Immunohistochemical image analysis of estrogen and progesterone receptors in breast cancer

Background  Recently objective quantification of immunohistochemical estrogen receptor (ER) and progesterone receptor (PgR) staining in breast cancer by image cytometry has been predominantly performed by measuring the area of positively stained cells. However, in sample preparations of immunostained hormone receptors, both the stained area and the intensity of staining vary. In this study, we performed quantification of the stained area by measuring, tailing intensity using image cytometry. Methods  Quantitative analysis of ER and PgR immunohistochemistry was performed using image cytometry. The obtained values were presented as % of positive staining (%PS). Comparison of %PS with values obtained by EIA and with clinicopathological features was performed. Results  The %PS values and the natural logarithm of the EIA levels of the hormone receptors showed a significant positive correlation for both ER and PgR. The concordance of the results obtained by the two methods was 96.3% for ER and 73.7% for PgR. The ER-%PS values of postmenopausal patients were significantly higher than those of premenopausal patients, whereas the PgR-%PS values of the former group were significantly lower than those of the latter group. Conclusions  The quantification of ER and PgR in immunostained preparations using %PS as a parameter was reproducible and showed a high correlation with values obtained by EIA. It was shown that only menopausal status affects hormone receptor levels when analyzing the relationship between %PS measurements and clinicopathological features.     

乳腺癌临床诊断中雌激素和孕激素受体表达的意义

目的探讨雌激素(ER)和孕激素(PR)受体的表达在乳腺癌临床诊断中的意义。方法选取2013年3月至2014年3月间大连大学附属中山医院收治的60例乳腺癌患者,另选取大连市红星社区体检健康人员60例为对照组。所有患者术前均进行ER和PR水平测定,分析彩色多普勒超声表现特征,采用免疫组织化学染色检测ER和PR在乳腺及其周围组织中的表达。结果年龄≥40岁的乳腺癌患者的雌二醇激素含量和孕酮激素含量均高于<40岁的患者,两者比较差异有统计学意义(P<0.05)。免疫组化反应测定中,与健康成年女性比较,乳腺癌患者ER和PR水平均高(均P<0.05),且集中分布于细胞质当中。乳腺癌肿块边缘性状和血流信号的分级与ER、PR均有统计学意义(均P<0.05)。结论年龄偏大,ER和PR水平高,彩色多普勒检查显示乳腺肿块边缘不清晰、周围组织血流不丰富,提示可能患有乳腺癌。     

Prognostic significance of progesterone receptors alone or in association with estrogen receptors in human breast cancer

Estrogen (ER) and progesterone (PgR) receptors were measured simultaneously in 1144 consecutive breast cancer patients to determine the distribution of patients according to receptor and menopausal status when receptor occurrence rates were considered. The prognostic significance of PgR, either alone or in association with ER, was studied on 187 consecutive breast cancer patients treated only by radical mastectomy. All the cases, as regards axillary node status, were pathologically assessed as node negative. These patients did not receive any adjuvant treatment after mastectomy. At 36 months after mastectomy, the follow-up indicated that PgR- patients have a worse prognosis than PgR+ ones. This is evident when PgR alone is considered as a prognostic factor as well as when it is used to identify, within ER+ cases, those with a higher probability of relapse. In conclusion, it can be stated that although PgR status is an independent prognostic factor, it is useful to evaluate ER and PgR simultaneously for better patient management.     

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     

Immunological quantitation of nuclear receptors in human breast cancer: relation to cytosolic estrogen and progesterone receptors

