Influence of estrogen receptor variants on the determination of ER status in human breast cancer

Determination of estrogen receptor alpha (ER) status in breast cancer is an important predictive factor for clinical response to endocrine therapy. We have recently shown that discrepancies in ER status determined by immunohistochemical assay (ER-IHA) can occur between amino-terminal (1D5) and carboxyl-terminal (AER-311) targeted ER antibodies and that those tumors which demonstrate discordance are associated with increased expression of truncated ER variant mRNAs. In this study, we have explored this observation to examine if ER variant expression can exert a direct effect on ER-IHA or whether this association is attributable to the characteristics of the antibodies. ER negative cos-1 cells were transfected with expression vectors containing wild type ER (wt-ER) and/or a frequently expressed truncated variant, ER-clone-4 variant. We found that ER-IHA performed with the same N- and C-terminal targeting ER antibodies on cos-1 cells expressing wt-ER alone demonstrated no difference in signals by western blot (P>0.1). However, co-expression of wt-ER and the truncated ER-clone-4 variant, resulted in discordant IHA results with relatively higher ER-IHA H-scores from N-terminal antibodies (P<0.03). Furthermore, re-examination of a subset of breast tumors previously studied by ER-IHA showed persistent concordance in 4/5 cases and persistent differences in 3/5 cases with a different pair of ER antibodies. We conclude that the presence of truncated ER variant proteins can interfere with the interpretation of ER status determined by IHA and that this may account for some of the inconsistencies between ER status and response to endocrine therapy.     

Tumour steroid hormone synthesis and estrogen receptor status in breast cancer patients

Summary The capacity of breast cancer to synthesise active androgens and estrogens has been related to estrogen receptor (ER) status in 79 postmenopausal patients with breast cancer. Although there was no quantitative relationship between levels of ER and steroid metabolism in ER positive tumours, there was (a) a positive correlation between estrogen synthesis and ER positivity and (b) increased androgen synthesis and ER negativity. This may imply an inherent difference in the handling of hormones in ER positive and negative tumours. Address for reprints: R.C. Mason, University Department of Clinical Surgery, Royal Infirmary, Edinburgh EH39YW, United Kingdom.     

65 and 47 kDa forms of estrogen receptor in human breast cancer: relation with estrogen responsiveness

Summary In breast cancer nearly 40% of estrogen receptor (ER) positive patients do not respond to hormone therapy. As several species of ER have been described, we examined 41 breast cancers for: (1) the presence of ER and progesterone receptor (PR); (2) the molecular weight (Mr) of ER; (3) estrogen responsiveness, appreciated by the ability of a piece of tumor transplanted in nude mice to show an estrogen-induced protein synthesis (PR synthesis). We found that there are: two species of ER with different Mr (65 and 47 kDa), and three species of tumors (36% containing the highest form of ER alone, 49% bearing the two components in variable amounts, and 15% bearing only the minor species). Eleven of these 41 tumors could be assayed for PR synthesis induction, showing that estrogen responsiveness is correlated with the major component. Due to the limited number of samples (11) the data are preliminary, but they strongly suggest that the different forms of ER could exist in the living cell with different functional abilities.     

Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism.

