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Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.

To elucidate the clinical importance of estrogen receptor (ER) beta in breast cancer, 29 archival primary breast cancer specimens, six locally recurrent cancers, and five benign mammary tumors were examined histochemically for ERalpha, ERbeta and the proliferation markers Ki67 and cyclin A. In benign tumors, most epithelial cells contained ERbeta, but ERalpha was rare. In primary cancers, both ERalpha and ERbeta occurred in epithelial cells, the presence of ERbeta being associated with elevated expression of Ki67 and cyclin A, and ERalpha with decreased levels. Thus, the highest content of proliferation markers was seen in primary cancers that were ERalpha(-) ERbeta(+). Most Ki67-containing cells coexpressed ERbeta, but few showed ERalpha. In locally recurring cancers, ERalpha, ERbeta, and Ki67 were more highly expressed than in the corresponding primary tumors, and many cells containing ERbeta, but few with ERalpha, expressed Ki67. Surprisingly, ERbeta, but not ERalpha, was seen in the stromal cells of both primary and recurrent cancers. Because the response of breast cancers to tamoxifen therapy is correlated with the presence of ERalpha, cancer cells that lack ERalpha but contain ERbeta and proliferation markers represent a novel population of apparently proliferating cells that probably are not targeted by the current antiestrogens. Thus, appropriate ERbeta-specific ligands, perhaps in combination with tamoxifen, may be useful in improving the treatment of breast cancers.

Background: We wanted to determine whether neoadjuvant systemic chemoendocrine therapy guided by the estrogen receptor (ER) status of the primary breast cancer, followed by conventional surgery and/or radiotherapy, reduces local and distant recurrence and improves survival compared with adjuvant treatment given conventionally postoperatively., Patients and Methods: Two hundred ten patients with primary breast cancer (T1-T4, N0, N1-2) were randomised to receive treatment with neoadjuvant chemoendocrine therapy or conventional post-operative chemoendocrine therapy. Systemic therapy was based on the estrogen receptor (ER) status of the primary tumour obtained by trucut core biopsy. ER-negative patients received MMM chemotherapy (methotrexate (30 mg/m2), mitozantrone (7 mg/m2) and mitomycin (7 mg/m2) three-weekly for three months and ER-positive patients who were premenopausal received goserelin (3.75 mg monthly), and post menopausal women formestane (250 mg every two weeks) over three months., Results: With a minimum of five years follow-up, there is no evidence of any survival benefit from the pretreatment neoadjuvant therapy regimen, with five year overall survival being 79% +/- 4.7% (neoadjuvant) and 87% +/- 3.4% (adjuvant). Similarly, there was no apparent benefit in terms of disease-free survival. There was, however, a significant reduction in the incidence of distant metastases in responders (4 of 51; 8%) compared with non-responders (17 of 49; 35%) (P < 0.01). There was a reduction in the need for surgery in responding patients with T1 and T2 tumours, since 10 of 74 (14%) had no detectable residual tumour, without any apparent increase in the risk of local or distant recurrence., Conclusion: In this study neoadjuvant treatment with endocrine or chemotherapy provided no obvious survival benefit to women with breast cancer. However, it does allow avoidance of surgery in some cases. Also, the patients whose tumours respond to neoadjuvant systemic therapy have a lower incidence of distant metastases after five year follow-up compared to those whose tumours fail to respond.

Phosphorylation of the estrogen receptor alpha (ERalpha) N-terminal transcription activation function AF1 at serine 118 (S118) modulates its activity. We show here that human ERalpha is phosphorylated by the TFIIH cyclin-dependent kinase in a ligand-dependent manner. Furthermore, the efficient phosphorylation of S118 requires a ligand-regulated interaction of TFIIH with AF2, the activation function located in the ligand binding domain (LBD) of ERalpha. This interaction involves (1) the integrity of helix 12 of the LBD/AF2 and (2) p62 and XPD, two subunits of the core TFIIH. These findings are suggestive of a novel mechanism by which nuclear receptor activity can be regulated by ligand-dependent recruitment of modifying activities, such as kinases.