Nuclear estrogen receptors (ERn) can now be reliably analyzed using the monoclonal estrogen receptor enzyme immunoassay. In a consecutive series of 135 breast cancer biopsies, ERn as well as cytosolic estrogen receptor (ERc) and progesterone receptor (PgR) concentrations were determined to evaluate whether ERn assays provide additional valuable information for the clinical management of the disease. Furthermore, by performing analyses on this relatively large number of patients, we sought explanations for the occurrence of the receptor profiles of ERc negative PgR positive and ERc positive PgR negative, which are found in a significant proportion of tumor biopsies. Eight-four % of all tumors are classified as ERn positive (greater than or equal to 10 fmol/mg nuclear extract protein) using the monoclonal assay technique. Two trends are evident: ERc positivity was found to be associated with ERn positivity (greater than or equal to 10 fmol/mg cytosol protein) in 98% of the cases investigated; and PgR positivity (greater than or equal to 10 fmol/mg cytosol protein) was found to be associated with ERn positivity in 95% of the cases investigated. However, a major proportion (approximately 28%) of ERn positive tumors are either ERc negative or PgR negative. The pattern of ERc negative ERn positive occurs almost exclusively among younger women, most of whom also had detectable amounts of PgR in their tumor tissues, while the pattern of ERn positive PgR negative occurs primarily among older women. ERn concentration was found to be significantly correlated to the concentration of both PgR and ERc. While the correlation between ERn and PgR was found to be strongest among women younger than 50 years of age, the correlation between ERn and ERc was strongest among women older than 50 years. Young women were found to have a significantly higher proportion of total tissue estrogen receptor present as ERn than older women (27 versus 14%). The information obtained by performing ERn analyses concurrently with or in place of ERc and PgR analyses does not appear to be valuable for the clinical management of the disease. However, this new method for determination of ERn is a significant advance in receptor technology that permits reevaluation of established enigmas concerning the biology and natural history of breast cancer.     

Prognostic value of estrogen and progesterone receptors in primary breast cancer

Estradiol receptor alone or estradiol and progesterone receptors (ER, PgR) have been measured in tumors from 307 women who were treated for primary breast adenocarcinoma. Patients received adjuvant therapy in relation to tumor stage independently of receptor status. Over a relatively short follow-up, more recurrences are recorded in patients with ER+ tumors, but at 7 years the same proportion of recurrences are registered in both groups of patients whose tumors were ER+ or ER-. Patients whose tumors were processed for both ER and PgR (148 cases) have now been evaluated after 5 years follow-up. Among 56 patients with PgR+ tumors 5 recurred, versus 22/92 in the PgR- group. 21/87 patients who had 1 to 4 invaded nodes at the time of surgery relapsed: 17/54 had PgR- tumors versus 4/33 PgR+. 6 recurrences were recorded in the 61 patients with negative nodes: 5 of them occurred in patients with PgR- tumors. In addition, recurrences observed in patients with negative receptor status occurred after a shorter disease-free interval. Analysis of the incidence of recurrences in relation to the combined ER/PgR status in patients who did not receive adjuvant therapy suggests that tumor PgR content is a more significant criterion than ER for long-term prognosis.     

Comparative studies of prolactin, estrogen, progesterone and androgen receptor levels in human breast cancers

A receptor for lactogenic hormones (prolactin of several species and human growth hormone) was characterized in crude plasma membrane preparations of adult female rat liver. The binding of 125J-labeled prolactin to receptors was specific, saturable, reversible, and of high affinity (Kd = 0.23 x 10(-10)M). The maximum amount of prolactin specifically bound to liver of untreated female rats was 45 fmol/mg protein, whereas no specific prolactin binding was detected in plasma membrane preparations of adult male or immature rats. With this assay, a specific and reversible binding of ovine prolactin was detected in plasma membrane preparations of 25 human breast cancers, ranging from 2-29% of total activity. The estrogen-, progesterone- and androgen receptor content was determined in the breast cancer specimens using the dextran-coated charcoal method. Specimens with the highest specific prolactin binding showed the lowest steroid receptor concentrations, but no significant correlation between estrogen-, progesterone-, androgen-, and prolactin receptor content and other prognostic factors was observed in our series. We conclude, that steroid and prolactin receptors are expressed independently in human breast cancer.     

