Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids.

UNLABELLED: LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37 degrees C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. IN CONCLUSION: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect.

Breast-conserving therapy: proteases as risk factors in relation to survival after local relapse.

PURPOSE: To evaluate whether cathepsin D, urokinase-type plasminogen activator (uPA), its inhibitor, plasminogen activator inhibitor-1 (PAI-1), or clinical factors can predict which patients are at risk for developing distant metastases after local recurrence (LR). PATIENTS AND METHODS: Of 1,630 patients treated with breast-conserving surgery and radiotherapy of the breast between 1980 and 1992, LR developed in 171 as a first event. From the available primary tumor tissues, we determined the cytosolic levels of cathepsin D, uPA and PAI-1. RESULTS: In patients with LR, a short (< or = 2 years) disease-free interval (DFI) and skin involvement of LR were associated with poor postrelapse distant metastasis-free survival (PR-DMFS, P = .001, both) and postrelapse overall survival (PR-OS; P < .0001 and P < .0002, respectively). The primary tumor levels of uPA and PAI-1 were elevated for patients with a short DFI (P < .01), but such a relation was not observed for patients with skin involvement. In univariate analyses, high levels of uPA and PAI-1 in the primary tumor were associated with poor PR-OS (P = .038 and P = .040, respectively) but not PR-DMFS. In Cox multivariate analyses for PR-DMFS and PR-OS, only a short DFI and skin involvement of the LR were independently associated with a poor clinical outcome. CONCLUSION: In patients treated with breast-conserving therapy who had LR as a first event, a short DFI and skin involvement were strong indicators for poor PR-DMFS and PR-OS. The proteases studied did not contribute significantly to the final multivariate model.

The human androgen receptor is a 110 kDa protein.

The androgen receptor in human prostate carcinoma cells (LNCaP) has been studied after in situ photolabeling with [3H]R1881. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole cell extracts revealed the presence of two specifically labeled proteins of 110 kDa and 43 kDa. Both photolabeled proteins were stable in cell homogenates and generated different chymotryptic maps, suggesting that the two proteins were different. From ligand binding specificity studies could be concluded that the 110 kDa protein represents the androgen receptor. The 43 kDa protein showed binding specificity only for R1881. Both photolabeled proteins were recovered from LNCaP nuclei, but the 43 kDa protein showed a relatively higher affinity for nuclei than the 110 kDa protein. The function of this protein is unknown. It is concluded that the human androgen receptor is a protein with a molecular mass of 110 kDa.

Androgens and androgen-receptors in prostate tissue from patients with benign prostatic hyperplasia: effects of cyproterone acetate.

Testosterone, 5 alpha-dihydrotestosterone and cyproterone acetate (CPA) were estimated in samples of prostate tissue, obtained from benign prostatic hyperplasia (BPH) patients who were or were not pretreated with CPA. Furthermore, these steroids were estimated in various fractions of the BPH tissue, and the number of nuclear androgen-receptor sites was determined. CPA-treatment caused a 4-fold, significant suppression of 5 alpha-dihydrotestosterone levels in total prostate tissue and its subfractions, without affecting testosterone levels or the androgen-receptor contents of the nuclear extracts. Nuclear concentrations of CPA were twice as high as those of 5 alpha-dihydrotestosterone. It is concluded that effects of CPA may have been caused through a combination of the following mechanisms: (1) suppression of peripheral androgen levels; (2) competition with androgens for (nuclear) androgen-receptors; and (3) suppression of prostatic 5 alpha-reductase.

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.

Nuclear androgen receptors in human prostatic tissue. Extraction with heparin and estimation of the number of binding sites with different methods.