Estrogens have long been recognized as being important for stimulating the growth of a large proportion of breast cancers. Now it is recognized that estrogen action is mediated by two receptors, and the presence of estrogen receptor alpha (ER alpha) correlates with better prognosis and the likelihood of response to hormonal therapy. Over half of all breast cancers overexpress ER alpha and around 70% of these respond to anti-estrogen (for example tamoxifen) therapy. In addition, the presence of elevated levels of ER alpha in benign breast epithelium appears to indicate an increased risk of breast cancer, suggesting a role for ER alpha in breast cancer initiation, as well as progression. However, a proportion of ER alpha-positive tumors does not respond to endocrine therapy and the majority of those that do respond eventually become resistant. Most resistant tumors remain ER alpha-positive and frequently respond to alternative endocrine treatment, indicative of a continued role for ER alpha in breast cancer cell proliferation. The problem of resistance has resulted in the search for and the development of diverse hormonal therapies designed to inhibit ER alpha action, while research on the mechanisms which underlie resistance has shed light on the cellular mechanisms, other than ligand binding, which control ER alpha function.

Phosphorylation provides an important mechanism by which transcription factor activity is regulated. Estrogen receptor alpha (ERalpha) is phosphorylated on multiple sites, and stimulation of a number of growth factor receptors and/or protein kinases leads to ligand-independent and/or synergistic increase in transcriptional activation by ERalpha in the presence of estrogen. Here we show that ERalpha is phosphorylated by protein kinase A (PKA) on serine-236 within the DNA binding domain. Mutation of serine-236 to glutamic acid prevents DNA binding by inhibiting dimerization by ERalpha, whereas mutation to alanine has little effect on DNA binding or dimerization. Furthermore, PKA overexpression or activation of endogenous PKA inhibits dimerization in the absence of ligand. This inhibition is overcome by the addition of 17beta-estradiol or the partial agonist 4-hydroxy tamoxifen. Interestingly, treatment with the complete antagonist ICI 182,780 does not overcome the inhibitory effect of PKA activation. Our results indicate that in the absence of ligand ERalpha forms dimers through interaction between DNA binding domains and that dimerization mediated by the ligand binding domain only occurs upon ligand binding but that the complete antagonist ICI 182,780 prevents dimerization through the ligand-binding domain. Heterodimer formation between ERalpha and ERbeta is similarly affected by PKA phosphorylation of serine 236 of ERalpha. However, 4-hydroxytamoxifen is unable to overcome inhibition of dimerization by PKA. Thus, phosphorylation of ERalpha in the DNA binding domain provides a mechanism by which dimerization and thereby DNA binding by the estrogen receptor is regulated.

The cloning of a novel estrogen receptor beta (denoted ERbeta) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930 and Mosselman, S., Polman, J. , and Dijkema, R. (1996) FEBS Lett. 392, 49-53). ERbeta is highly homologous to the "classical" estrogen receptor alpha (here referred to as ERalpha), has been shown to bind estrogens with an affinity similar to that of ERalpha, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERalpha and beta in gel shift assays. We show that DNA binding by ERalpha and beta are similarly affected by elevated temperature in the absence of ligand or in the presence of 17beta-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERalpha and beta is rapidly lost at 37 degrees C, while in the presence of 17beta-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182, 780 does not "protect" human ERalpha (hERalpha) from loss of DNA binding at elevated temperature in vitro, it does appear to protect human ERbeta (hERbeta), suggestive of differences in the way ICI 182, 780 acts on hERalpha and beta. We further report that ERalpha and beta can dimerize with each other, the DNA binding domain of hERalpha being sufficient for dimerization with hERbeta. Cell and promoter-specific transcription activation by ERalpha has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERalpha and beta add greater levels of complexity to transcription activation in response to estrogens.

Oestrone sulphatase is an important source of local synthesis of biologically active oestrogens in human breast cancer. The oestrone sulphatase enzyme in the particulate fraction of human breast carcinoma was characterised. The Km was 8.91 microM, and the Vmax was 0.022 nmol min-1 mg-1. Oestrone sulphatase activity was detected in 93 of 104 human breast carcinoma samples (89%), and mean activity was 0.041 nmol min-1 mg-1 (range 0-0.399 nmol min-1 mg-1). There was no significant correlation between intratumoral oestrone sulphatase activity and oestrogen receptor status, or with any other prognostic factors. Intratumoral enzyme levels were not associated with time to recurrence or with overall survival time. It thus appears that, although a useful source of intratumoral oestrogens, oestrone sulphatase activity is not of prognostic significance in breast carcinoma.

