Effect of tumor size on the association between ps2 and cathepsin-d in primary breast-cancer

Previous studies have shown that pS2 and cathepsin D are linked in lymph node positive (N+) tumours, but not in tumours from lymph node negative (N-) patients. The purpose of this study was to understand whether or not size would effect the relationship between pS2 and cathepsin D. Findings were further extended to some subgroups of tumours obtained stratifying for T and N and particularly to the small (TI) but aggressive (N+) cancers (T1/N+) and to those of size greater than 2 cm (T2 and T3) but yet node negative (T2+T3/N-). Oestrogen (ER) and progesterone (PR) receptors, pS2 and cathepsin D concentrations were therefore assayed in 355 primary breast cancers. ER, PR, pS2 and cathepsin D did not correlate to nodal status and size of the tumours; no significant differences in the expression of these four biological factors between infiltrating ductal carcinomas without special features (NOS) and non-NOS carcinomas were found. Multivariate analysis performed among cathepsin D, ER, PR and pS2 indicated that, in T1 tumours, pS2 was the most important variable and the best predictor in cathepsin D determination, while such association was absent in T2 and T3 tumours. pS2 and cathepsin D significantly associated also in tumours obtained from N+ patients, and such correlation was highest in T1 tumours with positive axillary nodes (N+/T1). pS2 and cathepsin D did not associate in tumours taken from N- patients. Considering the NOS carcinomas, correlation between pS2 and cathepsin D in the N+, T1 and N+/T1 subgroups was higher in the poorly differentiated grade 3 with respect to grade 1 and grade 2 cancers. The data suggest that pS2 could have a role in cathepsin D expression and we hypothesise that such control could be an early biological event occurring in the development and progression of particularly aggressive (N+/grade 3), small (T1) breast cancers.     

Automated and quantitative immunocytochemical assays of Bcl-2 protein in breast carcinomas.

Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients'' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P < 0.0001), Ki67/MIB1 antigen- (P = 0.0012), and P-gp- (P = 0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < or = 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB1) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp).     

PS2 in breast cancer--alternative or complementary tool to steroid receptor status? Evaluation of 446 cases.

The oestrogen induced pS2 protein was measured in the cytosol of 446 breast cancer samples by an immunoradiometric assay. The relationships between pS2 and several clinical and biological parameters were evaluated. pS2 was not correlated to age, pT and nodal status, while it was higher in pre- than in peri- and post-menopausal women. A statistically significant positive association was found between pS2 and ER, PgR and cathepsin D. However, the frequency of pS2 negative values in ER+ (25.6%), PgR+ (21.7%) and cathepsin D-(19.0%) cases suggests that pS2 provides information independent of the above parameters in a fairly high percentage of patients. The prognostic role of pS2 was evaluated in 267 cases (follow up time 24-102 months). pS2+ showed longer RFS (P = 0.016) and OS (P = 0.004) than pS2-. pS2+ cases were significantly associated with a better prognosis in N+ but not in N- cases. Multivariate analysis showed that pS2 is an independent prognostic factor being the second most effective indicator for OS after nodal status and the third for RFS after nodal status and cathepsin D. From the present findings, we conclude that pS2 probably provides additional biological information to steroid receptor status and cathepsin D in patients with primary breast cancer.     

pS2 protein: a marker improving prediction of response to neoadjuvant tamoxifen in post-menopausal breast cancer patients.

Tamoxifen as sole initial therapy is gaining importance in the management of post-menopausal breast cancer patients. Age oestrogen (ER) and progesterone (PR) receptor status are accurately considered to select patients for hormonal treatment. However, additional markers are needed. By immunohistochemistry (IHC), we studied tumour expression of ER, PR, pS2, c-erbB-2 and glutathione S-transferase pi (GST pi) on initial core biopsies of 208 post-menopausal patients with a non-metastatic invasive ductal carcinoma, treated by neoadjuvant tamoxifen therapy. A good response to tamoxifen was defined as tumoral regression > or = 50% (110 patients). Relationship between response and age, tumour size, T, N, histological grade, ER and PR contents evaluated by radioimmunoassay, ER, PR, pS2, c-erbB-2 and GST pi expression evaluated by IHC were studied. Univariate and multivariate analysis showed that tumoral regression was linked only to pS2 (P = 0.004) and ER (P = 0.018) IHC expression. According to the immunohistochemical profile, three groups could be defined: pS2- and ER-positive tumours, pS2- or ER-positive tumours and pS2- and ER-negative tumours with response rates of 60%, 45% and 8% respectively. Although prospective studies are needed to confirm these results, we conclude that pS2 and ER immunohistochemical status are useful tools for predicting tumour regression with neoadjuvant tamoxifen in post-menopausal breast carcinoma patients.     