A procedure for the estimation of nuclear androgen receptors in benign prostatic hyperplastic tissue is described, which employs extraction of receptors from nuclei with buffers containing heparin. Extraction of a nuclear pellet with a heparin-containing (1 g/l) buffer appeared to have definite advantages over 0.4 mol/l KCl extraction. Heparin appeared to be twice as efficient in extracting androgen receptors. In addition aggregated receptor proteins, formed after storage at -80 degrees C, were partly deaggregated by heparin. Specific isolation of the androgen receptor was performed using either agar gel electrophoresis, protamine sulphate precipitation or LH-20 gel filtration. A comparison was made between the amounts of estimated receptors with these different techniques. Protamine sulphate precipitation resulted in the highest estimates of receptor-bound 5 alpha-[3H]dihydrotestosterone (3H-DHT). Treatment of the labelled nuclear extracts with a charcoal suspension prior to the receptor assay resulted in lower amounts of estimated androgen receptors. A method for routine evaluation of nuclear androgen receptors in prostatic tissue has been evaluated, which involves extraction of nuclear pellets with a heparin-containing (1 g/l) buffer, exchange labelling of the nuclear extracts for 20 h at 10 degrees C and quantification of the receptors with protamine sulphate precipitation.

[Androgen receptor and 5 alpha-reductase activity in the epithelium and the stroma of human benign prostatic hyperplasia].

An average of 20 X 10(6) nucleated cells were obtained from 1 g tissue of human benign prostatic hyperplasia by mechanical separation technique. Of these cells, 96.2% showed acid phosphatase activity and this was 10 times higher than the remaining stromal fraction on a protein base. The total activity of 5 alpha-reductase was 81 times higher in stroma than epithelium and the total activity of 3(beta)-oxidoreductase was 29 times higher in stroma. Androgen receptor amount measured in total tissue, epithelium and stroma were 100, 29 and 62 fmol R1881/mg DNA, respectively. These results suggest that androgen metabolism takes place mainly in the stroma of human BPH tissue, and that BPH is probably the disease of prostatic stroma.

Hormone receptors in human prostate cancer.

The relative success with which the response of breast cancer to endocrine therapy can be predicted by assay of female sex steroid receptors has led to attempts to use measurement of androgen receptors in neoplastic prostate tissue for predicting the success of anti-androgen therapy in prostate cancer. Hitherto hopes have not been fulfilled. Androgen receptors are present in almost all prostate samples, but with inhomogeneous distribution. No relationship was found between androgen receptor levels in needle aspirate and prognosis in prostatic carcinoma. Receptors for oestrogen, progestin and prolactin were also studied for identification of possible prognostic indicators. Progestin receptors appear to be present in prostatic tissue. Lack of consensus regarding prostatic oestrogen and prolactin receptors is due partly to their low (if any) concentrations and partly to differing methodology and interpretation of results. Oestrogen, progestin and prolactin receptors seem to lack prognostic significance in prostatic cancer. These findings and the high initial response rate of prostatic carcinoma to endocrine therapy indicate that further studies should focus on elucidating how such tumours become hormone-independent.

In situ photolabelling of the human androgen receptor.

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.

Prediction of time to progression after orchiectomy by the nuclear androgen receptor content from multiple biopsy specimens in patients with advanced prostate cancer.

The nuclear androgen receptor (ARn) content of cancerous prostatic tissue has been investigated as a prognosticator for time to progression under endocrine therapy. In 1981 a prospective study was started to investigate whether the ARn content in biopsy specimens of patients with prostatic carcinoma predicts the duration of response following hormonal treatment. ARn was estimated by a microassay which involves extraction of nuclear pellets with a heparin-containing buffer, exchange labeling of the nuclear extract with 3H-R1881, and quantitation of the receptor with protamine sulphate precipitation. One hundred and fifteen patients with prostatic cancer entered this study; 47 patients had evidence of metastatic disease as proven by bone scan. Forty-two patients were treated by orchiectomy; 37 of these patients are evaluable with a minimal follow-up of 30 months. A relationship between the nuclear androgen receptor content and the time to progression following orchiectomy in these patients with metastatic disease of the prostate was not found. This could possibly be attributed to the heterogeneous nature of the prostatic tumor tissue with respect to the distribution of the ARn. We conclude that androgen receptor assay in needle biopsies, at least in this study, had no value for the prediction of the time to progression after orchiectomy.

