Cytosol and nuclear estrogen receptors (occupied and unoccupied sites) and progesterone receptors in human breast cancer

Summary Estrogen and progesterone receptor concentrations in cytosol and nucleus were measured in 21 primary breast cancer tumors. Twelve out of the 21 tumor samples were cytosol estrogen receptor positive, 8 of which contained only unoccupied estrogen binding sites in the cytosol, but 2 of the 9 estrogen receptor negative samples did contain cytosol binding sites already occupied by endogenous homone. Four other estrogen receptor negative tumors only showed nuclear binding sites. Only 3 of the 12 estrogen receptor positive tumors also contained progesterone receptors. All of these tumors also had estrogen receptor in the nucleus. However, three of the 17 progesterone receptor negative samples had progesterone receptor only in the nucleus. The present data indicate that 3 possible classes of false negative tumors can be encountered: 1) estrogen receptors occupied by endogenous hormone, 2) tumors containing only nuclear estrogen receptors, and 3) tumors having only nuclear progesterone receptors. Measurement of nuclear estrogen receptor together with the progesterone receptor provides further information on whether the estrogen receptor system is not only present but also functional, and should be of value in the prediction of hormone dependent breast cancer.     

Estrogen and progesterone receptor isoforms: clinical significance in breast cancer

The identification and exploitation of biomarkers that may predict response to anti-cancer treatments has the capacity to revolutionize the way that patients with cancer are treated. In breast cancer, the estrogen receptor (ER) and the progesterone receptor (PgR) are known to have a significant predictive value in determining sensitivity to endocrine therapies. Tumor expression of ER or PgR is known to affect clinical outcome and this information is often used to determine a patient's optimal treatment regimen. However, the measurement of ER and PgR alone is more complex than originally thought and the impact of the recently identified isoforms of ER (ERα and ERβ) and PgR (PgRA and PgRB), as well as several variant and mutant forms, upon the choice of treatment remains unclear. Therefore, ER and PgR expression alone are unlikely to determine a patient's optimal treatment regimen, particularly when the amount of ‘cross-talk’ between different pathways, such as the epidermal growth factor receptor pathway, is considered. In order to account for the complex cell-signaling environment that occurs in breast cancer, multifactorial techniques are needed to analyze tumor biomarker expression. The recent advances in genomic- or proteomic-based approaches has enabled molecular portraits of breast cancers to be painted, allowing biomarkers of response and prognosis to be identified and characterized more accurately than before. In the future, patients could be treated according to the molecular portrait of their tumor biomarker expression, maximizing the therapeutic benefit that each patient receives. This revised version was published online in August 2006 with corrections to the Cover Date.     

Estrogen sensitivity of normal human breast tissue in vivo and implanted into athymic nude mice: Analysis of the relationship between estrogen-induced proliferation and progesterone receptor expression

High serum concentrations of estradiol (E₂) equivalent tothose observed in the luteal phase of the menstrual cycle stimulate bothepithelial cell proliferation and progesterone receptor (PgR) expression innormal human breast tissue xenografted into athymic nude mice. We reporthere the results of further investigations designed to determine whether theinduction of PgR expression and proliferation require differentE₂ concentrations and whether proliferating cells expressedthe PgR. In untreated normal breast xenografts, the PgR was virtuallyundetectable and proliferation was at basal levels. Progesterone (Pg)treatment alone had no effect compared to no treatment. Treatment withE₂ at follicular phase serum concentrations maximallyincreased PgR expression but was without effect on proliferation. However,treatment with E₂ at luteal phase serum concentrations, aloneor in combination with Pg, significantly increased both the PgR content andthe proliferation of the breast epithelium. These experimentally deriveddata reflected the observations made on normal breast tissue at surgicalbiopsy where PgR content was similar in both halves of the menstrual cycle,whereas proliferation was significantly higher in the luteal phase. Finally,using double labelling techniques, it was demonstrated that proliferatingepithelial cells rarely expressed PgR in normal breast tissue obtained atsurgical biopsy. These results suggest that the threshold ofE₂ required to induce PgR expression in normal human breastepithelial cells is lower than that required to induce proliferation andthat the majority of proliferating breast cells do not express the PgR.     