BACKGROUND: To study the short-term biological effect of anastrozole on serum estrogens, androgens, 17-hydroxyprogesterone (17OH-PGR), gonadotrophins, sex hormone binding globulin (SHBG) and bone metabolism markers. MATERIALS AND METHODS: Thirty-four consecutive patients with advanced breast cancer received anastrozole 1 mg/day. Blood samples were taken before commencement of treatment and at 2, 4, 8 and 12 weeks during treatment to measure serum levels of estrogens (E(1), E(2) and E(1)-S), androgens [androstenedione (Delta(4)), dihydrotestosterone (DHT), testosterone (TST), free TST, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S)], 17OH-PGR, SHBG and gonadotrophins. As an indicator of bone resorption, we measured serum levels of C-terminal telopeptide of type I collagen (ICTP) and the cross-linked N-telopeptide of type I collagen (NTx), and for osteoblastic activity, intact osteocalcin (BGP) and bone alkaline phosphatase (BAP). RESULTS: After 2 weeks E(1 )and E(1)-S levels decreased on average by 56% (range 23.1-88.8%) and 75.8% (range 52.4-87.2%), respectively; E(2) decreased on average by 62% (range 31.4-89.6%). No significant changes were detected in levels of androgens or 17OH-PGR. There was a significant increase in gonadotrophins over time (P = 0.0001 for both luteinizing hormone and follicle-stimulating hormone), and a significant decrease in SHBG (P = 0.0001). A progressive significant increase in bone metabolism serum markers was detected in all patients: BAP, P = 0.039; BGP, P = 0.016; ICTP, P = 0.0021; and NTx, P = 0.0013. In particular, patients with bone metastases had a statistically significant increase of bone resorption markers (ICTP, P = 0.0019; NTx, P = 0.025) and borderline for bone formation markers. In patients without bone disease, BAP, BGP and ICTP remained unchanged, whereas serum NTx significantly increased (P = 0.019). CONCLUSIONS: Anastrozole is a selective aromatase inhibitor as it does not modify serum levels of androgens and 17OH-PGR. In our experience no relationship was found in the short-term period between serum estrogen suppression and bone metabolism.     

Complete remission after ovariectomy for advanced breast cancer correlated with estrogen receptor status and urinary androgen excretion

Summary Our previous work showed urinary androgen excretion (A) as well as estrogen receptor (ER) to predict clinical response and survival after ovariectomy for advanced breast cancer. We here compare the complete responders with the partial responders to ovariectomy. The likelihood of CR (55% of responders) rather than PR was not strongly dependent on the location of metastases or on the ER/A status (though as noted previously there were no responses at all in the ER-/A- group), but CR did appear to increase survival.Presented as an abstract at the 10th Annual Breast Cancer Symposium, San Antonio, Texas, December 11–12, 1987     

雌激素对人乳腺癌细胞株MCF7增殖的促进作用

目的：观察雌二醇对乳腺癌细胞增殖的促进作用和可能的机制。方法：经不同浓度的雌二醇处理乳腺癌细胞株MCF7后，用MTT比色试验观察雌二醇对MCF7增殖活性的影响，免疫印迹法测量多种与细胞周期、抑癌、增殖、凋亡相关的蛋白质。结果：经MTT比色试验检测，雌二醇能增加MCF7细胞株的增殖活性，细胞周期素-D1明显增加。但雌二醇对抑癌基因：PTEN的蛋白表达没有明显影响，以及对具有抗细胞凋亡作用的PKB蛋白量也无明显作用。结论：雌二醇可促进乳腺癌细胞的增殖，可能通过细胞周期素-D1介导的分子机制，而不涉及PKB抗凋亡作用。     

乳腺癌组织缺氧与雌激素受体-α的关系

目的:缺氧是乳腺癌常见的现象,并可能诱导肿瘤的进展。雌激素受体(ER)-α状态是乳腺癌预后和内分泌治疗疗效的重要预测指标。本研究旨在揭示乳腺癌中缺氧与ER-α表达的关系。方法:51例患者经配体结合法证实为ER阳性的乳腺癌。采用免疫组织化学染色的方法观察ER-α的异质性表达与多种缺氧标志的关系。结果:免疫组织化学染色发现本组有49例乳腺癌为ER-α阳性。无论乳腺导管内癌(n=29)还是浸润性癌(n=20),ER-α蛋白在邻近坏死灶的区域都出现了下降(P分别≤0.0001);ER-α的区域性下调还与缺氧诱导基因CA-Ⅸ和Glut-1的表达有关(P<0.0001)。结论:乳腺癌中ER-α区域性的表达下降与缺氧有关。因此缺氧可能促使乳腺癌在进展过程中向ER-α阴性的表型演变。     

Urinary androgens and tumor estrogen receptor as predictors of ovariectomy response and of survival in advanced breast cancer

