Estrogen receptor beta (ER beta) level but not its ER beta cx variant helps to predict tamoxifen resistance in breast cancer.

The antiestrogen tamoxifen, a major endocrine therapy of estrogen receptor (ER)-positive breast cancer, is nevertheless inefficient in 30 to 40% of cases for unknown reasons. We retrospectively studied 50 ER-positive primary breast carcinomas. All of the patients had received tamoxifen as the only adjuvant therapy. They were divided into two groups depending on whether they relapsed within 5 years (16 tamoxifen-resistant cases) or did not relapse within 5 years (34 tamoxifen-sensitive cases). The expression of total ER beta protein, and of ER beta cx protein, was estimated anonymously in formalin-fixed, paraffin-embedded tumor sections, by using specific antibodies and quantifiying nuclear immunostaining with a computer image analyzer. All of the tumors were found to be HER-2/neu-negative by immunohistochemistry. Univariate analysis showed that Scarff-Bloom-Richardsson grade modified by Elston (SBR grade; P < 0.001), tumor size (P = 0.042), and MIB-1 proliferation index (P = 0.02) were significantly higher in tamoxifen-resistant tumors. A low level of total ER beta, whether in percentage of positive cells or in quantitative immunocytochemical (QIC) score, was also associated with tamoxifen resistance (P = 0.004). ER beta cx expression and lymph node status were similar between the two groups. The expression of ER beta in the total population was positively correlated with ER beta cx (r = 0.63, P < 0.001), and was independent of the other parameters. In a multivariate analysis, ER beta expression was the most important variable (P = 0.001), followed by SBR grade (I+II versus III; P = 0.008), and MIB-1 (P = 0.016). To conclude, tamoxifen resistance is associated with classical variables of aggressive tumors (high SBR grade, proliferation index, and tumor size) but not with node invasiveness. Low ER beta level is an additional independent marker, better than ER alpha level, to predict tamoxifen resistance.     

Hormone receptor status in human endometrial adenocarcinoma

Steroid receptor levels were determined in 196 samples of endometrial adenocarcinoma: cytosol estradiol receptors (ERc) were measured in 171 samples, cytosol progesterone receptors (PRc) in all samples; nuclear estradiol receptors (ERn) and nuclear progesterone receptors (PRn) in 68 samples; total estradiol receptors (ERt = ERc plus ERn) and total progesterone receptors (PRt = PRc plus PRn) were measured in 68 samples. The ERc levels were 88.2 +/- 8.9 (mean +/- SEM) and ERn were 94.4 +/- 15.6 fmol/mg protein; PRc levels were 197.9 +/- 25.9 and PRn 178.3 +/- 55.9 fmol/mg protein. The ERt levels were 162.6 +/- 23.2 and PRt 249.8 +/- 75.7 fmol/mg protein. The presence of PRc was related to the ERc levels according to the cut-off used. Estradiol receptors (ER) and progesterone receptors (PR) were present in the cytoplasmic and nuclear fractions in 60.2% and 36.8% of cases, respectively. The simultaneous presence of both ERt and PRt was observed only in 27.9% of cases. In the normal endometrium ERc and PRc were negatively correlated (r = -0.525, P less than 0.005), whereas in endometrial adenocarcinoma the correlation was positive (r = 0.491, P less than 0.001). In contrast with the normal endometrium the correlation between ERc and ERn was positive (r = 0.582, P less than 0.001) in tumor tissue. In neoplastic tissue Scatchard analysis showed a single class of specific ERc sites with a dissociation constant (Kd) of 1.39 +/- 0.8 X 10(-9) mol/l, one tenth of that found in the normal premenopausal endometrium. Qualitative and quantitative analysis of the receptor status showed that in 30% to 40% of cases studied the behavior of the neoplastic cell was similar to that found in the normal endometrial cell. In a 4-year follow-up of patients affected by endometrial adenocarcinoma there is better survival in the groups of patients with a simultaneous presence of ERt and PRt than in the group with their absence.     

