CA IX is an independent prognostic marker in premenopausal breast cancer patients with one to three positive lymph nodes and a putative marker of radiation resistance.

PURPOSE: Hypoxia in breast cancer is associated with poor prognosis and down-regulation of the estrogen receptor. Carbonic anhydrase IX (CA IX) is a hypoxia-inducible gene that has been associated with poor outcome in many epithelial cancers. Previous studies of CA IX in breast cancer have been carried out on mixed cohorts of premenopausal and postmenopausal patients with locally advanced disease and varying treatment regimens. We examined the potential prognostic and predictive role of CA IX in premenopausal breast cancer patients. EXPERIMENTAL DESIGN: Using tissue microarrays, we analyzed CA IX expression in 400 stage II breast cancers from premenopausal women. The patients had previously participated in a randomized control trial comparing 2 years of tamoxifen to no systemic adjuvant treatment. Median follow-up was 13.9 years. RESULTS: CA IX expression correlated positively with tumor size, grade, hypoxia-inducible factor 1alpha, Ki-67, cyclin E, and cyclin A2 expression. CA IX expression correlated negatively with cyclin D1, estrogen receptor, and progesterone receptor. CA IX expression was associated with a reduced relapse-free survival (P=0.032), overall survival (P=0.022), and breast cancer-specific survival (P=0.005). Multivariate analysis revealed that CA IX was an independent prognostic marker in untreated patients with one to three positive lymph nodes (hazard ratio, 3.2; 95% confidence interval, 1.15-9.13; P=0.027). CONCLUSION: CA IX is marker of poor prognosis in premenopausal breast cancer patients and it is an independent predictor of survival in patients with one to three positive lymph nodes. As all these patients received locoregional radiation therapy, CA IX may be associated with resistance to radiotherapy.     

HIF-1alpha and CA IX staining in invasive breast carcinomas: prognosis and treatment outcome

Hypoxia stabilizes HIF-1alpha (Hypoxia Inducible Factor-1alpha), which then triggers the expression of several genes involved in many aspects of cancer progression, including metabolic adaptation, cell survival and angiogenesis. The aim of our study was to evaluate the impact of HIF-1alpha and CA IX (carbonic anhydrase IX) (one of its target genes) expression on prognosis and treatment outcome of patients with breast cancer. Because of the extreme O2-dependent instability of the protein, we first validated HIF-1alpha staining using xenograft tumours that were subjected to experimental conditions mimicking surgical clamping or sitting at room temperature under normoxic conditions after surgical excision but before fixation. Afterwards, the immunohistochemical staining of HIF-1alpha and CA IX was evaluated in 132 invasive breast carcinomas with a 10-year follow-up, and correlated to classical clinicopathological parameters and response to adjuvant therapy. No significant correlation was found between tumour size or nodal status and the expression of HIF-1alpha or CA IX. Statistically significant association was found between HIF-1alpha or CA IX staining and the grade, hormonal receptors loss and the presence of carcinoma in situ. Overexpression of HIF-1alpha and CA IX correlates with a poor prognosis in breast cancer. We show that HIF-1alpha is an independent prognostic factor for distant metastasis-free survival and disease-free survival in multivariate analysis. Furthermore, overexpression of HIF-1alpha or CA IX correlates with a poor outcome after conventional adjuvant therapy. CA IX is, however, a weaker prognostic and predictive factor than HIF-1alpha, and its association with HIF-1alpha does not modify the survival curve neither response to therapy, compared to HIF-1alpha alone.     

Cyclin D1 overexpression is a negative predictive factor for tamoxifen response in postmenopausal breast cancer patients

Antioestrogen treatment by tamoxifen is a well-established adjuvant therapy for oestrogen receptor-alpha (ERalpha) positive breast cancer. Despite ERalpha expression some tumours do not respond to tamoxifen and we therefore delineated the potential link between the cell cycle regulator and ERalpha co-factor, cyclin D1, and tamoxifen response in a material of 167 postmenopausal breast cancers arranged in a tissue array. The patients had been randomised to 2 years of tamoxifen treatment or no treatment and the median follow-up time was 18 years. Interestingly in the 55 strongly ERalpha positive samples with moderate or low cyclin D1 levels, patients responded to tamoxifen treatment whereas the 46 patients with highly ERalpha positive and cyclin D1 overexpressing tumours did not show any difference in survival between tamoxifen and no treatment. Survival in untreated patients with cyclin D1 high tumours was slightly better than for patients with cyclin D1 low/moderate tumours. However, there was a clearly increased risk of death in the cyclin D1 high group compared to an age-matched control population. Our results suggest that cyclin D1 overexpression predicts for tamoxifen treatment resistance in breast cancer, which is line with recent experimental data using breast cancer cell lines and overexpression systems.     