前列腺特异抗原和雌激素、孕激素受体在乳腺癌中的表达及其相关性

目的：研究前列腺特异抗原在乳腺癌组织中的表达及其与ＥＲ、ＰＲ表达的关系。方法：采用免疫组化法同１０２例乳腺癌的抗人体前列腺特异抗原（ＰＳＡ）,雌激素受体（ＥＲ）和孕激素受体（ＰＲ）。结果：２６例ＰＳＡ阳性（２５．５％）,ＰＳＡ＾＋和ＥＲ＾＋、ＰＲ之间存在密切关系（Ｐ〈０．０５）。ＰＳＡ＾＋、ＥＲ＾＋的乳腺癌液下淋巴结转移率明显低于ＰＳＡ＾－、ＥＲ＾－的乳腺癌（Ｐ〈０．０５）。结论：ＰＳＡ、ＥＲ、Ｐ     

The effect of tumor size and axillary lymph node metastasis on estrogen and progesterone receptors in primary breast cancer

We evaluated the estrogen receptors (Er) and the progesterone receptors (Pr) in 209 female patients with primary breast cancer. There is statistically a very highly significant negative correlation between the size of the tumor and the Pr (p = 0.007); large tumor contains low Pr. The same correlation was found with Er, but it was not statistically significant (p = 0.40). There is also a negative correlation between the number of axillary metastatic lymph nodes and Er and Pr, but this was not statistically significant.     

Response to primary chemotherapy in breast cancer patients with tumors not expressing estrogen and progesterone receptors

Background:We recently demonstrated that in premenopausalpatients with estrogen receptors (ER)-absent tumors, early initiation ofsystemic chemotherapy after primary surgery might improve outcome. These dataindicate a different responsiveness to chemotherapy for tumors not expressinghormone receptors. To test this hypothesis we evaluated the responsiveness topreoperative chemotherapy in patients with ER and progesterone receptors(PgR)-absent tumors. Patients and methods:Patients with biopsy-provenT₂–T₃, N_0–2 breast cancertreated at a single institution from January 1995 to August 1999 withpreoperative chemotherapy were retrospectively evaluated. ER and PgR weredetermined immunohistochemically and classified for this purpose as absent(0% of the cells positive) or positive (1% of the cells). Results:On 117 evaluable patients 72 had an objective response(61%). A significant difference in response was observed for patientswith ER and PgR absent compared with those with ER and/or PgR-positive tumors(82% vs. 57%,P = 0.03 Fishers's exact test).Pathological complete remission rates were also significantly different in thetwo groups (23% vs. 7%, respectively; P = 0.04). Conclusions:The different degree of response according to hormonereceptors expression supports the hypothesis that tumors not expressing bothER and PgR might represent a different clinical entity in terms ofchemotherapy responsiveness.     

Estrogen and progesterone receptor profile patterns in primary breast cancer

Summary Estrogen (ER) and progesterone receptor (PgR) analyses have been performed in 884 primary, malignant human breast tumor biopsies. Receptor contents were evaluated with respect to age and menopausal status. The frequency of ER + tumors was found to be significantly higher in postmenopausal than in pre/perimenopausal women. Age rather than menopausal status was found to be associated with this difference. The significant association with age was found in the post- but not the pre/perimenopausal women.The frequency of PgR + tumors was found to be significantly lower in the postmenopausal than in the pre/perimenopausal women. Neither age nor menopausal status alone could account for this difference, which appears to be due to a compound effect of the two factors.The distribution of receptor profile patterns is described according to menopausal status. The patterns differ significantly in pre- and postmenopausal women. PgR dominates in the premenopausal tumor while ER dominates in the postmenopausal tumor. This difference is apparent within the subgroup of ER + PgR + patients as well.The current tenets for prediction of recurrent disease utilizing steroid hormone receptor determinations are discussed for the group of ER + PgR + patients.sponsored by The Danish Cancer Society     

The Detection and Analysis of Estrogen and Progesterone Receptors in Breast Cancer （Report of 1,393 Patients）

Objective  To explore the distribution of estrogen receptors (ER) and progesterone receptors (PR) in patients with breast cancer and to compare the results with clinical parameters. Methods  Breast cancer specimens of 1393 cases were stained for the ER and PR by a SP Two -Step method, and analyzed with respect to age, menstrual status, histopathology and metastasis of axillary lymph nodes. Results  The correlation coefficients between ER and PR were positive-(P< 0.0001). The negative expression of ER in patients 39 years or less was the highest with a statistical significance (P<0.0001 ). There was no relationship between the patient’s age and positive expression of ER, PR and negative expression of PR (P>0.05). There were significantly higher positive rates of ER and lower positive rates of PR in post-menopausaf patients than in pre -menopausal cases(P<0.0001). There was no relationship between the status of ER, PR and the corresponding histopathology (P>0.05). The patients with no metastasis in the axillary lymph nodes had higher simultaneous positive rates of ER and PR (P<0.0001), and those with axillary lymph node metastasis had significantly higher rates of negative expression of ER and PR(P<0.0001). Conclusion  The positive and negative distributions of ER and PR have some regular patterns which may be used as a reference to choose combined therapy and to predict the prognosis for breast cancer patients.     

