British Journal of Cancer (1996) 73, 5-12
`© 1996 Stockton Press All rights reserved 0007—0920/96 $12.00
`
`Oestrogen receptor: a stable phenotype in breast cancer
`
`J FR Robertson
`
`Senior Lecturer in‘Surgery, City Hospital, Nottingham, NG5 IPB, UK.
`
`Summary Oestrogen receptor (ER) expression in breast cancer is regarded as a phenotype that may change
`during the natural history of the disease or during endocrine therapy. It has been suggested that in up to 70%
`of tumours that show acquired resistance the mechanism may be changed in ER status from positive to
`negative. This paper proposes an alternative hypothesis that ER expression is a stable phenotype in breast
`cancer. The paper reviews the literature on ER expression during the natural history of breast cancer in
`patients and also presents data on the efiect of endocrine therapy on ER expression. If the alternative
`hypothesis is true it has important implications for treatment from chemoprevention to acquired endocrine
`resistance in advanced disease. Equally, if the hypothesis is true, attempts to develop laboratory models of
`endocrine resistance where ER-positive tumours become ER negative need to be re-evaluated.
`
`Keywords: breast cancer; oestrogen receptor; stable phenotype
`
`
`The oestrogen receptor (ER) is a 65 kDa oestrogen-binding
`protein expressed by 46—77% of breast cancers (Walt et al.,
`1976; Knight et al., 1977; Maynard et al., 1978; Brooks et al.,
`1980; Osborne et al., 1980; Croton et al., 1981; Howell et al.,
`1984; Hawkins et al.,1987a; Williams et al., 1987; Clarke and
`McGuire, 1988). It is a generally held view that ER expres-
`sion is not a permanent phenotype in breast cancer cells
`(Allegra et al., 1980; Moolgavakar et al., 1980; Encarnacion
`et al., 1993; Morrow and Jordon, 1993; Nomura et al., 1985;
`Jordan, 1994; Paik et al., 1994). One reason for this view is
`the belief that patients with ER-positive primary breast
`tumours often develop ER‘negative metastases in regional
`lymph nodes or distant sites. This has been interpreted to
`show that ER negativity correlates with more aggressive
`tumour biology and loss of cellular control. A second reason
`is the strong correlation between ER and therapeutic res-
`ponse to primary endocrine therapy (Samaan et al., 1981;
`Howell et al., 1984; Williams et al., 1987), which formed the
`basis for early hypotheses of endocrine sensitivity and resis-
`tance. Up to 60% of ER-positive tumours respond to hor-
`mone
`therapy (e.g. Tamoxifen), while
`for ER-negative
`tumours the figure is around 10% (Allegra et al., 1980;
`Samaan et al., 1981; Williams et al., 1987). Therapeutic
`response to endocrine therapy is not permanent and even-
`tually all such tumours progress. As ER negativity is strongly
`associated with primary resistance to endocrine therapy it is
`generally accepted that when responding tumours subse-
`quently progress that in the majority of tumours this is due
`to loss of ER expression by the tumour (Allegra et al., 1980;
`Moolgavakar et al., 1980; Nomura et al., 1985; Encarnacion
`et al., 1993; Morrow and Jordan, 1993; Jordan, 1994; Paik et
`al., 1994). This paper proposes the alternative hypothesis that
`ER is a stable phenotype in breast cancer cells.
`The discovery of monoclonal antibodies (Kohler and Mils—
`tein, 1975) subsequently led to specific antibodies being
`raised to ER (Green and Jensen, 1982). H222 and H226
`identify different epitopes on the ER,
`the hormone-binding
`and the DNA—binding domain respectively. Neither antibody
`blocks the natural ligand, oestradiol, binding to ER. H222
`forms the basis for two commercially available ER assay kits
`— an enzyme immunoassay (EIA) and an immunocyto-
`chemistry assay (ICA). The ER-EIA measures the concentra-
`tion of ER and,
`like the ligand binding assay (LBA),
`is
`reported in fmol mg‘l cytosol protein (Nicholson et al.,
`1986). The ER-ICA allowed assessment of tumour tissue
`sections (King and Green, 1984; Walker et al., 1988).