VLA2 integrin expression in breast carcinomas evaluated by automated and quantitative immunohistochemistry.

VLA2 is thought to be involved in the metastatic process in malignant tumours, in particular in carcinomatous cell adhesion to vessel basement membrane. VLA2 expression was immunohistochemically investigated in 204 breast carcinomas. Frozen tissue sections were probed with monoclonal anti-VLA2 using automated (Ventana ES 320 System) and quantitative (SAMBA 2005 image processor) immunoperoxidase. A positive anti-VLA2 immunoreaction was observed in 48 tumours (23.5%), within epithelial carcinomatous cells. The VLA2-positive surface in tumours varied from 3% to 20% (mean 8.75, S.D. 7.17) and was correlated with histoprognostic indicators and tumour expression of various antigens detected using the same method as that for VLA2. The results show that VLA2 immunoexpression was independent of the tumour size, grade, type and aneuploidy, and of the nodal status. VLA2 significantly correlated with ELAM, VCAM, VLA3 and P-glycoprotein (P-gp) (P < 0.01) and inversely correlated with cathepsin D (P < 0.001), but was independent of Ki67/MIB1, p53, bcl-2, c-erbB-2, E cadherin, CD44v, CD31, oestrogen and progesterone receptors'' (ER, PR) antigenic sites and pS2. The exact role, if any, of VLA2 in tumour cell dissemination remains to be elucidated and the clinical relevance of VLA2 immunodetection in breast carcinomas requires further investigation of the correlation between VLA2 immunocytochemical expression and patients'' outcome and response to chemotherapy.     

Time at surgery during menstrual cycle and menopause affects pS2 but not cathepsin D levels in breast cancer

Many studies have addressed the clinical value of pS2 as a marker of hormone responsiveness and of cathepsin D (Cath D) as a prognostic factor in breast cancer. Because pS2 and Cath D are both oestrogen induced in human breast cancer cell lines, we studied the influence of the menstrual cycle phase and menopausal status at the time of surgery on the levels of these proteins in breast cancer. A population of 1750 patients with breast cancer, including 339 women in menstrual cycle, was analysed. Tumoral Cath D and pS2 were measured by radioimmunoassay. Serum oestradiol (E2), progesterone (Pg), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels at the day of surgery were used to define the hormonal phase in premenopausal women. There was a trend towards a higher mean pS2 level in the follicular phase compared with the luteal phase (17 ng mg(-1) and 11 ng mg(-1) respectively, P = 0.09). Mean pS2 was lower in menopausal patients than in women with cycle (8 ng mg(-1) and 14 ng mg(-1) respectively, P = 0.0001). No differences in mean Cath D level were observed between the different phases of the menstrual cycle, or between pre- and post-menopausal women. In the overall population, pS2 was slightly positively associated with E2 and Pg levels and negatively associated with FSH and LH, probably reflecting the link between pS2 and menopausal status. In premenopausal women, no association was found between pS2 and E2, Pg, FSH or LH levels. There were no correlations between Cath D level and circulating hormone levels in the overall population. However, in the subgroup of premenopausal women with ER-positive (ER+) tumours, E2 was slightly associated with both pS2 and Cath D, consistent with oestrogen induction of these proteins in ER+ breast cancer cell lines. There are changes in pS2 level in breast cancer throughout the menstrual cycle and menopause. This suggests that the choice of the pS2 cut-off level should take the hormonal status at the time of surgery into account. In contrast, the level of Cath D is unrelated to the menstrual cycle and menopausal status.     

Biphasic Scatchard plots of oestrogen receptors are associated with low pS2 levels in human breast cancers.

