Apolipoprotein-D: a novel cellular marker for HGPIN and prostate cancer.

BACKGROUND: High grade prostatic intraepithelial neoplasia (HGPIN) is a putative pre-malignant lesion of the prostate. While apolipoprotein-D (Apo-D), an androgen-regulated hydrophobic transporter protein, is expressed in prostate tumors, its expression in HGPIN is unknown. METHODS: Immunoreactivity for Apo-D and another androgen-regulated protein, prostate specific antigen (PSA), was investigated in 64 radical prostatectomy tissues by video image analysis. RESULTS: Eighty two percent of prostatectomy specimens demonstrated moderate to strong Apo-D immunoreactivity in areas of HGPIN. In comparison, weak Apo-D immunoreactivity was observed in non-malignant areas in only 24% of specimens. The median (range) percentage cellular area of HGPIN immunopositive for Apo-D (9.7%, 0-42.9), and the cellular concentration of Apo-D (MIOD 3.1, 0-13.3), were intermediate between that of normal (area 0%, 0-53.5%, MIOD 0, 0-12.6) and early stage prostate cancer tissues (area 29.2%, 0-90.8%, MIOD 6.7, 0-28.1). This increase in Apo-D expression from non-malignant, through HGPIN to prostate cancer was statistically significant (P < 0.001), and contrasted with the decrease observed in PSA staining between adjacent areas of normal glands, HGPIN, and cancer (P = 0.026). CONCLUSIONS: The presence of high levels of immunoreactive Apo-D in HGPIN and prostate cancer, but not in non-malignant epithelial cells, is consistent with HGPIN being an intermediate lesion in the transition to prostate cancer, and suggests that cellular Apo-D expression is a marker of malignant transformation of the prostate.

Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor beta1.

We have previously demonstrated that peritumoral stromal matrix derived from prostate cancer patients who relapse after radical surgery contains elevated levels of versican. The purpose of this study was to determine whether prostate cancer cells control stromal cell secretion of versican. Serum-free conditioned medium from three prostate adenocarcinoma cell lines, LNCaP, PC3, and DU145, was added to cultures of fibroblasts established from prostatic tissue of patients with benign prostatic hyperplasia, and the medium was harvested at 24, 48, and 72 h. Immunoblotting with an antiversican core protein antibody revealed that prostatic fibroblast medium harvested at 72 h contained increased levels of versican after treatment with either LNCaP-, PC3- or DU145-conditioned medium (2.5-, 4.5-, and 5-fold, respectively) compared with control cultures. This increase in versican in the culture medium was not observed after coincubation with transforming growth factor beta1-neutralizing antisera. The results of this study suggest that prostate tumor cells induce host stromal cells to secrete increased versican levels via a paracrine mechanism mediated by transforming growth factor beta1.

Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function.

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Androgen receptor agonist activity of the synthetic progestin, medroxyprogesterone acetate, in human breast cancer cells.

Medroxyprogesterone acetate (MPA), which is frequently used as second line hormonal therapy for the treatment of metastatic breast cancer, binds with high affinity to the progesterone receptor (PR). However, the androgenic side-effects of MPA suggest that it may also activate androgen receptor (AR) regulated pathways. Treatment of the human breast cancer cell lines MDA-MB-453, ZR-75-1 and T47-D with high dose (100 nM) MPA resulted in 26-30% inhibition of cell growth, which was partially reversed by co-treatment with a 10-fold excess of the synthetic antiandrogen, anandron. Scatchard analysis demonstrated specific, high affinity (non-PR) binding of [3H]MPA to cytosols prepared from the PR-/AR+ MDA-MB-453 and PR+/AR+ ZR-75-1, but not the PR-/AR- BT-20 breast cancer cell lines. Competition of [3H]MPA binding to MDA-MB-453 cytosols by equimolar concentrations of androgens (5alpha-dihydrotestosterone (DHT), R1881) and the antiandrogen, anandron was consistent with binding of MPA to the AR. In ZR-75-1 cell cytosol fractions, DHT, R1881 and anandron only partially competed out [3H]MPA binding, suggesting that androgens displace [3H]MPA binding to AR but not to PR. Induction by MPA of AR transactivation was demonstrated in MDA-MB-453 and ZR-75-1 cells, and in the CV-1 cell line transfected with a full-length AR. In these cell lines the increased activity of the androgen responsive reporter gene (MMTV-CAT) by 1 nM MPA was fully (MDA-MB-453, CV-1) or partially (ZR-75-1) inhibited by co-culture with 1 microM anandron. These findings indicate that MPA is an AR agonist and suggest that the in vivo effects of MPA in breast cancer patients may in part be mediated by the AR.