The detection and analysis of estrogen and progesterone receptors in breast cancer (report of 1,393 patients)

Objective To explore the distribution of estrogen receptors (ER) and progesterone receptors (PR) in patients with breast cancer and to compare the results with clinical parameters. Methods Breast cancer specimens of 1393 cases were stained for the ER and PR by a SP Two -Step method, and analyzed with respect to age, menstrual status, histopathology and metastasis of axillary lymph nodes. Results The correlation coefficients between ER and PR were positive-(P< 0.0001). The negative expression of ER in patients 39 years or less was the highest with a statistical significance (P<0.0001 ). There was no relationship between the patient’s age and positive expression of ER, PR and negative expression of PR (P>0.05). There were significantly higher positive rates of ER and lower positive rates of PR in post-menopausaf patients than in pre -menopausal cases(P<0.0001). There was no relationship between the status of ER, PR and the corresponding histopathology (P>0.05). The patients with no metastasis in the axillary lymph nodes had higher simultaneous positive rates of ER and PR (P<0.0001), and those with axillary lymph node metastasis had significantly higher rates of negative expression of ER and PR(P<0.0001). Conclusion The positive and negative distributions of ER and PR have some regular patterns which may be used as a reference to choose combined therapy and to predict the prognosis for breast cancer patients.     

Estrogen alpha and progesterone receptor expression in the normal mammary epithelium in relation to breast cancer risk

Estrogens play a central role in the etiology of breast cancer, and results from observational studies and randomized trials have also implicated progestins. The effects of these hormones in the mammary tissue are exerted through binding with specific receptor proteins in the cell nucleus. It has been proposed that higher estrogen receptor alpha expression in the normal breast epithelium may increase breast cancer risk. In a study in Greece, we determined estrogen alpha and progesterone receptor expression in normal mammary tissue adjacent to the pathological tissue from 267 women with breast cancer and 299 women with benign breast disease. Mouse monoclonal antibodies specific for estrogen receptor alpha and progesterone receptor were applied. The H-index, which incorporates frequency and intensity of staining of the cells, and can range from 0 to 300, was deemed positive when it exceeded 9. Among premenopausal women, there was no evidence for an association with breast cancer risk for expression of either type of receptors. Among postmenopausal women, breast cancer risk was inversely associated with expression of both estrogen alpha (odds ratio (OR)=0.39; p=0.015) and progesterone (OR=0.40; p=0.008) receptors. The hypothesis that overexpression of estrogen receptors alpha or progesterone receptors in normal breast epithelium may increase the risk of breast cancer was not supported by our data. Instead, we found evidence that overexpression of these receptors may be associated with reduced risk for breast cancer in line with the well-known association of expression of these receptors in the malignant tissue and better breast cancer prognosis.     

Estrogen and progesterone receptor levels are lower in specimens taken from previously biopsied breast tumor tissue

Estrogen and progesterone receptor (ER and PgR) levels and peroxidase activity were measured in the first biopsy and in tissue removed 4-10 days later during mastectomy of previously biopsied tumor. The proportion of ER- and PgR-positive tumors declined from 71% and 63%, respectively, in the biopsy group, to 46% and 36% in the mastectomy group. Peroxidase activity was low or absent in all biopsy specimens, but was present in 16 out of 25 tumors which had been biopsied previously. Receptor levels in paired specimens obtained from the same patient were lower in the mastectomy in 14 out of 18 receptor-positive cases. This decrease in the receptor level was accompanied by a significant increase in peroxidase activity. We suggest that an attempt be made to perform receptor analysis from "virgin" tumor tissues at initial biopsy. If this is not possible, measurement of peroxidase activity could be helpful in evaluating the degree of receptor deterioration.     

