A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen

Tamoxifen significantly reduces tumor recurrence in certain patients with early-stage estrogen receptor-positive breast cancer, but markers predictive of treatment failure have not been identified. Here, we generated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. Ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The HOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer.     

TAG-72 expression and clinical outcome in primary breast cancer.

Clinical data of 92 patients with primary breast carcinomas previously analysed for the pattern of immunohistochemical expression of three distinct carbohydrate epitopes of the TAG-72 molecule were reviewed. The clinical outcome of the patients after a median follow-up of 66 months was determined in 84 out of 92 patients. Clinicopathological characteristics of the tumours and clinical outcome of the patients were correlated with the TAG-72 epitope expression. TAG-72 was expressed more frequently in patients aged more than 50 years and in tumours of larger size, with lymph nodes metastasis, with low differentiation and with high proliferative activity. A statistical correlation was found with more advanced stages of the disease (35.7% vs 60% in stage I and in stage II-III, respectively, p=0.03). Disease-free survival and overall survival were estimated by the Kaplan-Meier method. The survival of the patients with tumours expressing TAG-72 was not statistically different from that of patients with tumours without TAG-72 expression. These data suggest that TAG-72 expression is associated with clinicopathological parameters of aggressiveness in primary breast cancer, but it does not appear to affect the clinical outcome of the patients.     

Gene expression preferentially regulated by tamoxifen in breast cancer cells and correlations with clinical outcome

The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERalpha-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERalpha and reversed by E2 or ICI 182,780. Introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy.     

Biologic markers as predictors of clinical outcome from systemic therapy for primary operable breast cancer.

PURPOSE: To determine whether pretreatment clinical features and molecular markers, together with changes in these factors, can predict treatment response and survival in patients with primary operable breast cancer who receive neoadjuvant therapy. PATIENTS AND METHODS: Mitoxantrone, methotrexate (with or without mitomycin), and tamoxifen chemoendocrine therapy was administered to 158 patients before surgery. Clinical response was assessed after four cycles of treatment. Fine-needle aspiration cytology for estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53, bcl-2, Ki67, S-phase fraction (SPF), and ploidy were performed pretreatment and repeated on day 10 or day 21 after the first cycle of treatment. RESULTS: Good clinical response (GCR, defined as complete response or minimal residual disease) was achieved in 31% of patients (49 of 158). Tumor size, nodal disease, response, ER, PgR, c-erbB-2, p53, bcl-2, Ki67, SPF, and ploidy were analyzed as predictors of survival. By univariate analysis, node-positive disease (P =.05), lack of ER (P <.05) and PgR (P <.05), and failure to attain GCR (P =.008) were associated with a significantly increased risk of relapse. A significantly increased risk of death was associated with node-positive disease (P =.02), lack of ER expression (P =.04), and failure to attain GCR. By multivariate analysis, GCR was an independent predictor for survival (P =.05). ER expression (P =.03), absence of c-erbB-2 (P =.03), and a decrease in Ki67 on day 10 or day 21 of the first cycle (P <.05) significantly predicted for subsequent GCR. CONCLUSION: Molecular markers may be used to predict the likelihood of achieving GCR, which seems to be a valid surrogate marker for survival.     

Biomarker prediction of clinical outcome in operable breast cancer patients treated with tamoxifen

The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in 25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.     

Cellular effects of tamoxifen in primary breast cancer.

We have investigated the effect of tamoxifen on the biological characteristics of the primary tumour in 33 patients with breast cancer. All patients had pre-treatment biopsy of the primary breast cancer and subsequent trucut biopsy of the primary tumour after 1-4 months on tamoxifen therapy. The staining patterns in the primary tumour of each of the tumour antigens 115D8, DF3, NCRC-11, and carcinoembryonic antigen (CEA) were unaffected by tamoxifen therapy: there was 16%-31% change before vs. during tamoxifen therapy, but this did not reach significance for any of the four antigens. Comparison of the pattern of staining between the four antigens showed 115D8, DF3, and NCRC-11 to be similar to each other both before and during tamoxifen therapy. All three antigens were significantly different from the staining pattern shown by CEA before and during therapy. Tumour antigen expression pre-treatment did not correlate with therapeutic response for any of the four antigens. However, NCRC-11 staining during tamoxifen therapy did correlate with response (p = 0.004). There was no significant difference in oestrogen receptor status before and during tamoxifen therapy, both of which correlated with response. DNA ploidy of the tumour before tamoxifen did not correlate with response to therapy, but there was a weak correlation between response and DNA ploidy measured during tamoxifen therapy. In only a minority of tumours (up to 30%) did tamoxifen exert an effect on the biological characteristics studied. Changes in these biological markers were mainly in tumours which responded to tamoxifen, and the changes seen were a tendency to greater expression of differentiation antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients

Introduction

A gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.

Methods

Gene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-??)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.

Results

Extratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-?? induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.

Conclusions

These results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.     

c-erbB2 expression predicts tamoxifen efficacy in breast cancer patients

c-erbB2 is a proto-oncogene that encodes the trans- membrane protein p185. This protein shares considerable sequence and structure homology with members of the epidermal growth factor receptor family and it is believed to cooperate with these receptors in the signal transduction process in order to control cell proliferation. Overexpression of c-erbB2, with or without gene amplification, is frequently found in breast cancer, and a body of evidence suggests it is implicated in the development of resistance to the anti-estrogen tamoxifen. Scientific evidence strongly supports a correlation between c-erbB2 overexpression and lack of efficacy of tamoxifen in both advanced and adjuvant settings. However, given the important therapeutic repercussion of this topic, further studies are required before c- erbB2 evaluation can be routinely used to select patients who are likely to benefit from tamoxifen administration.     

PRAME expression and clinical outcome of breast cancer

The tumour antigen PReferentially expressed Antigen of MElanoma (PRAME) is expressed in a variety of malignancies, including breast cancer. We have analysed PRAME gene expression in relation to clinical outcome for 295 primary breast cancer patients. Kaplan-Meier survival curves show a correlation of PRAME expression levels with increased rates of distant metastases and decreased overall patient survival. This correlation existed both for the entire patient group (n=295) and for the subgroup of patients (n=185) who did not receive adjuvant chemotherapy. Multivariable analysis indicated that PRAME is an independent marker of shortened metastasis-free interval in patients who did not receive adjuvant chemotherapy. PRAME expression was associated with tumour grade and negative oestrogen receptor status. We conclude that PRAME expression is a prognostic marker for clinical outcome of breast cancer, independent of traditional clinicopathological markers.     

Cellular effects of tamoxifen in primary breast cancer

We have investigated the effect of tamoxifen on the biological characteristics of the primary tumour in 33 patients with breast cancer. All patients had pre-treatment biopsy of the primary breast cancer and subsequent trucut biopsy of the primary tumour after 1–4 months on tamoxifen therapy.The staining patterns in the primary tumour of each of the tumour antigens 115D8, DF3, NCRC-11, and carcinoembryonic antigen (CEA) were unaffected by tamoxifen therapy: there was 16%–31% change before vs. during tamoxifen therapy, but this did not reach significance for any of the four antigens. Comparison of the pattern of staining between the four antigens showed 115D8, DF3, and NCRC-11 to be similar to each other both before and during tamoxifen therapy. All three antigens were significantly different from the staining pattern shown by CEA before and during therapy.Tumour antigen expression pre-treatment did not correlate with therapeutic response for any of the four antigens. However, NCRC-11 staining during tamoxifen therapy did correlate with response (p=0.004).There was no significant difference in oestrogen receptor status before and during tamoxifen therapy, both of which correlated with response. DNA ploidy of the tumour before tamoxifen did not correlate with response to therapy, but there was a weak correlation between response and DNA ploidy measured during tamoxifen therapy.In only a minority of tumours (up to 30%) did tamoxifen exert an effect on the biological characteristics studied. Changes in these biological markers were mainly in tumours which responded to tamoxifen, and the changes seen were a tendency to greater expression of differentiation antigens. It is uncertain whether these changes are a primary or a secondary effect of tamoxifen with respect to inducing tumour response.     