In a series of 100 breast tumours, oestrogen receptors (ER) were analysed by Scatchard plots, progesterone receptors (PR) by enzyme immunoassay and pS2 by an immunoradiometric assay. Scatchard analysis gave information on receptor heterogeneity in that there was a large variation in Kd values obtained, from 0.001-2.95 nM. This variation was largely confined to tumours containing less than 70 fmol receptor per mg protein, while tumours with higher receptor concentrations were a more homogeneous population with low Kd values. An obvious correlation between ER and PR was found; moreover, pS2 was correlated to both ER and PR. In addition, 20 of the 100 tumours gave biphasic Scatchard plots, indicating the presence of at least two oestrogen-binding moieties, with Kd values and concentrations both in the range of those of receptors. The tumours displaying biphasic Scatchard plots had very low pS2 expression, regardless of ER concentrations; this was not true for PR. It is suggested that variability in responses to endocrine therapy may be related to the heterogeneity of the ER present in breast tumours.     

Clinical relevance of biologic factors in male breast cancer

There is ample information on the clinical role of biologic factors in female breast cancer: urokinase-type plasminogen activator (uPA), its receptor uPAR, its inhibitors PAI-1 and PAI-2, cathepsin D and pS2-protein. However such reports are missing or very rare for male breast cancer. We determined the cytosolic levels of oestrogen receptor (ER), progesterone receptor (PgR), cathepsin D, pS2-protein, uPA, uPAR, PAI-1 and PAI-2 of the primary tumour tissues from 40 male breast cancer patients. The tumour levels were compared with those of 180 matched females and 4114 historic females with breast cancer. In male breast tumours the level of PgR was higher, those of uPA, PAI-1, PAI-2 and cathepsin D lower. The tumour level of ER in men was similar to those in the matched and postmenopausal women, but much higher than those in the historic women. Male breast cancer seems to be biologically different from female breast cancer. Correlation of the eight cell biologic factors with disease outcome showed that PAI-1 (p=0.03) was the only independent predictive factor for poor prognosis in male breast cancer.     

Oestrogen-regulated genes in breast cancer: association of pLIV1 with response to endocrine therapy.

Northern hybridization analyses of the oestrogen-inducible mRNAs pLIV1 and pS2 were compared with oestrogen receptor (ER) immunocytochemistry assessments in 40 untreated primary or early recurrent breast tumours. Significant associations were observed between pLIV1/ER (P < 0.03), pS2/ER (P < 0.001) and pLIV1/pS2 (P < 0.04) status. After disease recurrence, patients were treated with assessable courses of endocrine therapies. Positive pLIV1, pS2 and ER statuses in primary disease were consequently found to be predictive of endocrine responsiveness in the secondary lesions (P < 0.03, P < 0.02, P < 0.005 respectively). However, despite these associations, a number of pLIV1- and/or pS2-positive tumours failed to respond to therapy.     

Age-dependent changes in breast cancer hormone receptors and oxidant stress markers

Breast cancer incidence increases with age but this relationship has not been fully explored with regard to expression of estrogen receptor (ER) and ER-inducible genes (PR, pS2, Bcl2, cathepsin D), or the age-dependence of oxidant stress markers that also affect ER-inducible gene expression. In this three-part study, we first correlated age at diagnosis with expression of breast cancer markers ER, PR, pS2, Bcl2, and cathepsin D, quantitated by enzyme immunoassays from a European collective of 3000 cryobanked primary breast cancers and 300 adjacent non-malignant breast tissues. Results were then compared with ER and PR data reported to the SEER registry for 83,541 US cancers diagnosed during 1992–1997. Lastly, a homogeneous subset of 70 ER-positive tumors preselected from the European collective was blindly analyzed for age-specific changes in the DNA-binding content of redox-sensitive transcriprtion factors, AP1 and Sp1, and the oxidant stress-activated protein kinase, phosphorylated(P)-Erk5. Increases in breast tumor ER from patients aged <30 to >80 years mirrored 10-fold lower increases in non-malignant breast tissue ER content up to age 60, rising faster thereafter and reaching a near 25-fold differential between malignant and non-malignant breast tissue by age 80. ER-inducible markers PR, pS2, Bcl2, and cathepsin D were overexpressed in tumors relative to non-malignant breast tissue but, unlike ER, did not increase with patient age. While SEER data demonstrated that the increase in US breast cancer incidence rates after age 50 is confined to ER-positive tumors in patients of all ethnic subsets, these patients also showed a striking increase in the proportion of higher-risk ER-positive/PR-negative breast cancers arising after age 50. Mechanistically essential for ER-inducible PR expression, Sp1 DNA-binding function (but not Sp1 content) was lost with age in ER-positive tumors; and this functional defect correlated with increased tumor content of the oxidant stress marker, P-Erk5. Altogether these findings support two hypotheses: (i) dysregulated ER expression underlies the age-specific increase in breast cancer incidence after age 50; and (ii) oxidative stress and loss of Sp1 DNA-binding may contribute to an increasing incidence in higher-risk ER-positive/PR-negative breast cancers with aging.     

