89080 Clin Pathol 1995;48:890-895 Steroid hormone receptors and their clinical significance in cancer J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from

R I Nicholson, R A McClelland, J M W Gee

Introduction (aE, ,B and y) and so called "orphan" receptors,'0 Although all cells within the body are exposed which do not have recognised physiological to steroid hormones, their physiological actions ligands as yet (including HNF-4,'5 COUP,'6 are limited to those tissues which contain in- Rev-erb A-alpha,'7 Nur77,'8 NGFI-B,'9 TR3,2. tracellular binding proteins, termed receptors. retinoid X receptor,2' P-450 inducers," These proteins specifically bind the individual and the DHR3/MHR3 proteins22), show classes of steroid hormones with high affinity marked conservation of structure and contain and transmit the steroid signal to sensitive genes various functional domains, including those for located throughout the chromatin. The type of ligand and DNA binding.2324 In each instance response subsequently induced is dependent the steroid receptors act as ligand inducible on both the developmental stage and differ- nuclear transcription factors, with interactions entiation status of the target tissue, but may between activated receptors and hormone re- involve the control of such diverse end points sponse elements (HRE) on the DNA directly as cell proliferation, cell death, secretory ac- modifying gene expression. 1 2125 Although tivity, and cellular mobility. Importantly, the some differences exist with respect to the or- ability of steroid hormones to influence these ganisation and initial cellular localisation of characteristics is retained in many disease the individual classes ofsteroid hormone recep- states, notably in certain cancers originating tors, 2 they all ultimately act to stimulate or in steroid hormone sensitive tissues including repress mRNA production from sensitive carcinomas of the breast,' genital tract,23 gas- genes, leading to changes in protein synthesis trointestinaI tract,4 pancreas,5 lung,6 and also and finally cellular function.23-25 However, it intracranial tumours.7 This has been harnessed has become increasingly evident that steroid therapeutically, with drugs which modify the hormone receptors represent only part of the cellular levels or actions of steroid hormones transcriptional apparatus involved in the being commonly used in the treatment of regulation ofresponsive genes and that they act

breast, endometrial and prostate cancer.89 in concert with other inducers or suppressors of http://jcp.bmj.com/ These tumours frequently retain steroid hor- gene expression. 10 26 Thus, hormonally directed mone receptors and their measurement is cur- genes can show responsiveness to peptide rently used diagnostically in several centres to growth factors. This is exemplified by the oes- identify potential endocrine responsiveness. trogen regulated gene pS2 (pNR2) which has As steroid sensitive cancers are relatively been shown to be sensitive to the growth factors common and as cellular transitions associated transforming growth factor cz (TGFoc), epi-

with the loss of their hormone sensitivity in- dermal growth factor and also insulin-like on September 26, 2021 by guest. Protected copyright. variably worsen the prognosis for a patient, growth factor I.27 A further example is seen considerable effort has been afforded to their with PR, traditionally an oestrogen regulated study. The examination of steroid hormone protein, which can also be activated through receptors is pivotal to our understanding of dopaminergic and adrenergic signalling mech- endocrine sensitivity and therefore the current anisms.28 Such interactions raise interesting article attempts to summarise our present questions concerning the role of cross-talk be- knowledge ofthese proteins specifically in those tween steroid hormone receptors and other areas of cancer biology which we hope are of signalling pathways in the regulation of tran- particular interest to the pathologist. scriptional efficiency, constitutive action of the receptor and acquisition of endocrine re- sistance. A knowledge of such pathways may Molecular aspects of steroid hormone provide previously unrecognised opportunities receptors for tumour therapy.'° Breast Cancer The past decade has seen a remarkable increase Laboratory, in our knowledge of the structure and function Tenovus Cancer ofsteroid hormone receptors. This has occurred Methods of detection Research Centre, University of Wales through the isolation and sequence analysis of The observation that hormone sensitive tissues College of Medicine, recombinant DNA clones for the individual were able to take up and retain radiolabelled Cardiff CF4 4XX receptors. It is now established that the receptor steroids against a concentration gradient of the R I Nicholson proteins from each of the five classes of steroid steroid26 lead to the initial discovery of steroid R A McClelland J M W Gee hormones, notably oestrogens (ER)'0 and hormone receptors. This property was sub- progestins (PR),'0 androgens (AR), gluco- sequently exploited in "test tube" biochemical Correspondence to: 12 J M W Gee. corticoids (GR)," and mineralocorticoids, as assays, notably dextran coated charcoal (DCC) Accepted for publication well as the receptors for thyroid hormone," adsorption ligand binding assays (LBA' 26), 18 January 1995 1,25-dihydroxy vitamin D3 (VDR),'4 retinoids generating information on the concentration of Steroid hormone receptors and their clinical significance in cancer 891 steroid hormone receptors within solubilised Glasgow), providing new workers with vital tissue extracts, together with their binding information regarding steroid receptor QA

