Japanese Journal of Clinical Oncology Advance Access published online on October 11, 2007
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hym114
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© 2007 Foundation for Promotion of Cancer Research
Expression of Estrogen Receptor ß Wild-type and its Variant ERßcx/ß2 is Correlated with Better Prognosis in Breast Cancer
1 Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya
2 Breast and Endocrine Surgery, Kumamoto University, Kumamoto, Japan
For all reprints and correspondence: Hiroko Yamashita, Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: hirokoy{at}med.nagoya-cu.ac.jp
Received May 7, 2007; accepted July 23, 2007
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Abstract |
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 TOP Abstract INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Background: The clinical value of estrogen receptor (ER) ß and its variants has been investigated. However, reported results have frequently been discordant.
Methods: We investigated mRNA and protein expression of ERß wild type (ERß1) and its variant, ERßcx/ß2, by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry in 150 breast cancer tissues, and analyzed association between their expression and clinicopathological factors and prognosis.
Results: ERß1 mRNA expression was significantly correlated with progesterone receptor expression and low histological grade. ERß1 protein expression was significantly correlated with small tumor size, negative lymph node status and low histological grade. ERßcx/ß2 protein expression was significantly correlated with ER
expression and low histological grade. Patients with high expression of ERß1 or ERßcx/ß2 had a significantly better disease-free and overall survival than those with low expression. In multivariate analysis, ERßcx/ß2 mRNA expression was identified as a prognostic factor in disease-free and overall survival.
Conclusion: Our results indicate that ERßcx/ß2 mRNA expression is an independent prognostic factor in breast cancer. ERß expression status, both wild-type and the variant cx/ß2, might represent significant predictors of breast cancer prognosis.
Key Words: breast cancer • estrogen receptor ß • prognosis
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INTRODUCTION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Estrogens are important for growth and development of normal human mammary tissues. However, they are also thought to promote the growth of breast tumors through their mitogenic effects on breast cancer cells. This concept led to the development of selective estrogen receptor (ER) modulators, such as tamoxifen and toremifene, as endocrine therapy for breast cancer. These modulators bind to the estrogen receptor alpha (ER
), a member of the nuclear receptor superfamily, and thereby inhibit estrogenic action. For many years only one gene for ER had been recognized, but in 1996 a second ER subtype, estrogen receptor beta (ERß), was identified in rats (1), mice (2) and humans (3). ERß shows strong homology with ER in the DNA-binding domain (96%) and in the ligand-binding domain (53%), but little elsewhere (4). In particular it binds specifically to 17ß-estradiol with a high affinity (5). More recently, variants of ERß have been identified with, for example, exon deletion (6,7), insertion (7–9), or C-terminal splicing (5,10). ERßcx/ß2 is one such variant that has been characterized; it is C-terminal splicing, and has a poor binding affinity for 17ß-estradiol (5,10). However, ERßcx/ß2 forms heterodimers with ER, as well as wild-type ERß (ERß1), and mediates significant dominant negative activity against ER transactivation (5). This suggests that ERß1 and its variants, such as ERßcx/ß2, have different functions and behave in a manner different from ER in breast cancer.
Several groups have investigated the expression of ERß and its variants using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique (6,7,11–19) and immunohistochemistry (IHC) or western blotting (12–14,16,18–21). However, the results have been rather inconsistent. In a previous study, we used IHC techniques to show that breast cancer patients with ERß1-positive tumors tended to have a better prognosis than patients who were ERß1-negative. Using the RT-PCR method, some groups reported that ERß expression was increased in ER-negative tumors (7,15). More recently, expression of ERß1 and its variants has been examined separately by several investigators. One group showed that ERß1 mRNA expression was correlated with tumor grade, and that the variants ERßcx/ß2 and ERß5 were up-regulated during breast tumorigenesis and tumor progression (22). Using real-time RT-PCR, another group has reported that ERß mRNA was down-regulated during breast carcinogenesis and that changes in the relative proportions of ERß variants were implicated in this process (15). On the other hand, other investigators also showed that ERß expression was not associated with ER (16–18).
In this study, we investigated the expression of ERß1 and ERßcx/ß2 mRNA using quantitative real-time RT-PCR and the respective protein expression using IHC in 150 breast cancer patients and examined its relationship with clinicopathological factors and prognosis.