`The ER-ICA test revealed that in tumours measured as ER
`positive by ligand-binding assays or EIA not all the tumour
`
`
`Received 18 April 1995; revised 18 July 1995; accepted 28 July 1995
`
`cells expressed ER (McCarty et al., 1986; Walker et al.,
`1988). This led to studies defining the number of ER-positive
`cells that a tumour required to accurately predict therapeutic
`response to endocrine therapy (Walker et al., 1988; Gaskell et
`al., 1989; Nicholson et al., 1991; Robertson et al., 1992). The
`finding of ER-positive and ER-negative cells in most tumours
`appeared to strengthen the belief that endocrine-sensitive
`ER-positive cells, with an inherently better prognosis, would
`eventually change to ER negative cells, both as the disease
`progressed from primary to metastatic disease and also from
`endocrine sensitive to endocrine resistant.
`This concept has major implications, particularly in the
`area of acquired resistance to endocrine therapy when
`tumours initially respond and subsequently progress. ER exp-
`ression has been shown to correlate strongly with primary
`sensitivity or insensitivity of tumours to endocrine therapy.
`Previous hypotheses have tried to fit acquired resistance into
`the same explanation as primary insensitivity,
`i.e. ER
`negativity. The alternative hypothesis that ER is a stable
`phenotype in breast cancer cells is more consistent with both
`the clinical and laboratory data.
`
`Sources of variability in ER measurements
`
`Before ascribing reported changes in ER status to biological
`changes in breast
`tumours the extent of other potential
`influences on ER measurements should be considered. Im-
`proper handling of specimens and warm ischaemic time
`(Newsome et al.,
`1981) both affect ER measurements.
`Tumour heterogeneity is another source of error in ER
`measurements. Previous LBA studies of multiple biopsies of
`the same primary tumour at the same time had shown discor—
`dant results for ER status between 17% and 32% (Kiang and
`Kennedy, 1977; Tilley et al., 1978; Silfversward et al., 1980;
`Straus et al., 1982; Davis et al., 1984). This intra-tumour,
`intersample receptor variation was not time dependent as the
`multiple biopsies were taken from each tumour at the same
`time. Among other factors reported to aflect ER measure-
`ments are tumour cellularity (Hawkins et al., 1977; Davis et
`al., 1984) and tumour necrosis (Masters et al., 1978; Silfvers-
`ward et al., 1980; Euseli et al., 1981).
`Variation in ER assays is a major factor in the interpreta—
`tion of ER results. The potential sources of laboratory
`variability have been well reviewed by Thorpe (1987). Intra-
`laboratory variation in a number of studies using LBA is
`reported to range from 15% to 34% (Hawkins et al., 1975,
`1987b; Taylor et al., 1982; Davis et al., 1984; Bojar, 1986;
`Anderson et al., 1989). Inter-laboratory variation, which is
`usually higher
`than intra-laboratory variation,
`is also a
`significant
`factor, even where laboratories participate in
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`°‘R
`
`Oestrogen receptors in breast cancer
`JFR Robertson
`
`external quality control programmes. Even between large
`series in which differences might be expected to be small and
`in which one cut-ofl‘ value of 5 fmol mg‘l cytosol protein has
`been used to define ER positivity, significantly different rates
`of ER positivity, as low as 51% (Cooke et al., 1979) and as
`high as 76% (Hawkins et al., 1987a), have been reported.
`Even the newer monoclonal antibody-based EIA has high
`CVs.
`lntra—assay and interassay CVs range from 3.4% to
`14.3% (Jordan et al., 1986; Nicholson 6! al., 1986) and 3.7%
`to 16.7% (Bojar, 1986; Jordan et al., 1986; Leclerq et al.,
`1986; Nicholson et
`al.,
`1986)
`respectively. The
`inter-
`laboratory CV for EIA was reported to range between 10%
`and 19% (Bojar, 1986; Leclerq et al., 1986).