Summary Estrogen receptor (ER) status and urinary androgen (A) concentration were simultaneously determined in 50 premenopausal patients submitted to bilateral ovariectomy for advanced carcinoma of the breast. When both the hormonal parameters were positive (ER+ A+), the response to castration was favorable in 87.5% of the cases, with a survival rate of 39% at 5 years. No patient responded to the therapy when both the parameters were negative (ER– A–); none of them was alive at 5 years. An intermediate response (more than 50%) and survival rate at 5 years (more than 20%) was obtained in the group of patients with at least one of the two parameters positive (ER+ A–, or ER– A+). These responses were independent of the topography of neoplastic localizations and the length of the disease-free interval.     

芳香化酶抑制剂治疗乳腺癌的临床研究进展

综述芳香化酶抑制剂治疗乳腺癌的作用机制、常见药物和在乳腺癌内分泌治疗中的临床研究进展。     

Tumor response and estrogen suppression in breast cancer patients treated with aromatase inhibitors

Background:The rationale for the hormonal treatment of breastcancer (BC) is based on depriving tumor cells of estrogenic stimulation.Aromatase inhibitors (AIs) block the conversion of peripheral tissue androgensto estrogens with different levels of potency. In an attempt to investigatethe relationship between tumor response and estrogen suppression, we reviewedthe hormonal and clinical data of two previous studies with formestane (250and 500 mg i.m. fortnightly) in advanced BC patients. Patients and methods:Two hundred four BC patients were selectedon the basis of the availability of records concerning their plasma estrone(E1) and estradiol (E2) levels assessed at scheduled times. The degree ofestrogen suppression and the best clinical response of each patient during thetrials were considered. Results:There was a positive and significant (P <0.05) correlation between baseline and post-formestane E1 and E2 levels, witha decrease in the levels of both hormones irrespective of any antitumorresponse. In particular, the degree of plasma estrogen suppression was similarin the patients who experienced a complete remission and those withprogressive disease (PD). Conclusions:The plasma estrogen suppression induced by aromataseinhibition is not the only mechanism accounting for its clinical activity.Many clinical trials have demonstrated that all AIs induce a similar antitumorresponse regardless of their potency, and further investigations are warrantedin order to improve our understanding as to why the patients with PD also showa significant plasma estrogen suppression. It is possible that intratumoralaromatase activity may be a marker for selecting the BC patients most likelyto respond to AI treatment.     

乳腺癌药物预防的新进展

随着我国乳腺癌的发病率逐年增高,对乳腺癌进行预防干预显得尤为重要。使用自然或人工合成的化学药物可以干预乳腺癌发生、发展的过程,达到预防乳腺癌的目的。本文通过回顾乳腺癌药物预防相关的临床试验,对该领域研究新进展进行综述。      

Obesity, hormone therapy, estrogen metabolism and risk of postmenopausal breast cancer

Hormone therapy (HT) and body mass index (BMI) have been associated with postmenopausal breast cancer. Because estrogen metabolism may affect breast cancer risk and can be altered by weight and HT, it might play a role in the HT-BMI-breast cancer associations. We undertook a nested case-control study within the Observational Study of the Women's Health Initiative. Baseline levels of 2- and 16alpha-hydroxy estrone (2-OHE1 and 16alpha-OHE1) were measured in 200 women who developed breast cancer during follow-up and 200 healthy controls matched to cases by ethnicity, enrollment date, clinic site, type of HT and years since menopause. Wilcoxon nonparametric tests were used to compare estrogen metabolite levels between cases and controls. Conditional logistic regression was used to assess the relationship between BMI, estrogen metabolites and breast cancer risk. 16alpha-OHE1 levels were modestly but significantly higher in HT users among cases (median 356 pg/ml vs. 315 pg/ml) and controls (354 pg/ml vs. 298 pg/ml). 2-OHE1 levels were substantially and significantly higher in HT users among cases (369 pg/ml vs. 125 pg/ml) and controls (347 pg/ml vs. 134 pg/ml). For non-HT users only, greater BMI and higher 16alpha-OHE1 levels were individually and jointly associated with increased breast cancer risk (OR for women with high BMI and high 16alpha-OHE1 compared to those with low BMI and low 16alpha-OHE1 = 3.51, 95% CI = 1.34-9.16). No associations between BMI, estrogen metabolism and breast cancer risk were found for HT users. Estrogen metabolism differs according to both BMI and HT use, potentially explaining the interaction between BMI and HT in relation to breast cancer risk.     