Oestrogen receptor status, pathological complete response and prognosis in patients receiving neoadjuvant chemotherapy for early breast cancer

The aim of this study was to ascertain if oestrogen receptor (ER) status predicts for pathological complete response (pCR) to neoadjuvant chemotherapy in operable breast cancer, and the effects of pCR on survival. Using a single-institution database, 435 patients were identified, who received neoadjuvant chemotherapy for operable breast cancer and were eligible for the analysis. Patients whose tumours were ER negative were more likely to achieve a pCR than patients who were ER positive (21.6 vs 8.1%, P<0.001). Owing to a strong correlation between ER status and grade, these variables were not shown to be independent predictors of pCR. Overall survival (OS) was better in those patients who achieved a pCR compared to those who did not (5-year OS 91 vs 73%; P=0.02). This was still the case when only patients with ER-negative tumours were examined (5-year OS 90 vs 52%, P=0.005), but not in the subset of patients with ER-positive tumours (5-year OS 93 vs 79%; P=0.3). Therefore, patients with ER-negative tumours were found to be more likely to achieve a pCR to neoadjuvant chemotherapy than those with ER-positive tumours, and pathological response did not have prognostic significance in patients with ER-positive tumours.     

In vivo stimulation by tamoxifen of cathepsin D RNA level in breast cancer

We have previously shown that 3 weeks of treatment with tamoxifen, of patients with primary breast carcinomas, increased cytosolic cathepsin D protein in oestrogen receptor (ER) positive tumours [Maudelonde et al., Cancer 1989, 63, 1265–1270]. In order to investigate the mechanism of this increase and to eliminate a transient flare-up effect, we semi-quantified cathepsin D RNA levels by in situ hybridisation in 32 breast carcinomas from patients treated with tamoxifen for 3 weeks prior to surgery and in 35 breast cancer patients receiving no tamoxifen. We found that tamoxifen increased cathepsin D RNA level regardless of the ER status of the tumours. In ER positive tumours, tamoxifen increased the cathepsin D RNA level to the same extent as cytosolic cathepsin D protein but not in ER negative tumours. The induction of cathepsin D RNA by tamoxifen in ER positive tumours was probably due to its agonist activity, also observed in vitro in breast cancer cell lines. These results suggest that the cathepsin D gene is inducible by oestrogens in ER positive breast cancer as it is in breast cancer cell lines.     

Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer

Vascular endothelial growth factor (VEGF) receptors consist of three cell-membrane type receptors (VEGFR-1, VEGFR-2 and VEGFR-3), and soluble form of VEGFR-1 (sVEGFR-1), an intrinsic negative counterpart of the VEGF. In this study, we measured intratumoral protein levels of free and total VEGF, VEGFR-2 and sVEGFR-1 from 202 primary breast cancer tissues and examined their prognostic values. A significant inverse correlation was found between free or total VEGF and oestrogen receptor (ER) status (P=0.042 and 0.032, respectively). A univariate analysis showed that low sVEGFR-1 and high total VEGF were significantly associated with poor prognosis in disease-free survival (DFS) and overall survival (OS). The ratio of sVEGFR-1 to total VEGF was a strong prognostic indicator (DFS: P=0.008; OS: P=0.0002). A multivariate analysis confirmed the independent prognostic values of total VEGF and the ratio of sVEGFR-1 to total VEGF. In subgroup analysis, total VEGF was a significant prognostic indicator for ER-positive tumours but not for ER-negative tumours, whereas sVEGFR-1 was significant for ER-negative tumours but not for ER-positive tumours. In conclusion, the intratumoral sVEGFR-1 level, VEGF level and the ratio of sVEGFR-1 to total VEGF are potent prognostic indicators of primary breast cancer, and might be relevant to ER status.     

pS2 is an independent factor of good prognosis in primary breast cancer.