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PATIENTS AND METHODS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Patients and Tumor Samples
Primary invasive breast carcinoma specimens were obtained by surgical excision from 150 female patients at Nagoya City University Hospital between 1993 and 2000. Informed consent was obtained from all patients before surgery. The study protocol was approved by the institutional review board and conformed with the guidelines of the 1975 Declaration of Helsinki. The samples in this study were chosen from the continuous series of invasive carcinoma tissues. The median age of the patients was 53 years (range 34–88 years). The patients' tumors were classified with the International Union Against Cancer (UICC) staging system as follows: 39 cases were classified as stage I, 93 cases as stage II, and 16 cases as stage III. Patients were graded histopathologically according to the modified Bloom and Richardson method proposed by Elston and Ellis (23). As postoperative adjuvant treatment, tamoxifen was given to patients with ER- and/or progesterone receptor (PgR)-positive tumors. One of the following three chemotherapy regimens was administered, depending on tumor stage: oral 5-fluorouracil (5-FU 300 mg/m2), CMF (cyclophosphamide 100 mg p.o., days 1–14; methotrexate 40 mg/m2 i.v., days 1 and 8; 5-FU 500 mg/m2 i.v., days 1 and 8), or CEF (cyclophosphamide 500 mg/m2 i.v., epirubicin 60 mg/m2 i.v., 5-FU 500 mg/m2 i.v., every 3 weeks). Since 1995, postoperative treatment has been done with reference to the recommendation of St Gullen. After surgery, 42 patients (28%) received no additional therapy. Of the remaining 108 patients, 55 (37%) received systemic therapy consisting of endocrine therapy alone, 23 (15%) received chemotherapy alone and 30 (20%) received combined endocrine therapy and chemotherapy. Patients were observed for disease recurrence and death at least once every 6 months for 5 years after surgery and yearly thereafter. The median follow-up period was 58 months (range, 22–90 months). Samples were snap-frozen in liquid nitrogen and stored at –80°C until RNA extraction.
In 19 of the 150 cases, there was a recurrence, including lymph node metastasis and distant metastasis, and these patients received endocrine therapy, such as tamoxifen or a luteinizing hormone-releasing hormone (LHRH) analog with tamoxifen. All these patients had ER and/or PgR positive tumors. Recurrence of their disease occurred either after adjuvant tamoxifen had been stopped, in which case they were re-challenged with tamoxifen, or while they were taking adjuvant tamoxifen. Patients were assessed monthly for clinical response, which was defined according to World Health Organization criteria as complete response, partial response, no change (stable disease) or progressive disease. Clinical benefit was recorded in seven out of 19 recurrent cases: complete response (one patient), partial response (four patients) and stable disease continuing over 24 weeks (two patients). These patients were classified as ‘responders’ to endocrine therapy. The remaining 12 patients with recurrent tumors did not respond to endocrine therapy, and these were classified as ‘nonresponders’.
Isolation of Total RNA and Reverse Transcription
Total RNA was extracted from approximately 500 mg of frozen breast cancer tissue using TRIZOL reagent (Life Technologies Inc., Tokyo, Japan) according to the manufacturer's instructions. Reverse transcription reactions were performed as previously described (24). Briefly, each 20 µl cDNA synthesis reaction contained 1 µg of total RNA, buffer (250 mM Tris–HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2), 0.1 M dithiothreitol (DTT), 1 mM each of deoxynucleotide triphosphates (dNTP), 25 units of RNAguard RNase inhibitor (Amersham Pharmacia Biotech Inc., Tokyo, Japan), 200 units of Superscript II reverse transcriptase (Life Technologies Inc.) and 100 ng of pd(N)6 random hexamer (Amersham Pharmacia Biotech Inc.).