Estrogen receptors,progesterone receptors,and cell proliferation in human breast cancer

Summary The breast is a target organ for estrogens and progesterone. These hormones control several functions of the normal and abnormal mammary epithelium including cell proliferation. Most of the actions of estrogens and progesterone are mediated via specific steroid receptors, and one would expect that proliferating cells should contain estrogen receptors (ER) and/or progesterone receptors (PR). However, the correlation between receptor expression and cell proliferation is still controversial. In the present study we have examined 29 human breast cancer samples; in 17 of them we evaluated the simultaneous ER and PR localization with that of proliferating cell nuclear antigen (PCNA) and silver-stained nucleolar organizer regions (AgNORs) in a cell-by-cell study. We found that in almost 50% of the tumor biopsies examined, the cells expressing ER were significantly associated with elevated cell proliferation. In another group (38%) there were not significant differences between ER expression and cell proliferation. In only one of the samples (6%) the cells expressing ER showed lower cell proliferation. The study also revealed that in 44% of the tumors the PR expressing cells were associated with elevated cell proliferation. In a second group the PR expression was not significantly associated with cell proliferation (33% of the cases). Finally, in 22% of the samples the cells carrying PR showed lower cell proliferation. We also detected lower ER immunoreactivity in 30% of the breast cancer biopsies with one of the monoclonal antibodies against ER (antibody 1D5 directed against the A/B domain). This group of tumors was PR-negative (or very weakly positive) and had high proliferation. The presence of tumors with abnormal ER proteins and displaying ER/PR significantly associated with elevated cell proliferation could have implications in human breast cancer treatment.     

Immunohistochemical determination of androgen receptors in relation to oestrogen and progesterone receptors in female breast cancer.

The expression of oestrogen (ER), progesterone (PR) and androgen (AR) receptors in female breast cancer was investigated by immunohistochemistry on snap-frozen tissue specimens of a series of 100 breast cancers. For detection of the AR we used a recently developed mouse monoclonal antibody specific for the N-terminal domain of the human AR. Expression of AR was compared with that of ER and PR as well as with tumour grade and age. Of the breast cancers investigated, 76% were AR-positive. This high percentage corresponds well with previous data on AR expression in breast cancer determined with ligand-binding assays. In 53% of the tumours AR, ER and PR were present, while 9% of the tumours were positive for AR and negative for ER and PR. In 13% of the tumours no ER, PR or AR expression was seen; these were all grade-III tumours. A positive correlation was found between age and ER expression, but no correlation was seen between age and PR or AR. Future studies should establish the prognostic value of the combination ER, PR and AR determinations on female breast cancer with regard to biological behaviour and response rate to hormonal therapy.     

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.     

A clinical evaluation of nuclear estrogen receptors combined with cytosolic estrogen and progesterone receptors in breast cancer

Breast cancer tissue from 95 women was simultaneously assayed for three receptors: cytosolic estrogen (CER), cytosolic progesterone (CPR), and nuclear estrogen (NER). The main objective was to determine whether the addition of NER assay to the currently accepted practice with only CER and CPR could improve the predictive capacity of receptors. Forty-two patients were studied for response to hormone therapy and 95 patients were studied for survival; the median follow-up period was 73 months (range, 8 to 300 months). The incidence of CER+, CPR+, and NER+ was 74%, 70%, and 52%, respectively. Each receptor appeared more frequently, although not significantly so, in higher age groups. Forty percent of tumors had all three receptors positive and 14% had all negative; the remaining tumors showed all possible combinations of receptors. Both the rate of response and survival curves among 70 patients with CER+ did not show any significant difference whether NER was positive or negative. Also, among 38 patients with CER+, CPR+, and NER+, there was no significant difference in the clinical outcome as compared to 17 patients with CER+, CPR+, and NER-. Among 25 patients with CER- the rare occurrence of NER+ in only three patients did not suggest any clinical implication. It is concluded, therefore, that on overall clinical grounds the current series does not support the addition of NER assay whenever data is available on both CER and CPR.     

Current status of estrogen receptors in human breast cancer.

In estrogen target tissues and hormone-dependent tumors, the steroid enters the cells and binds to a cytoplasmic protein called the estrogen receptor (ER). The steroid-receptor complex then migrates to the nuclei, where it initiates the biochemicial events characteristic of estrogen stimulation. Since ER is absent in tissues not responsive to estrogen, recent studies have asked whether ER assays in human breast cancer tissue might be used to identify those patients likely to respond to endocrine therapy. Data on 436 clinical trials contributed from a dozen centers around the world now clearly indicate that if a patient's tumor does not contain ER, there is virtually no chance of tumor regression following endocrine therapy. A large number of patients can be thus spared unrewarding major endocrine ablative therapy if ER assays are performed routinely. Of tumors with positiev ER, 55-60% respond to endocrine therapy. This single piece of data, when coupled with available clinical prognostic factors such as menopausal status, disease free interval, site of the dominant lesion, and especially response to previous hormonal therapies, should be practicing oncologist to select or reject endocrine therapy with considerable confidence.     