In breast cancer, oestrogen regulated genes, such as pS2, may be expressed in well differentiated tumours with a good prognosis. We have examined pS2 mRNA expression in 78 primary, untreated breast cancers and related pS2 expression to disease behaviour and known prognostic factors. pS2 mRNA expression was detected in 25/78 (32%) of cancers and was significantly associated with a moderate/high oestrogen receptor content (P = 0.045, Chi Square test). pS2 mRNA expression was associated with freedom from disease at median 31 months clinical and radiological follow-up (P = 0.015, Fisher''s exact test, odds ratio 8.6). Using multiple logistic regression analysis of six potential prognostic factors only pathological axillary node status (P < 0.01) and pS2 mRNA expression (P < 0.05) provided independent prognostic information. Furthermore, pS2 was associated with a good prognosis in the axillary node positive patients where only 1/13 (8%) with pS2 mRNA expression compared with 13/29 (45%) without detectable expression had recurrence of their disease. These data provides strong support for pS2 as a useful independent prognostic factor in primary breast cancer.     

Estrogen receptor (ER) mRNA expression and molecular subtype distribution in ER-negative/progesterone receptor-positive breast cancers

We examined estrogen receptor (ER) mRNA expression and molecular subtypes in stage I–III breast cancers that are progesterone receptor (PR) positive but ER and HER2 negative by immunohistochemistry (IHC) or fluorescent in situ hybridization. The ER, PR, and HER2 status was determined by IHC as part of routine clinical assessment (N = 501). Gene expression profiling was done with the Affymetrix U133A gene chip. We compared expressions of ESR1 and MKI67 mRNA, distribution of molecular subtypes by the PAM50 classifier, the sensitivity to endocrine therapy index, and the DLDA30 chemotherapy response predictor signature among ER/PR-positive (n = 223), ER-positive/PR-negative (n = 73), ER-negative/PR-positive (n = 20), and triple-negative (n = 185) cancers. All patients received neoadjuvant chemotherapy with an anthracycline and taxane and had adjuvant endocrine therapy only if ER or PR > 10 % positive. ESR1 expression was high in 25 % of ER-negative/PR-positive, in 79 % of ER-positive/PR-negative, in 96 % of ER/PR-positive, and in 12 % of triple-negative cancers by IHC. The average MKI67 expression was significantly higher in the ER-negative/PR-positive and triple-negative cohorts. Among the ER-negative/PR-positive patients, 15 % were luminal A, 5 % were Luminal B, and 65 % were basal like. The relapse-free survival rate of ER-negative/PR-positive patients was equivalent to ER-positive cancers and better than the triple-negative cohort. Only 20–25 % of the ER-negative/PR-positive tumors show molecular features of ER-positive cancers. In this rare subset of patients (i) a second RNA-based assessment may help identifying the minority of ESR1 mRNA-positive, luminal-type cancers and (ii) the safest clinical approach may be to consider both adjuvant endocrine and chemotherapy.     

Effect of steroid receptors,pS2 and cathepsin D on the outcome of elderly breast cancer patients: An exploratory investigation