A simple index using video image analysis to predict disease outcome in primary breast cancer.

Image analysis was used to investigate the prognostic significance of immunostaining for oestrogen receptor (ER), p53 tumour-suppressor protein and tumour cell proliferation (MIB-1) in a random cohort of 200 primary breast cancer patients with between 4 and 7 years of clinical follow-up. Image measurements of the percentage of immunopositive cancer cell nuclei (% positive nuclear area) were recorded for the above tumour features for each patient. Assessment of relative risk using Cox's univariate analysis indicated that tumour size, number of cancer-involved nodes, MIB-1 and ER % positive nuclear area were significantly associated with breast cancer disease outcome, i.e., relapse-free survival and overall survival. In multivariate analysis, tumour size, number of involved nodes, ER and MIB-1 % positive nuclear area were retained as independent predictors of prognosis, depending on the image measurement cut-point used. A prognostic model, which can be used without reference to nodal involvement, was constructed for tumour size, ER cut-point of 30% positive nuclear area and MIB-1 cut-point of 10% positive nuclear area. Kaplan-Meier analysis of this image-based prognostic index identified 4 risk groups with predicted 5-year overall survival rates of 93%, 83%, 76.7% and 61.5%. We conclude that image measurements of ER and proliferative rate can be combined with tumour size to construct a prognostic index which reliably predicts disease outcome in primary breast cancer without knowledge of the nodal status of the patient.

Elevated levels of peritumoral chondroitin sulfate are predictive of poor prognosis in patients treated by radical prostatectomy for early-stage prostate cancer.

The disease course of localized prostate cancer is highly variable, and patients potentially curable by aggressive management are not readily identified by current clinical practice. Chondroitin sulfate (CS) glycosaminoglycan is a candidate biomarker as elevated levels of CS in peritumoral stroma of prostate cancer have been associated with prostate-specific antigen (PSA) failure. Immunoreactive CS was measured using image analysis of archived radical prostatectomy tissues, obtained from 157 men with a median of 47 months (range, 16-111 months) clinical follow-up. CS level, Gleason score, and preoperative serum PSA levels were independent predictors of PSA failure by Cox's multivariate analysis. Patients with low CS levels had significantly fewer PSA failures after radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot; 32% PSA failures at 5 years for CS mean integrated absorbance cut point < 7.0 versus 50% for CS > or = 7.0, P = 0.0001). In the subgroup of patients with preoperative serum PSA levels < 10 ng/ml, CS was particularly useful in discriminating retrospectively those patients most suited for surgery (Kaplan-Meier plot; 14% PSA failures at 5 years for CS mean integrated absorbance cut point < 7.0 versus 47% for CS > or = 7.0, P = 0.0001). We conclude that measurements of CS level can assist in predicting patient outcome after surgery. Additionally, our data suggest that the combination of CS and PSA measurements may improve outcome prediction for patients with intermediate Gleason scores.

Immunolocalization of apolipoprotein D, androgen receptor and prostate specific antigen in early stage prostate cancers.

PURPOSE: To determine the cellular distribution and levels of immunohistochemical staining for apolipoprotein D (Apo-D), prostate specific antigen (PSA) and androgen receptor (AR) in early stage prostate cancers. MATERIALS AND METHODS: Cellular distribution of Apo-D, PSA and AR in 30 stage A/B prostate cancers and in non-malignant glandular tissue contained in the same sections was detected immunohistochemically, and staining was evaluated by computerized video image analysis. RESULTS: Staining for Apo-D (percentage positive cellular area) was significantly increased in tumor cells of early stage prostate cancers compared with non-malignant glandular tissue. PSA and AR were present at high levels in both early stage prostate tumors and non-malignant prostate. CONCLUSIONS: Malignant transformation in the prostate is associated with increased cellular levels of Apo-D.