affinity constants. Unfortunately, the ease of materials, QA schemes as well as a reference J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from performing these assays and also their sub- directory of expert laboratories currently en- sequent clinical value varies between tumour gaged in such assays. types and surgical procedures used. Thus, while Early immunocytochemical studies using breast and endometrial cancer samples are rel- antibodies directed against ER (for example, atively accessible enabling valuable data to be H222 antibody, available in the ER-ICA kit" readily obtained, it is often essential to remove from Abbott Diagnostics, North Chicago, Il- prostate tumour specimens by transurethral linois, USA) showed that the protein is localised sectioning, a procedure which generates el- exclusively within the epithelial cell nuclei in evated temperatures and consequently is po- suitably fixed cryosections" (that is, frozen tentially damaging to any receptor proteins.26 specimens stored at - 700C, sectioned and Furthermore, the assays are also influenced by fixed for 10 to 15 minutes in 3-7% form- both the cellularity of the cancer and the con- aldehyde/phosphate buffered saline at room tent and associated steroid hormone receptor temperature; post-fixed in methanol (five min- status ofnormal or benign tissue. These factors utes) and acetone (three minutes) at - 200C) may contribute towards falsely negative or of normal and cancerous genital tract, breast, positive receptor results. pituitary, and liver. This observation was at More recently developed methodologies rely odds with the previously held view of this re- on direct antigen recognition rather than ster- ceptor as a cytosolic protein which was sub- oid binding activity, utilising monoclonal sequently translocated to the nucleus following antibodies29 specific for the various steroid re- its activation by oestrogens. It is now believed ceptors in both enzyme immunoassays (for ex- that, with the exception of glucocorticoid re- ample, oestrogen receptor enzyme immuno- ceptors, each class of steroid hormone receptor assay (ER-EIA)"0) and also immunocyto- resides predominantly in the nuclei of steroid chemistry (for example, oestrogen receptor sensitive cells.' immunocytochemical assay (ER-ICA) 2631). Using such antibodies, ER (H222 and 1D5 The value of the latter technique lies in its (Dako, High Wycombe, UK)) and PR may relevance to histological sections and thus the now also be reliably localised in 10% buffered procedure has numerous advantages for the formalin fixed (exposed to formalin for no pathologist. Steroid receptors can be directly longer than 24 hours as prolonged fixation visualised by a colour reaction within individual results both in decreased receptor immuno- cell types. This is not possible using bio- reactivity and in the appearance ofnon-specific chemical methods, which require that tissues cytoplasmic staining), paraffin wax embedded should be homogenised before being assayed, material following the re-exposure of masked with an associated loss ofinformation regarding antigenic determinants by controlled protease