Estrogen receptor (ER) beta1 and ERbetacx/beta2 inhibit ERalpha function differently in breast cancer cell line MCF7

Estrogen receptor (ER) alpha plays an important role in the proliferation and progression of breast cancer. In order to explore the function of wild-type ERbeta (ERbeta1) and its variant form, ERbetacx/beta2, stable transformants of ERalpha-positive breast cancer MCF7 cells with ERbeta1 or ERbetacx/beta2 expression vector were established. Constitutive expression of ERbeta1 or ERbetacx/beta2 reduced the S phase population of the cell cycle in dish culture and the number of colonies in an anchorage-independent assay. DNA-protein complexes of ERE with nuclear extracts from ERbeta1 transformants were observed in the electrophoretic mobility shift assay, while no complex was observed for ERbetacx/beta2 transformants. Reporter gene assay using estrogen-responsive element (ERE)-luciferase showed less responsiveness to estrogen in these transformants compared with parental cells. Endogenous mRNA expression of two known estrogen-responsive genes, cathepsin D and IGFBP4, was weakly induced by estrogen in ERbeta1 and ERbetacx/beta2 transformants compared with parental cells. A comprehensive gene expression analysis using our custom-made cDNA microarray showed that MCF7 and ERbeta1 transformants had a similar gene expression profile, whereas ERbetacx/beta2 showed a distinct profile from others. These results indicate that ERbeta1 and ERbetacx/beta2 inhibit ERalpha function differently in MCF7 cells.     

SULT1E1、ERβ在子宫内膜腺癌组织中的表达及其相关性

目的:探讨SULT1E1及ERβ蛋白与子宫内膜腺癌的发生、发展之间的关系。方法:采用Western blot方法检测正常增生期子宫内膜组织、不典型增生子宫内膜组织及子宫内膜腺癌组织中SULT1E1和ERβ蛋白的表达含量。结果:SULT1E1和ERβ蛋白在正常增生期子宫内膜组织、不典型增生子宫内膜组织及子宫内膜腺癌组织中的表达含量逐渐降低,三者比较差异有统计学意义(P<0.05)。不典型增生子宫内膜组织及子宫内膜腺癌组织与正常增生期子宫内膜组织比较差异均有统计学意义(P<0.05)。SULT1E1和ERβ蛋白与子宫内膜腺癌组织学分级、肌层浸润深度、手术病理分期及淋巴结转移等无关(P>0.05)。在子宫内膜腺癌组织中,SULT1E1蛋白与ERβ蛋白表达呈明显正相关(r=0.66,P<0.01)。在正常增生期和不典型增生子宫内膜组织中,SULT1E1蛋白与ERβ蛋白表达无明显相关性(r=0.035,P>0.05;r=0.466,P>0.05)。结论:在子宫内膜腺癌的渐近演变过程中,SULT1E1、ERβ表达降低或缺失可能与子宫内膜癌的早期形成有一定的关系,ERβ蛋白含量越多,SULT1E1蛋白对雌激素的调节作用越强,但不能作为子宫内膜腺癌病情严重程度和预后进行评价的指标。     

子宫内膜中PTEN、p16蛋白的表达及临床意义

目的:研究子宫内膜组织中PTEN、p16蛋白的异常表达,探讨其与子宫内膜癌变的关系和早期诊断及评判预后的可能性.方法:应用免疫组化S-P法对20例正常子宫内膜组织、40例子宫内膜增生症组织、30例内膜腺癌组织中PTEN、p16蛋白的表达进行研究.结果:在正常增生期子宫内膜、子宫内膜增生症(单纯型增生、复杂型增生、不典型增生)、子宫内膜腺癌组织中PTEN p16蛋白的阳性表达率呈递减趋势,子宫内膜腺癌与除不典型增生外的子宫内膜增生症组织及正常子宫内膜组织的PTEN蛋白表达差异有显著性(P＜0.05),正常子宫内膜、单纯型增生与不典型增生组织的PTEN蛋白表达差异有显著性(P＜0.05),子宫内膜腺癌与正常子宫内膜及单纯型增生子宫内膜p16 蛋白表达差异有显著性(P＜0.05),正常子宫内膜与增生症子宫内膜差异无显著性(P＞0.05) 增生症子宫内膜各组间p16蛋白表达无显著性差异(P＞0.05),PTEN P16蛋白的表达与子宫内膜癌的手术分期、组织学分级,肌层浸润有关(P＜0.05).PTEN、p16蛋白表达存在正相关性(r=0.978,P＜0.05).结论:PTEN、p16蛋白的异常表达与子宫内膜的癌变过程相关.     

Estrogen receptor alpha and beta profiling in human breast cancer.