Tumor microvascularity has no independent prognostic significance for breast cancer.

There is a continuing controversy regarding the value of estimating degree of intra-tumor vascularity to predict prognosis in breast cancer. In order to resolve this controversy, primary tumors from a cohort of 519 women with breast cancer were analysed to determine whether association exists between degree of vascularity and prognosis. Tumor vascularity was estimated by immunohistochemistry using a monoclonal antibody to the antigen CD31. The tumor area showing the highest degree of vascularity was chosen to score the number of microvessels per unit area. Issues such as the reproducibility of the microvascularity score and its association with tumor parameters including size, histological grade and hormone receptor levels were investigated. Although previously agreed criteria were used, consensus between two pathologists' estimations of the degree of vascularity was only moderate. There was no statistically significant association between tumor vascularity score and other currently established parameters of prognosis. After a median follow up of 71 months for axillary node negative patients, there was no association between tumor vascularity score and increased risk of relapse or death from breast cancer. In axillary node positive patients, tumor vascularity score was associated with increased risk of relapse and death from breast cancer. This association was not however independent of other established parameters of prognosis.

Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer.

Patients with clinically localized prostate cancer who might be cured by aggressive management are not easily identified using current clinical information. Additional, more accurate, biomarkers of tumor behavior need to be identified to improve clinical outcome. Our previous studies indicated that the concentration of the glycosaminoglycan chondroitin sulfate in prostatic stroma might be a useful biomarker of disease progression in early-stage prostate cancer. In this study, two chondroitin sulfate proteoglycans, versican and decorin, were investigated. Versican and decorin were immunolocalized to the periacinar and peritumoral fibromuscular stroma in sections of nonmalignant and malignant human prostate tissues. Video image measurements indicated that the concentrations of both proteoglycans were increased in the prostatic tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P = 0.0006). Cox's univariate analysis indicated that increases in versican concentration but not in that of decorin were associated with increased risk of prostate-specific antigen (PSA) progression. Versican concentration was compared with other clinical or biological features of prognosis in two-variable regression analyses. Versican and serum PSA concentrations were independent predictors of PSA progression. Versican was a stronger prognostic factor than tumor grade, and it could predict outcome for patients with moderately differentiated tumors. Patients with low versican concentration had significantly better progression-free survival than patients with high levels of versican (Kaplan-Meier plot, 89% versus 27% PSA progression-free at 5 years, respectively; P = 0.0001). We conclude that the measurement of prostatic concentrations of versican, a molecule with reported anticellular adhesive properties, may be a useful marker of disease progression in patients with early-stage prostate cancer and that further study of versican in other patient cohorts is warranted.

A simple index to predict prognosis independent of axillary node information in breast cancer.

BACKGROUND: Since the course of breast cancer is often unpredictable, we wished to develop a model using characteristics of the primary tumour alone to predict prognosis. METHODS: Several tumour features were determined, and after a median follow-up duration of 65 months, multivariate analysis identified tumour size and grade, oestrogen receptor concentration, axillary lymph node metastasis and tumour cell proliferation fraction (MIB-1 count) as being independently associated with increases in risk for both relapse and death from breast cancer. A prognostic model was constructed using tumour size and grade, oestrogen receptor concentration and MIB-1 count only. A score of 1 for each was given to tumour size > 20 mm, tumour grade 2 or 3, oestrogen receptor concentration < 10 fmol/mg cytosol protein and MIB-1 count > 9%. Five groups established by assigning a combined score of 0, 1, 2, 3 or 4 for each patient were analysed for their associations with disease-free and overall survivals. RESULTS: This preliminary model predicted 5-year survival rates of 97, 91, 85, 68 and 50% for the five groups. The model was further simplified by excluding tumour grade from the analysis. The revised model identified four risk groups with predicted 5-year survival rates of 91, 86, 66 and 52%. This model, the Adelaide prognostic index, was also able to identify four risk groups in both node-negative and node-positive patients. CONCLUSIONS: The Adelaide prognostic index can be used to predict prognosis even in the absence of axillary lymph node information.

EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer.