heterogeneity ofreceptor expression in normal, digestion of the tissue sections'6 or microwave http://jcp.bmj.com/ benign and malignant components of the treatment.'7 These procedures maintain ex- tumour.26 Immunohistochemical assays can cellent tissue preservation, and thus accurate also be applied to biopsied or cytological ma- receptor determinations can be made in the terial' and thus have the ability to generate invasive component of archival tumours, as accurate results on low cellular tumours. Fur- well as within small, early cancers which are thermore, the technique allows several different usually processed entirely as formalin fixed, proteins to be investigated on the multiple paraffin wax embedded material. on September 26, 2021 by guest. Protected copyright. sections obtained from each tissue sample, The cloning of the steroid receptor genes readily permitting the construction of a larger and the generation ofspecific cDNA and oligo- biological profile of the tumour. Scoring of nucleotide probes has allowed northern hy- nuclear heterogeneity after immunocyto- bridisation analysis to be applied to tumour chemical staining is possible either manually extracts. However, this procedure, while semi- or using an image analyzer, both methodologies quantifiable, is subject to many of the prob- correlating with each other when carried out lems described for steroid receptor biochemical by experienced workers.32 Manual assessment assays which utilise tumour lysates. For- involves the construction of a H score" which tunately, several groups have begun to use in takes into account both the percentage pos- situ hybridisation technologies to study hor- itivity and staining intensity, while image anal- mone receptor mRNA levels in histological yzers can be used to assess the percentage sections. Indeed, both isotopic and non-iso- of nuclear area immunopositivity."2 In breast topic (notably digoxigenin) probe labelling pro- cancers the use of cut off values32 aids the cedures can now be used successfully to study examination of relations between the degree the cellular expression of steroid receptor of ER/PR immunostaining and response to mRNA within frozen and paraffin wax em- endocrine therapies.'4 Intra- and inter- bedded tumour material.'839 Although in situ laboratory studies35 monitoring immuno- hybridisation is often less amenable to routine cytochemical performance and also external use than immunohistochemistry, a recent quality assurance (QA) schemes for steroid study'8 has illustrated its potential value in hormone receptor immunocytochemistry are understanding defects in the steroid receptor finally emerging. The UK Steroid Receptor transcription/translation pathway. The ex- Quality Assessment Group has recently been quisite sensitivity ofthis technique has revealed established, spear-headed by Dr R Leake (De- that more mRNA positive results can be re- partment of Biochemistry, University of corded for ER than for the resultant protein,38 892 Nicholson, McClelland, Gee

implying significant post-transcriptional re- characteristics, including a well differentiated gulation of ER mRNA, assuming that any neg- histology, low nuclear grade, low cellular pro-

ativity recorded for the receptor protein is liferation, prominent elastosis, slight or absent J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from genuine and not merely the result of poor assay lymphocytic infiltration, and minimal tumour sensitivity. necrosis.'26 For the first five to 10 years after primary surgery, patients with ER rich tumours have a more favourable prognosis that those Steroid hormone receptor expression in with receptor negative disease.2650 Un- the breast fortunately, however, with a longer follow up Steroid hormones, notably oestrogens, are of time and in the absence of adjuvant endocrine central importance in growth regulation of the treatment, the disease free intervals converge.' breast.40 This requirement is often maintained In adjuvant endocrine trials greatest benefit is in the various disease states, including neo- predictably observed in those women with ER/ plasia. Receptors for the steroid hormones PR rich cancers.'26 51 oestrogen and progesterone can thus be In the UK the use of these assays to tailor demonstrated immunocytochemically in the therapy to the tumour characteristics of in- normal mammary luminal epithelium, but dividual patients is only sparingly applied and staining is usually sparse (7% of cells) and a more pragmatic approach is frequently un- largely confined to the lobular elements in the dertaken which uses the relatively non-toxic premenopausal breast."'4 This is paradoxical antioestrogenic drug tamoxifen to assess en- as the mammary parenchyma is usually re- docrine responsiveness clinically.42 This ap- garded as exquisitely steroid hormone sensitive, proach has arisen primarily from inaccuracies suggesting that such ER negativity should per- observed in the prediction of clinical response haps be viewed with caution. It may merely by steroid receptor assessment.52 An im- represent the limits of detection of the assay munohistochemical approach to steroid hor- used or may indicate cellular quiescence.4'42 mone receptor measurements, however, ER concentrations in the breast are suppressed facilitates the construction of a broader bio- at ovulation when there is a surge of oestrogens logical view of the tumour and demonstrates in premenopausal women so that the ER con- that further elements other than steroid re- centrations are lowest during the luteal phase.' ceptors can influence and correlate closely with This sharply contrasts with PR, which is under clinical response to endocrine treatments. regulation by both oestrogen and progesterone Thus, the increased expression of various pep- and is relatively constant throughout the tide growth factors (notably TGFc(53) and their cycle.4344 receptors (EGFR5455), proto-oncogene prod- Steroid hormone receptors are largely main- ucts (c-erbB254) and also markers indicative of tained in benign breast conditions, ''45 while cell proliferative activity (Ki6754) have been elevated expression is often associated with associated with loss of endocrine sensitivity40

breast neoplasia.'26 Heterogeneity of ex- despite positivity for steroid receptors often http://jcp.bmj.com/ pression is marked both between patients and being maintained. within an individual tumour.'26 Approximately It is also possible that further anomalies could 70% of primary breast cancers are ER positive arise in the prediction of clinical response to while 50% are PR positive, and the proportion endocrine therapy using steroid receptor pro- of ER positive tumours increases with age.' 42 files if these receptors were occasionally non- ER positive tumours are more likely to spread functional. An interesting approach to the iden-