`
`ER and the natural history of breast cancer
`
`Many studies have reported no correlation between ER
`status and axillary lymph node status (Maynard et al., 1978;
`Hahnel et al., 1979; Mason et al., 1983; Williams et al., 1987;
`Hawkins 9! al., 1987a). Nevertheless,
`it has been assumed
`that ER-negative cells have more metastatic potential. As a
`corollary,
`it has been believed that metastatic deposits from
`ER-positive primary tumours may be ER negative, reflecting
`the change to a more metastatic phenotype. This view is not
`supported by review of the literature.
`Hahnel and Twaddle (1985) reviewed 20 published studies
`in the literature on ER status of synchronous primary and
`secondary concurrent breast
`cancer. Of
`the
`516 cases
`reviewed, the average discordance rate was 18%. The study
`also reported that changes between primary and metastases
`could be either ER positive changing to negative or vice
`versa. In addition, it was also noted that when both primary
`and metastases provided ER positive tumours the concentra-
`tion of ER could be higher or lower in metastases. Hahnel
`and Twaddle (1985) also reviewed ER status in sequential
`primary and secondary breast carcinoma paired biopsies in
`405 cases. In 18 studies reviewed they found a 21% major
`discordance rate. In these asynchronous tumours the concen-
`tration of ER was higher in the primary tumour or the
`metastasis in an equal number of cases. Other studies have
`shown similar results (Bishop, 1982; Peetz et al., 1982; Har-
`land et al., 1983).
`Studies of ER in paired primary and metastatic tumours
`have a discordance rate that can be explained by the sources
`of variation in ER measurements discussed above. The most
`striking findings are that the rate of discordance between the
`primary tumour and metastases is particularly low and that
`any changes in ER status between primary and metastases
`can be either ER positive to ER negative or vice versa. These
`findings argue strongly against phenotypic drift of ER
`(positive to negative) during the natural history of the un-
`treated disease.
`
`That phenotypic drift does not occur between primary
`invasive cancer and metastatic disease raises the issue of
`whether ER expression changes from preinvasive to invasive
`breast cancer. Studies of ER in ductal breast carcinoma in
`situ (DCIS) have reported positivity in between 32% and
`80% of tumours depending on the cut-ofl level chosen for
`positivity (Giri et al., 1989; Malafa et al., 1990; Bur et al.,
`1992; Pallis et al., 1992; Soomro et al., 1992; Poller et al.,
`1993; Wilbur and Barrows, 1993; Murphy et al., submitted).
`The positivity rates for DCIS are similar to reported rates for
`invasive breast cancer, which argues against phenotypic drift
`as tumours progress biologically from preinvasive to invasive
`carcinoma. In the study that reported the highest rate of ER
`positivity, 80% in 100 cases of DCIS, only 38 had pure in
`situ cancer without associated invasive carcinoma (Bur et al.,
`1992). In the group of pure in situ cancers the ER positivity
`rate was 65% (25/38). In the group with associated invasive
`carcinoma the ER positivity rate was (91%). This finding by
`Bur et al. may be important as it suggests that invasion does
`not correlate with ER-negative tumours, rather if anything
`with ER-positive tumours. This challenges concepts of the
`comparative invasive potential of ER-positive tumours. How-
`
`ever, while there may be a statistical correlation between
`ER-positive tumours and invasion we know that virtually all
`ER-positive tumours contain a varying proportion of ER-
`negative cells. The relative importance of ER-negative or
`-positive cells or their interaction in tumour invasion requires
`further investigation.
`The expression of ER in normal breast tissue has also been
`examined. Walker et al. (1992) reported that ER negativity
`(<2% cells staining) was more common in the normal
`breast tissue of premenopausal compared to post-menopausal
`women. Normal ductal structures had a higher number of
`ER—negative cells (>50% ER negative) in premenopausal
`patients (88%) compared to post-menopausal
`(62%). The
`paper suggested that these ER-negative cells may be a dor-
`mant hormone-responsive cell population down-regulated by
`circulating oestrogens. A cut-ofi‘ level for ER positivity of
`2% cells for ER may still underestimate the low level of ER
`expression in normal tissue. In a recent publication Howell et
`al. (1994) reviewed the literature and reported that in 94% of
`normal breast
`tissue specimens one or more epithelial cells
`was reported as ER positive.