The levels of oestrogen receptor (ER), progesterone receptor (PR) and transforming growth factor-beta 1 (TGF-beta 1) were measured by immunocytochemistry in 19 patients prior to and 1 month after the start of endocrine therapy (tamoxifen 10 patients; aromatase inhibition 9 patients). A complete or partial response was observed in 10 patients. The proportion of cells showing ER staining was higher in responding patients, but there was no change observed with endocrine therapy in either responding or non-responding patients. In contrast, cells staining for PR in responding patients were significantly reduced following therapy (59 +/- 9% to 24 +/- 9%: P < 0.05). There was no reduction in immunocytochemical PR in non-responding patients, although the numbers of these patients with initially positive PR levels was small. Stromal tissue adjacent to tumour cells stained with the antibody to TGF-beta 1, with particularly intense staining at the periphery of tumour cell aggregates. There was no correlation between the degree of TGF-beta 1 staining and ER or PR status, and no evidence of a change with endocrine therapy. It is concluded that neither tamoxifen nor aromatase inhibitors produce a change in the ER content or TGF-beta 1 content of breast tumours as detected immunocytochemically, but PR levels are significantly reduced after therapy in responding patients.

Histological sections obtained from 70 patients with breast cancer, all of whom had received endocrine therapy for metastatic or locally advanced disease, were assessed for specific immunocytochemical staining of oestrogen receptor and the oestrogen-induced protein pS2. There was also sufficient material from 25 patients for an assessment of progesterone receptor by immunocytochemistry. We found that, when using a "cut-off" point of 50% for ER and PR, and of 25% for pS2, ER was positive in 22/29 responders and in 12/41 non-responders, and thus was significantly associated with response to endocrine therapy. Similarly, in those subjects in whom PR was measured, PR was positive in 5/14 responders and negative in all 11 non-responders, again being significantly correlated with response. However, pS2 did not relate to response, being only positive in 10/29 responders and negative in 26/41 non-responders. The different response categories varied in their "percentage of positivity" as determined by the 2 tests. Thus, for ER and pS2 we observed: complete response--71% for ER compared with 14% for pS2; partial response--77% compared with 41%; stable disease--36% compared with 64%; and progressive disease--27% compared with 27%. We conclude that at the present time ER appears to be the most reliable indicator for predicting response to endocrine therapy in patients with breast cancer.

New tamoxifen analogues were tested for their antiproliferative activity both in vitro and in vivo. Binding studies showed that both 4-iodotamoxifen and pyrrolidino-4-iodotamoxifen and 2.5-fold higher affinities for the estrogen receptor compared with tamoxifen. Pyrrolidino-4-iodotamoxifen was also 1.5-fold more effective in causing inhibition of estrogen-induced growth of MCF-7 cells compared with tamoxifen at 10(-6) M. The 4-iodotamoxifen analogue was similar to tamoxifen in its inhibitory action at 10(-6) M. Antiproliferative activities of these drugs were tested using the nitrosomethylurea-induced rat mammary tumor model. Pyrrolidino-4-iodotamoxifen caused regression in 92% of rats, whereas tamoxifen caused regression in 75% of rats. The agonist activity of the analogues was determined using the immature rat and mouse uterotrophic assays. Both tamoxifen and 4-iodotamoxifen had similar partial agonist activity, and this was greater than that seen with pyrrolidino-4-iodotamoxifen. Furthermore, pyrrolidino-4-iodotamoxifen caused a dose-dependent inhibition of estrogen-induced vaginal cornification, whereas tamoxifen and 4-iodotamoxifen did not. These studies demonstrate that pyrrolidino-4-iodotamoxifen is more effective than tamoxifen in inhibiting tumor regression and that its reduced uterotrophic activity and increased estrogen receptor binding may give it significant clinical advantages over the parent compound.

We have studied estrogen receptor (ER) and progesterone receptor (PR) in normal breast by immunocytochemistry using tissue biopsies and fine needle aspirates (FNA) and, in the case of ER, by enzyme immunoassay. For ER we found a high degree of reproducibility for biopsies taken from the upper outer quadrant: FNA, r = 0.56 (P less than 0.002); tissue section immunocytochemistry, r = 0.89 (P less than 0.0001); and enzyme immunoassay, r = 0.76 (P less than 0.0001). For PR, FNA (r = 0.56, P less than 0.002) and tissue section (r = 0.97, P less than 0.0001) were also found to be reproducible techniques. Using enzyme immunoassay, we were able to measure ER accurately in normal breast tissue. In 59 samples we found a range of 0-37 fmol/mg cytosol protein (mean, 4 fmol/mg). In an age-matched group of 126 women with breast cancer, we found a significantly higher ER [range, 0-139 fmol/mg; mean, 37 fmol/mg (P less than 0.001)]. We then analyzed the ER and PR content of FNAs obtained from the upper outer quadrant of the normal breast in 143 normal women. We found that in only 23 of 143 samples (16%) were greater than or equal to 50% epithelial cells stained. There was a relationship between ER and PR (P = 0.03) and a higher ER content in European women than in non-European women (P less than 0.03). The PR content was related to high body mass index (P less than 0.02) and family history of breast cancer (P = 0.04). Samples tended to be more frequently ER positive by FNA if taken in the follicular phase of the menstrual cycle. We conclude that, although the levels of ER and PR are low in normal breast, they can be accurately measured. There is significant variation of ER and PR with several clinical parameters.