to bone, while ER negative tumours often tification of functionality of steroid receptors on September 26, 2021 by guest. Protected copyright. metastasise to the liver, brain and regional has been to measure their gene products. It is lymph nodes.' 46 The measurement of ER and fair to say, however, that the identification of PR concentrations in breast cancer specimens target genes involved in the hormonal control is of therapeutic value, providing a means of of cell behaviour has lagged behind the analysis preselecting patients for various endocrine of the structure and function of these receptors treatments (primarily antioestrogens), with as transcription factors. Most progress has been approximately 75% of advanced breast cancer made with oestrogen regulated genes, with sev- patients with tumours positive for ER and PR eral groups reporting successful cloning strat- achieving favourable clinical remission.'2647 egies. PR52, pS256, pLIV1,57 and cathepsin D58 This compares with only 5-10% of patients have been described as oestrogen regulated with steroid receptor negative disease showing proteins, with all but cathepsin D showing an objective response.26 Tumours containing enrichment within ER positive disease.565759 higher steroid receptor concentrations are more Where studied, the expression of oestrogen likely to respond than those with reduced regulated gene products in clinical breast can- concentrations. 26 PR has been reported to be cer, although often associated with endocrine an independent predictive factor for response responsiveness and invariably also having prog- to the antioestrogen tamoxifen48 and the pre- nostic value,56 does not add substantially to dictability of response is reported to be in- the information that may be gained from ER creased to 90% by measuring in vivo induction measurements alone. Where the expression of of this receptor resultant from the partial ag- several oestrogen regulated genes has been onistic activity shown by tamoxifen acting via studied within the breast cancer population, it an intact ER mechanism.49 Such receptor as- is evident that they are not always co-expressed says also provide prognostically useful in- in ER positive disease. Their differential ex- formation. The presence of ER has been pression may reflect differing biological be- correlated with a series offavourable prognostic haviour patterns of the tumours, with-for Steroid hormone receptors and their clinical significance in cancer 893 example, ER/pLIVl dual positivity significantly gesterone are low, and subsequently falls under correlating with lymph node involvement in the inhibition of rising progesterone during

contrast to ER/pS2 positivity.60 the secretory phase. PR is induced during the J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from Potentially, the control of such activities may proliferative phase by elevated oestrogen con- reside in variations in the promoters of in- centrations. dividual genes or in the sensitivity of the pro- As in the breast, steroid hormone receptors moters to mutations in the ER. Possibly the are maintained in the female reproductive tract most important value of polymerase chain re- during the process of neoplasia.2 Similarly, action (PCR) amplification technology in there is a loss of these receptors in the genital steroid receptor determination is the highly tumour epithelial cells with diminished sensitive detection of rare defects in the steroid tumour differentiation and advancement of receptor genes and their mRNAs.61 An ex- malignancy.26667 Thus, in the endometrium, panding number of ER mutations have now PR (and to a lesser extent ER) concentrations been documented, including those which can are inversely related to tumour grade and stage, lead to either a loss of either the hormone or while they are usually diminished in associated DNA binding domain. Several mutations have metastases.2266667 been successfully identified, albeit at a low Although not of established value as yet, frequency, in human breast cancers in vivo.6263 steroid receptor analyses look promising for the These frequently occur through frame shift/ stratification of endometrial cancer patients for termination mutations in the ER gene, gen- endocrine therapy (primarily progestational erating what has been appropriately described agents26). Thus, PR is proving to be a par- by the group from Texas as "outlaw re- ticularly valuable index ofresponse to progestin ceptors".62 The functional significance of these therapy in such tumours. There is an overall mutants has been demonstrated by Fuqua et response rate of 89% for PR positive metastatic a164 who showed that ER lacking exon 5, en- endometrial adenocarcinomas compared with coding the hormone binding domain, con- only 17% for receptor negative cancers.26 Un- stitutively activated transcription of a normally fortunately, the therapeutic usefulness of ER oestrogen dependent gene construct in yeast is thought to be more limited.226 Indeed, in cells. These observations show that a mutant ovarian cancers the concentrations of ER re- ER could potentially dramatically affect oes- ported are on average lower than those recorded trogen responsiveness of a cancer by acting as in breast cancers,2 and this may explain the a constitutive activator of gene expression even overall poor and often conflicting objective re- in the absence of oestrogens,62 and thus such sponse rate of ovarian cancer to antioestrogen mutations could potentially be ofsome import- therapy. ance in controlling breast cancer growth ob- The assessment of steroid receptors is also served following the acquisition of hormone/ potentially of important prognostic value in antihormone resistance.63 Similarly, mutations female genital cancers.2 This is most apparent

