considered a "purer" ERß profile, showed a similar degree of nonoverlap with the ER{alpha} gene list from all cases: 1% in top 100 and 7% in top 1,000.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this is the first study to show an ER{alpha} status–specific survival benefit of ERß expression in tamoxifen-treated breast cancer patients. In light of a previous study showing no prognostic value of ERß in untreated patients (15), our results suggest that ERß is a predictive marker for response to tamoxifen in ER{alpha}-negative patients.

Although ER{alpha} positivity is a well-established predictor of response to tamoxifen and ER{alpha}-negative patients are considered nonresponders, it has been noted that 5% to 10% of ER{alpha}-negative tumors do benefit from adjuvant tamoxifen (5–7). Several theories have been put forward to explain these cases: Failures or inconsistencies in the performance or evaluation of ER{alpha} measurements result in miscategorization of what are actually ER{alpha}-positive tumors, or that tamoxifen acts through mechanisms independent of ER{alpha}. Our results support the latter and suggest that an ER{alpha}-independent alternative mechanism of action of tamoxifen is via ERß (of note, 17% of ER{alpha}-negative tumors were strongly positive for ERß). Furthermore, there was a similar distribution of ER{alpha} and PgR protein values in the three ERß subgroups within the ER{alpha}-negative tumors, suggesting that misclassification of ER{alpha}-positive tumors did not confound our results.

Although ER{alpha} did not have significant predictive value for tamoxifen response in the entire patient group over the complete long follow-up period, ER{alpha} was predictive within the ERß-negative group (P = 0.05, Fig. 1E; HR, 0.44; 95% CI, 0.21-0.89; P = 0.02, extrapolated from Table 2). Moreover, it appeared that ER{alpha} negativity may confer a better outcome within the ERß strongly positive group (P = 0.09, Fig. 1G; HR, 5.2; 95% CI, 0.67-40; P = 0.12, Table 2). These findings raise an intriguing hypothesis that the best tamoxifen response is achieved when a tumor is expressing either ER{alpha} or ERß, but not both. A biological explanation for this could be that the two receptors modulate the function of each other, so that when coexpressed the effect of tamoxifen becomes less pronounced or, alternatively, is growth promoting. This postulation is supported by the following observations: The two receptors can be coexpressed within individual breast carcinoma cells (33); they can form heterodimers (11–13); ERß can function as an inhibitor of ER{alpha} activity under certain conditions (34); and when coexpressed with ER{alpha}, ERß has been suggested to be associated with tumor characteristics indicative of a poorer prognosis (35).

Despite the similar utility of ERß or ER{alpha} as markers for benefit from tamoxifen therapy in this study group, the two receptors show many dissimilarities. Within all cases, ER{alpha} expression correlates strongly with the majority of other standard clinicopathologic markers, whereas ERß expression did not, corroborating the results of recent studies (15, 16). ERß did, however, correlate with high S-phase fraction within the ER{alpha}-negative group, which is consistent with the literature showing an association between ERß and the proliferation marker Ki67 (16, 36). An ERß-dependent high proliferation rate of these tumor cells may render them more sensitive to tamoxifen therapy (37).

We have previously shown that ER{alpha} status in breast cancer is associated with a robust gene expression signature (38), and the most readily apparent subdivision of breast cancers based on gene expression data is according to ER{alpha} status (39, 40). The present study is the first to identify an ERß-associated gene expression profile in human tumor biopsies. The lack of ERß-associated signal in ER{alpha}-positive tumors suggests that ERß seems to be less influential on gene expression, and hence tumor biology, in cancers also expressing ER{alpha}. However, this does not necessarily rule out that ERß under certain conditions can have some modulating effect on ER{alpha}. Our results indicate that the ERß gene expression signature was markedly different from the ER{alpha} gene expression signature as the genes included in the two profiles showed minimal overlap. However, due to the limited sample size in this study, these findings call for further validation in data sets including larger number of tumors. Together, our data suggests that ERß, in the absence of ER{alpha}, is not simply a surrogate marker for ER{alpha}, but rather ERß may affect growth and proliferation of breast cancer cells through modulation of different downstream target genes.