Effect of long-term hyperprolactinemia on the prolactin receptor content of the rat ventral prostate.

The rat ventral prostate contains prolactin receptors, and during sexual maturation prolactin stimulates the growth of this gland. The aim of the present investigation was to evaluate whether prolactin is involved in the regulation of the number of its own receptor sites in the rat ventral prostate. To this end, the content of prolactin receptors was estimated in prostate membranes of control and chronically hyperprolactinemic rats both before and after in vitro desaturation with 4 M MgCl2. Hyperprolactinemia resulted in a 40% increase in the number of available prolactin receptors (P less than 0.05). In vitro desaturation of receptors resulted in loss of 84% of protein and 36 +/- 6% and 52 +/- 6% of prolactin receptors from ventral prostate membranes of control and hyperprolactinemic rats respectively (P less than 0.05). We have concluded that the rat ventral prostate membranes are not suited to in vitro desaturation of prolactin receptors with MgCl2. From the increase in the number of available prolactin receptors after hyperprolactinemia we have concluded that prolactin is involved in the regulation of the number of its own receptors in the rat ventral prostate.

Nuclear androgen receptor assay in biopsy-size specimens of human prostatic tissue.

Nuclear androgen receptors in benign and malignant human prostatic tumors were estimated with a nuclear exchange assay to evaluate the applicability of the assay to biopsy size tissue samples. The mean nuclear androgen receptor content of six different prostate samples was not dependent on the amount of tissue used. In contrast, however, there was no significant correlation between the results obtained for individual prostates when large samples (500 mg) and samples weighing 100, 50(r = 0.38, n = 14), and 25 mg were compared. This lack of correlation could not be attributed to variations in the assay nor to differences in the percentage of epithelium in the samples. There was no effect of the presence of molybdate on the estimated nuclear androgen receptor level. We concluded that androgen receptors are distributed nonhomogeneously over prostatic tissue and that androgen receptor assays on multiple biopsies are required to obtain a proper estimate of the true androgen receptor content of the tissue.

Transplantable human prostatic carcinoma (PC-82) in athymic nude mice. II. Tumor growth and androgen receptors.

Androgen receptor (AR) levels were measured in PC-82 tumor tissue grown in hormonally manipulated nude mice. In the nuclei of tumor tissue from intact male mice a relatively low concentration (mean 25 fmol/mg protein) of androgen receptors (ARn) was found, while no receptors for estrogens or progestins were detected. The total number of androgen receptors in the PC-82 tumor tissue (measured in the nuclei 1 h after injection of a single high dose of testosterone (T) was found to be 100 fmol/mg protein. The antiandrogen cyproterone acetate, administered in combination with the high dose of T, significantly lowered the amount of ARn in the tumor tissue. In the nuclei of tumor tissue from intact tumor-bearing male mice with T-containing Silastic implants, a 4-times higher amount of tightly associated AR was found. In addition, an increased growth rate of the tumor was observed following T implantation. This finding suggests that the increased growth rate of the PC-82 tumor is associated with a continuous occupancy of AR in the nuclei of the tumor tissue. Castration of tumor-bearing male mice, which arrests the growth of this tumor, did not affect the concentration of ARn in the tissue compared to that of tissue in the intact control situation. In addition, the total amount of AR measured after T injection was not affected by castration. Therefore, the availability of a sufficient and steady level of T in the plasma and consequently the duration of the presence of AR in the nucleus of the PC-82 tumor tissue, rather than the total concentration of AR, appear to be the limiting factors in the modulation of hormonal responses in this androgen target tissue.

Nuclear androgen receptor content in biopsy specimens from histologically normal, hyperplastic, and cancerous human prostatic tissue.