`In normal breast tissue ER-negative cells predominate over
`ER—positive cells in terms of numbers. This is in contrast to
`breast tumours in which 70% of tumours show expression of
`ER in >5% of tumour cells. Premenopausal patients in
`particular appear to maintain most normal breast epithelial
`cells in an ER-negative phenotype as assessed by immuno-
`cytochemistry. Walker et al. (1992) suggest that there is a
`physiological control of cellular ER negativity and certainly
`in premenopausal patients ER negativity increases during
`weeks 2—3 of the cycle (Markopoulos et al., 1988; Walker et
`al., 1992; Howell et al., 1994) just after the peak of serum
`oestradiol has been reached. However, it is clear that the vast
`majority of normal breast epithelial cells are phenotypically
`ER negative. This too argues against phenotypic drift from
`normal
`tissue through preinvasive to invasive and then
`metastatic tumour tissue.
`
`ER and Endocrine Therapy
`
`Laboratory data
`lines have been established
`Most human breast cancer cell
`from ER-negative rather than ER-positive tumours, presum-
`ably reflecting biological differences between such tumours
`important in establishing in vitro cell lines. ER-negative cell
`lines have not been reported to spontaneously change in
`culture and express ER. The most common ER-positive cell
`lines are MCF-7 and its numerous derivatives, T47D and
`ZR-75 and its sublines. It is striking that with these cell lines
`and in particular MCF-7, which is the most widely inves-
`tigated breast cancer cell
`line, that there are no reports of
`spontaneous change in ER phenotype when the cells are
`being passaged in serum-free or
`fetal calf serum (FCS).
`Moreover, even when selection for endocrine resistance in
`MCF-7 cells has been successfully achieved it would appear
`that in very few cases has this involved loss of ER (Van den
`Berg et al., 1989; Murphy et al., 1990).
`Other authors have reported loss of oestrogen sensitivity in
`T47D and ZR-75 cell lines (Daly and Dabre, 1990) and in
`T47D and LY2 (a derivative of MCF-7) cell lines (Mullick
`and Chambon, 1990), without loss of ER. Furthermore,
`in
`both T47D and LY2 structurally the ER was wild type
`(Mullick and Chambon, 1990). Another group started with
`ER-positive MCF-7 cells that were both oestrogen sensitive
`and inhibited by the partial anti-oestrogen,
`tamoxifen, and
`the specific anti-oestrogen, ICI 182,780; from this ‘parental’
`cell line various sublines have been established (Clarke et al.,
`1994). MCF7/LCC1 was derived from a variant MCF-7
`xenograft, MIII, which grows without E2 in nude mice but is
`sensitive to the mouse’s endogenous E2 in that ablation of
`ovarian function results in the tumour xenografts regressing
`(Yano et al., 1992). MCF7/LCC1,
`the ex vivo culture of
`MIII, is insensitive to E2 in vitro culture but is sensitive to
`tamoxifen and ICI 182,780. MCF7/LCC2 was derived from
`
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`LCCl cultures grown in increasing concentrations of tamox-
`ifen. It
`is therefore resistant
`to tamoxifen but not
`to ICI
`182,780 (Clarke et al., 1994). MCF7/LCC9 was derived from
`MCF-7 cells grown in the presence of ICI 182,780.
`It
`is
`resistant to [CI 182,780 with cross-resistance to tamoxifen,
`yet it remains sensitive to E2. These cell lines retain levels of
`ER expression similar to the parental MCF-7 line, despite
`successful selection for endocrine resistance (Brunner et al.,
`1993a,b).
`Two groups have reported in vivo experiments with MCF-7
`xenograft tumours in which the tumour growth was initially
`inhibited by tamoxifen but subsequently tamoxifen, which is
`known to have oestrogenic properties, stimulated tumour
`growth (Osborne et al., 1987; Gottardis and Jordan, 1988;
`Osborne et al.,
`1991). The ER was normal
`in these
`tamoxifen-resistant tumours (Osborne, 1993). Introduction of
`the specific anti-oestrogen ICI 164,384 or ICI 182,780 at that
`point inhibited the tamoxifen-stimulated growth (Gottardis et
`al., 1989; Osborne et al., 1994). It would appear that in the in
`vivo model too, resistance to tamoxifen does not involve loss
`of a functioning ER, demonstrated by the subsequent inhibi-
`tion of tumour growth by the pure anti-oestrogens.