As far as we are aware, this is the largest survival study of ERß in breast cancer specimens to date. It will be important to confirm our results in other cohorts containing large numbers of ER{alpha}-negative tumors treated uniformly with tamoxifen. It remains to be tested whether a predictive effect of ERß exists for patients treated with other selective ER modulators or other types of endocrine therapies, such as aromatase inhibitors. Moreover, analysis of large series of untreated patients is necessary to confirm that ERß does not have prognostic value (15) and is specifically a predictive marker for therapeutic response to tamoxifen. In this regard and suggesting that the ERß benefit may indeed be related to antiestrogen therapy, a recent study of ER{alpha}-negative breast tumor patients that received varied therapies (<35% received unspecified hormonal therapies alone or in combination) found no recurrence-free survival benefit for ERß expression (16).

In the present study, we categorized ERß status into three groups with the goal of understanding the influence of different levels of ERß expression on survival for tamoxifen-treated patients. Further refinement will be needed to identify optimal and standardized methods for determining ERß content, scoring, and thresholds, and whether different ERß variants modulate response dissimilarly.

From our data, it can be estimated that in the United States alone, ~10,000 patients per year will be diagnosed with ER{alpha}-negative/ERß-positive breast carcinoma. Given the relatively low toxicity and cost of tamoxifen, our results have striking clinical implications that motivate further studies to explore the efficacy of tamoxifen to treat ER{alpha}-negative/ERß-positive breast tumors.


   Acknowledgments
 
We thank the participating departments of the South Sweden Breast Cancer Group for providing us with breast cancer samples, and Karolina Holm and Kristina Lövgren for excellent technical assistance.


   Footnotes
 
Grant support: Swedish Cancer Society, Gunnar, Arvid, and Elisabeth Nilsson Foundation (Å. Borg and M. Fernö); Mrs. Berta Kamprad Foundation, John and Augusta Persson Foundation for Medical Science, and University Hospital of Lund Research Foundation (S.K. Gruvberger-Saal, Å. Borg, and M. Fernö); Knut and Alice Wallenberg Foundation via the SWEGENE Program (C. Peterson and Å. Borg); IngaBritt and Arne Lundberg Foundation (Å. Borg); Swedish Research Council (C. Peterson); Swedish Foundation for Strategic Research through the Lund Center for Stem Cell Biology and Cell Therapy (P. Edén, C. Peterson, and Å. Borg); and the NIH Medical Scientist Training Program (L.H. Saal).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Current address for S.K. Gruvberger-Saal: Institute for Cancer Genetics, Columbia University, New York, NY 10032.