Chromosome 11q13 is amplified in about 13% of primary breast cancers. CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding a filamentous actin binding protein, are favoured candidate onocogenes, whereas INT-2 is an unexpressed gene at this locus. In this study we tested the possibility that different regions of this large amplicon could be independently amplified and subsequently defined the phenotype of EMS1 amplified tumours in a series of 961 primary breast carcinomas. Using DNA slot blots, EMS1 was amplified in 15.2% of samples: 5.4% were coamplified for CCND1; 7.9% coamplified for INT-2 and 6.7% showed EMS1 amplification alone. The degree of amplification of CCND1 and INT-2 was highly correlated (P =0.0001). In contrast, no such relationship existed between EMS1 and CCND1 or INT-2 amplification, demonstrating independent amplification of EMS1 in 44% of amplified tumours. EMS1 amplification (> or = twofold increase in copy number) was positively correlated with patient age > or = 50 years (P = 0.025), ER positivity (P = 0.022), PgR positivity (P = 0.018), and was negatively correlated with HER-2/neu (c-erbB2) amplification (P = 0.01). In common with CCND1/INT-2, EMS1 amplification was associated with increased risk of relapse in patients with lymph node-negative disease (P = 0.028). In contrast, EMS1 and CCND1/INT-2 amplification appeared to confer different phenotypes in ER positive and negative tumours. A > or = threefold increase in EMS1 copy number was associated with an apparent increased risk of relapse and death in patients with ER negative tumours, but was without effect in ER positive tumours. In contrast, CCND1/INT-2 amplification had no effect in the patients with ER negative tumours but was associated with early relapse in ER positive patients. Thus EMS1 amplification may identify subgroups of breast cancer patients with increased probability of relapse and death distinct from those identified by CCND1/INT-2 amplification. Further studies are required to more clearly determine the functional consequences of EMS1 overexpression and a biological basis for the relationship between EMS1 amplification and phenotype in breast cancer.

Elevated stromal chondroitin sulfate glycosaminoglycan predicts progression in early-stage prostate cancer.

Curative therapies for clinically localized prostate cancer have significant morbidity, and those patients who might be cured by aggressive management are not easily identified using current clinical information. Better biomarkers of tumor behavior need to be identified to improve clinical outcome. Chondroitin sulfate (CS), a glycosaminoglycan, may be a potentially useful biomarker as it is known to influence cell growth and differentiation and might influence malignant progression. In this study, CS was immuno-localized to the periacinar and peritumoral fibromuscular stromal tissue of nonmalignant and malignant prostates. The CS concentration was increased in the prostate tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P < 0.0001). Using Cox's univariate analysis, CS concentration, tumor grade, preoperative serum prostate-specific antigen (PSA), extracapsular extension of disease, positive surgical margins, and patient age were associated with an increased risk of PSA failure. The CS concentration was compared with the other features in two-variable regression analyses. CS and preoperative serum PSA concentrations were independent predictors of PSA failure. CS was a stronger prognostic feature than tumor grade and could predict outcome for patients with moderately differentiated tumors. Patients with a low CS concentration had significantly better progression-free survival following radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot, 91% versus 49% PSA progression free at 5 years, respectively, P = 0.0038). Only postoperative pathological indices (extracapsular extension, surgical margins) were stronger predictors than CS. We conclude that measurement of prostatic CS concentrations at diagnosis may allow stratification of patients with early-stage prostate cancer for adjunctive or alternate therapies.

Expression of the androgen receptor and an androgen-responsive protein, apolipoprotein D, in human breast cancer.

Little is known regarding the activity and function of the androgen receptor (AR) in human breast cancer. In the present study AR was evaluated in untreated primary breast cancers using antisera to the amino- and carboxy-termini of the receptor and quantitated using colour video image analysis. A strong correlation between tissue concentration and percentage AR-positive cells was observed for each antiserum. However, comparison of percentage positive cells using the amino- and carboxy-terminal AR antisera in individual breast cancer specimens revealed a subset of tumours with discordantly increased staining for the carboxy terminus. These findings suggest the presence of amino-terminal-truncated AR in a proportion of breast cancer cells or presence of AR mutations or associated protein alterations that affect binding of the amino-terminal AR antiserum. Immunohistochemical expression of the androgen-regulated glycoprotein, apolipoprotein D (apo-D), was also evaluated in the breast cancer specimens. Focal positivity of apo-D staining, which did not always co-localise with AR-positive cells, was observed within breast tumours. Furthermore, no correlation was evident between percentage positive cells stained for AR and apo-D in breast cancer specimens. These findings indicate that, although apo-D expression is androgen regulated in human breast cancer cell lines in vitro, its expression in primary breast cancers may be regulated by other factors. The expression of AR in primary breast cancers also suggests that the receptor may be involved in tumour responsiveness or in abnormal responses to endocrine therapies.