`
`Clinical data
`
`ER and therapeutic response ER expression in primary
`breast tumours correlates strongly with response to first-line
`hormone therapy,
`the most commonly reported being the
`anti-oestrogen,
`tamoxifen. Response rates of between 30%
`and 65% have been reported in ER-positive tumours, wheth-
`er by ligand binding assays (McGuire et al., 1975; Walt et al.,
`1976; Roberts et al., 1978; Lippman and Allegra, 1980;
`Osborne et al., 1980; Paridaens et al., 1980; Campbell et al.,
`1981; Williams et al., 1987; Anderson et al., 1989; Robertson
`et al., 1989) or the newer monoclonal antibody-dependent
`EIA (Robertson et al., 1992) and the ICA (Jonat et al., 1986;
`McClelland et al., 1986a,b; Coombes et al., 1987; Hawkins et
`al., 1988; Robertson et al., 1992). Within the group of ER-
`positive tumours the response rate increases as the tumour
`ER concentration (McGuire et al.,
`1978; Lippman and
`Allegra, 1980; Osborne et al., 1980; Campbell et al., 1981;
`Williams et al., 1987; Anderson et al., 1989) or ER expres-
`sion (Coombes et al., 1987; Gaskell et al., 1989) increases.
`The response rate is also higher in tumours in which the ER
`is
`functional, as assessed indirectly by the expression of
`progesterone (PgR) (Brookes et al., 1980; Osborne et al.,
`1980; Brenner et al., 1988).
`The response rate for ER-negative tumours has varied for
`LBA between 0% and 17% (Walt et al., 1976; Lippman and
`Allegra, 1980; Osborne et al., 1980; Paridaens et al., 1980;
`Williams et al., 1987; Anderson et al., 1989), for EIA 8%
`(Robertson et al., 1992) and for ICA between 0% and 11%
`(Jonat et al., 1986; McClelland et al., 1986a,b; Robertson et
`al.,
`1992). As noted above PgR subdivides ER-positive
`tumours. A more powerful
`factor
`for
`subdividing ER-
`negative
`tumours
`is
`epidermal growth factor
`receptor
`(EGFR) expression, which is inversely related to ER expres-
`sion (Sainsbury et al., 1985; Toi et al., 1989; McClelland et
`al., 1993). ER-negative tumours which do not express EGFR
`are usually more responsive to primary endocrine therapy
`(66% response rate) compared with ER-negative/EGFR-
`positive tumours (5% response rate), although the degree of
`expression of EGFR (i.e. percentage of cells EGFR positive)
`did not affect
`the response rate or post-metastases survival
`(McClelland et al., 1993).
`In overtly ER-positive tumours ER and EGFR expression
`is mutually exclusive on individual tumour cells (Sharma et
`al., 1994).
`In overtly ER-positive tumours there exists a
`population of tumour cells (approximately 25%) that are ER
`negative/EGFR positive, a phenotype that
`in overtly ER-
`negative tumours is a marker of endocrine unresponsiveness.
`Lower objective response rates,
`increased static disease and
`tumour progression rates are found as the percentage of
`ER-negative cells (presumably also EGF R positive) increases
`in overtly ER-positive tumours.
`It may be that
`the ER-
`
`Oestrogen receptors in breast cancer
`JFR Robertson
`
`negative/EGFR-positive subpopulation of cells simply does
`not respond to endocrine manipulation, accounting for the
`higher tumour progression rate and also the poorer quality
`of response (static disease, partial remission) when it occurs
`in such tumours. However, individually some such tumours
`do undergo complete response and the precise cellular
`mechanism that may include
`response
`in ER-negative/
`EGFR-positive tumour cells is not clearly understood.