Primers and Probes
Blast searches (GenBank) were used to confirm the specificity of nucleotide sequences chosen for the primers and probes and to confirm the absence of DNA polymorphism. To avoid detection of contaminating genomic DNA, the primers were targeted to the junction between different exons. The sequences of the primers were as follows: sense primer, 5'-GAT GCT TTG GTT TGG GTG AT-3' (1401–1420); antisense primer, 5'-CTT GTT ACT CGC ATG CCT GA-3' (1492–1511) for ERß1, and sense primer, 5'-GAT GCT TTG GTT TGG GTG AT-3' (2578–2597); antisense primer, 5'-GTT TGA GAG GCC TTT TCT GC-3' (2683–2702) for ERßcx/ß2. The donor probe for ERß1, fluorescein-labeled at its 3' end, was 5'-TCT CCT CCC AGC AGC AAT CCA-3'; the acceptor probe for ERß1, with LC Red 640 at its 5' end, was 5'-GCG CCT GGC TAA CCT CCT GAT-3'. The donor probe for ERßcx/ß2 was 5'-CAA TCC ATG CGC CTG GCT AAC-3'; and the acceptor probe for ERßcx/ß2 was 5'-TCC TGA TGC TCC TGT CCC ACG-3'. The close proximity of the two probes hybridized to adjacent internal sequences for each specific primer allows specific amplicons to be detected, which provides sequence-specific detection of the amplified target sequence.
To ensure the fidelity of mRNA extraction and reverse transcription, all samples were subjected to PCR amplification with oligonucleotide primers and probes specific for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and normalized. GAPDH primers were as follows: forward primer, 5'-AAA TCA AGT GGG GCG ATG CTG-3' and reverse primer 5'-GCA GAG ATG ATG ACC CTT TTG-3'. The sequences of GAPDH probes for LightCycler real-time PCR were 5'-AGA AGG CTG GGG CTC ATT TGC AGG G-3' and 5'-GTC CAC TGG CGT CTT CAC CAC CAT G-3'. All primers and probes were purchased from the Japanese Gene Institute (Saitama, Japan).
Real-time PCR with Hybridization Probes
Real-time PCR was performed using a LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) as previously reported (25). The PCR reaction for ERß1 and ERßcx/ß2 was carried out in a final volume of 20 µl containing 2.4 µl of 25 mM MgCl2; 0.5 µl of 20 pmol/µl sense primer and antisense primer; 0.4 µl of 10 pmol/µl donor and acceptor probe; 2 µl of PCR master mix; 1.5 µl of cDNA, and made up to 20 µl with water. After an initial denaturation step at 95°C for 60 s, temperature cycling was initiated. Each cycle consisted of denaturation at 95°C for 0 s, hybridization at 57°C for 5 s, and elongation at 72°C for 6 s. The fluorescence signal was acquired at the end of the hybridization step. A total of 45 cycles were performed. The cycling conditions for GAPDH were as follows: initial denaturation at 95°C for 60 s, followed by 50 cycles at 95°C for 0 s, 60°C for 5 s and 72°C for 8 s.
Standard Curves and Presentation of Results
For each PCR run, a standard curve was constructed from serial dilutions of cDNA obtained from COS-7 cells transfected with either expression plasmid encoding ERß1 or ERßcx/ß2 (kindly donated by Masami Muramatsu, Saitama, Japan) for detection of ERß1 and ERßcx/ß2, respectively. The levels of expression of ERß1 and ERßcx/ß2 were given as relative copy numbers normalized against GAPDH mRNA. A nontemplate negative control was included in each experiment. All of the nontemplate negative controls, the standard cDNA dilutions from the COS-7 cells and the tumor samples were assayed in duplicate.
Generation of Specific antibodies for ERß Proteins
To detect specific ERß1 and ERßcx/ß2 proteins, rabbit polyclonal antibodies were generated against synthesized peptides of the C-terminal region of ERß1 (CSPAEDSKSKEGSQNPQSQ) and ERßcx/ß2 (MKMETLLPEATMEQ), according to the method of Ogawa et al. (5) and purified on affinity columns bound with each synthetic peptide as described previously (26). To confirm the specificity of these polyclonal antibodies, immunoblot analysis was performed using COS-7 cells transfected with either expression plasmid encoding ERß1 or ERßcx/ß2 (kindly donated by Masami Muramatsu, Saitama, Japan) as previously described (26). Immunoblotting with specific anti-ERß antibodies demonstrated that the polyclonal antibody for ERß1 detected a specific band at 60 kDa only in the lysates of COS-7 cells transfected with an ERß1 expression plasmid but not in those transfected with an ERßcx/ß2 expression plasmid. Conversely, the polyclonal antibody for ERßcx/ß2 detected a specific band at 55 and 51 kDa only in the lysates of COS-7 cells transfected with an ERßcx/ß2 expression plasmid but not in those transfected with an ERß1 expression plasmid, as described previously by Ogawa et al. (5).