We have determined the estrogen receptor, progesterone receptor (PR), and 1,25-dihydroxyvitamin D3 receptor content of 136 breast carcinomas by an immunocytochemical method. The presence of the three receptors was not related to clinical features of presentation such as T-stage or to age or menopausal status. However, each of the three receptors has a different relationship to the course of the disease in these patients. The presence of PR was significantly associated with an improved overall survival (chi 2 = 4.61, P = 0.032). Patients whose tumors contained immunocytochemically detectable 1,25-dihydroxyvitamin D3 receptor had a longer disease-free interval than those patients with negative tumors (chi 2 = 4.01, P = 0.045). The presence of estrogen receptor and PR were found to correlate with an increased survival between relapse and death (P = 0.027 and P = 0.09, respectively). The relationships between estrogen receptor and PR and prognosis are more apparent when the degree of cell staining is considered. Combined receptor analysis improves our ability to predict the course of the disease and may therefore facilitate better management of the patients.

Estrogen and progesterone receptors were demonstrated immunocytochemically in the lung tissue of two cases of pulmonary lymphangioleiomyomatosis. In one case both receptors were localized in the proliferative smooth muscle nodules and diffusely in the interstitial tissue in close proximity to the muscle. The pulmonary tissue in the other case contained the estrogen receptor only. The authors conclude that the response to hormonal treatment seen in this disease might be optimized in the future by monitoring for the presence of steroid hormone receptors.

In order to determine the mechanisms of relapse following response to endocrine therapy, we have measured the oestrogen receptor (RE) content of biopsies of breast cancer in patients receiving various types of endocrine treatment. RE content fell in responding (means of 260.2 to 12 fmol/mg protein) and in nonresponding (means of 155.1 to 31.8 fmol/mg protein) patients who had measurable receptor at the start of treatment. Some of these patients, and a further group of responders to endocrine therapy, were monitored until relapse. Tumour biopsies at the time of relapse showed that 10/14 tumour samples contained significant RE (mean of 86.7 fmol/mg protein; range less than 10-271 fmol/mg protein) after successful endocrine therapy. No relationship could be found between RE content and plasma gonadotrophin or steroid-hormone concentration, but the fall in RE content correlated with reduced numbers of tumour cells in the biopsy. These results indicate that relapse following successful endocrine therapy in breast cancer does not appear to be due to the emergence of RE-negative tumour cells. The fall in RE content during response to endocrine therapy may be due to reduced tumour-cell content of the biopsy.

We have used an immunoperoxidase technique utilizing a monoclonal antibody to the estradiol receptor to identify immunoreactive estradiol receptor in breast carcinomas and have examined the relationship between the immunoreactive estradiol receptor and response to therapy in patients with advanced breast cancer. Fifty-six patients were found to be assessable for response to endocrine therapy. Twenty-two showed an objective response to some form of endocrine manipulation, and all these had positively stained carcinomas. None of the 17 patients with negatively stained carcinomas responded to endocrine therapy. We conclude that the monoclonal antibody to estradiol receptor can help identify breast cancer patients who may respond to endocrine therapy.

We developed an immunoperoxidase technique using a monoclonal antibody to the estradiol receptor (ER) to identify immunoreactive ER (iER) in breast carcinomas and compared this with the conventional dextran-coated charcoal (DCC) steroid binding assay. We also examined the relationship between the iER and response to therapy in patients with advanced breast cancer. We found iER-positive cells in 60 of 90 carcinomas (66.7%); this correlated with the DCC assay (r = 0.76; P less than .001). Of these, 56 patients were found to be assessable for response to endocrine therapy. Twenty-two showed an objective response to some form of endocrine manipulation, and all these had positively stained carcinomas. By deriving a staining intensity index (SII) we observed that 21 of 22 responders (95%) had an SII of greater than or equal to 0.5, whereas only 8 of 34 nonresponders (24%) had an SII of greater than or equal to 0.5. This difference is highly significant (P less than .001). None of the 17 patients with negatively stained carcinomas responded to endocrine therapy. We conclude that the monoclonal antibody to ER can help identify breast cancer patients who may respond to endocrine therapy.