6 http://www.ncbi.nih.gov/geo/ Back

Received 7/24/06; revised 12/22/06; accepted 1/ 3/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Green S, Walter P, Kumar V, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 1986;320:134–9.[CrossRef][Medline]
  2. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science 1986;231:1150–4.[Abstract/Free Full Text]
  3. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci U S A 1996;93:5925–30.[Abstract/Free Full Text]
  4. Osborne CK. Tamoxifen in the treatment of breast cancer. N Engl J Med 1998;339:1609–18.[Free Full Text]
  5. McGuire WL. Current status of estrogen receptors in human breast cancer. Cancer 1975;36:638–44.[CrossRef][Medline]
  6. Early Breast Cancer Trialists' Collaborative Group. Systemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials involving 31,000 recurrences and 24,000 deaths among 75,000 women. Lancet 1992;339:1–15.[Medline]
  7. Early Breast Cancer Trialists' Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomised trials. Lancet 1998;351:1451–67.[CrossRef][Medline]
  8. Kuiper GG, Carlsson B, Grandien K, et al. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 1997;138:863–70.[Abstract/Free Full Text]
  9. Mosselman S, Polman J, Dijkema R. ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 1996;392:49–53.[CrossRef][Medline]
  10. Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 1997;277:1508–10.[Abstract/Free Full Text]
  11. Cowley SM, Hoare S, Mosselman S, Parker MG. Estrogen receptors {alpha} and ß form heterodimers on DNA. J Biol Chem 1997;272:19858–62.[Abstract/Free Full Text]
  12. Pettersson K, Grandien K, Kuiper GG, Gustafsson JA. Mouse estrogen receptor ß forms estrogen response element-binding heterodimers with estrogen receptor {alpha}. Mol Endocrinol 1997;11:1486–96.[Abstract/Free Full Text]
  13. Ogawa S, Inoue S, Watanabe T, et al. The complete primary structure of human estrogen receptor ß (hERß) and its heterodimerization with ER{alpha} in vivo and in vitro. Biochem Biophys Res Commun 1998;243:122–6.[CrossRef][Medline]
  14. Esslimani-Sahla M, Simony-Lafontaine J, Kramar A, et al. Estrogen receptor ß (ERß) level but not its ERß cx variant helps to predict tamoxifen resistance in breast cancer. Clin Cancer Res 2004;10:5769–76.[Abstract/Free Full Text]
  15. Hopp TA, Weiss HL, Parra IS, Cui Y, Osborne CK, Fuqua SA. Low levels of estrogen receptor ß protein predict resistance to tamoxifen therapy in breast cancer. Clin Cancer Res 2004;10:7490–9.[Abstract/Free Full Text]
  16. Skliris GP, Leygue E, Curtis-Snell L, Watson PH, Murphy LC. Expression of oestrogen receptor-ß in oestrogen receptor-{alpha} negative human breast tumours. Br J Cancer 2006;95:616–26.[CrossRef][Medline]
  17. Speirs V, Carder PJ, Lane S, Dodwell D, Lansdown MR, Hanby AM. Oestrogen receptor ß: what it means for patients with breast cancer. Lancet Oncol 2004;5:174–81.[CrossRef][Medline]
  18. Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 1998;139:4252–63.[Abstract/Free Full Text]
  19. Swedish Breast Cancer Cooperative Group. Randomized trial of two versus five years of adjuvant tamoxifen for postmenopausal early stage breast cancer. J Natl Cancer Inst 1996;88:1543–9.[Abstract/Free Full Text]
  20. Ryden L, Jonsson PE, Chebil G, et al. Two years of adjuvant tamoxifen in premenopausal patients with breast cancer: a randomised, controlled trial with long-term follow-up. Eur J Cancer 2005;41:256–64.[CrossRef][Medline]
  21. Lundin M, Lundin J, Helin H, Isola J. A digital atlas of breast histopathology: an application of web based virtual microscopy. J Clin Pathol 2004;57:1288–91.[Abstract/Free Full Text]
  22. Ferno M, Stal O, Baldetorp B, et al. Results of two or five years of adjuvant tamoxifen correlated to steroid receptor and S-phase levels. South Sweden Breast Cancer Group, and South-East Sweden Breast Cancer Group. Breast Cancer Res Treat 2000;59:69–76.[CrossRef][Medline]
  23. Baldetorp B, Bendahl PO, Ferno M, et al. Reproducibility in DNA flow cytometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates. Cytometry 1995;22:115–27.[CrossRef][Medline]
  24. Sigurdsson H, Baldetorp B, Borg A, et al. Indicators of prognosis in node-negative breast cancer. N Engl J Med 1990;322:1045–53.[Abstract]