Immunohistochemical Staining of ER, PgR, ERß1, and ERßcx/ß2
Immunohistochemical staining of ER, PgR, ERß1 and ERßcx/ß2 was done using monoclonal mouse antihuman ER antibody (1D5, DAKO) at 1:100 dilution for ER, monoclonal mouse antihuman PgR antibody (636, DAKO) at 1:100 dilution for PgR, polyclonal rabbit anti-ERß1 antibody at 1:10 000 dilution for ERß1 and polyclonal rabbit anti-ERßcx/ß2 antibody at 1:2000 dilution for ERßcx/ß2 as previously described (26). The expression of proteins was estimated in accordance with the procedure of Allred et al. (27). In brief, a proportion score represented the estimated proportion of tumor cells staining positive, as follows: 0 (none); 1 (<1/100), 2 (1/100 to 1/10), 3 (1/10 to 1/3), 4 (1/3 to 2/3) and 5 (>2/3). Any brown nuclear staining in invasive breast cancer cells counted towards the proportion score. An intensity score represented the average intensity of the positive cells, as follows: 0 (none), 1 (weak), 2 (intermediate) and 3 (strong). The proportion and intensity scores were then added to obtain a total score, which could range from 0 to 8. Staining status by immunohistochemistry was then assessed as negative (scores of 0 and 2) or positive (scores of 3–8) for ER and PgR, and assessed as negative (scores of 0, 2 and 3) or positive (scores of 4–8) for ERß1 and ERßcx/ß2; the cut-off level was set between a score of 3 and 4, in order to detect the most significant difference between patient groups, which will be described in the Results section.
Statistical Analysis
Correlation between mRNA expression of ERß1 and ERßcx/ß2 and correlation between mRNA and protein expression of ERß1 and ERßcx/ß2 were evaluated using the Spearman rank correlation test. Correlations between the expression of ERß1, ERßcx/ß2 and clinicopathological factors were evaluated using the 
2 test. The Mann–Whitney U-test was used for the statistical analyses of the correlation between IHC scores and ERß mRNA expression levels, and of the correlation between ERß mRNA expression levels and response to tamoxifen. Disease-free and overall survival curves were generated by the Kaplan–Meier method and verified with the Breslow–Gehan–Wilcoxon test. Cox's proportional hazards model was used for univariate and multivariate analyses of prognostic values. Differences were considered significant when a P < 0.05 was obtained.
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RESULTS |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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Evaluation of ERß1 and ERßcx/ß2 mRNA Expression in Breast Cancer Tissues
The amount of ERß1 mRNA in the tissue samples from 150 patients ranged from 12 to 846 relative copy numbers (mean, 104.2), whereas the amount of ERßcx/ß2 mRNA ranged from 0 to 298 relative copy numbers (mean, 27.1). To identify a clinically meaningful cutoff point for levels of ERß1 and ERßcx/ß2 mRNA expression that could be used in disease prognosis analysis, various levels of ERß1 and ERßcx/ß2 mRNA expression were tested using the Kaplan–Meier method and verified by the Breslow–Gehan–Wilcoxon test. When analyzing disease-free and overall survival, the cutoff points for the levels of ERß1 and ERßcx/ß2 mRNA were set at 95 and 16, respectively. Interestingly, ERß1 mRNA expression was strongly correlated with ERßcx/ß2 mRNA expression (P < 0.0001; Fig. 1).
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Immunohistochemical Staining of ERß1 and ERßcx/ß2 Protein
Staining with IHC techniques was used to investigate the expression of ERß1 and ERßcx/ß2 proteins. Various patterns of the proportion and intensity of positive nuclear staining were observed in the tissue samples from the 150 breast cancer patients. In addition, in some samples, normal mammary duct cells were also stained with ERß1 and ERßcx/ß2 polyclonal antibodies. However, because many samples in the study did not contain any normal tissue part at all, it is not possible to evaluate the significance of this staining of normal mammary duct (Fig. 2). ERß1 protein expression was strongly correlated with ERß1 mRNA expression (P = 0.0016). ERßcx/ß2 protein expression was also significantly correlated with ERßcx/ß2 mRNA expression (P = 0.035).