Vimentin expression is not associated with poor prognosis in breast cancer.

The clinical significance of vimentin intermediate filament (VIF) expression was studied in relation to other established prognostic parameters in primary breast cancer. Archival tumour samples embedded in paraffin were examined by immuno-histochemistry with monoclonal antibodies (MAbs) to VIF, p53 protein and cell proliferation marker MIB-I. The vimentin staining pattern was heterogeneous, but in vimentin-positive areas > 80% of the tumour cells were positive. There was no association between vimentin expression and tumour size or the number of axillary lymph nodes involved. Vimentin expression was significantly associated with high-grade tumours, absence of hormone receptors, increased p53 expression and high tumour proliferation fraction as estimated by MIB-I count. Despite these associations with several recognised features of tumour aggressiveness, vimentin expression was not associated with increase in risk of relapse or death from breast cancer.

Cyclin DI amplification is not associated with reduced overall survival in primary breast cancer but may predict early relapse in patients with features of good prognosis.

Amplification of chromosome 11q13 is frequently observed in human malignancies, including breast cancers. A candidate oncogene at this locus is the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. Because published data on the relationship between 11q13 amplification and prognosis in breast cancer have been controversial, we investigated the clinical significance of CCND1 amplification and its association with established clinicopathological features of prognosis in 1014 primary breast cancer patients. Amplification of the CCND1 gene and the INT-2/FGF-3 gene, which also maps to 11q13, was 10% and 17%, respectively. There were no associations between CCND1 or INT-2 amplification and patient age, tumor size, tumor grade, axillary lymph node status, HER/neu amplification, MIB-1 monoclonal antibody to Ki67 antigen count, or p53 expression. CCND1 amplification was predominantly observed in hormone receptor-positive tumors; at a copy number >/=3, CCND1 amplification was significantly correlated with both estrogen receptor (ER; P = 0.036) and progesterone receptor (P = 0.012) positivity. After a median follow-up period of 66 months, CCND1 or INT-2 amplification was not associated with significant increases in relapse or death from breast cancer. However, in the node-negative and ER-positive subgroups, there was a trend for an increased relapse rate in patients with INT-2 or CCND1 amplification. Thus, in this study, assessment of CCND1 or INT-2 amplification at 11q13 by slot-blot hybridization was of little use in determining phenotype or disease outcome in the whole group of patients but had a potential role in identifying a subset of poor-prognosis patients within the node-negative or ER-positive, good-prognosis groups. Because the prevalence of CCND1 amplification is much lower than the reported prevalence of cyclin D1 overexpression, additional studies are required to determine the true prognostic significance of altered cyclin D1 expression in breast cancer.

Relationship between p53 gene abnormalities and other tumour characteristics in breast-cancer prognosis.

The prognostic significance of p53 gene abnormalities was investigated in 919 primary breast-cancer patients. p53 expression and tumour-cell proliferation fraction determined by MIB-1 count, p53 exon 5 and 6 mutations and HER-2/neu oncogene amplification were detected by immunohistochemistry, PCR-SSCP and slot-blot hybridization, respectively. Increased MIB-1 count, p53 expression, HER-2/neu oncogene amplification and p53 mutations were detected in 33%, 29%, 10% and 8% of tumours, respectively. Statistically significant associations were observed between p53 expression or MIB-1 count and age below 50 years, high-grade tumours, medullary carcinomas, and absence of hormone receptors. p53 mutations were associated with increased MIB-1 count, HER-2/neu oncogene amplification and absence of hormone receptors, but not with age, tumour size or grade, histological subtype, or the number of axillary nodes involved. After a median follow-up of 66 months, p53 expression was observed to be associated with significant increases in risk of both relapse and death from breast cancer, but not after adjusting for the effect of other parameters. In these analyses, MIB-1 count, and not HER-2/neu oncogene amplification, was an independent predictor of prognosis. In node-negative patients, only p53 exon 5 and 6 mutations and MIB-1 count were associated with a statistically significant increase in risk of death from breast cancer, independent of tumour size and ER concentration. We conclude that tumour-cell proliferation fraction, as measured by MIB-1 count, is the most useful parameter of breast-cancer prognosis, with the exception of ER, tumour size and the number of axillary nodes involved.