BACKGROUND: The identification of a second estrogen receptor (ER-beta) has significant implications for therapeutic strategy in breast cancer management arising from the potential agonist effect of Tamoxifen at estrogen receptor sites and as such, antiestrogen therapy may be inappropriate in patients with a dominance of ER-beta. METHODS: To determine the proportion of breast cancer patients who may be so at risk, we developed a novel multiplexed RT-PCR technique to establish the relative ER-alpha and ER-beta levels in 53 primary breast cancers, 11 normal breast tissues and six cell lines. We further assessed the prognostic significance of receptor status relative to the Nottingham prognostic index (NPI). The ER-alpha and ER-beta status was also determined by immunohistochemistry using previously published and 'in-house' scoring systems. RESULTS: Using RT-PCR analysis, 46 tumours were hormone receptor positive (ER+) with 42 displaying ER-alpha predominance. Comparison with immunohistochemistry demonstrated 44/53 (ER-alpha) and 27/50 (ER-beta) concordance rates. There was no significant difference in the NPI between ER-alpha and ER-beta predominant cohorts or between ER+ and ER- cohorts. CONCLUSION: This study identifies the existence of a subgroup of ER+ patients in whom Tamoxifen therapy may be inappropriate and has significant implications for adjuvant therapy of primary breast cancer.     

Expression of estrogen receptor (ER) (beta)cx protein in ER(alpha)-positive breast cancer: specific correlation with progesterone receptor

Estrogen receptor (ER) (beta)cx, a splice variant of ERbeta, is a dominant repressor of ER(alpha) function. In this study we investigated the possibility that because the progesterone receptor (PR) gene is a downstream target of activated ER(alpha), in ER(alpha)-positive breast cancers, expression of ER(beta)cx would result in repression of PR. In ER(alpha)-positive MCF-7 cells, stable transfection of an ER(beta)cx expression vector resulted in reduced expression of PR without affecting ER(alpha) expression. In breast cancers, immunohistochemical evaluation of ER(alpha)-positive foci for the expression of PR and ER(beta)cx revealed a significant correlation between a PR-negative phenotype and the presence of ER(beta)cx within the foci. However, when entire lesions were evaluated by Allred scoring in 115 ER(alpha)-positive breast cancer specimens, the presence of two distinct groups of patients could be discerned. One group expressed ER(beta)cx and had very reduced levels of PR expression, as expected. The second group showed both ER(beta)cx and high levels of PR. To evaluate the role of ER(beta)cx in sensitivity to tamoxifen, 18 core needle biopsies, obtained before preoperative treatment with tamoxifen, were investigated. The results show that expression of ER(beta)cx in primary lesions correlated with a poor response to tamoxifen, especially in cancers with a low PR expression in Allred score. This is the first evidence that evaluation of ER(beta)cx along with PR may contribute to a better characterization of ER(alpha)-positive breast cancers.     

雌、孕激素受体与子宫内膜癌相关性的研究

目的　通过对雌、孕激素受体表达水平的检测分析 ,探讨雌、孕激素受体的表达水平与子宫内膜癌的发生、发展的关系。方法　收集我科经手术治疗的新鲜活体组织标本 ,并经病理组织学确诊的子宫内膜癌 30例。采用较新的LSAB免疫组织化学方法检测雌激素受体 (ER)和孕激素受体 (PR)的表达水平。结果　ER和PR的阳性率一致 ,均为 6 6 6 7% :组织学分级高分化腺癌 (Ⅰ级 )的阳性率为 4 0 % ,明显高于中、低分化者 (Ⅱ、Ⅲ级 ) ;临床分期早期 (Ⅰ期 )的阳性率为 4 3 33% ,明显高于中、晚期 (Ⅱ、Ⅲ期 )患者 (P <0 0 1)。在同一标本不同部位的癌灶、癌旁和正常组织中ER和PR的阳性率显示癌灶组织明显低于癌旁和正常组织(P <0 0 5 )。结论　ER和PR的表达水平与子宫内膜癌的组织学分级和临床分期有一定的关系 ,高分化癌的ER和PR表达水平高 ,低分化癌的ER和PR含量低 ;临床分期早期患者的ER和PR含量高 ,中、晚期患者的ER和PR含量低。笔者认为 :ER和PR的检测对子宫内膜癌的组织学分化趋势、分级及临床分期和内分泌治疗与对预后的监测有指导意义     

Estrogen receptor alpha (ERα) mRNA copy numbers in immunohistochemically ERα-positive-, and negative breast cancer tissues

Background  

The presence of ERα is the basis for treating breast cancer patients with targeted molecular therapies that block estrogen stimulation of breast cancer cell division. To select patients for the above therapies, currently, the ERα presence in breast cancer tissues is determined in clinical laboratories by microscopically scoring the slides subjected to immunohistochemistry (IHC). This method is not quantitative, highly subjective and requires large amount of tumor tissue, therefore, cannot be applied to sterotactic and ultrasound guided biopsy samples. To circumvent these problems, we previously developed quantitative real-time PCR based molecular assay that can be applied to determine mRNA copies of ERα in picogram amounts of total RNA from tumor samples. However, it is not known how the mRNA copy numbers correlate to IHC positive and negative status.     