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Staining status by IHC was assessed as negative (scores 0, 2 and 3) or positive (scores 4–8) for ERß1 and ERßcx/ß2 using a cut-off level that allowed us to obtain the most significant difference between patient groups in disease-free and overall analyses. Out of these 150 samples, 103 (69%) and 76 (51%), respectively, stained positive for ERß1 and ERßcx/ß2 polyclonal antibodies.
Relationship between mRNA and Protein Expression of ERß1 and ERßcx/ß2 and Clinicopathological Factors
ERß1 mRNA expression was significantly correlated with positive PgR status (P = 0.0004) and low histological grade (P = 0.037; Table 1). Whereas ERß1 protein expression was strongly correlated with small tumor size (P = 0.029), negative lymph node status (P = 0.03) and low histological grade (P = 0.007). ERßcx/ß2 protein expression was significantly associated with positive ER status (P = 0.040) and low histological grade (P = 0.007), although ERßcx/ß2 mRNA expression did not correlate with clinicopathological factors (Table 1).
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Patients whose Tumor Expressed Higher Levels of ERß1 and ERßcx/ß2 Expression had Better Disease-free and Overall Survival
The correlation between ERß1 and ERßcx/ß2 mRNA expression and prognosis is shown in Fig. 3. Patients with high expression of ERß1 mRNA experienced significantly longer disease-free survival (Fig. 3a) and overall survival (Fig. 3b) than those with low expression (P = 0.02 and P = 0.04, respectively). Similarly, high expression of ERßcx/ß2 mRNA was also significantly associated with better disease-free survival (Fig. 3c) and overall survival (Fig. 3d; P = 0.005 and P = 0.005, respectively).
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The correlation between ERß1 and ERßcx/ß2 protein expression and prognosis is shown in Fig. 4. Patient samples staining positive for ERß1 protein expression experienced significantly longer disease-free survival (Fig. 4a) and overall survival (Fig. 4b) than those with negative expression (P = 0.01 and P = 0.002, respectively). Patients who were positive for the expression of ERßcx/ß2 protein had significantly better overall survival than negative patients (Fig. 4d, P = 0.03), although there was no benefit in favor of ERßcx/ß2-positive patients with respect to disease-free survival (Fig. 4c).
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ERßcx/ß2 mRNA Expression is an Independent Prognostic Factor of Disease-free and Overall Survival in Breast Cancer
Univariate analysis of the impact of clinicopathological parameters on disease-free survival revealed that ERß1 and ERßcx/ß2 mRNA expression status, PgR status, lymph node status and histological grade were all statistically significant prognostic factors, although tumor size and ER
status were not significant (Table 2). Moreover, multivariate analysis identified ERßcx/ß2 mRNA expression (P = 0.002) as well as PgR and lymph node status as a statistically significant prognostic factor for disease-free survival (Table 2). For overall survival, univariate analysis (Table 3) showed significant associations between overall survival and ERßcx/ß2 mRNA expression (P = 0.01), PgR status (P = 0.0002), lymph node status (P = 0.001) and histological grade (P = 0.0005). In multivariate analysis, patients with high ERßcx/ß2 mRNA expression (P = 0.03), positive PgR status and negative lymph node status had significantly increased overall survival (Table 3). We concluded from these analyses that ERßcx/ß2 mRNA expression is an independent prognostic factor for disease-free and overall survival in breast cancer.
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Tamoxifen Response and ERß1 and ERßcx/ß2 mRNA Expression
There were 19 patients with recurrent breast cancer who received tamoxifen for metastatic breast cancer; seven (36.8%) patients responded. We analyzed whether ERß1 and ERßcx/ß2 mRNA expression in the primary breast tumors affected the response to tamoxifen. No significant differences were found between ‘responders’ and ‘nonresponders’ in the levels of expression of either ERß1 mRNA (P = 0.44) or ERßcx/ß2 mRNA (P = 0.86; Fig. 5).