Intratumoral androgen metabolism and actions in invasive lobular carcinoma of the breast

Invasive lobular carcinoma (ILC) accounts for approximately 10% of all breast carcinomas and is characterized by higher levels of androgen receptor (AR) compared to invasive ductal carcinoma (IDC). Despite this potentially androgen‐responsive environment, the combined importance of AR and androgen metabolism in non‐neoplastic lobules and lobular carcinoma remains unknown. Therefore, in this study, we evaluated the status of pivotal androgen‐producing enzymes 17β‐hydroxysteroid dehydrogenase type 5 (17βHSD5) and 5α‐reductase type 1 (5αRed1) in 178 cases of ILC and surrounding histologically non‐neoplastic lobular tissue using immunohistochemistry. Androgen receptor prevalence was higher but androgenic enzymes lower in ILC than non‐neoplastic lobules. In ILC cases the status of 5αRed1 and 17βHSD5 was inversely correlated with tumor size (P = 0.0053) and nuclear grade (P = 0.0290), and significantly associated with better overall survival of the patients (P = 0.0059). Based on these findings, we hypothesized that androgen signaling could act as a tumor suppressor. As previous studies suggested that androgens might partially act by increasing levels of the estrogen inactivating enzyme 17β‐hydroxysteroid dehydrogenase type 2 (17βHSD2) in IDC tissues, this was reasonably considered a potential mechanism of androgen actions. Significantly positive correlation was detected between the status of androgenic enzymes and 17βHSD2 (P < 0.0001) and intratumoral 17βHSD2 was inversely correlated with tumor size in ILC (P = 0.0075). These correlations suggest one protective mode of androgen action could be through modulation of estrogen metabolism. Results of our present study indicated that androgen‐producing enzymes could play pivotal protective roles in AR‐enriched ILC cases.     

Impairment of stimulation by estrogen of insulin-activated nitric oxide synthase in human breast cancer

Nitric oxide (NO) is reported to have several important effects in the control of neoplasm. We have reported before the presence of an insulin-activated constitutive form of membrane-bound nitric oxide synthase (IANOS) in various cells. Since the insulin-induced NO synthesis by IANOS could have important consequences on the pathophysiology of neoplastic cells, the role of estrogen on the activity of IANOS in malignant and nonmalignant breast tissue as well as in erythrocytes in breast cancer patients was determined. It was found that the IANOS activity of nonmalignant breast tissue was maximally stimulated by 4-fold over the basal activity in the presence of physiologic amounts of estrogen (8-32 nM). The enzymic activity was, however, inhibited by estrogen both below and above this range when compared to appropriate controls. In contrast, both the basal IANOS activity and the stimulatory effect of estrogen was markedly impaired in malignant breast tissue and in erythrocytes in these patients. It was also noted that tamoxifen, a widely used nonsteroidal compound in breast cancer, mimicked estrogen both in the stimulation and in the inhibition of IANOS activity in both of the tissues. These results indicated the probable existence of a novel pathway for estrogen effect independent of nuclear receptor for the stimulation of IANOS activity that might have important consequences in breast cancer and suggested that some of the beneficial effects of tamoxifen could be related to its estrogen-mimicking effect on IANOS independent of hormone-responsive elements sequence in the DNA.     