An immunocytochemical technique has been developed to detect the oestrogen receptor (ER) in samples obtained by fine-needle aspiration of primary or secondary breast carcinoma tissue. 45 (75%) of 60 samples contained sufficient cells for analysis. 19 (90%) of 21 patients who responded to endocrine therapy had samples with ER-positive cells compared with only 7 (39%) of 18 of those who did not respond (p less than 0.001). This test, which causes little pain to the patient and does not need a surgical biopsy, is as accurate as any other method of predicting the response to endocrine treatment in breast cancer.

To examine the presence and distribution of oestrogen receptors in the normal breast during the menstrual cycle cytological samples obtained by fine needle aspiration from 69 premenopausal women with normal breasts were analysed immunocytochemically with a monoclonal antibody to oestrogen receptor; samples from 15 postmenopausal women were also analysed. The receptor content of breast cancers from 83 premenopausal women was also determined in relation to when during the menstrual cycle excision was performed. In the normal premenopausal women oestrogen receptors were detected in the nuclei of epithelial cells in 21 out 68 (31%) assessable samples. All 21 of these samples were obtained from the 35 women who were studied during the first half of their menstrual cycle (days 28 to 14). None of the 33 samples obtained during the second half of the cycle contained oestrogen receptors. Samples were assessable in eight of the postmenopausal women, six giving a positive result for oestrogen receptor. Fifty one of the 83 carcinomas were positive for oestrogen receptor, 24 having been excised during the first half of the cycle and 27 during the second half. Production of oestrogen receptor protein is suppressed at the time of ovulation in the normal breast epithelium of premenopausal women. In contrast, breast carcinoma cells either synthesise this protein continuously throughout the cycle or fail to express it despite fluctuations of serum hormone concentrations.

We devised a method of detecting estrogen receptors (ER) in bone marrow metastases from patients with breast cancer. The method involves a sequential double-staining immunocytochemical technique, with a monoclonal antibody to ER and a polyclonal antibody recognizing epithelial membrane antigen to confirm the epithelial nature of suspected tumor cells. Twenty-seven patients were assessed: ten were found to have ER-positive tumor cells in the bone marrow; ten had ER-negative cells; and the remaining seven patients had no tumor cells in the bone marrow smears. Of the ten patients with ER-positive cells, eight (80%) either had a response to endocrine therapy, implying that they possess ER-positive breast cancers, or had ER-positive primary tumors as determined by the dextran-coated charcoal biochemical assay (DCC). Of the ten patients with ER-negative cells in the bone marrow, eight failed to respond to endocrine therapy. This technique therefore provides a means of predicting which patients will respond to endocrine therapy, and is particularly important in those patients whose ER status is unknown.

Frozen sections of breast cancer specimens from 114 patients were assayed for the presence of estrogen receptor (ER) with an immunocytochemical method (ERICA). Significant positive correlations were found between receptor status and various pathologic features, including better histologic degrees of differentiation (P less than 0.001), smaller tumor cell size (P = 0.001), and lower levels of either tumor necrosis (P less than 0.001) or lymphocytic infiltration (P less than 0.001). There was a significant relationship between tubular formation and ER positivity (P = 0.003). This study confirms the results from some previous studies in which ER, assessed by biochemical means, has shown a correlation with good-prognosis pathologic features.

The importance of estrogen receptor (ER) determination in breast cancer is well established. Approximately 70% of ER-positive tumors are hormone responsive compared to 5-10% of ER-negative tumors. However, one-third of ER-positive tumors fail to respond, and the reasons for this are unclear. To further investigate these relationships we have determined levels of ER protein and mRNA in a number of human breast cancer biopsies. ER protein was estimated by the dextran-coated charcoal steroid binding method and by an ER immunocytochemical assay using a specific monoclonal antibody. A complimentary DNA clone (lambda OR3) encoding part of the human ER was used to determine mRNA levels. Dot blot analysis of twenty-seven tumors revealed a close agreement between ER mRNA and the dextran-coated charcoal assay (rs = 0.9; P less than 0.001). ER immunocytochemical assay staining also correlated with ER mRNA in twenty-five cases (rs = 0.75; P less than 0.001). Tumors from postmenopausal patients contained much higher levels of ER mRNA and ER protein than their premenopausal counterparts. ER-negative tumors produced no measurable ER mRNA. Northern blot analysis revealed a 6.4- and 3.7-kilobase species in ER-positive tumors and also in the human breast cancer cell line MCF-7. No differences in transcript sizes were found in tumors from hormone-responsive patients compared to nonresponding patients. We have also demonstrated, in tissue sections of normal and malignant breast, localization of ER mRNA by in situ hybridization to the same population of cells which exhibit immunoreactive ER.

