In order to resolve the question of which ovarian steroid stimulates normal human mammary epithelial cell proliferation, we have implanted pieces of normal human breast tissue subcutaneously into athymic nude mice. These mice were then treated with slow-release pellets containing estradiol (E2) or progesterone (P) such that serum levels of E2 and P were increased to those seen in normal women. The proliferative activity of the tissue implants was assessed by uptake of tritiated thymidine and steroid receptor expression was measured immunocytochemically. Insertion of a 2 mg E2 pellet 14 days after tissue implantation increased the thymidine labeling index (TLI) from a median of 0.4% (n = 34) to a median of 2.1% after 7 days (n = 43; P < 0.001 by Mann Whitney U test). In contrast, treatment with a P pellet (4 mg) had no effect upon the TLI whereas P (4 mg) in combination with E2 (2 mg) had no effect over and above that of E2 alone. There was a significant correlation between the increase in TLI and either the E2 content of the pellets (P < 0.001 by linear regression) or the serum E2 levels achieved (P < 0.001). Expression of the P receptor was increased 15- to 20-fold by E2 treatment. We conclude that E2 is sufficient to stimulate human breast epithelial cell proliferation at physiologically relevant concentrations and that P does not affect proliferation either alone or after E2 priming.

Summary This study aimed to investigate the effect of tamoxifen on breast tumour levels of oestrogen and progesterone receptor (ER and PR) and proliferation as defined by the Ki67 antibody. A group of primary breast cancer patients was randomised to receive either tamoxifen (n = 59) or placebo (n = 44) treatment in the interval between clinic and surgery (median 21 days). Frozen (McGuire & Clark, 1983 .Tumour proliferative activity is also related to the prognosis of breast cancer and a negative correlation has been observed between presence of ER and/or PR and the growth fraction as measured by a variety of methods. These methods include measurement of thymidine incorporation (Meyer et al., 1977;, estimation of the S phase fraction by DNA flow cytometry (Olszewski et al., 1981;Raber et al., 1982) and immunohistological staining using the mouse monoclonal antibody Ki67 (McGurrin et al., 1987) which recognises a proliferation-associated nuclear antigen present in the late Gl, S, G2 and M, but not in the Go, phases of the cell cycle (Gerdes et al., 1983;1984). As the endpoint of antioestrogen action is inhibition of tumour cell proliferation, a more functional approach to predicting response would be the measurement of proliferative activity before and during administration of a short course of tamoxifen.In the present study, we have used immunohistological methods to study breast tumour tissue before and after treatment with tamoxifen or a placebo in order to correlate and compare levels of expression of ER and PR with the proliferating cell-associated antigen defined by the monoclonal antibody Ki67, and with other histological data. The aim of the study was to elucidate the relationships between these factors before and after tamoxifen treatment in order to achieve a better prediction of response to endocrine treatment in individual patients. Patients and methods Patients

The aims of this study in 227 premenopausal women were (a) to determine the mitotic index (MI), the thymidine labelling index (LI), and the apoptotic index (AI) within the epithelial cells of histologically 'normal' human breast biopsy material removed away from the site of either a fibroadenoma or a carcinoma; and (b) to relate differences in the kinetic indices of the 'normal' epithelium to the pathology in the same breast diagnosed as fibroadenoma alone (125 patients), fibroadenoma with accompanying mild fibrocystic change (79 patients), or carcinoma (23 patients). Ratios of the average indices (AI/MI, AI/LI, MI/LI) were also calculated to minimize uncertainties related to the total cell population counted, the denominator in the LI, MI, and AI. All indices and ratios of indices were corrected for age, averaged over the cycle, and expressed as log-transformed values for analysis. Significant (P less than 0.001) reductions in AI and in apoptosis relative to mitosis (reduced AI/MI) were found in 'normal' epithelium from breasts having fibrocystic change (AI = 0.17 +/- 0.02; AI/MI = 1.01 +/- 0.18) and carcinoma (AI = 0.19 +/- 0.04; AI/MI = 0.88 +/- 0.29), compared with breast with fibroadenoma alone (AI = 0.27 +/- 0.03; AI/MI = 1.29 +/- 0.39). In the absence of significant differences in MI and LI between the 'normal' tissue groups, this finding raises the possibility that reduced epithelial cell apoptosis might be causally associated with the development of fibrocystic change and with an increased risk of development of carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy.

The correlation with vorozole of Ki67 with volume and clinical response supports this as an intermediate marker. The nonsignificant effects on bone and lipid metabolism by the aromatase inhibitor may be important to consider for adjuvant and potential prevention strategies.