In 83 elderly breast cancer patients, oestrogen and progesterone receptors (ER, PgR), pS2 and cathepsin D (CathD) were evaluated for their possible prognostic role on disease-free survival (DFS). The biomarkers were determined on the same cytosol by using immunoradiometric assays, and the variables were considered on a continuous scale. Univariate analysis indicated a linear relationship between logarithmic hazard ratio (log(HR)) and the log(ER) and log(PgR) concentration, but a non-linear relationship between log(HR) and CathD. As regards pS2, there was no evidence of a relationship with log(HR). In multivariate analysis, log(ER) content did not have a significant prognostic role, whereas log(PgR) retained a significant prognostic role. As regards the predictive ability, log(PgR) was the best discriminator of outcome followed by CathD, whereas the contribution of log(ER) was negligible. In multivariate analysis, 2 models were considered: one with log(ER), pS2, CathD and the interaction between pS2 and CathD, and another with log(PgR), pS2, CathD and the interaction between pS2 and CathD. In the first model, log(ER) content did not have a significant prognostic role, whereas in the second model log(PgR) retained a significant prognostic role. Our findings indicate that the quantitative determination of pS2 and CathD, in addition to steroid receptors, on the same cytosolic fraction could be a complementary tool to describe all breast cancer patients rather than just the elderly and that the use of a continuous scale, instead of a simple dichotomous “status”, may improve the biological information supplied by the variables. Int. J. Cancer (Pred. Oncol.) 79:305–311, 1998. © 1998 Wiley-Liss, Inc.     

Expression of ps2 protein in endometrial carcinomas: Correlation with clinicopathologic features and sex steroid receptor status

Using immunohistochemistry, we examined pS2 expression in 64 samples of endometrial carcinoma, 11 samples of endometrial hyperplasia and 15 samples of normal endometrium, and compared them with clinicopathological data, estrogen receptor (ER) expression and progesterone receptor (PR) expression. Of the 64 samples of endometrial carcinoma, 45 (70%) expressed the pS2 protein. The average age of the patients with pS2-positive carcinomas (54.8 ± 8.6 years) was significantly lower than that of the patients with pS2-negative carcinomas, and all premenopausal patients were positive for the pS2 protein. Among histological types, pS2 expression was observed in 33 (92%) of the 36 G1 carcinomas, but in none of the 5 nonendometrioid carcinomas. Of the 48 ER-positive carcinomas, 43 (90%) were pS2-positive and 5 were pS2-negative. Of the 40 PR-positive carcinomas, 37 (93%) were positive for pS2. There were significant associations between pS2 expression and ER/PR expression (p < 0.001). Staining of the pS2 protein was also observed in the samples of normal endometrium. We found a progressive increase in immunoreactivity of pS2 protein from normal endometrium to endometrial hyperplasia and still more in well-differentiated carcinoma. All 11 cases of endometrial hyperplasia were strongly positive for pS2. Furthermore, patients with pS2-positive carcinomas had a better survival rate than those with pS2-negative carcinomas (p < 0.05). Our data suggest that pS2 expression is likely correlated with estrogen-related endometrial carcinoma and is possibly involved in early disease progression. Int. J. Cancer 74:237-244, 1997. © 1997 Wiley-Liss, Inc.     

Close correlation between progesterone receptor concentration and hormonal sensitivity in DMBA-induced mammary tumours of the rat

Cyclic administration of oestrogen alone and with progesterone to ovariectomized rats bearing DMBA-induced tumours resulted in more uniform cytosol oestrogen and progesterone receptor (ER and PgR) concentrations in the uteri and a stronger correlation between ER and PgR concentrations in the tumours than in a group of naturally cycling animals. Using this model, the concentration of PgR was a better indicator of tumour response to ovariectomy and hormonal restimulation than the concentration of ER. In ten unequivocally responsive adenocarcinomas receptor values, expressed as fmol/mg cytosol protein, were: ER 47 ± S.D. 19, PgR 246 ± 134, PgR:ER 5.9 ± 3.0, compared with ER 18 ± 2, PgR 33 ± 8 and PgR:ER 1.8 ± 0.3 in three autonomous adenocarcinomas. All 3 parameters were significantly different between the two groups (0.02 < P < 0.05), but there was overlap in the ER values, whereas no overlap occurred in the PgR values or the PgR:ER ratios. Ten other adenocarcinomas showed a different sensitivity to restimulation than to ovariectomy and six tumours were fibroadenomas or of mixed histopathology. For the whole tumour population the response to hormonal restimulation at the time of excision suggested that a PgR concentration >60 fm/mg protein combined with an ER concentration >20 fm/mg protein is necessary for the maintenance of hormonal sensitivity. Using these criteria, response was related to receptor concentrations in 93% of the tumours.