Using a rat monoclonal antibody raised against human progesterone receptor (PR) we have developed an immunocytochemical technique to detect PR in human normal and malignant breast tissue and have compared the distribution of this with that obtained by the conventional dextran-coated charcoal steroid-binding assay. Immunoreactive PR was detected exclusively in the nuclei of epithelial cells in 29/51 (56.9%) of breast cancers studied. There was an excellent correlation between the immunocytochemical and dextran-coated charcoal techniques, with concordance in 43/51 (84.3%) cases [regression coefficient (Spearman) = 0.78; P less than 0.001]. The relationship between PR and estrogen receptor (ER) was also examined immunocytochemically using a monoclonal antiserum to ER. Twenty-eight out of 51 (54.9%) tumors were positive for both receptors and 13/51 (25.5%) negative for both. ER-positive, PR-negative tumors were found in 9/51 (17.6%) cases whereas only one case (2%) was PR-positive, ER-negative.

We have developed an immunocytochemical staining procedure (ERICA) using a monoclonal antibody to the estrogen receptor (ER) to determine ER status from samples obtained by fine needle aspiration of primary and recurrent breast cancer tissue (cyto-ERICA). ER status was assessable on 214 of 246 smeared aspirates from breast cancer patients. In 143 (66.8%) assessable smears positive nuclear staining was observed but was completely absent in 71 (33.2%) cases. In 107 cases we were able to compare results with those obtained with the quantifiable dextrancoated charcoal (DCC) radioligand binding technique using surgically excised material. We observed qualitative agreement in 53 of 62 (85.5%) of primary specimens and 16 of 16 (100%) recurrent samples compared to the subsequent DCC result on the same sample. Aspirates obtained from new secondary deposits were also assessed and in 16 of 19 (84.2%) cases results agreed with that established previously by DCC on the primary breast tumor. In a further 6 of 10 (60%) cases the cyto-ERICA result obtained from recurrent samples qualitatively agreed with that determined by DCC on a previous recurrent lesion. A comparison of staining of aspirates was also made against frozen tissue sections stained with the monoclonal antibody (tissue-ERICA). Where comparison was made of primary tumor specimens agreement was observed in 40 of 45 (88.9%) of cases while specimens from secondary lesions agreed qualitatively in 14 of 17 (82.3%) of cases. In a small number of samples where tissue-ERICA was performed on an earlier lesion to that aspirated for cyto-ERICA an agreement of 4 of 5 (80%) was observed. This technique shows good sensitivity in demonstrating ER in aspirate specimens, should therefore permit us to determine ER status before surgery for primary breast cancer, and may also mean that surgery for recurrent disease to determine receptor status is no longer necessary.

Derivatives of tamoxifen (1) and 4-hydroxy-2-methyltamoxifen (2) in which the basic side chain has been modified by N-oxidation or by quaternization have been investigated with respect to the effects on affinity for the oestrogen receptor and on cytostatic activity towards the MCF-7 cell line in vitro. In addition to the conventional cytosol assay for receptor binding affinity (RBA) a recently developed whole-cell assay was employed. N-oxidation (e.g. 2----3) produced no significant alteration in RBA value either in cytosol or in whole cells, nor in activity towards the MCF-7 line. Quaternization with methyl iodide (1----4, 2----6) or ethyl bromide (1----5, 2----7b: the cis isomer 7a also formed) did not alter receptor binding in the cytosol assay but almost abolished binding in the whole cell and cytostatic activity. The whole-cell RBA values for 2 (0.45) and 3 (0.5) were lower than those for 4-hydroxytamoxifen (2.9), suggested to be due to the lower oestrogenicity of the 2-methyl derivatives since activity against MCF-7 cells was unimpaired. The even lower values of whole-cell RBA (0.01-0.02) for the quaternary ethyl bromide derivatives 7a and 7b were ascribed to poor penetration into the cell since these compounds had minimal cytostatic activity.