Antioestrogen treatment by tamoxifen is a well-established adjuvant therapy for oestrogen receptor-alpha (ERa) positive breast cancer. Despite ERa expression some tumours do not respond to tamoxifen and we therefore delineated the potential link between the cell cycle regulator and ERa co-factor, cyclin D1, and tamoxifen response in a material of 167 postmenopausal breast cancers arranged in a tissue array. The patients had been randomised to 2 years of tamoxifen treatment or no treatment and the median follow-up time was 18 years. Interestingly in the 55 strongly ERa positive samples with moderate or low cyclin D1 levels, patients responded to tamoxifen treatment whereas the 46 patients with highly ERa positive and cyclin D1 overexpressing tumours did not show any difference in survival between tamoxifen and no treatment. Survival in untreated patients with cyclin D1 high tumours was slightly better than for patients with cyclin D1 low/moderate tumours. However, there was a clearly increased risk of death in the cyclin D1 high group compared to an age-matched control population. Our results suggest that cyclin D1 overexpression predicts for tamoxifen treatment resistance in breast cancer, which is line with recent experimental data using breast cancer cell lines and overexpression systems.

Cyclins D1 and A2 are cell cycle regulators that also have the ability to interact with the estrogen receptor (ER) and consequently interfere with antiestrogen treatment in breast cancer. Experimental data support this concept, but the clinical relevance needs to be further established. In this study, we evaluated cyclin D1 and A2 protein expression by immunohistochemistry and cyclin D1 gene (CCND1) amplification by fluorescence in situ hybridization in 500 primary breast cancers arranged in tissue microarrays. Patients had been randomized to 2 years of adjuvant tamoxifen or no treatment with a median follow-up of 14 years, allowing for subgroup analysis of treatment response defined by cyclin status. We found that both cyclin D1 and A2 protein overexpression was associated with an impaired tamoxifen response, although not significant in multivariate interaction analyses, whereas tamoxifen-treated patients with CCND1-amplified tumors had a substantially increased risk for disease recurrence after tamoxifen treatment in univariate analyses [relative risk (RR), 2.22; 95% confidence interval (95% CI), 0.94-5.26; P = 0.06] in contrast to nonamplified tumors (RR, 0.39; 95% CI, 0.23-0.65; P < 0.0001). Consequently, a highly significant interaction between tamoxifen treatment and CCND1 amplification could be shown regarding both recurrence-free survival (RR, 6.38; 95% CI, 2.29-17.78; P < 0.001) and overall survival (RR, 5.34; 95% CI, 1.84-15.51; P = 0.002), suggesting an agonistic effect of tamoxifen in ERpositive tumors. In node-positive patients, the disparate outcome according to gene amplification status was even more accentuated. In summary, our data implicate that despite a significant correlation to cyclin D1 protein expression, amplification status of the CCND1 gene seems a strong independent predictor of tamoxifen response, and possibly agonism, in premenopausal breast cancer. (Cancer Res 2005; 65(17): 8009-16)

Purpose: Hypoxia in breast cancer is associated with poor prognosis and down-regulation of the estrogen receptor. Carbonic anhydrase IX (CA IX) is a hypoxia-inducible gene that has been associated with poor outcome in many epithelial cancers. Previous studies of CA IX in breast cancer have been carried out on mixed cohorts of premenopausal and postmenopausal patients with locally advanced disease and varying treatment regimens.We examined the potential prognostic and predictive role of CA IX in premenopausal breast cancer patients. Experimental Design: Using tissue microarrays, we analyzed CA IX expression in 400 stage II breast cancers from premenopausal women. The patients had previously participated in a randomized control trial comparing 2 years of tamoxifen to no systemic adjuvant treatment. Median follow-up was 13.9 years. Results: CA IX expression correlated positively with tumor size, grade, hypoxia-inducible factor 1a, Ki-67, cyclin E, and cyclin A2 expression. CA IX expression correlated negatively with cyclin D1, estrogen receptor, and progesterone receptor. CA IX expression was associated with a reduced relapse-free survival (P = 0.032), overall survival (P = 0.022), and breast cancerŝ pecific survival (P = 0.005). Multivariate analysis revealed that CA IX was an independent prognostic marker in untreated patients with one to three positive lymph nodes (hazard ratio, 3.2; 95% confidence interval, 1.15-9.13; P = 0.027). Conclusion: CA IX is marker of poor prognosis in premenopausal breast cancer patients and it is an independent predictor of survival in patients with one to three positive lymph nodes. As all these patients received locoregional radiation therapy, CA IX may be associated with resistance to radiotherapy.

Hypoxia is common in many solid tumours, including breast cancer. Hypoxia triggers the expression of hypoxia inducible factor1a (HIF-1a), and HIF-1a has been associated with an impaired prognosis in breast cancer and down-regulation of the oestrogen receptor (ER), potentially affecting the treatment efficiency of antioestrogens. The role of HIF-1a regarding prognostic and treatment predictive information in breast cancer has not been established and we therefore analyzed HIF-1a using immunohistochemistry in a cohort of 377 premenopausal stage II breast cancers arranged in a tissue microarray. The patients were included in a randomized trial with either 2 years of tamoxifen or no adjuvant treatment. The tamoxifen treatment effect could be studied in subgroups of breast cancer and pure prognostic information could be scrutinized for untreated control patients. HIF-1a was scored as positive in 24% of the tumours and correlated positively to tumour size, Nottingham histological grade (NHG), Ki-67, Her2 and cyclin E expression and negatively to lymph node status, cyclin D1, ER and PR (progesterone receptor) expression. Surprisingly, there was no difference in tamoxifen response for patients with high or low HIF-1a expressing tumours. In lymph node-positive patients as well as NHG 1/2 tumours, high HIF-1a protein expression was significantly associated with an impaired recurrence-free survival (p 5 0.014, 0.018). When analyzing the subgroup of NHG 1/2 tumours, a high HIF-1a expression was the only independent significant prognostic marker in multivariate analysis, including standard prognostic markers, suggesting that HIF-1a might be a useful prognostic marker in this subgroup of breast cancer, with a rather good but diverse prognosis. ' 2005 Wiley-Liss, Inc.