Hormone receptors and obesity in Japanese women with breast cancer

Summary An association between hormone receptors in primary breast cancer and obesity determined prior to mastectomy was investigated in 128 Japanese women. The following criteria for obesity were used: (1) weight 60 kg (132 lbs), (2) weight kg/height cm–105 1.3, (3) weight lbs/height in 2.30, (4) body surface area 1.56 m². In view of the 4 criteria, tumor estrogen receptor (ER) values 4 fmol/mg were observed in obese patients more often than in nonobese patients, though the difference was not statistically significant. The same tendency was observed in the postmenopausal subgroup, 62 patients, especially in the 36 patients more than 5 years beyond menopause. However, there was still no statistical difference between obese and nonobese patients because the number of subjects was small. The same tendency was observed in the case of progesterone receptor (PgR) (6 fmol/mg) as observed in the case of ER. Address for reprints: Dr. Keijiro Kuno, Department of Surgery, Cancer Institute Hospital, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo, Japan 170     

Colocalization of estrogen and progesterone receptors with an estrogenregulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue

Summary We have studied by immunocytochemistry and monoclonal antibodies the presence and localization of estrogen receptors, progesterone receptors, and a 24-kD estrogen-regulated heat shock protein in biopsies from breast and endometrial cancer patients. Three different tissue processing protocols were used to colocalize the antigens in the same tissue sections: a) frozen sections, b) formalin fixation with routine paraffin embedding, and c) picric acid-formaldehyde (PAF) fixation with a rapid embedding in paraffin. Frozen sections showed good receptor staining but poor 24-kD protein immunoreactivity, while routine paraffin sections (with or without DNase pretreatment) were inadequate to reveal the nuclear receptor proteins at the same level seen in frozen sections. On the other hand, all three proteins could be detected satisfactorily in PAF-fixed paraffin-embedded tissue. Using this procedure we were able to visualize 24-kD protein and estrogen receptor or progesterone receptor in individual cells in paraffin sections. The study revealed that in all of the estrogen receptor positive breast and endometrial tumor samples, almost 90% of the cells expressing the cytoplasmic 24-kD protein contained estrogen receptor in the cell nucleus. In contrast, 24-kD immunoreactive cells did not express progesterone receptors in almost 40% of the progesterone receptor positive tumor samples.     

Estrogen and progesterone receptors in malignant mixed mesodermal tumors of the ovary

Malignant mixed mesodermal tumors of the ovary occur in less than 1% of cases of ovarian cancer. They have a dismal prognosis and the most effective type of therapy is still not known. All cases of malignant mixed mesodermal tumor of the ovary between Jaunary 1, 1985 and May 1, 1994 operated on by the gynecologic oncology service are the subject of this report. Data were obtained from the hospital and office records of the patients. Nine patients who had their primary surgery by the gynecologic oncology service were found to have the diagnosis of malignant mixed mesodermal tumor of the ovary. Homologous tumors were found in five patients and heterologous tumors in four. Homologous tumors (mean survival 15.2 months) showed a better survival than heterologous tumors (mean survival 6.5 months; P = 0.001). An elevated estrogen receptor status was shown to correspond to longer survival (p < 0.0001). Six specimens were considered to be estrogen receptor positive and three were receptor negative. The mean survival in those patients who had a positive estrogen receptor status, 13.7 months, with a median of 7.5 months was significantly higher than those who were not positive, 6.7 months (P = 0.019) with a median of 6.25 months. All specimens were progesterone receptor negative. Malignant mixed mesodermal tumors of the ovary have a dismal prognosis, no effective therapy, and controversial prognostic indicators. Increasing estrogen receptor status appeared to correlate with longer mean survival. Larger, multi-institutional studies need to be done to determine the overall significance of these findings.     

Changes in hormone receptors and proliferation markers in tamoxifen treated breast cancer patients and the relationship with response