in the DNA binding domain could alter the for endometrial cancer, where PR positivity is http://jcp.bmj.com/ DNA specificity of the receptor leading to pro- associated with an increased disease free in- miscuous activation of genes not normally re- terval and improved survival.68 The usefulness sponsive to oestrogens, or result in the loss of ER assessment in genital cancers is again of DNA binding. Most striking is the recent more controversial,2 266 although dual as- observation showing that ER variants can in- sessment of a high tumour ER and PR content terfere with the function of the wild-type is, however, usually an indicator of longer

receptor,62 64 either by forming inactive survival.7071 on September 26, 2021 by guest. Protected copyright. heterodimer complexes or by binding to the oestrogen response element (ERE) in a re- dundant conformation. These observations suggest that mutations can act in a rather in- Steroid hormone receptor expression in sidious way as dominant negative oncogenes.62 the male genital tract It is known that as with the female, the male reproductive system is also under strict steroid Steroid hormone receptor expression in hormone control in both normal and disease the female genital tract states. Thus, the prostate gland contains re- The female genital system, like the breast, is a ceptors for androgens (AR) in its secretory prime target organ for the steroid hormones epithelia.72 In contrast to the breast, however, progesterone and oestrogen. Steroid receptors there have been difficulties in the development are present throughout the reproductive tract of accurate biochemical and immunocyto- in the ovaries, endometrium, cervix, and vulva.2 chemical assays for prostatic AR,26 although Steroid hormone dependence is particularly research in this field is now expanding rapidly marked in the endometrium, which in the pre- as antibodies to the receptor become more menopausal woman contains glandular epi- readily available.7' The steroid receptors ER thelial cells (and to a lesser extent stroma) and PR have also been identified in the uro- with both ER and PR.26' In common with the thelium of the central prostatic ducts and the normal breast, receptors in the endometrium prostatic urethra, in the periglandular fibro- vary noticeably in concentration with the fluc- cytes, smooth muscle cells, interglandular tuations that occur in their respective ligands stromal cells, and also associated with the throughout the menstrual cycle.265 Thus, ER stroma surrounding the ejaculatory ducts and peaks in the proliferative phase of the cycle seminal vesicles, although the secretory epi- when the circulating concentrations of pro- thelia are thought to be ER and PR negative.74 894 Nicholson, McClelland, Gee All three of these steroid hormone receptors3 ceptors would ultimately provide data that have been shown to be more apparent in benign might be used to aid in selecting primary or