`The ER-negative/EGFR-positive cell population in overtly
`ER-positive tumours may be controlled indirectly through
`paracrine-mediated effects from the hormone-sensitive ER-
`positive/EGFR-negative tumour cells. ER-mediated pathways
`can initiate transcription of growth factors (e.g. transforming
`growth factor alpha) which interact with EGFR (Roberts et
`al., 1983). Other studies have shown that endocrine therapy
`can influence expression of both these receptors (Ewing et al.,
`1989). The dual receptor phenotype may not be irreversibly
`fixed. Alteration in the receptor expression may be an alter-
`native explanation why overtly ER-positive tumours with
`ER-negative/EGFR—positive cell subpopulations do some-
`times respond completely to endocrine therapy. Sharma et a1.
`(1994) have suggested that the mutually exclusive staining for
`ER or EGFR on individual tumour cells raises the possibility
`that ER and EGFR expression have either a common
`regulating mechanism or both pathways interact to regulate
`the expression of the other receptor. Either of these poss-
`ibilities may be relevant in controlling the growth of popula-
`tions of ER-negative/EGFR-positive cells.
`A number of factors are known to regulate the level of ER
`expression in human breast tumours and cell
`lines without
`involving permanent
`loss of ER. The potential
`interaction
`between ER and EGFR expression in subpopulation of cells
`does not negate the hypothesis that ER is a stable phenotype
`in breast tumours. However, the effect of endocrine therapy
`on the co-expression of these two receptors will be an inter-
`esting observation and one that is currently being evaluated.
`
`Effect of endocrine therapy on ER
`
`Studies of ER expression in sequential tumour biopsies from
`patients on tamoxifen have not reported consistent results.
`Tamoxifen has been calculated to have a half-life of 5.3 days
`(Wilkinson et al., 1980) and it is still measurable in patients’
`blood 6 weeks after stopping tamoxifen therapy (Fabian et
`al., 1981). In early studies virtually all
`the repeat
`tumour
`biopsies were taken with the patients on tamoxifen treatment.
`It was subsequently recognised that tamoxifen could compete
`with the labelled oestradiol in the LEA giving a false ER-
`negative result. Undoubtedly these early ligand binding
`studies using LBAs (Namer et al., 1980; Waseda et al., 1981;
`Taylor et al., 1982; Noguchi et al., 1988) contributed to the
`concept of ER-positive cells becoming tamoxifen and anti-
`oestrogen resistant by becoming ER-negative tumours and
`are therefore difficult to interpret.
`Monoclonal antibodies to ER made it possible to assess
`ER status of tumours even when patients were on tamoxifen.
`An early study reported that tumours biopsied after first-line
`endocrine therapy were as
`likely to be ER positive as
`tumours biopsied before first-line endocrine therapy (Coom-
`bes et al., 1987). This study gave indications even at this
`early stage that ER expression was a stable phenotype,
`although this was not commented on by the authors. One of
`the first studies to report on ER in sequential tumour biop-
`sies using ICA reported on 23 tumours biopsied before and
`during (1—4 months) tamoxifen therapy (Robertson et al.,
`1991). There was no significant difference in the ER expres-
`sion of the paired biopsies. Six tumours were negative on
`both biopsies; two patients had static disease and four had
`progressive disease. Seventeen were positive on initial biopsy:
`14 of these were positive on repeated biopsy and three
`negative. The clinical responses of these latter patients was
`not published in the original
`report but have now been
`reviewed.
`In the latter three patients one had a complete
`response, one a partial response and the third progressive
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`Oestrogen receptors in breast cancer
`JFR Robertson
`
`disease. Only I of the 16 patients with tumours ER positive
`on both biopsies had progressive disease after 6 months on
`tamoxifen therapy.
`In this particular patient
`the repeat
`biopsy was performed after 2 months on tamoxifen while the
`patient’s disease was static; progression of disease in this
`patient was diagnosed after 6 months’ tamoxifen treatment.
`In none of these patients were repeat biopsies taken at the
`time of disease progression. Another study reported no
`change in ER but this was on short-term tamoxifen therapy
`A median 21 days (range 6—65) (Clarke et al., 1993). A
`further study examining the etTect of short-term tamoxifen
`therapy (<1 month) in 19 patients also reported no change
`in ER expression (Murray et al., 1994). However, again few
`if any tumours would be progressing at
`the time of the
`second biopsy in the latter two studies. They therefore do not
`answer whether there is a change between tumour ER status
`pretreatment and at progression on tamoxifen.
