Changing estrogen and progesterone receptor patterns in breast carcinoma during the menstrual cycle and menopause

BACKGROUND: Estrogen receptor (ER) and progesterone receptor (PgR) status at the time of breast carcinoma surgery is used as a marker of both prognosis and hormone dependency to guide adjuvant therapy. The authors studied the influence of hormonal milieu at the time of surgery on ER and PgR levels. METHODS: A population of 2020 patients with breast carcinoma, including 575 premenopausal women, was analyzed. ER and PgR levels were determined by radioligand binding assays (cutoff values, 10 fmol/mg). Serum estradiol (E2), progesterone (Pg), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels obtained on the day of surgery were used to define the menstrual cycle phase in premenopause. RESULTS: In premenopause, there was a higher proportion of ER positive (ER+) tumors in the follicular phase (62%, n = 316) than in the ovulatory phase (51%, n = 59) and the luteal phase (53%, n = 200, P = 0.03). The mean ER level was also higher in the follicular phase (30 fmol/mg) than in the ovulatory phase (20 fmol/ mg) and the luteal phase (25 fmol/mg, P < 0.001). The percentage of PgR positive (PgR+) tumors tended to be higher in the ovulatory phase (85%) than in the follicular (78%) and luteal (72%) phases (P = 0.11). The mean PgR was also higher in the ovulatory phase (177 fmol/mg) than in the follicular and luteal phases (134 and 92 fmol/mg, respectively; P < 0.001). The percentage of ER+ tumors was higher among menopausal women than among premenopausal women (67% vs. 59%, respectively; P < 0.001). Conversely, the percentage of PgR+ tumors was lower among menopausal women than among premenopausal women (65% vs. 78%, respectively; P < 0.001). In premenopause, there was a weak negative correlation between ER and E2 levels. No correlations were found between levels of ER and Pg and levels of FSH and LH or among levels of PgR and E2, Pg, and FSH and LH in premenopausal and menopausal women. CONCLUSIONS: Changes in ER and PgR levels in breast carcinoma during the menstrual cycle and menopause suggest that interpretations of hormone dependency on the basis of steroid receptor values should take into account hormonal status at the time of surgery.     

Female sex hormone receptor status in advanced hepatocellular carcinoma and outcome after surgical resection

BACKGROUND: There is a limited amount of data regarding the estrogen receptor (ER) and progesterone receptor (PgR) status of hepatocellular carcinomas (HGCs), and the relationship between receptor status and clinicopathologic features of tumors has not been reported. METHODS: Between April 1992 and December 1993, cancerous tissues for cytosolic preparation and receptor quantification in a monoclonal solid-phase enzyme immunoassay were obtained from 28 patients undergoing resection, three patients with total hepatectomy and subsequent liver transplantation, and two patients suffering from nonresectable HCC. RESULTS: ER and PgR were detected in the HCCs of 13 (39%) and 6 patients (18%), respectively. A lower age was observed among the female patients whose receptor status was negative for ER or PgR or both, as compared with the respective receptor-positive groups. No significant differences with respect to tumor stage and grading could be observed. There was one perioperative death (3%). In patients undergoing curative resection, 1-year survival in the ER(+) group was significantly lower than in the ER(-) group (40% versus 79%, p < 0.05). The 2-year survival rates in the ER(+) and ER(-) groups were 40% and 71%, respectively. A comparable trend did not become evident for PgR(+) and PgR(-) patients. CONCLUSIONS: Our data suggest a negative effect of an ER(+) tumor on patient survival after curative resection of advanced HCC.     

Relationship of standardized mitotic indices to other prognostic factors in breast cancer

OBJECTIVE: To examine the relationship between mitotic index (MI), calculated from direct microscopic counts, and other prognostic features in breast cancer. DESIGN: Mitotic index was based on direct microscopic observations of mitotic figures in 10 consecutive microscopic fields, and the average cell number was determined by counts of population density in three of those fields. Tumor grade and type were established from tissue sections, whereas metastases were detected in lymph node biopsy, chest roentgenograms, and bone scan. Estrogen receptor (ER) and progesterone receptor (PgR) levels were determined by flow cytometry. RESULTS: The MI for 242 patients ranged from 0.2 to 37.6, with a mean of 5.8 mitoses per 1000 cells. More than 85% of the tumors with an MI below 1.0 were diploid and contained an S-phase fraction of 6.7% or less. In contrast, more than 75% of tumors with an MI above 5.0 were aneuploid with more than 6.7% of cells in S-phase. There was an inverse relationship between ER and PgR status and MI. Eighty percent of tumors with an MI less than 1.0 were both ER and PgR positive while only 25% of those with an MI above 10.0 were both ER and PgR positive. Receptor-positive tumors with high S-phase and MI values had ER and PgR levels below 100 fmol/mg. CONCLUSIONS: Lower MI values calculated from direct cell counts are correlated with negative node status, diploid DNA content, low S-phase fraction, and positive receptor status. Thus, there is a significant relationship between objective MI values and several other factors that predict the probability of breast tumor recurrence.     