  25. Isola J, Tanner M, Forsyth A, Cooke TG, Watters AD, Bartlett JM. Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization. Clin Cancer Res 2004;10:4793–8.[Abstract/Free Full Text]
  26. Khan J, Saal LH, Bittner ML, et al. Gene expression profiling in cancer using cDNA microarrays. Methods Mol Med 2002;68:205–22.[Medline]
  27. Saal LH, Troein C, Vallon-Christersson J, Gruvberger S, Borg A, Peterson C. BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data. Genome Biol 2002;3:SOFTWARE0003.[Medline]
  28. Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999;286:531–7.[Abstract/Free Full Text]
  29. Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery rate in behavior genetics research. Behav Brain Res 2001;125:279–84.[CrossRef][Medline]
  30. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116–21.[Abstract/Free Full Text]
  31. Saeed AI, Sharov V, White J, et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 2003;34:374–8.[Medline]
  32. Skliris GP, Parkes AT, Limer JL, Burdall SE, Carder PJ, Speirs V. Evaluation of seven oestrogen receptor ß antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue. J Pathol 2002;197:155–62.[CrossRef][Medline]
  33. Fuqua SA, Schiff R, Parra I, et al. Estrogen receptor ß protein in human breast cancer: correlation with clinical tumor parameters. Cancer Res 2003;63:2434–9.[Abstract/Free Full Text]
  34. Hall JM, McDonnell DP. The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 1999;140:5566–78.[Abstract/Free Full Text]
  35. Speirs V, Parkes AT, Kerin MJ, et al. Coexpression of estrogen receptor {alpha} and ß: poor prognostic factors in human breast cancer? Cancer Res 1999;59:525–8.[Abstract/Free Full Text]
  36. Jensen EV, Cheng G, Palmieri C, et al. Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci U S A 2001;98:15197–202.[Abstract/Free Full Text]
  37. Ferno M, Baldetorp B, Bendahl PO, et al. Recurrence-free survival in breast cancer improved by adjuvant tamoxifen-especially for progesterone receptor positive tumors with a high proliferation. Breast Cancer Res Treat 1995;36:23–34.[CrossRef][Medline]
  38. Gruvberger S, Ringner M, Chen Y, et al. Estrogen receptor status in breast cancer is associated with remarkably distinct gene expression patterns. Cancer Res 2001;61:5979–84.[Abstract/Free Full Text]
  39. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000;406:747–52.[CrossRef][Medline]
  40. van't Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415:530–6.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 JCOHome page
N. Honma, R. Horii, T. Iwase, S. Saji, M. Younes, K. Takubo, M. Matsuura, Y. Ito, F. Akiyama, and G. Sakamoto
Clinical Importance of Estrogen Receptor-{beta} Evaluation in Breast Cancer Patients Treated With Adjuvant Tamoxifen Therapy
J. Clin. Oncol., August 1, 2008; 26(22): 3727 - 3734.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. A. Greenberg, S. Somme, H. E. Russnes, A. D. Durbin, and D. Malkin
The Estrogen Receptor Pathway in Rhabdomyosarcoma: A Role for Estrogen Receptor-{beta} in Proliferation and Response to the Antiestrogen 4'OH-Tamoxifen
Cancer Res., May 1, 2008; 68(9): 3476 - 3485.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Pathol.Home page
S Borgquist, C Holm, M Stendahl, L Anagnostaki, G Landberg, and K Jirstrom
Oestrogen receptors {alpha} and show different associations to clinicopathological parameters and their co-expression might predict a better response to endocrine treatment in breast cancer
J. Clin. Pathol., February 1, 2008; 61(2): 197 - 203.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. R. Yang, R. M. Pfeiffer, M. Garcia-Closas, D. L. Rimm, J. Lissowska, L. A. Brinton, B. Peplonska, S. M. Hewitt, R. W. Cartun, D. Mandich, et al.
Hormonal Markers in Breast Cancer: Coexpression, Relationship with Pathologic Characteristics, and Risk Factor Associations in a Population-Based Study
Cancer Res., November 1, 2007; 67(21): 10608 - 10617.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplementary Data, Gruverberger-Saal, et al.
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gruvberger-Saal, S. K.
Right arrow Articles by Fernö, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gruvberger-Saal, S. K.
Right arrow Articles by Fernö, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online