Aim: To determine the effects of tamoxifen on the levels of hormone receptors and proliferation markers in the early phase of treatment and the relationship of the changes with tumor response in patients with primary breast cancer. Methods: Twenty-one women with primary, operable breast carcinomas were treated with tamoxifen 20 mg daily. Fine needle aspiration (FNA) was used to obtain samples prior to the start and at 14 days and 8-weeks post-treatment. From these samples estrogen receptor (ER), progesterone receptor (PgR), and Ki67 levels were determined using immunocytochemistry and ploidy and S-phase fraction (SPF) using flow cytometry. Tumor response was measured clinically according to UICC criteria. Results: There were 12 responders (2 CR, 10 PR) and 9 non-responders (2 NC, 7 PD). Responders were more likely to be ER + (p=0.002), PgR + (p=0.006), and low SPF (p=0.06). At 14 days post-tamoxifen, the median decrease in Ki67 (% cells staining) for responders was – 4.8 and for non-responders – 0.15 (p=0.005). This decrease was seen predominantly in ER + tumours. The difference in SPF was not significant. A decrease in ER was seen in 3/15 patients all of whom were responders. A rise in PgR was seen in 7/17 patients and all but one were responders. Similar changes for ER and PgR were seen at 8-weeks post-tamoxifen, although the reductions in Ki67 and SPF at that time point were not related to response. Conclusion: We have observed a decrease in Ki67 and ER and a rise in PgR after 14 days of treatment with tamoxifen that was related to subsequent response. This is the first study in which an early decrease in a proliferation marker has been shown to relate to subsequent clinical response.     

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D

[³H]Tamoxifen Aziridine ([³H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [³H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [³H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [³H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [³H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/mg p.). The 4S peak of [³H]TAZ was partially inhibited by both estrogens and antiestrogens. When [³H]E₂ was used instead of [³H]TAZ, only an 8S peak was detected. [³H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [³H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [³H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [³H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [³H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.     

Presurgical radiotherapy decreases the concentrations of estrogen and progesterone receptors in human breast cancer: A 200-patient study

Summary The concentrations of cytosol estrogen (RE) and progesterone (RP) receptors were evaluated in human breast cancer either pretreated or untreated by radiotherapy. Receptor sites were assayed within a week following surgery by using the classical dextran-coated charcoal technique with three saturating concentrations of (³H) 17 estradiol and (³H) R5020 and by correcting for nonsaturable binding estimated in parallel with an excess of nonradioactive ligand. Only tissue containing cancer cells as determined by a pathologist was assayed for receptors.Two nonrandomised groups were compared: a control group of 91 patients which were not treated before surgery, and an irradiated group of 108 patients which received radiotherapy (60 to 70 grays) about 2 to 4 months before surgery. Radiotherapy significantly reduced the percentage of receptor positive tumors ( 10 fmoles/mg protein) from RE 82% and RP 67% in the control group to RE 54% and RP 28% in the irradiated group.The mean concentrations of the positive receptors were also decreased by irradiation, from 238 fm/mgP and 146 fm/mgP to 55 fm/mgP and 54 fm/mgP for RE and RP respectively. This decrease was observed for all histopathological stages studied in both pre- and postmenopausal patients, and was also not dependent on cell density. Since the irradiated patients mostly had tumors of TNMT₂ and nonirradiated patients had smaller tumors (TNM T₂), we checked that the same effect of radiotherapy was found in the homogeneous group of TNM = T₂.We conclude that presurgical radiotherapy of breast cancer decreases the percentage of estrogen receptor and progestin receptor positive patients. Therefore, the assay of steroid receptors in breast cancer remains useful in predicting hormone dependency and prognosis of breast cancer when receptor concentrations are positive. But the assay is of no value when receptor concentrations are negative. New micro techniques must therefore be developed for assaying receptor before any therapy.     

Epidermal growth factor receptors in human breast cancer

Summary The capacity for specific binding of¹²⁵I-epidermal growth factor (EGF) was studied in crude membrane fractions from 95 human breast carcinomas. About 42% of the samples showed saturable, high affinity, specific binding of EGF. In 21% of the tumors we were able to demostrate high (above 10 fmoles/mg protein) binding capacity. Moreover, high EGF receptor values were associated with a low content of estradiol receptor. These studies are related to the definition of new biochemical markers in human breast cancer.     

Estrogen and estrogen receptors of breast cancer.

Human breast cancer can be divided into a group that contains specific receptor sites for estrogen and a group without such specific estrogen-binding sites. The presence of specific estrogen receptors in some tumors indicating hormonal dependency has been shown to be of predictive value for endocrine treatment. This would greatly improve therapeutic planning for patients with breast cancer. Tumor tissue from 52 patients was investigated for content of both cytosol estrogen and estrogen receptor. In addition, the total tumor estrogen was also determined in 14 of these tumors. The results of this investigation show two distinct groups: one group containing both estrogen receptor and estrogen and a second group with no receptor but with measurable amount of estrogen. Tumors with estrogen receptors have higher tissue levels of estrogen than tumors without specific estrogen receptor. Even in the absence of estrogen recptor, however, most tumor tissue examined contained a measurable amount of estrogen.