Evaluation of a displacement assay with tamoxifen as prognostic indicator in breast-cancer patients with estrogen-receptor-positive tumors

A displacement assay with tamoxifen, based on the relative binding affinity of tamoxifen and estradiol for the estrogen receptor (ER), was proposed in 1990 as prognostic indicator for breast-cancer patients. Validation of its predictive results in relation to the outcome of 73 patients with ER+ tumors is analyzed. ER, progesterone receptor (PgR) determinations and other conventional prognostic factors in relation to the displacement assay, were considered. Displacement assay results allowed ER+ tumors to be grouped as displaceable (D) or weakly displaceable (WD), with the implication that D tumors should respond better to tamoxifen (Tam) administration. Survival and disease-free interval curves showed highly significant differences between patients with ER+ D and ER+ WD tumors. For survival, including all tumor stages, 73.9% of patients were alive at 9 years after surgery in the group with D tumors and 37.0% in the group with WD tumors (p < 0.005); relative contribution of the different stages is analyzed. Addition of axillary-node number increased the prognostic significance of displacement categories for survival and disease-free interval. PgR determination as another ER functional expression failed to show significant differences for survival and disease-free interval between ER+ PgR+ and ER+ PgR- tumors. Thus, results from the displacement assay and from PgR determinations reflect 2 independent ER functional expressions. Displacement assay data appear as reliable prognostic indicators of breast-cancer outcome, and contribute to more appropriate treatment decisions in this pathology.     

Urokinase-type plasminogen activator and its inhibitor PAI-1: predictors of poor response to tamoxifen therapy in recurrent breast cancer

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a proteolytic enzyme thought to be involved in processes leading to tumor cell invasion of surrounding tissues. Its activity during metastasis may be regulated by an inhibitor, PAI-1. Previous work has shown that high levels of uPA and PAI-1 are associated with poor prognosis in primary breast cancers. PURPOSE: In this pilot study, we explored possible associations between the expression levels of uPA or PAI-1 and the efficacy of tamoxifen treatment in breast cancer patients with relapsed disease. METHODS: Levels of uPA, PAI-1, estrogen receptor (ER), and progesterone receptor (PgR) were assayed in cytosolic extracts derived from the primary breast tumors of 235 tamoxifennaive patients who had recurrent disease. The extracts were classified as positive or negative for each assayed factor. In some analyses, ER and PgR levels were evaluated together. In these analyses, three ER/PgR subsets were defined: low, intermediate, and high. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 57 months). Association of the tested factors with the response to tamoxifen treatment was studied by logistic regression analysis. Association of the factors with progression-free and overall survival was further evaluated by Cox univariate and multivariate regression analyses. RESULTS: Patients with uPA-negative tumors exhibited a better response (tumor regression or stable disease, maintained for more than 6 months) to tamoxifen treatment than those with uPA-positive tumors (51% versus 26% response; odds ratio [OR] = 0.34; 95% confidence interval [CI] = 0.18-0.65). The response rate was also better for patients with PAI-1-negative tumors than for those with PAI-1-positive tumors (49% versus 35% response; OR = 0.57; 95% CI = 0.32-1.01). In addition, patients with uPA-positive or PAI-1-positive tumors showed shorter progression-free survival (P = .001 and P < .05, respectively) and total survival after relapse (P = .005 and P < .005, respectively). When patients were stratified by ER/PgR status, the only statistically significant association between uPA levels and reduced tamoxifen response was seen in the subset whose tumors possessed intermediate levels of ER/PgR (16% response in uPA-positive versus 60% response in uPA-negative tumors; OR = 0.13; 95% CI = 0.04-0.41). Overall, uPA status appeared independent of association with ER/PgR status in its ability to predict response to tamoxifen treatment. The association of PAI-1 expression and the response to tamoxifen was less pronounced when patients were stratified by ER/PgR status. CONCLUSION: Measurement of primary breast tumor uPA levels may be useful in predicting the overall response of metastatic disease to tamoxifen therapy.     