`One study reported tamoxifen concentrations in serum and
`tumour tissue in patients with primary resistance (n= 16)
`and acquired resistance (n= 17). The authors commented
`that
`the percentage of tumours ER positive in these two
`groups were 37% and 88% respectively (Johnston et al.,
`1994). BR expression pretamoxifen was not
`reported,
`although the high expression of ER in the acquired resistance
`group supports the hypothesis that ER expression is stable.
`In a recent study of ER expression tumour biopsies were
`obtained from 37 patients pretreatment, after 6 weeks and
`after 6 months on tamoxifen therapy. On each sample an
`H-score was calculated =(percentage of cells staining with
`intensity of staining l><1)+(percentage of cells staining
`with intensity score 2 x 2) + (percentage of cells staining with
`intensity score 3 x 3). The range for H-score is 0—300. Three
`patients showed an l-I-score of zero on initial measurements
`and these remained unchanged on all sequential biopsies.
`One patient showed an I-I-score of 10 initially but the two
`subsequent measurements showed H—scores of zero. In three
`of these four patients the tumour progressed within 6 months
`and in the fourth stable disease was recorded for 1 month
`before tamoxifen was discontinued.
`In the remaining 33
`patients tumours that were ER positive before tamoxifen
`remained positive on sequential biopsies: ER expression was
`either down-regulated (though detectable) or unchanged in
`all three categories of partial response, static disease or prog-
`ressive disease (Table I). In 6 of the 33 patients who prog-
`ressed and in whom tumour biopsies were taken at the time
`of progression on tamoxifen, ER was still present by ICA.
`It
`is difficult
`to be certain whether the change in the
`percentage of ER-positive cells is as a result of marked
`down-regulation of ER in previously positive cells or whether
`the balance between ER positive and ER negative has
`changed. for example because of apoptosis in ER-positive
`cells. In some tumours there is a decreased expression of ER
`on the biopsy after 6 weeks' tamoxifen and this is maintained
`at 6 months. If the decreased expression of ER at 6 weeks
`was as a result of individual cells changing from ER positive
`to ER negative or even as a result of an uncontrolled pro-
`gressive growth of ER-negative cells, one would not expect to
`see such tumours going on to a partial response at 6 months
`as many achieve. The clinical results suggest that the change
`in ER expression is caused by a down-regulation mechanism.
`
`that
`that ER-positive tumours
`findings
`These clinical
`become tamoxifen resistant do not lose ER expression are in
`keeping with the laboratory data described above and have
`implications for our understanding of acquired resistance to
`tamoxifen. While ER status predicts for sensitivity or insen-
`sitivity to first—line endocrine therapy. it appears to play little
`or no part in predicting or determining acquired resistance.
`This implies that the mechanisms of primary endocrine insen—
`sitivity and acquired (secondary) endocrine resistance are
`difierent. The former appears to be mediated via the ER (or
`lack of it), the latter not. The second point arising from the
`data and consistent with the point above is
`that
`these
`findings explain the clinical studies reporting that tamoxifen
`resistance does not necessarily mean complete endocrine
`resistance.
`In a
`study of the synthetic progestogen, megace, 97
`patients had tumours that
`initially responded or remained
`static on tamoxifen and then subsequently progressed, of
`whom 60 (62%) were reported to show a further period of
`response or static disease on second-line endocrine therapy,
`megace. In contrast. of 66 patients whose tumours progressed
`de novo on tamoxifen. only 17% showed an objective res-
`ponse or static disease on megace (Robertson et al., 1989).
`Response to megace was better predicted by response to
`first-line tamoxifen than by tumour ER status. Similar res-
`ponse rates following tamoxifen therapy have been reported
`for
`second-line
`aromatase
`inhibitor
`therapy in
`post-
`menopausal patients (Smith et al., 1981; Buzdar et al., 1982;
`Harvey et al.. 1982; Kaye et al., 1982; Murray and Pitt, 1982)
`and for oopherectomy in premenopausal patients (Margreiter
`and Wiegele, 1984; Sawka et al., 1986).
