Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age-and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P ¼ 0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P ¼ 0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (Po0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (Po0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer.

S_ary The use of chemotherapy as a form of primary treatment for breast cancer is increasing and, as a result, more resection specimens contain tumours which have been exposed to cytotoxic drugs. We have studied the effects of chemotherapy on the tumour morphology and various biological features of breast carcinoma in a group of 35 patients. These were a group who responded to treatment in a clinical study of the use of primary chemotherapy designed to reduce tumour bulk prior to surgery. Characteristic morphological changes, temporally related to the administration of cytotoxic agents, are seen. The malignant cells become enlarged with vacuolated cytoplasm and vesicular nuclei containing prominent nucleoli; occasionally the nuclei were angular and hyperchromatic. These features are interpreted as degenerative in nature. In 15 cases sufficient material was present in the pretreatment biopsies to compare the grade of the tumours before and after chemotherapy: changes were found in six tumours. Cytotoxic drugs do not induce a consistent pattern of change in the proliferation and apoptotic indices of individual tumours, but there is a tendency to reduce proliferative activity over all the tumours as a group. It was also found that chemotherapy is capable of modifying the expression of the oncoproteins c-erbB-2 and p53 in a minority of cases of breast cancer, usually resulting in an acquisition of immunoreactive oncoprotein. It is important to be aware of these effects when studying breast carcinomas removed after chemotherapy.

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Materials and methodsSurgical specimens of breast tissue containing carcinoma were received fresh in the laboratory 15 min after removal from the patient. After standard pathological assessment and sampling for routine histology and hormone receptor assay, and if sufficient tumour was available, multiple slices, on average measuring 1 cm2, were taken and placed in a variety of fixatives (Table I). Cytosols were prepared from frozen tissue after homogenisation using a microdismembrator (Braun, Melsungen, Germany) according to the instructions in the Abbott enzyme immmunoassay kit (Abbott Laboratories, Maidenhead, Berkshire, UK). The protein concentration of the cytosol extracts was determined by a dye-binding assay (Bradford, 1976 ethanol (six changes), clearing in xylene (three changes) and paraffin wax impregnation (four changes), the entire process taking 11 h. The tissues were then embedded and sectioned for routine histology and immunohistochemistry. Subsequent to the fixation study tissue from 95 consecutive cases of infiltrating mammary carcinoma and 20 cases of pure in situ or predominantly in situ carcinoma with minimal invasion (<2 mm maximum diameter) was fixed in phenol formol saline for evaluation of the different staining patterns seen with the p53 antibody.Tumour typing and grading Infiltrating tumour types were classified according to a modification of the WHO (1982) system. Tumour grading was carried out on all infiltrating ductal carcinomas and tumours of special type. Infiltrating lobular carcinomas were not graded. The histological grading was based on the method of Bloom and Richardson as modified by Elston and Ellis (1991). Cases of in situ ductal carcinoma were classified as comedo when composed of large pleomorphic cells, usually with areas of extensive necrosis, or non-comedo when composed of small or intermediate-sized cells with minimal or no necrosis, usually with a cribriform or micropapillary pattern (Bobrow et al., 1993). ImmunohistochemistrySections of 3 ym were cut and floated onto glass slides coated with poly-L-lysine and allowed to dry overnight. Heat was not used to stick the tissue sections on to the glass as this can

S_q In this single-centre study of 881 patients, S-phase fraction (SPF) was shown to be a sgnifiant prognostic marker in terms of ovrll suvival (OS), rdapse-free survival (RFS) A further stimulus to publish our data came from two recent reports which failed to find a prognostic role for SPF in breast cancer (Stanton et al., 1992; Silvestini et al., 1993