Measurement of oestrogen receptor mRNA levels in human breast tumours

Abstract

Summary A sensitive single-stranded hybridisation probe for the oestrogen receptor mRNA was synthesised using T3 polymerase from oestrogen receptor cDNA cloned in the Bluescript vector. This probe was used to measure oestrogen receptor mRNA in total RNA extracted from breast tumours. Oestrogen receptor mRNA was detected in 41 of 47 (87%) tumours whereas cytosolic oestrogen receptor protein was detected in only 18 out of 39 (46%). There was a significant correlation between the levels of the oestrogen receptor, as measured by 3H-oestradiol binding, and the oestrogen receptor mRNA. Breast cancer has long been known to be oestrogen responsive and the expression of the oestrogen receptor in a proportion of tumours is well recognised (McGuire et al., 1975). Different studies find the proportion of breast tumours expressing oestrogen receptor to be variable, ranging from 60% to 80% (Hawkins, 1985). Most studies indicate that patients with oestrogen receptor-positive tumours have better disease-free, post-relapse and overall survival, compared to those with oestrogen-receptor negative tumours (reviewed by Hawkins, 1985). A higher proportion of oestrogen receptor-positive tumours respond to antioestrogen therapy: paradoxically, a small number of oestrogen receptor-negative tumours also respond (Desombre et al., 1978).A variety of technique' have been used to determine oestrogen receptor protein levels in breast tumours, including tritiated hexoestrol binding in vivo (Folca, 1961), sedimentation assays (Desombre et al., 1978), dextran-coated charcoal assays (Korenman & Dukes, 1970), radio-immunoassay (Thorpe, 1987 logically. RNA was also prepared from combined endometrium and myometrium from the uterus of a premenopausal woman. Patient age ranged from 24 to 95 years: 14 were less than 50 years old and considered to be premenopausal. RNA extractionRNA was extracted from the 47 tumour biopsies and the uterus using a modification of the method of Auffrey & Rougeon (1980). Tumours frozen in liquid nitrogen were reduced to a powder using a Braun mikro dismembrator. Tumour powder was transferred to a Corex centrifuge tube, suspended in 5ml of 3M LiCl, 6M urea, 0.5% SDS and 50mM sodium acetate (pH5.6) and the DNA was sheared with an Ultraturrax homogeniser. RNA was precipitated at 4°C for 18h and recovered by centrifugation at 10,OOOg for 20 min. The supernatant was discarded and the resultant pellet was washed in 3 ml of 3 M LiCl, 6 M urea and 50mM sodium acetate and recentrifuged at 10,000 g for 0 min. The pellet was then resuspended in 1.125ml lOmM Tris buffer pH 8.0, 0.2% SDS, at room temperature, Tris buffer pH 8.0 and EDTA were added to 0.1 M and 5mM, respectively in 1.25ml total volume and then extracted with phenol:chloroform (50:50) and chloroform:isoamyl alcohol (24:1). The organic phases were back extracted with 1.25 ml 100mM Tris pH9. Following the addition of 6.25ml 100% ethanol and 50 pl 5 M NaCl, RNA was precipitated at -20°C. After centrifugation, the pellet was washed in 70% cold ethanol, recentrifuged, dried...

pNR-2/pS2 immunohistochemical staining in breast cancer: correlation with prognostic factors and endocrine response

“…The apparent expression of pNR-2 by oestrogen receptor 'negative' tumours is probably due to the relative insensitivity of cytosolic ligand binding assays and the somewhat arbitrary nature of rigid cut-off points for oestrogen receptor positivity rather than constitutive nonoestrogen regulated pNR-2 expression in breast cancer cells. Using highly sensitive Northern transfer techniques, we have detected oestrogen receptor mRNA expression in a much larger proportion of tumours than considered positive by ligand binding assay (Henry et al, 1988 (Rio et al, 1987;Henry et al, 1990).…”

“…response to hormonal therapy on relapse was disease which did not progress for at least 6 months. Samples of fresh tumour tissue were taken at the time of primary surgery for oestrogen receptor assay by dextran coated charcoal ligand binding assay as described previously (Henry et al, 1988). Statistical analysis was performed using the programme CSS (Statsoft, USA): survival was analysed using the Log-Rank test (Peto et al, 1977 Figure Id) There was no significant association between menopausal status and pNR-2 expression when the median level of pNR-2 expression was chosen for defining pNR-2 positive tumours.…”

Identification of ERF-1 as a member of the AP2 transcription factor family

“…ER-positive tumors are more common in postmenopausal women and many of these tumors over-express ER as compared with ER levels in normal mammary epithelium (4). Tumors with abundant ER mRNA levels tend to be well-differentiated tumors which are PRpositive and have a lower nuclear grade as compared with tumors with low or absent ER mRNA (5,6). It has been assumed that the phenotypic differences between hormonally responsive and unresponsive tumors is due to expression of genes regulated by ER (7).…”

PS2 mRNA expression adds prognostic information to node status for 6-year survival in breast cancer

“…Furthermore, the data presented here suggest that a combination of axillary node status and pS2 expression measured at the time of diagnosis can define patients at high and low risk of death for medium-term follow-up. As pS2 expression is a good indicator of endocrine responsiveness both in the primary cancer and on relapse (Henry et al, 1988;Ramm et al, 1988;Skilton et al, 1989;Schwartz et al, 1991;Westley and May, 1991;Coradini et al, 1996;Soubeyran et al, 1996), the detection of pS2 expression in the primary cancer, whether at the mRNA or the protein level, may be useful both as prognostic information and as a guide to therapeutic intervention. In this study, patients with tumours that were oestrogen receptor moderate/rich at diagnosis (therefore including the tumours that express pS2) were treated with tamoxifen.…”

“…Conversely, pS2-negative patients have a significantly shorter relapse-free survival (Gion et al, 1993;Foekens et al, 1994;Speiser et al, 1994;Schmidt et al, 1996) and overall survival (Gion et al, 1993;Speiser et al, 1994). pS2 also predicts a subsequent response to hormone manipulation as first-line therapy and on disease relapse (Henry et al, 1988(Henry et al, , 1990(Henry et al, , 1991Schwartz et al 1991;Soubeyran et al, 1996). Thus, the detection of pS2 expression in breast cancers may define a subset of cancers with functional oestrogen receptors and hence patients with a good prognosis who are more likely to respond to endocrine manipulation.…”