`Two clinical studies have been reported using the specific
`anti—oestrogen ICI 182,780. In the first study patients with
`primary operable (Stage I/Il) breast cancer were treated with
`ICI 182,780 for 7 days between diagnosis and definitive
`surgery (DeFriend et al., 1994). Patients were randomised to
`receive no treatment (n= 19), 6mg of ICI 182,780 daily
`(n=21) or
`18 mg of ICI 182,780 daily (n=16). Tumour
`specimens were available before randomisation to either no
`treatment or to ICI 182,780 and from the resected tumour at
`definitive surgery. There was down-regulation of ER on ICI
`182,780 both at the 6 mg and the 18 mg dose. At the higher
`dose of ICI 182.780 (18mg day") five out of ten tumours
`showed absence of ER expression immunocytochemically in
`the primary tumour after 7 days’ treatment (Nicholson et al.,
`1994). The majority of ICI 182,780-treated tumours therefore
`continued to express ER, although at reduced levels. In the
`five tumours that did not express ER, it is much more likely
`that after such short-term treatment the absence of ER exp-
`ression is as a result of down-regulation rather than true loss
`of ER. Down-regulation of ER can be induced in vitro by
`short-term treatment of ER-positive MCF-7 cells by pure
`anti-oestrogens (Nicholson et (11., 1994) without actual long-
`term loss of ER as already noted (Brunner et al., 1993a,b;
`Clarke et
`(11., 1994). Similarly, down-regulation was also
`noted for oestrogen—inducible gene products PgR and p82.
`These findings are qualitatively similar to those reported with
`tamoxifen except that reported down-regulation on tamox-
`ifen was after 6 weeks. Nicholson reported that the fall in ER
`expression after 7 days on 18 mg of ICI 182,780 was greater
`
`Table I Changes during tamoxifen therapy of ER expression in 33 tumours ER positive on
`pretreatment biopsy (repeat biopsy on tamoxifen vs pretreatment)
`Time (months) from pretreatment to repeat biopsy
`
`<6 months
`6 months
`
`UICC assessment at 6/12
`
`Change in ER Expression
`Down-regulated (but present)
`No change
`Up-regulated
`
`PR
`3
`
`3
`—
`—
`
`SD
`4
`
`3
`1
`
`PD
`1
`
`—
`1
`
`PR
`13
`
`9
`4
`
`SD
`7
`
`5
`2
`
`PD
`5
`
`3
`2
`
`PR, partial response; SD. static disease; PD, progressive disease.
`
`AstraZeneca Exhibit 2009 p. 4
`
`

`

`m9
`
`Oestrogen receptors in breast cancer
`JFR Robertson
`
`than after tamoxifen therapy. The tamoxifen-treated tumours
`were essentially the same group of tumours reported by
`Clarke et a]. (1993) when the median duration of tamoxifen
`was 21 days w there was no significant down-regulation of
`ER by that time. One explanation for the reported differences
`between tamoxifen and [CI 182,780 could be the markedly
`different affinity of the two compounds for ER.
`It may
`therefore take longer for tamoxifen, which binds less avidly
`-to the ER,
`to induce down-regulation.
`The second study with ICI 182,780 was a phase II study to
`assess therapeutic efficacy (Howell er al., 1995). Patients who
`had previously received tamoxifen as adjuvant therapy or as
`initial therapy for advanced disease and deemed to have been
`tamoxifen responsive were entered into the study. A total of
`13/19 (69%) showed objective response or static disease on
`ICI 182,780, 12/19 (63%) for more than 6 months’ duration.
`These second-line response rates are in keeping with those
`referenced above for
`second-line megace or aminoglute-
`thamide in post-menopausal patients or ovarian ablation as
`second-line in premenopausal patients. With the median
`duration of response not yet having been reached at
`18
`months (Howell et al., 1995) early indications are that ICI
`182,780 may produce a longer duration of response than
`megace or aminoglutethamide, with the possibility of a fur-
`ther
`response if megace or aminoglutethamide is subse-
`quently introduced. However, this will require co