A comparison between radioligand and immunohistochemical assay of hormone receptors in primary breast cancer

The detection of oestrogen and progesterone receptor (ER and PgR) levels in human breast carcinoma has traditionally been performed using a biochemical radioligand binding method. This method has several disadvantages including the requirement for generous tissue samples, the production of radioactive waste products and the inability to exclude non-malignant cellular material from the assay process. An alternative method for detecting hormone receptors is available with the use of a monoclonal antibody specific for the ER or PgR receptor using immunocytochemical assay (ER-ICA or PgR-ICA). Although designed for use on frozen section material, with modifications this method can be used on paraffin sections of routinely fixed and processed tissue, on archival material and on very small specimens. Further, an objective assessment or scoring of staining intensity is possible using computerized video-image analysis. Forty-three cases of primary breast carcinoma, treated from 1989 to 1991 at Goulburn Valley Base Hospital, Shepparton were assessed for ER and PgR content using both the radioligand method and immunohistochemistry with video-image analysis, and the results were compared. Of the 43 cases, ER-ICA and ER had a concordance of 81% (P < 0.001, r = 0.58) and in 39 cases, PgR and PgR-ICA had a concordance of 87% (P < 0.001, r = 0.54). Because the sample for radioligand assay is of uncertain composition and the immunohistochemical stain can be scored specifically for malignant epithelium, a degree of discordance is thought to be mostly attributable to the limitations of the radioligand assay.     

Ki-67 staining in benign, borderline, malignant primary and metastatic ovarian tumors: correlation with steroid receptors, epidermal-growth-factor receptor and cathepsin D

Ki-67 immunoreactivity was studied in relation to immunohistochemically assessed expression of epidermal-growth-factor receptor (EGFR), estrogen receptor (ER) progesterone receptor (PgR) and cytosolic levels of cathepsin D in advanced human ovarian adenocarcinomas, borderline and benign cystadenomas and normal ovaries. A significantly higher number of Ki-67-positive cells were found in metastatic tumors vs. primary adenocarcinomas and in the total group of adenocarcinomas vs. benign/borderline cystadenomas. Cathepsin-D levels were also significantly higher in metastatic tumors than in primary adenocarcinomas, which in turn presented higher levels than were found in normal ovaries. However, no significant difference was observed between cathepsin-D levels in malignant adenocarcinomas and borderline/benign cystadenomas. Immunohistochemically assessed expression of ER and PgR was detected in variable percentages of epithelial tumor cells, and stromal cells were occasionally positive as well. In the group of primary adenocarcinomas, 46% were ER-positive and 34% were PgR-positive, although there was no significant difference between primary and metastatic lesions with respect to ER or PgR expression. Concordance between immunohistochemically assessed ER or PgR data and cytosolic ER and PgR levels measured with enzyme immunoassay was relatively low. EGFR, immunohistochemically assessed with MAb-EGFRI, was positive in 76% of the primary and in 78% of the metastatic adenocarcinomas. A strong positive association was detected between ER and PgR, and EGFR was observed to present a weak positive correlation with Ki-67 and ER. Cathepsin-D levels were not found to be significantly correlated with the expression of ER, PgR, EGFR or Ki-67.     

Estrogen receptor, progesterone receptor, and HER-2/neu protein in breast cancers from pregnant patients

BACKGROUND: Because the occurrence of breast cancer during pregnancy is uncommon and because the high levels of estrogens and progestins associated with pregnancy could cause false-negative results from ligand binding assays (LBA), the actual incidence of steroid hormone receptor positivity in tumors from this subset of women is unclear. METHODS: Estrogen receptor (ER) and progesterone receptor (PgR) were determined using LBA methods in 15 tumors from 15 pregnant patients with breast cancer. In addition, immunohistochemistry was done for ER, PgR, pS2, heat shock protein 27 (hsp27), and HER-2/neu on 12 of the 15 tumors. RESULTS: Five of 15 (33%) tumors were positive for ER by LBA, compared with 52% of tumors from age-matched nonpregnant patients. Six of 12 (50%) were ER-positive by immunohistochemistry. For PgR, 7 of 15 (47%) tumors were positive by LBA, compared with 42% of tumors from nonpregnant patients. Ten of 12 (83%) stained positive for PgR. By LBA, 67% of tumors studied were positive for ER or PgR or both, as opposed to 57% of tumors from the nonpregnant comparison group. Two other estrogen receptor-mediated proteins, pS2 and hsp27, were present by staining in 8 of 12 (67%) and 10 of 12 (83%) of tumors, respectively. Seven of 12 tumors (58%) had positive staining for HER-2/neu, whereas only 16% of age-matched nonpregnant patients had positive-staining tumors. CONCLUSION: By LBA, the incidence of ER and PgR in breast tumors from pregnant women was not significantly different from that of tumors from nonpregnant age-matched patients. Some ER-negative tumors were PgR, pS2, or hsp27 positive, indicating that an intact estrogen response system was operative although ER was not detectable by standard LBA.     