p27Kip1 is a predictive factor for tamoxifen treatment response but not a prognostic marker in premenopausal breast cancer patients

The cell-cycle regulating protein p27(Kip1) (p27) has dual roles by acting as both a cdk inhibitor and as an assembly factor for different cdk complexes. Loss of p27 has been linked to malignant features in tumours; however, the exact role of p27 deregulation in breast cancer regarding prognostic and treatment predictive information has not been fully clarified. We have evaluated p27 expression in 328 primary, Stage II breast cancers from premenopausal patients who had been randomised to either tamoxifen treatment or no adjuvant treatment after surgery. p27 was associated with the oestrogen receptor and cyclin D1, and p27 downregulation was associated with high proliferation. There was no association between recurrence-free survival (RFS) and p27 (HR = 0.800, 95% CI 0.523-1.222, p = 0.300), indicating that p27 is not a prognostic marker. The predictive value of p27 was analysed by comparing RFS in tamoxifen-treated and untreated patients in subgroups of low and high p27 expression (HR = 0.747, 95% CI 0.335-1.664, p = 0.474 and HR = 0.401, 95% CI 0.240-0.670, p < 0.001, respectively). Only patients with p27-high tumours benefited from tamoxifen (multivariate interaction analysis p = 0.034). Our study suggests that p27 downregulation is associated with tamoxifen resistance in premenopausal breast cancer but is not linked to impaired prognosis.     

Effects of cyclin D1 gene amplification and protein expression on time to recurrence in postmenopausal breast cancer patients treated with anastrozole or tamoxifen: a TransATAC study

INTRODUCTION: Gene amplification of CCND1 is observed in a subgroup of breast cancers with poor prognosis, whereas overexpression of the protein cyclin D1 has been linked to both worse and better clinical outcome. CCND1 amplification and protein overexpression have also been associated with resistance to treatment with tamoxifen or even to a potentially detrimental effect of tamoxifen. METHODS: To clarify these challenging and partly contrasting treatment predictive and prognostic links for cyclin D1 we analysed a large cohort of postmenopausal breast cancer patients randomised to receive either adjuvant anastrozole or tamoxifen, as part of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. The CCND1 amplification status and protein expression of cyclin D1 were assessed by chromogenic in situ hybridisation and immunohistochemistry, respectively, in 1,155 postmenopausal, oestrogen-receptor-positive breast cancer patients included in the TransATAC substudy. RESULTS: Amplification of CCND1 was observed in 8.7% of the tumours and was associated with increased risk of disease recurrence (hazard ratio = 1.61; 95% confidence interval, 1.08 to 2.41) after adjustment for other clinicopathological parameters. In contrast, nuclear expression of cyclin D1 protein was associated with decreased recurrence rate (hazard ratio = 0.6; 95% confidence interval, 0.39 to 0.92). The intensity of nuclear or cytoplasmic expression was not of prognostic value. There was no significant interaction between cyclin D1 status and treatment efficacy, ruling out any major detrimental effect of tamoxifen in CCND1-amplified postmenopausal breast cancer. CONCLUSIONS: In summary, CCND1 amplification and low nuclear expression of cyclin D1 predicted poor clinical outcome in postmenopausal breast cancer patients treated with either anastrozole or tamoxifen. TRIAL REGISTRATION: Current Controlled Trials ISRCTN18233230.     

Hypoxia-inducible factor-1alpha expression predicts a poor response to primary chemoendocrine therapy and disease-free survival in primary human breast cancer.