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DISCUSSION |
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TOPAbstractINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONReferences |
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In the present study, we showed that elevated levels of expression of both ERß1 and ERßcx/ß2 were significantly associated with superior disease-free and overall survival. These results were in agreement with our earlier work, where we showed that patients with ERß1-positive cancers tended to have a better prognosis (12,28). Palmieri et al. also showed that patients whose tumors expressed ERßcx/ß2 had a longer survival time (29). Furthermore, in multivariate analysis, we found that a high level of expression of ERßcx/ß2 mRNA was a significant prognostic factor, indicating that it is a superior prognostic factor in breast cancer than ER
. It is not clear why ERßcx/ß2 is a better prognostic factor than ERß1 in our analysis, although ERß1 expression is strongly correlated with ERßcx/ß2 expression. Functional differences between ERß1 and ERßcx/ß2 in breast cancer, such as response to endocrine therapy, may affect the prognosis. Our data suggest that, in breast cancer, ERß expression is correlated with low malignancy. Roger et al. reported that the ERß/ER ratio in breast cancer was lower than that in benign disease, and that the ratio in breast benign disease was lower than that in breast normal tissue. This led them to propose that the expression of ERß markedly decreases in the early stage of mammary carcinogenesis (30). It is hypothesized that both ERß1 and ERßcx/ß2 might act as repressors of ER function, and that this would result in the suppression of hormone-dependent tumor growth. We showed that, while high expression of ERßcx/ß2 protein was correlated with superior overall survival benefits, it was not associated with superior disease-free survival. This could suggest that the patients with high protein expression ERßcx/ß2 might respond to endocrine therapy after recurrence in order to further improve their survival rate.
Zhao et al. recently reported that ERßcx/ß2 negatively regulates the transactivation of ER in human breast cancer cells and that expression of ERßcx/ß2 significantly correlated with ER-negative phenotype in human breast cancer samples, although their study only included a total of 37 individual human breast cancer samples (31). Our results also showed that ERßcx/ß2 protein expression positively correlated with ER expression in 150 breast cancer samples, although no correlation was found between ERßcx/ß2 mRNA expression and ER status. On the other hand, because ERß is expressed not only ER-positive phenotype but also ER-negative phenotype, ERß may have the potential to become a therapeutic target in the specific subcohort of ER-negative breast cancers (32).
We analyzed the correlation between the expression of ERß and response to endocrine therapy in breast cancer. In this study we analyzed 19 cases treated by tamoxifen, but there was no significant correlation between ERß expression and response to tamoxifen. Hopp et al. showed that high levels of ERß predict an improved survival in patients treated with adjuvant tamoxifen therapy (33). Another study showed that low ERß level was an additional independent marker to predict tamoxifen resistance (34). Furthermore, from previous data of 11 evaluated cases, we showed that tamoxifen responders tended to express higher level of ERß protein than nonresponders (35). However, we reported in another study that protein expression of ERß1 and ERßcx/ß2 did not affect response to endocrine therapy in 75 metastatic breast cancer patients (27). Other groups have reported that ERß expression was not predictive of response to antiestroge therapy (36,37). Murphy and Watson recently reported that there were two ERß expressing groups, one in which ERß was co-expressed with ER and the other where ERß was expressed alone, and that reports supported the hypothesis that increased expression of ERß was associated with increased likelihood of response to endocrine therapy (38). However, the association between presence of ERß and response to endocrine therapy is still controversial, and therefore further studies are required.
In conclusion, we carried out quantitative real-time RT-PCR and IHC to examine the expression of ERß1 and ERßcx/ß2 and have demonstrated that breast cancer patients with positive expression of ERß1 and ERßcx/ß2 had a better prognosis than those with negative expression. Moreover, our study suggests that the expression of ERßcx/ß2 mRNA, in particular, is a strong candidate for consideration as a positive prognostic factor, together with confirmed factors such as ER expression, lymph node status and histological grade. However, the significance of ERß status is still controversial and further analysis of the role it plays in the pathogenesis of breast cancer is required. A better understanding of its function will ultimately allow for more accurate treatment for patients with this disease.
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Acknowledgments |
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We are grateful for the technical assistance of Mrs Mariko Nishio, Second Department of Surgery, Nagoya City University Medical School, Japan. This work was supported in part by a grant-in-aid (project numbers 14370362) for scientific research from the Ministry of Education, Sports, Science and Technology of Japan.
Conflict of interest
None declared.
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Footnotes |
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Abbreviations: ER, estrogen receptor; IHC, immunohistochemistry; PgR, progesterone receptor; RT-PCR, reverse transcriptase-polymerase chain reaction.
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References |
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