The prognostic significance of steroid receptor activity in tumor tissues of patients with primary breast cancer

The prognostic significance of steroid-receptor activity is still debatable. Discrepancies in results are probably attributable to few patients, heterogeneous patient populations, and short follow-up. We investigated the prognostic significance of estrogen- and progesterone-receptor (ER and PgR, respectively) activity as a continuous variable in a homogeneous patient population. The prognostic significance of steroid-receptor activity was examined in 329 node-negative and 320 node-positive unselected breast cancer patients. In node-negative patients, ER values of primary tumors between 100 and 400 fmol/mg protein appeared to be a significant predictor for low risk of recurrence, whereas high ER (> 400) revealed an unfavorable prognosis. The classic cutoff level of ER (< 10 fmol/mg proteins) had no prognostic significance, however. In patients receiving adjuvant chemotherapy--the node-positive breast cancer patients--the classic cutoff value of ER (10 fmol/mg protein) predicts significantly distant metastases-free survival and overall survival only in the first 4 years of follow-up after diagnosis. Progesterone receptor is a time-dependent prognosticator in node-negative breast cancer patients (cutoff point for PgR, 80 fmol/mg). In node-positive breast cancer patients treated with chemotherapy or a combination of chemo- and hormonal therapy, PgR values lower than 60 fmol/mg had a worse prognosis. The results show the poor performance of standard cutoff points for ER and PgR positivity in predicting prognosis. Better prognosis is related to higher receptor levels but this relation is predominantly time-dependent. Moreover, patients who have high ER levels have a prognosis that is worse when compared with intermediate ER levels. Standard cutoff points for steroid receptors should not be used to select patients for prognosis.     

Is steroid receptor profile in contralateral breast cancer a marker of independence of the corresponding primary tumour?

We compared oestrogen receptor (ER) and progesterone receptor (PgR) profiles between primary and corresponding contralateral breast cancer (CBC) to investigate whether CBC should be considered relapse of a primary or as a feature of the multicentric origin of breast cancer. We adjusted for patient age, menopausal status, histology and adjuvant therapy. In spite of the general application of a cut-off value to dichotomise ER and PgR, we considered them as continuous variables. Moreover, we considered as synchronous cancers only simultaneously occurring lesions. For 399 patients, ER and PgR receptor levels in primary and CBC did not differ significantly, but were significantly correlated within the same patient. The correlation was higher for synchronous than for metachronous lesions when considering ER, but not PgR. The correlation between ER and PgR levels in the same tumour (primary or CBC) appeared stronger than the correlation of either receptor type (ER or PgR) between primary and CBC. Age, histology and adjuvant treatment affected ER concentration, whereas age, menopausal status and histology affected PgR concentration. The analysis indicated that primary and CBC tend to be characterised by a similar steroid receptor profile. The finding may support the hypothesis of CBC as a second primary arising in a common predisposing milieu, rather than a primary-dependent contralateral lesion. In this light, the clinical management of patients with a bilateral breast cancer should be similar to that of a unilateral breast cancer.     

New p53-based anti-cancer therapeutic strategies

We used immunological methods to determine cytosolic and nuclear steroid receptors to evaluate the advantages of nuclear receptor measurement in the selection of breast cancer patients for treatment. Around 75% of tumors showed coincidence between nuclear and cytosolic receptors (+/+ or -/-) for estrogen receptor (ER) and for progesterone receptor (PgR). Only cytosolic receptors were detected in around 20% of tumors. Distributed in the ER/PgR phenotypes according to the nuclear or cytosolic receptors, 64% of tumors remained in the same subgroup, whereas 16% of tumors were classified as hormone dependent according to cytosolic and independent according to nuclear receptors, which could be considered as 'false-positive' results. 6% of tumors would be classified as negative according to cytosolic receptors but positive according to nuclear receptors and would correspond to 'false-negative' results by conventional methods. Cytosolic receptor results may overrate the hormone dependence and cause some 'misclassifications' of patients. This could partially explain the lack of response to therapy in some cases.     

Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

The prognosis of patients with gastric cancer possessing sex hormone receptors

Estrogen receptors (ER) and progesterone receptors (PgR) were immunohistologically investigated in 107 patients with gastric cancer who underwent curative resection. Both ER and PgR were detected only in the cancer cell nucleus. The ER positive rate was 27.7% for males and 31.0% for females, while the PgR positive rate was 9.2% for males and 11.9% for females. Clinicopathologically, the ER positive rate was slightly higher in young females and in cases of poorly differentiated gastric cancer. When cumulative survival rates were analyzed in relation to the presence or absence of receptors, the 10-year cumulative survival rate after surgery was significantly lower in the ER positive cases, being 15.7% cent, than in the ER negative cases, being 62.7%, and also significantly lower in the PgR positive cases, being 18.2%, than in the PgR negative cases, being 48.3%. The coexistence of ER and PgR in gastric cancer tissues suggests that the ER is physiologically active, or that ER positive gastric cancer is hormone-dependent. The poor prognosis of patients with receptor positive gastric cancer suggests that gastric cancer with these receptors is highly malignant.     

Adenosine 3'':5''-cyclic monophosphate induces regulated secretion of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells

The effects of the short-term pre-operative administration of tamoxifen (TAM, 20 mg once daily) on the tumor levels of steroid receptors and the nuclear proliferation Ki-67 antigen, were investigated in 32 elderly patients with hormone-sensitive, operable primary breast cancer by means of fine-needle aspiration biopsy (FNAB). The FNAB smears before (pre-TAM) and after six weeks of treatment (post-TAM) were stained immunocytochemically in order to obtain an H-score for steroid receptors, and the percentage of cellular nuclei containing Ki-67. The mean oestrogen receptor (ER) score between the pre- and post-TAM specimens fell from 181.2 9.7 ( SEM) to 148.1 7.9 (Wilcoxon's matched-pairs signed-rank test, p=0. 01) and there was also a significant decrease in both the mean progesterone receptor (PgR) score (178.4 10.6 vs 148.5 10.6; p=0.01) and mean Ki-67 index (8.2% 1.2 vs 4.9% 0.9; p=0.0002). The reliability of FNAB as a sampling method was checked by comparing the results of the immunocytochemical assay (ICA) of the post-TAM biopsies with those of the immunohistochemical assay (IHA) of the corresponding excised tumors. There was a positive correlation between the ICA and IHA scores: ER (Spearman's correlation coefficient, rho=0.66, p<0.001), PgR (rho=0.84, p<0.001) and Ki-67 (rho=0.96, p<0.001). We conclude that the sequential use of FNAB is a reliable means of assessing the behaviour of within-tumor biomarkers during endocrine therapy.     

Short recurrence-free survival associated with high oestrogen receptor levels in the natural history of postmenopausal, primary breast cancer

The ability of oestrogen and progesterone receptor (ER and PgR, respectively) status to discriminate recurrence-free survival (RFS) among a cohort of consecutively accrued 952 postmenopausal patients has been studied. None of the cohort members investigated were treated with adjuvant therapy. Using a graduated scale of receptor status [low, intermediate and high receptor levels (< 10 vs. 10-107 vs. > or = 108 fmol/mg cytosol protein, respectively)] instead of the more commonly used dichotomous subdivision (positive vs. negative), ER level significantly discriminated between groups of patients with long vs. short RFS. Contrary to our expectations, patients with highest ER levels have as poor a prognosis as ER-negative patients, while patients with intermediate ER levels have longest RFS. The group of patients with ER levels > or = 108 fmol/mg cytosol protein comprises 47% of the cohort. The independent significance of overexpression of ER as a prognostic factor among this patient group is demonstrated in multivariate analysis where ER level is more significant than either grade of anaplasia or tumour size. PgR status did not significantly predict RFS among these patients. While the highest ER levels predispose for poorer prognosis among postmenopausal patients, it is precisely this group that experiences greatest benefit from adjuvant treatment with tamoxifen. Thus, patients who might otherwise go untreated due to their node-negative status can be readily identified and offered adjuvant treatment.     