PURPOSE: To investigate the relationship of hypoxia-inducible factor-1alpha (HIF-1alpha) tumor expression in predicting the response to epirubicin and disease-free survival (DFS) in patients with breast cancer enrolled in a single institution trial of primary anthracycline and tamoxifen therapy. EXPERIMENTAL DESIGN: The expression of HIF-1alpha was assessed by immunohistochemistry in 187 patients with T(2-4) N(0-1) breast cancer enrolled in a randomized trial comparing four cycles of single agent epirubicin versus epirubicin + tamoxifen as primary systemic treatment. All patients postoperatively received four cycles of the four weekly i.v. CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Patients with estrogen receptor (ER)-positive primary tumors also underwent 5 years of treatment with adjuvant tamoxifen. Carbonic anhydrase IX (CAIX) was also scored as a marker of HIF activity. RESULTS: Overall response to therapy progressively decreased with increasing tumor HIF-1alpha (P < 0.05), and HIF-1alpha was an independent predictor of response (P < 0.048). HIF-1alpha expression was also associated with a significantly shorter DFS (P < 0.02) in all patients and in ER-positive but not in ER-negative patients. Furthermore, CAIX positivity conferred a significantly shorter DFS (P = 0.02) compared with CAIX-negative tumors in patients with HIF-1alpha-negative tumors. CONCLUSIONS: HIF-1alpha expression in patients with breast cancer is a marker of poor therapy response and outcome, especially in ER-positive patients. The combination of two hypoxia markers has greater utility than assessing just one, and patients with hypoxia markers in their tumors may be suitable for administration of drugs that reduce HIF-1alpha expression and increase oxygen delivery to the tumor bed before starting neoadjuvant therapies.     

细胞周期蛋白D1、CDK4在乳腺癌中的表达及其临床意义

目的：研究乳腺癌组织周期蛋白D1、CDK4的蛋白表达定位、表达水平,及与P21蛋白表达、与临床病理指标的关系及其预后意义。方法：①应用免疫组织化学染色方法,检测106例乳腺癌组织石蜡切片上的周期蛋白D1、CDK4的蛋白定位和蛋白表达,比较其与P21的蛋白表达,与临床病理学指标,如病理类型、组织学分型、组织学分级、淋巴结状态、雌激素受体状态、TNM分期等之间的关系;②对周期蛋白D1、CDK4、P21及临床病理指标如肿瘤的组织学分级、淋巴结状态、TNM分期、ER状态等,应用Kaplan-Meier法Log-rank检验进行单因素生存分析,应用Cox比例风险模型进行多因素生存分析。结果：①周期蛋白D1表达定位于细胞核、CDK4的表达位于细胞质和细胞核,周期蛋白D1的阳性率为30.2%,CDK4的阳性率为53.8%。②周期蛋白D1及CDK4的表达与乳腺癌的病理诊断组织学分型、组织学分级、ER状态、临床TNM分期、淋巴结状态、P21蛋白表达水平无关（P＞0.05)。周期蛋白D1的蛋白表达与DCK4的蛋白表达相关（P＜0.05)。③单因素分析提示影响140个月无瘤生存率及总生存率的因素有：临床TNM分期、组织学分级、腋淋巴结状态、周期蛋白D1的蛋白表达（P＜0.05)。在无腋淋巴结转移亚组周期蛋白D1蛋白表达单因素生存分析无统计学意义（P＞0.05),在腋淋巴结有转移亚组周期蛋白D1是影响140个月无瘤生存率和总生存率的因素（P＜0.01)。④多因素生存分析结果表明周期蛋白D1、P21蛋白表达、组织学分级是影响乳腺癌患者无瘤生存率和总生存率的独立预后因素（P＜0.05)。结论：周期蛋白D1、P21的蛋白表达水平及组织学分级可能是判断乳腺癌术后生存的有效指标,综合应用这些指标可能更有帮助。     

Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma

Hypoxia and pH influence gene expression in tumours, and it is becoming increasingly clear that the pattern of genes expressed by a tumour determines its growth and survival characteristics. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the cellular response to hypoxia and high HIF-1 expression has been identified as a poor prognostic factor in tumours. Recently, we identified the tumour-associated carbonic anhydrases (CA), CA9 and CA12 as hypoxia-inducible in tumour cell lines. Furthermore, we identified CA IX to be a poor prognostic factor in breast cancer. The aim of this study was to assess the prognostic significance of CA XII. CA XII expression was studied by immunohistochemistry in a series of 103 cases of invasive breast cancer and any association with recognised prognostic factors or relation with the outcome was examined. CA XII expression was present in 77 out of 103 (75%) cases and was associated with lower grade (P=0.001), positive estrogen receptor status (P<0.001), and negative epidermal growth factor receptor status (P<0.001). Furthermore, although CA XII expression was associated with an absence of necrosis (P<0.001), expression of CA XII in some high-grade tumours was induced in regions directly adjacent to morphological necrosis. Additionally, using univariate analysis, CA XII positive tumours were associated with a lower relapse rate (P=0.04) and a better overall survival (P=0.01). In conclusion, CA XII expression is influenced both by factors related to differentiation and hypoxia in breast cancer in vivo and CA XII expression is associated with a better prognosis in an unselected series of invasive breast carcinoma patients.     

Role of cyclin D1 in ErbB2-positive breast cancer and tamoxifen resistance

Cyclin D1 plays an important role in the regulation of the G1 phase in the cell cycle. In mammary epithelial cells the expression of cyclin D1 is regulated through the oestrogen receptor and via ErbB2 signalling. Here we investigated the prognostic significance of cyclin D1 among 230 breast cancer patients randomised for tamoxifen, CMF chemotherapy and radiotherapy. The importance of combined cyclin D1 and ErbB2 overexpression was also analysed. Immunohistochemical analysis of the cyclin D1 expression resulted in 69 (29.8%) weakly positive, 107 (46.5%) moderately positive and 54 (23.7%) strongly positive cases. The prognostic importance of ErbB2 was significantly greater for patients whose tumours overexpressed cyclin D1 than for other patients (p = 0.026). In the former group, ErbB2 overexpression was strongly associated with increased risk of recurrence (RR = 4.7; 95% CI, 2.1–10.4) and breast cancer death (RR = 5.4; 95% CI, 2.3–12.6). This result is in accordance with experimental studies demonstrating a link between cyclin D1 and ErbB2 in oncogenesis. Among oestrogen receptor positive patients, those with moderate cyclin D1 expression significantly did benefit from tamoxifen treatment (RR = 0.42; 95% CI, 0.21–0.82) whereas those with weak or strong expression did not. Therefore cyclin D1 might be a predictive marker for tamoxifen resistance.     

HER2 status in hormone receptor positive premenopausal primary breast cancer adds prognostic, but not tamoxifen treatment predictive, information

BACKGROUND: Overexpression of human epidermal growth factor receptor 2 (HER2) or amplification of its gene is a prognostic factor in primary breast cancer and a predictor for tamoxifen treatment efficacy in oestrogen receptor (ER) positive disease. In the present study we explored a defined cohort of breast cancer patients included in a randomised trial in order to assess prognostic and tamoxifen treatment information yielded by HER2 status. METHODS: Premenopausal breast cancer patients with stage II tumours (n = 564) were included and allocated to 2 years of adjuvant tamoxifen treatment versus no adjuvant treatment. ER, progesterone receptor (PR) status and HER2 status was determined by immunohistochemistry using a tissue microarray. HER2 amplification was analysed by fluorescent in situ hybridisation and tumours being amplified and/or HER2 3+ were considered HER2+. HER2 status was evaluable in 83% of the patients and 12.6% were HER2+. In untreated patients, HER2 was a negative prognostic factor in ER+ patients, HR 2.95; 95% CI: 1.61-5.38, p < 0.001, but not in ER- patients, HR 0.67; 95% CI: 0.28-1.61, p = 0.4, and a significant interaction between the two markers was found, p < 0.01. HER2 status was not related to tamoxifen treatment efficacy in ER+ patients (term of interaction p = 0.95). When stratifying for PR status, similar results were achieved. DISCUSSION: HER2+ and ER+ breast cancer constituted a subgroup of tumours with poor prognosis in premenopausal breast cancer, whereas no treatment interaction was found between HER2 status and tamoxifen in ER+ tumours. The poor prognosis in HER2+ and ER+ patients may interfere with the interpretation of HER2 data in non-randomised trials of adjuvant tamoxifen.     