Androgen receptors (ARn) were assayed in nuclear extracts of prostatic biopsies from 60 patients with benign prostatic hyperplasia (BPH) and 82 patients with prostatic cancer (PC), with an exchange assay using heparin extraction, labelling with 3H-R1881, and protamine sulphate precipitation. The content of ARn of BPH biopsies (38 +/- 34 fmol/mg protein [mean +/- SD]; n = 70) was not different from that of PC biopsies (39 +/- 32 fmol/mg protein; n = 115). Biopsies showing essentially normal prostatic tissue had a lower ARn content (12 +/- 13 fmol/mg protein; n = 6). The content of ARn was independent of the age of the patient and of the histological grade of the carcinomas. A considerable variation in ARn content within tumors of individual patients was found, indicating that ARn are not uniformly distributed over prostatic tissue; ie, cells with high and low receptor content may coexist in different proportions in different regions of the prostate. Therefore, assays on multiple biopsies may be required for a proper estimation of the mean receptor content. The question remains, however, whether the behavior of the tumor is adequately predicted by the mean receptor level or, for instance, by the region with the lowest receptor content.

Androgen receptor heterogeneity and phosphorylation in human LNCaP cells.

Androgen receptor heterogeneity and phosphorylation were studied in the human LNCaP cell line. Fluorography after photoaffinity labeling as well as immunoblotting with a specific polyclonal antibody revealed that the human androgen receptor migrated as a closely spaced 110 kD doublet on SDS-polyacrylamide gels. A time-dependent change in the ratio between the two isoforms was not observed after R1881 treatment of intact cells. In nuclear extracts of LNCaP cells that were incubated with [32P]orthophosphate in the presence of 10 nM R1881, a 110 kD phosphorylated protein was demonstrated after immunopurification using a monoclonal antibody against the human androgen receptor. Only a very small amount of this phosphoprotein was detected in the nuclear fraction from cells not treated with R1881. These results indicate that the human androgen receptor in LNCaP cells can be phosphorylated.

Circulating testosterone, prostatic nuclear androgen receptor and time to progression in patients with metastatic disease of the prostate treated by orchiectomy.

The content of nuclear androgen receptors (ARn) in prostatic carcinoma biopsies is not predictive for the duration of response of the tumor to endocrine therapy. Recently pre-treatment plasma testosterone has been suggested to be predictive in this respect. Therefore, pre-treatment plasma testosterone (T) and sex hormone binding globulin (SHBG) levels were studied in 31 patients aged 72 +/- 10 years (range: 45-87) with stage D2 carcinoma of the prostate treated by orchiectomy. In 26 of these patients, the ARn level of the carcinoma was also known (61 +/- 41 fmol/mg protein; range 0-169). Plasma T levels (mean: 13.7 +/- 6.1 nmol/l) varied widely (range: 2.4-25.4), as did plasma SHBG (32.5 +/- 19.3 nmol/l; range 4.4-78.8), and time to progression (TTP; 14.6 +/- 11.2 months; range 1-48). Plasma T was found to be correlated to age (Rs = 0.537; P less than 0.01) and TTP (Rs = 0.4495; P less than 0.02). Tissue ARn and plasma SHBG did not correlate to any of the parameters studied.

Effect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis.