prostatic hyperplasia (BPH), while AR con- adjuvant therapy, evaluating new therapies, es- J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from centrations are usually reduced in carcinomas timating prognosis, and assessing outcome.80 compared with those seen in BPH,73 with an inverse correlation existing between receptor 1 Rayter Z. Steroid receptors in breast cancer. BrJ Surg 1991; and 78:528-35. expression grade.5 While the ER and the 2 Soutter WP, Leake RE. Steroid hormone receptors in gynae- PR status are of some predictive value in many cological cancers. In: Bonnar J, ed. Recent advances in obstetrics and gynecology. Vol 15. New York: Churchill neoplasms of the female reproductive system Livingstone, 1987:175-94. and the breast, it is the AR that is of primary 3 Mobbs BG, Liu Y Immunohistochemical localization of progesterone receptor in benign and malignant human importance in prostate carcinoma. Indeed, this prostate. Prostate 1990;16:245-51. forms the rationale for treatment of the disease 4 Wu CW, Tsay SH, Chang TJ, Chang HM, Hsieh MC, with and AR tu- Lui WY, et al. Clinicopathologic comparisons between antiandrogenic drugs positive estrogen receptor-positive and -negative gastric cancers. J mours usually have a better response to such Surg Oncol 1992;51:231-5. Recent data that the 5 Ladanyi M, Mulay S, Arseneau J, Bettez P. Estrogen and therapies.26 suggest scoring progesterone receptor determination in the papillary cystic of AR immunostaining intensity rather than neoplasm of the pancreas. With immunohistochemical and ultrastructural observations. Cancer 1987;60: 1604-11. percentage positivity" may possibly dis- 6 Colley MH, Geppert E, Franklin WA. Immuno- criminate good responders. histochemical detection of steroid receptors in a case of As seen with ER in the a pulmonary lymphangioleiomyomatosis. Am J7 Surg Pathol breast, number of 1989;13:803-7. AR mutations have been detected in clinical 7 Whittle IR, Hawkins RA, Miller JD. Sex hormone receptors cancer and also in intracranial tumours and normal brain. EurJ Surg Oncol prostate specimens7778 within 1987;13:303-7. the LNCaP prostate carcinoma cell line.79 Im- 8 Nicholson RI, Walker KJ, Davies P. Hormone agonists and the AR mutation in LNCaP antagonists in the treatment of hormone sensitive breast portantly, cells and prostate cancer. Cancer Surv 1986;5:463-86. greatly influences the specificity of hormone 9 Nicholson RI, Manning DL, Gee JMW. New antihormonal in that approaches to breast cancer therapy. Drugs of Today 1993; binding, resulting receptors have an 29:363-72. increased binding affinity for both oestradiol 10 Wakeling AE. Steroid antagonists as nuclear receptor block- and with the ers. Cancer Surv 1992;14:71-85. progesterone, antiandrogen 11 Gustafsson JA, Dahlman-Wright K, Stromstedt PE, Wright hydroxyflutamide acting as an agonist.79 In- T, Carlstedt-Duke J. 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Orphan antagonists.79 receptor HNF-4 and bZip protein C/EBP alpha bind to ical material AR mutations are thought to occur overlapping regions ofthe apolipoprotein B gene promoter as late events and are thus and synergistically activate transcription. Jf Biol Chem quite prevalent in 1993;268:16831-8. advanced disease where they have been linked 16 Tran P, Zhang XK, Salbert G, Hermann T, Lehmann JM, http://jcp.bmj.com/ with the of Pfahl M. COUP orphan receptors are negative regulators acquisition androgen insensitivity.77 of retinoic acid response pathways. Mol Cell Biol 1992;12: 4666-76. 17 Chawla A, Lazar MA. Induction of Rev-ErbA alpha, an orphan receptor encoded on the opposite strand of the Conclusions alpha-thyroid hormone receptor gene, during adipocyte It is that steroid hormone differentiation. J Biol Chem 1993;268:16265-9. apparent receptors 18 Davis IJ, Hazel TG, Chen RH, Blenis J, Lau LF. 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New concepts in steroid hormone importance in genital cancers in both women action: transcription factors, proto-oncogenes, and the and men It is cascade model for steroid regulation of gene expression. (PR) (AR). becoming increasingly Crit Rev Eukaryot Gene Expr 1992;2:19-63. obvious that a wider knowledge of the mol- 26 Merkel DA, Osborne CK. Use of steroid receptor assays in ecular and cellular of these cancer management. Rev Endocrinol Rel Cancer 1988;30: biology receptors 5-12. in their functional and non-functional forms 27 Chalbos D, Philips A, Galtier F, Rochefort H. Synthetic is an essential to the full under- antiestrogens modulate induction of pS2 and cathepsin- prerequisite D messenger ribonucleic acid by growth factors and ad- standing of pathways of steroid hormone enosine 3',5'-monophosphate in MCF-7 cells. 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assay measurement of estrogen receptors in human breast 54 Nicholson RI, McClelland RA, Finlay P, Eaton CL, Gullick cancer: a European multicentre study. Cancer Res 1986; WJ, Dixon AR, et al. Relationship between EGF-R, c- 46:4233-6. erbB-2 protein expression and Ki-67 immunostaining in 31 Walker KJ, Bouzubar N, Robertson J, Ellis IO, Elston CW, breast cancer and hormone insensitivity. Eur Jf Cancer Blamey RW, et al. Immunocytochemical localization of 1993;29:1018-23. J Clin Pathol: first published as 10.1136/jcp.48.10.890 on 1 October 1995. Downloaded from estrogen receptor in human breast tissue. Cancer Res 1988; 55 Nicholson RI, McClelland RA, Gee JMW, Manning DL, 48:6517-22. Cannon P, Robertson JFR, et al. Epidermal growth factor 32 McClelland RA, Finlay P, Walker KJ, Nicholson D, Rob- receptor expression in breast cancer: association with re- ertson JF, Blamey RW, et al. Automated quantitation sponse to endocrine therapy. 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