MUC1 expression in primary breast cancer: the effect of tamoxifen treatment

This was a non-randomised single institution retrospective study. Forty-six banked frozen tumour specimens were obtained from a group of patients who had undergone 3 weeks of neoadjuvant treatment with tamoxifen between biopsy and surgery. Fifty-one comparison specimens were randomly selected from a group of concomitantly treated primary breast cancer patients who did not receive neoadjuvant tamoxifen. Specimen selection was not based on prognostic factors: hormone receptor status, patient age, or menopausal status. MUC1 expression and cell cycle distribution were assessed by flow cytometry. S-phase fraction of MUC1 positive and MUC1 negative cells were compared. A lower percentage of cells expressed MUC1 following 3-week tamoxifen treatment 18.2% versus 28.5% (p=0.03, Mann-Whitney) and lower levels of MUC1 expression were seen following tamoxifen treatment 31,519 molecules/cell versus 39,387 (p=0.04, Mann-Whitney). MUC1 positive cells, irrespective of treatment group, had a greater proportion of cells in S-phase of the cell cycle 27.9% versus 16.8% (p=0.0004, Mann-Whitney) and demonstrated more cases of aneuploidy 80.65% versus 42.6% (p<0.0001). MUC1 levels in primary tumours treated neoadjunctively with 3 weeks of tamoxifen were lower than a comparison group which did not receive tamoxifen. MUC1 should be explored further as an intermediate biomarker for assessment of treatment and prognosis.     

Association between HER-2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients.

PURPOSE: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a). evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b). compare the prognostic significance of VEGF isoforms; and (c). analyze the combined effects of HER-2/neu and VEGF on clinical outcome. EXPERIMENTAL DESIGN: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF(121-206) ELISA uses antibodies that bind to VEGF(121) and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF(165-206) ELISA uses antibodies that bind to VEGF(165) and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF(121-206) and VEGF(165-206) were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. RESULTS: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF(121-206) and VEGF(165-206) expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (chi(2) test: P < 0.001 for both VEGF(121-206) and VEGF(165-206)). VEGF(121-206) and VEGF(165-206) demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF(121-206), P = 0.0068; VEGF(165-206), P = 0.0046; multivariate analysis: VEGF(121-206), P = 0.1475; VEGF(165-206), P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF(121-206) and VEGF(165-206) were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF(121-206), P = 0.0003; VEGF(165-206), P = 0.0038; multivariate analysis: VEGF(121-206), P = 0.0103; VEGF(165-206), P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF(121-206), P = 0.0280; VEGF(165-206,) P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF(121-206)/VEGF(165-206) results in additional prognostic information for survival (VEGF(121-206), P = 0.0133; VEGF(165-206), P = 0.0092). CONCLUSION: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.     

TSC22D1 and PSAP predict clinical outcome of tamoxifen treatment in patients with recurrent breast cancer

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04–1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03–1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06–0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29–3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance. Danielle Meijer and Maurice P.H.M. Jansen contributed equally to this work.     

Tumor-specific HMG-CoA reductase expression in primary premenopausal breast cancer predicts response to tamoxifen

Introduction  

We previously reported an association between tumor-specific 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR) expression and a good prognosis in breast cancer. Here, the predictive value of HMG-CoAR expression in relation to tamoxifen response was examined.     

Impact of cyclins E, neutrophil elastase and proteinase 3 expression levels on clinical outcome in primary breast cancer patients