Adult rats were treated with ethane dimethane sulphonate (EDS) to eliminate the Leydig cells. This treatment resulted in very low levels of testosterone in the blood and in the testis. Furthermore, histological evaluation of spermatogenesis showed no marked differences between control and EDS-treated animals. In the ventral prostate, 5 days after EDS-treatment, a 4.0 +/- 0.3-fold up-regulation of androgen receptor (AR) mRNA was observed, together with a 2.2 +/- 0.2-fold increase in actin mRNA. In the epididymis, a 2.0 +/- 0.5-fold increase in AR mRNA level was observed, without a change in actin mRNA level. In the testes of EDS-treated rats, the AR mRNA level was not changed (1.02 +/- 0.17-fold of controls), and there was also no change in actin mRNA level at 5 days after EDS-treatment. These results indicate that AR mRNA expression in the ventral prostate and epididymis is regulated differentially by testosterone when compared to regulation in the testis. Testicular androgen binding sites were assayed by Scatchard analysis of the binding of 3H-R1881 to a nuclear fraction, that was isolated by a method which involved the use of liquid nitrogen and high sucrose buffer. The number of specific binding sites per testis in EDS-treated rats with testosterone-implants, remained unaltered compared to control rats (9.1 +/- 1.4 pmol/testis). In these rats, 20% of the normal testicular testosterone level was sufficient to maintain the androgen receptor in a tight nuclear binding (transformed) form. In testes from EDS-treated rats without testosterone-implants, the AR did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals was close to control levels, as measured by nuclear 3H-R1881 binding after receptor transformation through injection of a high dose of testosterone (10 mg) 2 h before killing the rats (testosterone pulse). In the different experimental groups, FSH was not required to maintain the total testicular AR content (ligand binding). Immunoprecipitation and Western blotting of the testicular AR using specific monoclonal and polyclonal antibodies indicated that the total testicular amount of immunodetectable AR protein in long-term testosterone deprived rats was very low when compared to that in control rats or rats with testosterone-implants. This is in disagreement with results obtained in the ligand binding assay, and may point to a structural modification of the AR in the testis that possibly occurs in the prolonged absence of androgens.

Effect of hormone treatment on prostatic acid phosphatase in a serially transplantable human prostatic adenocarcinoma (PC-82).

The influence of endocrine manipulation on the tissue concentration of prostatic acid phosphatase (PAP) was studied in the hormone dependent transplantable human prostatic tumor line PC-82. Tumor bearing nude mice were left intact, castrated or treated for a 5-day period with a subcutaneous implant containing testosterone or estradiol. The concentration of PAP in castrated mice was not different from that in the controls. The DNA content of PC-82 tumor tissue obtained from 5-day castrated animals was significantly lower than that of tissue from intact animals. Therefore the concentration of PAP in tissue from castrated mice was significantly elevated when expressed per mg. of DNA (p less than 0.05). Treatment of the mice with testosterone or estradiol did not affect the PAP concentration in the tumor tissue. A significant correlation was observed between the concentration of PAP in the serum and the tumor burden of the mice. Long-term withdrawal of androgens resulted in a decrease of the concentration of PAP in the serum, as well as in a decrease of the tumor burden. The concentration of PAP in the tumor tissue remaining after castration of these animals was not significantly different from that in controls. The present data from the tumor line PC-82 do not support the hypothesis that the concentration of PAP in prostatic tumor tissue is controlled by androgens, but are in agreement with the concept that the level of PAP in plasma is related to the tumor mass.

Cathepsin-D in primary breast cancer: prognostic evaluation involving 2810 patients.

There is controversy regarding the prognostic value of cathepsin-D in primary breast cancer. An increased level of cathepsin-D in tumour extracts has been found to be associated with a poor relapse-free and overall survival. Studies performed with immunohistochemistry or Western blotting have produced diverse results. We have analysed 2810 cytosolic extracts obtained from human primary breast tumours for cathepsin-D expression, and have correlated their levels with prognosis. The median follow-up of the patients still alive was 88 months. Patients with high cathepsin-D levels had a significantly worse relapse-free and overall survival, also in multivariate analysis (P < 0.0001). Adjuvant therapy which was associated with an improved prognosis in node-positive patients in univariate analysis, also significantly added to the multivariate models for relapse-free and overall survival. There were no statistically significant interactions between the levels of cathepsin-D and any of the classical prognostic factors in analysis for relapse-free survival, suggesting that the prognostic value of cathepsin-D is not different in the various subgroups of patients. Indeed, multivariate analyses in subgroups of node-negative and -positive patients, pre- and post-menopausal patients, and their combinations, showed that tumours with high cathepsin-D values had a significantly poor relapse-free survival, with relative hazard rates ranging from 1.3 to 1.5, compared with tumours with low cathepsin-D levels. The results presented here on 2810 patients confirm that high cytosolic cathepsin-D values are associated with poor prognosis in human primary breast cancer.