Ligand activation of peroxisome proliferator-activated receptor beta/delta (PPAR beta/delta) inhibits chemically induced skin tumorigenesis

Peroxisome proliferator-activated receptor (PPAR)beta/delta-null mice exhibit enhanced tumorigenesis in a two-stage chemical carcinogenesis model as compared with wild-type mice. Previous work showed that ligand activation of PPARbeta/delta induces terminal differentiation and inhibits proliferation of primary keratinocytes, and this effect does not occur in the absence of PPARbeta/delta expression. In the present studies, the effect of ligand activation of PPARbeta/delta on skin tumorigenesis was examined using both in vivo and ex vivo skin carcinogenesis models. Inhibition of chemically induced skin tumorigenesis was observed in wild-type mice administered GW0742, and this effect was likely the result of ligand-induced terminal differentiation and inhibition of replicative DNA synthesis. These effects were not found in similarly treated PPARbeta/delta-null mice. Ligand activation of PPARbeta/delta also inhibited cell proliferation and induced terminal differentiation in initiated/neoplastic keratinocyte cell lines representing different stages of skin carcinogenesis. These studies suggest that topical administration of PPARbeta/delta ligands may be useful as both a chemopreventive and/or a chemotherapeutic approach to inhibit skin cancer.     

p27、PCNA在子宫内膜增生过长、腺癌中的表达及意义

目的　探讨 p2 7、PCNA在子宫内膜增生过长、腺癌中的表达及意义。 方法　应用免疫组化SP法检测子宫内膜单纯和复杂性增生过长、不典型增生、腺癌中p2 7、PCNA蛋白表达。结果　p2 7蛋白高表达 (++)率在子宫内膜单纯和复杂性、不典型增生过长、腺癌中分别为 6 6 .7% (2 4 / 36 )、35 % (7/ 2 0 )、30 % (10 / 33) ,在单纯和复杂性增生过长、腺癌间差异有显著性 (P<0 .0 5 )。p2 7蛋白表达与腺癌组织分化程度、有无肌层浸润无关 (P >0 .0 5 )。PCNA表达在子宫内膜单纯和复杂性、不典型增生及腺癌中逐级递增 ,差异有显著性 (P <0 .0 1)。子宫内膜增生过长组中PCNA与 p2 7蛋白表达负相关 (P <0 .0 1) ,腺癌中p2 7与PCNA不相关。 结论　p2 7蛋白表达减少与子宫内膜腺癌发生相关 ,腺癌的发生、发展与子宫内膜细胞增殖活性提高有关。     

Analysis of the potential contribution of estrogen receptor (ER) beta in ER cytosolic assay of breast cancer

Estrogen receptor (ER) content is the most useful parameter for predicting hormone response therapy in breast cancer. Assays available for detecting ER in breast tumor cytosol are ligand-binding assay (LBA), which detects both ERalpha and ERbeta, and the enzymatic immunoassay (EIA), in which monoclonal antibodies are directed against ERalpha. As shown in several studies, the 2 assays correlate and both are used routinely. However, some discrepancies between the 2 assays were found and explanations remain controversial. We evaluated ERalpha and ERbeta mRNA coexpression in breast tumors in order to study whether the presence of ERbeta could account for differences between LBA and EIA in the determination of ER protein level. Using HeLa cell lines transfected with either ERalpha or ERbeta, we confirmed that EIA, using H222 and D547 monoclonal antibodies, recognizes only ERalpha expression, whereas LBA detects both isoforms. In 119 breast tumor cytosols, the correlation between ER-EIA and ER-LBA was high (r = 0.72), although some discrepancies were found. When analyzing ER mRNA expression of samples with higher LBA values, no overexpression of ERbeta mRNA relatively to ERalpha mRNA were observed. There was a difference in ERbeta/ERalpha ratio between ER-negative and ER-positive samples, with a 10-fold increased median ratio in ER-negative samples (p = 0.01). We thus confirmed that the major form of ER in breast cancer is the ERalpha at both the protein and mRNA levels. Moreover, our data do not support the hypothesis that ERbeta expression could explain differences between LBA and EIA in the determination of ER protein level.