Hypoxia-inducible factor 1α is closely linked to an aggressive phenotype in breast cancer

PURPOSE: The aim of this study is to clarify characteristics of invasive breast cancer with expression of Hypoxia-induced factor 1alpha (HIF-1alpha) which is induced by hypoxia and signal transduction of growth factors. EXPERIMENTAL DESIGN : We examined, by immunohistochemical analysis, the expression of HIF-1alpha in normal breast tissue, benign disorders and breast cancer. In invasive breast cancer, we investigated the correlation between expression of HIF-1alpha and clinicopathological and biological factors. We also studied the prognostic value of HIF-1alpha in breast cancer. RESULTS : HIF-1alpha was mainly detected in tumor cell nuclei. In the 171 cases of invasive breast cancer examined, nuclear HIF-1alpha expression was detected in 63 (36.8%) cases. Immunoreactive nuclear HIF-1alpha was correlated with tumor size (p = 0.0013), lymph node metastasis (p = 0.0005), tumor stage (p = 0.0031) and histological grade (p = 0.0074). Elevated HIF-1alpha levels was also associated with hormone receptor negativity (p = 0.0025), HER2 overexpression (p = 0.0053), high Ki67 labeling index (p = 0.0002), increased levels of VEGF (p < 0.0001), COX-2 overexpression (p = 0.0029) and increased nuclear p53 (p = 0.0048). In terms of the possible use of HIF-1alpha as a prognostic indicator, patients who had increased HIF-1alpha levels in their tumor showed shorter disease-free survival (DFS) (p < 0.0001) and overall survival (OS) (p = 0.0002) than those lacking HIF-1alpha in univariate analysis. In multivariate analysis of DFS and OS, HIF-1alpha was identified an independent prognostic factor. CONCLUSIONS: These findings suggest that HIF-1alpha was closely linked to an aggressive phenotype in breast cancer. It may be useful to study the expression of HIF-1alpha using immunohistochemical analysis for better understanding of the tumor characteristics of breast cancer.     

Measuring IGF-1, ER-α and EGFR expression can predict tamoxifen-resistance in ER-positive breast cancer

In vitro studies have suggested that tamoxifen resistance may be due to altered expression and downstream signalling of insulin-like growth factor-1 (IGF-1) receptor (IGF-1l), oestrogen receptor-alpha (ERα), epidermal growth factor receptor (EGFR) and HER-2. We investigated which gene expressions could predict tamoxifen resistant breast cancer. Expression of IGF-1R, IGF-1 ligand (IGF-1), ERα, EGFR and HER-2 in 91 ER-positive breast cancer tumours were measured using real-time PCR and correlated with clinical outcome. The tamoxifen resistant group (n=20) consisted of: i) tumours which were resistant to neoadjuvant tamoxifen treatment and ii) tumours which were excised from patients who later developed recurrence or metastasis during adjuvant tamoxifen treatment. These were compared with tamoxifen sensitive tumours which were surgical excision specimens from patients who did not develop recurrence/metastasis during adjuvant tamoxifen treatment. Tumours with higher IGF-1 ligand and ERα expression took longer to develop tamoxifen resistance. Tamoxifen resistant tumours had lower IGF-1 and ERα expression compared to tamoxifen-sensitive tumours. IGF-1 expression strongly correlated with ERα expression in the tamoxifen sensitive group only. ERα inversely correlated with EGFR expression in the tamoxifen resistant group only. We conclude that IGF-1 ligand and ERα expression in breast carcinomas can be measured to predict tamoxifen resistance. Measuring ERα expression using RT-PCR may be more sensitive than immunohistochemistry in determining anti-oestrogen sensitivity.     

Cyclin D1 expression and patient outcome after tamoxifen therapy in estrogen receptor positive metastatic breast cancer

Cyclin D1 expression is closely related with ER status in breast cancer. We executed this study to evaluate whether therapeutic response to tamoxifen varies with levels of cyclin D1 expression in 66 ER positive breast cancer patients having solitary bone metastasis. Treatment response to tamoxifen and correlation between cyclin D1 expression and biologic data of the patients were analyzed. Cyclin D1 expression was detected in 46 patients (69.7%) and significantly reduced in poorly differentiated cancer (p=0.023). Patients with cyclin D1-expressing tumors showed better response to tamoxifen but the difference was not statistically significant. Cyclin D1 expression was associated with differentiation of the breast cancer but not useful in discriminating a good responder to tamoxifen treatment.     