Uncontrolled cell proliferation is one of the hallmarks of cancer and the transition from the G1 to S phase is the most commonly reported cell cycle abnormality in tumors. It has been shown that the oncogenic activity of G1 cyclin E (CCNE) can be amplified by generating hyperactive low molecular weight forms (LMW) through elastase-mediated proteolytic processing. Neutrophil elastase (NE) and proteinase 3 (PR3) are 2 proteases that are aberrantly expressed in breast cancer cells and seem to be involved in cell proliferation. In this study, we evaluated the effect of the expression of these 2 proteases in addition to 2 potential intracellular targets of NE (CCNE1 and CCNE2) on clinical outcome in a population of 205 primary breast cancer patients. By univariate analysis, CCNE1, CCNE2, estrogen receptor and grade significantly predicted relapse free interval (RFI). NE and PR3 did not achieve statistical significance. In a multivariate analysis, elevated CCNE2 [hazard ratio (HR) 2.10, p = 0.008] predicted shorter RFI. In subgroup analyses of the tamoxifen-only treated patients, high CCNE1 levels predicted treatment resistance, while high levels of CCNE2 were associated with poor RFI in untreated patients. Investigation of the relationship between CCNE1, CCNE2 and NE did not show any impact on RFI. To conclude, this study was the first to evaluate these markers at the mRNA level by RT-PCR in a series of primary breast cancer patients, and our results confirmed the impact of high CCNE levels on clinical outcome in systemically untreated and of CCNE1 in tamoxifen-only treated early breast cancer patients.     

Prognostic significance of biologic markers in node-negative breast cancer patients: a prospective study

It is generally thought that future advances in the treatment and cure of breast cancer patients will be made possible through a deeper understanding of tumor biology and an improved capability to define the prognosis of each single patient. This will lead to the formulation of new, more selective, and patient-tailored therapies. It is therefore important, when studying potential prognostic factors, to follow methodologic requirements and guidelines which involve the carrying out of prospective studies as confirmatory steps. Repeatedly or recently investigated prognostic markers (tumor size, menopausal status, ER, PgR, ³H thymidine labeling index, c-erbB-2 and p27 expression) were evaluated on a series of 286 prospectively recruited node negative breast cancer patients who underwent loco-regional treatment alone and were closely followed. The individual and relative prognostic contribution of each variable with respect to other factors, as well as their ability to identify node negative patients at risk, were assessed by univariate and multivariate analysis. At a five-year follow-up, only tumor size (p=0.021) and TLI (p=0.016) individually proved to be significant prognostic indicators of relapse-free survival. Conversely, p27 expression was not related to RFS and c-erbB-2 expression appeared to have only a short-term effect on patient prognosis. TLI and tumor size, tested in multivariate analysis along with ER and menopausal status, maintained their independent prognostic relevance. The study, performed on a large series of node-negative patients given loco-regional treatment alone, for the first time prospectively recruited, showed the prognostic relevance of TLI and its independence from other clinico-pathologic and biologic factors over a five-year period.     

Free circulating mRNA in plasma from breast cancer patients and clinical outcome

We studied by real-time PCR cyclin D1 and thymidylate synthase (TS) mRNA in plasma as possible markers of clinical outcome in breast cancer. We observed poor outcome in patients with presence of cyclin D1 mRNA in good-prognosis groups, such as negative vascular invasion. Presence of both markers was associated with non-response to treatment after relapse. In patients treated with tamoxifen, a trend to significant relation between poor outcome and cyclin D1 mRNA was found. Cyclin D1 mRNA in plasma could identify patients with poor overall survival in good-prognosis groups and patients non-responsive to tamoxifen.     

Induction of apoptosis by tamoxifen and ICI 182780 in primary breast cancer

Hormonal breast cancer therapies have traditionally been considered cytostatic, but recent pre-clinical data suggest that anti-oestrogens can induce apoptosis. The aim of this study was to assess whether tamoxifen (TAM) and ICI 182780 (ICI) could induce apoptosis in human breast cancer, and whether this was related to oestrogen receptor status. We measured apoptosis in primary breast cancer patients before and after pre-surgical treatment with 20 mg/day TAM (study 1) or 6 or 18 mg/day ICI (study 2). In each study there was a randomised non-treatment (NT) control group. TAM significantly increased apoptotic index (AI) in ER⁺ but not in ER⁻ tumours. There was a significant increase in AI following treatment with ICI. Insufficient pairs of samples were available to determine whether this change was confined to ER⁺ tumours, but in a cross-sectional analysis AI was significantly higher in excision biopsies for ICI-treated than NT patients for ER⁺ but not ER⁻ tumours. Our results provide clinical evidence that apoptosis may be induced in ER⁺ primary breast cancer by both non-steroidal and steroidal anti-oestrogens. Int. J. Cancer 72:608–613, 1997. © 1997 Wiley-Liss, Inc.