The importance of steroid receptors for the prognosis and hormone treatment of breast cancer patients: a retrospective study in Southeastern Netherlands

OBJECTIVE: To assess the effect of oestrogen (ER) and progesterone (PgR) receptors on the prognosis of patients with operable breast cancer and the decision to treat these patients with adjuvant tamoxifen. DESIGN: Retrospective. SETTING: Eight community hospitals in the Southeast Netherlands. METHOD: Using the registry of the Comprehensive Cancer Centre South, 2862 breast cancer patients were identified with stage I, II or IIIA tumours, treated during the period 1984-1992. RESULTS: ER and PgR status were known for 2393 (84%) and 1761 (62%) patients respectively. From 1991, over 80% of the postmenopausal, lymph node positive patients had received tamoxifen, irrespective of the steroid receptor status. Of all lymph node negative patients fewer than 3% received adjuvant systemic treatment. Among the lymph node negative patients the steroid receptor status was not a significant predictor of survival. Among the lymph node positive patients whose tumours were both ER-negative and PgR-negative, a 2.8-fold increased risk of death was found during the first four years after primary treatment. The risk of death was not increased if only the ER or only the PgR status was negative. CONCLUSION: This study shows that ER and PgR receptors are significant prognostic factors for survival in breast cancer patients with involved axillary lymph nodes. The prognostic effect appeared to be restricted to the first four years after primary treatment. Selection of patients for endocrine treatment should be based on the steroid receptor status, considering the importance of the steroid receptors for predicting the response to endocrine treatment.     

Predicted anti-oestrogen resistance in BRCA-associated familial breast cancers

There is controversy concerning the prognosis of breast cancers arising in women carrying loss of function mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. This study was carried out to assess the likely hormone dependence of this group of tumours in comparison with an age and grade matched group of control sporadic tumours. We used quantitative immunohistochemical analysis for the oestrogen receptor (ER), progesterone receptor (PgR), cyclin D1 and pS2 on sections of primary tumours and ductal carcinoma in situ (DCIS). Expression of PgR (P < 0.05) and cyclin D1 (P < 0.01) was low in the BRCA1- and BRCA2-associated cancers compared with sporadic cases. The low frequency of expression of ER (9/40), PgR (2/40) cyclin D1 (5/36) and pS2 (5/36) in the familial tumours indicates that the majority of such tumours will be oestrogen insensitive and unlikely to respond to hormonal manipulation even at the in situ stage in their evolution. The low level of PgR (2/40 cases) suggests that there may be some abnormality of transactivating function of the ER in these tumours.     

The effect of lyophilization on steroid receptor behavior, as defined by sucrose density gradient analysis

The effects of lyophilization of both cytosol and tissue powder were examined by sucrose density gradient analysis (SDGA) of uterine estrogen receptor (ER) and progesterone receptor (PgR) from pig and calf. Cytosol prepared from lyophilized material (both cytosol and tissue powder) was compared to that prepared concomitantly from frozen, pulverized powders of the same tissue. It was found that both ER and PgR responses to SDG ionic strength and the ability of ER to demonstrate temperature-dependent 4S leads to 5S transformation were unimpaired by lyophilization. In addition, lyophilized cytosol and lyophilized tissue powder gave equivalent results. A few experiments examining lyophilization effects on steroid receptors from human breast biopsies did note some diminution of the 8S form of both ER and PgR following lyophilization, although this effect was variable in extent. It is nevertheless concluded that much lyophilized material exhibits many of the same responses as frozen, and may be used in lieu of the latter, at least within the confines of the parameters described here.     

Association of bone mineral density with apolipoprotein E phenotype

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47-82 years (64.0 +/- 1.0 years, mean +/- SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4-/- (E3/2 and E3/3, n = 217, Apo E4 +/- (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4-/- (0.91 +/- 0.01 g/cm2), E4 +/- (0.85 +/- 0.02 g/cm2), and E4+/+ (0.83 +/- 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 +/- 5.7 ng/ml) was higher than in E4-/- (7.7 +/- 0.3 ng/ml) or E4 +/- (7.7 +/- 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (-0.493 +/- 0.152) was not significantly different from that in the AAB of VDR (-0.616 +/- 0.225) or PPxx of ER (-0.785 +/- 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.