Targeting tumour hypoxia in breast cancer

Breast cancer is the most common malignancy in women. Hypoxia occurs in breast cancer and in other solid tumours due to the tumour outgrowing the existing vasculature. Hypoxia leads to an adaptive response, orchestrated by HIF-1 (hypoxia-inducible factor-1), that is crucial for tumour progression and therapy resistance responsible for poor patient outcome. In several studies, downstream targets of HIF-1alpha were considered as hypoxia markers. The biological heterogeneity of breast cancer has been investigated through genome profiling technologies. The recent data suggest that treatment outcome depends on individual genetic features and that the hypoxia signature is a significant prognostic factor. The identification of molecular biomarkers with the potential to predict treatment outcome is essential for selecting patients to receive the most beneficial therapy, and in the future may drive stratification in clinical trials.     

Adverse effect of adjuvant tamoxifen in premenopausal breast cancer with cyclin D1 gene amplification

Cyclins D1 and A2 are cell cycle regulators that also have the ability to interact with the estrogen receptor (ER) and consequently interfere with antiestrogen treatment in breast cancer. Experimental data support this concept, but the clinical relevance needs to be further established. In this study, we evaluated cyclin D1 and A2 protein expression by immunohistochemistry and cyclin D1 gene (CCND1) amplification by fluorescence in situ hybridization in 500 primary breast cancers arranged in tissue microarrays. Patients had been randomized to 2 years of adjuvant tamoxifen or no treatment with a median follow-up of 14 years, allowing for subgroup analysis of treatment response defined by cyclin status. We found that both cyclin D1 and A2 protein overexpression was associated with an impaired tamoxifen response, although not significant in multivariate interaction analyses, whereas tamoxifen-treated patients with CCND1-amplified tumors had a substantially increased risk for disease recurrence after tamoxifen treatment in univariate analyses [relative risk (RR), 2.22; 95% confidence interval (95% CI), 0.94-5.26; P = 0.06] in contrast to non-amplified tumors (RR, 0.39; 95% CI, 0.23-0.65; P < 0.0001). Consequently, a highly significant interaction between tamoxifen treatment and CCND1 amplification could be shown regarding both recurrence-free survival (RR, 6.38; 95% CI, 2.29-17.78; P < 0.001) and overall survival (RR, 5.34; 95% CI, 1.84-15.51; P = 0.002), suggesting an agonistic effect of tamoxifen in ER-positive tumors. In node-positive patients, the disparate outcome according to gene amplification status was even more accentuated. In summary, our data implicate that despite a significant correlation to cyclin D1 protein expression, amplification status of the CCND1 gene seems a strong independent predictor of tamoxifen response, and possibly agonism, in premenopausal breast cancer.     

Lack of G protein-coupled estrogen receptor (GPER) in the plasma membrane is associated with excellent long-term prognosis in breast cancer

G protein-coupled estrogen receptor (GPER), or GPR30, is a membrane receptor reported to mediate non-genomic estrogen responses. Tamoxifen is a partial agonist at GPER in vitro. Here, we investigated if GPER expression is prognostic in primary breast cancer, if the receptor is treatment-predictive for adjuvant tamoxifen, and if receptor subcellular localization has any impact on the prognostic value. Total and plasma membrane (PM) GPER expression was analyzed by immunohistochemistry in breast tumors from 742 postmenopausal lymph node-negative patients subsequently randomized for tamoxifen treatment for 2–5 years versus no systemic treatment, regardless of estrogen receptor (ER) status, and with a median follow-up of 17 years for patients free of event. PM GPER expression was a strong independent prognostic factor for poor prognosis in breast cancer without treatment-predictive information for tamoxifen. In the tamoxifen-treated ER-positive and progesterone receptor (PgR)-positive patient subgroup, the absence of PM GPER (53 % of all ER-positive tumors) predicted 91 % 20-year distant disease-free survival, compared to 73 % in the presence of GPER (p = 0.001). Total GPER expression showed positive correlations with ER and PgR and negative correlation with histological grade, but the correlations were biphasic. On the other hand, PM GPER expression showed strong negative correlations with ER and PgR, and strong positive correlation with HER2 overexpression and high histological grade. GPER overexpression and PM localization are critical events in breast cancer progression, and lack of GPER in the PM is associated with excellent long-term prognosis in ER-positive and PgR-positive tamoxifen-treated primary breast cancer.