Variants in estrogen metabolism and biosynthesis genes and urinary estrogen metabolites in women with a family history of breast cancer

We examined associations between polymorphisms in genes related to estrogen metabolism (CYP1B1 codon 432G → C rs#1056836, CYP1B1 codon 453A → G rs#1800440, COMT codon 158G → A rs#4680) and biosynthesis (CYP17 T → C promoter rs#743572, CYP19 exon 4 TTTA repeat) and urinary estrogen metabolites (2-hydroxyestrogens (2-OHE), 16α-hydroxyestrone (16α-OHE1), and their ratio) in a pilot study of 64 pre- and post-menopausal women with a family history of breast cancer. Women were participants in the Metropolitan New York Registry of Breast Cancer Families, one of six international sites of the National Cancer Institute’s Breast Cancer Family Registry. We used linear regression to examine the effects of genetic variants on log-transformed urinary estrogen metabolites. After adjusting for menopausal status, BMI, and age, carriers of the CYP1B1 codon 453G variant allele had 31.0% lower levels of 2-OHE (P-value = 0.05) and 40.2% lower levels of 16α-OHE1 (P = 0.01). Results were similar after restricting the analyses to pre-menopausal women (n = 41). Consistent with other studies, among pre-menopausal women, carriers of the COMT codon 158A variant allele had increased 2-OHE levels (P = 0.03) and an increased 2-OHE/16α-OHE1 ratio (P = 0.04); carriers of the CYP17 C promoter variant allele had increased 2-OHE levels (P = 0.08). To our knowledge this is the first report showing associations between the CYP1B1 codon 453G variant allele and urinary 2-OHE and 16α-OHE1 metabolites. Further larger studies should be conducted to confirm these results. Future identification of individuals with genetic polymorphisms that affect estrogen metabolism and biosynthesis may help characterize women at higher breast cancer risk and could guide breast cancer prevention strategies for those individuals.     

Secreted growth factors from estrogen receptor-negative human breast cancer do not support growth of estrogen receptor-positive breast cancer in the nude mouse model

Estrogen receptor (ER)-negative MDA-231 human breast cancer cells have been shown to secrete high concentrations of several growth factors including transforming growth factor-alpha and insulin-like growth factor I, which could have important autocrine or paracrine growth regulatory functions and, additionally, could explain the rapid autonomous growth of these cells. In contrast, the hormone-responsive, ER-positive MCF-7 cells secrete low levels of these factors constitutively. Since estrogen treatment increases secretion of these growth factors in MCF-7 cells, it has been postulated that these growth factors mediate estrogen's growth effects through an autocrine mechanism. To test this hypothesis we reasoned that growth factors supplied by MDA-231 cells should support growth of MCF-7 cells in an estrogen-depleted environment. Inoculation of castrated female athymic nude mice with MDA-231 cells resulted in rapid tumor growth. However, MDA-231 tumors did not support growth of MCF-7 cells inoculated on the opposite flank by an endocrine mechanism; MCF-7 tumors required estrogen supplementation for growth. To determine if MDA-231 cells could support MCF-7 growth by a paracrine mechanism, various mixtures of the two cell lines were coinoculated at the same site in castrated or in estrogen-supplemented mice. ER was not detectable in tumors derived from a mixed inoculum, indicating the absence of MCF-7 cell growth. Furthermore, DNA flow cytometry of these tumors revealed only a single G₁ peak representative of MDA-231 cells in estrogen-deprived mice. On the other hand, two distinct G₁ peaks representing both MDA-231 and MCF-7 cells were detected in tumors grown in estrogen-supplemented mice. These data demonstrate that growth factors from estrogen-independent MDA-231 cells are not capable of replacing estrogen for growth stimulation of MCF-7 cells. Either estrogen-stimulated growth of MCF-7 cells requires other secreted factors not supplied by MDA-231 cells, or it involves a different mechanism.     