Prospective study of the prognostic significance of epidermal growth factor receptor in primary breast cancer.

Bearing in mind the continuing controversy over the prognostic significance of epidermal growth factor receptor (EGF-r) expression, we investigated its clinical significance prospectively in 345 primary breast cancer patients. The prognostic significance of EGF-r expression, as measured by a radioligand binding assay, was determined by Cox's multivariate analysis using EGF-r concentration as a continuous or dichotomous variable. Increased EGF-r expression was detected in 20-32% of tumours, depending on the cut-off in concentration used. EGF-r expression, irrespective of the cut-off, was not associated with tumour size or grade or the number of axillary nodes involved. There was, however, a strong inverse association between EGF-r expression and the absence of hormone receptors. After a median follow-up period of 57 months, multivariate analysis suggested that EGF-r expression was associated with increases in risk for both relapse and death from breast cancer, even after adjusting for oestrogen receptor (ER) concentration, tumour size and the number of axillary nodes involved. Patients with ER-positive tumours, which also expressed EGF-r, had increases in risk for both relapse and death from breast cancer compared with tumours without EGF-r. Expression of EGF-r was not a predictor of poor prognosis in either node-negative or ER-negative subgroups of patients.

Differential expression of apolipoprotein-D and prostate specific antigen in benign and malignant prostate tissues.

PURPOSE: To investigate Apolipoprotein-D (Apo-D) and prostate specific antigen (PSA) immunohistochemical staining of nonmalignant and malignant human prostate tissues. MATERIALS AND METHODS: Apolipoprotein-D and PSA immunoreactivity were evaluated by video image analysis in nonmalignant prostates and in 30 stage D2 prostate cancers. RESULTS: Apolipoprotein-D was detected in all 30 tumors, and the level of staining was elevated in comparison to age-matched nonmalignant prostates (p < 0.05). In contrast, the level of PSA staining in tumors was less than that detected in nonmalignant prostates. CONCLUSIONS: Apolipoprotein-D is expressed in normal human prostate. Elevated Apo-D staining is associated with advanced prostate cancer.

Medroxyprogesterone acetate therapy in advanced breast cancer: the predictive value of androgen receptor expression.

PURPOSE: To determine the predictive value of androgen receptor (AR) levels in primary tumors of women who undergo medroxyprogesterone acetate (MPA) therapy for advanced breast cancer after relapse on tamoxifen adjuvant therapy. METHODS: Between 1984 and 1987 at Flinders Medical Centre, South Australia, 136 postmenopausal women received adjuvant tamoxifen therapy for lymph node-positive breast cancer. Estrogen receptor (ER), progesterone receptor (PgR), and AR levels, tumor size, and degree of axillary node involvement were determined at the time of diagnosis. The median follow-up period was 81 months; 89 women developed metastatic disease, 83 of whom subsequently received MPA (500 mg/d). The objective response rate ([RR] ie, complete response [CR] and partial response [PR]) and progression-free interval (PFI) were assessed in response to MPA therapy. Associations between RR, PFI, and primary tumor characteristics including ER, PgR, and AR levels were examined using the Mann-Whitney U test, Kaplan-Meier product-limit estimator, and Cox proportional hazards regression, as appropriate. RESULTS: Thirty-two of 83 patients (38.6%) responded to MPA. RR was significantly associated with the presence of AR (P < .001), but not with other primary tumor characteristics or duration of tamoxifen therapy. After initiation of MPA treatment, PFI increased with increasing concentration of AR in the primary tumor. CONCLUSION: Response to MPA after adjuvant tamoxifen treatment for lymph node-positive breast cancer was positively associated with AR level in the primary tumor. This finding suggests that MPA action in breast cancer may be mediated in part by the AR.

Androgens induce divergent proliferative responses in human breast cancer cell lines.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.