固定后乳腺癌细胞雌激素受体和孕激素受体的FCM和Western blot检测

背景与目的：雌激素受体(estrogen receptor,ER)和孕激素受体(progesterone receptor,PR)水平的定性、定量检测,对乳腺癌患者预后判断和内分泌治疗效果的评价具有重要意义。Western blot法和流式细胞术(flow cytometry,FCM)是蛋白质定性、定量分析的重要手段,但常规Western blot法要求提取新鲜样本的蛋白质。本研究拟建立固定后乳腺癌细胞ER和PR的Western blot检测方法,探索Western blot法和FCM对同批固定样本ER、PR进行同步分析的可行性。方法：取不同乳腺癌细胞株对数生长期新鲜细胞和固定细胞的蛋白提取物,分别用ERα单克隆抗体1D5和PR单克隆抗体PgR636以Western blot法对．ER、PR的表达情况进行检测,并与同期固定细胞的FCM检测结果进行比较。结果：经Westernblot法检测,T-47d、MCF-7、ZR-75-l细胞可见分子量正确的ERα清晰条带,固定T-47d和ZR-75-1细胞的ERα条带较新鲜细胞的条带浓,MM23l细胞ERα检测为阴性;T-47d和ZR-75-1细胞可见清晰且分子量正确的PR条带,固定细胞的PR条带较新鲜细胞的条带浓,MM23l和,MCF-7细胞PR检测为阴性;同期固定细胞ER、PR阳性表达的FCM检测结果与Western blot检测结果一致。结论：不同乳腺癌细胞在经0．25％多聚甲(paraformaldehyde,PFA)和75％乙醇固定后,可用于ER、PR的FCM定量检测,也可用于ER、PR的Western blot分析。     

Distribution of estrogen and progesterone receptors in primary tumor and lymph nodes in individual patients with breast cancer

Primary breast cancer tissue and lymph nodes were obtained from 48 patients. Estrogen receptors (ER) and progesterone receptors (PgR) were determined by a dextran-coated charcoal assay. ER were present in 72.9% of the primary tumors and in 62.4% of the malignant lymph nodes, whereas PgR were present in 73.0% and 50.0% of the cases, respectively. The primary tumor and the corresponding malignant lymph nodes showed an identical ER and PgR status, i.e., both tumor sites were receptor positive or both receptor negative in 89.6% and 77.1%, respectively. However, 10.4% of the patients had ER-positive tumors but ER-negative lymph nodes and 22.9% had PgR-positive primaries with PgR-negative lymph nodes. No receptor-positive lymph nodes showed a combination with receptor-negative primary tumor. This preliminary data shows that receptor-positive malignant lymph nodes mostly display the same receptor status as the corresponding primary tumor, whereas receptor-negative lymph nodes may have a receptor-positive primary tumor.     

Estrogen, progesterone, and androgen receptors in breast cancer in the Japanese: brief communication

Estrogen, progesterone, and androgen receptors (ER, PgR, and AR, respectively) were determined by dextran-coated charcoal assay analyzed with Scatchard plots in cytosols from 81 Japanese patients with breast cancer. For the estimations of PgR and AR, the following compounds were used: a synthetic progestin, [3H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), not bound by corticosteroid-binding globulin; and a synthetic androgen, [3H]R1881 (17beta-hydroxy-17-methylestra-4,9,11-trien-3-one), not bound by human sex steroid plasma-binding protein. Forty-two of 81 of the cancers revealed measurable amounts of ER (greater than 2 fmoles/mg cytosol protein), whereas the rates of measurable amounts of PgR (greater than 5) and AR (greater than 5) were 27% (18/67) and 36% (17/47), respectively. PgR were found in 45% of cancer patients with ER, but in only 9% of the cancer patients without ER. AR were found in 50% of the cancer patients with ER, but in only 22% of the cancer patients without ER. These results are consistent with those reported by Western investigators.