Incidence of an estrogen receptor polymorphism in breast cancer patients

Summary We previously identified a polymorphism in the human estrogen receptor (ER) gene, within the coding region for the protein's amino terminal B-domain. In estrogen receptor-positive (ER⁺) breast tumors, the variant allele was preferentially associated with lower levels of ER, and was clinically correlated with frequent spontaneous abortions. DNA sequencing revealed a point mutation that changes codon 86 from Ala to Val and a silent mutation in codon 87. Because we initially detected the variant allele by analyzing RNA, only those tissues in which the ER gene is actively expressed were suitable for genotype analysis. We now describe an assay that uses genomic DNA as the substrate for determining the ER B genotype; DNA containing the polymorphic region of the ER gene is amplified by the polymerase chain reaction, then the amplified DNA is hybridized with radiolabeled oligonucleotide probes complementary to the wild type and variant ER alleles. This method allowed us to determine the ER B genotype of women with ER⁺ and ER^– tumors, starting with minute amounts of DNA from frozen or paraffin embedded tissues. ER B genotyping was also performed on women without breast cancer using DNA extracted from blood cells. The combined results from analyses of RNA and DNA from 300 breast cancer patients showed that 12% were heterozygotes. In the ER⁺ group (n=183), 11.5% carried the variant gene compared to 12.8% in the ER-negative group (n=117) (²=0.11; df=1; p>0.25). No link to tumor histology could be established. Preliminary data on DNA from blood of healthy women over the age of 50 (n=64) yielded a slightly lower ER B-variant frequency (9.4%); this frequency was not significantly different than that in the breast cancer groups. Thus, while the variant ER allele is associated with low ER levels in ER positive breast tumors, its frequency is not different in the ER⁺ and ER^– tumor groups and may be unrelated to breast cancer development.     

芳香化酶抑制剂治疗相关的骨丢失及其防治

临床研究显示对于激素受体阳性的绝经后乳腺癌患者芳香化酶抑制剂的疗效优于他莫昔芬,但芳香化酶抑制剂进一步降低循环中雌激素水平,对骨代谢产生不良影响,导致骨成分丢失增加,增加骨质疏松症和骨折的风险,尤其是高龄女性及低骨密度者。本文就芳香化酶抑制剂治疗与骨丢失的关系及其监测和防治进行综述。     

Association between estrogen receptors and weight in women with breast cancer

A significant association between body weight and estrogen receptor protein (ERP) was noted in 83 women with primary and metastatic breast cancer. Thirteen of 34 (54%) women with weight >150 lb had low or absent receptor protein (<10 femtomoles/mg of cytoplasmic protein) compared to 15 of 59 (25%) women with weight <150 lb (P <0.025). The observed association was stronger in the group of 62 postmenopausal women (P <0.01). The findings suggest that the endocrine and metabolic milieu in women with increased weight favors autonomous growth of breast cancer, and adjuvant treatment in this group should be planned accordingly.     

Influence of endocrine status on biochemical and immunocytochemical estrogen and progesterone receptor assays in breast cancer patients

Summary Breast cancer tissue from 190 patients was studied for immunocytochemically reactive estrogen and progesterone receptors (ER, PR). Parallel cytosol ER and PR assays were performed on 159 of these patients using the dextran-coated charcoal (DCC) method. For the immunocytochemical determination, monoclonal antibodies to ER (ER-ICA kit) and PR were used in an immunoperoxidase procedure. Agreement between the two techniques in postmenopausal patients was better than in the premenopausal group (ER, kappa = 0.597 vs. 0.398; PR, kappa = 0.460 vs. 0.329). The median ER cytosol concentration in receptor-positive postmenopausal patients was significantly higher than in receptor-positive premenopausal patients (87 vs. 31 fmol/mg cytosol protein, p<0.001). A similar trend was also found in the immunocytochemical ER assay (270 vs. 207 histoscore units, p>0.05). Significantly higher cytosol ER contents were found in patients with low serum estradiol concentration. The proportion of ER-negative tumors was slightly higher in the premenopausal patients by both methods. In the PR assays (biochemical or immunocytochemical) there were no significant differences between the two patient groups in the proportion of PR-negative tumors or in the median PR content in PR-positive tumors.